wisconsin center for dairy research

University of Wisconsin—Madison1605 Linden Dr.Madison, WI 53706-1565

608/262-5970fax 608/262-1578http://www.cdr.wisc.edu

CDR

Center for Dairy ResearchWisconsin

annual report 1999

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CDR Annual Report 1999

CDR Annual ReportPublished March 1, 2000, by the Wisconsin Center for Dairy Research.

Our annual report is a technical overview of CDR funded research and otherCenter activities during fiscal year 1999. This document was prepared fororganizations funding CDR and for fellow dairy researchers. Although it

describes projects in progress and interpretations of data gathered to date, it isnot a peer-reviewed publication.

Please seek the author's written consent before reprinting, referencing, orpublicizing any reports contained in this document.

For more information call Karen Paulus at (608) 262-8015.(E-mail: [email protected])

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Overview

ContentsChapter 1, Milkfat

Investigation of baked milkfat flavor development in milkfat ingredients for thebakery and food industries ..................................................................................................................................3

Improvement of functionality, flavor, and stability of butter and milkfat fractions ........................... 9

Use of immobilized lipases to prepare dairy products enriched in conjugatedlinoleic acid (CLA) ............................................................................................................................................... 10

Using immobilized esterases/lipases to modify the composition of milkfat ................................... 15

Determination of caloric bioavailability and apparent lipid digestibility ofliquid milkfat fractions ....................................................................................................................................... 18

Milkfat applications research program ......................................................................................................... 19

Use of butterfat fractions and emulsifiers in dairy-based reduced-fat spreads .............................. 21

Rheological and structural properties of dairy-based lipids .................................................................. 24

Effects of milkfat source and composition on crystallization kinetics ................................................ 29

Chapter 2, Cheese

Intensified flavors in Cheddar cheese and cheese ingredients for enhancedapplications in foods .......................................................................................................................................... 37

Characterization of interactions between ingredients and cheese constituentsfor improved functionality of fat-free processed cheese ........................................................................ 40

Improvement of Cheddar cheese quality through identification and characterizationof microbial enzymes responsible for the production or degradation of bitter peptidesin cheese ................................................................................................................................................................ 46

Succinate production by Lactobacillus casei: pathways responsible and developmentof strategies to control its accumulation. ..................................................................................................... 48

Glutathione and Cheddar cheese flavor development ........................................................................... 50

Growth of nonstarter lactic acid bacteria in reduced fat Cheddar cheese ....................................... 54

Optimizing the standardization of milk to manufacture 50% reduced fatCheddar cheese ................................................................................................................................................... 56

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Cheese applications program .......................................................................................................................... 61

Extending the cheese net paradigm to include economic evaluation andoptimization in cheese manufacture ............................................................................................................. 64

Technology for improving the flavor and consumer acceptability offat-free Cheddar cheese .................................................................................................................................... 65

Whey applications research program ............................................................................................................ 66

Dairy marketing and economics program ................................................................................................... 68

A multi-country analysis of household food demand: Implications for U.S.food exports (phase I) ....................................................................................................................................... 71

Development and application of a cheese shred/texture map delineated bycheese rheological, sensory and chemical analysis ................................................................................. 76

CDR specialty cheese applications program ............................................................................................... 77

Chapter 2, Section 2 Cheese Safety

Microbiological safety of reduced fat and fat free pasteurized process cheese products .......... 81

Safety/quality applications program ............................................................................................................. 83

Chapter 3, Fluid Milk

Identification and characterization of components of the proteolytic enzyme system of Lacto-bacillus helveticus which affect bioactive peptide accumulation ........................................................ 87

Application of milk powders in milk chocolate, butter and butter spreads ..................................... 88

Growth and biocontrol of enterotoxigenic Bacillus cereus in infant formula and processedcheese prepared with milk powder ............................................................................................................... 89

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Overview

Reports unavailable at publication date:

Cheese

Minimizing the watering-off of unripened lower fat and no fat Mozzarella cheeseCarol M. Chen

Pizza cheese II: Shelf-life evaluation and tailor manufacturing of pizza cheeseCarol M. Chen

Fractionation of κ-casein glycomacropeptide from whey for nutraceutical uses: scale up of theion exchange membrane technologyMark. R. Etzel

Improved quality of shredded cheese—antimycotics, oxygen scavengers and modified atmo-sphere packagingJoseph E. Marcy

Effect of water distribution on physical properties of pizza cheese and LMPS Mozzarella cheeseduring early stages of maturation and freezing and thawingS. Gunasekaran

Investigating reasons for hardening of reduced-fat Cheddar cheese during heatingS. Gunasekaran

Effect of Water Distribution on Physical Properties of Pizza cheese and LMPS Mozzarellacheese during early stages of Maturation and Freezing and ThawingSundaram Gunasekaran

Large Amplitude Nonlinear Viscoelastic Behavior of Mozzarella Cheese During Twin-ScrewExtrusionSundaram Gunasekaran

“Whey Refinery” for producing proteins for beverages and nutraceuticals.Mark Etzel

Prevention of germination and growth by gas-forming Clostridium tyrobutyricum in high-pHcheesesSteven C. Ingham

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Alto DairyBeatrice CheeseChris Hansen LabsDairy Management Inc(DMI)Dean Foods Technical CtrForemost FarmsGist-brocades Dairy Ingredient GroupGrande CheeseHilmar CheeseInternational Flavors and Fragrances (IFF)Kraft Foods Technology CtrLand O’ Lakes IncPlymouth Cheese/DFARhodia IncSaputo CheeseSchreiber Foods IncSKW Biosystems IncWisconsin Milk Marketing Board (WMMB)

CDR’s Cheese Industry Team

CDR staffJ. Russell Bishop, director

Administration

Tom Szalkucki, administrative coordinatorCurtis BlevinsJoe BissonnetteCarmen HustonLisa LokkenJackie UtterSandra Sekel

Applications Staff

Carol Chen, cheese applications coordinatorKim Burrington, whey applications coordinatorBrian Gould, marketing and econ. coordinatorJohn Jaeggi, cheese applications coordinatorKerry Kaylegian, milkfat apps coordinatorJim Path, specialty cheese coordinatorJuan Romero, analytical coordinatorMarianne Smukowski, safety & qual.coordinator

Program Area Coordinators

Research Staff

Gene BarmoreAmy DikkeboomRani Govindasamy-LuceyBill HoeslyKristen HouckMark JohnsonKaren SmithWilliam TricomiMatt Zimbric

Communications Staff

Mary Thompson, coordinatorJoanne GauthierTim HogensenKaren Paulus

Cheese— Robert Lindsay, Dept. of Food Science, University of WI-MadisonMilkfat—Rich Hartel, Dept. of Food Science, University of WI-MadisonWhey—Mark Etzel, Dept. of Food Science, University of WI-MadisonQuality and Safety—Eric Johnson, Food Research Institute, University of WI-Madison

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Overview

Barmore, Gene (608)262-5798 [email protected], Rusty (608)265-3696 [email protected], Curtis (608)265-6194 [email protected], KJ (608)265-9297 [email protected], Carol (608)262-3268 [email protected], Amy (608)265-2271 [email protected], Joanne (608)263-1874 [email protected], Brian (608)263-3212 [email protected], Rani (608)265-5447 [email protected], Bill (608)263-3215 [email protected], Tim (608)265-2133 [email protected], Kristen (608)265-6346 [email protected], Carmen (608)262-3416 [email protected], John (608)262-2264 [email protected], Mark (608)262-0275 [email protected], Kerry (608)265-3086 [email protected], Lisa (608)265-9113 [email protected], Jim (608)262-2253 [email protected], Karen (608)262-8015 [email protected], Juan (608)265-9242 [email protected], Sandra (608)262-5970 [email protected], Karen (608)265-9605 [email protected], Marianne (608)265-6346 [email protected], Tom (608)262-9020 [email protected], Mary (608)262-2217 [email protected], Bill (608)262-1534 [email protected], Jackie (608)265-2117 [email protected]

Zimbric, Matt (608)265-2271 [email protected]

CDR Directory

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Our Mission StatementThe Wisconsin Center for Dairy Research will serve as a

national leader in strategic research to improve thecompetitive position of the dairy industry by linking Center/

University faculty, staff, students and the dairy/food industriesto address key issues resulting in transfer of technology and

communication of information.

Investigation of baked milkfat flavor development in milkfat ingredients for the bakery and food industries ..............3

Improvement of functionality, flavor, and stability of butter and milkfat fractions ...................................................................9

Use of immobilized lipases to prepare dairy products enriched in conjugated linoleic acid (CLA) ................................ 10

Using immobilized esterases/lipases to modify the composition of milkfat ........................................................................... 15

Determination of caloric bioavailability and apparent lipid digestibility of liquid milkfat fractions ................................. 18

Milkfat applications research program .................................................................................................................................................. 19

Use of butterfat fractions and emulsifiers in dairy-based reduced-fat spreads ...................................................................... 21

Rheological and structural properties of dairy-based lipids .......................................................................................................... 24

Effects of milkfat source and composition on crystallization kinetics ........................................................................................ 29

Milkfat

chapter 1

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3

Milkfat

FINAL REPORT

Investigation of baked milkfat flavor development inmilkfat ingredients for the bakery and food industriesPersonnelRobert C. Lindsay, professor, Dept. of FoodScience

FundingWisconsin Milk Marketing Board

DatesFebruary 1993—January 1995(Extended to 6/30/96)

Objectives1. To establish a sensory panel trained in therecognition and descriptive sensory analysis ofbaked milkfat flavor in baked products (NZDRI).

2. To establish a model baking system for theassessment of baked milkfat flavor in baked goods(NZDRI).

3. Using the model baking system, undertake andcorrelate chemical and sensory analyses to iden-tify the key compounds responsible for bakedmilkfat flavor in baked products (UW andNZDRI).

4. To define the specific chemical reactionsinvolved in the development of enhanced bakedmilkfat flavor (UW and NZDRI).

5. To formulate and prepare milkfat based ingre-dients which provide enhanced baked milkfatflavor (UW and NZDRI).

Summary

Establishment of Sensory Analysis ProceduresThe NZDRI team established a sensory paneland appropriate guidelines for descriptive sensoryanalysis procedures for evaluating baked-milkfatflavors in baked products. Using an ADL-typedescriptor profile generation technique, theNZDRI team identified aroma and flavor charac-teristics of butter shortbreads (called buttercookies in North America) which they felt theirtrained panelists could describe baked butterflavors using 9-point intensity scales. The NZsensory descriptor terms and their technicaldefinitions are listed in Table 1.

Concurrently, the UW-Madison team usedexperienced panelists to evaluate butter cookies(shortbreads), and found that panelists haddifficulty in meaningfully separating the flavorsinto the flavor categories proposed by theNZDRI team. Thus, at the UW, a ballot wasadapted which permitted a focus on the primarykey flavor attributes of baked-butter flavor inbutter cookies.

The UW primary scale for characterizing thedistinct flavor of baked butter as selected at“baked butter flavor intensity” (7-point scale) andmilkiness, a term that was ultimately returned tothe baked butter flavor intensity. Because subtleflavors with less desirable connotations (animal,cowy, etc.) were encountered in evaluations of

Buttery cooked butter, melted butter, overcooked butter, and butterscotch.Oxidized rancid butter, dripping, and plasticky; stale.Vegetable oil food cooked in oil, salad oil, margarine, beany, and oily.Sweetness sweetness provided by sucrose.Saltiness saltiness provided by sodium chloride.Cereal/Wheatin bran, stale bran, floury, uncooked flour, oat cereal, husky, porridge.Browned flour flavor of flour on bottom of scones,

browned flour from greasing a baking dish.Caramel/Butterscotch caramelized sweetened condensed milk.Milk Solids condensed milk, milk powder, and creamy.Cultured cream cheese, sour cream, and cheesecake.

Table 1. Aroma and flavor characteristics of butter shortbreads

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concentrated butter flavor ingredients, a generaloff-flavor intensity scale was incorporated rou-tinely. For broad scale sampling from unguidedexperiments, it was found that use of the broaderrange of terms assigned at NZDRI gave morecomplete information. Thus, standardized proce-dures were developed and adopted.

Establishment of a model baking system forbaked butter flavorsThe establishment of a standardized, modelbaking system was pursued simultaneously withthe development of descriptive sensory analysisprotocols, and the formulas evaluated by NZDRIserved as the basis for selection of the modelshortbread baking systems at NZDRI and UW-Madison. This was a simple standard, shortbread(butter cookie) formulation consisting of thefollowing:

The NZDRI formula (i.e., for very low saltbutters) applied directly to US butter gaveshortbreads that were too salty because of typicalUS salting rates of about 2%, and thus omission ofadded salt brought the saltiness into an appropri-ate intensity for UW-Madison formulations.

When shortenings or anhydrous milkfat weresubstituted as part of the experimental formula-tions, the basic formula was adjusted to accom-modate the shifted constituents according to thefollowing:

Because skim milk powder was found to containimportant precursors (UW-Madison—alkyl phenolconjugates) for baked butter, the UW-Madisonformulation omitted this ingredient to preventinterference from uncontrolled introduction ofthese important flavor substances.

Mixing protocols for the cookie dough alsoaffected the characteristics of the cookies, andwere standardized. The New Zealand protocoladopted an aerated dough (50-150 rpm) in mixer(to a fixed density) which produced a Danish styleshortbread when dispensed with a piping bag.The UW-Madison protocol used a less vigorousmixing (low speed-Hobart) followed by flatteningthe dough which produced a more dense, disc-shaped butter cookie of the Scottish shortbread-type. Shortbread cookies were baked at 205°Cfor 16 min, and the length of time was varied tomodulate the degree of Maillard browningoccurring. Both styles of preparation yieldeddistinct baked-butter flavors when prepared withregular butter, and were very applicable forstandardized experimentation.

Identification of key flavor compoundsBoth the NZDRI and the UW carried out flavorchemistry studies on the volatile flavor com-pounds in heated butter and butter cookies whichwere isolated by headspace, vacuum steamdistillation, carbon dioxide and solvent extrac-tions. Compounds identified by gas chromatogra-phy and mass spectrometry included free fattyacids, methyl ketones, γ- and δ-lactones, alde-hydes, esters, furanones, pyrazines, and pyrroles,the latter of which are Maillard browning prod-ucts.

Two other groups around the world also havebeen researching heated butter flavors (G.Reiniccius, U. of Minnesota, Personal Communi-cation; and P. Schieberle, U. Wuppertal, Personal

Communication). Both these groupshave used similar isolation methods,but also used aroma-dilution extractanalysis to determine the most-potentaroma compounds in flavor isolates.However, unfortunately the methoddoes not address the key aspect offlavor quality (i.e., identifiable flavorcharacteristics), and the compounds .They have found essentially the samegroups of compounds identified at

Ingredients % of Formula % of Formula NZDRI UW-Madison

Butter 36.1 36Sugar 12.9 14Salt 1.0 —Flour 49.9 50

Ingredients % of Formula % of FormulaNZDRI UW-Madison

Shortening/Milkfat 29.1 30.0Skim Milk Powder 0.7 ——Water 5.8 5.0Sugar 13.0 14.0Salt 1.0 1.0Flour 50.0 50.0

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Milkfat

NZDRI and the UW, and they have indicatedseveral that should contribute to heated butterflavors according to their methods. These included-decalactone (coconut-like), (E)-2-nonenal(green, fatty), (Z)-2-nonenal (fatty, tallowy), 4,5-epoxy-(E)-2-decenal (metallic), 2-acetyl-1-pyrroline (roasty), (ZZ)-2,4-decadienal (fatty,tallowy), skatole (fecal), (E)-6-dodecenolactone(fruity), 1-octen-3-one (mushroom), 2,5-dimethyl-4-OH-3(2H)-furanone, g-octalactone (peach-coconut), 1-hexen-3-one (ketone), and methional(cooked potato).

At the UW-Madison, in-depth flavor chemistrystudies were pursured to investigate the chemicalreactions involved in the development of bakedbutter flavors. Since elevated heat is involved inbaking (205°C), the role of heat-acceleratedoxidation of butterfat on baked butter flavor wasinvestigated. When variously oxidized (none toodistinctly) milkfat was used in butter cookies, thelower levels and less oxidized samples gaveblended desirable, browned flavors, but higherlevels and more extensively oxidized butterfatsresulted in oxidized flavors. Notably, the truebaked butter flavor in butter cookies was notintensified by additions of oxidized milkfat.

New literature reports from Germany had indi-cated that some new volatile compounds hadbeen found in oxidized milkfats, and that thesemight be important in butter flavors. Thesereports had indicated that milkfat contained 3-methyl-2,4-nonadione (MND) and bovolidewhich have potent flavors, and they were derivedvia oxidation from minor furanoid fatty acids inmilkfat. However, isolation of crude fractionscontaining the furanoid fatty acids from milkfatusing an approach involving methanolysis andurea adduct fractionation followed by oxidationdid not provide flavor concentrates that intensi-fied butter or baked butter flavors.

Role of alkyl phenols in baked butter flavorsResearch at the UW prior to the initiation of thisproject had revealed that alkyl phenols present inskim milk caused milky flavors, and that theybroadly greatly enhanced dairy flavors. Thesedairy-type flavors were greatly intensified in milk-based systems when free forms were releasedfrom bound forms by conjugase enzyme activity.(Lopez and Lindsay, J. Agric. Food Chem.41:446. 1993.) When concentrations of alkyl

phenols were increased sufficiently, their flavorcontributions were perceived as cowy, but never-theless unmistakably characteristic of milk anddairy products, and were suspected as contribu-tors in some manner to heated butter flavors.

Since the general survey approach to the flavorchemistry of baked butter flavors did not yield aviable direction for further intensive research,attention at the UW-Madison turned to investigat-ing the alkyl phenol flavor system. Remarkably,adding alkyl phenols to butter cookies confirmedthat they provided the missing note—the truebaked butter flavor in butter cookies.

Chemical reactions in the baked butter flavorsThe UW research then centered on studying thealkyl phenols that occur in butter, and definingthe specific chemical reactions involved in thedevelopment of baked butter flavors. The free,active alkyl phenols are high boiling in nature,and are present in dairy products at very lowparts per billion (ppb) concentrations. However,higher concentrations are present in bound ormetabolically-conjugated forms which can bereleased by using the conjugases, sulfatase,phosphatase, and glucuronidase. Studies werethen carried out using conjugase treatments ofbutter and churning cream, and these ingredientprototypes were evaluated in butter cookies

The conjugase enzymes that are commerciallyavailable are crude enzyme preparations withgood specific activity (for example, that fromHelix pomatia), but they also contain other en-zymes, including some lipase activity whichreleases free fatty acids. Thus, after specifiedreactions times, it was necessary to heat thesamples to inactivate the enzymes. Because of thisneed, it was also discovered that applying theheat treatment in the preparation of intensely-flavored butter ingredients gave more full, sweetbaked butter flavors than without the heat treat-ment.

The supporting flavors for the baked butter alkylphenol flavor notes in the baked butter flavorconcentrates were quite familiar, however, andincluded lactones, methyl ketones and volatilefree fatty acids. It is well-established that heatingmilkfat yields several methyl ketones and lactonesfrom the hydrolysis of hydroxy- and keto-fattyacids present in milkfat. Similarly, using lipolyzed

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butter oil ingredients to provide butter-typeflavors is a well-established commercial practice.

The collaboration with the NZDRI researchersprovided several samples of New Zealand butter,buttermilk, and butter cookies for comparisonwith U.S. dairy products. These samples wereanalyzed with available quantitative (semi-quanti-tative) methods along with selected U.S. productsfor key baked butter flavor components.

The concentrations of methyl ketones as support-ing-flavor compounds for alkyl phenols in bakedbutter flavor ingredients are missing from un-treated U.S. butter, very low in N.Z. butter, butnotably elevated in a heated, baked butter flavoringredient. Similarly, the concentrations of keysupporting lactone flavor components are sub-stantially elevated in the baked butter ingredient.Contrary to what was hypothesized, crudeconjugase (H. pomatia) did not provide an activelipase, but instead the final heat treatment notablyelevated some of the longer-chain free fatty acids,but did not elevate the level of butyric acid.These results are useful in understanding theabsence of rancid, butyric acid flavor notes in theintensely-flavored baked butter flavor ingredients.The analytical data illustrate the notable elevationof the alkyl phenols in the enzymically-derivedbaked butter flavor ingredient compared to thestarting anhydrous milkfat employed in its manu-facture.

Analysis methods for alkyl phenolsThe initial methods that were available for analy-sis of alkyl phenols were extremely time-consum-ing, and subject to experimental error because ofthe nature of the flavor compounds and theirconjugate precursors. Basically, the initial meth-ods available were adsorptions, extractions,distillations, high-sensitivity capillary gas chroma-tography, and SIM mass spectrometry.

The literature indicated that some solventscaused alkyl phenol conjugates to spontaneouslyhydrolyze. Thus, solvent selection for extractionsused in anlaysis was critical because functionallevels of flavor-active free alkyl phenols in dairyproducts and ingredient were extremely low (<20ppb) while the concentrations of non-flavorfulbound alkyl phenols reached levels up to 2,000 to3,000 ppb. As a result, any incidental hydrolysiswould grossly distort the values obtained fromsubsequent instrumental analysis. A detailed

study (Han and Lindsay, 1995. J. Food Science,60:1100) provided the basic information neededto permit the selection of solvent systems forextracting free alkyl phenols from alkyl phenolflavor concentrates and dairy products. Thesewere avoidance of polar solvents held againstacidic aqueous phases, and instead usingdiethylether at nearly neutral pH along withexcess water, saturation with sodium chloride, atambient temperatures, and short solvent-exposuretimes.

Similarly, a detailed study was conducted for thedevelopment of a rapid high performance liquidchromatography analysis procedure, and themethod is described in detail in a chapter in Q.Zeng’s thesis (1996). Generally, free alkyl phenolsare extracted from the sample, and then areanalyzed by fluorescence detection usingprecolumn derivatization with dansyl chloride.When analysis of total alkyl phenols (free +conjugate-bound) is desired, the extracted conju-gates are first hydrolyzed by a combination ofenzyme and acid hydrolysis before derivatization.The detection limit of dansylated alkyl phenolswas about 0.2 nanogram absolute amount. Cali-bration curves were linear for dansylated alkylphenols from the detection limit to about 60nanograms for phenols and cresols injected, and80 nanograms for other alkyl phenols injected,and overall the correlation coefficient was 0.98 orhigher. The reproducibility of the method wasfrom 5.5 to 18%, and the recovery of alkylphenols from skim milk ranged from 78-106%.

Our basic approach in this segment of the projectwas to use all-dairy starting materials except forenzymes and processing chemicals. Additionally,the intensely baked-butter flavor ingredients werestructured to assume the form of a high-fat ingre-dient, such as anhydrous milkfat, recombinedbutter, or similar product. In these forms theywould represent new generation butter ingredi-ents.

Several approaches to the preparation of intenselyalkyl phenol-flavor ingredients were used, includ-ing conjugase treatment of cream, milk, butter,nonfat milk solids, whey solids, and buttermilksolids. All of these ingredients provide a reservoirof alkyl phenols in the form of conjugates, but thedried products provide much more concentratedsources.

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Milkfat

The difficult to describe “baked butter” flavorfound in authentic butter cookies was routinelydescribed as rich, milky, buttery. Generally, N.Z.butter yielded cookies with stronger baked butterflavors than those made with unmodified U.S.butter, and the concentrations of free alkylphenols typically present in butter cookies in thecookies correlated with the more intense bakedbutter flavors.

Intensely-flavored baked butter flavor concen-trate produced intensification of the milky flavorcharacteristic of milk chocolate. Similar intensifi-cation of the rich dairy-milky flavor attribute inbutter toffee confection was seen and the overallacceptability was also significantly improved. Thedata has shown to date that the alkyl phenolconcentrations must be carefully controlled atlevels below where they produce slightly cowy oranimal-like notes. The natural variablity of alkylphenol concentrations in dairy ingredients usedto prepare baked butter flavor concentratesmakes this a very formidable task. To achieveconstant flavor, the alkyl phenol profile must beblended to within some as yet undefined param-eters. However, the basic contributions of alkylphenols to baked butter flavors have now beenestablished and shown to produce desirablebaked butter flavors under controlled conditions.Further applications work should lead to over-coming the intensity and variability problemswith alkyl phenols in flavor concentrates, andcommercialization of concentrated baked buttermilkfat-based ingredients should quickly follow.

Publications/PresentationsHan, L.-H., and Lindsay, R. C. 1995. Stability ofMetabollically Conjugated Precursors of Meatand Milk Flavor Compounds in Various Solvents.Journal of Food Science 60:1100-1103.

Lindsay, R. C. 1992. The Chemistry of FlavoursAssociated with Milkfat. Presented to the NewZealand Dairy Research Institute Flavour Forum,Palmerston North, NZ, March 3-4, (Abstract).

Lindsay, R. C. 1993, Effects of Feeds on MilkFlavors. Presented at the Technical WorkshopParallels in Dairy Grazing in New Zealand andthe Midwest, UW Babcock Institute for Interna-tional Dairy Research and Development,AgResearch Corporation of New Zealand, andUSDA Forage Research Laboratory, Arlington,WI, August 25-28, (Proceedings)

The preferred method for manufacture of in-tensely baked butter flavor ingredients employsenzyme treating a solution of dairy (approxi-mately 30%) to effect the primary release of freealkyl phenols. This treatment is then followed bycombining equal amounts of the enzymically-treated dairy solids solution and anhydrousmilkfat, and then heating to effect extraction offreed alkyl phenols into the milkfat.

Alkyl phenols in dairy ingredients Analysis of dairy ingredients for free and conju-gated alkyl phenols revealed that considerablevariation occurred between samples from bothNew Zealand and the U.S. Presumably, thisreflects the feeding regime (New Zealand ispastured; U.S. is dry-lot) because alkyl phenolsare generally accepted to derive from feed ingre-dients although the mechanisms are still incom-pletely understood (R. Lindsay, UnpublishedResults; D. Rowan, NZDRI, Personal Communi-cation).

Data for levels of free alkyl phenols in U.S. andN.Z. butters show that N.Z. butter has higherlevels of flavorful free alkyl phenols, especiallythe summer N.Z. butter from pastured cows. Thiscontrast in concentrations of alkyl phenols be-tween U.S. and N.Z. butters is also observed forthe reservoir of bound alkyl phenols in buttersamples where the p/m-cresol levels are particu-larly higher than that found in dry-lot fed U.S.butter.

Further analyses of U.S. and N.Z. raw ingredients,including buttermilk powders and whey productsused in the preparation of baked butter flavorconcentrates, show similar trends of elevatedconcentrations in N.Z. pastured dairy products.This is especially note- worthy for the bound p/m-cresols in the N.Z. buttermilk powders. On theother hand, the 4(3)-ethylphenols are generallyhigher in the U.S buttermilk powders.

Influence of baked butter flavorIntensely alkyl phenol-flavor baked butter con-centrates have been prepared into a variety ofbutter-compatible foods, including butter, buttercookies, pound cakes, butter toffee, and milkchocolates, and their introduction intensifiedbaked butter flavors. Free-choice descriptivesensory paneling was used to evaluate selecteddairy or butter-compatible foods.

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Lindsay, R. C. 1996. Bake-Through Butter Flavor.Presented at the Wisconsin Center for DairyResearch Open House Program, University ofWisconsin-Madison, March, Madison (Abstract).

Lindsay, R. C. 1996. Butter Flavor Technologiesand Applications. Presented at the Milkfat Tech-nology Forum ’96, April 23, 1996, Madison; Alsoin: Proceedings of the Milkfat Forum ’96, Na-tional Milkfat Program Consortium, Madison,pages 59-63, (Text).

Lindsay, R. C. 1996 Milkfat Flavor. Presented atthe “Milkfat as a Food Ingredient Short Course,Center for Dairy Research, University of Wiscon-sin-Madison, October 22-23, Madison, (Slides &Course Information).

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Milkfat

INTERIM REPORT

Improvement of functionality, flavor, and stability of butterand milkfat fractionsPersonnelR. C. Lindsay, professor, Ann Han, graduatestudent, and Sungjoon Jang, graduate student,Department of Food Science, University ofWisconsin-Madison

FundingDairy Management Inc.

DatesJuly, 1997—December, 1999 (Extended to June,2000)

Objectives1. To develop an expanded understanding of theflavor properties of alkylphenols in butter andmilkfat fractions to enhance the functionality ofbutter and milkfat fractions in food ingredientapplications.

2. To develop information on the biochemicalorigin of alkylphenols in milk and butter throughstudies of alkylphenol precursors and theirformation in milk.

3. To investigate the oxidative stability of milkfatfractions, and determine the effectiveness ofantioxidant strategies for extending the stabilityand flavor quality of milkfat fractions and recom-bined butters.

Summary

Temperature-gradient milkfat fractionationproduces a range of ingredient milkfats thatexhibit enhanced physical properties that areapplicable for specific applications, includingcold-spreadable butter and more temperature-resistant pastry butters. Because of the inherentnature of temperature-gradient milkfat fraction-ation, soluble functional components of butter,including flavors, remain in the melt as crystallinefractions are harvested. Thus, higher meltingfractions are characterized by low flavor intensi-ties while lower melting fractions contain adisproportionate amount of the initial butterflavor.

In order to compensate and enhance the flavorsin the higher melting fractions, the functionalities

of a variety of butter-derived flavor componentswere investigated, including lactones,methylketones and short-chain fatty acids, fortheir ability to interact with alkylphenolflavors. From these studies, the role ofalkylphenols in enhancing accurate sweet creambutter flavors was reinforced, and research hasbeen centered on developing a further under-standing of the flavor contributions ofalkylphenols to butter flavors.

The flavor properties of naturally-occurringalkylphenols (9 isomers) have been determined ina variety of media, including water, butteroil, andsalt and sugar solutions. At low concentrations(0.1 to 10 parts per billion), the phenolic flavorsassocated with neat or pure compounds are notdetectable, but instead a key butter flavor-enhanc-ing effect is provided. These alkylphenols inten-sify butter flavors when present at appropriateconcentrations through an apparently previouslyunrecognized mechanism.

This flavor effect is distinctly different from theumami flavor of monosodium glutamate, but thenewly recognized flavor effect potentiates umamisensations. Combinations of alkylphenols andother milkfat flavor compounds provide en-hanced flavors to milk chocolate, baked goods,and butter ingredients. Research is continuing onthe flavor effects of the alkylphenols when intro-duced into the foods listed above.

Studies employing controlled feeding of cow andsheep ruminant models have been used to exam-ine the importance of individual feed constituentsupon the biochemical formation of alkylphenolsin milk. Results have shown that the type of dietis influential in determining the specificalkylphenols that occur naturally in milk, andtherefore the flavor functionality of butter de-pends on the feed. High forage or pasture dietscontribute much lower levels of the key flavorfunctional alkylphenol isomers than certainconcentrate diets. Therefore, an opportunity toenhance specifically enhance butter flavorintensity and functionality is provided throughconcentrate selection.

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PersonnelCharles G. Hill, Jr., professor of Chemical Engi-neering, Hugo S. Garcia, visiting scientist, associ-ate professor, Department of Food Technology,Centro de Graduados, Instituto Tecnologico deVeracruz, Veracruz, Mexico; Prima Sehanputri,research assistant, Colin Crowley, researchassistant, Jose Arcos, postdoctoral fellow, KurtKeough, undergraduate, Carlos Torres,postdoctoral fellow, Department of ChemicalEngineering


DatesJuly 1996—December 1999

Objectives1. To effect the synthesis of glycerides containingresidues of conjugated linoleic acid (CLA) usingimmobilized lipases (e.g., Candida sp. orRhizomucor miehei). Both batch and continuousflow reactor configurations will be employed tobring about these reactions. Two synthetic routesare being investigated, viz., a] direct synthesis ofthe glyceride via the reactions between CLA andglycerol to obtain mixtures of monoacylglycerides(MAG), diacylglycerides (DAG) andtriacylglycerides (TAG); and b] directinteresterification (acidolysis) of butteroil orbutteroil fractions with free CLA.

2. To generate the experimental data necessary tocharacterize the rates of the reactions of interestover a limited range of conditions. The resultingrate expressions will be employed to developmathematical models for process simulation,optimization and economic analysis. Such infor-mation will be necessary to conduct a preliminaryassessment of the commercial feasibility of pro-ducing butteroils enriched in CLA. The resultantbutteroils could be used in the formulation ofdairy products designed for consumers seekingfoods with both nutritional and medical/healthbenefits.

3. To assess whether the results obtained in thispreliminary study indicate that more comprehen-sive studies are merited. The expanded workwould encompass such aspects as studies ofexpanded ranges of experimental conditions (e.g., type of reactor, enzyme source, temperature,pH), nutritional/animal feeding work, determina-tion of physical and functional properties, andengineering/economic analyses. The futurestudies would provide the information necessaryfor implementation of this technology for com-mercial production of dairy products containingglycerides enriched in CLA residues.

Summary

Triacylglycerols containing conjugated linoleicacid residues have properties that make thesematerials appropriate for use in nutraceuticals asanti-oxidants, anti-atherogenic and anti-carcino-genic agents. These attributes make CLA veryattractive for inclusion in the human diet, espe-cially since the consumption level necessary toachieve efficacy is anticipated to be only ca. 3.5grams per day for a 70 kilogram person. A par-ticularly attractive route for incorporation of CLAin the human diet involves modification ofconventional dairy spreads (or cheese) by eitherreplacement of some of the fatty acid residuesnaturally present in the triglycerides whichconstitute milkfat or by addition of syntheticglycerides containing CLA residues to butter,butterine, and other dairy products which are richin milkfat. Studies pursuing the avenues estab-lished by the proof-of-concept work reported inearlier progress reports confirmed the technicalviability of several routes proposed for incorpora-tion of CLA residues in triacylglycerols.

Our efforts in the past year have focused on twoprimary activities:1. Studies of the kinetics of the reactions of CLAwith glycerol and butterfat in the presence ofimmobilized lipases in order to determine thereaction conditions and types of reactors that willbe most useful for commercial implementation ofthis technology. We have persuasively demon-

FINAL REPORT

Use of immobilized lipases to prepare dairy productsenriched in conjugated linoleic acid (CLA)

11

Milkfat

strated the technical feasibility of both routes forobtaining glycerides that can be incorporated inany dairy product that contains milkfat. Thiswork will continue past the expiration of theCDR grant. Publications in archival journals andpresentations at professional society meetings arebeing used to disseminate the results that we haveobtained to date. It is important to note thatsubstantially higher levels (by a factor of 10 to100) of CLA residues in milkfat acylglycerols canbe obtained by either of these immobilizedenzyme routes than can be achieved by modifica-tion of the diets of dairy cows.

2. Studies of the production of CLA from linoleicacid using a bioconversion process involvingLactobacillus ruteri. To date we have demonstratedthat we can accomplish this isomerization reac-tion in a manner which gives significant improve-ments in the yield of the desired biologicallyactive cis-9, trans-11 isomer of CLA. Work in thisarea is continuing with a view towards obtainingfurther improvements in the yield of the bioactiveisomer. This phase of the research will be fol-lowed by studies (with concomitant economicimplications) involving immobilized cells. Inaddition, we have fabricated two chemostats thatcan be operated either independently or in aseries flow configuration. These units have beenemployed in studies of the kinetics of cell growthand CLA production.

The work on producing acylglycerols containingCLA residues has been carried out primarily byDr. Hugo S. Garcia (a visiting faculty member)and Jose Arcos (a postdoctoral fellow) and under-graduate students working with these individuals.Dr. Garcia’s work focused on acidolysis reactionsof conjugated linoleic acid with butteroil. Dr.Arcos concentrated on the direct synthesis ofacylglycerols from CLA and glycerol.

The work by Dr. Garcia has led to several papersand presentations at professional society meetingsthat clearly demonstrate the technical feasibilityof using immobilized lipases to catalyze acidolysisreactions of CLA and milkfat. These reactions, aswell as other forms of transesterification reactions,serve to substitute a beneficial fatty acid residuefor some of the fatty acid residues present in thenative triacylglycerols that constitute milkfat,thereby producing a product with nontraditionalhealth benefits.

An important result of this aspect of our researchhas been experimental validation of the workinghypothesis that one can employ immobilizedlipases to produce fats and oils which are substan-tially enriched in conjugated linoleic acid resi-dues. For example, we have employed a Candidaantarctica lipase (Novozym 435) in a substrates-only medium to increase the conjugated linoleicacid content of milkfat acylglycerols from thenative value of 0.6 to 15 g/100 g fat. While wehave not conducted experiments to assess thefunctional and sensory attributes of dairy spreadsand other traditional products obtained fromthese modified milkfats, we believe that therewould not be insurmountable problems in thisregard, because the large majority of said prod-ucts consists of traditional milkfat.

In an investigation of the direct synthesis ofglycerides from glycerol and CLA, Dr. Arcosdetermined rates and product distributions for theconsecutive esterification reactions of conjugatedlinoleic acid (CLA) with glycerol in the presenceof an immobilized form of a Mucor miehei lipase(Lipozyme IM, kindly provided by NovoNordisk). In a solvent-free environment (a sub-strates only medium), both rates and productdistributions are affected by the ratio of reactants,temperature, and hydration level. Incorporationof up to 95 % of the original CLA into the prod-uct acylglycerols occurred at 50˚C. Typical dataindicating the observed product distributions areshown in Figure 1. (next page)

Inspection of Figure 1 reveals that the rates ofesterification of CLA were comparable in magni-tude, but increased slightly with increasing ratioof CLA to glycerol. The most evident disparitiesare those associated with formation of the triester.When the stoichiometric ratio of CLA to glycerolrequired for formation of the triacylglycerol isemployed, both the original glycerol and themonoester are rapidly consumed. However, thesubsequent reaction of the diester to form thetriacylglycerol occurs much more slowly becauseit primarily involves esterification of a secondaryhydroxyl group on the 1,3-diester. Because Mucormiehei is a 1,3-specific lipase, isomerization of the1,3-diester to the 1,2- or 2,3- form must occurprior to esterification of the third hydroxyl group.This isomerization is responsible for the low rateof reaction observed after ca. 2/3 of the originalCLA has reacted.

12


Figure 1. Percentage of CLA incorporated in various acylglycerols and unreacted CLA for differentratios of CLA to glycerol. A) 1/1, B) 1.5/1 C) 2/1, D) 3/1. Conditions: 2000 mg CLA, 300 mgLipozyme IM, 50 °C, and 800 mg molecular sieves.

The most significant aspect of this portion of ourwork is that it clearly demonstrates the feasibilityof obtaining a wide range of acylglycerol compo-sitions that could be employed in a variety ofsituations to manufacture nutraceuticals. Via thedirect synthesis route, we can obtain products inwhich all of the ester bonds in acylglycerolsinvolve CLA residues. Via the acidolysis route,we can obtain very substantive degrees of substi-tution of CLA residues for the residues present inthe original milkfat. The extent of substitutiondepends on the ratio of CLA to milkfat employedin the reaction, but extents as high as 60% should

be readily achievable, although this level is notnecessarily the level which gives the best processeconomics. That remains to be determined on thebasis of future studies. Our technology permitsone to obtain triacylglycerols characterized by afactor of 10 to 100 greater enrichment in CLAresidues than can be achieved by modification ofthe diets of dairy cows. Moreover, by appropriatechoice of operating conditions, one can obtainproducts covering a wide range of CLA contents.

In the light of the growing demand fornutraceuticals and the increasing health aware-

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Milkfat

ness of consumers, dairy products containingmilkfat enriched with CLA present an intriguingmarketing option for the dairy industry. Suchproducts could be able to partially counteract thenegative image that milkfat has developed inrecent years because of its relatively high propor-tion of saturated fatty acids, particularly those thathave demonstrated hypercholesterolemic effectson humans. In practice, any dairy product thatmay be formulated using milkfat as an ingredientcould be a potential product of the technology onwhich our research was based; in particular,butter, butterine, butteroil, and reconstituteddairy products prepared from skim milk andanhydrous (modified, CLA-rich) milkfat such asfluid milks, cream, cheese and frozen productsrepresent very attractive marketing options. Themodified milkfat products of interest thus havesignificant dietary implications with respect to notonly nutrition, but also with respect to anti-atherogenic and anti-cancer activity. The use ofimmobilized enzyme technology for the produc-tion of tailor-made triacylglycerols offers theintriguing possibility of being able to producespecially designed foods for selected segments ofthe population, in particular, those individualswho are especially health conscious from adietary standpoint or who are high risk candi-dates for cancer, atherosclerosis, hypertension, orother health problems. These products representa very significant long-term marketing opportu-nity for the dairy industry.

Publications/presentations“Enzymatic Synthesis of Glycerides Enriched inConjugated Linoleic Acid: Batch and Packed BedReactor Studies,” by J. A. Arcos and C. G. Hill,Jr., presented at the 1999 annual meeting of theAmerican Oil Chemists’ Society.

“Production of a Food-Grade Linoleic Acid viaHydrolysis of Corn Oil in a Hollow Fiber ReactorContaining an Immobilized Lipase,” by P. S.Sehanputri and C. G. Hill, Jr., presented at the1999 annual meeting of the Institute of FoodTechnologists.

“Enrichment of Butteroil in Conjugated LinoleicAcid Residues in a Continuous Flow ReactorContaining an Immobilized Lipase.” By H. S.Garcia, K. J. Keough, J. A. Arcos, and C. G. Hill,Jr., paper presented at the 1999 annual meeting ofthe Institute of Food Technologists.

“Lipase-Catalyzed Interesterification (Acidolysis)of Corn Oil and Conjugated Linoleic Acid in anOrganic Solvent,” by C.E. Martinez, J.C. Vinay,R. Brieva, C.G. Hill, Jr., and H.S. Garcia, paperpresented at the 1999 annual meeting of theInstitute of Food Technologists.

“Immobilized Lipase-Mediated Acidolysis ofButteroil with Conjugated Linoleic Acid: BatchReactor and Packed Bed Reactor Studies,” byH.S. Garcia, K.J. Keough, J.A. Arcos, and C.G.Hill, Jr., poster presented at Biotrans ‘99, theFourth International Symposium on Biocatalysisand Biotransformations.

“Rapid Solvent-Free Esterification of ConjugatedLinoleic Acid and Glycerol in a Packed-BedReactor Containing an Immobilized Lipase,” byJ.A. Arcos and C.G. Hill, Jr., 12th InternationalCongress on Catalysis.

“Continuous Interesterification of Butteroil andConjugated Linoleic Acid in a Tubular ReactorPacked with an Immobilized Lipase,” by H.S.Garcia, K.J. Keough, J.A. Arcos, and C.G. Hill,Jr., Biotechnology Techniques, 13, 369-373 (1999).

“Lipase-Catalyzed Interesterification (Acidolysis)of Corn Oil and Conjugated Linoleic Acid inOrganic Solvents,” by C.E. Martinez, J.C. Vinay,R. Brieva, C.G. Hill, Jr., and H.S. Garcia, FoodBiotechnology, 13, 183-193.

“Biotechnology for the Production ofNutraceuticals Enriched in Conjugated LinoleicAcid: 1. Uniresponse Kinetics of the Hydrolysis ofCorn Oil by a Pseudomanas sp Lipase Immobi-lized in a Hollow Fiber Reactor,” by P. S.Sehanputri and C. G. Hill, Jr., Biotechnology andBioengineering, 65, 568-579.

“Enzymatic Synthesis and Hydrolysis Reactionsof Acylglycerols in Solvent-Free Systems,” by C.Otero, J. A. Arcos, H. S. Garcia, and C. G. Hill,Jr., invited manuscript accepted for publication inthe next volume of the series of books entitledMethods in Biotechnology.

“Continuous Enzymatic Esterification of Glycerolwith (Poly)unsaturated Fatty Acids in a PackedBed Reactor,” by J.A. Arcos, H. S. Garcia, andC.G. Hill, Jr., paper accepted for publication inBiotechnology and Bioengineering.

14


“Interesterification (Acidolysis) of Butterfat withConjugated Linoleic Acid in a Batch Reactor,” byH. S. Garcia, K. J. Keough, J. A. Arcos and C. G.Hill, Jr., accepted for publication in the Journal ofDairy Science.

Papers Submitted for Publication

“Immobilized Lipase-Mediated Acidolysis ofButteroil with Conjugated Linoleic Acid: BatchReactor and Packed Bed Reactor Studies,” byH.S. Garcia, K.J. Keough, J.A. Arcos, and C.G.Hill, Jr., submitted for publication in the Journalof Molecular Catalysis: B. Enzymatic.

“Biotechnology for the Production ofNutraceuticals Enriched in Conjugated LinoleicAcid: II. Multiresponse Kinetics of the Hydrolysisof Corn Oil by a Pseudomonas sp Lipase Immobi-lized in a Hollow Fiber Reactor,” by P.S.Sehanputri and C.G. Hill, Jr., submitted forpublication in Biotechnology and Bioengineering.

.”Increased Production of Conjugated LinoleicAcid by Modification of a Reaction MediumContaining Free Lactobacillus ruteri Cells, “ by C.P.Crowley and C.G. Hill, Jr., submitted for publica-tion in Enzyme and Microbial Technology.

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Milkfat

PersonnelCharles G. Hill, Jr., professor of Chemical Engi-neering, Hugo S. Garcia, visiting scientist, associ-ate professor, Department of Food Technology,Centro deGraduados, Instituto Tecnologico deVeracruz, Veracruz, Mexico; Louis Lessard,research assistant, Souheil Ghannouchi, researchassistant, Department of Chemical Engineering


DatesJuly 1997 — December 1999

Objectives1. Generate the experimental data necessary tocharacterize rates of reactions constituting thereaction networks of interest. Determine theeffects of temperature and pH on both the overallrate of lipolysis and the reaction specificity foreach esterase.

2. Utilize these kinetic data to develop bothuniresponse and multi-response mathematicalmodels of the reaction network which can beused for purposes of process design and simula-tion, control, and optimization.

3. Establish the nature of the dependence of thecomposition of the lipolyzed dairy product on theprocess conditions (reactor space time, pH,temperature, source of enzyme).

4. Assess the commercial viability of proposedprocesses in terms of technical and economicconsiderations.

SummaryThis project was an extension of an earlier projectfunded in part through the Center for DairyResearch and in part through a grant from theNational Science Foundation. Efforts in ourlaboratory focused on the use of pregastric es-terases derived from the salivary glands of suck-ling animals (calf, kid goat and lamb) to effect thelipolysis of butteroil. We developed experimentalprotocols for partial purification of these en-

FINAL REPORT

Using immobilized esterases/lipases to modify thecomposition of milkfat

zymes, beginning with the crude preparationgenerously supplied by Systems Bio-Industries,Inc. Subsequent immobilization of these enzymesin a hollow fiber reactor provided a vehicle forobtaining lipolyzed butteroil products withsignificantly different sensory attributes thaneither typical commercial products or the effluentfrom a reactor containing an immobilized A. nigerlipase. The three pregastric esterases gave prod-ucts that differed in fatty acid composition fromone another.

Two PhD candidates (Louis Lessard and SouheilGhannouchi), a visiting professor (Hugo S.Garcia), and a visiting scholar ( Julio Vinay)conducted the experimental work and developedkinetic models to characterize the performance ofthe reactor in terms of both the total amount offree fatty acids released (the uniresponse model)and the amounts of the individual free fatty acidspresent in the effluent stream (the multiresponsemodel). Funding for these activities was split ca.50-50 between NSF and the University of Wis-consin Center for Dairy Research.

HPLC analyses of the product streams indicatedthat all three pregastric esterases have highspecificities for release of butyric (C4) and caproic(C6) acid residues, but lower specificities forcaprylic (C8), capric (C10) and longer-chain fattyacid residues. None of these enzymes releasedsignificant amounts of intermediate length or longchain fatty acids. While the lamb and kid lipasesgive high ratios for the C4 to C6 fatty acidsreleased by lipolysis, the calf lipase gave moreeven proportions of these acids. These resultssuggest that reactors containing immobilizedlipases from different sources could be used totailor-make lipolyzed butter oils with specificflavor notes. For example, high values of C4/C6and C4/C8 correspond to intense, but desirableflavors. By contrast, low values of C4/C12 can beutilized as indicators of soaplike (undesirable)flavors. In studies with an immobilized kid goatlipase, we employed variations in buffer pH andthe reactor space time to manipulate the composi-tion of the reactor effluent. In several cases we

16


were able to approximate the C4-C10 fatty acidcontent of commercial lipolyzed butteroils whilereducing the C12-C18 content by an order ofmagnitude or more. Varying the pH and tempera-ture at which the reactor operates permits addi-tional manipulation of the chemical composition(and hence the apparent flavor notes) of theproduct mixture. Our results clearly demonstratethat you can use an immobilized pregastricesterase reactor to tailor the product compositionfor specific applications by selecting operatingconditions and source of the enzyme.

Louis Lessard focused on characterizing thekinetics of the lipolysis of anhydrous milkfat in ahollow fiber reactor containing an immobilizedcalf pregastric esterase, in particular, the effects ofpH on reaction rates and product distribution.Butyric and capric acids (together with othershort chain fatty acids) are largely responsible fordesirable flavor components of the lipolyzedbutteroil product. By contrast, palmitic and oleicacids, the two fatty acids present in highestconcentrations in the product, are associated withundesirable flavor notes. At low conversions, theimmobilized calf pregastric esterase favors releaseof butyric acid during hydrolysis. As the conver-sion increases, the relative rate of release ofbutyric acid decreases because fewer residuesremain to be released.

Souheil Ghannouchi concentrated his efforts onan investigation of the lipolysis of anhydrousbutterfat using immobilized forms of pregastricesterases derived from the salivary tissues oflambs and kid goats. Both hollow fiber and batchreactor experiments indicate that the optimumconditions (maximum conversion) correspond toa pH of ca. 6.0 (see Figure 1) and a temperature of40 °C. A mathematical model based onMichaelis-Menten kinetics and a ping pong bi bimechanism provides an appropriate fit of thedata. Mr. Ghannouchi also employed a neuralnetwork model to analyze his data.

The results of our research have been describedin several publications in the archival literature,presentations at professional society meetings,and the PhD thesis of Souheil Ghannouchi (seebelow). Additional publications and another PhDthesis will be generated in the future.

The thrust of this research project addressed thatcomponent of the 1996 National Milkfat Planwhich was intended to create new uses formilkfat, modified milkfat and/or its components.Specifically, it focused on enzymatic modificationof milkfat to produce lipolyzed butteroils and/ordiacyl- and monoacyl-glycerides that can beemployed as food grade emulsifiers. The variouspregastric esterases produce lipolyzed butteroilswhich could find applications as flavoring agentswithin the food industry. Each type of enzymeproduces a butteroil with somewhat differentflavor notes and odors because of differences inthe specificities of the different enzymes.

This research project was intended to establish arational scientific basis for employing immobi-lized enzyme technology for the manufacture oflipolyzed dairy products with specified free fattyacid profiles and unique sensory and functionalcharacteristics. This research has direct implica-tions with respect to the production of lipolyzeddairy products that find applications as flavoringagents within the food industry.

Representatives of a producer of flavoring agentsmet with us on several occasions to explore thecommercial potential of this technology.

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Milkfat

Presentations/publications

“Enzymatic Synthesis and Hydrolysis Reactionsof Acylglycerols in Solvent-Free Systems,” by C.Otero, J. A. Arcos, H. S. Garcia, and C. G. Hill,Jr., invited manuscript submitted for publicationin the next volume of the series of books entitledMethods in Biotechnology.

Manuscripts submitted“Effect of pH on the Production of LipolyzedButteroil by a Calf Pregastric Esterase Immobi-lized in a Hollow Fiber Reactor: I. UniresponseKinetics” by L. P. Lessard and C.G. Hill, Jr.,paper submitted for publication in Biotechnologyand Bioengineering.

“Effect of pH on the Production of LipolyzedButteroil by a Calf Pregastric Esterase Immobi-lized in a Hollow Fiber Reactor: II. MultiresponseKinetics” by L. P. Lessard and C.G. Hill, Jr.,paper submitted for publication in Biotechnologyand Bioengineering.

“Hydrolysis of Butteroil by Pregastric EsterasesImmobilized in a Hollow Fiber Reactor,” PhDthesis of Souheil Ghannouchi, University ofWisconsin - Madison (1999).

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INTERIM REPORT

Determination of caloric bioavailability and apparent lipiddigestibility of liquid milkfat fractionsPersonnelDenise M. Ney, professor, Dept of NutritionalSciences



Objectives1. To determine apparent lipid digestibility, andthe concentration of cholesterol andtriacylglycerol in liver and plasma of rats fed dietscontaining liquid milkfat fractions, intact milkfator corn oil.

Summary

The Center for Dairy Research provided 5 kg of avery low melting milkfat fraction (dropping point< 10° C) and intact anhydrous milkfat in August1998. The liquid milkfat fraction contains adecreased proportion of 16:0 and 18:0 saturatedfatty acids and an increased proportion of 18:1monosaturated fatty acid compared to the intactmilkfat. Both fractions contain approximately10% of fatty acids with less than or equal to10carbon atoms. During the last year we haveobtained a profile of the triacylglycerol speciespresent in the milkfat fractions using high tem-perature capillary gas chromatography andconducted an animal feeding study to determineapparent lipid digestibility.

The liquid milkfat fraction contains higher levelsof triacylglycerols with unsaturated fatty acids,especially 18:1 and the intact milkfat containshigher levels of triacylglycerols with trisaturatesincluding: tripalmitate, myristate-myristate-palmitate, myristate-palmitate-palmitate andstearate-stearate-myristate. The lower levels oftrisaturated triacylglycerols in liquid milkfatcompared to intact milkfat may improve the lipiddigestibility of the liquid milkfat. An animalfeeding study comparing the apparent lipiddigestibility of diets containing corn oil, liquidmilkfat, intact milkfat and medium chain

triacylglycerols was conducted to test this con-cept. Liquid milkfat showed improved digestibil-ity associated with the lower levels of trisaturatedtriacylglycerols such that the apparent lipiddigestibility of liquid milkfat was not significantlydifferent from corn oil (96%) and significantlyimproved compared to intact milkfat (90%).These data demonstrate that temperature frac-tionation of intact milkfat to reduce the propor-tion of trisaturated triacylglycerols significantlyimproves the lipid digestibility of milkfat.

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Milkfat

APPLICATIONS PROGRAM REPORT

Milkfat applications research programPersonnelKerry E. Kaylegian, researcher, Gene Barmore,research specialist, Kathy Nelson, research spe-cialist, Center for Dairy Research

FundingWisconsin Milk Marketing BoardDairy Management, Inc.

DatesJanuary 1999—December 1999

Objectives1. To provide technical support on butter andmilkfat fractions to the dairy, bakery, confection-ery, and food industries:a. through direct inquiries, consultations, and on-site supportb. through the milkfat fractionation and specialtyingredient pilot plant programc. through research trials to evaluate the func-tional properties of milkfat ingredients, andinvestigate potential new applications for spe-cialty milkfat ingredients

Summary

Technical support questions come through ouroffices on a regular basis and run the gamut oftopics related to butter, milkfat fractions, andother dairy ingredients. We provide answers andtechnical information to these inquiries fromdairy and food manufacturers, universityreseachers, trade organizations, the media, andconsumers.

The milkfat fractionation and specialty ingredientpilot plant program continues to support the U.S.commercialization efforts of these products. Theprogram provides samples of milkfat fractionsthat have a wide range of physical and chemicalproperties. We also use these fractions to makespecialty ingredients, such as cold spreadablebutters for consumer type markets and highmelting pastry butters for the food processors.This past year we evaluated several productionschemes to determine which sequence providesgood manufacturing characteristics and valuablemilkfat fractions. Some processing sequencesresult in difficulties in the separation of the solid

and liquid fractions, and have low yields in thedesirable fractions. Other schemes providefractions with desirable characteristics but may beimpractical for commercial use. We are currentlyevaluating the data from these experiments sothat we can make recommendations to the indus-try for optimal production of new milkfat ingredi-ents.

Cold spreadable butter and pastry butter proto-types were essentially finished this year. We wentthrough multiple processing and evaluation trialsof these products to obtain prototypes with thedesired performance characteristics. Cold spread-able butter was evaluated for its spreadabilitydirectly from the refrigerator, flavor characteris-tics, and performance in a consumer home-typesetting. Consumer-type testing involves cooking(e.g., frying eggs) and baking with the butter tosimulate how a consumer might use this productat home in addition to using it as a bread spread.Some of the prototypes were spreadable butburned easily in a frying pan, others did not havegood baking characteristics. This information willbe used to highlight the benefits of spreadablebutter, and also to provide warnings if necessary,such as “do not soften prior to use in baking,”because most recipes call for “softened” butter.

The pastry butter prototype performs very well inpuff pastry applications. We achieved significantdifferences in plasticity and height of the pastriescompared with conventionally churned butter.We think that the pastries made with our experi-mental butter showed better characteristics thanpastries made with vegetable margarines that aredesigned for pastry use. The experimental butterwas also evaluated using only 75% of the normallevel for puff pastries, and the finished productswere very similar to those made with 100% fat.This may allow a manufacture to use less of thespecialty pastry butter and still have the charac-teristics they desire compared with the full fatproduct.

We began trials on an all-purpose bakery orcookie-type butter. Our first round of prototypesdid not perform well, but gave us a good indica-

20


tion of what direction to take. These butters werebased on solely on fractions that did not haveother highly valued uses, largely the middle andlow melting fractions that melt slightly lower thanintact milkfat. It seems that we may not be able torely only on these fractions, and our next roundof prototypes will use a combination of these andother fractions to provide the correct characteris-tics for the target applications.

Other applications for milkfat fractions that wehave begun to investigate, at industry’s request,include soups, sauces, and cheese applications.We continue to respond to requests for samples ofmilkfat fractions, specialty butters, and productevaluations as the program allows.

Publications and PresentationsKaylegian, K.E. Milkfat Fractions in Ice Cream.Invited presentation at the International IceCream Association Technical Council Meeting,Scottsdale, AZ. March, 1999.

Kaylegian, K.E. Properties of Milkfat Fractions.Laboratory demonstration at the UW AppliedDairy Chemistry Short Course, Madison. May1999.

Kaylegian, K.E. Dairy Ingredients in ChocolateProducts. Invited lecture for the American Asso-ciation of Candy Technologists Chocolate ShortCourse, Chicago, IL. October, 1999.

Kaylegian, K.E. The Production of Specialty MilkFat Ingredients. J. Dairy Sci. 82:1433-1439.

Kaylegian, K.E. Contemporary Issues in Milk FatTechnology. Lipid Technol. 11:132-136.

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Milkfat

INTERIM REPORT

Use of butterfat fractions and emulsifiers in dairy-basedreduced-fat spreadsPersonnelKerry E. Kaylegian, researcher, Center for DairyResearch; Kirk L. Parkin, professor, Wade N.Schmelzer and Melanie Dineen, research assis-tants, Department of Food Science

FundingDairy Management, Inc.


Objectives1. Screen for formulations that yield stable, dairy-based, reduced-fat, water-in-oil emulsions.2. Optimize formulations and processing proto-cols for preparing dairy-based, reduced-fat tablespreads on a pilot scale.

Summary

The project continues to proceed along two linesof inquiry viewed as critical to success in reachingthe objectives. We have almost completed anevaluation of interfacial dynamics at an oil/waterinterfaces, focusing on issues specific to usingmilkfat fractions and evaluating the effect ofsurface-active agents, primarilymonoacylglycerols (MAG). The other areainvolves developing a bench-test method forscreening table spread formulations in a mannerthat is predictive of processing efficacy on a pilotscale Gerstenberg & Agger texturizer.

Interfacial relationships between oil and waterphases were evaluated for a liquid milkfat fractionusing interfacial tension measurements, and for asolid milkfat fraction using contact angle measure-ments. Milkfat fractions were obtained from theUW-Madison CDR milkfat fractionation pilotplant, and are classified as 8L (liquid fractionisolated at 8oC) and 21S (solid fraction isolated at21oC). Preliminary experiments indicated that asolvent extactable or sedimentable endogenouscomponent of the 21S fraction was polar in natureand may be responsible for modulating thetemperature-solidity profile (solid fat content, orSFC) of the 21S fraction. An extracted 21S

fraction was prepared (E21S) and was found bythin-layer chromatography analysis to be dimin-ished in polar components that constituted <0.1%of the native 21S fraction. Initial interfacialtension measurements between water and milkfatfractions ranged from 18-19 mN/m for the 8Lfraction (at 25o and 50oC) to 19-21 mN/m (at50oC) for a 8L/21S blend (82.5:17.5, w/w, suitablefor a dairy spread formulation). These values areless than those (27-28 mN/m) recorded for acanola oil/water system for comparison. Thisindicates that surface active constitutents arepresent in the milkfat fractions, and these compo-nents may facilitate the stable incorporation ofmilkfat into table spreads compared to othernative oils. In addition, surface tension valuesdeclined during the 15 minute analysis period to13-15 mN/m for the milkfat fractions, implyingdynamic and competitive interfacial absorptionprocesses among surface active agents in themilkfat blends (no similar observation was madefor the canola oil/water system).

The addition of MAG preparations at 0.25%,0.75% and 1.5% levels of addition (w/w of oilphase) to the milkfat blends decreased surfacetension values to 12-14 mN/m, 8-11 mN/m, and4-6 mN/m, respectively. A time-dependentdecrease in surface tension was again noted, butminimum values for surface tensions wereachieved faster when blends contained the E21S(extracted 21S) compared to the native 21Smilkfat fraction. This indicates that the removal ofthe trace component in the E21S fraction maylead to enhanced rates at which water can bedispersed in the milkfat blend during the dynamicprocess of table spread production. At equivalentlevels of addition, MAG prepared from a milkfatfraction (12S fraction, crystallized at 12oC) by anenzyme process developed in this laboratory wasas equally effective as two commercial MAGpreparations.

Dynamics of interaction between solid milkfatwater phases was assessed by contact angle (θ)measurements (the lower the θ measured, thegreater the wetting of the lipid surface by an

22


applied drop of water). For the 8L/21S and 8L/E21S blends, q was 79-83o at 12oC, and no differ-ence was evident between the two blends. For the21S and E21S fractions, θ ranged from 95o to 107-108o as temperature of measurement was reducedfrom 35oC to 12oC, again with no differenceobserved between the fractions. Studies arecontinuing on the influence on θ of MAG addedto these solid lipid systems. When these studiesconclude, a set of predictions will be possible interms of what compositional factors may beresponsible for controlling the ease of dispersingwater in the milkfat blends during table spreadproduction. These predictions will then be testedusing the bench-top (and possible pilot scale)studies that constitute the balance of the project.

The first of two phases of development of abench-top scale system to evaluate efficacy ofpreparing table spreads has been completed. Ascraped-surface heat exchanger with a thermostatwas simulated with a batch processing scale ofabout 200 g. This series of studies focused onpreparing 60% (reduced fat) spreads using the 8L/21S blend (82.5:17.5, w/w). MAG levels (0-3% ofthe milkfat phase) and the processing parametersof cooling rate, final product temperature (10-16oC) and extent of working were evaluated.Quality of the prepared products was indexed bymeasurement of emulsion stability (by a centrifu-gation technique), color/appearance (reflectancecolorimetry), morphology (photomicroscopy),and textural analysis (total and peak force re-quired to “spread” the product).

A central finding of this first phase of studies wasthat a compromise between the degree/rate ofcooling and a need to “work” the spreads appearsto be necessary to yield high/uniform quality,stable spreads. Although the conditions thatafford this in the bench-top apparatus are empiri-cal, an attempt will be made to relate these “near-optimum” conditions (chilling the product withdyanamic agitation from 50oC to 13oC in about a5 minute time frame) to the scale of the pilotplant. Processing to a greater end-point tempera-tures (16˚C vs. 13oC) led to a great variability inthe finished products, and generally losses inproduct quality. Processing to lower producttemperatures (10˚C vs. 13oC) and greater processtimes (greater working) were associated with anapparent collapse of the product structure andlosses in product quality/uniformity. A surprising

finding was that within the range of processingparameters evaluated, MAG levels had no signifi-cant impact on the quality/uniformity of the 60%milkfat spreads. This may indicate that the levelof polar/surface active lipids endogenous to themilkfat fractions are sufficient to stabilize thewater/oil interface, obviating the need to addexogenous emulsifiers. We anticipate that addedMAG may have a greater role in product/processperformance as the level of fat in the spread isreduced further (to 40% and 20%).

At a constant heat removal rate during process-ing, statistically significant relationships werefound between 1) processing time and lightness ofappearance (positive correlation, based on reflec-tance “L” values), 2) processing time and bothtextural force parameters associated withspreadibility (negative correlations), and 3)between the two textural force parameters (posi-tive correlation). When comparing processes atdifferent rates of heat removal to a 13oC finalproduct temperature, statistically significantrelationships were found between 1) processingtime and lightness of appearance (positive corre-lation), and 2) the two textural force parametersassociated with spreadability (positive correla-tion). The relationships between the two texturalparameters measured for the finished productsappeared to be modeled by fairly simple relation-ships, yielding different models under differentprocessing conditions. We speculate that within arange of process conditions that can yield highquality/uniform products, the specific processingconditions may be manipulated to providedifferential control over the various textural at-tributes of spreadability. This may allow a givenspread formulation to be processed in a mannerthat meets the specific needs (conferred by end-use) of a variety of finished table spread products.

Future work will focus on evaluating formulationsof 40% and possibly 20% milkfat-based tablespreads, and the effect of hydrocolloids andemulsifiers that are anticipated to be required forproducts most reduced in fat. Finally, the most(and least) successful formulations based onbench-scale testing will be processed on the pilotscale apparatus, to confirm the ability of thebench-top procedure for predicting processingefficacy of various table spread formulations intofinished products of high and uniform quality.

23

Milkfat

Table spread products constitute an establishedand expanding global market. Much of thecurrent focus on development of these products ison reduced-fat formulations, however, they arealso being considered as vectors for delivering“nutraceuticals” and other health-promotingingredients. Milkfat holds advantages over otherfats and oils in this type of product because ofinherent flavoring properties and low trans fattyacid content relative to hydrogenated vegetableoils. The objective of this project is to developentirely dairy-based, reduced-fat table spreadformulations, with the ultimate goal of trying toexpand the use of milkfat.

24


FINAL REPORT

Rheological and structural properties of dairy-based lipidsPersonnelRW Hartel, professor, Baomin Liang, assoc.researcher, M. Lidia Herrera, Visiting Scientist,Dept of Food Science


DatesJuly 1997—June 1999

ObjectivesThe primary objectives of this project were:1. To determine the effects of processing condi-tions (time, temperature) on crystalline structureof mixed lipids of importance to dairy-basedspreads.

2. To correlate the rheological properties of mixedlipids to their crystalline structure, based onprocessing conditions, types of fats mixed togetherand storage conditions.

Summary

Mixtures of high-melting (HMF) milkfat fractions(30, 40 and 50%) with low-melting (LMF) frac-tions or canola oil (CNL) have been crystallizedunder different processing conditions. The moltenfat was cooled to crystallization temperatures (25to 30°C) at different cooling rates (fast and slow).The fats were allowed to crystallize for severalhours at different agitation speeds (50 to 300RPM). The slurry in this vessel was then cooledto 10°C for 24 hours for further analysis. Imagesof the crystalline structure of the set product wererecorded using Confocal Scanning Laser Micros-copy (CSLM). The rheological properties of theproduct fats were determined using DynamicMechanical Analyzer (DMA). The effects ofstorage time on crystalline microstructure andmechanical properties were also studied.

Each of the processing conditions influenced bothcrystalline microstructure and mechanical proper-ties for both model systems (HMF in CNL andHMF in LMF). The crystalline microstructure ofthe semi-solid samples contained dense, primarycrystals (formed during agitation at crystallization

temperature) surrounded by a matrix of second-ary crystals (formed during cooling to 10°C).Rapid cooling of the sample with 30% high-melting milkfat fraction (HMF) resulted in manysmall primary crystals, as compared to slowcooling, and this led to slightly lower elasticmodulus (from DMA). Increasing agitation speed(from 50 to 300 RPM) also resulted in decreasingprimary particle size, which again led to lowerelastic modulus. Lower crystallization tempera-ture (from 30 to 25°C) also led to lower primarycrystal size and lower elastic modulus. Note thateven though the primary crystallization tempera-ture was different, the final solid fat content (SFC)for these samples (all 30% HMF) was the samesince they were all equilibrated to 10°C. How-ever, increasing the ratio of HMF to low-meltingfraction (LMF) caused an increase in SFC andresulted in an increase in elastic modulus. Longerstorage times led to increased elastic modulus, asexpected. Over time, the secondary crystallinestructure appeared to get more dense, whichcould potentially have led to the higher elasticmodulus. Some small differences in behaviorwere observed for the two systems (HMF in LMFand HMF in CNL). The secondary crystallinestructure in the HMF-CNL system was lessstructured and more diffuse than the secondarycrystalline structure of the HMF-LMF system.This suggests that the triacylglycerols (TAG) ofthe two fats were sufficiently different to promotediffuse crystallization. Differences in mechanicalproperties due to operating parameters were alsosmaller for the HMF-CNL system. In addition,the HMF-CNL samples did not undergo signifi-cant restructuring during storage so there was noincrease in hardness during storage.

The effects of lipid composition and processingconditions on lipid crystalline microstructurehave been studied for two model lipid systems(HMF in LMF and HMF in CNL). To preparethe samples for crystallization, mixtures of HMFand either LMF or canola oil were melted at60°C for one hour to remove any crystal memory.The molten fat was cooled to crystallizationtemperature, where crystallization is allowed toproceed for several hours in the presence ofagitation. The crystal slurry from the crystallizer

25

Milkfat

100 µm

Figure 1. Confocal microscope images of a 50-50% blend of high-melting milk fat fraction (HMF) inlow-melting milk fat fraction (LMF) crystallized first at 25°C (agitation rate, 50 RPM; cooling rate,0.2°C/min) and stored for 24 h at 10°C. Images were taken at 3 µm increments from the surface: a)surface, b) 3 µm, c) 6 µm, d) 9 µm, e) 12 µm, f) 15 µm.

A B

C D

E F

26


was then allowed to cool quiescently to 10°C andset into a semi-solid product. This process simu-lates commercial processing of lipids, whereprocessing occurs in two steps, although theconditions are not exactly translatable to com-mercial conditions.

The variables studied in this experiment in-cluded:

• rate of cooling to crystallization temperature(fast, 5.3°C/min, and slow, 0.2°C/min),• crystallization temperature (25 to 30°C),• agitation rate (50 to 300 RPM),• lipid formulation:

- 30, 40 and 50% high-melting in low- melting milkfat fractions,- 30, 40 and 50% high-melting milkfat

fraction in canola oil,• storage time at 10°C (1 day to 3 weeks).

The following analyses were performed on eitherthe slurry or the semi-solid sample.

• solid fat content of slurry (NMR),• solid fat content of semi-solid product (NMR),• optical microscopy of slurry crystals,• confocal microscopy of crystals in semi-solid product,

A small amount of dye was added to themolten product prior to crystallization toenhance the image from confocal microscopy. Initial experiments were performedto verify that this dye had no impact oncrystallization kinetics of the fat systems.

• mechanical properties of semi-solid product(DMA).

The HMF-LMF systemRepresentative confocal microscope images of thecrystalline microstructure in the semi-solid prod-uct are shown in Figures 1 and 2 for slow (0.2°C/min) and fast (5.5°C/min) cooling rates, respec-tively. These images clearly show two crystallinephases. The dense primary crystals were formedat the elevated crystallization temperature (25-30°C) under agitation. The primary crystals weresurrounded by a more diffuse secondary crystal-line structure, which formed as the slurry cooledunder stagnant conditions to molding tempera-ture of 10°C. The interaction between thesecrystalline structures, and the liquid matrix inwhich they are contained, gives rise to the charac-teristic mechanical properties of that semi-solid

material. For comparison, the DMA frequencyscan for the samples crystallized at differentcooling rates is shown in Figure 3. These clearlyshow that crystalline structure influenced me-chanical properties (elastic modulus) even thoughall of the samples had identical solid fat content at10°C. Note that elastic modulus (e’) represents thesolid-like nature of the semi-solid material.Higher elastic modulus generally correlates withharder, more solid-like materials.

The results may be summarized as follows.Increasing RPM gives rise to more smallerprimary crystals and this leads to a decrease inelastic modulus. Rapid cooling also led to highernumbers of smaller primary particles and this alsoresulted in lower elastic modulus, althoughdifferences were fairly small. The lower crystalli-zation temperature (25°C) led to smaller primarycrystal sizes, which, again, led to lower elasticmodulus. In contrast, addition of higher levels ofhigh-melting milkfat fraction (up to 50%) resultedin more, larger and more dense primary crystals.This led to higher elastic modulus; however, thehigher level of high-melting fraction also meanthigher solid fat content.

The HMF-CNL systemSimilar results for crystalline microstructure werefound when HMF was added to canola oil. Thesecondary crystalline microstructure seemed tohave more distinct characteristics than the diffusenature found in the HMF-LMF system. Thissuggests that the milkfat crystallized separatelyfrom the canola oil over the entire range oftemperatures. One main difference between theHMF-LMF and HMF CNL systems appears tothe effect of crystalline microstructure on me-chanical properties of the solidified product. Inthe HMF-CNL system, no significant differencesin elastic modulus were found for any of theoperating conditions, despite the distinct differ-ences in crystalline microstructure. The onlyparameter that lead to a difference in elasticmodulus was the amount of HMF added tocanola oil. In this case, the elastic modulusincreased as HMF content increased according tothe increase in solid fat content of the product.There were also no changes in elastic modulus inthe HMF-CNL system during storage, whereaselastic modulus of the HMF-LMF product in-creased substantially over the first few weeks ofstorage.

27

Milkfat

Figure 2. Confocal microscope images of a 50-50% blend of high-melting milk fat fraction (HMF) inlow-melting milk fat fraction (LMF) crystallized first at 25°C (agitation rate, 50 RPM; cooling rate,5.5°C/min) and stored for 24 h at 10°C. Images were taken at 3 µm increments from the surface: a)surface, b) 3 µm, c) 6 µm, d) 9 µm, e) 12 µm, f) 15 µm.

A

FE

D

B

C

28


This work has led to substantial progress in ourunderstanding of the effects of processing condi-tions on lipid crystallization kinetics, the lipidcrystalline structure that is formed under theseconditions and how these structures influence themechanical properties of the product. However,this project has barely scratched the surface.Much more work is necessary to understand therelationships between lipid crystalline structureand the mechanical properties of lipid-baseddairy foods. In addition, further work is necessaryto correlate the mechanical properties with theorganoleptic (sensory) properties, such asspreadability, of lipid-based dairy products.

Publications and PresentationsHerrera, M.L. and R.W. Hartel, Crystallization ofa Model Milkfat System, J. AOCS (accepted).

Herrera, M.L. and R.W. Hartel, Effect of Process-ing Conditions on Physical Properties of a MilkfatModel System I. Rheology, J. AOCS (accepted).

Herrera, M.L. and R.W. Hartel, Effect of Process-ing Conditions on Physical Properties of a MilkfatModel System II. Microstructure, J. AOCS(accepted).

Herrera, M.L. and R.W. Hartel, Kinetics ofCrystallization of a Model Milkfat System, paperpresented at AOCS Conference, Orlando, FL(May, 1999).

Herrera, M.L. and R.W. Hartel, CrystallineMicrostructure in a Model Milkfat System, paperpresented at AOCS Conference, Orlando, FL(May, 1999).

Hartel, R.W. and B. Liang, Applications ofMilkfat Fractions: Interactions With Other Fats,paper presented at International Society of FatResearch, Brighton, UK (Oct, 1999).

Hartel, R.W., Relationships Between CrystallineMicrostructure and Mechanical Properties inLipid Foods, paper presented at Eastern Analyti-cal Science Symposium, Somerset, NJ (Nov.,1999).

0

0.5

1

1.5

2

2.5

3

3.5

1 10

Frequency (Hz)

E*

E'

E*

E'

E"

E"

2 4 6 8

slow rate

fast rate

Figure 3. Effect of cooling rate on storage (E’),loss (E”) and complex (E*) modulus values mea-sured for a 50-50% mixture of high-melting(HMF) in low-melting (LMF) milk fat fractioninitially crystallized at 25°C (50 RPM) and storedfor 24 h at 10°C. Frequency scan from 1 to 10 Hz.

29

Milkfat

FINAL REPORT

Effects of milkfat source and composition oncrystallization kineticsPersonnelRW Hartel, professor, Colleen Kubitz, researchassociate, Yuping Shi, assoc. researcher,Dept of Food Science


DatesJune 1997— June 1999

ObjectivesThe overall objective is to correlate the variabilityin anhydrous milkfat with fractionation efficiency.Specific objectives are:

1. To analyze and identify the key differences inchemical composition and physical properties ofanhydrous milkfat produced from differentsources (seasonality, regionality, etc.) and materi-als (cream vs. butter).

2. To correlate the differences found betweenAMF samples (Objective 1) with differences in

crystallization kinetics and fractionation effi-ciency.

Summary

Milkfat samples were collected from varioussources throughout the year. In particular, AMFproduced from fresh cream obtained from thesame source (in Minnesota) has been analyzedover the past year. In addition, AMF samplesproduced from whey cream were obtained fromthe same supplier. Further, several AMF sampleswere obtained from German, Irish and NewZealand sources for comparison.

All samples were analyzed for fatty acid profiles(GC analysis), acyl carbon profiles (GC analysis)and minor lipid content (TLC and HPLC analy-ses). Clear point, Mettler dropping point, solid fatcontent (SFC) curves and melting profiles (DSC)were also measured for each sample. Crystalliza-tion kinetics were measured by cooling themolten samples to 28°C in a temperature-con-trolled spectrophotometer in an agitated (250RPM) cuvette. Change in turbidity was used to

Table 1. Milkfat samples used in this study

Sample Identity Date Received Source Identification

Sweet Cream AMF* January 1998 United States 1Sweet Cream AMF* March 1998 United States 2Whey Cream AMF March 1998 United States 3Sweet Cream AMF* April 1998 United States 4Sweet Cream AMF* May 1998 United States 5Winter AMF May 1998 Ireland 9Winter AMF June 1998 New Zealand 10Winter AMF June 1998 Germany 11Sweet Cream AMF* June 1998 United States 6Whey Cream AMF June 1998 United States 7Sweet Cream AMF* August 1998 United States 8Winter AMF December 1998 Germany -Summer AMF December 1998 Germany -Whey Cream AMF March 1999 United States 12Sweet Cream AMF* March 1999 United States 13Butter April 1999 Ireland 14

* Sequence for Sweet cream AMF through 15 months of collection

30


characterize crystallization rates for each sample.Since the turbidity technique does not giveinformation about nucleation rate, a separateexperiment was performed to determine thenumber of nuclei formed over a specified periodof time under controlled conditions. In thisprocedure, the samples were cooled from 70 to27.8°C. Before they crystallized, they were agi-tated for 30 s at 200 RPM to induce nucleation.Samples were then incubated at 30.5°C to allownuclei to grow without new nuclei forming. After3.5 h of incubation, a sample was prepared on acustom-built microscope slide and crystals werecounted in a sample of known volume. Anaverage nucleation rate was calculated from thenumber of nuclei counted per unit volume pertime of inducing action (30 s).

Somewhat surprisingly, the AMF samples ob-tained from sweet cream from the same originthroughout the year showed only slight differ-ences in chemical composition with no obvioustrends that could be attributed to seasonal fluctua-tions. Even the whey cream samples showed onlyminor differences in chemical composition. Thesamples that stood out as having significantlydifferent composition were the samples fromIreland. These had lower melting points, whichwere attributed to higher levels of unsaturatedfatty acids. The Irish samples also had slightly (3-4%) lower levels of short-chain triacylglycerols(TAG) with acyl carbon number greater than 40and higher free fatty acid content to go along withthe lower melting points than the other samples.

Also somewhat surprisingly, no significant differ-ences in crystallization rate were found amongsamples when using the turbidity technique, withthe exception again of the Irish samples. For allsamples except the Irish samples, the inductiontimes and crystallization rates as measured byturbidity change were within standard deviationsof the measurement. The Irish samples hadsignificantly longer induction times before onsetof crystallization (by turbidity). However, whenthe crystalline microstructure of all samples wasinvestigated by using confocal microscopy, somedifferences became apparent. This led to utiliza-tion of a more accurate measure of nucleationrate for the milkfat samples and correlation ofthese nucleation rates against the minor differ-ences in chemical composition (particularlyTAG).

Based on nucleation rate data, the samples inTable 1 were ordered from 1 to 14. The relation-ships between nucleation rate and some indica-tors of chemical composition are shown in Figure1. More specific differences in TAG compositionare shown in Figure 2. Our conclusions are thatthere was not a single TAG component thatcorrelated with nucleation rate. However, thesum of differences in composition led to signifi-cant differences in the solid to liquid (S/L) ratio,which correlated well with nucleation rate (Figure2). For the purposes of this report, solid-like TAGwere those with acyl carbon number betweenC46 to C54, excluding the C54 trans contribu-tion. The liquid-like TAGs included those withacyl carbon number less than C40 plus the C54cis contribution. As S/L increased, the nucleationrate increased dramatically. Samples with low S/L, such as the Irish samples, had the lowestnucleation rate, whereas samples with higher S/L,primarily the whey cream samples, had muchhigher nucleation rates.

It has been well documented that the compositionof anhydrous milkfat (AMF) can vary based onsource and processing conditions, and that thesedifferences can have considerable effect oncrystallization kinetics and fractionation effi-ciency. However, our seasonal analysis showedthat there were no distinct trends in chemicalcomposition among AMF samples made fromsweet cream obtained from the same locationover a 15 month period. Also, only slight differ-ences were found in crystallization kinetics andthese could not be correlated with seasonalfluctuations. Significant differences in crystalliza-tion kinetics were found for AMF samples ob-tained from whey creams and for some of theinternational samples. There are many composi-tional factors that influence crystallization kinet-ics, although no clear trends have been delineatedin the past. These results show that the ratio ofTAGs with solid-like characteristics to those withliquid-like characteristics provides a good indica-tor of crystallization kinetics of different milkfatsamples.

31

Milkfat

Fig. 1 Major TAG components contained in

AMFs from different sources

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00A

MF

14

AM

F9

AM

F1

0

AM

F4

AM

F1

1

AM

F1

AM

F5

AM

F8

AM

F1

3

AM

F6

AM

F2

AM

F3

AM

F1

2

AM

F7

AMF ID

TA

G C

on

ten

t (g

./1

00

g.

ide

nti

fie

d T

AG

s)

C34

C36

C38

C40

C46

C48

C50

C52

C54

32


Fig. 2 Content of TAGs and nucleation rate for different AMFs

0

10

20

30

40

50

60

70

80

90

Sample ID of AMFs

0

20

40

60

80

100

120

140

160

<=C40 (L)

C46-C52

C46-C54d

S/L

Rate(Nu)

33

Milkfat

Fig. 3 Major TAG components contained in AMFs from different sources

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

AMF ID

C34

C36

C38

C40

C46

C48

C50

C52

C54

34


Cheese

chapter 2

Intensified flavors in Cheddar cheese and cheese ingredients for enhanced applications in foods .............................. 37

Characterization of interactions between ingredients and cheese constituents for improved functionalityof fat-free processed cheese ..................................................................................................................................................................... 40

Improvement of Cheddar cheese quality through identification and characterization of microbial enzymesresponsible for the production or degradation of bitter peptides in cheese .......................................................................... 46

Succinate production by Lactobacillus casei: pathways responsible and development of strategies tocontrol its accumulation. ............................................................................................................................................................................ 48

Glutathione and Cheddar cheese flavor development .................................................................................................................... 50

Growth of nonstarter lactic acid bacteria in reduced fat Cheddar cheese ............................................................................... 54

Optimizing the standardization of milk to manufacture 50% reduced fat Cheddar cheese .............................................. 56

Cheese applications program ................................................................................................................................................................... 61

Extending the cheese net paradigm to include economic evaluation and optimization in cheese manufacture ...... 64

Technology for improving the flavor and consumer acceptability of fat-free Cheddar cheese ......................................... 65

Whey applications research program .................................................................................................................................................... 66

Dairy marketing and economics program ............................................................................................................................................ 68

A multi-country analysis of household food demand: Implications for U.S. food exports (phase I) .............................. 71

Development and application of a cheese shred/texture map delineated by cheese rheological,sensory and chemical analysis ................................................................................................................................................................. 76

CDR specialty cheese applications program ....................................................................................................................................... 77

36


37

Cheese

FINAL REPORT

Intensified flavors in Cheddar cheese and cheeseingredients for enhanced applications in foodsPersonnelRobert C. Lindsay, professor, Department ofFood Science

FundingWisconsin Milk Marketing Board and DairyManagement, Inc.


Objectives1. To determine the flavor systems and constitu-ents of Cheddar cheese that are responsible forproviding the desirable cook-through cheeseflavors in foods, especially bakery products.

2. To develop technological means for intensify-ing desirable cook-through Cheddar cheeseflavors in prepared foods, especially bakeryproducts, through the selection and use of ingre-dients which interact during processing to provideproducts that amplify the impact of cheese flavorsin these products.

3. To develop adjunct lactic acid bacteria cultur-ing procedures for cheese ingredients which yieldflavor compounds that intensify Cheddar cheeseflavor in prepared foods, especially bakeryproducts.

Summary

To assess the baked flavors of cheeses we modi-fied a non-fermented dough batch-method toprepare cheese crackers. This model crackercontained levels of fat provided by butter and thefat from cheeses, and thus was characterized as ahigh fat cheese cracker. The high-fat modelcracker system was selected because it amplifiedthe cheese flavor notes that were formed eitherduring baking or provided directly by the cheeseingredients for exposure to the baking conditions.

Descriptive sensory analysis ballots were devel-oped and evaluated for effectively indexing bake-through cheese flavors. The most effective scalesfor documenting bake-through cheese flavors

included: toasted flavor intensity (very weak tovery strong), overall cheesiness flavor intensity(not cheesy to very cheesy), sourness/tartness(absent to very pronounced),varietal cheese flavorintensity (absent to pronounced; panelists pro-vided cheese variety believed present), off-flavorintensity (absent to very pronounced). Sometimesoverall acceptability (unacceptable to very accept-able) was also scaled.

Evaluating bake-through flavorsA wide range of cheese varieties, representing arange of ages, were added to cheese crackers.Then, expert flavor assessors and formal descrip-tive analysis sensory panels evaluated them toestablish the types of cheese flavors that could bedeveloped in baked cheese crackers. The cheesecracker varieties evaluated along with theirdistinguishing flavor characteristics as determinedby expert flavor assessors included Cheddar-type,Swiss-type, hard Italian-type, and blue-typecheeses. These cheeses represented some mild,medium, and intensely flavored cheeses.

From a combination of the descriptive sensoryanalysis data and the expert assessor profiles ofthe baked cheese crackers, nine categories ofbake-through cheese flavors were formulated. Inaddition to the categories, specific flavor noteswere noted for the characterizing flavors in thecheese crackers, and supporting or modifyingflavor notes were also identified. The maincategories were baked true-Cheddar-type flavor,generic baked aged cheese flavor, bakeddimethyldisulfide-type flavor, baked sweet Swiss-type flavor, baked blue-fatty acid-type flavor,baked fresh goat-type flavor, baked white moldsurface-ripened type, and baked hard sheep’smilk-type cheese flavors.

Cheese slurry systemsModified cheese slurry systems were developedwhich were based on the initial work of Singhand Kristopherson (1969; Factors Affecting theFlavor Development in Cheddar Cheese Slurries,J. Dairy Sci. 55: 536) for use in systematic evalua-tions of both lactic bacterial adjunct strategies and

38


chemical flavor precursor additions. The overallslurry was composed of 60-62% Cheddar cheesecomponent, and the remainder was added waterand water-based ingredients. The Cheddar cheesecomponent can be selected from freshly-madecurd to very aged Cheddar cheese, and thecheese component can be prepared from mix-tures of Cheddar cheeses with differing ages.Slurry components (Cheddar cheese, brine [to38%], and other dissolved ingredients) are thor-oughly mixed with a stomacher to homogeneityin sterile pouches, and then the pouches areincubated under a variety of oxygen-tensionconditions (air exposure to anaerobic with GasPacs) for periods up to 21 days at 30°C.

Since contamination by advantitious innocuousmicroorganisms, as well as potential pathogens,could occur during manufacture, particularattention was given to the evaluation and selec-tion of acceptable antimicrobial food additives forthe cheese slurry systems. To accomplish this, thebasic cheese slurry was prepared by adding anappropriate amount of sterile 5.2% sodiumchloride to provide an elevated salt content toimprove overall microbial stability. Then, througha series of trials, it was established thatsupplemention with 2% w/w MicrogardR (as aspoilage inhibitor; Wesman Foods, Inc), 1% w/wcitric acid (to lower the pH; Mallinckrodt U.S.P.),and 0.2% w/w potassium sorbate (as a moldinhibitor; Pfizer) greatly enhanced the microbialstability of the cheese slurries.

Trials to determine the influence of pH conditions(4.4-7.1) on slurry aroma and flavor developmentwere carried out employing pH adjustments witheither phosphoric acid, citric acid, or Lb. caseigrowth and incubating for up to 72 hours at 30°C.A culture of Lactobacillus casei10, obtained fromthe culture collection of the Department of FoodScience- University of Wisconsin where it wasstored at -40 ˚C before cultivation in MRS broth(Difco) for 18 h at 32˚C, was used to evaluateslurry culturing conditions. It was found that goodflavor development occurred for combinationsemploying at least 20% of the total cheese ingre-dient as aged Cheddar cheese, when held at pH5.0 and under carbon dioxide-nitrogen or aerobicatmospheres. However, accentuated cheeseflavors were found to develop for samples pre-pared only from mild cheese and held undereither aerobic or anaerobic conditions.

Studies were conducted on the susceptibilty of theCheddar cheese slurry system to listeria hazards(objectives 2 and 3). Slurry samples were inocu-lated with Listeria monocytogenes Petite Scott A orNCTC 7973 at a level of 4 x 105 cells per gram,and then were incubated with 0.0, 0.5, and 1.0%citric acid at 30°C for 14 days with periodicexamination for listeria counts. Listeria grew inthe slurry system when the pH was adjusted to7.1, but at lower pH values (pH 4.4 and 5.2), thelisteria cells did not proliferate, and were depletedafter about 150 hours. The inclusion of Lactobacil-lus casei in the cheese slurries provided someprotection to the listeria, but cells were soondestroyed at pH 4.4 and 5.2. Based on the resultsof these studies, it was concluded that the cheeseslurry system adopted provided a listeria-safeapproach to production of concentrated cheeseflavors.

Studies of adjunct cultures producing key bake-through cheese flavors identified in the bakingtrials were carried out, initially focusing on α-dicarbonyl production by lactic acid bacteriawhich had been earlier identified as an importantroute to overall cheese flavor development. Theα-dicarbonyls, glyoxal, methylglyoxal, anddiacetyl, react with amino acids to producereaction flavor compounds that contribute to thegeneral cheesiness category. These studiesshowed that selected lactic acid bacteria pos-sessed the capability for producing elevated levelsin cheese slurries used as ingredients in cheesecrackers and other applications. These cheeseswere made into cheese crackers, and higher α-dicarbonyl-containing samples which gave stron-ger toasted, baked cheese flavors.

Cheese flavors in crackersResearch was continued on the bake-throughflavors of cheese crackers, and the data revealedthat most commercial cheese crackers rely princi-pally upon a generic methional, cheesinesscharacter. These data also showed that the trueCheddar flavor was largely missing from thecrackers, and they lacked much of the savoryflavor provided by succinic acid. Using Cheddarcheese slurry systems prepared with intensifiedlevels of succinic acid, aged Cheddar cheese, a-dicarbonyls, and volatile fatty acids, we producedCheddar cheese crackers with substantiallyenhanced cheesiness. They were more intenselycheese-flavored than most commercial cheesecrackers.

39

Cheese

Investigations of the addition of selected ingredi-ents or chemicals to mild Cheddar cheese-basedcheese cracker doughs showed that methionineand a reducing sugar, such as glucose, provideddistinct baked generic cheesiness to the resultingcheese crackers. Addition of succinic acid (75-300ppm) greatly improved the savory cheesinessflavor, and it was stable to elevated temperaturesencountered during baking. The discovery ofsuccinic acid as a savory cheese flavor ingredientin Cheddar cheese flavors in this project hasinitiated substantial commercial activity by cheeseculture companies, and it is the current focus ofseveral University research projects involvingadaptation during cheesemaking and/or geneticengineering to produce greater quantities incultures.

Producing bake-through cheese flavorSince volatile free fatty acids are key forcheesiness flavors (discovered in a parallelproject), studies were carried out to demonstratethe production of volatile fatty acids by a varietyof lactic acid bacteria in the cheese slurry system.Cell-associated lipases of adjunct lactobacillus andrelated bacteria produce substantial flavor poten-tial in the cheese slurries. Levels of butyric acidprovide an index for cheesiness, and concentra-tions over 150 ppm butyric acid in cheese ingredi-ents provide desirable cheesiness. When selectedslurries were incorporated into cheese crackers,distinctively elevated cheesiness flavors wereobtained when compared to a mild Cheddarcheese control sample.

True Cheddar flavorThe chemistry of the true sulfury Cheddar com-pound was intensively studied using a variety oftechniques for attempting to stablize the com-pound for analysis. Using an enclosed glove boxprovided an approach to maintain cheeses in anatmosphere free of oxygen, and it was conclu-sively shown using descriptive analysis that thetrue Cheddar flavor, hence compound, wasgreatly suppressed or destroyed upon exposure tooxygen. Only methanethiol could be demon-strated to be present regularly, but evidence wascollected which indicated that the compound wasan oxygen-heat sensitive compound that formedfrom diacetyl/acetoin, hydrogen sulfide, andmethanethiol. For this reason, the adopted use ofglutathione in the cheese slurry system washypothesized to result from such a reaction.

Specific analysis of aged Cheddar cheeses de-scribed as “catty” by dairy products judgesyielded the identification of 4-methyl-4-mercapto-pentan-2-one in low concentrations. This com-pound is produced by the condensation of ac-etone to mesityl oxide, and then a subsequentreaction with hydrogen sulfide from amino acidsor glutathione yields the mercaptoketone. Ac-etone is produced by some lactic acid bacteria,and light catalyzes the condensation to mesityloxide. Based on its flavor properties, it washypothesized that the mercaptopentanone mayaccompany and potentiate the as yet unidentifiedtrue Cheddar flavor compound.

Publications/presentationsLindsay, R. C. 1997. Recent Advances in theFlavor Chemistry of Cheddar Cheese. In: TheNational Cheese Technology Forum, Proceedings’97, December 9-10, Rosemont, IL, pp. 1-16.

40


FINAL REPORT

Characterization of interactions between ingredients andcheese constituents for improved functionality of fat-freeprocessed cheesePersonnelWilliam L. Wendorff, associate professor, Dept. ofFood Science, Brad Swenson, graduate researchassistant


DatesJuly 1997— June 1999

Objectives1. Determine the interactive effect of stabilizers,emulsifying salts and other dairy ingredients onthe functionality of no-fat pasteurized processedcheese spreads.

2. Evaluate effect of stabilizers and other dairyingredients on skin formation during heating ofno fat processed cheese products.

3. Evaluate water retention in the protein matrixwhen fat is eliminated in processed cheese prod-ucts versus full-fat processed cheese.

Summary

Manufacture of process cheesesPasteurized process cheeses were manufactured ina Blentech twin screw pilot cooker (BlentechCorp., Rohnert Park, CA) equipped with variableagitation and indirect steam heating capabilities.Batches of cheeses were produced by the formula-tion shown in Table 1. Disodium phosphateduohydrate was used as the emulsifying saltexcept where indicated. Throughout the study, fatand moisture compositions of fat-free processcheeses were measured, and mean values arereported in Table 2. Little variation in thesevalues was observed throughout the study, whichsupported the view that the results reflectedexperimental treatments rather than composi-tional variations. Similarly, pH values of all fat-free process cheeses were monitored, generallyshowing little variation throughout the study.

Emulsifying saltsEffects of different emulsifying salts, at 3%, ontextural attributes of fat-free process cheeses arereported in Table 3. Trisodium citrate and diso-dium phosphate produced significantly softercheeses and melted more easily than those pre-pared with condensed phosphate Joha brandsalts. For these trials, trisodium citrate producedthe softest cheese, which also melted slightlymore readily than the full-fat reference. In thecase of Joha brand emulsifying salts, firm cheeseswith limited melt and minimal spreadabilityresulted. Increasing the amount of emulsifying

Ingredient %

Hard skim milk cheese 59.8Water 26.8Dried sweet whey 5.3Nonfat dry milk 4.1Emulsifier 3.0Salt 1.0Hydrocolloid 0

Total 100.0

Table 1. Fat-free process cheese base formulation.

Component %

Fat 0.6 + 0.2Moisture 58.5 + 1.0pH 5.6 + 0.4

Table 2. Typical compositiona ofexperimental fat-free process cheeses.

a Mean values + standard deviation of duplicatedeterminations for fat-free trials (n=88)

41

Cheese

Table 3. Effect of emulsifying salts used at a 3 % level on the functional propertiesof fat-free process cheeses.

Hydrocolloid Firmnessf Meltabilityg Spreadabilityh

peak force (N) flow (mm) total force (N’s)

Gelatin 45.0b 34.3b 524.0b

Carrageenan 45.1b 9.3c 485.3c

Locust bean gum 53.0a 12.0c 593.1a

Guar gum 38.1c 6.5c 228.9d

Full-fat referencei 12.2d 145.5a 192.1e

a,b,c,d,e Means within a column with no common superscripts differ significantly (p<0.05).f Means of triplicate determinations for two trials (n=6).g Means of duplicate determinations for two trials (n=4).h Means of duplicate determinations for two trials (n=4), smaller values equal greater spreadability.i Full-fat reference contained 3% disodium phosphate duohydrate as the emulsifying salt.

Table 4. Effect of hydrocolloids used at a 2 % level on the functional properties of fat-freeprocess cheeses.

Emulsifying Firmnessf Meltabilityg Spreadabilityh

salt peak force (N) flow (mm) total force (N’s)

Trisodium citrate 21.3d 155.8a 521.6a,b

Disodium phosphate 25.4c 46.5c 493.5b

Joha S9 33.0b 9.5e NSTC Joha SE 32.9b 8.0e NSTC Joha C New 66.0a 19.5d 552.2a

Full-fat referencei 12.2e 145.5b 192.1c

a,b,c,d,e Means within a column with no common superscripts differ significantly (p<0.05).NSTC = Not spreadable under test conditions.f Means of triplicate determinations for two trials (n=6).g Means of duplicate determinations for two trials (n=4).h Means of duplicate determinations for two trials (n=4), smaller values equal greater spreadability.

salt from 0.5 to 3% generally resulted in increasedfirmness, decreased melt and decreasedspreadability in all cases (data not shown). Addi-tionally, various mixtures of the emulsifiers didnot provide advantages over single emulsifierusage for any of the textural attributes. The highdegree of firmness for cheeses containing Johabrand emulsifying salts might be explained by anincrease in the amount of protein-protein interac-

tion facilitated by greater Ca++ sequesteringabilities of these polyphosphate-containingingredients. The similar consequences of in-creased firmness and decreased melt correspond-ing to increasing concentrations of emulsifyingsalts mentioned earlier also would be in agree-ment with this.

42


Cook Time Firmnesse Meltabilityf Spreadabilityg

(min at 75˚C) peak force (N) flow (mm) total force (N*s)

0 31.7a 48.8b 518.0a

5 27.1b 57.5b 504.1a

10 26.3b,c 70.3a 500.2a

15 25.3c,d 78.0a 484.8a

20 24.1d 77.5a 485.5a

a,b,c,d Means within a column with no common superscripts differ significantly (p<0.05). e Means of triplicate determinations for two trials (n=6). f Means of duplicate determinations for two trials (n=4). g Means of duplicate determinations for two trials (n=4), smaller values equal greater spreadability.

Table 5. Effect of cook time on the functional properties of fat-free process cheeses.

HydrocolloidsResults of additions of commercial hydrocolloidsto fat-free process cheeses formulated with 3%disodium phosphate are summarized in Table 4.Overall, an increase in the firmness of the cheeseand a decrease in meltability occurred for alltreatments compared to process cheese controlsamples without added hydrocolloids. Guar gumproduced the softest texture of all hydrocolloidsstudied with gelatin exhibiting the greatest overallmeltability. The heat reversible property ofgelatin gels above 48.8∞C undoubtedly contrib-uted to the melt characteristics of cheeses contain-ing gelatin. However, the low meltability ofcheeses containing carrageenan indicated thatsome polymers yielding heat meltable gels per-form differently in fat-free process cheeses than inmodel aqueous systems.

In regards to spreadability, guar gum additionsyielded significantly more spreadability com-pared to control cheeses prepared without guargum. This may be due to the thixotropic nature ofguar gum gels and may indicate that other hydro-colloids possessing this characteristic couldenhance the spreadability of fat-free products.While addition of hydrocolloids did not providefat-free process cheeses with textural propertiessimulating full-fat cheeses, qualitative observa-tions indicate that products incorporating hydro-colloids had more uniform, smooth consistencies.Thus, certain hydrocolloids might be useful inprocess cheeses, especially when used at lowerlevels or in process cheeses with high watercontents.

Cook timeThe amount of time fat-free process cheeses wereheld in the cooker at 75˚C was examined duringincrementally increasing times (Table 5). In allcases, approximately four and one-half minuteswas required to reach cook temperature. Cooktime affected firmness and meltability. As cooktime increased, significant decreases in the firm-ness of finished cheeses were observed. On theother hand, meltability tended to increase up to10 minutes of cook time, after this further en-hancement of melt was not observed.Spreadability was not significantly affected bycook time, although absolute values showedslightly greater spreadability resulted with longercooking times.

Since the cheeses in this study were fat free, theconsequences of increased breakdown of youngcheese proteins as cooking progressed might beexplained by a different mechanism. Perhaps lessstructure-building capability remained, resultingin less opportunity for protein-protein interaction.While this explanation appears consistent withthe observations of this study, further investiga-tion on molecular weight profiles of cookedcheese should be conducted to verify this hypoth-esis.

Cook temperatureThe effect of cook temperature on the texturalattributes of fat-free process cheeses is presentedin Table 6. In all trials it took approximately fourand one-half minutes to reach specified cooktemperatures. Our results show that as the cooktemperature was increased, firmness generally

43

Cheese

Table 7. Effect of pH on the functional properties of fat-free process cheeses.

pH Firmnessd Meltabilitye Spreadabilityf

peak force (N) flow (mm) total force (N*s)

5.26 26.0c 42.8b 381.8c

5.64 27.1c 47.5b 472.8b

6.09 32.8b 85.0a 568.2a

6.88 46.8a 82.3a NSTC

a,b,c Means within a column with no common superscripts differ significantly (p<0.05). NSTC = Not spreadable under test conditions. d Means of triplicate determinations for two trials (n=6). e Means of duplicate determinations for two trials (n=4). f Means of duplicate determinations for two trials (n=4), smaller values equal greater spreadability.

Cook Temperature Firmnesse Meltabilityf Spreadabilityg

(˚C) peak force (N) flow (mm) total force (N*s)

60 32.9a 18.5d 533.5a

70 28.2b 37.8c 493.7b

80 23.9d 89.3b 431.9c

90 26.2c 98.0a 342.7d

a,b,c,d Means within a column with no common superscripts differ significantly (p<0.05). e Means of triplicate determinations for two trials (n=6). f Means of duplicate determinations for two trials (n=4). g Means of duplicate determinations for two trials (n=4), smaller values equal greater spreadability.

Table 6. Effect of cook temperature on the functional properties of fat-free process cheeses.

decreased over the temperature range studied(60˚- 90˚C), except in the case of the 90˚C trialwhere cheese firmness increased slightly.A marked increase in the ease of meltability, aswell as spreadability, was observed as cooktemperatures were raised from 60 to 90˚C. Theseresults clearly show that fat-free process cheesesproduced at higher cooking temperatures exhib-ited enhanced melting and spreading characteris-tics. Observations in the present study indicatethat, in the absence of fat, protein structuralinteractions and modifications, and not the fatemulsifying capability of proteins, govern thetextural properties of fat-free process cheese.

pHThe effect of pH on cheese texture was evaluatedby adding glacial acetic acid or powdered sodiumbicarbonate to adjust the pH of the fat-freeprocess cheese formulations. Physical propertiesof cheeses were examined over the range of pH5.26 to 6.88, and results are reported in Table 7.As pH values increased, a corresponding increasein cheese firmness resulted, with significantincreases above 6.0. In the case of meltability,cheeses produced with higher pH values meltedmore readily than those produced with lower pHvalues. Results of experimental process cheesesshowed those produced at lower pH values were

44


Trial % Moisture % Fat Skin Formation1

Fat-Free2 59.0 0.4 +++ Full-Fat2 49.1 20.8 ++ Full-Fat3 49.2 20.9 +

1 + = slight, ++ = moderate, +++ = heavy. 2 Sealed in two-pound high-density polyethylene tubs. 3 Sealed in two-pound loaf box with foil wrapping.

Table 8. Apparent skin-formation on experimental processed cheeses.

more spreadable than those produced at higherpH values. Steady decreases in spreadability werenoticed as pH values increased from 5.26 to 6.88.These observations may indicate the isoelectricpoint of cheese proteins plays a large role inresulting textures of process cheeses. At higherpH values, cheese proteins are further from theirnormal isoelectric point of about 5, and tend toexist in a more open conformation. This wouldfacilitate protein-protein interactions that shouldresult in increased firmness and decreasedspreadability. However, the increased meltabilityof fat-free process cheeses at higher pH valueswould not be explained by this theory, indicatingthat other factors also must be involved.

Changes in pH, relative to the isoelectric point ofcheese proteins, would affect the water-bindingcapacity of fat-free process cheeses. Presumably,cheese proteins function to bind greater amountsof free water at increased pH values. Thesechanges in the ability to absorb water have beenshown to play a major role in the textural perfor-mance of various foods and may have influenceson the texture of fat-free process cheeses. Over-all, the influence of pH on cheese texture occursthrough a number of mechanisms and furtherinvestigation is needed to clarify its effects.

Results of this study indicate that fat-free processcheese physical functionality resulted fromprotein-protein interactions and protein modifica-tion mechanisms. Evaluation of emulsifying salts,hydrocolloids, cook time, cook temperature, andpH showed that all affected final texture to somedegree. Emulsifying salt-type produced largevariations in functionality, with trisodium citrateproducing the most functional textural properties.Emulsifying salts containing polyphosphatesincreased firmness, while decreasing melt and

spreadability, possibly by allowing for increasedprotein interactions after more extensive calciumchelation. Addition of hydrocolloids was not aneffective means for enhancing the functionalproperties of the experimental fat-free processcheeses (49-60% moisture). However, hydrocol-loids may be valuable for texture modification inhigher moisture fat-free process cheese products.

Results from cook time and cook temperaturetrials revealed that increased heat and agitationyielded samples with enhanced functional proper-ties. These treatments would disrupt and possiblybreak down existing protein (casein) structuresproducing softer textures with greater meltabilityand spreadability. Studies of the influence of pHon fat-free process cheese properties revealed thatcheeses produced in the pH range of 5.0 to 6.0had the softest and most spreadable textures.Elevated pH values above 5.6 (6.0-6.9) greatlyenhanced the meltability of fat-free processedcheeses. A mechanism involving properties ofproteins (caseins) at pH values around the isoelec-tric point was proposed to account for the effectsof pH on firmness and spreadability of fat-freeprocess cheeses.

Skin formation on process cheeseIn recent years, industry personnel have reporteda defect in pasteurized processed cheese referredto as skin-formation. Skin-formation is the pres-ence of a shiny, rubber-like appearance on thesurface of processed cheeses following manufac-ture. However, the problem has also been widelyreported in low-fat and non-fat products duringthe heating or cooking process.

Observations on degree of skin formation onpasteurized processed cheeses, along with mois-ture and fat analysis, are given in Table 8. Quali-

45

Cheese

Trial % Moisture (Bulk) % Moisture (Surface)2

Fat-Free3 59.0 45.6 Full-Fat3 49.1 48.2 Full-Fat4 49.2 49.1

1 Means of duplicate determinations (n=2). 2 Determined by removing approximately 1 mm cheese from surface with a cheese cutter. 3 Sealed in two-pound high-density polyethylene tubs. 4 Sealed in two-pound loaf box with foil wrapping.

Table 9. Comparison of bulk versus surface moisture content1 in experimental processed cheeses.

tative observations showed that of all samplesmonitored, fat-free processed cheeses exhibitedthe most skin formation. Of the full-fat cheesesexamined, those stored in high-density polyethyl-ene tubs showed a greater tendency to skin-overthan the same product stored in foil-wrappedloaves. Therefore, initial observations on cheesesproduced in this study indicate that moisture lossmay play a role in the skin formation phenom-enon.

Presumably, in the full-fat processed cheeseshaving the least amount of observable skin, foilwrapping efficiently slowed moisture migrationfrom outer surfaces. Small degrees of skin-forma-tion were still observed, which was probablycaused by moisture losses through folded edges ofthe foil wrapping material. In the case of cheesesstored in high-density polyethylene tubs, moisturemigration and loss from exposed surfaces wasmuch more extensive through the openheadspace and loose covers. Fat-free experimen-tal samples stored in tubs exhibited more exten-sive skin formation than full-fat samples in tubs,apparently in part because of a lack of sufficientfree milk-fat along surfaces to inhibit moisturevaporization.

Surface moisture losses were evidenced byremoving noticeable areas of skin from cheesesurfaces with a cheese cutter and analyzing andcomparing their moisture contents to those ofbulk cheese (Table 9). While clear moisture losseswere observed in the case of fat-free samples,smaller losses were observed in full-fat samples.The small loss of moisture in full-fat cheeses couldbe attributed to increased proportion of bulkcheese removed by the cheese cutter in processedcheeses with thinner skin formation. Overall,

losses of moisture from experimental processedcheeses may have facilitated skin-formationthrough physical alterations of protein structuresin dehydrating cheese surfaces. Furthermore,losses in moisture may have had subsequenteffects on the glass transition temperature ofsurface proteins, functionally causing the skin.

Observations made in this study have relatedmoisture migration from cheese surfaces to theskinning-over phenomenon in processed cheeses.Modification of protein orientations and struc-tures may be partially responsible for the forma-tion of skin. Additionally, shifts in state relative toglass transition temperatures may be important tothe development and overall control of skinformation. However, the results of this study areconsidered preliminary, and further study isneeded to clarify the skin-formation mechanismin processed cheeses.

46


INTERIM REPORT

Improvement of Cheddar cheese quality throughidentification and characterization of microbial enzymesresponsible for the production or degradation of bitterpeptides in cheesePersonnelJames L. Steele, professor, Dept. of Food Science,Mark E. Johnson, senior scientist, Center forDairy Research, Yo-Shen Chen, research assis-tant, Dept of Food Science, Jeff Christensen,research assistant, UW-Madison Bacteriology, JeffBroadbent, associate professor, CharlotteBrennand, assoc. professor, Marie Strickland,research associate, Utah State Univ.


DatesJune 1997—June 2000

Objectives1. Define the contribution of starter proteinasespecificity on peptide pools and bitterness inCheddar cheese.

2. Develop a cheese-based test for bitterness inCheddar cheese and establish factors that influ-ence sensory perception of bitterness in Cheddarcheese.

3. Determine the bitter taste threshold for ß-CN(f193-209) and αs1-CN(f1-9).

4. Define the contribution of Lactobacillus helveticusCNRZ32 peptidases to the degradation of ß-CN(f193-209) and αs1-CN(f1-9).

5. Construct Lactococcus lactis derivatives withenhanced activity of the peptidases demonstratedto be important in the hydrolysis of ß-CN(f193-209) and αs1-CN (f1-9) (UW).

Summary

Variability in the degree of autolysis and intracel-lular peptidase activity among strains ofLactococcus lactis limited our initial effort todefine the relationship between proteinase speci-ficity and bitterness. To overcome this limitation,

we constructed a series of isogenic strains whichdiffer only in proteinase specificity and whichlack the gene for the major lactococcal autolysin,AcmA. The proteinases which we evaluatedincluded the L. lactis Wg2 group e proteinase,CEP, the L. lactis SK11 group a proteinase, andthe group h proteinase from the bitter starter L.lactis S3. The proteinase specificity of eachisogenic construct was confirmed by in vitroincubation of whole cells with αs1-CN(f1-23) atpH 5.2 in 4% NaCl. Reduced fat Cheddar cheeseswere then manufactured using these isogenicstrains which differed only in their proteinasespecificity. HPLC analysis has confirmed thatpeptide accumulation in the experimental cheesesis occurring as predicted by the CEP specificity ofeach starter. Trained sensory analysis of theexperimental cheeses after 2, 4, and 6 mo ofripening has established a clear role for protein-ase specificity in bitterness. As expected, strainscarrying the group a, e, or h proteinase had low,intermediate, or high propensities for bitterness,respectively. These results confirm our previousfindings that starter culture proteinase specificityis a key determinate of whether or not a cheesewill develop bitterness.

In the past, researchers seeking to determine thecontribution of specific peptides to bitterness incheese have relied on sensory evaluation ofpeptides in aqueous solutions to measure bitter-ness. However, sensory studies have clearlyestablished that taste thresholds for a compoundincrease when viscosity increases or when com-peting tastes are present. For this reason, thequantity of any peptide necessary to evoke abitter response will always be much higher incheese than in water, so water dispersion datacannot be reliably applied to cheese. Our grouphas demonstrated that dispersal of bitter com-pounds in a cheese model system is a representa-tive and effective means to study bitterness incheese.

47

Cheese

To our knowledge, we are the first group to studythe contribution of individual peptides to bitter-ness in model cheese system, and our work onbitter taste thresholds for ß-CN (f193-209) andαs1-CN(f1-9) has provided valuable new insightinto the role of specific peptides in bitterness. Inthe case of both peptides the bitter taste thresholdwas approximately 10-fold higher in the modelcheese system than in water. When the bitter tastethresholds of these peptides in the model cheesesystem were compare to the levels of thesepeptides observed in a bitter cheese, it wasconcluded that the αs1-CN(f1-9) was primarilyresponsible for bitterness in this cheese. While theß-CN (f193-209) peptide likely had a complemen-tary function, rather than a dominant role, in theperception of bitterness in this cheese.

The peptidase system of Lactobacillus helveticusCNRZ32, an adjunct that reduces bitterness incheese, has been investigated in detail by Dr.Steele’s laboratory. Genes for ten peptidases havebeen cloned and sequenced from this organism.Of these enzymes, the contribution of 2 generalaminopeptidases (PepC and PepN), a proline-specific aminopeptidase (PepX), and two en-dopeptidases (PepO and PepE) to the hydrolysisof the known bitter peptides ß-CN (f193-209) andαs1-CN(f1-9) have been evaluated. Growth studiesand studies with cell-free extracts (CFEs) ofCNRZ32 and isogenic strains lacking one of thefive peptidases mentioned above revealed that allof the mutants hydrolyzed these peptides com-pletely to free amino acids. These results indi-cated that overlapping specificities in CNRZ32peptidases were masking the effect of individualpeptidases. To overcome this problem, we evalu-ated the rate of hydrolysis and the transitionpeptides formed by cell-free extracts of CNRZ32and the five isogenic peptidase-deficient deriva-tives described above. Differences in the hydroly-sis of ß-CN (f193-209) were only observed be-tween CNRZ32 and the mutant lacking PepNactivity. These results indicated that PepC, PepX,PepO, and PepE have no detectable role in thehydrolysis of ß-CN (f193-209) and that PepNinitiates the N-terminal hydrolysis of this peptide.The observation that 50% of the transition pep-tides identified from ß-CN (f193-209) had either aC-terminal Pro204 or Pro206 residue suggestedthat a post-proline endopeptidase was also in-volved in the hydrolysis of this peptide. Confir-mation of a post-proline endopeptidase inCNRZ32 was obtained by the ability of CNRZ32

CFEs to hydrolyze C- and N-blocked ß-CN(f203-209). The identification of a post- prolineendopeptidase in CNRZ32 is significant, as thisenzyme’s substrate specificity suggests it maycontribute to the hydrolysis of numerous bitterpeptides. Hydrolysis of the αs1-CN(f1-9) by CFEsfrom CNRZ32 and its isogenic derivatives lackingone of the five peptidases previously describedwas evaluated. The primary peptide produced byall CFEs was αs1-CN(f1-7), suggesting either thatan endopeptidase distinct from PepO and PepEor a carboxypeptidase was responsible for theformation of this peptide. Currently, the possibleinvolvement of the post-proline endopeptidase inthe formation of this peptide is under investiga-tion.

Publications/PresentationsChristensen, J.E., E.G. Dudley, and J.R.Pederson, J.L. Steele. 1999. Peptidases and aminoacid catabolism in lactic acid bacteria. Antonievan Leeuwenhoek 76:217-246.

Steele, J.L. (1999). Peptidases and amino acidcatabolism. Institute Food Technol. Abstr., 1999,53-4.

“Peptidases and amino acid catabolism”. Sympo-sium on “Dairy Flavors and Biotechnology” at the1999 IFT Annual Meeting. July 1999.

“Peptidases and amino acid catabolism.” At theSixth Symposium on Lactic Acid Bacteria. Sep-tember 1999 in The Netherlands.

48


INTERIM REPORT

Succinate production by Lactobacillus casei: pathwaysresponsible and development of strategies to control itsaccumulation.PersonnelJames L. Steele, Professor, Dept. of Food Science,Ed Dudley, Research Assistant, Bacteriology


DatesJuly 1997— September 2000

Objectives1. Screening strains of Lactobacillus casei for theability to metabolize citrate and produce succi-nate.

2. Construction and characterization of Lb. caseimutants defective in lactate dehydrogenase andoxaloacetate decarboxylase.

3. Evaluate the effect of the lactate dehydrogenaseand oxaloacetate decarboxylase mutations on theability of Lb. casei to produce succinate in a modelcheese ripening system.

Summary

Succinate is an organic acid known to affect theflavor of fermented foods and beverages. Non-starter lactobacilli are primarily responsible forthe production of succinate in Cheddar cheese,however limited information exists concerningthe pathways utilized.

Previously we reported the screening of twostrains of Lb. plantarum, twelve strains of Lb. casei,and eight strains of Lactobillus rhamnosus (for-merly Lb. casei subsp. rhamnosus) for succinateproduction. Cultures were grown to carbohydrateexhaustion in a complex medium under anaero-bic conditions, and were resuspended in phos-phate buffer saline pH 7.0 containing one ofthe following: 10mM citrate, 10mM L-lactate,10mM citrate plus 10mM L-lactate, or 10mMAsp. After 3 days incubation at 37˚C, succinateproduction was detected under all four conditionsfor Lb. plantarum ATCC 14917, and under allconditions except for Asp for Lb. plantarumATCC 14431.

No succinate production was detected with anyLb. casei or Lb. rhamnosus strains studied. Wholecells of ATCC 14917 and ATCC 14431 convertedapproximately 44% and 15% of the citrate and33% and 5% of the lactate to succinate, respec-tively. Additionally for both strains, the amount ofsuccinate produced in the presence of both citrateand L-lactate was higher than the sum of theamounts produced by citrate and L-lactate alone.Therefore, this screen suggested Lb. plantarumpossesses at least three distinct biochemicalpathways for succinate production, and thesestrains are able to cometabolize citrate and L-lactate.

Additionally, the above screen was repeated withthree strains of Lb. plantarum (ATCC 14917,ATCC 14431 and RL3), two strains of Lb. casei(ATCC 393 and ATCC 334), and one strain ofLb. rhamnosus (ATCC 7469). This second screenwas performed similarly to the first screen, exceptall growth media and solutions included 0.5 g/l L-Cys and 10 mg/ml resazurin, were spargedwith 02-free N2 prior to autoclaving, and werestored in sealed bottles or tubes under a pressur-ized N2 atmosphere. Resazurin is a redox-indicat-ing dye that is colorless when Eh<-110 mV. Thus,this screen was done under redox conditionsmore typical of those found in Cheddar cheeses.Under these conditions, ATCC 14917, ATCC14431 and RL3 produced 0.26mM, 1.94mMand 0.34mM succinate in 24h from 10mM citrateat pH 7.0 and 37˚C, respectively. These strainsalso produced 0.75mM, 2.77mM and 1.0 mMsuccinate from the combination of 10mM citrateand 10mM L-lactate, respectively. No succinateproduction was detected from 10mM L-lactate or10mM Asp. Additionally, no succinate productionwas detected from 10mM isocitrate, a precursorto succinate in other bacteria. No succinateproduction was detected from the Lb. casei or Lb.rhamnosus strains.

The above data suggests Lb. plantarum producessignificant levels of succinate from citrate, whileLb. casei and Lb. rhamnosus likely divert citrate to

49

Cheese

other products. While this suggests Lb. plantarummay be added to cheese during the manufactur-ing process to increase the level of succinate,recent research suggests Lb. plantarum does noteffectively outcompete other nonstarter lactic acidbacteria found in Cheddar, including Lb. casei.Therefore, we have chosen the approach ofengineering Lb. casei to produce succinate. Asoxaloacetate is a precursor to succinate in otherorganisms, we began this strategy by targetinggenes whose inactivation might increase intracel-lular pools of oxaloacetate. Two such genes arelactate dehydrogenase and oxaloacetate decar-boxylase (oadA). We have recently isolated oadAfrom Lb. casei ATCC 393 using a degenerate PCRapproach. The gene encodes a putative protein of467 amino acids with sequence identity to oxalo-acetate decarboxylases from other bacteria.Unlike previously described genes from Salmo-nella and Klebsiella, the ATCC 393 oadA is notassociated with two other genes (oadB and oadG)which are involved in associating oadA with thecell membrane and the pumping of Na+ ionsacross the membrane to generate a chemicalpotential. Upstream of oadA are the genes encod-ing the three subunits of citrate lyase and aprotein with identity to the Escherichia coli CitWwhich currently has no defined function. Thesefour genes appear to be cotranscribed with oadA.More sequence information is being obtainedupstream of oadA to isolate the remaining gene(citrate lyase-ligase) expected to be associatedwith the citrate lyase gene cluster. Downstream ofoadA two additional open reading frames (ORFs)were identified. One of these ORFs has identityto a family of transcriptional regulatory proteins.The second ORF has identity to CitG, a proteinof unknown function found associated with thecitrate lyase clusters of Salmonella and Klebsiella.Whether the putative transcriptional regulatoryprotein is functional on the citrate lyase clusterremains to be determined.

Currently, 13C-NMR spectroscopy is being usedto deduce the citrate catabolic pathways of Lb.casei and Lb. plantarum. Enzymatic assays will beused to support the 13C-NMR data. Also, genetictechniques are being developed to inactivate oadAin Lb. casei and to determine the effect of thisinactivation on citrate metabolism and succinateproduction. The results from this study willaddress industry needs outlined under Objective1, Goal 1.1, Tactic 2 of the National Dairy Re-search Plan.

Publications/PresentationsDudley, E.G., M.W. Atiles, and J.L. Steele. (1999).Characterization and physiological role of thebranched-chain and aspartate aminotransferasegenes of Lactococcus lactis. FEMS Sixth Sympo-sium on Lactic Acid Bacteria Abstracts, 1999,H27.

Dudley, E.G. and J.L. Steele. (1999). Productionof succinate by Lactobacillus plantarum, Lactobacil-lus casei and Lactobacillus rhamnous. FEMS SixthSymposium on Lactic Acid Bacteria Abstracts,1999, G54.

Dudley, E.G. and J.L. Steele. (1999). Productionof succinate by Lactobacillus plantarum, Lactobacil-lus casei and Lactobacillus rhamnous. 99th GeneralMeeting of the American Society for Microbiol-ogy, 1999, O-72.

50


FINAL REPORT

Glutathione and Cheddar cheese flavor developmentPersonnelJames L. Steele, associate professor, UW-MadisonFood Science, Bart Weimer, associate professor,Utah State Univ., Debbie Mikesell, ResearchAssistant, UW-Madison Food Science



Objectives1. Construct derivatives of Lactococcus lactis 1228lacking either -glutamyl transpeptidase activity orthe ability to transport glutathione.

2. Evaluation of the ability of Lc. lactis 1228derivatives lacking either -glutamyl transpeptidaseactivity or the ability to transport glutathione toproduce volatile sulphur compounds in a definedmedium which simulates Cheddar cheese ripen-ing conditions.

3. Determine if starter cultures ability to transportglutathione influences the production of volatilesulphur compounds in cheese slurries.

4. Determine if starter culture encoded -glutamyltranspeptidase activity influences the productionof volatile sulphur compounds in cheese slurries.

Summary

A variety of lactic acid bacteria were screened forthe ability to obtain glutamic acid from glu-tathione (-glutamyl-cysteinyl-glycine) and for -glutamyl transpeptidase (-GTP) activity (Table 1).The results of this screen indicated that the abilityto obtain glutamic acid from glutathione is straindependent among lactic acid bacteria; -GTPactivity is also strain dependent; and -GTPactivity is necessary but insufficient for lactic acidbacteria to obtain glutamate from GSH. It islikely that the ability to transport GSH is alsorequired for lactic acid bacteria to obtainglutamate from GSH. Additionally, the observa-tion that -GTP activity was only detected inpermeabilized cells and cell-free extracts indicatesthat -GTP is an intracellular enzyme.

Due to its -GTP activity and ability to obtainglutamic acid from GSH (suggesting the presenceof a mechanism to transport GSH), Lactococcuslactis 1228 was selected for further investigation.The temperature sensitive integration vectorpGH9::ISS1 was introduced into 1228 byelectroporation. Integration of the vector follow-ing propagation at the non-permissive tempera-ture was demonstrated to occur randomly into Lc.lactis 1228 chromosomal DNA by Southernhybridizations utilizing a probe derived fromISS1. These 1228 derivatives were then screenedfor their ability to obtain glutamic acid fromGSH. Although more than 5,000 individualintegrants were screened, no derivatives lackingthe ability to obtain glutamic acid from GSHwere obtained. This result was surprising since weanticipated that this screen would identifyintegrants either -GTP activity or the ability totransport GSH. Currently, we are unable toexplain this result. Subsequently, attempts weremade to identify the gene encoding -GTP activityby complementation of a strain of Escherichia colilacking this enzyme; this approach also failed toidentify the -GTP gene. The inability to identifythese genes made it impossible to constructisogenic strains differing only in their ability tometabolize GSH.

The ability of three strains of Lc. lactis to producehydrogen sulfide from cysteine (Table 2) andGSH (Table 3) was investigated. These strainswere chosen based on their ability to transportand hydrolze GSH. Lc. lactis 1228 is able to bothtransport and hydrolyze GSH (Table 1). Lc. lactisLM0230 possesses -GTP activity but is unable toutilize GSH as the sole source of glutamate (Table1), which suggests it is unable to transport GSH.Lc. lactis Z8 is known to be able to transport GSH(previous published research) and lacks -GTPactivity (Table 1). The results presented in Table 2indicate that while all three strains were able toproduce hydrogen sulfide from cysteine, differ-ences exist both in the relative rates and quantityof hydrogen sulfide produced. The results pre-sented in Table 3 indicate that all three strainswere able to produce similar quantities of hydro-gen sulfide from GSH; however, Lc. lactis 1228

51

Cheese

Table 1. Ability of lactic acid bacteria to use reduced glutathione (GSH) as the sole source of glutamateand g-glutamyl transpeptidase (g-GTP) activity.

Absorbance 600 values 1 g-GTP Activity 2

Strain Growth on Glutamate 3 Growth on GSH 4 Permeablized cells 5

Lactococcus lactis 6 SD SD SD1363 1.43 ± 0.054 0.02 ± 0.004 0.73 ± 0.0701228 1.46 ± 0.032 0.97 ± 0.022 1.08 ± 0.1181362 1.25 ± 0.114 0.81 ± 0.028 0.87 ± 0.0851361 1.21 ± 0.051 0.73 ± 0.045 1.36 ± 0.165ATCC 11454 0.99 ± 0.222 0.61 ± 0.003 1.14 ± 0.109C2O 1.07 ± 0.051 0.13 ± 0.112 0.77 ± 0.048C2 1.13 ± 0.006 0.13 ± 0.006 0.69 ± 0.029LM0230 1.40 ± 0.015 BQL 7 0.70 ± 0.017MG1614 1.51 ± 0.035 0.08 ± 0.002 0.44 ± 0.058S3 1.49 ± 0.032 BQL 0.07 ± 0.01611007 1.19 ± 0.107 0.53 ± 0.011 1.14 ± 0.0781816 1.33 ± 0.036 BQL ND 8DL16 1.05 ± 0.0 0.08 ± 0.015 0.32 ± 0.052Z8 0.98 ± 0.063 BQL NDStreptococcus thermophilus9ATCC 19258 0.93 ± 0.040 BQL 0.02 ± 0.008ATCC 19987 0.98 ± 0.019 0.35 ± 0.053 0.10 ± 0.006Lactobacillus helveticus910386 2.77 ± 0.075 BQL NDCNRZ32 2.78 ± 0.037 BQL 0.40 ± 0.011

1 Absorbance readings at 600nm were taken after a 42hr incubation period2 Absorbance readings at 405nm correlating to g-GTP activity3 Cultures grown in defined media (Christensen & Steele, unpublished) containing 2.7mM Glutamic acid4 Cultures grown in defined media containing 2.7mM GSH5 Cells permeablized with Triton X followed by a 25min incubation with g-GTP synthetic substrate6 propagated at 30°C7 Absorbance 600 values of < 0.018 Absorbance 405 values of < 0.019 propagated at 37°C

did so at a significantly faster rate. These resultsindicate that neither GSH transport nor hydroly-sis is required for the production of hydrogensulfide from GSH; however, strains capable ofboth transport and hydrolysis of GSH are likelyto produce hydrogen sulfide at a significantlyfaster rate.

In summary, GSH is likely the primary source ofcysteine in the cheese matrix and cysteine hasbeen shown to play a critical role in the produc-tion of volatile sulphur compounds involved inthe development of Cheddar cheese flavor. Theability of starter cultures to transport GSH, which

is water soluble and hence most milk-derivedGSH will be lost in the whey, is likely to increasethe level of GSH in the cheese matrix. Addition-ally, lactococcal strains capable of both transportand hydrolysis of GSH are likely to producevolatile sulphur compounds at an enhanced rateresulting in more rapid Cheddar cheese flavordevelopment.

Publications/presentationsMikesell, D. and J.L. Steele. 1998. EnhancedCheddar cheese flavor via starter culture uptakeand hydrolysis of glutathione. J. Dairy Sci., Vol.81, Suppl. 1, 1998, 16.

52


Table 2. Hydrogen sulfide production from lactococcal strains incubated with cysteine.

picomoles of H2S/g (dry weight)

4hrs 8hrs 12hrs 24hrs

Lactococcus lactis SE SE SE SE

Z8 BQL 1b BQL c 36,000 c 2600 89,000 c 95001228 180,000 a 9000 280,000 a 9500 250,000 a 23000 500,000 a 35500LM0230 BQL b 120,000 b 24000 200,000 b 33500 290,000 b 36000

Christensen, J.E., E.G. Dudley, and J.R.Pederson, J.L. Steele. 1999. Peptidases and aminoacid catabolism in lactic acid bacteria. Antonievan Leeuwenhoek 76:217-246.

Steele, J.L. 1999. Peptidases and amino acidcatabolism. Institute Food Technol. Abstr., 1999,53-4.

Mikesell, D. and J.L. Steele. 2000. Production ofH2S from glutathione and cysteine by lactic acidbacteria. Manuscript in preparation

“Production of cheese flavor compounds byamino acid metabolism.” By Dr. Jim Steele atDairy Management, Inc’s National CheeseTechnology Forum. December 1997


48hrs 72hrs

Lactococcus lactis SE SE

Z8 97,000 c 20500 95,000 c 290001228 500,000 a 50000 600,000 a 50000LM0230 470,000 b 55000 490,000 b 47500

1 Below quantifiable limits < 33,000 picomoles of H2S/g (dry weight)a,b,c Means within the same column followed by no common superscript letter differ (P < 0.05)

“Enhanced Cheddar cheese flavor via starterculture uptake and hydrolysis of glutathione.” ByDebbie Mikesell at the 1998 ADSA AnnualMeeting. June 1998.

“Peptdases and amino acid catabolism”. By Dr.Jim Steele at the 1999 IFT Annual Meeting aspart of the symposium on “Dairy Flavors andBiotechnology”. July 1999.

“Peptidases and amino acid catabolism”. By Dr.Jim Steele at the Sixth Symposium on Lactic AcidBacteria. September 99 in The Netherlands.

53

Cheese

Table 3. Hydrogen sulfide production from lactococcal strains incubated with reduced glutathione (GSH).


4hrs 8hrs 12hrs 24hrs 48hrs

Lactococcus lactis SE SE SE SE

Z8 BQL a BQL b BQL b 36,000 b 4950 110,000 b 100001228 BQL a 40,000 a 1800 44,000 a 3700 140,000 a 8500 150,000 a 3050LM0230 BQL a BQL b BQL b 38,000 b 7000 120,000 b 5000

1 Below quantifiable limits < 33,000 picomoles of H2S/g (dry weight)a,b,c Means within the same column followed by no common superscript letter differ (P < 0.05)

54


INTERIM REPORT

Growth of nonstarter lactic acid bacteria in reduced fatCheddar cheesePersonnelMark E. Johnson, senior scientist, Kristen Houck,research specialist, Wisconsin Center for DairyResearch, James Steele, professor, Bilal Dosti,research associate, Vidya R. Sridar, researchassociate , Department of Food Science, Univer-sity of Wisconsin, Jeff Broadbent, professor,Rebekah Allen, research specialist, Utah StateUniversity Food Science Department


Dates July 1997— December 2000

Objectives1. To establish the population dynamics betweenstarter, nonstarter, and adjunct bacteria duringripening of 50% reduced fat Cheddar cheese.

2. To construct derivatives of the adjunct Lactoba-cillus casei subsp. pseudoplantarum that are unableto co-metabolize citrate and lactate and to test theinfluence of the loss of this metabolism on theability of the adjunct to grow in cheese.

3. To establish the impact on the sensory at-tributes of reduced fat Cheddar cheese to whichadjunct bacteria have been added by monitoringthe relationship between growth of starter, ad-junct and nonstarter bacteria and flavor attributesduring aging of the cheese.

Summary

Microbial studies of ripening cheese reveal thatnumbers of starter bacteria decline during matu-ration while those of, while those of nonstarterbacteria (NSLAB; in particular lactobacilli)increase to levels of 107-108 CFU per gram ofcheese. It is well established that starter, adjunct,and NSLAB can have a profound effect on thedevelopment of flavor in Cheddar cheese. Thecause and effect relationship between thesebacteria, however, has not been studied, nor ismuch known about mechanisms that enable thesebacteria to maintain viability or proliferate incheese. While the type and numbers of adjunct

and starter bacteria can be controlled, the types ofNSLAB still remain a matter of chance. It is thehypothesis of this project that certain adjunctbacteria can be used to control the NSLABpopulation to ensure proper flavor development.To test this hypothesis, we are investigating theeffect of adjunct bacteria on the numbers andtypes of NSLAB in ripening cheese and theinfluence of cheese environment on NSLAB andadjunct populations.

The ability to address population dynamicsbetween starter, non-starter, and adjunct bacteriaduring cheese ripening requires methodology thatcan detect and follow changes in that population,over time, at the strain level. Dr. Broadbent’sgroup has found that random amplified polymor-phic DNA (RAPD) fingerprinting by the poly-merase chain reaction (PCR) can be used todifferentiate between individual strains ofLactococcus lactis, Lactobacillus casei, and Lactobacil-lus helveticus. We have also been able to isolatebacterial DNA from commercial cheese and usethis DNA as a template for the amplification of16S rRNA genes to search for sequences fromNSLAB population that cannot be cultured in thelaboratory. These methodologies are now beingused to analyze isolates collected from duplicatevats of 50% reduced-fat Cheddar and Colbycheeses that were manufactured with or without aLactobacillus paracasei (lila) adjunct by Dr. MarkJohnson’s group at the Center for Dairy Research.To date, we have prepared template DNA forPCR from isolates collected from each cheese byplating on Rogosa and Elliker’s agar (8 vats total)at time 0 (press), 2 wks, and after 1, 2, 3, 4 and 6mo of ripening. RAPD analysis of isolates from t= 2, 4 and 6 mo show that essentially all Elliker’sisolates are the starter bacterium, L. lactisSCO213. In cheeses where lila was added, theadjunct clearly dominates NSLAB populations at2 mo, but represents only 50% of isolates at 4 moand was not detected after 6 mo. Heterogeneityclearly exists in the NSLAB population of ourripening cheeses, and DNA sequence analysis of16S rDNA from some of these strains indicatesthat they are predominantly Lb. paracasei, butother species of lactobacilli are also present.

55

Cheese

Lactobacillus casei strains capable of transformingD (-) -lactic acid to L (+)- lactic acid (racemasepositive strains) were tested to establish specificenzyme activity; L and D-lactate dehydrogenases.There is evidence that both L and D-lactatedehydrogenase enzymes are responsible for theconversion of L-lactate to D- lactate. This needsto be confirmed. A separate racemase enzymewas not found. Both lactate dehydrogenaseenzymes of Lactobacillus casei have been se-quenced and work is now progressing to confirmthat the sequences are identical to these enzymesfound in other lactobacilli. Confirmation willhelp establish the roles of these enzymes in thegrowth of lactobacilli in cheese.

Lactobacillus casei is a predominant NSLAB foundin Cheddar cheese, and is often added as starteradjunct in cheese manufacture. The substratesthat enable Lb. casei to grow in ripening Cheddarcheese have yet to be determined. However,citrate (Cit) is thought to be a likely substrate forgrowth. We are investigating the possibity thatcitrate metabolism is required for growth of Lb.casei in ripening Cheddar cheese. A differentialmedia has been developed that can screen Cit +/Cit — phenotypes. Reaction between ferric ionsand potassium ferricyanide due to utilization ofcitrate in the media gives a light to dark bluecolor to the Cit+ strains, and Cit- remain white.Ten strains of Lb. casei were identified by 16SrRNA sequencing, and were screened using theabove media.

All of the 10 strains were determined as Cit+when incubated at 37˚C for 48 hours underanaerobic conditions. Lb. casei ATCC 334 wasdetermined to be transformable and having astrong Cit+ phenotype on the differential media.

Identification of a known citrate permease citPgene was attempted in Lb. casei via PCR. Degen-erate primers resulted in amplification of afragment of expected size from Lb. casei LB26R,ATCC 334 and Lila. Some of the subunits ofcitrate lyase were identified and sequenced fromLb. Casei ATCC 393 (Ed Dudley, Jim Steele’sLab). Work is now in progress to sequence theputative citP fragments obtained via PCR andalso the other subunits of the citrate lyase. Subse-quently, citP and citrate lyase will be inactivatedvia gene disruption resulting in a Cit - mutant ofLb. casei ATCC 334. Competition studies will be

conducted to determine the ability of the Cit -mutant to grow in ripening Cheddar relative to itswild-type strain.

We will be establishing knowledge matricesrelating flavor and the role of adjunct and non-starter microorganisms. We will do this by investi-gating the impact of adjunct bacteria on thegrowth of non-starter bacteria and flavor inreduced fat cheese.

56


FINAL REPORT

Optimizing the standardization of milk to manufacture50% reduced fat Cheddar cheesePersonnelCarol M. Chen, researcher, Mark E. Johnson,senior scientist, Brian Gould, senior scientist,Amy L. Dikkeboom, research specialist, BillHoesly, research cheesemaker, Kristen Houck,research specialist, John J. Jaeggi, assistant re-searcher, Juan Romero, associate researcher,William A. Tricomi, assistant researcher, Matt G.Zimbric, research specialist, Center for DairyResearch



Objectives1. To determine the influence of a constantcoagulant to casein ratio in the manufacture of 50% reduced fat Cheddar cheese in which the milkwas standardized by the addition of reconstitutednonfat dry milk.

Summary

This summary reports results from the last trial ofthis project. In previous trials, we observed adecrease in 12% soluble nitrogen when NDM wasused to standardize whole milk for the manufac-ture of 50% reduced fat Cheddar cheese. In thosetrials, the amount of coagulant used was based onweight of milk. Therefore, less coagulant was usedon a coagulant to casein basis as the amount ofcasein in the standardized milk increased. In thistrial, the amount of coagulant was based on theamount of casein. Each vat of cheese was madewith the same coagulant to casein ratio.

The final cheese making experiments wereconducted in May 1999. Whole milk was stan-dardized by the addition of reconstituted NDM(approximately 20% total solids). Total solids ofthe standardized milks were adjusted by addingwater. Control milks were standardized by creamremoval. The cheese making schedules were thesame with one exception. The control milk

(standardized by cream removal) was cut after a50-minute set while the milks standardized by theaddition of reconstituted NDM were cut in 40minutes (to decrease moisture). Milk and cheesecompositions are listed in Tables 1, and 2. Fat,nitrogen and solids recovery increased withincreasing milk solids via NDM addition. How-ever, fat recovery in the cheese was highest andnitrogen recovery in the cheese was lowest inmilk standardized by cream removal (Table 6).

As expected, there is an increase in lactose in themilk with added reconstituted NDM, a concomi-tant increase in lactic acid in the cheese and adecreased pH (Table 3). Descriptive taste panelsdid detect an increase in acid flavor intensity withthe higher acid cheeses (Table 7). Cheeses madefrom milk standardized by the addition of recon-stituted NDM (above 10% solids) tended tobecome more grainy (mouthfeel) with age, softer(6 mo) but less smooth than cheese made frommilk standardized by cream removal. There wasno difference in Cheddar flavor intensity orpreference between the cheeses at any tasting.However, these cheeses made with NDM wereless preferred in both flavor and texture catego-ries. Comments on the score sheets indicated thecheeses were less preferred due to brittle, grainy,crumbly body and unclean (stale casein) flavors.

The increase in milk solids retained in the cheeseincreased slightly when NDM was added. Most ofthis increase can be attributed to a higher nitro-gen (protein) recovery.

As expected there was an increase in solublenitrogen as the cheese aged but there were nodifferences in the 12 % TCA soluble nitrogen(indication of the extent of proteolysis) betweenthe cheeses of similar age (Table 5).

The use of reconstituted NDM to standardizewhole milk for the manufacture of 50% reducedfat Cheddar cheese has economic benefits but thismust be weighed against potential decreased

57

Cheese

Table 1. Pasteurized milk composition of whole milk standardized with reconstituted NDM for themanufacture of 50% reduced-fat Cheddar cheese.

Treatment (% solids) Solids Fat Total

Protein Casein1 Lactose C:F Ratio

(%)

control 10.26 1.49 3.14 2.46 4.56 1.65

NDM (10) 9.83 1.47 3.03 2.40 4.32 1.63

NDM (12) 11.49 1.79 3.61 2.85 4.98 1.59

NDM (14) 12.76 1.94 4.01 3.21 5.65 1.65

Table 2. Composition of 50% reduced fat Cheddar cheese made with whole milk standardized withreconstituted NDM. Compositional analysis completed on 2 week old cheese.

Treatment (% solids) Moisture Fat Protein1 Salt

Lacticacid Lactose Galactose

(%)

control 47.73 16.31 29.12 1.43 1.92 .01 .01

NDM (10) 48.30 15.77 28.25 1.63 1.72 .07 .04

NDM (12) 48.15 16.20 28.55 1.38 1.84 .43 .02

NDM (14) 48.88 16.00 28.57 1.24 1.90 .65 .03

Table 3. Cheese pH of 50% reduced fat Cheddar cheese made with whole milk standardized withreconstituted NDM.

Treatment (% solids) 1day 2 wk 6 wk 13 wk 26 wk

control 5.11 5.05 5.05 5.13 5.25

NDM (10) 5.20 5.12 5.14 5.15 5.19

NDM (12) 5.17 5.07 4.98 4.96 5.08

NDM (14) 5.15 5.01 4.97 4.91 4.88

58


Table 4 Mean r-value, actual yield, and Van Slyke numbers for 50% reduced fat Cheddar cheesewith whole milk standardized with skim milk or condensed skim milk.

Van Slyke Recovery Values

Actual Yield RF RC RS

control 8.20 89.75 .96 1.158

NDM (10) 8.06 86.90 .96 1.167

NDM (12) 9.77 88.41 .96 1.173

NDM (14) 10.88 89.62 .96 1.153

Table 5 12% TCA Soluble N as a percent of total nitrogen in cheese

2 weeks 6 weeks 13 weeks 26 weeks

control3.55 7.11 12.72 15.20

NDM (10)3.61 7.56 13.28 16.15

NDM (12)3.40 7.48 13.27 16.14

NDM (14)3.09 6.67 12.05 14.36

preference by the consumer. Use of NDM (andincreased solids) resulted in cheeses that did notage well (texture and flavor preferences werelower). Lower total solids could overcome someof the texture problems but not eliminate them.The results indicate that the decreased proteolysisobserved in previous trials was due to lowercoagulant levels.

59

Cheese

Table 6. Mean percentages of mass, fat, and N recoveries of 50% reduced fat Cheddar cheese withwhole milk standardized with skim milk or reconstituted NDM milk.

Treatment (% Solids) Cheese Whey

Pressedwhey Total

Mass recovery (%)

control 8.20 88.87 2.35 99.42

NDM (10) 8.06 88.97 2.30 99.33

NDM (12) 9.77 86.73 2.55 99.05

NDM (14) 10.88 84.89 2.35 98.12

Fat recovery (%)

control 89.75 9.90 .39 100.04

NDM (10) 86.90 12.43 .72 100.05

NDM (12) 88.41 10.78 .63 99.82

NDM (14) 89.62 9.83 .72 100.17

N Recovery (%)

control 75.37 23.37 .64 99.38

NDM (10) 75.36 22.05 .59 98.00

NDM (12) 77.18 22.51 .67 100.36

NDM (14) 77.60 21.95 .61 100.16

Solids Recovery (%)

control 40.58 57.14 .84 98.71

NDM (10) 41.10 56.77 1.33 99.20

NDM (12) 42.92 56.73 1.53 101.18

NDM (14) 42.47 56.34 2.03 100.84

60


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61

Cheese


Cheese applications programPersonnelCarol Chen, coordinator of cheese applicationsprogram, John Jaeggi, coordinator of CDR/industry projects, Mark Johnson, seniorscientist,Amy Dikkeboom, research specialist,Rani Govindasamy-Lucey, researcher, BillHoesly, research cheese maker, Kristen Houck,research specialist, Juan Romero, associateresearcher, William Tricomi, assistant researcher,Matt Zimbric, research specialist, WisconsinCenter for Dairy Research



Objectives1. Provide technical support for the use of com-modity and specialty cheeses in food applicationsystems through consultations, pilot plant trials,application lab evaluations and plant visits.

2. Conduct industry directed cheese applicationsresearch - modifying manufacturing processes oringredients during cheese making to produce afunctionally specific cheese.

3. Direct contact with industry to meet informa-tional needs.

Summary

In addition to Wisconsin cheese industry activi-ties, the 1999 Cheese Applications programannual report includes national cheese industryinteractions. Approximately 75% of the workconducted by the Cheese Applications Program isfor Wisconsin-based companies. Table 1 summa-rizes the Cheese Applications Program clients. In1999, we worked with 60 Wisconsin and 42national cheese industry clients. For Wisconsin,75% of those clients are cheese manufacturers,which is similar to 1998 figures. On a nationallevel, 35% of our interactions involve cheesemanufacturers. The large number of interactionsdemonstrates the commitment between theWisconsin Center for Dairy Research and thecheese industry.

A summary of technical transfer activities(cheesemaking, laboratory work, visits, consulta-tions) can be found in Table 2. This past year weworked directly with Wisconsin cheese manufac-turers to develop manufacturing protocols forcheeses, which target specific flavor profiles,texture and/or functional characteristics. Forexample, we outlined manufacturing protocols,demonstrated cheesemaking in the CDR pilotplant, then assisted in the commercial scale-up ofseveral specialty Italian, English and other variet-ies of cheese. We worked directly with cheesemanufacturers and end users to tailor manufac-ture Cheddar and Mozzarella cheeses for appe-tizer and pizza applications. For these projects itwas critical to clearly understand the desired meltcharacteristics to ensure cheese functionality.

This past year we noticed an increase in labora-tory work. The Cheese applications group con-ducts analytical, microbiological, applications andsensory testing on various cheese samples. Wis-consin 1998 and national 1999 figures weresimilar; 75% of laboratory work being completedin conjunction with CDR pilot plantcheesemaking. However, for Wisconsin compa-nies in 1999, more than half of the cheeses ana-lyzed were commercially manufactured. Thisshows that the cheese industry is placing a greateremphasis on understanding how the cheesecomposition/age affects the physical propertiesand thus the functionality of the cheese in the endapplication.

In 1999 we noted an increase in the number ofcompanies visiting the CDR, and in CDR person-nel traveling within the state of Wisconsin. Wehosted several industry groups at the CDR todiscuss application programs and current cheeseresearch topics. CDR personnel traveled tocheese plants to provide one-on-one technicaltransfers of cheesemaking protocols, milk stan-dardization and other cheese technology issues.

We noted several trends in cheese industrytechnology transfer requests. Controllingcheesemaking parameters, milk standardization,cheese yield, cheese defects and developingprotocols for specialty cheeses continue to be of

62


industry concern. Increasingly, questions aredirected toward the use of UF technology incheesemaking, methods of controlling and mea-suring cheese melt, and enhancing cheese func-tionality through the addition of whey proteins.

Publications and PresentationsMembers of the Cheese Applications Programteam provided technical information at severalnational and regional meetings or conferences.The staff plays an important role in the CheeseTechnology Short Course, sponsored by the UWFood Science Department and CooperativeExtension, held in March and October. Through-out the year, the CDR provides tours for variousjournalists, councils, academia and industrygroups.

“Wisconsin Center for Dairy Research CheeseApplications Program.” by John Jaeggi at theNorthwest Dairy Technology Society. January1999.

“A newly developed melt test procedure.” byMatt Zimbric, Carol Chen, S. Gunasekaran, JuanRomero at the 1999 Annual ADSA Meeting,Memphis, TN. June 1999.

“Lower fat Swiss cheese: Development of accu-rate Swiss-type flavors through adjunct / cultur-ing.” by Amy Dikkeboom, Carol Chen, KristenHouck, Mark Johnson, Robert Lindsay at the1999 Annual ADSA Meeting, Memphis, TN.June 1999.

“Comparative study of milk standardizationmethods and initial milk solids levels in themanufacture of 50% reduced-fat Cheddarcheese.” by Carol Chen, Amy Dikkeboom, MarkJohnson at the 1999 Annual ADSA Meeting,Memphis, TN. June 1999.

“Wisconsin Center for Dairy Research CheeseApplications Program” by John Jaeggi at theSouthwestern Wisconsin Cheesemakers Associa-tion Annual Meeting. October 1999.

Table 1. Cheese Applications Program Clients

Client Wisconsin National

Cheese Manufacturer 47 15End User 9 8Ingredient Supplier 5 7Communications 4 1Equipment Manufacturer 4 0Organization 3 2Consultant 3 1Contract Lab 2 1Milk Producer 1 1Total 60 42

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Cheese

Table 2. Cheese Applications Program Technical SupportActivit y Wisconsin NationalCheesemaking in theCDR pilotplant

Worked with 9 companies: 5 manufacturers, 3ingredient suppliers, 1 equipment manufacturer.

Sixteen cheese making dates: 30%manufacturers, 70% in gredient suppliers &equipment manufacturers.Manufactured a wide variety of cheeses:Beaufort, Caerphilly, Cheddar, Cheshire, Fontina,LMPS Mozzarella

Worked with 3 companies: 2ingredient suppliers, 1 consultant.Six cheese making datesEvaluated new ingredient functionalit yand demonstrated Mexican-stylecheese manufacturing protocols.

Analytical,applications orsensory work

Worked with 22 companies: 14 manufacturers, 3ingredient suppliers, 2 equipment manufacturers,2 end users, 1 consultant.

Thirty-eight sets of analyses: 40% of cheesemanufactured in CDR pilot plant, 60% of cheesescommercially manufactured.

Types of analyses: Composition, chemical,sensory, microbiological, physical properties,cheese functionality in end application.

Worked with 5 companies: 2 end users,2 ingredient suppliers, 1 consultant.

Eight sets of analyses: 75% of thecheese manufactured in the CDR pilotplant, 25% of the cheeses commerciallymanufactured.

Types of analyses: Composition,chemical, sensory, physical properties,cheese functionality in end application.

CDR orOnsite visits

Met with 34 companies: 25 cheese manufacturers,4 equipment manufacturers, 2 in gredientsuppliers, 2 end users and 1 milk producer.

Forty-four visits: 50% visits to the CDR, 50%onsite visits.

CDR visits included discussion on currentresearch, cheese applications, general cheesetechnology and methods of evaluating physicalproperties of cheese.

Onsite visit included assisting in the scale-up ofspecific cheese varieties, discussions of milkstandardization and cheese yield and generalcheese technology questions.

Met with 4 companies: 3 ingredientsuppliers, 1 end user.

Four visits: 100% visits to the CDR.

CDR visits included discussion oncurrent research, cheese applicat ionprogram, genera l cheese technology

Consultations Worked with 57 companies (34 manufacturers, 7end user, 4 ingredient suppliers, 4communications, 3 organizations, 3 consultants, 2contract laboratories, 1 equipment manufacturer)

Ninety-one different cheese topics discussed.

Discussed general cheese technology issues, milkstandardization, cheese yield, controlling themeltability of cheese, cheese defects, cheeseprocess control, UF and RO technology,labeling/nutrient claims issues.

Worked with 31 companies (15manufacturers, 6 end user, 4 ingredientsuppliers, 2 communications, 2organizations, 1 contract laboratories, 1milk producer.

Forty-three different cheese topicsdiscussed.

Discussed general cheese technologyissues, milk standardization, cheeseyield, controlling the meltability ofcheese, cheese defects, cheese processcontrol , UF and RO technology.

64


FINAL REPORT

Extending the cheese net paradigm to include economicevaluation and optimization in cheese manufacturePersonnelJohn P. Norback, professor, Dept. of Food Science


DatesJanuary 1998—January 1999

Objectives1. Devise an economic context for technicalinformation about cheese.Exploit this context bydeveloping and testing the models for usefulnessand accuracy.

2. Create optimization models to assist in deci-sion-making regarding the formulation of cheeseand other dairy products.

3. Build a spread sheet implementation of theoptimization models.

Summary

The cheese net flow of materials paradigm pro-vides a context for determining materials used indairy processing and the order of processing. Thisinformation is a base for organizing ingredientinput costs and selling prices for cheese process-ing outputs, including cheese and various formsof processed whey. This approach, coupled withtechnical information about converting dairyinputs to cheese, has produced an optimizationmodel for dairy product manufacture.

Interviews with dairy experts provided initialvalues for optimization coefficients, necessary tomake a model. The challenge has been to inter-pret and extract the information necessary foroptimization.Constraints that represent a massbalance are a key feature to any optimization ofthis sort.

We have built prototype optimization models andplaced them in a spreadsheet to represent theCheddar cheese making process. After creatingtest objective functions and applying optimizationmethods, in this case linear programming, themodels look very promising. In addition, mass

balance and component balance constraints havebeen built into these optimization models. Themass balance approach means that all outputsfrom processing are considered simultaneouslyduring optimization. No output is considered a“by-product.” In particular, whey is consideredone of the valuable outputs from the cheesemaking process.

A model for Cheddar cheese has been created.Component balance constraints have beendeveloped to allow the decision-maker to controlmeasurable qualities of Cheddar cheese. Forexample, constraints for moisture, fat, varioussolids and fat in the dry matter are all part of theoptimization model. The outputs from thesemodels are realistic and show promise to helpdairy processors make more efficient use of theirresources. This model demonstrates the impor-tance and economic impact of cheese milk stan-dardization. It also provides a way to value wheyoutput products.

In addition, we created a model for ice cream.This model includes constraints that allow theuser to manipulate quality measures such as fatcontent, protein content and freezing pointdepression. The cheese and ice cream modelshave been coupled with other constraints toprovide a ‘product mix’ optimization model. Thismodel allows the incorporation of many suchmodels into one overarching context. This willallow the decision maker to analyze plant wideutilization of incoming resources to achieveoptimal production amount targets.

Publications/presentationsNorback, J. P., “Making Cheese Choices” pre-sented at Cheese Technology Meeting, April,1999, LaCrosse, Wisconsin

Barcenas, C. and J. P. Norback, “Spreadsheetstrategies for diary optimization.” Presented at theIFT annual meeting, July, 1999.

Spread Sheet Strategies for Optimal Managementof a Dairy Plant,Copyright © by CandelariaBarcenas, 1999.Available at the Memorial Li-brary, University of Wisconsin, Madison.

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INTERIM REPORT

Technology for improving the flavor and consumeracceptability of fat-free Cheddar cheesePersonnelR. C. Lindsay, professor, Meral Kilic, researchassistant, Department of Food Science, Universityof Wisconsin-Madison


DatesJanuary, 1997—December, 1999Extended to June, 2000

Objectives1. To identify the consumer acceptance-limitingbrothy/umami off-flavor substance in the watersoluble fraction of aged fat-free Cheddar cheese,determine its mechanism for formation, anddevelop means for its control in fat-free Cheddarcheese.

2. To develop basic technology for optimizing thedesirable flavor properties, especially the fattyacid-based cheesiness flavor, in fat-free Cheddarcheese that is developed by the unique lipase-positive lactobacilli, Lactobacillus casei Lila strain.

Summary

Fat-free Cheddar cheese initially exhibits a blandflavor and rubbery texture, and upon aging itdevelops protein-like or brothy/umami typeflavors that are often accompanied by variousother off-flavors that are often associated withwild lactobacillus fermentations. Studies showedthat quite high levels of glutamic acid generallyaccumulate in aged full-fat Cheddar, and similarlevels also accumulate in fat-free Cheddar wherethe glutamate flavor dominates, partly because ofthe lack of balancing flavors. Thus, the absence orinadequate amount of influential flavor com-pounds in fat-free Cheddar cheese appears to bethe key to consumer-acceptance limiting brothyflavor in fat free cheese.

66



Whey applications programPersonnelKimberlee J. Burrington, coordinator, KarenSmith, researcher, Center for Dairy Research

FundingWisconsin Milk Marketing, Board, Dairy Man-agement, Inc.


Objectives1. Enhance the value of whey-derived ingredientsby providing technical support to the wheyprocessing industry. Provide processing andapplications support for whey, permeate, lactose,whey protein concentrate, whey protein isolate,and whey protein fractions.

2. Conduct industry directed whey applicationsprojects which evaluate the functional attributesof specific whey ingredients in finished foodsystems. Areas of food applications for wheyingredients are dairy and bakery products, bever-ages, soups, sauces, meats, neutraceuticals, andinfant formula.

3. Initiate development of a pilot plant facilitywhich provides the ability to conduct wheyprocessing projects with industry, for the evalua-tion of existing and new processing conditions.The pilot plant should be able to process wheyfrom the cheese vat to the spray dried ingredient.

Summary

This year completed the second year of the WheyApplications program. In1999, the Whey Appli-cations program was in contact with 17 Wiscon-sin-based companies and 41 national companies,consisting of whey processors, ingredient suppli-ers, and end-users. Activities were increased bothin applications and processing support.

Whey applications were developed and presentedat the following events, seminars, and companies:World Wide Food Expo, World Dairy Expo,Producer Value Showcases, the USDEC LatinAmerican/CDR mission, Snaxpo, Pillsbury, WICheesemakers conference, CDR Mexican Cheese

Seminar, and IFT. Applications developmentfocused on energy bars, marshmallow, caramels,dulce de leche, yogurt, and smoothies. Generalwhey processing, functionality, and applicationsinformation was presented 9 times over thecourse of the year.

Membrane processing support was initiated forthe UF Cheese project commissioned by theCheese Industry team this year. A project involv-ing processing support for further processing ofwhey and quality improvements of whey for amember of the CDR Cheese Industry team wasalso in progress and will continue this year. Otherprocessing support has involved further develop-ment of the whey processing pilot plant, with thepurchase of a microfiltration/ultrafiltration unit,repair of an existing high temperature short timeunit, and the ordering of ion exchange equipmentand a spray dryer. Funding approval is underwayfor a pilot scale evaporator also. A cream cheeseseparator was also purchased to do cream cheesedevelopment with the CDR Cheese group. Manyof the needs of the whey processors and end-usershave been informational needs. Typical requestsare for standard methods for chemical and func-tional analysis, specifications, whey ingredientsources, literature searches, formulations forspecific applications, and processing trouble-shooting questions.

PresentationsWhey Applications in Cultured Dairy Products,Chr. Hansen Cultured Dairy Products Sympo-sium, Kimberlee J. Burrington. May 11-12, 1999,Milwaukee, WI.

Composition of Whey Ingredients, Latin Ameri-can Candy Mission, Whey Applications in Con-fections, Karen Smith. March 15, 1999, Madison,WI.

Functionality of Whey Ingredients, Latin Ameri-can Candy Mission-Whey Applications in Con-fections, Kimberlee Burrington, March 15, 1999,Madison, WI.

Whey Processing Effects on Functionality, DMISeminar, Pillsbury Company, Karen Smith, July15, 1999, Minneapolis, MN.

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Cheese

Functionality of Whey Ingredients, DMI Semi-nar, Pillsbury Company, Kimberlee Burrington,July 15, 1999, Minneapolis, MN.

Processing of Whey into Value-added Ingredients,USDEC Asian Mission on Whey Processing andApplications, Karen Smith, August 8, 1999

Whey Ingredient Functionality and Applications,USDEC Asian Mission on Whey Processing andApplications, Kimberlee Burrington, August 8,1999.

Membrane Processes, CDR Seminar, KarenSmith, November 24, 1999, Madison, WI.

Ultrafiltration for Cheesemaking, Grande CheeseAnnual Meeting, December 8, 1999, Fond duLac, WI.

68



Dairy marketing and economics programPersonnelBrian W. Gould, senior scientist, WisconsinCenter for Dairy Research, Reyes Aterido, gradu-ate student, Ulan Asanov, graduate student, Dept.of Agricultural and Applied Economics

FundingWisconsin Milk Marketing Board, University ofWisconsin-Madison, Hatch Funds

DatesDecember 1998—December 1999

Summary

In 1992 researchers at the Wisconsin Center forDairy Research developed a computer program(CHYIELD©) to analyze the use of alternativemilk standardization procedures. CHYIELD©

included a number of features that allowed for asensitivity analysis of variables associated with thecheese making process. However, CHYIELD©

had several weaknesses. In July, 1998 we startedto upgrade and improve CHYIELD©. Theresulting program, EACY© is near completion. Allof the features contained in the originalCHYIELD© program are incorporated withinEACY© . Figure 1, contains an overview of thecharacteristics of this new and improved softwareprogram.

We currently have a test version of EACY©.Several cheese plants have been given copies ofthis software and we plan to incoporate sugges-tions from these plants when we convert the betainto a final version. The final step of this projectconsists of the development of an extensive on-line help system. A computer programmer iscurrently assisting with this phase of softwaredevelopment. We anticipate that we will have theprogram available for purchase by the end of thefirst quarter of 2000.

Development of Market-Related InformationSystems for Dairy Industry ParticipantsThere is no doubt that the marketing environ-ment faced by both dairy manufacturers and farmoperators has changed dramatically over the lastdecade. Figure 2 shows the MW/BFP since 1965.Prior to 1988, this figure shows the close corre-

spondence between the BFP and U.S. milk pricesupports. With a more market oriented U.S. dairypolicy, over the last decade the variability (risk)faced by dairy farm operators and processors isobvious. In response to this increased market risk,dairy industry participants are using some newtools to help control this risk —using forward milkpricing arrangements to lock in raw milk inputcosts (output price) and using dairy-based futuresand options. Through the support of the Wiscon-sin Milk Marketing Board we established theUniversity of Wisconsin Dairy Markets web site,(http://www.aae.wisc.edu/future) in 1998. Theobjective of this web site was to house within asingle location information resources, data andspecialized software that can be used by theWisconsin dairy industry to more effectivelyproduce market both raw milk and manufactureddairy products.

During 1999, this web site has been greatlyexpanded to include a more extensive collectionof current and historical data associated withdairy markets. We have also included a moredetailed graphical analysis of the U.S. dairymarket which is updated daily. These analysesprovide a quick and easy means of understandingcurrent trends that influence milk and manufac-tured dairy product pricing.

Besides being a data archive for dairy producersand processors looking for information, this website contains a number of software applicationsthat assist with controlling dairy price risk andmarketing of milk and dairy-based products.These applications can be divided into twogeneral areas; spreadsheet models to help under-stand pricing under Federal Order Reform andmodels designed to help the dairy industry usedairy-based futures and options to control theirprice risk. For example, an interactive tutorialsystem can be downloaded from the above site toallow both producers and manufacturers under-stand how dairy-based futures and options can beused to control both input and output price risk.There are also a number of spreadsheet-basedmodels covering the use of alternative price riskmanagement strategies that can be downloadedfrom our site.

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Cheese

EACY © can generate a variety of analysesof your cheese yield. Examples include:

♦ Sensitivity analysis: Changes in milkcomposition and the impact on standard-ization methods

♦ Economic impact of product changes:cheeses produced, types of by-products

♦ Yield impacts of alternative standardiza-tion targets: fat content, FDB, casein-to-fatratio

Figure 1. About Eacy©

EACY© is a user-friendly computer pro-gram designedto:

♦ Identify the components of your raw milk& track them in primary & secondaryproducts

♦ Predict your cheese yield for alternativemilk composition & standardization proce-dures

♦ Evaluate the economic consequences ofchanges in milk quality, cheese characteris-tics or market conditions

♦ Allow you to control your cheese yieldsby adopting alternative standardizationstrategies

EACY© is designed to be flexible. Users ofthis software can enter many types of dataincluding:

♦ Raw milk components: total protein,casein, fat & lactose

♦ Cheese characteristics: fat, casein & othersolids retention factors, final moisturecontent, price

♦ Standardization agent profiles: totalprotein, casein, fat, other solids, price

♦ Characteristics of your whey-basedproducts: price, product composition,availability as a primary or secondaryproduct, compatibility with other by-prod-ucts

EACY © can help you manage yourcheese yield data within a single databasesystem.

♦ Enter data only once

♦ Easily modify previously entered data

♦ Match current & future productionprofiles

EACY © can accommodate futurechanges:

♦ Design new cheeses and whey by-products

♦ Specify alternative standardizationprocedures

♦ Alternative dairy-based ingredients assuggested under proposed CODEX rules

70


Adoption of HACCP by the cheese industryThe third area of research undertaken in theDairy Marketing and Economics Program is ananalysis of the principles of Hazard Analysis ofCritical Control Points (HACCP) and how theycan be an integral part of the dairy productproduction process. The first step towards adopt-ing these principles is the development of aHACCP plan reflecting individual productionenvironment. Over the last year we developed aspreadsheet based system that will help small/

medium dairy product manufacturers examinetheir product processes for critical control points(CCP’s) and then develop a HACCP plan tomonitor these CCP’s. This software system isbuilt on a number of menu driven options thatallow the user to enter both production relateddata and points where production hazards mightoccur. We are close to having a prototype modelcompleted and will be testing the spreadsheetmodel in the near future.

Figure 2. Relationship between support and MW/BFP

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Cheese

INTERIM REPORT

A multi-country analysis of household food demand:Implications for U.S. food exports (phase I)PersonnelBrian W. Gould, senior scientist, WisconsinCenter for Dairy Research, D. Dong, researcher,formerly with the Wisconsin Center for DairyResearch,W.S. Chern, professor, Ohio StateUniversity, B.K. Goodwin, professor, NorthCarolina State University, R. Mittlehammer,professor, Washington State University,T.I. Wahl,professor, Washington State University

FundingU.S. Department of Agriculture, National Re-search Initiative Competitive Grants Program,Babcock Institute for International Dairy Devel-opment, University of Wisconsin Madison

DatesOctober 1998—September 2001

Objectives1. Review the literature regarding alternativemethods for estimating disaggregated food de-mand systems that incorporate limited dependentvariables.

2. Identify an appropriate methodology to ana-lyze disaggregated food (dairy product) demandin developing and developed countries thatovercomes the limitations imposed by earlier two-stage estimation procedures.

3. Develop the necessary econometric software toapply this methodology to household level foodexpenditure data.

4. Apply this methodology to a single county toverify its ability to accurately describe the struc-ture of international food demand.

Summary

Understanding how food consumption respondsto changes in relative prices, income, householdcomposition and other exogenous factors isimportant for U.S. farm operators, processors,policy analysts and policy makers looking toexpand the markets for U.S. farm products. Thiscan be said not only with respect to domestic butalso foreign markets. Given the recent economic

problems in the former Soviet Union, LatinAmerica and Asia, an understanding of thestructure of food demand in current and poten-tially important export markets is more importantthan ever.

A good example of the importance of under-standing the nature of international food demandcan be found in the U.S. dairy sector wheregrowth in domestic demand is relatively flat.TheU.S. share of world dairy trade is small andcompeting dairy producing countries are attempt-ing to increase their dairy export efforts. Previousresearch has indicated that the U.S. dairy industrywill increase its export activity under tradeliberalization via the passage of the North Ameri-can Free Trade Agreement and the UruguayRound Agreements of the General Agreementson Tariffs and Trade and the elimination ofdomestic dairy price supports after 1999. Withreduced trade barriers, we need to understand thestructure of dairy product demand in potentiallynew export markets. It will be important toquantify the sensitivity of consumption levels tochanges in household income, the impact ofchanges on market price, the role of age/sexcomposition of households on dairy productconsumption and the implications of futurechanges in these variables. This research projectattempts to answer such questions not only fordairy products, but also for other foods thatcompete for dairy products’ share of theconsumer’s food budget.

Previous analyses focused on identifying thedeterminants of the type of food (dairy products)and how much was purchased have used histori-cal time-series (annual, quarterly, or in somecases, monthly) data and prices, incomes, andper-capita consumption. The inferences yieldedby such analyses are important and useful. How-ever, these inferences may be influenced bysignificant structural adjustments characterizingindividual economies. An alternative approach tothe estimation of demand system parameters usescross-sectional data collected from individualhouseholds. This approach to demand analysishas several advantages. Cross-sectional analyses

72


make possible richer, more detailed inferencesdrawn from more detailed disaggregated house-hold level data. In particular, the use of cross-sectional surveys enables the researcher to evalu-ate demographic and socioeconomic factors thatare relevant to food demand issues. Perhaps themost important factor is that cross-sectionalconsumption data often provide the degrees offreedom necessary to permit an analysis to beconducted using very recent data. This advantageis important when considering food demand incountries that have undergone significantchanges, such as the Eastern European countries,Latin America and China.

This project uses a number of internationalhousehold expenditure/purchase survey data setsto evaluate, compare, and contrast dairy productand other food demand conditions in severalcountries important to US agricultural exports.The following general approach to the analysisand estimation of food demand parameters willprovide a coordinated research effort across teammembers. First, a demand system comprised ofaggregate commodity classes (e.g., beef, pork,vegetables, fruits, grains, dairy products) will beestimated. Second, less aggregated models per-mitting a greater degree of detail will be evalu-ated for the commodities of particular interest inthis study. Demographic effects and the censoringimposed by observed zero purchases will beconsidered in the estimation process. Accountingfor the censored nature of disaggregated fooddemand is essential for obtaining unbiasedestimates of the structure of such demand.

For this project we emphasize the use of a de-mand system approach to characterize the foodchoices made by consumers. We use the systemapproach as it allows one to quantify the trade-offs associated with the consumption of one typeof food versus another. Using household-leveldata for demand analysis requires the appliedresearcher to account for the censoring of com-modity purchases. When analyzing food demandone commodity at a time, there are a number offairly standard econometric techniques that canbe used to address this censoring. The statisticalissues become much more complex when ex-panding the analysis from a single equationapproach to a demand system framework. Thisproject will extend basic research to an analysis offood (dairy product) demand in a number ofdeveloped and developing countries.

Since starting this project we have developed aunique econometric model that accounts for boththe censoring of commodity demands within asystems framework as well as addressing the issueof how to evaluate commodity prices for non-consuming households. An increasing number ofcross-sectional surveys of food purchase behavior,especially those associated with developingcountries, include both quantity and expendituredata. Division of observed expenditures byquantity (here referred to as unit-value) is oftenused as an estimate of a commodity’s price. Thismethod of calculating price reflects not onlydifferences in market prices faced by each house-hold but also differences in endogenously deter-mined commodity quality. For example, observeddifferences in price paid for cheese across house-holds may be reflecting not only local marketconditions but also final product form. House-holds purchasing cheese in block form would beexpected to pay a lower price than householdspurchasing cheese that is pre-sliced or shredded.The portion of product price determined bymarket forces is obviously beyond the control ofthe consumer.

During Phase I of this project we developed amethodology that will allow researchers to consis-tently estimate purchase price for non-consuminghouseholds along with the other food demandparameters. Using our demand system approach,researchers can investigate the determinants ofthe multi-stage purchase process faced by theconsumer: whether-or-not to purchase a particu-lar commodity versus the decision as to theamount to purchase given that an individual doesindeed purchase the commodity. A detailedreview of our methodology can be found inGould and Dong (1999) and in Dong and Gould(2000). Some preliminary investigations of thestructure of food demand in Mexico and theFormer Soviet Union undertake as part of theproject can be found in Gould and Kim (1998a,b).

Using our econometric structure, we have under-taken some preliminary model testing usingCanadian food expenditure data. In this test casewe present the results obtained from a five-commodity system composed of aggregate beef,pork, poultry, cheese and fluid milk. The exampleshown here is meant only to be illustrative. Theexpenditure data used in this preliminary testingwas obtained from the nationwide 1992 CanadianFamily Food Expenditure Survey. This survey con-

73

Cheese

tains two-week diaries of food expenditures andquantity purchased where each expenditure itemis coded according to a four-digit food code. Inaddition to purchase information, other dataincluded in the survey are household member agedistribution, pre-tax household income, male andfemale head country of birth, residential prov-ince, degree of urbanization and month duringwhich the survey was undertaken. There are10,848 households in the base data set. For thisanalysis we use a random sample of approxi-mately 3,000 households. Table 1 provides anoverview of data used in the analysis.

From the implementation of our econometricmodel to Canadian food demand, we estimateuncompensated price and expenditure elasticities(Table 2). These elasticities quantify the percentchange in quantity purchased of each food as aresult of a percent change in a variety of explana-tory variables such as own price, other goodsprices, and household income. All of the own-price elasticities are negative, statistically signifi-cant and of reasonable values compared toprevious studies. We are currently developing themethods to separate the total impact of price

Table 1. Characteristics of Canadian Data Used With Preliminary Model Specification

Purchase CharacteristicsCommodity Percent Bi-Weekly Expenditure Bi-Weekly Expenditures ($) Price

Purchasing Shares( %) (CND$)

Uncond. Conditional Uncond. ConditionalMilk (litre) 92.5 33.2 35.9 10.29 11.12 1.10Cheese(kg) 72.1 19.7 27.4 7.42 10.29 8.97Beef (kg) 68.0 24.5 36.0 12.09 17.78 6.45Pork (kg) 39.2 8.6 22.0 4.20 10.73 6.19Poultry (kg) 51.1 13.9 27.3 6.78 13.25 5.21

Characteristics Used in Household Characteristics Used In Unit-Value EquationsTranslating Functions

Variable Mean Variable Mean Description

QRTR 1 0.251 HH Inc ($) 42,350 Annual Pre-Tax Income(28,555)

QRTR 2 0.252 HH Size 2.68 Household residents eating out(1.39) of household food supply

QRTR 3 0.246 Metro 0.618 Household located in urbanarea with population 100,000+

Single HH 0.210 Quebec 0.197 Dummy variables identifyingprovince of residence

Two NOKD 0.252 Ontario 0.239Manitoba 0.062Sask 0.081Alberta 0.081BC 0.099

Note: The conditional expenditure shares do not sum to one as these values are conditional on therebeing positive shares. The unconditional shares and expenditures are calculated as the mean acrosshouseholds. QRTR variables identify quarter data was collected. Single HH identifies single personhouseholds. Two NOKD identifies two-person households with no children.

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(expenditure) changes on unconditional demandinto the component impacts on conditionaldemand and market entry. This is similar to thedecomposition shown by McDonald and Moffittin their analysis of Tobit results. This decomposi-tion will allow us to identify the relative impor-tance of the market entry versus conditionaldemand price (expenditure) responses.

Comparing the results obtained under our ap-proach, we present price and expenditure elastici-ties obtained from the estimation of two alterna-tive model specifications: traditional LinearApproximate Almost Ideal Demand System (LA/AIDS) without accounting for censored quantitydistributions and Heien and Wessells (1990) two-step censored demand system. Under the firstalternative model we estimate the LA/AIDSmodel using the traditional SUR specification(referred to as the “uncensored” model). Similarto other researchers, we estimate missing pricesusing independent unit-value regression equationsfor consuming households. Under the Heien andWessells (1990) specification, a SUR estimationprocedure is used where all (including observa-tions with zero-valued shares) are used. A seriesof explanatory variables calculated fromunivariate probit models of the decision-to-purchase stage are used as explanatory variablesto account for censoring. Again, missing unit-values are calculated from a set of exogenousprice regressions.

We obtained significant and negative own-priceelasticities under the two alternative models. Themost surprising result was the similarity of theown-price elasticities in spite of significantlydifferent parameter values generated under ourcensored demand system. The similarity of theresults between the censored versus uncensoredsystems is in contrast to the results obtained byHeien and Wessells (1990) who found, for anumber of commodities, reduced own-priceelasticities for their censored versus uncensoredcommodities with the difference being propor-tional to the degree of censoring. These are alsosurprising in light of the fact that we are usingendogenous unit-values to estimate demandfunction parameters under our censored systemwhile the other two procedures use the moretraditional exogenously estimated unit-values.The cross-price elasticities were also very similar

across model specification with the exception ofthe influence of milk price on cheese and beefpurchases. Under all three specifications wefound a complementary relationship but the sizeof the price response was much larger under ourspecification. Considering the similarity of theresults obtained under the three specificationsversus the differences found by Heien andWessells (1990) in their analysis, points to theneed for examining the robustness of our resultsto more disaggregated commodity definitions.

Future researchOne problem that needs to be addressed isreducing the computation time required toestimate the econometric model. Our currentalgorithm is developed using the GAUSS soft-ware system. Given the short time since our initialfunding (5 months) we developed the codenecessary to obtain parameter estimates with littleconsideration for computational speed. We plan anumber of approaches to improving computationspeed during the last half of our current funding.First, we will modify our current computer code.Under the current configuration there are majorsections of the GAUSS code that could be im-proved by the eliminating numerical loops andreplacing them with algorithms based on fastermatrix procedures. Second, we are currentlyusing an algorithm developed by Breslaw toevaluate higher order integrals. We will experi-ment with alternative approximating algorithmsto determine if there could be significant reduc-tions in computational time. Finally, we willinvestigate whether computational time could bereduced if an alternative software system such asMATLAB or Mathematica were used to optimizethe models likelihood function.

We currently have obtained food (dairy product)expenditure/purchase data for Canada, Mexico,Brazil, China and Eastern Europe. We are at-tempting to obtain 1996 data for Argentina. Withthis data, we will use the econometric lessonslearned under Phase I of this project to betteridentify the important determinants of the struc-ture of food (dairy product) consumption inthesecountries.

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Cheese

Presentations/publications

B.W. Gould and D. Dong, 1999. Estimation ofCensored Demand Systems withEndogenous Unit Values, Working Paper, Depart-ment of Agricultural and Applied Economics,University of Wisconsin-Madison, October

D. Dong and B.W. Gould, 2000. Quality VersusQuantity in Mexican Household Poultry and PorkPurchases, forthcoming, Agribusiness.

Brian W. Gould and J. Kim, 1998. The Structure ofMeat, Poultry and Dairy Product Demandin the Former Soviet Union, Babcock InstituteDiscussion Paper, Babcock Institute forInternational Dairy Development, University ofWisconsin-Madison, October.

Table 2. Comparison of estimated uncompensated price and expenditure elasticities under our prelimi-nary censored system with other published model specifications

Commodity Price Elasticities Expenditure Elasticities

Fluid Milk Cheese Beef Pork Poultry

Censored SystemFluid Milk -0.737* -0.027* -0.039* 0.008 0.023* 1.023*

Cheese -1.456* -0.464* -0.038 0.105 -0.095 0.637*

Beef -1.341* -0.040 -0.690* -0.104* 0.078 1.032*

Pork 0.325 0.021 -0.200* -0.849* -0.161* 1.100*

Poultry 0.307 -0.075* 0.021 -0.077* -1.081* 1.221*

Heien and WessellsFluid Milk -0.832* -0.021 -0.163 0.117 -0.156 1.058*

Cheese -0.097* -0.581* -0.094* 0.092 -0.252* 0.462*

Beef -0.142* 0.039 -0.679* -0.173* -0.174* 0.975*

Pork 0.019 0.067 -0.072* -0.896* -0.078 0.882*

Poultry -0.006 0.033 0.033 -0.021 -1.079* 1.740*

Uncensored SystemFluid Milk -0.808* -0.109 -0.201* 0.075 0.005 1.058*

Cheese -0.136* -0.571* -0.106* 0.012 -0.105 0.542*

Beef -0.153* 0.047 -0.686* -0.312* -0.060 1.093*

Pork 0.022 0.055 -0.103* -0.804* -0.069 1.125*

Poultry 0.017 0.037 0.002 -0.096 -1.039* 1.268*

Note: * identifies elasticities significant at the 0.01 level of significance. Elasticity variances are calcu-lated using Monte Carlo methods. The elasticities were evaluated at the mean values of the exogenousvariables. The price elasticities reflect the impacts on unconditional quantity demanded.

Brian W. Gould and J. Kim, 1998. Characteristics ofCanadian and Mexican Dairy ProductPurchases: A Comparison Using Household Expendi-ture Data, Babcock Institute DiscussionPaper, Babcock Institute for International DairyDevelopment, University of Wisconsin-Madison,July.

Mcdonald, J.F., and R.A. Moffitt, The Uses ofTobit Analysis, Review of Economicsand Statistics, Vol.62:318-321.

Heien, D., and C. R. Wessells. Demand SystemsEstimation with Microdata: ACensored Regression Approach. J. Bus. and Econ.Statis. 8(1990):365-71.

Breslaw, J.A., Evaluation of Multivariate NormalProbability Integrals Using a LowVariance Simulator, Review of Economics andStatistics, Vol. LXXVI:673-683.

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INTERIM REPORT

Development and application of a cheese shred/texturemap delineated by cheese rheological, sensory andchemical analysisPersonnelCarol M. Chen, researcher, Juan E.Romero,associate researcher, Mark Johnson,senior scientist, Brian Gould, senior scientist,Wisconsin Center for Dairy Research, SundaramGunasekaran, professor, Biological SystemsEngineering

FundingWisconsin Milk Marketing Board and DairyManagement Inc.

DatesJuly 1999—December 2001

Objectives1. To develop a shred / texture map of cheesesbased on rheological, sensory and chemicalmeasurements.

2. To define manufacturing protocols of Cheddarand Mozzarella, tailored for shredding.

Summary

We started to work on this project in Novem-ber,1999 by procuring the cheese shreddingequipment. Electrical installation was then com-pleted in the dairy plant. In order to do our tests,we are now setting up a controlled environment.The rheometer has been set up and we havebegun optimizing our testing parameters (tem-perature, capstan diameter) using commercialcheese samples. We anticipate beginning work onobjective 2 during calendar year 2000.

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Cheese

APPLICATIONS PROGRAMREPORT

CDR specialty cheese applications programPersonnelJim Path, outreach specialist, John Jaeggi, assistantresearcher



Objectives1. Continue developing the artisan workshops, amodule of the Wisconsin Master Cheesemaker®program.

2. Provide technical support to cheesemakers,including workshops, consulting, and on sitemanufacturing trials.

3. Manage the Wisconsin Master Cheesemaker®program.

4. Develop a cheese database.

Summary

The third class of certified Wisconsin MasterCheese makers graduated and were honored atthe CDR/WCMA ceremony in La Crosse,Wisconsin on April 14, 1999. Eight cheesemakersreceived this honor during the ceremony, whichwas widely covered by the media. CDR hasfeatured cheeses by Wisconsin MasterCheesemakers at several meetings and events.

Eight more Wisconsin Master Cheesemakers(class of 2000) were certified by the MasterCheese maker Board on December 9, 1999. Theywill be officially recognized at the CDR/WCMAceremony on April 27, 2000. The class of 2001has completed the second round of cheese sampletesting in the apprenticeship phase of the pro-gram. The board received 12 new applicationsand approved 12. These 12 (class of 2002) havecompleted the oral exam and plant visit phase ofthe apprenticeship.

A 2nd Cheeses from Mexican/Latin AmericaCheese Seminar was held on April 22-24, 1999.Instructors from Mexico, CDR and a consultant

with experience in Costa Rica and Mexicopresented information about the manufacture ofMexican cheeses. Production of cheeses wasdemonstrated in the dairy plant.

On Feb 22-23,1999 the Wisconsin Process CheeseCourse was held as part of the Wisconsin MasterCheese makers curriculum. This unique course,the only course of it’s type in the USA, was filledto capacity.

On May 4-5, 1999 the Wisconsin Dairy PlantWater & Waste Management Short course washeld at Babcock Hall in Madison. This is a jointeffort between CDR and Agricultural Extension.

The CDR World Cheese Exchange is now avail-able on CDR’s website. This database lists over1400 different varieties of cheese and we encour-age submissions from cheesemakers as we con-tinue adding photos and data.

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chapter 2,section 2Cheese safety

Microbiological safety of reduced fat and fat free pasteurized process cheese products ................................................. 81

Safety/quality applications program ...................................................................................................................................................... 83

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Cheese safety

INTERIM REPORT

Microbiological safety of reduced fat and fat freepasteurized process cheese productsPersonnelEric A. Johnson, professor, Kathleen A. Glass,researcher, Food Research Institute, University ofWisconsin-Madison

FundingDairy Management, Inc.

Dates January 1997—December 1999

Objectives1. Evaluate the effect of fat and fat-replacers ongrowth of Clostridium botulinum in full fat, reducedfat, and fat free process cheese products.

2. Determine the efficacy of antimicrobials in fullfat, reduced fat, and fat free process cheeseproducts.

3. Identify factors that inhibit botulinal toxinproduction in full fat, reduced fat, and fat freeprocess cheese products.

4. Develop the foundation to expand the FRImodel to predict growth and toxin production byClostridium botulinum in reduced fat and fat freeprocess cheese products.

5. Evaluate the effect of moisture-fat free andnonfat solids in process cheese products madewith skim milk cheese, disodium phosphate, NaCland water.

Summary

Previous research in our laboratory revealed that5% fat and fat free pasteurized process cheeseproducts delayed toxin production by Clostridiumbotulinum when compared with full fat productswith similar moisture, pH, and total salts. Theobjective of this study was to evaluate the effectsof cheese-base type, fat, 0.05% monolaurin, 1.5%Cheddar enzyme modified cheese (EMC), 1.5%sodium lactate, and 3% ß-glucan fat replacer onthe botulinal toxin production in pasteurizedprocess cheese products.

To evaluate the effect of type of natural cheesefrom which it was derived, process cheese prod-ucts were formulated using full fat Cheddar, 30%reduced fat Cheddar, or skim milk cheese, respec-tively, and standardized to 59% moisture, pH 5.8,3 or 4% total salts (sodium chloride+disodiumphosphate), and 15-19% fat using anhydrous milkfat. Subsequent trials evaluated the effect ofadjunct ingredients in process cheese productsformulated to <1, 10, and 20% fat when madewith skim cheese, reduced fat and full fat cheese,respectively.

In trials evaluating cheese-base type (fat standard-ized to 15-19%) botulinal toxin production wasdelayed several days in 15-19% fat productsformulated with skim cheese compared withreduced fat or full fat cheese. However, the effectwas not statistically significant (p>0.05). When fatlevels were not standardized, botulinal toxinproduction was significantly delayed in productsmade with skim cheese (<1% fat) compared withreduced fat (10% fat) or full fat (20% fat) cheese.Reducing fat in skim milk-process cheese productformulations from 15 to <1% fat resulted in a 2-week delay for botulinal toxin production.

In a previous reporting period, we formulated fatfree process cheese products with high moistureskim milk cheese. The formulation was adjustedto 68% moisture, pH 5.8, and 3% total salts(disodium phosphate+sodium chloride). Inocu-lated formulations were supplemented with 1%EMC, 1.5% sodium lactate, 0.05% monolaurin or4% ß-glucan fat-replacer. No difference in time todetectable toxin production was observed amongthe four treatments and the control withoutsupplement, likely due to the high moisture of theproduct. We repeated the tests in fat free, reducedfat, and full fat products formulated to 62%moisture, pH 5.7%, 3 or 4% total salts, and 1.5%Cheddar EMC (sample previously shown toinhibit botulinal growth in media), 1.5% sodiumlactate, 0.05% monolaurin or 3% ß-glucan fat-replacer.

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Sodium lactate significantly delayed toxin produc-tion for all cheese types tested, however the fat-replacer did not delay growth. Botulinal toxinproduction was delayed 11, 14, and 47 d for fullfat, reduced fat, and fat free process cheese prod-ucts formulated with 1.5% sodium lactate, respec-tively. No delay in toxin production was detectedin products formulated with ß-glucan. Monolaurinand EMC significantly delayed toxin productionin skim cheese products, but had less effect inreduced fat and full fat products. Monolaurin didnot delay time to toxin production in reduced fatand full fat products but the number of toxicsamples was fewer. However, botulinal toxinproduction was delayed two weeks in fat freeproduct formulated with monolaurin. Addition of1.5% EMC did not delay toxin production in fullfat cheese, but delayed toxin production 3 and 67days for reduced fat and fat free products, respec-tively. One should use caution in interpreting theeffect of EMC because antibotulinal activity maybe dependent on the method used to produce theEMC.

These results verify that reduced fat processcheese products manufactured with fat free andreduced fat cheese may exhibit greater stabilitythan full fat products and that safety may beenhanced by using certain adjunct ingredients.

Process cheese and related foods and spreadsaccount for over 2 billion pounds of dairy food inthe United States. Traditionally, the microbiologi-cal safety of these products relies on formulationto inhibit toxin production by Clostridium botuli-num. In order to produce organoleptically accept-able reduced fat and fat free process cheeseproducts, microbial control factors such asmoisture, salt, and pH are often adjusted to morepermissive conditions. This raises safety concernsamong the dairy industry and regulators.

Preliminary research at FRI suggests that C.botulinum toxin production is delayed in reducedfat and fat free process cheese products com-pared with full fat products with similar levels ofmoisture, salt, and pH. However, the mechanismof inhibition is unknown. The objective of ourstudy is to identify factors that control botulinaltoxin production in process cheese productswhich will permit greater flexibility in formulat-ing safe products that appeal to the consumer.This in turn, will increase dairy consumption.

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Cheese safety

Safety/quality applications programPersonnelMarianne Smukowski, coordinator



Objectives1. Maintain and improve HACCP-based safety/quality programs used by manufacturers andproducers

2. Continue strong relationships with DMI,IDFA, and FDA for implementation of HACCP-based applied technologies

3. Assist in executing national safety/qualityprogram

4. Conduct technology development targeted atWI cheese manufacturers

5. Assist the Wisconsin Master CheesemakerProgram®

6. Participate and assist in UW and industrysponsored courses

Summary

The Safety/Quality Applications Program assistsWisconsin dairy manufacturers in the followingareas: safety/quality audits, GMP reviews,developing HACCP plans, aid the WI MasterCheesemaker program®, and provide technicalsupport in regulatory matters. A total of 30 plantvisits were made this year and numerous phonecalls were answered to address S/Q audits,HACCP implementation, and regulatory issues. Iam a member of the NCIMS laboratory commit-tee, which addresses the use of FDA 2400 formsand laboratory practices. I reviewed severalgrade standards and specifications for cheeseincluding Swiss and Muenster. These changes tothe grade standards would affect WI manufactur-ers.


One of the major accomplishments of the Safety/Quality program was the offering of a dairyHACCP workshop. The class enrollment wasoriginally 45 people due to the breakout sessions.However, the enrollment had to be expanded to55 to accommodate the overwhelming responsefor this first time workshop.

Publications and presentationsWI cheese grading short course, Italian cheeseevaluation (twice a year)

Intercollegiate Dairy Products Evaluation Contest,Lead Butter Judge

WI CIP Workshop, Plant Sanitation Audits

WI Dairy Products Assoc. Cheese and butterevaluation clinic, Overview of butter Grading

GMP presentation, Kerry Ingredients, Vesper, WI

Dairy HACCP Workshop, Phase II, Greencounty, WI

1999 WI State Fair Judge for butter and cheeseproducts

Intro to Codex, WDATCP All Employee Meeting,WI Dells, WI

Importance of HACCP Implementation, Manag-ing Dairy Food Safety Workshop, Madison, WI

Overview of HACCP, USDA Dairy Division AllEmployee meeting, Mpls, MNHACCP and theDairy Industry: An Overview of Internationaland U.S. Experiences by B. Gould, M.Smukowski and J. R. Bishop

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Identification and characterization of components of the proteolytic enzyme system of Lactobacillus helveticuswhich affect bioactive peptide accumulation ..................................................................................................................................... 87

Application of milk powders in milk chocolate, butter and butter spreads ............................................................................. 88

Growth and biocontrol of enterotoxigenic Bacillus cereus in infant formula and processed cheese prepared withmilk powder ................................................................................................................................................................................................... 89

chapter 3Fluid milk

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Fluid milk

INTERIM REPORT

Identification and characterization of components of theproteolytic enzyme system of Lactobacillus helveticuswhich affect bioactive peptide accumulation

PersonnelJames L. Steele, professor, Jeff Pederson, post-doctoral researcher, Dept of Food Science,UW-Madison, Bart Weimer, associate professor,Jeff Broadbent, assoc. professor, Utah State Univ.



Objectives1. To screen strains of Lactobacillus helveticus forthe type and level of bioactive peptides/bioactivepeptide precursors which accumulate as the resultof the organism’s growth in milk.

2. Determine which components of the pro-teolytic systems of the selected strains of Lb.helveticus are essential for the accumulation ofbioactive peptides/bioactive peptide precursorsfrom milk.

3. Construct strains of Lb. helveticus which accu-mulate elevated levels of the bioactive peptides/bioactive peptide precursors of interest.

Summary

Our progress towards objective one involveddeveloping analytical techniques for the rapididentification of peptides. Specifically, progresshas been made in the area of coupling capillarychromatography with mass spectroscopy. Thecoupling of these pieces of equipment shouldallow us to rapidly screen strains of Lactobacillushelveticus for the type and level of bioactivepeptides which accumulate as a result of organismgrowth in milk. Research towards objective twohas focused on the cell-envelope proteinasespecificity of various Lactobacillus helveticus strains.To date, the proteinase specificity of eight strainshas been determined using the alpha S1-caseinfragment (f1-23) as a substrate for hydrolysis.However, Lactobacillus helveticus has unique cell

surface proteinase specificity. The gene encodinga cell-envelope proteinase from Lactobacillushelveticus CNRZ32 was cloned by PCR amplifica-tion. Primers were designed based on the nucle-otide sequence of the proteinase gene fromLactobacillus delbruickii subsp. bulgaricus. The entireLactobacillus helveticus CNRZ32 proteinase gene(called prtH) has been sequenced. It shares 45%identity at the amino acid level with the protein-ases of lactococci and prtH has been classified asa new group, designated group I. A prtH-negativeCNRZ32 strain has been constructed via genereplacement. Importantly, there still is proteinaseactivity on the surface the prtH-negative CNRZ32derivative and there is no difference in acidifica-tion or growth rate in milk compared to wild-typeCNRZ32. This will allow us to examine theseisogenic derivatives which differ only in cellsurface proteinase activity/specificity for theaccumulation of bioactive peptides during growthin milk. Then, we can determine if proteinasespecificity has an essential role in theaccumulation of bioactive peptides/bioactivepeptide precursors from casein during growth ofLactobacillus helveticus CNRZ32 in milk.

The intent of this project is to begin developingthe knowledge required to select/construct strainof lactic acid bacteria which will enhance thelevel of casein-derived bioactive peptides pro-duced by digestion of fermented milk products.

Publications/PresentationsChristensen, J.E., E.G. Dudley, and J.A.Pederson, J.L. Steele. 1999. Peptidases and aminoacid catabolism in lactic acid bacteria. Antonievan Leeuwenhoek 76:217-246.

Pederson, J.A. and J.L. Steele. 1999. Characteriza-tion and physiological role of a cell-envelopeassociated proteinase from Lactobacillus helveticusCNRZ32. J. Bacteriol. 181:4592-4597.

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INTERIM REPORT

Application of milk powders in milk chocolate, butter andbutter spreads

In this project, the effects of type of milk powderon milk chocolate qualities will be investigatedand documented. A variety of powders will beobtained with a range of free fat and particlestructure. Chocolates will be evaluated for a rangeof physical and chemical, sensory and storage(bloom) properties.

PersonnelRW Hartel, professor, Baomin Liang, assoc.researcher, Dept of Food Science



ObjectivesUse of milk powders as specialized ingredients inchocolate and confectionery products can beenhanced through a better understanding of thefactors that influence the physical and chemicalproperties, sensory qualities and storage stabilityof chocolates. The specific objective is:

1. To compare the effects of free fat and particlestructure in milk powders on chocolate quality,processing requirements and storage stability. Thiswill involve measurement of molten chocolaterheology, conditions needed to properly temperthe chocolates, measurement of chocolate hard-ness, sensory characteristics and stability to fatbloom.

Summary

The milk chocolate industry is a major user ofmilk powders, with the most common ingredientin the US being spray-dried milk powder. How-ever, the chocolate industry prefers roller-driedwhole milk powder since it gives better and moreeconomic chocolate. The qualities of a milkpowder important to milk chocolate include freefat and particle structure (size, shape, air content,crystallinity, etc.). These properties have a largeimpact on the economics of milk chocolateproduction as well as on the physical, sensory andstorage characteristics.

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Fluid milk

FINAL REPORT

Growth and biocontrol of enterotoxigenic Bacillus cereusin infant formula and processed cheese prepared withmilk powderPersonnelAmy C. Lee Wong, associate professor, John B.Luchansky, associate professor, Amy B. Ronner,research specialist, Alan J. Degnan, senior re-search specialist, Dept. of Food Microbiology andToxicology; Mark E. Johnson. senior scientist,Center for Dairy Research



Objectives1. Determine the potential for growth of B. cereusand enterotoxin production in rehydrated infantformula and processed cheese spread at refrigera-tion and abuse temperatures.

2. Validate the effectiveness of bacteriocins againstB. cereus in rehydrated infant formula and inprocessed cheese spread during storage.

Summary

Five B. cereus strains were used for these studies:strains B4-ac, F4433/73, and FM-1 were originallyisolated from diarrheal outbreaks and were testedas a three-strain cocktail; strains HRM44 and D1were isolated from dairy products and were testedindividually. Three types of powdered infantformula [low iron (“S”), and iron-fortified with(“GS”) and without (“SF”) maltodextrin] andsteam distilled “infant drinking water” werepurchased from a local grocery store chain.Formulas were rehydrated with infant drinkingwater according to label directions, which wasapproximately 10 g powder per 60 ml water. Eachof the three types of rehydrated infant formulawas added to sterile 500 ml glass bottles, inocu-lated with two target levels of spores, 10 cfu/gramand 1000 cfu/gram powder, and incubated atthree refrigeration temperatures (4, 8, and 12°C)

and one abuse temperature (25°C). Inoculatedformulas were assayed for bacterial numbersimmediately after inoculation, at 8 hr, 24 hr, and2, 3, 5, 7, and 10 days.

Both the three-strain cocktail and strain HRM44grew readily in the three formulas at 25°C . Cellnumbers increased by at least 4 logs by 24 hr.Rehydrated formula containing the three-straincocktail became overtly spoiled after 48 hr,however formula containing HRM44 did notbecome noticeably spoiled until 72 hr. Anotherdifference between HRM44 and the three-straincocktail was the ability of HRM44 to grow at12°C. HRM44 grew most rapidly in the iron-fortified formula and most slowly in the low-ironformula, however by day 10 all bacterial countswere 106/ml or higher. After 10 days at 4 and 8°C,counts of both the three-strain cocktail andHRM44 either remained the same or decreasedslightly. Like HRM44 and the three-strain cock-tail, strain D1 was unable to grow in any formulaat 4°C. However, this strain did grow readily at12°C in all formulas, and at 8°C in SF and GSformulas by day 5. These data indicate that thereare strain differences in the growth characteristicsof B. cereus. Although none of the strains weexamined could grow in rehydrated formula at4°C, one strain did grow at 8°C and thus couldpotentially produce enterotoxin even whilerefrigerated, if stored for long periods. Analysiswith an ELISA we developed showed that hemol-ysin BL, a diarrheal enterotoxin produced by B.cereus, could be detected when growth exceeded106 cfu/ml. We also inoculated all three types offormula with a low level of spores (5.9 cfu/grampowder) and incubated the samples at 25°C.Growth to 105/ml and higher occurred by 24 hr.

We considered the possibility that consumersmight rehydrate infant formula but not use itimmediately, or that unused portions of formulamight be left unrefrigerated, and outgrowth of

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any B. cereus spores present in the powder couldoccur. We conducted pre-incubation experimentsusing one type of rehydrated infant formula (SF)at a spore inoculum target level of 1000 cfu/gram,and held the inoculated formula at 25°C for 6 hrbefore storing it at 4, 8, or 12°C. Pre-incubation ofthe three-strain cocktail allowed outgrowth of thespores to greater than 105 cfu/ml after 48 hr ofsubsequent storage at 12°C, even though growthwould not normally occur at this temperature.Similarly, strain HRM44, which grew less than 1log in 10 days when incubated at 8°C, grew to 106

cfu/ml or higher when pre-incubated at 25°C. Nogrowth was observed at 4°C.

We investigated the use of several bacteriocinsactive against B. cereus, and found that nisin wasthe most widely effective against the strains of B.cereus used in our studies. It is also the easiest touse since it is commercially available in a stan-dardized formulation. We examined the effective-ness of a commercial nisin preparation, whichcontains 2.5% active nisin in milk solids, against1000 cfu/gram B. cereus spores in rehydrated SFformula. We found that 0.05% nisin was sufficientto prevent outgrowth of 103 spores of strainHRM44 per gram of formula for 5 days. Thethree-strain cocktail was slightly more resistant tonisin, and although the spores were not com-pletely inactivated, a level of 0.1% nisin couldreduce outgrowth to less than 102 cfu/ml at up to5 days at 25°C. Our data show that the additionof nisin to powdered infant formula can reducethe potential for B. cereus growth in rehydratedformulas.

We produced processed cheese spread from acheese blend containing aged and new Cheddar,unsalted butter, nonfat dry milk, whey powder,and whey protein concentrate. The cheese blendwas inoculated with spores of strain HRM44 orthe three-strain cocktail, to concentrations of 7.5 x104 cfu/gram to 2.4 x 105 cfu/gram, respectively,and heated in a steam-jacketed cooker to amaximum temperature of 88°C. The meltedcheese was poured into sterile glass vials inapproximately 20 ml portions, tightly capped,and incubated at 8. 12, or 25°C for up to 6months. At regular intervals the cheese sampleswere assayed for B. cereus. Neither HRM44 northe three-strain cocktail grew in the processedcheese during 6 months of storage. Becausespores of these strains were unable to grow in theproduct, studies examining the effect of nisin inprocessed cheese spread were not conducted.

wisconsin center for dairy research

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