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Meat Fermentation Technology

FIDEL TOLDRÁ, YOLANDA SANZ, and MÓNICA FLORES

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Burjassot (Valencia),Spain

I. INTRODUCTION

II. PROCESSING OF FERMENTED SAUSAGESA. IngredientsB. AdditivesC. TechnologyD. Microbiology of the Indigenous Flora

III. METABOLISMA. Sugar MetabolismB. ProteolysisC. Amino Acid MetabolismD. LipolysisE. Nitrate and Nitrite Reductase ActivityF. Catalase Activity

IV. MICROBIOLOGY OF STARTER CULTURESA. Starter Cultures Used for Meat FermentationB. Requirements for Starter CulturesC. Production, Quality Control, and Application of Starter CulturesD. Strain Improvement

V. CONTRIBUTION OF FERMENTATION TO SENSORY ATTRIBUTES AND SAFETYA. Sensory AttributesB. Safety

VI. TRENDS FOR ACCELERATION

REFERENCES

Copyright © 2001 by Marcel Dekker, Inc. All Rights Reserved.

I. INTRODUCTION

Fermentation by microorganisms is one of the oldest food preservation practices of hu-mankind. The development of a strain of microbial flora succeeds, dominating and dis-placing other undesirable microorganisms, and producing a fermented meat in which thegenerated metabolites contribute to the product’s appropriate sensory characteristics. Thereare many varieties of fermented meats, varying according to region/country, climate, her-itage, and culture. Thus, different amounts of raw materials, spices, and condiments andprocessing lengths are used for fermentation. Traditionally, fermented sausages were driedin the Mediterranean countries and dried/smoked in central and northern Europe. Today,most fermented meat products are produced and consumed in Europe, mainly Germany andthe Mediterranean area, although in many other countries, such as the United States, de-mand as well as production are increasing.

II. PROCESSING OF FERMENTED SAUSAGES

A microenvironment has to be created during sausage fermentation in order to control thegrowth of pathogenic and/or spoilage bacteria (i.e., Salmonella spp., Escherichia coli,Clostridium perfringens). So, anaerobic conditions in combination with the presence ofcuring salts (salt and nitrite), lowered pH, reduced water activity, and drying will contributeas a hurdle to bacterial growth (Leistner, 1992). The main ingredients, additives, and stagesin the processing of fermented sausages are described below.

A. Ingredients

The main ingredients in sausages are chilled raw meat from skeletal muscle tissue (usuallyporcine alone or mixed with bovine, although other species may be used) and frozen fat tis-sue, preferably firm pork backfat, with low content of polyunsaturated fatty acids. The useof fat with high unsaturated fat content might oxidize the color, give a turbid appearance ofmelting fats on the cut surface, and contribute to the development of rancid flavors.

B. Additives

Salt, with levels in the range of 2% to 3%, exerts a partial bacteriostatic action, an initialreduction in water activity to 0.96, an improvement in protein solubilization and imparts atypical salty taste. On the other hand, it may cause some undesirable effects such as pro-moting oxidation of pigments and fats, contributing to off-colors and rancid taste.

Nitrite, and sometimes nitrate, is added to the curing mixture. The main role of nitriteis as a microbial preservative with a specific protective effect against pathogens, especiallyC. botulinum. It also prevents oxidation and contributes to the cured meat flavor (Gray andPearson, 1984), although the full chemical mechanisms are not fully understood because ofthe number of complex compounds in the sausage and the high reactivity of nitrite (Cassens,1994). Nitrite also plays an important role in the development of the typical cured meat color.Several mechanisms are involved in the formation of the cured pigment nitrosomyoglobin ornitric oxide myoglobin, which gives the pinky color, more reddish with dehydration. The useof older animals with a higher myoglobin content also contributes to a more intense color. Ni-trate is used in Mediterranean countries for the processing of long-ripened products typical ofthat area. Sodium or potassium nitrate is reduced to nitrite by bacteria with nitrate reductaseactivity (i.e., Micrococcaceae). These bacteria may be naturally present in the meat or addedas starter cultures. However, pH must be kept above 5.4 during the first hours because lowervalues would inhibit the nitrate reductase activity. A cold resting period before increasing the

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temperature is advisable in these cases. The use of nitrate in curing is being reduced becauseof the uncertainty in its conversion to nitrite (Cassens, 1995). In addition, because of the po-tential toxic effects of N-nitroso compounds, the levels of nitrite have been reduced to thosestrictly necessary for protecting against botulism (Cassens, 1995). In this sense, the EuropeanDirective (1995) allows maximum and residual amounts of nitrates and nitrites in meat prod-ucts. Sodium ascorbate and/or erythorbate are used as curing adjuncts to ensure the reductionof nitrous acid to nitric oxide and low residual levels to avoid nitrosamine generation.

Carbohydrates are used as a substrate for microbial growth and fermentation to lacticacid with subsequent pH reduction. The rate and extent of lactic acid formation is stronglydependent on the type and amount of carbohydrate added (Lücke, 1985). Thus, the pH maydrop very fast if readily metabolizable sugars (such as glucose or saccharose) are added, in-hibiting acid-sensitive bacteria. The use of slowly metabolized carbohydrates (such as dex-trins) reduces the rate of lactic acid generation and thus the pH reduction. A combination ofboth kind of sugars is used for controlling the rate of the pH reduction in specific productswhere other enzymatic reactions are looked for. The amount of carbohydrates is also veryimportant because they are directly related to the final amount of lactic acid. Therefore, car-bohydrate levels of 0.5% to 0.7% are usually added for reducing the pH to values slightlylower than 5.0. An excessive amount of carbohydrates reduces the pH to values near 4.5,which results in products with a noticeable, at most times unpleasant, acid taste. Smallamounts of carbohydrates (below 0.3%) do not produce so discernible pH reduction.

The description of microbial starter cultures and their role during the processing offermented sausages is given below. Finally, the use of spices and condiments such asground pepper, paprika, garlic, red pepper, and mace is a very extensive practice that con-tributes to the final specific flavor of the product.

C. Technology

The chilled meat pieces and frozen fat tissues are comminuted in a meat grinder or cutterand then the additives (salt, nitrate/nitrite, carbohydrates, microbial starters, spices, andsodium ascorbate or erythorbate) are incorporated. The ground mass is mixed for homog-enization under vacuum for removing as much oxygen as possible. The homogenized massis stuffed into natural, restructured collagen, by using vacuum-filling devices. The sausagesare placed in artificial ripening chambers with control of temperature, relative humidity,and air flow rate or in natural ripening rooms when producing traditional sausages in an ar-tisanal way.

The conditions for fermentation vary depending on the kind of microbial starters andtype of product. For instance, there is a clear difference in meat fermentation technology be-tween the United States and most of the European countries. In the United States, the goal israpid acid production through a fast fermentation in order to inhibit spoilage microorganisms.Starters such as Lactobacillus plantarum or Pediococcus acidilactici are used for fermentingup to 40°C. The product reaches high lactic acid accumulation, pH drops below 5.0 to 4.6,and the flavor formation is restricted because of the high percentage of inhibition of exopep-tidases and lipolytic enzymes. Milder fermentation temperatures, around 22° to 26°C, areused in European countries, although other differences may be found within Europe.

The ripening/drying period, the length of which is variable depending on the kind ofproduct and its diameter, usually takes from 20 days to 3 months. In general, and dependingon the total processing time, three main groups of fermented sausages can be established(Flores, 1997): (a) rapid (less than 7 days), (b) regular (around 3 weeks), and (c) slow (up to3 to 4 months). The length and conditions of the process as well as the optional smoking have

Meat Fermentation Technology 539


a strong and definitive influence on the sensory properties. Some typical Mediterraneansausages are French saucisson, Spanish chorizo, and Italian salami. On the other hand, Ger-man- or Hungarian-style salamis represent some of the typical north European products.There are basic differences between both groups of products (Flores, 1997). The Mediter-ranean sausages, which are not smoked, undergo a slow process with nitrate addition andvery mild temperatures. On the other hand, only nitrite is used in north European sausages,with faster processes and final smoking in most cases—up to 95% of German raw sausagesare smoked (Leistner, 1995). The growth of a mold layer on the outer surface, typical ofsome Mediterranean dry-cured sausages, may contribute to flavor and appearance. Based onthe moisture content, most fermented meat products may be classified as dry (weight losshigher than 30%) or semi-dry (weight loss lower than 20%) fermented sausages.

D. Microbiology of the Indigenous Flora

The origin of the microflora of the raw sausage mix is diverse and its composition variesdepending on meat manipulation, the microorganisms present in the environment, and theadditives used for manufacture. However, there are a number of factors that impose selec-tivity in favor of the development of the desirable flora (Micrococcaceae and lactic acidbacteria) and preventing growth of pathogenic and spoilage microorganisms (mainly gram-negative aerobic bacteria). These selective factors include low pH, reduction in water ac-tivity, temperature, oxygen depletion, accumulation of metabolic products, and presence ofadditives (salt and nitrite). The succession of microbial changes during ripening of differ-ent varieties of sausages has been described by several authors (Lücke, 1985; Roncalés etal., 1991; Samelis et al., 1994; Sanz et al., 1997, 1998a). An example of the evolution ofthe main bacterial groups during the processing of a fermented sausage is shown in Fig. 1.

Total counts of aerobic mesophilic bacteria initially reach values of from 104 to 106

colony forming unit, CFU/g. The levels of lactic acid bacteria and Micrococcaceae arecommonly around 103 to 105 CFU/g. Initial counts of gram-negative bacteria (Enterobac-teriaceae, Pseudomonas, Achromobacter, etc.) are around 103 to 104 CFU/g. The initiallevels of yeast and molds are a bit lower, with values of 102 to 103 CFU/g or cm2 (Roncaléset al., 1991). The fermentation stage is characterized by a general exponential growth of ev-ery microbial group parallel to a decrease in pH as a result of carbohydrate fermentation.Lactic acid bacteria dominate the microflora, reaching levels of 107 to 109 CFU/g that re-main almost constant during the drying period. The evolution of this group is, in fact, par-allel to that showed by total aerobic mesophilic bacteria. Members of the genus Lacto-bacillus are the most competitive among lactic acid bacteria, followed by Leuconostoc,Pediococcus, and Streptococcus. The species L. sakei and L. curvatus dominate the flora oftraditional European products fermented at temperatures around 20° to 25°C, whereas L.plantarum is found in sausages fermented at higher temperatures. Moreover, strains ofother species such as L. alimentarius, L. farciminis, and L. pentosus have also been isolated.Leuconostoc (Lc) and heterofermentative lactobacilli usually do not represent more than10% of lactic acid bacteria. Among those we can mention L. viridescens, L. brevis, Lc.mesenteroides, and Lc. paramesenteroides (recently reclassified as Weissella paramesen-teroides; Samelis et al., 1994; Kröckel, 1995). Micrococcaceae also gain importance in thefermentation stage, reaching levels of 106 to 107 CFU/g. Members of the Micrococcaceaefamily (Staphylococcus and Micrococcus; now divided into different genera, Stackebrandtet al., 1995) are, however, acid-sensitive and tend to decline during the drying period. Thedevelopment and survival of this microbial group greatly depends on the degree of acidifi-

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cation reached in the product. The colonization of staphylococci over micrococci is the re-sult of the ability of the former to grow and metabolize in anaerobic conditions. The strainsfound in natural fermented products belong mainly to the species S. xylosus and S.carnosus; strains of S. saprophyticus, S. simulans, or S. sciuri constitute a minor proportionof the isolates. Levels of yeast and molds also increase to 106 to 107 CFU/g or cm2 in thefermentation stage. Yeasts are anaerobic facultative, and therefore they are able to grow inthe inner and superficial part of sausages, whereas growth of molds is restricted to the sur-face. Debaryomyces hansenii is the yeast most frequently isolated from natural fermentedmeats, although species of Candida, Cryptococcus, Pichia, Rhodotorula, and Trichosporonhave also been detected. The mycoflora of mold-fermented sausages is mainly dominatedby Penicillium spp.; species of the genera Eurotium and Aspergillus develop more exten-sively in dry-cured hams. Enterobacteriaceae can experience a slight increase in the fer-mentation stage, reaching values above 105 CFU/g that dramatically decrease during thedrying period. In general, levels of gram-negative bacteria (enterobacteria and psy-chrotrophs) become almost negligible at the end of ripening (less than 103 CFU/g). Thegrowth of pathogenic bacteria such as Salmonella spp. is prevented mainly by the presenceof nitrite in the initial stages and the further reduction of water activity and pH. Listeriamonocytogenes is inhibited by the low pH, competitive flora, and accumulation of antimi-crobial compounds. An adequate fermentation process prevents the growth of and toxinproduction by Staphylococcus aureus. The presence of Clostridium botulinicum and


Figure 1 Microbiology of the indigenous flora during the processing of a typical fermentedsausage. (•) Mesophilic aerobic bacteria, (�) Lactic acid bacteria, (�) Micrococcaceae, (�) Enter-obacteriaceae, (�) Yeast. (Adapted from Sanz et al., 1997.)


C. perfringens is excluded by the effect of nitrite combined with other selective factors suchas presence of sodium chloride and low pH.

III. METABOLISM

A. Sugar Metabolism

Lactic acid is the main product of carbohydrate fermentation. The enantiomers L and D lac-tic acid are usually present in the final product and their ratio depends on the species of lac-tic acid bacteria present, more specifically on the action of L and D lactate dehydrogenase,respectively, and the presence of lactate racemase. Usually, the ratio is near 1, a racemicmixture.

Sugar metabolism starts after glucose is transported into the cell and metabolizationoccurs via the glycolytic or Embden-Meyerhof pathway. Some of the key enzymes (seeFig. 2) are (a) aldolases, which generate glyceraldehyde-3-phosphate, (b) pyruvate kinase,which forms pyruvate (the central intermediate in fermentation) from phosphoethanolpyruvate, and (c) lactate dehydrogenase, which generates lactic acid from pyruvate.NADH, originated during the hydrolysis of glyceraldehyde 3-phosphate, is oxidized in thelatter step. By far, the greatest part of glucose is decomposed in a homofermentative way.However, although sugar metabolism is primarily homofermentative, trace amounts ofother end products, such as acetate, formate, ethanol, and acetoin may result from alterna-tive metabolic pathways.

The pH drop, a consequence of lactic acid accumulation, is of paramount importancefor preservation of sausages. The pH drop has other interesting contributions, such as fla-vor due to the formation of metabolites and the consistency of the product because of wa-ter holding capacity reduction and protein coagulation as pH approaches the isoelectricpoint of most of the meat proteins. The combination of the muscle and the lactic acid bac-

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Figure 2 Simplified scheme of homofermentative metabolism of glucose in lactic acid bacteria.


teria enzyme systems also contribute to keeping the environment anaerobic by reducing theredox potential during lactic acid fermentation.

B. Proteolysis

An important hydrolysis of myofibrillar and sarcoplasmic proteins takes place duringsausage fermentation and ripening. This hydrolysis is brought about by the combined ac-tion of muscle proteinases (cathepsins and calpains) and exopeptidases (dipeptidylpepti-dases and alanyl-, arginyl-, leucyl-, and pyroglutamyl-aminopeptidases) and starter pro-teases. One of the major challenges is the correct establishment of the relative role ofendogenous and microbial enzymes to proteolysis. This would help optimize the process-ing conditions, but it is extremely difficult because of the high variety of microorganismswith different enzymatic activities used as starter cultures. The major steps are shown inFigure 3. This proteolytic process finally contributes to product consistency by the degra-dation of the myofibrillar structure and to flavor by the accumulation of small peptides andfree amino acids directly related to taste or indirectly as precursors of flavor compoundsthrough amino acid degradation reactions that will be later described. The extent of prote-olysis varies depending on the processing conditions and type of starters added to thesausage, but it might be so intense that levels of non-protein nitrogen up to 20% of the to-tal nitrogen content may be easily reached.

The percentage of contribution of each group of enzymes is not fully clarified, but ac-cording to recent reports (Molly et al., 1997), it appears that protein degradation, especiallyof myosin and actin, is initiated by cathepsin D, an acid muscle proteinase that is favored bypH decrease. The activity of cathepsins B, H, and L would be restricted more to actin and itsdegradation products. Serine-, trypsin-like, and metallo-proteinases were concluded to be ofno importance during dry sausage ripening. As muscle aminopeptidases have optimal activ-ity at neutral or basic pH (Toldrá and Flores, 1998), the latter stages of proteolysis would bedominated by bacterial peptidases (Sanz and Toldrá, 1999; Flores et al., 1998a).


Figure 3 Major steps in proteolysis.


C. Amino Acid Metabolism

Free amino acids may be subject to a number of chemical transformations such as decar-boxylation, deamination, and transamination, producing different compounds that will af-fect the sensory characteristics of the product (Ordoñez et al., 1999). The microorganismspresent in the product constitute the main enzyme source for most of these reactions. Freeamino acids, generated through the proteolysis of muscle proteins, act as a substrate forthese reactions, as shown in Fig. 4.

1. Degradation Reactions

There are nonenzymatic pathways for amino acid conversion such as the Strecker degra-dation of amino acids that is an oxidative deamination-decarboxylation reaction producingbranched aldehydes. The generation of 3-methylbutanal, 2-methylbutanal, and phenylac-etaldehyde from leucine, isoleucine, and phenylalanine, respectively, has been found indry-fermented sausages. The Strecker degradation of sulfur-containing amino acids suchas methionine, cysteine and cystine that leads to the production of sulfur compounds thatare characterized by low threshold values and therefore, exert a high aromatic impact inmeat products (Shahidi et al., 1986). The enzymatic degradation of the amino acid sidechain is another reaction occurring in fermented products. The side chain degradation oftyrosine and tryptophane by tyrosine-phenol-lyase and tryptophane-indole-lyase leads tophenol and indole formation (Molinard and Spinnler, 1996). These indole-derived com-pounds such as 3-methylindole (skatole) may be responsible for unpleasant odors in meatproducts.

2. Decarboxylation

Biogenic amines are produced by the microbial decarboxylation of amino acids (Ordoñezet al., 1999). The enzymatic decarboxylation of the amino acids tyrosine, tryptophane, andphenylalanine produce tyramine, tryptamine, and phenylethylamine, respectively. Simi-larly, lysine, histidine, and ornithine give cadaverine, histamine, and putrescine, respec-tively, the last one being a precursor of spermine and spermidine. The presence of thesesubstances not only can affect the flavor but also constitutes a risk for consumers’ health.Selection of appropriate raw material, processing temperature, and starter strains without

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Figure 4 Main reactions involved in the metabolism of free amino acids.


potential for amine generation are important factors in controlling the level of biogenicamines (Ordoñez et al., 1999).

3. Deamination

The oxidative deamination of amino acids is produced by several bacterias generating am-monia (Ordoñez et al., 1999). Glutamate dehydrogenase and alanine dehydrogenase gen-erate �-ketoglutarate and pyruvate, respectively, and ammonia in the presence of NAD� orNADP�. There is also another pathway that consists of the nonoxidative deamination ofamino acids. The enzymes involved in these reactions are deaminases, whose action is fa-cilitated by the presence of substituent in the carbon atom of the amino acid. The ketoacid also can be transformed to aldehyde by decarboxylation; then, this aldehyde can be re-duced to the corresponding primary alcohol or oxidized to acid.

4. Transamination

In this reaction, the �-amino group of the first amino acid is transferred to the � carbonatom from an �-keto acid, generating a new amino acid. Amino transferases and transam-inases present in bacteria catalyze this reaction.

amino acid 1 � keto acid 2 ⇔ keto acid 1 � amino acid 2

D. Lipolysis

The major steps in lipolysis of fat tissue are shown in Fig. 5. Lipolysis has an importantcontribution to flavor development through the generation of free fatty acids, and those thatare unsaturated will act as substrates for oxidation to form volatile compounds with aromaproperties. Adipose tissue and intermuscular fats are mainly composed of triglycerides; in-tramuscular fat also contains phospholipids, rich in polyunsaturated fatty acids. Initialbreakdown of triglycerides would be the result of endogenous lipases such as lysosomalacid lipase, usually present in muscle and very active at a pH around 5.0, and neutral lipase,naturally occurring in fat tissue (Motilva et al., 1992). The latter is probably most impor-tant for the final lipolysis because fat tissue constitutes the major fraction in the sausage


Figure 5 Major steps in lipolysis.


(Toldrá, 1992). Most of the lactic acid bacteria (LAB) cannot hydrolyze triglycerides butthey can act on mono and diglycerides, thus contributing to hydrolysis of fat and genera-tion of free fatty acids. As in the case of proteolysis—and although it is difficult to estab-lish the relative role of endogenous and microbial enzymes in lipolysis—the percentage ofthe contribution of endogenous lipolytic enzymes to total fat hydrolysis is estimated to bearound 60% to 80%, the rest being due to microbial lipases (Molly et al., 1997). In the caseof phospholipids, which constitute a minor fraction of the total fat, muscle phospholipasesare the only lipases responsible for their hydrolysis.

E. Nitrate and Nitrite Reductase Activity

Micrococcaceae are endowed with nitrate reductase activity that is essential for preserva-tion, color development, and aroma formation in cured meat products (Flores and Toldrá,1993). This enzyme reduces nitrate into nitrite and also recycles nitrite that was convertedinto nitrate in the sequential reactions with myoglobin. Then, nitrite may be reduced by bothnitrite reductases from Micrococcaceae or by chemical degradation at pH 5.4 to 5.5. The ni-trate and nitrite reductase activities of S. carnosus have been studied in more detail. The ni-trate reductase is a membrane-bound enzyme involved in respiratory energy conservation,whereas the nitrite reductase is a cytosolic enzyme involved in NADH reoxidation(Neubauer and Götz, 1996). The expression of these activities is stimulated by anaerobio-sis, nitrate, and nitrite. Molybdenum appears to be an essential cofactor for nitrate reduction(Pantel et al., 1998; Neubauer et al., 1999). Aerobic gram-positive bacteria (Enterobacteri-aceae and psychrotrophs) also possess nitrate reductase activity but their prevalence, andthus their role in nitrate reduction, is limited in fermented meat products. Lactic acid bacte-ria are poor contributors to nitrate/nitrite reduction. Lactobacillus plantarum can reduce ni-trate in vitro but not under conditions of meat fermentation (Lücke, 1985). Also, two typeof nitrite reductase activity has been described in lactic acid bacteria: (a) a heme-dependentactivity with ammonia as sole product, which has been found in strains of L. plantarum, L.pentosus, and P. pentosaceus, and (b) a heme-independent activity that generates NO andN2O, which was found in L. plantarum (Lücke, 1985, Wolf et al., 1990). This activity is notpresent in L. curvatus and rarely in L. sake strains (Wolf and Hammes, 1988).

F. Catalase Activity

Micrococcaceae possess catalase activity that mediates the degradation of hydrogen per-oxide responsible for color and flavor defects. This activity is characteristic of aerobic andmost facultative aerobic bacteria and together with the superoxide dismutase is involved inthe degradation of metabolically toxic compounds derived from oxygen. Moreover, gram-positive bacteria have more catalase activity per cell than gram-negative bacteria. Instaphylococci, catalase is maximally expressed at the onset of the stationary phase, in aer-obic conditions and at low glucose concentration (Baier et al., 1995). Lactic acid bacteriacan also synthesize two different type of enzymes for peroxide degradation: a heme-con-taining catalase, produced only in the presence of hematine, and a pseudocatalase or man-ganese-dependent catalase that is found in a few species. In lactic acid bacteria, these en-zymes are physiologically involved in resistance to oxidative stress (Hertel et al., 1998).Recently, the genes encoding the manganese catalase of L. plantarum and the heme-de-pendent catalase of L. sakei have been characterized (Igarashi et al., 1996; Hertel et al.,1998). In L. sakei, catalase activity is induced by aerobic conditions and the presence of hy-drogen peroxide in anaerobic conditions (Hertel et al., 1998). Nevertheless, the contribu-

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tion to reduction of peroxides by lactic acid bacteria is of minor technological importancecompared with that of Micrococcaceae.

IV. MICROBIOLOGY OF STARTER CULTURES

Traditional practices rely on the selection of desirable indigenous flora or the inoculation ofup to 5% of a previous fermentation mixture (back-slopping). These techniques are still inuse but have been progressively replaced by the application of well-defined starter cultures.The first commercialized starter culture (United States, 1957) consisted of a single strain ofPediococcus acidilactici (Niven et al., 1959; Everson et al., 1970). The first starter cultureavailable in Europe also consisted of a single strain of Kocuria, named M53 (Niinivaara etal., 1964). This market evolved with the development of mixed cultures consisting of differ-ent strains that could cover wider spectra of metabolic properties. Currently, the applicationof mixed starter cultures constitutes a common industrial practice, with the objective of ac-celerating the fermentation process and ensuring the products hygienic and sensory quality.

A. Starter Cultures Used for Meat Fermentation

The main microorganisms used in meat fermentation and their relevant properties are sum-marized in Tables 1 and 2, respectively. Lactic acid bacteria (Lactobacillus and Pediococ-cus) are essential components of the starter cultures usually accompanied by Micrococ-


Table 1 Example of Some of the Most Important Starter Cultures for Meat Fermentation

Microorganism Genera Species

BacteriaLactobacillus L. sakei, L. curvatus, L. plantarum, L. pentosusPediococcus P. pentosaceus, P. acidilacticiKocuria K. variansStaphylococcus S. xylosus, S. carnosus

Yeasts Debaryomyces D. hanseniiCandida C. famata

Molds Penicillium P. nalgiovense, P. chrysogenum

Source: Adapted from Hammes et al. (1990), Geiues et al. (1992), and Hammes and Kuauf (1994).

Table 2 Properties of Starter Cultures

ProteolyticCatalase Nitrite-reductase activity

Heme- Pseudo- Nitrate- Heme- Heme- LipolyticMicroorganism containing catalase reductase dependent independent Endo- Exo- activity

L. sake � � � � � � ��

L. curvatus � � � � � � ��

L. plantarum � � � � � � ��

P. acidilactici � � � � � � ��

P. pentosaceus � � � � � � ��

Kocuria ��

Staphylococcus ��

Yeast � � � � � � � �

Molds � � � � � � � �

Source: Adapted from Hammes et al. (1990) and Hammes and Kuauf (1999).


caceae (Kocuria and Staphylococcus). The application of yeasts or molds also constitutesan alternative but has been less exploited so far.

1. Lactic Acid Bacteria

The lactic acid bacteria used as starter cultures belong to the genera Lactobacillus andPediococcus. The species commercially available are listed in Table 1. L. sakei and L.curvatus are the most competitive microorganisms in this environment. These speciesare psychrotrophic with optimal growth (Ta25° to 30°C) closer to traditional Europeanfermentation temperatures (20° to 24°C). L. plantarum and Pediococcus spp. aremesophilic, showing optimal growth at 30° to 35°C (up to 40°C for P. acidilactici) and,therefore, their development is favored in fermentations at higher temperatures, typi-cally used in the United States. The major role evolved by lactic acid bacteria is relatedto carbohydrate metabolism that results in the acidification of the meat mixture. Thisprocess has the following desirable effects: (a) it ensures hygienic stability by the re-duction in pH itself and the generation of organic acids; (b) it imparts characteristic acidtaste, (c) it causes coagulation of meat proteins (at pH 5.4 to 5.5) reduction in waterholding capacity and facilitates the drying process with consequences in texture andfirmness, and (d) it contributes to the development of desirable red color by favoringthe reaction of nitrogen monoxide with myoglobin (pH 5.4 to 5.5). Lactobacilli used asstarters in meat are all facultative heterofermentative organisms that utilize glucose andhexose-phosphate via the Embden-Meyerhof-Parnas pathway (glycolysis), generatinglactic acid as the major fermentation product. Besides aldolase, the enzyme involved inglycolysis, these organisms also possess phosphoketolase, which decomposes pentosesinto lactate, acetate (or ethanol), and carbon dioxide. The carbohydrates are usually me-tabolized via glycolysis but the heterofermentative pathway can be activated in certainconditions, resulting in the production of undesirable fermentation products (acidacetic, hydrogen peroxide, carbon dioxide, acetoin, formic acid, etc). For instance, L.plantarum is known to oxidize lactic acid into acetate and carbon dioxide under aero-bic conditions. L sakei and L. curvatus use oxygen to generate hydrogen peroxide andpyruvate. The heterofermentative metabolism of these lactobacilli can also be activatedin glucose depletion conditions under anaerobiosis, resulting in the generation of for-mate, acetate, and small amounts of ethanol. Pediococcus are also homofermentativeorganisms that generate lactic acid from sugars. However, P. pentosaceus also produceacetate and ethanol from hexoses and pentoses (Kröckel, 1995). The presence of cata-lase and nitrate/nitrite reductase activities is desirable in selected starter cultures in or-der to avoid color and flavor defects. These activities are not attributed mainly to lac-tic acid bacteria but to Micrococcaceae. Despite that, lactic acid bacteria can synthesizea heme-containing catalase and a pseudocatalase or manganese-dependent catalase(Table 2). Nitrate and nitrite reductase activity has also been found in lactic acid bac-teria, one heme-dependent, the other heme-independent (Table 2). Proteolytic activityof lactic acid bacteria is thought to partially contribute to flavor development by re-leasing small peptides and free amino acids (Verplaetse, 1994; Molly et al., 1997). Sev-eral exopeptidases have been purified and characterized from L. sakei, showing theirpotential role in peptide degradation in meat fermentation (Montel et al., 1995; Sanzand Toldrá, 1997; Sanz et al., 1998b). The lipolytic activity from lactic acid bacteria isof limited interest, although lipolytic bacterial enzymes potentially active at the tem-peratures and pH values typical of meat fermentation process have been described (Pa-pon and Talon, 1988; Naes et al., 1991).

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Lactobacilli can also decarboxylate amino acids, generating biogenic amines withadverse biological activity in humans. This activity is strain-dependent. So far, decarboxy-lase activity has not been detected in strains of L. sakei, whereas some strains of L. curva-tus potentially produce up to four different amines (Hammes and Knauf, 1994; Straub etal., 1995). The selection of strains without ability to decarboxylate amino acids that com-petitively eliminate amine-producing strains is also essential to avoid health risks. Bacte-riophage infection of starter cultures may account for failures in fermentation. Sensitivityto bacteriophages and presence of prophages has been described in L. sakei and L. plan-tarum (Nes et al., 1988; Leuschner et al., 1993) but never in pediococci. Nevertheless, in-fections by phages do not constitute a practical problem, because the semi-solid matrix ofthe fermenting meat mixture does not seem to be suitable for phage propagation.

The synthesis of bacteriocins against undesirable organisms constitutes a trait of ut-most importance in the selection of starter cultures. Bacteriocinogenic strains of L. sakei, L.curvatus, L. plantarum, and Pediococcus have been found but are not in use as starter cul-tures yet. The introduction of these strains could contribute to a reduction in hygienic risks.

2. Micrococcaceae

Kocuria (exMicrococcus) and Staphylococcus strains are commercialized as starter cul-tures (Table 1). Staphylococci are, however, more competitive, mainly because of theirmetabolic activity under anaerobic conditions. The major functions evolved by this mi-crobial group comprise color formation and stabilization and aroma development bymeans of their catalase and nitrate and nitrite reductase activities and implication in lipidmetabolism (Table 2). Nitrate reductases are enzymes associated with the cytoplasmicmembrane that carries out the dissimilation of nitrate at very low oxygen concentrationsor in anaerobiosis. The enzymatic activity has been characterized in S. carnosus and it isdemonstrated to be active in conditions typical of meat fermentation (Neubauer and Götz,1996). This activity also has been studied in strains of K. varians showing optima thatvary depending on the strain. The nitrite generated from nitrate can be reduced by nitritereductases or chemically transformed.

The compounds responsible for the cured red color are susceptible to oxidation, re-sulting in color defects. The catalase activity of this microbial group plays an important rolein color stabilization by the degradation of the hydrogen peroxide. In this way lipid oxida-tion and rancidity are also prevented. Proteolytic activity is not significant in Micrococ-caceae, although some endo- and exoproteolytic activity has been detected in K. varians, S.sciuri, S. xylosus, and S. carnosus (Montel et al., 1992; Fransen et al., 1997). Micrococ-caceae seem to be more active in lipid metabolism and generation of volatile aroma com-pounds (Johansson et al., 1993; Stahnke, 1995).

Phage infection of S. carnosus has been detected in some cases but, in practice, theindustrial impact is of limited significance.

3. Yeast

Debaryomyces hansenii is the predominant species in fermented meat, and together withCandida famata, constitutes the only yeast available as starter culture so far. They have anaerobic and weak fermentative metabolism, allowing their growth in both the surface andthe inner part of meat products. The application of selected yeast strains mainly contributesto color stabilization and flavor generation by means of their catalase and lipolytic activity,respectively. Yeast also metabolizes organic acids and produces deaminase activity thatmay result in a pH increase.



4. Mold

Penicillium nalgiovense and P. chrysogenum constitute the fungi available as starter cul-tures. They mainly contribute to the appearance, flavor, and safety of fermented products.Molds have an aerobic metabolism, which restricts their growth to the surface. Apart fromthe external appearance, the implantation of molds exerts a protective effect against adverseeffects of oxygen and light, such as discoloration and rancidity; also, drying occurs moreevenly.

The contribution to flavor is mediated by the activity of lipolytic and proteolytic en-zymes. Also, the ability to metabolize organic acids resulting from lactic fermentationcauses a decrease in the acidification level and tangy taste. This is also the result of deam-inase activity that generates ammonium from amino acids. The application of nontoxicstrains protects the product from the adverse effects of the implantation of mycotoxigenicmolds.

B. Requirements for Starter Cultures

Strains used as starter cultures must be “generally regarded as safe” (GRAS) because theyare considered to be food additives. Laws regulating the market of starter cultures may varydepending on the country but, overall, there are some requirements for the starter cultures;they must:

1. Be neither pathogenic, toxic, nor allergenic2. Have phenotypical and genotypical stability3. Be competitive in the typical conditions of the process (tolerance to salt, nitrite,

low pH and water activity, considerable growth at manufacturing temperatures,etc.)

4. Provide some technological benefits; for instance, on acidification, preservation,flavor formation, quality assurance, etc.

5. Resist phage infection6. Be identifiable by specific methods

C. Production, Quality Control, and Application of Starter Cultures

The composition of the media and growth conditions (temperature, pH, aeration, etc.) arecritical for the production of starter cultures, and they must be selected taking into ac-count their cost and benefits in terms of biomass, enzymatic activity, resistance to freeze-drying, and stability during storage. Cells are usually collected at the end of the expo-nential growth phase or at the beginning of the stationary phase and cooled beforeprocessing. Concentration of the culture is carried out by centrifugation or ultrafiltration.The cultures are supplied frozen or lyophilized with or without previous immobilizationof the cells. Mold cultures are supplied as freeze-dried spore suspensions, and yeast cul-tures as freeze-dried cells. The product then is packaged according to a declared activityor cell number.

The quality control division must attend to the declared shelf life of the starter cul-ture. Generally, tests of acidification are introduced for lactic acid bacteria and tests of ni-trate reduction for Micrococcaceae. The absence of pathogenic or spoilage microorganismsas well as toxic contaminant compounds also must be controlled. Apart from controls of themetabolic activity, the stability of the genetic characteristics must be established by DNAfingerprinting and plasmid profile analysis.

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Lyophilized cultures must be reconstituted in water before their addition to themeat mixture. The cultures already reconstituted can be directly incorporated into themix but, in advance, they are usually activated by keeping the culture at room tempera-ture for 18 to 24 hours. The inoculum reaches between 106 and 108 CFU/g. The highestlevels are inoculated for the production of rapid fermented sausages. Cultures of lacticacid bacteria and Micrococcaceae are usually combined to inoculate the meat mixture,whereas the application of mold and yeast is only on the outer part, normally by immer-sion of the sausage into a solution containing the starter culture or by spraying the sus-pension on the surface. The application of yeast is also combined with the use of bacte-rial starter cultures.

D. Strain Improvement

There is an increasing demand for strains with improved properties that fully cover the re-quirements for meat processing. Efforts have been made to develop strategies for selectionof new strains from the meat environment. On the other hand, genetic manipulation ofstarter cultures is an alternative method of strain improvement, and research is currently inprogress. The DNA coding for desirable traits can be part of a plasmid or chromosome. Theproperties that reside on a plasmid can be transfered by conjugation that is generally re-garded as safe (GRAS). However, the use of gene cloning strategies is still controversial inthe food industry. Most work on genetic modification of meat starter cultures has beenmade in molds in order to eliminate the production of mycotoxins, regulate the metabolicactivity (proteolytic and lipolytic enzymes), or promote biopreservation (Leistner et al.,1991; Geisen, 1993). In staphylococci, the main interest is the antibiotic resistance of S. au-reus and the possibility of transferring genes involved in flavor generation (lipases) fromother gram-positive bacteria into the nonpathogenic S. carnosus (Goetz, 1990; Al-Masaudiet al., 1991). Within the genus Lactobacillus, one of the major goals is related to the de-velopment of strains that overproduce bacteriocins. Special interest has been focused on in-hibition of acid-resistant strains of Listeria monocytogenes by bacteriocins produced by L.sakei (Leistner et al., 1991). The expression of the lysostaphin gene of S. simulans into meatlactobacilli has also been investigated as a means to enhance the antimicrobial potential ofthese strains (Cavadini et al., 1996).

V. CONTRIBUTION OF FERMENTATION TO SENSORY ATTRIBUTESAND SAFETY

A. Sensory Attributes

The sensory characteristics of fermented products are achieved by the interaction of mi-crobial, physical, and biochemical reactions (Verplaetse, 1994). During the fermentationprocess, acidification produces reactions and changes that ensure the development of color,texture, and flavor specific to the fermented products as described in Figure 6.

1. Color

Visual appearance is a key factor that influences consumers when they are assessing thequality and palatability of meat and meat products. Certain colors influence food accep-tance, although the color of the meat itself may be influenced by its moisture and fat con-tent and also by the content of hemoprotein, particularly myoglobin and its relationshipwith the surrounding environment. The development of the characteristic color of fer-



mented products is the result of the action of nitrite with myoglobin, producing the red color(Pegg and Shahidi, 1997).

Some of the main additives in the formulation of fermented sausages are nitrate andnitrite. Nitrate is transformed into nitrite by microbial nitrate reductase activity. A largeproportion of nitrite reacts with meat constituents and induces desirable changes. The for-mation of curing color in fermented sausages is obtained through several steps (Lücke,1985): (a) oxygenated myoglobin (red) reacts with nitrous acid to give metmyoglobin(brown) and nitrate; (b) indigenous and exogenous reductants (e.g., ascorbate) reduce ni-trous acid to nitric oxide, and metmyoglobin to myoglobin; (c) both combine to form nitricoxide myoglobin (red). The rate of its formation increases with falling pH, and therefore itis accelerated by the activity of lactic acid bacteria in fermented sausages. During ripening,the protein moiety of nitric oxide myoglobin is denatured, giving the formation of nitric ox-ide myochromogen. This process improves color stability because the nitric oxide dissoci-ates less readily from the heme group. Nevertheless, nitric oxide myochromogen can be at-tacked by oxidants in several conditions, such as at low pH values and low redox potential.In fermented sausages, the oxidants are peroxide groups from the fatty tissue or are formedby lactic acid bacteria in the presence of oxygen. Therefore, it is worth emphasizing the im-portance of using fresh firm fat and of introducing as little oxygen as possible into thesausage mixture during its processing. The color defects appear when the peroxides oxidizethe iron within the porfirin ring; then, the curing color changes to gray or brown. In addi-tion, the presence of other enzymatic activities such as catalase are responsible for the elim-ination of peroxides, resulting in stabilization of the color and flavor, avoiding the attackon the porfirin ring and green discoloration (Demeyer, 1992).

2. Texture

As mentioned above, microbial activity produces a decrease in pH value. When the pH ap-proaches the isoelectrical point of meat proteins, the water-holding capacity is reduced,

552 Toldrá et al.

Figure 6 Contribution of fermentation to sensory attributes in fermented meat products.


producing an increase in consistency that will be also accelerated during the drying proc-ess. Therefore, the acidification process is necessary to achieve the sliceability typicalof the product. The salt added in the formula gives a suitable cohesion and texture dur-ing drying by solubilizing proteins, which act as a bridge between the constituting meatfragments.

3. Flavor

The characteristic flavor of fermented sausages mainly originates from the breakdown ofcarbohydrates, lipids, and proteins through the action of microbial and endogenous meatenzymes. But other substances added to the sausage, such as salt and spices, should betaken into account because of their important contribution to flavor. Additionally, there areother pathways, such as autoxidation, that form flavor compounds without direct enzymaticparticipation.

The carbohydrate fermentation is responsible for the typical tangy or sour taste(Lücke, 1985). The interactions between carbohydrate and protein metabolism during meatfermentation determine the rate of pH decline and flavor development (Demeyer, 1992).During carbohydrate fermentation, significant amounts of acetic acid, besides lactic acid,are generated. On the other hand, pH is partially neutralized during drying as the result offurther ammonia and free amino acids generation. All of these compounds have an impacton flavor.

There are internal and external parameters that influence flavor (Verplaetse, 1994).The internal parameters are chemical (added sugars or spices) or microbiological (startercultures); external parameters are physical, such as the temperature and humidity during theprocess.

a. Nonvolatile Fraction of Dry Sausage Aroma. The fermentation of carbohy-drates, proteolysis, and lipolysis generate many nonvolatile compounds that play an im-portant role in the taste impression (Verplaetse, 1994):

1. Glycolysis results in production of organic acids, the major products being lac-tate and acetate, which will contribute to the acid taste. However, the excessive productionof these acids is not desirable, because of the suppression of global aroma by the acid taste.An excessive production of the D-lactic acid isomer is undesirable because of its unpleas-ant spicy taste (Ramihone et al., 1988).

2. Lipolysis is carried out by either endogenous meat enzymes and/or starter cul-tures. This process generates free fatty acids and diglycerides. The free fatty acids gener-ated have a small impact on taste because it is necessary to have a high concentration ofthese compounds to produce a perceptible effect on sausage taste. The further oxidation ofthe free fatty acids generates many different compounds responsible for the aroma of theproduct as will be described. Lipolysis is mainly caused by endogenous enzymes, but lipidoxidation is caused by microbial action; therefore, they must be considered different pro-cesses.

The presence of yeasts and molds in fermented sausages contributes to the fermentedflavor. Their lipolytic enzymes contribute to flavor by generating carbonyl compounds. Inthe presence of oxygen, molds and yeasts do not only form flavor compounds but also ox-idize lactic acid.

3. Proteolysis of meat proteins produces polypeptides, peptides, and free aminoacids that are important for taste development in dry sausage (Nishimura et al., 1988, Katoet al., 1989). The extent of proteolysis depends on the acidity of the sausages. In low-acid-



ity sausages, the proteolytic activity is low and no major proteins are broken down. Inmedium- and high-acidity sausages, myosin and actin are clearly degraded to fragments of135 and 38 KDa, showing a pattern similar to that produced by endogenous cathepsins(Verplaetse, 1994). During ripening, there is a change in the composition of hydrophilicpeptides that is correlated with sausage taste. The generation of high amounts of hy-drophobic peptides is responsible for bitter taste and off-flavors. The amino acids releasedduring proteolysis can be decarboxylated, deaminated, or even further metabolized. There-fore, the generated ammonia and amines cause an increase in pH, which is observed dur-ing drying of sausages (Lücke, 1985), and enhance sausage taste by neutralizing the finalacidity.

A rapid decrease in pH during initial steps of sausage production, as occurs whenstarters are used, positively affects color development, texture, and homogeneity of drying,although taste may be negatively affected (Flores et al., 1997). Therefore, it is necessary tofind an equilibrium between acid production and taste and, last, ammonia production mustbe intensified to neutralize final acidity, enhancing sausage taste.

b. Volatile Compounds in Fermented Sausage Aroma. Many volatile compoundshave been identified in fermented products (Berdagué et al., 1993; Edwards et al., 1999)belonging to the following classes: alkanes, alkenes, aldehydes, ketones, alcohols, aromatichydrocarbons, carboxylic acids, esters, terpenes, sulfur compounds, furans, pyrazines,amines, and chloride compounds. Different pathways are responsible for the formation ofthese volatile compounds. However, the impact of an odor component on the total aromadepends on a number of factors, such as odor threshold, concentration, solubility in wateror fat, and temperature as reported for dry-cured ham flavor (Flores et al., 1998b). The dif-ferent pathways of the volatile compounds and their impact on sausage aroma are asfollows:

1. Lipid oxidation accounts for the generation of nonbranched aliphatic compoundssuch as alkanes, alkenes, methyl ketones, aldehydes, alcohols, and several furanic cycles.The contribution of alkanes to flavor is almost irrelevant because of their high thresholds.The flavor of alcohols was considered unimportant in comparison with other carbonyl com-pounds. The straight-chain primary alcohols are relatively flavorless but as the carbonchain increases, the flavor becomes stronger (Shahidi et al., 1986), giving greenish, woody,and fatty floral notes. C3 and C4 aldehydes have sharp and irritating flavors; intermediate(C5-C9) have green, oily, and fatty flavors; and the higher (C10-C12) have a citrus flavor(Forss, 1972).

2. The fermentation process release compounds of low molecular weight such asdiacetyl, acetoin, butanediol, acetaldehyde, ethanol, and acetic acid. The generation of thespecific volatile compounds during the carbohydrate fermentation depends on the starterused. The generation of compounds such as diacetyl, acetoin, or butanediol imparts a but-ter and yogurt aroma described in fermented sausage.

3. The catabolism of branched amino acids such as valine, leucine, and isoleucinegenerates 2- and 3-methylbutanal, 2- and 3-methylbutanol, 2- and 3-methylpentanoic acids,respectively, dimethyldisulfide from cysteine, and benzeneacetaldehyde from phenylala-nine. These sulfur compounds are important contributors to meat flavor because of theirlow threshold values.

4. Animal feedstuffs and contaminants constitute another source of volatile com-pounds. For example, toluene and xylene isomers are currently found in plants, giving

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sweety and fruity notes (Shahidi et al., 1986). The chloride compounds may originate frompesticide residues ingested by animals.

5. The spices and condiments added in the manufacture of fermented products con-tribute to a particular flavor, depending on local traditions. In fact, there are many specificflavors due to the high number of available aromatic plants such as pepper, paprika, mus-tard, nutmeg, cloves, oregano, rosemary, thyme, garlic, and onion (Ordoñez et al., 1999).These compounds have a high impact on the aroma of fermented products. For instance,the high content of terpene hydrocarbons or sulfur compounds found in the headspace offermented sausages comes from pepper or garlic, respectively.

However, it is important to note the diverse pathways leading to the same volatilecompounds. This is the case with acetic acid and ethanol, which also can be produced inthe catabolism of lipids or amino acids, and some methyl compounds also generated in thedegradation of branched amino acids by Strecker degradations. Sulfur compounds such asdimethyl disulfide and methyl-propyl disulfide have been found in the headspace of fer-mented sausages. Although these compounds can come from the Strecker degradation ofcysteine and methionine in instances when garlic is added as a spice, many of the sulfurcompounds come from their previous degradation in the garlic itself (Viallon et al., 1996).

The volatile-compound content depends on the sausage type. Italian and Spanishsausages mainly contain the following dominant compounds: terpenes (from spices), ke-tones and aldehydes (from lipolysis and lipid oxidation), and esters. Certain low-acidsausages contain aldehydes, ketones, alcohols, and esters and low quantities of N-contain-ing volatiles, indicating a low proteolysis in the product. On the other hand, medium-acidsausages (pH 5.1 to 5.3) contain aldehydes and ketones (constituents of the 60% of totalvolatile), furans, sulfur compounds, pyrazines, and amines, indicating a high proteolysis inthe product (Verplaetse, 1994).

B. Safety

Lactic acid bacteria play a critical role in safety and preservation by the fermentative con-version of carbohydrates to organic acids (lactic and acetic acids). The production of or-ganic acids has two concomitant effects: (a) the inhibition of the acid-sensitive spoilage mi-croorganisms by the pH drop and (b) the intracellular inhibition of microbial metabolicprocess by the penetration of the nondissociate acid form across the cell membrane. In thissense, acetic acid has a greater antibacterial activity than lactic acid due to differences indissociation constants of these two acids (Weber, 1994). The presence of lactobacilli is notthe only factor necessary for producing safe and stable sausages. There are many other fac-tors, such as anaerobic conditions, salt, nitrite, and low water activity, interacting with eachother and exerting a hurdle effect (Leistner, 1992).

The overall inhibitory functions of lactic acid bacteria are, however, due to a morecomplex antagonist system, which also includes production of other inhibitory substances.These antibacterial metabolic products exert a protective effect against a wide spectrum ofmicroorganisms and are produced in fewer amounts than lactic and acetic acids (De Vuystand Vandamme 1994b). Antibacterial compounds include formic acid, free fatty acids, am-monia, ethanol, hydrogen peroxide, carbon dioxide, diacetyl, acetoin, 2,3-butanediol, ac-etaldehyde, benzoate, D-amino acids, bacteriolytic enzymes, bacteriocins, and antibiotics,as well as several unidentified inhibitory substances (Daeschel, 1989; Piard et al., 1992).

The competition for essential nutrients also constitutes a factor of selection in favorof lactic acid bacteria. Food-borne pathogens such as Staphylococcus aureus and Listeria



monocytogenes can be inhibited by depletion of essential amino acids and vitamins (Ian-dolo et al., 1965; Degnan et al., 1992).

The production of bacteriocins by lactic acid bacteria is considered of great impor-tance for future applications in food preservation. Bacteriocins can be defined as biologi-cally active proteins or protein complexes (protein aggregates, lipocarbohydrate proteins,glycoproteins) displaying a bactericidal action, against other, mostly closely related, mi-croorganisms (De Vuyst and Vandamme, 1994b). These compounds are usually smallcationic peptides with high isoelectric points and amphiphilic characteristics, which are ac-tive at micromolar concentrations (Leroy and Vuyst, 1999).

The bacteriocins produced by lactic acid bacteria can be divided in three majorgroups on the basis of primary structure, molecular mass, and heat stability:

1. Lantibiotics or lanthionine-containing bacteriocins, which are composed of un-usual amino acids (lanthionine and methyllanthionine). These are heat-stablecompounds of small size (19–37 amino acid residues) (e.g., nisin and lactocin S).

2. Non-lantibiotic bacteriocins, which are composed of common amino acids, aresmall (�15,000 Da), and heat-stable (e.g., sakacin A, pediocin PA-1, andcarnobacteriocins).

3. Non-lantibiotic bacteriocins, which are composed of common amino acids, arelarge (�15,000 Da) and heat sensitive (e.g., helveticin and caseicin).

Many strains of lactic acid bacteria associated with meat and meat products, be-longing to the genera Lactobacillus, Pediococcus, Leuconostoc, Carnobacterium, andEnterococcus, are important bacteriocin producers (Aymerich et al., 1998). Two group ofbacteriocins produced by lactic acid bacteria also have been defined according to their in-hibitory spectrum: (a) bacteriocin, with a narrow inhibitory spectrum (Klaenhammer,1988), active only against bacteria belonging to the same genus, and (b) bacteriocins ac-tive against other bacteria genera. In the latter, antibacterial activity has been describedagainst gram-positive bacteria, such as Listeria monocytogenes, Staphylococcus aureus,Clostridium perfringens, C. botulinicum, and Brochothrix thermosphacta. In only a fewcases, bacteriocins have been reported to inhibit the gram-negative Aeromonas hy-drophila and Pseudomonas putida. The inhibition of other gram-negative bacteria suchas E. coli and Salmonella by bacteriocins requires the addition of chelating-like agents(Aymerich et al., 1998).

The mode of action involves the adsorption of bacteriocins to specific or nonspecificreceptors on the cell surface, resulting in cell death. The primary target of bacteriocins isthe cytoplasmic membrane, initiating changes in membrane permeability, disturbing mem-brane transport, or dissipating the proton motive force. Ultimately, energy production andbiosynthesis of macromolecules are inhibited, causing cell death (De Vuyst and Van-damme, 1994a).

The application of bacteriocins, as naturally occurring antimicrobial compounds,is promising given the trend to avoid additives in food. Many bacteriocins are heatstable; have a bactericidal and irreversible mode of action; and are stable, digestible,biodegradable, safe to health, and active at low concentrations (De Vuyst andVandamme, 1994b). However, their application has some limitations: for instance, re-stricted spectrum of antimicrobial activity, susceptibility to tissue proteolytic enzymes,and significant lower activity in meat systems due to limited diffusion in the matrix andunspecific binding to meat components such as fat particles (Daeschel, 1993; Holzapfel,et al., 1995).

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VI. TRENDS FOR ACCELERATION

Large-scale production requires the application of techniques to enhance flavor and re-duce ripening time. The use of higher fermentation temperatures constitutes the simplestapproach to achieving these objectives, but it may imply hygienic risks and flavor de-fects. Other strategies have consisted of reducing the water content of raw meat byfreeze-drying or its binding capacity by including pale, soft, exudative (PSE) meat. Cur-rently, the inoculation of starter cultures constitutes the most successful way to speed upand control the fermentation process. Most attention has been paid to the acidificationability of lactic acid bacteria, although their exclusive use results in a fast pH reductionthat may result in unpleasant acid tastes. This may occur because of an unbalanced de-velopment of the complex set of reactions that contributes to the overall flavor. Thus, fla-vor enhancement still constitutes one of the major challenges for which two intense fieldsof research are initiated: use of enzymes and use of whole cells from new or modifiedstarter cultures.

Lipolysis and proteolysis are directly related to flavor development and, therefore,the incorporation of lipolytic and/or proteolytic enzymes has been considered a way foracceleration and flavor improvement. The first attempts to use proteinases in meat hadthe sole aim of increasing tenderness, but also a bitter taste impression was associatedwith enzyme-treated meats. Further, proteases of different origins have been assayed infermented meat products. The incorporation of commercially available proteinases suchas pronase E from Streptomyces griseus, an aspartyl proteinase from Aspergillus ozyzae,papain from Carica papaya, and neutrase from Bacillus subtilis have been tested (Diazet al., 1997; Zapelena et al., 1997). However, the effect of these enzymes on sensorycharacteristics is not clearly positive. Despite the fact that proteolysis was stimulated, anexcessive softening and bitter taste was frequently found. More promising results havebeen obtained with the use of the cell-envelope proteinase of Lactobacillus paracasei(Blom et al., 1996). The increased production of amino acids and peptides stimulates themetabolism of lactic acid bacteria, causing a rapid pH drop that results in accelerated gelformation and drying. On the other hand, flavor development is also promoted, reachingscores comparable to those of long-ripening sausages. The application of exoproteolyticenzymes in fermented meats is still a neglected area, as well as the application of mi-croencapsulated enzymes that can be progressively released in the sausage mix. Studieson the addition of exogenous lipases have not revealed acceleration so far (Blom et al.,1996).

The second alternative is directed to improving the enzymatic properties developedby meat-related bacteria, an area in which little has been done so far. Most advances havebeen made on cell modification of dairy organisms. Bacterial cells have been attenuatedby physical methods (freezing, heating, chemical treatments, and/or drying) or mutagen-esis, with the purpose of inactivating undesirable enzymes (e.g., those related to acidifi-cation) and promoting the activity of desirable enzymes (e.g., peptidases). These tech-nologies have been proved to enhance flavor in shorter ripening times. The role ofautolysin of lactic acid bacteria is also critical for flavor development through the re-lease of intracellular enzymes. The application of autolytic strains is being exploited forcheese-making but is still obscure in meat fermentation. Therefore, substantial researchis needed on all these topics as well as further studies on selection of new starter strainsand all physiological and biochemical aspects related to flavor generation in fermentedmeats.



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