a new innovative process to produce lactose-reduced skim milk

Journal of Biotechnology 119 (2005) 212–218

A new innovative process to produce lactose-reduced skim milk

Senad Novalina, Winfried Neuhausb,∗, Klaus D. Kulbea

a Division of Food Biotechnology, Department of Food Sciences and Technology,University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria

b Department of Medicinal/Pharmaceutical Chemistry, University of Vienna,Pharmacy Centre, Althanstrasse 14, A-1090 Vienna, Austria

Received 17 June 2004; received in revised form 29 March 2005; accepted 31 March 2005

Abstract

The research field for applications for lactose hydrolysis has been investigated for some decades. Lactose intolerance, improve-ment for technical processing of solutions containing lactose and utilisation of lactose in whey are main topics in development ofbiotechnological processes. In this article, the establishment of a hollow fiber membrane reactor process for enzymatic lactosehydrolysis is reported. Mesophilic�-galactosidases were circulated abluminally during luminal flow of skim milk. The mainproblem, microorganisms growth in the enzyme solution, was minimised by sterile filtration and UV irradiation. In order tocharacterise the process parameters, such as skim milk concentration, enzyme activity and flow rates were varied. In comparisonto a batch process, enzyme activity could be used longer and enzyme rest into the product should not occur. Furthermore, thethree-dimensional separation of the substrate from the enzyme solution minimise blocking and washing out effects, which restrictpa©

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rocesses with immobilised enzymes. A conversion rate of 78.11% was achieved at a skim milk flow rate of 9.9 l h−1, enzymectivity of 120 U ml−1 and a temperature of 23± 2 ◦C in a hollow fiber reactor with a membrane area of 4.9 m2.2005 Elsevier B.V. All rights reserved.

eywords: Enzyme technology; Lactose hydrolysis;�-Galactosidase; Diffusional reactor

. Introduction

Every year 3.2 million tonnes of lactose, dissolved inhey, is accrued by the cheese production world-wide

Ruttloff, 1994). Almost half of this amount is used foruman and animal nutrition. The rest is waste that is

∗ Corresponding author. Tel.: +43 1 4277 55089;ax: +43 1 4277 9551.

E-mail address: [email protected] (W. Neuhaus).

difficult to dispose of and adds to the environmepollution. Therefore, there is a need for investigaabout further utilisation possibilities of lactose frowhey. One of these applications with a high technoical and dietetic interest is the enzymatic hydrolysilactose, whose economic importance has been incing ever since the 1960s.

Next to the medical aspect of lactose intolerasome very important technological advantages refrom the lactose hydrolysis into glucose and galact

168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2005.03.018

S. Novalin et al. / Journal of Biotechnology 119 (2005) 212–218 213

For example, the solubility increases from 18 to 55%(w/v) at 80% conversion and the sweetness rises up to70% related to sucrose. Furthermore, there is a lower-ing of the freezing point, an increase of the probabilityfor non-enzymatic browning reactions and a fasterfermentation process in lactose hydrolysed medium.Thus, the production of self-sweetening products orproducts with less sucrose addition would be possibleby using lactose hydrolysed milk or whey. Also, posi-tive effects on the crystallisation and process propertieswould be achieved after lactose hydrolysis (Zadow,1992).

In general, there are several technologies for enzy-matic hydrolysis of lactose (Pivarnik et al., 1995;Mahoney, 1985; Gekas and Lopez-Leiva, 1985). Theeasiest way is the discontinuous batch-process. Afterreaching the aimed conversion, the reaction is stoppedby heating, which causes enzyme denaturation and con-sequently the loss of enzymatic activity. Furthermore,the enzymes become product components after the pro-cess.

The immobilisation can be employed to use theenzyme’s activity for as long as possible. It can beaffected by physical or chemical binding on a solidmatrix like glass surfaces, cellulose acetate or oxiran-gel (Richmond et al., 1981). High cost of the immo-bilising steps, the activity loss during immobilisationand the occurrence of hygienic problems because of thefat and protein content in milk and whey (Reimerdes,1985) has a detrimental effect on the decision to chooset

a-t ratefl .,1 ymefi ee con-t d thes es.H ace-m andl ssi-b theh ctora

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tion are too expensive for reaching the economic aimto be at least as cheap as the batch process. Thus, it isnecessary to find another solution to control the micro-biological growth in the system.

2. Material and methods

2.1. Functional principles of the bioreactor system

An ultrafiltration unit (called module) consists of abundle of small hollow fibers. The outside (shell side)compartment is constructed as a closed circulation andis filled with the enzyme solution. A continuous flowof the substrate (skim milk) is applied to the inside(tube side) of the hollow fiber membranes. The spatialenzyme separation from the substrate solution is guar-anteed by the selected membrane cut-off value (Fig. 1).

In this way, continuous, enzymatic lactose hydrol-ysis is possible without inherent problems of immo-bilised enzymes. The driving force for this system isthe lactose diffusion, which mainly depends on the con-centration gradient, the temperature and the flow ratesof the substrate and the enzyme solution.

Fig. 2 shows the standard flow sheet of the newlydeveloped plant. The reactor is a hollow fiber module(43 in. module, 10 kDa cut-off and 4.9 m2 membranearea), which consists of a bundle of capillaries made ofpolysulfone membrane. The shell side volume is about2.5 l and the tube side volume is about 0.65 l. Skim milki oree berm ome-t , thel

rcu-l stedw hells a-fi em-b ed tob atchp utiont . Ino adi-a edi theu ia)

his process.The third possibility is the “physical immobilis

ion” by separating enzyme solution from the substow via ultrafiltration membranes (Czermak et al988). This system causes workable and cheap enzxation with little loss of the catalytic activity of thnzyme. Further advantages of this process are the

inuous operation of the reactor at low pressure anelectivity control by selection of suitable membranigh enzyme concentration application, easy replent and regeneration of spent enzyme solution

ittle loss of enzymes due to wash out effects are pole. A drawback is, that the diffusion resistance ofollow fiber membrane seems to be the limiting fat high conversion rates.

Ultrafiltration modules were constructed as steterilisable plastic membranes byCzermak et al. (1990ndCzermak (1992), whose development and prod

s pumped through the hollow fiber module. Befnzymatic conversion takes place in the hollow fiodule, skim milk passes a heat exchanger, a man

er and a thermometer. After the end of the moduleactose-hydrolysed product can be collected.

The enzyme solution is pumped in a closed ciation. Temperature of enzyme solution was adjuith a waterbath located before the hollow fiber’s side.Czermak and Bauer (1990)have constructed ultrltration modules as steam sterilisable plastic mranes. Development and production costs seeme too high for commercial use compared to the brocess. Thus, it is necessary to find another sol

o control the microbiological growth in the systemrder to sufficiently reduce germs presence a UV irrtion module and a sterile filtration unit were includ

n the enzyme circulation. To our best knowledge,se of a commercially available UV-unit (Visa, Austr

214 S. Novalin et al. / Journal of Biotechnology 119 (2005) 212–218

Fig. 1. Principle of lactose hydrolysis in the hollow fiber module by�-galactosidase denoted as�-gal in the scheme. While skim milk ispumped through the hollow fiber, molecules with lower size than themembrane cut-off value, such as small proteins, salts and lactose passthrough into the shell side. There, lactose is enzymatically convertedto glucose, galactose and a small amount of oligosaccharides by�-galactosidase. The product transfer back into the tube side is alsoeffected by concentration gradient driven diffusion.

is a novelty in the enzyme technology field. In orderto investigate the effects of UV irradiation on enzymeactivity and germ number, enzymes were dissolvedin milk buffer and pumped into a closed circulationthrough the UV module at a flow rate of 25 l h−1 usingdifferent UV intensity levels. Enzyme activity and totalgerm number (carried out by Koch plating process withstandard count agar from Merck, Germany) were deter-mined from samples taken at several time points.

2.2. Material

Apparatus: Hollow fiber module RomiPro5 in.× 43 in. PM 10 was from Koch (MA, USA),sterile filter FG-50 and FG-30 were purchasedfrom Millipore (MA, USA) and the sterile filtration

module type MD 020 GP 2N was obtained fromMicrodyn (Germany).Chemicals: o-Nitrophenyl-�-d-galactopyranoside(ONPG),o-nitrophenole (ONP) andd-glucose werepurchased from Sigma (MO, USA), lactose was fromMerck (Germany). Milk buffer described byNovoNordisk (1977)should imitate the milk’s salt systemand was used in enzyme experiments, when milk hadto be excluded. For skim milk “medium heat extraskim milk powder” was dissolved in bidistilled water.The powder was produced by Lactoprot (Austria)and consisted of following components: 35% protein,1.25% fat, 51% lactose, 8% ashes and less than 4%water.β-Galactosidases: The systematic name for thisenzyme is �-d-galactoside-galactohydrolases andits EC-number is 3.2.1.23. Maxilact L 2000 andMaxilact LX 5000 fromKluyveromyces lactis wereobtained from Gist-brocades (Netherlands). Theycatalyse the lactose hydrolysis.

2.3. Analytics

2.3.1. Determination of enzyme activityThis method was described byPetzelbauer et al.

(1999)for thermophilic�-galactosidase and was usedwith slight modification for mesophilic variants. ONPGwas utilised as a substrate and the measurements ofenzyme activity were carried out at a constant concen-tration of ONPG. By adding 20�l of the enzyme solu-tsO pH6 NPc whichw

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ion to 980�l of the substrate solution (860�l 50 mModium phosphate buffer, pH 6.5 and 120�l 25 mMNPG diluted in 50 mM sodium phosphate buffer,.5) the hydrolysis was initiated. The release of Oaused an increase in the absorbance at 405 nm,as measured for 5 min at 25◦C.One unit of ONPG activity is defined as 1�mol

eleased ONP per minute under the reaction coions described above. Whenever it was necessarnzyme solution was diluted in milk buffer.

.3.2. Sugar analysisQuantitative glucose determination was acc

lished with an Ebio glucose analyser from EppenGermany). The measurement range lies betweend 50 mM glucose. Consequently, the milk samere diluted with bidistilled water in order to prepauitable concentrations.


Fig. 2. Scheme of hollow fiber reactor for lactose hydrolysis by�-galactosidase: (1) hollow fiber module, (2) UV module, (3) sterile filtrationmodule, (4) heat exchanger, (5) peristaltic pump, (6) heating bath, (7) storage tank for the substrate, (8) tank for enzyme solution, (9) tank forsterile filtration circulation, (10) sampling port, (11) three-way cock, (12) waste line and (13) product collecting.

Samples from the enzyme solution in the reactor’sshell-side were incubated at 100◦C for 2 min to pre-vent interference of�-galactosidase. Afterwards, thehot solution was centrifuged at 10,000 rpm for 5 min.Twenty microlitres of appropriately diluted supernatantwas mixed with the Ebio buffer solution before the mea-surement. For one-point-calibration a 12 mM standardglucose solution from Eppendorf was used.

HPLC was used to quantify lactose, galactose andglucose concentrations. Samples had to be precipi-tated by Carrez clarification with 85 mM K4[Fe(CN)6]and 250 mM ZnSO4·7 H2O followed by centrifu-gation at 11,000 rpm for 10 min. Concentrations ofthe supernatants were measured with a HPLC-systemfrom Merck Hitachi (HPX 87-C-column from Aminex,85◦C, 0.7 ml min−1 flow rate, bidistilled water as elu-ent, ERC 7512 IR-detector from Erma Cr Inc., USA).External standard solutions of 10 g l−1 lactose, galac-tose and glucose were used for calculation.

3. Results and discussion

The aim of the present work was to establish an alter-native membrane bioreactor process for hydrolysing

lactose in complex media like skim milk. We focusedon the development of a cheap and easy way to enhancelong-term stability of both the process and enzyme’sactivity. The high costs for a steam sterilisable systemwould not allow to reach our aim to develop a processwith at least comparable costs to a batch process. Inorder to find another way for sufficient germ reductionin the enzyme solution, a combination of sterile filtra-tion and UV irradiation was developed.Fig. 3 showsthe results of an experiment, where an UV intensityof 45% of a 25 W lamp has led to both a satisfyinggerm reduction and enzyme stabilisation at the sametime. After 90 min, germ number was sufficientlyreduced from 104 to 102 germs ml−1 and only 10%less enzyme activity was detected compared to theblank solution. Blank solution was supplemented with0.02% NaN3 in order to suppress microbiologicalgrowth.

Enzyme stability is one reason to favour packed-bedreactors instead of hollow fiber reactors (Pivarnik et al.,1995). One main cause for loss of enzyme activity inhollow fiber reactors is the absorption of the enzymesby the membranes. In order to avoid high enzyme activ-ity loss by this, pre-absorption processes with proteinsolutions in the shell side were carried out. The cheap-


Fig. 3. UV experiment at 45% irradiation intensity of 25 W UV lampsituated in a UV module. Enzyme solution (120 U ml−1 Maxilact L2000, milk puffer pH 6.5, 106 mM lactose) was circulated at 25 l h−1.Relative enzyme activity (©), relative enzyme activity of a blanksolution (�) which was supplemented with 0.02% (w/v) NaN3 andtotal germ numbers (�) are depicted vs. time.

est and most practical way to cover the membranesurface with proteins was to fill the shell side (enzymecirculation) by ultrafiltration of skim milk. This wasfollowed by circulation of this whey for 1 h beforeenzyme solution was added into the shell side. For theseexperiments, commercially available enzymes, Maxi-lact (Gist Brocades) and Lactozym (Novo Nordirsk,Denmark) were chosen as suitable enzymes due to theiroptimum temperature between 35 and 45◦C and pHvalue about 6.5–6.8. Several tests showed that Maxi-lact was more stable and did not lose as much activitylike Lactozym. Thus, Maxilact was used for the furtherstudies.

In order to develop a cost-effective process weaimed at gaining 75% lactose hydrolysis in skimmilk at 10 l h−1 (360 g l−1 h−1 productivity) flow rate.Czermak et al. (1988)showed that this kind of pro-cess is mainly limited by lactose diffusion and notby enzyme concentration. Accordingly, dependence oflactose hydrolysis on enzyme concentration was inves-tigated (Fig. 4). Skim milk was supplemented with0.02% NaN3 and was pumped with 10± 0.5 l h−1.Enzyme activity was adjusted by dosages to the enzymetank. It is very important to recognise that the enzymeactivity of 100 U ml−1 was still a limiting factor forthe conversion rate. Further studies resulted in deter-mining optimum enzyme concentration at 120 U ml−1.Adding more enzyme led to a minimal increase of lac-tose hydrolyis. Therefore, all further experiments werecarried out with 120 U ml−1 Maxilact.

Fig. 4. Dependence of lactose conversion (%) on enzyme activity(U ml−1) at 10± 0.5 l h−1 skim milk flow, 0.02% (w/v) NaN3, lactoseconversion (%) means the formed glucose related to lactose, whichis determined by HPLC, in skim milk (�) and enzyme solution (©).

In order to optimise and characterise the process,parameters like skim milk flow rate and concentrationwere varied. At the beginning of every experiment,a steady-state of the diffusing substances had to beset between the enzyme solution and the skim milk.After steady-state was reached, constant measure val-ues and production were possible. Using an experimen-tal design where skim milk (supplemented with 0.02%NaN3) was pumped with a flow rate of 10.5 l h−1 andthe enzyme solution (120 U ml−1, Maxilact L 2000)circulated at 25 l h−1, measured lactose conversion inskim milk levelled at 81% after 2 h. Steady-state in theenzyme solution was reached after 3 h at 96%. Inter-estingly, after 90 min measured lactose conversion inskim milk was still higher than in the enzyme solution.

Experiments with constant enzyme activity and var-ied skim milk flow rates were also important for under-standing this process.Fig. 5summarises the influenceof varied skim milk flow rates and skim milk concen-trations on lactose conversion rate at same enzymeactivity (120 U ml−1). Firstly, the correlation betweenskim milk flow rate and lactose conversion was investi-gated. Lactose conversion in skim milk decreased from92.48% at 5.04 l h−1 to 82.95% at 7.83 l h−1 and furtherto 78.11% at 9.9 l h−1, conversion rates determined inenzyme solution were always higher at the same flowrate. In order to improve process productivity, severalexperiments with 1.5:1 and 2:1 concentrated skim milkwere accomplished. Although higher blocking effects


Fig. 5. Influence of skim milk flow rate and concentration on lac-tose conversion (%). All experiments were carried out at 23± 2◦C,120 U ml−1 and 25 l h−1 enzyme solution flow rate. Lactose conver-sion means the formed glucose related to lactose, which is determinedby HPLC, in skim milk (�) and enzyme solution (�) Maxilaxt L2000. Skim milk was supplemented with 0.02% (w/v) NaN3. Alsoflow rate dependent lactose conversion (%) in 1.5:1 concentratedskim milk (�) and enzyme solution (�) Maxilact LX 5000, insteadof NaN3 45% UV irradiation 10 min h−1 and continuous microfiltra-tion and in 2:1 concentrated skim milk (�) and enzyme solution (©)Maxilact LX 5000, instead of NaN3 45% UV irradiation 10 min h−1

and continuous microfiltration are depicted.

on the membranes were expected, higher substanceconcentration should result in increased diffusionrates and consequently in enhanced productivity.For the first test, 1.5:1 concentrated skim milk wasnot supplemented with NaN3. The enzyme solution(120 U ml−1 Maxilact LX 5000) was treated for 10 minfor every hour with UV irradiation at 45% intensityand was continuously sterile filtered by microfiltration.Lactose conversion in skim milk decreased as expecteddepending on flow rate of substrate from 80.09% at6.5 l h−1 to 72.68% at 8.6 l h−1 and further to 68.54%at 10.4 l h−1, conversion rates in enzyme solutionwere always higher at the same flow rate. For the nexttest, 2:1 concentrated skim milk was also not supple-mented with NaN3. The enzyme solution (120 U ml−1

Maxilact LX 5000) was treated for 10 min for everyhour with UV irradiation at 45% intensity and wascontinuously sterile filtered by microfiltration. Lactoseconversion rate in skim milk decreased also dependingon substrate flow rate. In general, productivity (hydrol-ysed lactose in g l−1 h−1) increased at higher flow ratesand higher skim milk concentrations. Five hundred and

nineteen grams per litre per hour lactose conversioncan be achieved with two-fold concentrated skim milkat 8.46 l h−1 flow rate and 63.99% lactose hydrolysisin contrast to 311.76 g l−1 h−1 for 1:1 skim milk at asimilar flow rate. If highest productivity is the aim forthe process, two-fold concentrated skim milk should bepreferred, if long-term stability under these conditionsis proved. Using high concentrated skim milk includesthe danger of blocking effects due to fat or proteinadsorption. This might harm the cartridges leadingto cost explosion. Also, higher concentration of skimmilk means more nourishment for microorganismsin the enzyme circulation and consequently fastergrowth and loss of enzyme activity due to digestion.All experiments lasted between 6 and 8 h. Future workwill carry out long-term experiments over severaldays.

Finally, two different procedures were compared toeach other in order to establish the most suitable onefor maximum lactose conversion. Under the same con-ditions, it was investigated whether lactose conversionrate would be higher at a flow of 5 or at 10 l h−1, if thesame amount is pumped through the reactor a secondtime. The idea was that the procedure with the higherflow rate would cause a higher concentration gradientand consequently higher diffusion rates. At a flow rateof 5 l h−1 a lactose conversion rate of 92.48% in skimmilk and 97.08% in enzyme solution was achieved.Whereas running the second procedure (at a flow rateof 10 l h−1, collecting the product and pumping it as wasp allyu mes ve-m

4

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• toreyelynec-

achiallyfirst

econd time through the hollow fiber apparatus) itossible to increase lactose conversion rate minimp to 94.35% in skim milk and to 99.14% in enzyolution. This procedure may lead to a small improent of the economic efficiency.

. Conclusions

The major conclusions of this study are as follo

It is well known that a membrane-diffusion reacis applicable for the hydrolysis of lactose in whor milk using enzymes. Because of the relativlow mass transfer, large membrane areas areessary, which therefore, makes it difficult to reeconomic goals. In the actual case, a commercmembrane module was used overcoming this


problem. Results show high conversion rates at highflow rates and consequently high productivity.

• The second problem lies in the growth of microor-ganism within the enzyme solution (the shell side ofthe module). Two different processes were appliedin order to minimise this issue: microfiltration andUV irradiation of the enzyme solution. As the experi-ments have shown, this technological idea could leadto a definite solution of this second problem.

In any case, the applicability of membrane-diffusionreactors needs to be tested commercially. We wereable to introduce a process for direct lactose hydrol-ysis in skim milk without any ultrafiltration step beforeenzymatic conversion. Thus, our system can easily beconnected directly with milk storage tanks in dairyindustry as an inline installation. Compared to otherpublished data (Splechtna et al., 2002) productivityseems to be much higher. Also it should be possi-ble to use other substrates for this process like wheyor high concentrated whey. Future investigations willfocus on optimising this process considering temper-ature influence on long-term stability and conversionperformance. Furthermore, we will try to develop amathematical possibility to predict lactose conversionrate depending on lactose concentration and flow ratefor this system.

Acknowledgement

omt 96-1

R

C n amring

Czermak, P., Bauer, W., 1990. Optimization of the continuoushydrolysis of lactose in the dialysis enzyme membrane reactor.DECHEMA Biotechnol. Conf. 4 (B), 763–766.

Czermak, P., Bahr, D., Bauer, W., 1990. Verfahrenstechnische Opti-mierung der kontiniuerlichen enzymatischen Laktose-Hydrolyseim Dialyse-Membranreaktor. Chem.-Ing.-Tech. 62, 678–679.

Czermak, P., Eberhard, G., Konig, A., Tretzel, J., Reimerdes, E.H.,Bauer, W., 1988. Dialysis membrane reactors for enzymaticconversions in biotechnical processes: functional principles andexamples for application. In: Behrens, D. (Ed.), DECHEMA.Biotechnol. Conf. 2, VCH Verlagsgesellschaft, Weinheim, NewYork, pp. 133–145.

Gekas, V., Lopez-Leiva, M., 1985. Hydrolysis of lactose: a literaturereview. Process Biochem. 20, 2–12.

Mahoney, R.R., 1985. Modification of lactose and lactose-containingdairy products with�-galactosidase. In: Fox, P.F. (Ed.), Develop-ments in Dairy Chemistry-3. Elsevier Applied Science PublishersLtd., Amsterdam, pp. 69–108.

Novo Nordisk, 1977. Determination ofK. fragilis lactase. NovoIndustri AS, Analytical Method Nr. AF 171/1-GB-a.

Petzelbauer, I., Nidetzky, B., Haltrich, D., Kulbe, K.D., 1999. Devel-opment of an ultra-high-temperature process for the enzymatichydrolysis of lactose. I. The properties of two thermostable beta-glycosidases. Biotechnol. Bioeng. 64 (3), 322–332.

Pivarnik, L.F., Senecal, A.G., Rand, A.G., 1995. Hydrolytic andtransgalactosylic activities of commercial�-galactosidase (lac-tase) in food processing. In: Kinsella, J.E., Taylor, D.L. (Eds.),Advances in Food and Nutrition: Research, vol. 3. Academic,NY, pp. 1–101.

Reimerdes, E.H., 1985. Entwicklung von Enzymreaktorenzur Lact asebehandlung von Milch- und Folgeprodukten.Abschlußbericht BM f. Forschung und Technologie, Referencenumber 038491, Follow up report PTB 0382628, Bonn, 3–63.

Richmond, M.L., Gray, J.I., Stine, C.M., 1981.�-Galactosidase:review of recent research, related to technological applica-

64,

R ehrs

S zky,romfymeture-88.

Z is ins Ltd.,

We gratefully acknowledge financial support frhe European Commission under Contract EU CT048.

eferences

zermak, P., 1992. Scale up von Enzym-MembranreaktoreBeispiel der kontinuierlichen Lactosehydrolyse. Bioenginee1/92 8.Jg., Lebensmitteltechnologie, 47–52.

tion, nutritional concerns and immobilization. J. Dairy Sci.1759–1771.

uttloff, H., 1994. Lactase. Industrielle Enzyme, second ed. BVerlag, pp. 766–777.

plechtna, B., Petzelbauer, I., Kuhn, B., Kulbe, K.D., NidetB., 2002. Hydrolysis of lactose by beta-glycosidase CelB fhyperthermophilic archaeonPyrococcus furiosus: comparison ohollow-fiber membrane and packed-bed immobilized enzreactors for continuous processing of ultrahigh temperatreated skim milk. Appl. Biochem. Biotechnol. 98–100, 473–4

adow, J.G., 1992. In: Zadow, J.G. (Ed.), Lactose HydrolysWhey and Lactose Processing. Elsevier Science PublisherAmsterdam, pp. 2–21.

a new innovative process to produce lactose-reduced skim milk

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