ORIGINALRESEARCH Viability and a- and b-galactosidase activity of

Bifidobacterium breve and Lactobacillus reuteri inyoghurt products supplemented with shiitakemushroom extract during refrigerated storage

OSMAN A HASSAN,1* AMER A ABUGHAZALEH,2 SALAM AIBRAHIM,1 OMOANGHE S ISIKHUEMHEN,3 SADDAM S AWAISHEH4

and REZA TAHERGORABI11Food and Nutritional Sciences, NC A&T State University, Greensboro, NC 27411, 2Department of Animal Science,Food and Nutrition, Southern Illinois University, Carbondale, IL 62901, 3Department of Natural Resources, NC A&TState University, Greensboro, NC 27411, USA, and 4Department of Nutrition and Food Science, Al-Balqa AppliedUniversity, 19117 Salt, Jordan

This study examined the impact of shiitake mushroom extract (ME) on the viability and a/b enzymeactivity of Bifidobacterium breve ATCC 15701 and Lactobacillus reuteri DSM20016 in yoghurt dur-ing refrigerated storage. The presence of ME significantly enhanced the viability of both strains.L. reuteri DSM20016 exhibited better stability in yoghurt than B. breve ATCC 15701. L. reuteriDSM20016 showed the highest viability in yoghurt samples containing 4% ME, followed byB. breve ATCC 15701, and then control samples. Results suggest that ME can be used as a naturaladditive to dairy products to improve the growth and viability of L. reuteri DSM20016 and B. breveATCC 15701 strains.

Keywords Probiotics, Bacterial survival, Probiotic yoghurt, Enzymology, Lactobacillus, Prebiotics.

INTRODUCTION

The popularity of probiotics has been increasingrapidly worldwide (Benkouider 2004; Rafter2004). Probiotics are defined as live microbialfeed supplements that beneficially affect the hostby improving intestinal microbial balance (Fuller1989). In recent years, probiotics have drawnthe attention of the food industry (Saarela et al.2002; Salminen and Gueimonde 2004). Forexample, the food industry is increasingly manu-facturing different kinds of foods containingprobiotic bacteria. These foods are referred to asfunctional foods. Dairy products, one group offunctional foods to which probiotics such asLactobacillus strains and Bifidobacterium spp.are added, constitute a significant amount ofcommercially available probiotic foods (Shah2001; Reid et al. 2003; Awaisheh 2011). Main-taining the viability of probiotic bacteria in dairyproducts remains a major challenge to fermenteddairy producers because of the inability of many

strains to grow well in milk (Awaisheh et al.2005, 2012). Thus, ensuring the survivability ofprobiotic bacteria until the host products areconsumed has been of much interest to thoseseeking healthy lives.Ibrahim and Carr (2006) reported that probiot-

ic cultures in many commercial yoghurt brandswere not viable after 3–4 weeks of storage at4 °C. Therefore, there have been many sugges-tions regarding how to use nutrients to enhancethe viability and survival of probiotic strains.Shiitake mushroom (Lentinus edodes) extract

(ME) contains many oligosaccharides and poly-saccharides (Kawakami et al. 2004) and istherefore considered to be a prebiotic whichsupports the viability of probiotics. Hassan et al.2011 studied the impact of ME on the growthof several lactic acid bacteria and bifidobacteriausing MRS as a laboratory growth medium andfound that 4% of ME supported the growth ofthe tested strains. The results also showed thatthe effect of ME is strain-dependent and most

*Author forcorrespondence. E-mail:oahassan@ncat.edu

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effective with both L. reuteri DSM20016 and B. breveATCC 15701. Based on these observations, we expandedour study and investigated the effectiveness of ME on theviability of L. reuteri DSM20016 and B. breve ATCC15701 in yoghurt products during refrigerated storage.

MATERIALS AND METHODS

Culture conditionsOne strain of L. reuteri DSM20016 and one strain ofB. breve ATCC 15701 obtained from the culture collectionof the food microbiology laboratory at North Carolina A&TState University were used in this study. Both strains weremaintained on de Man, Rogosa and Sharpe (MRS) (DifcoLaboratories, Becton Dickinson, Sparks, MD, USA) agarPetri dishes at 4 °C before use and activated by placing onebacterial colony of each strain into fresh MRS broth inanaerobic culture tubes at 37 °C for 18 h.

Preparation of shiitake mushroom extractFresh shiitake mushroom was obtained from a local marketin Greensboro, North Carolina. The mushroom was freeze-dried (Labconco Freeze dryer, Kansas City, MO, USA),ground into a powder and then sieved (particle size 1 mm)to produce a fine powder. Four grams of the fine powderwere mixed with 100 mL of deionised water and thenheated on a hot plate at 80 °C for 3 h to make ME (4%).After cooling to room temperature, the mixture was first fil-tered through Whatman number 1 filter paper (WhatmanCorp., Clifton, NJ, USA) and then through a Nalgene filterunit with 0.2 lm pore size (Nalge Nunc International,Rochester, NY, USA) to obtain sterilized extract (ME).

Fermentation and storageTwo sets (2400 mL each) of pasteurised skim milk (0.1%milk fat) obtained from a local store in Greensboro, NC,were warmed in a water bath to 45 °C, and each set wasmixed with 5% (v/v) of active yoghurt starter culture(obtained from the Food Microbiology Laboratory at NorthCarolina A&T State University) to obtain 5.0 log CFU/mL.The yoghurt cultures used in this project consisted of Strep-tococcus thermophilus (ST) and Lactobacillus delbrueckiisubsp. bulgaricus (LB1). The yoghurt cultures were addeddirectly to the mix as frozen stock. Shiitake mushroomextract at 4% of ME was added to one set. One set withoutME was used as a control. Each set was then dividedequally into two containers and then inoculated with a sin-gle strain of the probiotic cultures of B. breve ATCC 15701or L. reuteri DSM20016 to achieve a 5.0 log CFU/mL. Allsamples were poured into 250-mL bottles, incubated at42 °C for 10 h and kept in a refrigerator at 4 °C for latertesting (0, 1, 2, 3, 4 and 5 weeks). Each experiment wasconducted in triplicate using three different lots of milk andshiitake mushroom extract.

Bacterial population in yoghurt samplesTo determine the effect of ME on the viability of L. reuteriDSM20016 and B. breve ATCC 15701 in yoghurt samples,samples were serially diluted in sterile 0.1% (w⁄ v) peptonewater at the end of the incubation period, and bacterial popu-lations were determined by surface plating on MRS agar.Plates were incubated at 37 °C for 48 h before colonies werecounted. The counts were recorded as CFU/g of sample.

Determination of pH values and titratable acidityA pH meter (model 410A; Orion, Boston, MA, USA) wasused for the measurement of the pH value of yoghurtsamples. Titratable acidity was determined by titrating 10-mL samples with 0.1 N NaOH with an endpoint of pH 8.6under constant stirring. Titratable acidity was recorded asthe percentage equivalent of lactic acid (AOAC 1999).

Enzyme assay

a-Galactosidase assaya-Galactosidase activity was assayed according to the FoodChemicals Codex (2003) protocol. a-galactopyranosideactivity was tested by adding 1 mL of p-nitrophenyl-a-D-ga-lactopyranoside substrate to 0.5 mL of each sample andtransferring all the samples into a shaker water bath at37 °C for 15 min. All reactions were stopped by adding2.5 mL of borax buffer. A blank for each sample was pre-pared by sequentially adding 0.5 mL sample, 2.5 mL boraxbuffer and 1 mL of substrate solution. A spectrophotometer(Model Genesys 10 Vis, Thermospectronic, Rochester, NY,USA) was used to measure absorbance at 405 nm (Ibrahimet al. 2010).

b-Galactosidase assayb-Galactosidase activity was assayed according to themethod described by Nagy et al. (2001). The reaction mix-ture was comprised of 0.5 mL of enzyme source and0.5 mL of 15 mM o-nitrophenyl-b-D-galactopyranoside in0.03 M sodium phosphate buffer (pH 6.8). Reactions weremaintained in a shaker water bath at 37 °C for 10 min, andthen, 2.0 mL of 0.1 M Na2CO3 was added to stop the reac-tion. The optical density for each of the samples was mea-sured at 420 nm with a spectrophotometer (Model Genesys10Vis, Thermo Spectronic). Units of activity were expressedas micro-moles of o-nitrophenyl released per minute,1U = 1 lmol/min (Hughes and Hoover 1995).

Statistical analysisEach experiment was independently replicated three timesfrom three lots of milk and shiitake mushroom extract in acompletely randomized design. Values were expressed asmeans and standard deviation. Mean values of treatmentswere compared by t-test SAS Institute Inc (1999). Differ-ences were considered significant at P < 0.01.

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RESULTS AND DISCUSSION

Viability of Lactobacillus reuteri DSM 20016 andBifidobacterium breve ATCC 15701 during refrigeratedstorageFigures 1 and 2 (a) show the viability of L. reuteri DSM20016 and B. breve ATCC 15701 in yoghurt samples by thepresence of ME. In the control samples with L. reuteriDSM20016, the initial population was 8.3 log CFU/mL. After1 week, the population dropped by ~ 0.6 log CFU/mL andcontinued to decline during the storage period. By the thirdweek, the population reached ~ 7.6 log CFU/mL, and after5 weeks, the population reached 7.3 log CFU/mL. When theME was added to the yoghurt samples, the initial populationof L. reuteri DSM20016 was 8.3 log CFU/mL. After 1 week,the population was 8.2 log CFU/mL. Thereafter, the popula-tion level was stable until the fourth week, when a very slightdrop in the bacterial population was observed. The bacterialpopulation reached 7.9 log CFU/mL by week 5.The initial population in control samples with B. breve

ATCC 15701 was 8.2 log CFU/mL. After 1 week, the pop-ulation dropped by ~ 0.5 log CFU/mL and continued todecline during the storage period. By the third week, thepopulation reached 7.5 log CFU/mL, and by week 5, it

reached ~ 7.3 log CFU/mL. In the yoghurt sample withME, the initial population of B. breve ATCC 15701 was 8.2log CFU/mL. After 1 week, the population was 8.1 logCFU/mL, after which the population remained in thesame range until the third week (8.1 log CFU/mL), reaching8.0 log CFU/mL by week 5. Compared with the controlsamples, the addition of 4% ME significantly enhanced theviability of bacterial strains (P < 0.01) during the storageperiod at 4 °C. These results suggest that ME could be usedto sustain the viability of L. reuteri DSM20016 andB. breve ATCC 15701 in yoghurt products.Overall, the bacterial population of B. breve ATCC 15701

and L. reuteri DSM20016 strains in control samples showeda significant decrease (P < 0.01) after 5 weeks of storage at4 °C, while the yoghurt samples with 4% ME maintained theviability level of both strains at 4 °C. The results suggestedthat ME in yoghurt could maintain the viability of L. reuteriDSM20016 and B. breve ATCC 15701 in fermented prod-ucts. The results indicated that the addition of 4% ME exerteda stabilising effect on B. breve ATCC 15701 and L. reuteriDSM20016 strains in pasteurised skim milk compared withthe control. Shin et al. (2000) and Akalin et al. (2004)showed similar results where a maximum effect was observedwhen they used 5% of fructooligosaccharide or inulin.

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Figure 1 Effect of mushroom extract on (a) viability of Lactobacillus reuteri DSM20016 (Log CFU/mL), (b) lactic acid production, (c) a-galactosi-dase activity (Gal U/mL) and (d) b-galactosidase activity during the incubation at 4 °C for 35 days.

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Change in pH of yoghurtThe pH values of yoghurt samples containing ME withL. reuteri DSM20016 and B. breve ATCC 15701 duringrefrigerated storage are shown in Table 1. The initial pHvalue in the yoghurt samples with ME and L. reuteriDSM20016 was 4.2. The pH of yoghurt samples decreasedslightly during storage and did not drop below 3.6 at theend of the storage (week 5), possibly due to low acidicactivity of the yoghurt and strain cultures. However, theinitial pH value in the control samples was 4.3 anddropped to 4.1 by the end of storage period.Table 1 also shows the initial pH value of 4.2 in the

yoghurt samples supplemented with ME in the presence ofB. breve ATCC15701. The pH value of yoghurt samplesdecreased at a steady rate of 0.2 units weekly until thefourth week and remained near a pH of 3.6 until the end ofstorage period. However, the initial pH value in the controlsamples with B. breve ATCC 15701 decreased by ~ 0.05units every 2 weeks and had dropped to 4.0 by the end ofthe storage period. As shown in Table 1, pH valuesdecreased when ME was added to yoghurt samples, thusindicating the stability of bacterial population throughoutthe study. Our result is in agreement with Shah et al.’sstudy (1995), who found that there was a relationshipbetween organism viability and pH value; the lactobacillus

and bifidobacteria used in his study were active even atrefrigerated temperatures and produced small amounts oflactic acid by fermentation of lactose. Also, Kailasapathyand his group (2007) reported that there was higher viabilityin bacteria when the pH value declined in range of 4.1–4.5,and this was presumably due to continued fermentation bylactic acid bacteria.

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Figure 2 Effect of mushroom extract on (a) viability of Bifidobacterium breve ATCC 15701 (Log CFU/mL), (b) lactic acid production, (c) a-galacto-sidase activity (Gal U/mL) and (d) b-galactosidase activity during the incubation at 4 °C for 35 days.

Table 1 Changes in pH values of yoghurt samples with mushroomextract during refrigerated storage at 4 °C for 5 weeks

Day

Yoghurt samplea

Lactobacillus reuteriDSM20016

Bifidobacteriumbreve ATCC 15701

Mushroom extract Mushroom extract

0.0% 4.0% 0.0% 4.0%

0 4.29 4.21 4.39 4.237 4.25 4.13 4.30 4.1314 4.21 4.03 4.23 4.0421 4.14 3.80 4.15 3.8028 4.08 3.70 4.11 3.6035 4.01 3.60 4.00 3.60a� standard deviation: SD 0.05.

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Titratable acidityThe development of acidity is necessary in yoghurt produc-tion for a well-balanced, desirable sensory characteristic andfor the viability of bacterial strain activity. This study of acidproduction of L. reuteri DSM20016 and B. breve ATCC15701 was conducted at 42 °C in skim milk culture mediafor 10 h, with and without the addition of the ME, afterwhich time the samples were refrigerated at 4 °C for5 weeks. According to Smith et al. (1975), acidity values canbe used as a measure of lactic acid bacteria growth in milk.Acidity measurements were taken over 5 weeks at 1 weekintervals. L. reuteri DSM20016 and B. breve ATCC 15701containing ME showed a significant amount of acid growth at~ 1.75 and 1.40%, respectively (Figures 1 and 2b). In controlsamples, L. reuteri DSM20016 and B. breve ATCC 15701without ME had minimal amounts of acid production at ~1.35 and 0.9%, respectively (Figures 1 and 2c). The largestamounts of acid production were observed in strain L. reuteriDSM20016. Statistical analysis showed that there was a sig-nificant difference in acid production between L. reuteriDSM20016 and B. breve ATCC 15701 (P < 0.01) and alsobetween samples with and without ME. This was well corre-lated with pH by increasing the storage time.

Enzyme activity

a-Galactosidase assayResults in Figures 1 and 2(c) show the enzyme activity ofL. reuteri DSM20016 and B. breve ATCC 15701 in yoghurtsamples with and without ME. In the control samples withL. reuteri DSM20016, the initial a-galactosidase activity was3.0 Gal U/mL. After 1 week, the a-galactosidase activitydropped to 2.6 Gal U/mL and continued to decrease duringthe storage period. By the third week, the a-galactosidaseactivity reached ~ less than 2.0 Gal U/mL, and after week 5,this activity significantly decreased and reached 1.1 gal U/mL. Whereas the initial a-galactosidase activity in yoghurtsamples with ME was 4.5 G U/mL and maintained this leveluntil the second week, thereafter, the a-galactosidase activitydecreased and reached 3.3 Gal U/mL by the end of the study.In the control yoghurt samples with B. breve ATCC

15701, the initial a-galactosidase activity was ~ 1.6 G U/mL. After 1 week, the a-galactosidase activity started todrop and continued to decrease during the storage period.The production of a-galactosidase in control yoghurt sam-ples with B. breve had reached less than 1 G U/mL by thethird week, and at the end of storage period, the a-galactosi-dase was ~ 0.8 G U/ml. In yoghurt samples with B. breveATCC 15701 and ME, the initial amount of a-galactosidasewas 3.3 G U/mL, which slightly decreased until it reached2.2 G U/mL by week 5 of storage at 4 °C.Generally, both strains of probiotic bacteria with ME used

in this study showed stability and a significant productionof a-galactosidase activity compared with the control until

the fifth week. These results agree with those of Awaisheh(2011), who reported that a-galactosidase activity in L. reu-teri (0.067–0.603 Gal U/mL) was higher than that producedby bifidobacteria (0.013–0.396 Gal U/mL) and a-galactosi-dase activity of L. reuteri strains DSM20016 and MM2-3was higher in comparison with the other bifidobacteria theyinvestigated when carbohydrate was added to the medium.Yeo and Liong (2009) found that the optimal level of a-galactosidase was 0.085–0.94 UmL when they used FOS asa prebiotic. Garro et al. (2006) also reported that raffinosein a high pH environment leads to an increase in a-galacto-sidase activity in B. longum. These results were expected asa-galactosidase hydrolyses the a-galactoside bonds in amonosaccharide such as raffinose. The results obtained fromour study might be due to ME high raffinose content (Has-san et al. 2011).

b-Galactosidase assayIn the control yoghurt samples without ME with L. reuteriDSM20016, the initial b-galactosidase was 4.0 G U/mL, butdropped to 3.6 G U/mL after 1 week (Figure 1e). In the sec-ond week, the level of b-galactosidase production was stableand then decreased until it reached 3.0 G U/mL by week 5.The initial level of b-galactosidase production in L. reuteriDSM20016 with ME was 4.5 G U/mL, but by the first week,b-galactosidase activity had significantly increased to 5.2 GU/mL and maintained this level until the second week.Thereafter, the b-galactosidase activity of L. reuteriDSM20016 decreased slightly by the third week to 5.1 G U/mL and reached 5.0 G U/mL, which is significantly higher(P < 0.01) compared with the control samples (Figure 1e).In the control samples with B. breve ATCC 15701, the

initial b-galactosidase activity was 3.2 G U/mL, whichdropped to 2.9 G U/mL after 1 week, and then dramaticallydecreased to 2.5, 2.1 and 1.8 G U/mL for the second, thirdand fourth weeks, respectively, until it reached ~ 1.5 by theend of the storage period (Figure 2e). With regard toB. breve ATCC 15701 in yoghurt samples with ME, the ini-tial b-galactosidase activity was ~ 3.7 G U/mL and thenincreased to 4.1 G U/mL after 1 week. The b-galactosidaseactivity of B. breve ATCC 15701 in the second, third andfourth weeks was 4.1, 4.08 and 4.0 G U/mL, respectively,and by the end of the storage period, the b-galactosidaseactivity was 4.0 G U/mL (Figure 2e).Several scientists have been studying the possibility of

increasing the activity of a- and b-galactosidases in differentbacterial strains using various prebiotics. Dumortier et al.(1994), for example, observed that b-galactosidase activityof B. bifidum occurred at higher temperatures. Othersshowed that a- and b-galactosidase activity changes are dueto the differences in cell mass, cell growth or variable num-ber of cells per gram of culture (Occhino et al. 1986; Hsuet al. 2005). Hsu et al. (2005) showed that carbohydrateand nitrogen sources, the pH of the media, and mineral

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additives have a significant effect on increasing the b-galac-tosidase activity in bifidobacteria.

CONCLUSIONS

The viability of L. reuteri DSM 20016 and B. breve ATCC15701 in yoghurt samples prepared using 4% ME during35 days of storage at 4 °C compared with samples withoutME showed stability in viability (7.9 to 7.3 log CFU/mL,respectively). The results indicated that addition of 4% ME tothe samples showed growth-prompting effect of L. reuteriDSM 20016 and B. breve ATCC 15701 compared with sam-ples without ME. Postfermentation acidification was reducedby increasing the concentration of ME in skim milk as itreached the lower pH in 35 days significantly (P < 0.05) ascompared to the control. After 35 days of storage at 4 °C,samples containing 4% ME showed a significant amount ofacid production in strain, L. reuteri DSM 20016 and B. breveATCC 15701 (1.75 and 1.40%, respectively), while in con-trast, samples without ME had minimal amounts of acid pro-duction was observed in L. reuteri DSM. Thus, ME couldserve as a functional food ingredient to maintain the viabilityof probiotic bacteria in food products as well as in the humanintestinal tract. Current work is undergoing in our laboratoryto determine the consumer acceptability and physical proper-ties of yoghurt fortified with ME.

ACKNOWLEDGEMENTS

This project was supported in part by Grant Number NC.X-234-5-09-170-1 from the National Institute of Food andAgriculture, and its contents are solely the responsibility of itsauthors and do not necessarily represent the official views ofthe National Institute of Food and Agriculture. We would liketo express our gratitude to Dr. Keith Schimmel (Energy andEnvironmental systems at NC A&T) for his support.

REFERENCES

Akalin A S, Fenderya S and Akbulut N (2004) Viability and activity ofbifidobacteria in yoghurt containing fructooligosaccharide duringrefrigerated storage. International Journal Food Science and Technol-ogy 39 613–662.

AOAC (1999) Official Methods of Analysis, 16th edn. Arlington, VA:Association of Official Analytical Chemists.

Awaisheh S S (2011) Development of probiotic soft cheese manufacturedusing goat’s milk with the addition of thyme. Milchwissenschaft 6651–54.

Awaisheh S S, Haddadin M S Y and Robinson R K (2005) Incorporationof selected nutraceuticals and probiotic bacteria into a fermentedmilk. International Dairy Journal 15 1184–1191.

Awaisheh S S, Al-Dmoor H, Omar S, Hawari A and Alroyli M (2012)Impact of selected nutraceuticals on viability of probiotic strains inmilk during refrigerated storage at 4°C for 15 days. InternationalJournal Dairy Technology 65 268–273.

Benkouider C (2004) Functional foods: a global overview. InternationalFood Ingredient 5 66–68.

Dumortier V, Brassart C and Bouquelet S (1994) Purification and proper-ties of a beta-D-galactosidase from Bifidobacterium bifidum exhibit-ing a transgalactosylation reaction. Biotechnology and AppliedBiochemistry. 19 341–354.

Food Chemicals Codex (2003) Committee on Food Chemicals Codex,Food and Nutrition Board, Institute of Medicine, National Academyof Sciences Pages 905–906, 5th edn. Washington, DC: NationalAcademy Press.

Fuller R (1989) Probiotics in man and animals. Journal of Bacteriology66 365–378.

Garro M S, Aguirre L and Savoy de Giori G S (2006) Biological activityof Bifidobacterium longum in response to environmental pH. AppliedMicrobiology and Biotechnology 5 612–617.

Hassan O A, Song D, Ibrahim S A, Isikhuemhen O S, Shabazi A and Abu-ghazaleha A A (2011) Shiitake mushroom (Lentinus edodes (Berk.)Singer) extract enhances the growth of lactic acid bacteria and bifido-bacteria in vitro.Milchwissenschaft 66 310–313.

Hsu C A, Yu R C and Chou C C (2005) Production of b-galactosidaseby bifidobacteria as influenced by various culture conditions. Interna-tional Journal of Food Microbiology 2 197–206.

Hughes D B and Hoover D G (1995) Viability and enzymatic activity ofbifidobacteria in milk. Journal of Dairy Science 2 268–276.

Ibrahim S and Carr J P (2006) Viability of bifidobacteria in commercialyogurt products in North Carolina during refrigerated storage. Inter-national Journal Diary Technology 4 272–277.

Ibrahim S A, Alazzeh A Y, Song D, Shahbazi A and AbuGhazaleh A A(2010) Enhancement of a- and b-galactosidase production in Lacto-bacillus reuteri by the addition of different metal ions. BiologicalTrace Elements Research 136 106–116.

Kawakami S, Minato K, Tokimoto K, Fujitake N and Mizuno M (2004)Changes of Lentinan contents and Glucanase activity in Lentinus ed-odes stored under controlled atmosphere. International JournalMedicinal Mushrooms 6 57–62.

Nagy Z, Kiss T, Szentirmai A and Bir�o S (2001) ß-Galactosidase of Pen-icillium chrysogenum: production, purification, and characterizationof the enzyme. Protein Experimental Purification 1 24–29.

Occhino L A, Morris H A and Savaiano D A (1986) A comparison ofbeta-galactosidase specific activities in strains of Streptococcus ther-mophilus. Journal Dairy Science 69 2583–2588.

Rafter J (2004) The effects of probiotics on colon cancer development.Nutritional Research Review 17 277–284.

Reid G, Sanders M E, Gaskins H R, Gibson G R, Mercenier A, RastallR, Roberfroid M, Rowland I, Cherbut C and Klaenhammer T R(2003) New scientific paradigms for probiotics and prebiotics. Jour-nal of Clinical Gastroenterology 37 105–118.

Saarela M, Rantala M, Hallamaa K, Nohynek L, Virkajarvi I and MattoJ (2002) Stationary phase acid and heat treatments for improvementof the viability of probiotic lactobacilli and bifidobacteria. Journal ofApplied Microbiology 96 1205–1214.

Salminen S and Gueimonde M (2004) Human studies on probiotics:what is scientifically proven. Journal of Food Science 69 137–140.

SAS Institute Inc (1999) SAS/STAT User’s Guide. Cary, NC: SAS Insti-tute.

Shah N P (2001) Functional foods from probiotics and prebiotics. FoodTechnology 55 46–53.

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Shah N, Lankaputhra W E V, Britz M and Kyle W S A (1995) Survivalof L. acidophilus and Bifidobacterium bifidum in commercial yoghurtduring refrigerated storage. International Dairy Journal 5 515.

Shin H S, Lee J H, Pestka J J and Ustunol Z (2000) Growth and viabil-ity of commercial Bifidobacterium spp in skim milk containing oligo-saccharides and inulin. Journal of Food Science 65 884–887.

Smith J S, Hillier A J and Lees G J (1975) The nature of the stimulationof the growth of Streptococcus lactis by yeast extract. Journal DairyResearch 42 123–138.

Yeo S K and Liong M T (2009) Effect of prebiotics on viability andgrowth characteristics of probiotics in soymilk. Journal of the Scienceof Food and Agriculture 90 267–275.

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