Effect of Inulin and Lactulose on Survival of LactobacillusAcidophilus LA-5 and Bifidobacterium Bifidum BB-02 in

Acidophilus-Bifidus Yoghurt

D. Özer,1 S. Akin2 and B. Özer2,*

1Harran University Faculty of Agriculture, Department of Agricultural Economics, 63040, S�anlıurfa, Turkey2Harran University Faculty of Agriculture, Department of Food Engineering, 63040 S�annlıurfa, Turkey

The effect of supplementation of lactulose and inulin as prebiotics on the growth of L. acidophilus LA-5and B. bifidum BB-02 in Acidophilus-Bifidus (AB) yoghurt and some quality parameters of the resultingproducts were studied. Yoghurts produced from milks with added inulin at levels of 0.5% and 1.0%, orwith lactulose at levels of 0.25% and 2.5% were compared with classical yoghurt and AB yoghurt(without added prebiotics). The results showed that inulin and lactulose did not affect the growth ofyoghurt starter bacteria, but stimulated the growth of B. bifidum BB-02 to a great extent. Lactulose wasfound to be more effective on the growth of both probiotic strains than inulin. Inulin did not stimulatethe growth of L. acidophilus LA-5. The cell counts of B. bifidum BB-02 and L. acidophilus LA-5 weredependent upon concentrations of lactulose and inulin used. No significant (p�0.05) difference betweenthe samples were observed with regard to the pH and lactic acid values. However, lactulose or inulinadded samples had lower acetaldehyde and tyrosine contents than the untreated samples.

Key Words: yoghurt, probiotics, prebiotics, Lactobacillus acidophilus, Bifidobacterium bifidum

INTRODUCTION

A probiotic is defined as a “living organism whichupon ingestion in certain numbers, exert health bene-fits beyond inherent basic nutrition” (Guarner andSchaafsma, 1998). Owing to this property, incorpora-tion of the probiotic microorganisms in dairy foods hasincreased rapidly during the last two decades. Con-sumption of probiotic bacteria via food products is anideal way to re-establish the balance of intestinalmicrobiota (Hattingh and Viljoen, 2001). Yoghurt isthe most popular dairy product due to its nutritionaland physiological properties. In recent years, the popu-larity of yoghurt has increased, accentuating the rele-vance of incorporating probiotic bacteria (e.g.,Lactobacillus spp. and Bifidobacterium spp.) to addextra nutritional and physiological values.

Bio-yoghurt, containing Lactobacillus acidophilusand Bifidobacterium bifidum, is a potential vehicle by

*To whom correspondence should be sent(e-mail: bozer@e-kolay.net).Received 19 February 2004; revised 11 May 2004.

Food Sci Tech Int 2005; 11(1):019–6© 2005 Sage PublicationsISSN: 1082-0132DOI: 10.1177/1082013205051275

which consumers can take probiotic cells (Hattinghand Viljoen, 2001). Although there is no evidenceregarding the minimum level of probiotic bacteria infermented milk to produce therapeutic benefits, it hasbeen suggested that a product should contain morethan 106 cfu/g to exert a probiotic effect (Hoier, 1992;Rybka and Kalisapathy, 1995). Several factors havebeen reported to affect the viability of probiotic cul-tures in fermented milks. Acidity, pH, dissolvedoxygen content, redox potential and hydrogen perox-ide have been identified to have an effect during man-ufacture and storage of yoghurt (Lankaputhra andShah, 1996). Dissolved oxygen and acidity weredemonstrated to be the key factors for bifidobacteria(Dave and Shah, 1997). During cold storage of fer-mented bio-products, the number of viable probioticcells often drops far below the minimum therapeuticlevel (Rybka, 1994). Therefore, efforts have beenintensified recently to find efficient means to stimulatethe growth and survival of probiotic microorganismsin fermented bio-products. In this regard, reducing theconcentration of dissolved oxygen was reported to bean effective way to keep high numbers of viable probi-otic cells in yoghurt (Dave and Shah, 1997). It was alsoreported that using the ruptured (Shah and Lanka-puthra, 1997) or microencapsulated (Adhikari et al.,2000) cells of probiotic strains in the manufacture offermented milk products was successful in keeping the

19

number of viable cells high enough for a suggestedprobiotic effect.

One of the approaches utilised to achieve suffi-ciently high numbers of desirable microorganisms inthe ecosystem of the human gastrointestinal tract is viathe use of “prebiotics” (Gopal et al., 2001). Prebioticsare specific dietary components that are used to modifyselectively the composition of resident microbiota ofthe colon. Prebiotic oligosaccharides stimulate thegrowth and colonisation of probiotic bacteria havingbeneficial health effects when ingested (Rastall andMaitin, 2002). Today, there are many prebioticoligosaccharides in the markets including fructo-oligosaccharides, inulin, galacto-oligosaccharides, lac-tulose and isomalto-oligosaccharides (Playne andCrittenden, 1996). Inulin, fructo-oligosaccharides andlactulose are probably the most commonly used prebi-otics (Holzapfel and Schillinger, 2002). Inulin wasfound to support larger total bacterial counts but lowerpopulations of bifidobacteria and lactobacilli thansoybean oligosaccharides, isomalto-oligosaccharides orlactulose (Rycroft et al., 2001). The largest increase inbifidobacteria was seen on xylo-oligosaccharides andlactulose, the largest in lactobacilli was on fructo-oligosaccharides (Rastall and Maitin, 2002).

Most of the studies carried out on the prebiotics areon the basis of clinical experiments and the number ofstudies on the survival of probiotic strains in fermenteddairy products containing prebiotics and on thecharacteristics of such products are rather limited. Inthe present study, therefore, the effects of inulin andlactulose on the survival of L. acidophilus and B.bifidum in AB yoghurt and some quality properties ofthe resulting products were studied.

MATERIALS AND METHODS

Cultures and Media

The yoghurt starter culture was obtained from Chr.Hansen (Peyma Chr. Hansen, Istanbul, Turkey) andconsisted in a blend of Streptococcus thermophilus andLactobacillus delbrueckii subsp. bulgaricus (code CH-1). Probiotic cultures used in the manufacture ofyoghurt were blends (1:1) of B. bifidum BB-02 and L.acidophilus LA-5 (Peyma Chr. Hansen, Istanbul,Turkey). The working cultures were prepared byadding a few milligrams of freeze-dried culture to100mL of previously reconstituted and sterilised(121°C, 2min) skimmed milk with total solids of100g/kg. This mixture was then incubated at 37°C untilthe onset of gelation. The probiotic cultures were pre-pared separately and then inoculated into milk prior toyoghurt-making.

20 D. ÖZER ET AL.

YOGHURT TRIALS

Yoghurt samples were prepared using bovine milksupplied from Harran University Dairy (Harran Uni-versity Faculty of Agriculture, Eyyubiye Campus,63040, S�anlıurfa, Turkey) according to the proceduredescribed by Tamime and Robinson (1999). The totalsolids content of the milk was adjusted to 16% (w/v) byskim milk powder addition. After heat treatment at85°C for 20min, the milk was cooled to 43°C by circu-lating chilled water, then divided into six equal por-tions and treated as follows:

a) Trial A: Inoculation with yoghurt starter cultureonly (2% v/v): classical yoghurt.

b) Trial B: Inoculation with yoghurt starter culture(2%, v/v) plus probiotic cultures (5% v/v): AByoghurt

c) Inoculation with yoghurt starter culture (2%, v/v)plus probiotic cultures (5%v/v) and addition ofinulin at a level of 0.5% (w/v): Trial C, or 1.0%(v/v): trial D, AB yoghurt.

d) Inoculation with yoghurt starter culture (2% v/v)plus probiotic cultures (5% v/v) and addition of lac-tulose at a level of 0.25% (v/v): trial E, or 2.5%(v/v): trial F, AB yoghurt.

The levels of probiotic cultures inoculated the milkbefore the yoghurt making was calculated by using thestandard McFarland method (Gurgun and Halkman,1990). A 5% (v/v) inoculum corresponded to anaverage of 2.1�109 cfu/g for each probiotic bacteriainitially. Samples (pH 6.6) were incubated at 43°Cuntil pH 4.6 was reached. After the incubation, thesamples were cooled down to room temperature andstored at �6°C for 14 days. Samples were analysed at0, 7 and 14 days of storage.

The pH was measured with a combined electrodepH-meter (Orion, 420 A, Orion Research Inc., Boston,USA). The tyrosine (Tunail, 1978), lactic acid (Özerand Atamer, 1999) and acetaldehyde (Lees and Jago,1969) contents were determined spectrophotometri-cally (UV-VIS spectrophotemeter, model 1650, Shi-madzu Deutschland GmbH, Duisburg, Germany).

Microbiological groups were determined by thepour plate method in duplicate. In the count of L. bul-garicus and Str. thermophilus, MRS agar (de Man,Rogosa and Sharp agar, Mann et al., 1960) and M17agar (Terzaghi and Sandine, 1975) were used, respec-tively. Aerobic incubation for Str. thermophilus andanaerobic incubation for L. bulgaricus were achievedat 37°C for 72h.

For counting L. acidophilus LA-5, 10mL of 10%(w/v) D-Sorbitol was added into MRS agar as sug-gested by Dave and Shah (1997). For counting B.bifidum BB-02 20ml of NNLP mixture (neomycin sul-

phate (100mg/L), nalidixic acid (50mg/L), lithiumchloride (3,000mg/L) and paramomycin sulphate(200mg/L)) were added into MRS agar (Dave andShah, 1997). Incubations were carried out anaerobi-cally at 37°C for 72 hours. Anaerobic conditions wereprovided using Gas Generating Kits (Unipath Ltd,Basingstoke, Hants., UK, Anaerobic System BR038B).

The study was repeated three times (n �3). Analysisof variance was performed on data obtained andsignificant differences between groups were deter-mined by Duncan’s Multiple Range test (Steel andTorrie, 1980).

RESULTS AND DISCUSSION

Incubation Period

Incubation of the experimental yoghurts was ceasedwhen the pH of the samples reached ca. 4.6. Theresults obtained showed that the incubation periods ofyoghurts differed significantly (p � 0.05, data notshown). While the trial A (classical yoghurt containingno probiotic strains) reached the target pH within3.5 h, the incubation of the AB yoghurt containing nogrowth-promoters (inulin or lactulose, trial B) took 8hours. The addition of inulin and lactulose at varyingconcentrations reduced the incubation period of thetrials C to F (between 7.2 h and 7.3 h). The incubationperiod of the trials C to F was found to be independ-ent from the type or concentration of the prebioticsused.

pH

At the beginning of storage, the pH values of theprobiotic yoghurts (trials B to F, pH 4.64 to 4.67) weresignificantly different (p�0.05, data not shown) fromthe sample A (pH 4.42). However, no significant differ-ences (p�0.05) among the probiotic samples withregard to the pH values were observed. During thestorage period of 14 days, the pH continued to declinein a similar way for all the samples. After 14 days ofstorage, the pH dropped to 4.03 for the sample A, andto 4.27–4.42 for the samples B to F.

Lactic Acid Contents

Changes in lactic acid contents of the experimentalyoghurts did not show, in overall, marginal differencesamong samples (Figure 1). At 0 days, the lactic acidcontents of the samples varied between 0.99 and1.18g/100g. These figures increased to 1.33–1.58g/100gafter 14 days storage. The effect of lactulose and/orinulin on the lactic acid values were found to beinsignificant (p�0.05)

Effect of Inulin and Lactulose on Survival of Microorganisms in Yoghurt 21

Figure 1. Changes in the lactic acid contents of theexperimental yoghurts (p�0.05). A: Classicalyoghurt. B: AB yoghurt with no prebiotics. C: AByoghurt with added inulin (0.5%). D: AB yoghurt withadded inulin (1.0%). E: AB yoghurt with added lactu-lose (0.25%). F: AB yoghurt with added lactulose(2.5%). (�) 0-day, (�) 7-day, (�) 14-day.

Tyrosine Contents

The addition of lactulose and inulin reduced theproteolysis development in yoghurt samples. Through-out storage, samples of trials A and B without addedoligosaccharides had higher tyrosine contents than therest of the samples (trials C to F, Figure 2). Supple-mentation of lactulose or inulin did not affect the tyro-sine contents of the samples significantly (p�0.05).However, it was noted that the inulin-added yoghurtsamples had higher tyrosine contents than those con-taining lactulose. The probiotic strains used in themanufacture of experimental yoghurt are known to beweakly proteolytic and they require some amino acidsfor their growth (Tamime et al., 1995). Rastall andMaitin (2002) reported that inulin supports highernumbers of total bacteria but lower numbers of bifi-dobacteria and lactobacilli than lactulose.

Acetaldehyde Contents

Among the samples analysed, those of the trial Ahad significantly higher (p�0.05) acetaldehyde contentthan the other samples (Figure 3). At 0 and 14 days,sample A had 28.0 and 17.0mg/kg acetaldehyde,respectively. The samples supplemented with lactuloseand inulin (samples C to F) had close acetaldehydelevels to each other but lower values than the sampleB. The acetaldehyde contents at 0 days were 18.0, 14.0,15.3, 13.6 and 14.0mg/kg for the samples B to F,respectively. In general, carbonyl compound produc-tion capacity of bifidobacteria and lactobacilli arelimited compared to the classical yoghurt starter bacte-ria (Tamime et al., 1995). It was reported that L. aci-dophilus had lower acetaldehyde synthesising capacitythan B. bifidum (Tamime et al., 1995). Throughoutstorage, in all samples, the levels of acetaldehyde

declined significantly (p�0.05). The time-dependentdecrease in acetaldehyde level may be linked to themetabolic activity of yoghurt starter bacteria. Bothyoghurt starter bacteria (L. bulgaricus and Str. ther-mophilus) are able to synthesise alcohol dehydroge-nase, an enzyme which converts acetaldehyde toethanol (Tamime and Robinson, 1999).

Changes in the Counts of Yoghurt Starter Bacteria

The experimental yoghurts made with the commer-cial yoghurt starter (classical yoghurt) contained lacticacid bacteria in numbers exceeding 109 cfu/g. At day 0,the cfus of Str. thermophilus ranged between7.6–9.1�109 cfu/g. The counts of Str. thermophilusincreased in all samples at day 7 which coincided withthe pH decrease at day 7 of storage. At the later periodof storage, slight decreases were noted in the counts ofStr. thermophilus, which indicated better stability ofthis organism under the conditions prevailing in thecorresponding samples (Figure 4). These findings wereconsistent with those reported by Dave and Shah(1997).

The counts of L. bulgaricus decreased in all samplesby some 2.5 to 4.2-fold during 14 days of storage

22 D. ÖZER ET AL.

Figure 2. Changes in the tyrosine contents of theexperimental yoghurts (p�0.05). (�) 0-day, (�) 7-day, (�) 14-day (see Figure 1 for samples notation).

Figure 3. Changes in the acetaldehyde contents ofthe experimental yoghurts (p�0.05). (�) 0-day, (�)7-day, (�) 14-day (see Figure 1 for samples nota-tion).

Figure 4. Changes in the counts of Str. thermophiluscells in the experimental yoghurts (p�0.05). (�) 0-day, (�) 7-day, (�) 14-day (see Figure 1 for samplesnotation).

Figure 5. Changes in the counts of L. delbrueckiisubsp. bulgaricus cells in the experimental yoghurts(p�0.05). (�) 0-day, (�) 7-day, (�) 14-day (seeFigure 1 for samples notation).

(Figure 5). It has been demonstrated that lower countsof L. bulgaricus was advantageous for the viability ofprobiotic organisms as it lowers the pH during storageto a level that may affect negatively the viability of pro-biotic organisms (Holcomb and Frank, 1991).However, it appeared that the viability of L. bulgaricuswas not affected by lactulose and inulin (p�0.05) inthis study. The decrease in L. bulgaricus cfus was moreobvious than that of Str. thermophilus.

Changes in the Counts of L. acidophilus LA-5 and B.bifidum BB-02

The counts of L. acidophilus LA-5 ranged between6.3x108–1.1�1010 cfu/g at day 0 depending on the typeof the prebiotic supplement used (Figure 6). Overall,inulin was found to be less effective on the viability ofL. acidophilus LA-5. However, the counts of L. aci-dophilus LA-5 in the samples of trial B (without addedprebiotics) reached 6.3 �108 and 2.2�108 cfu/g at days0 and 14, respectively. In the samples of trial C (con-taining 0.5% inulin) and D (containing 1.0% inulin)the counts of this bacterium were 7.0 �108–3.5�108

and 8.8�108–4.2�108 cfu/g. Conversely, in yoghurtsamples supplemented with lactulose, the counts of L.acidophilus LA-5 were significantly (p�0.05) higherthan the ones containing inulin. Also, the concentra-tion of lactulose was found to be a parameter thataffects significantly (p�0.05) the growth of this micro-organism. At day 0, the counts of L. acidophilus LA-5in samples of the trail E (with added 0.25% lactulose)and F (with added 2.5% lactulose) were 7.0 �109 and1.1�1010 cfu/g, respectively. These figures, however,decreased to 3.2 �108 and 6.9�108 cfu/g after 14 daysstorage, respectively. Although continuous decreaseswere noted in the counts of L. acidophilus LA-5, therecommended level of 107 cfu/g (Hoier, 1992) wasmaintained throughout the whole period of storage.

As seen in Figure 7, at the first day, both inulin andlactulose additions caused a significant increase(p�0.05) in the cell counts of B. bifidum BB-02. B.bifidum BB-02 showed ca. 4.6- to 7.5-fold increase inthe numbers for the inulin-added samples at levels of0.5% (trial C) and 1.0% (trial D), respectively. Thiswas even more pronounced for the samples supple-mented with lactulose at levels of 0.25% (trial E) and2.5% (trial F) 10- to 14-fold, respectively. Lactulosewas shown to be more effective in stimulating thegrowth of B. bifidum BB-02 than inulin. Throughoutstorage, the number of viable B. bifidum BB-02 cells inthe samples examined declined. The cumulative effectof storage and concentrations of prebiotics were foundto be significant (p�0.05). Similar results werereported by Lankaputhra and Shah (1996). It wasthought that the decrease in the counts of B. bifidumBB-02 during storage stemmed from the antagonisticeffect(s) of Str. thermophilus on this organism as statedby Dave and Shah (1997). On the other hand, B.bifidum was reported to grow better in the presence ofL. bulgaricus due to their symbiotic relation (Shankarand Davies, 1976). The free amino acids resulting fromthe proteolytic activity of L. bulgaricus may promotethe growth of bifidobacteria (Singh et al., 1980). Vin-

Effect of Inulin and Lactulose on Survival of Microorganisms in Yoghurt 23

Figure 6. Changes in the counts of L. acidophilusLA-5 cells in the experimental yoghurts (p�0.05). (�)0-day, (�) 7-day, (�) 14-day (see Figure 1 forsamples notation).

Figure 7. Changes in the counts of B. bifidum BB-02cells in the experimental yoghurts (p�0.05). (�) 0-day, (�) 7-day, (�) 14-day (see Figure 1 for samplesnotation).

derola et al. (2000) reported that pH values of 4.5 orlower affect negatively the cell viability of the probioticorganism in yoghurt at 5 °C.

According to Saarela et al. (2003), lactulose was thefavoured lactose derivative when Lactobacillus strainswere grown anaerobically at substrate concentrations1% or 2%. However, according to the same authors,lactulose did not promote the viability of Lactobacillusspp. during storage at 4 °C. Our results corroboratedsuch findings. On the contrary, Rastall and Maitin(2002) reported that the most significant increase inbifidobacteria numbers was obtained with lactuloseand oxylo-oligosaccharides while fructo-oligosaccha-rides stimulated the growth of lactobacilli. The sameauthors showed that inulin was less stimulatory on thegrowth of bifidobacteria.

Supplementation of milk with inulin and especiallywith lactulose as growth-promoters for B. bifidum BB-02 and L. acidophilus LA-5 could be a satisfactory wayof keeping the number of viable probiotic cells in AByoghurt above the suggested therapeutic minimum(�107 cfu/g) during cold storage. Results obtainedshowed that regarding the metabolic activities of theprobiotic bacteria, i.e. acetaldehyde, lactic acid andtyrosine production, addition of inulin and/or lactulosecould be an appropriate alternative to the microencap-sulation and rupture cell techniques.

ACKNOWLEDGEMENTS

The authors wish to thank the Harran UniversityResearch Fund (Turkey) for financial support.

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