viability of probiotic bacteria in fermented skim milk produced with different levels of milk powder...

ORIGINALRESEARCH Viability of probiotic bacteria in fermented skim milk

produced with different levels of milk powder andsugar

LUCIANA C MAGANHA,1 ROICE E ROSIM,1 CARLOS H CORASSIN,1

ADRIANO G CRUZ,2 JOS �E A F FARIA2 and CARLOS A F OLIVEIRA1*1Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade deS~ao Paulo, Av. Duque de Caxias Norte, 225, CEP, 13635-900, Pirassununga, and 2Departamento de Tecnologia deAlimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, Brazil

In this work, the effect of skim milk powder (0, 5%, 10%, 15% w/v) and sugar (0%, 10% w/v) onthe viability of probiotic micro-organisms and on the quality of fermented milks was investigated.Fermented milks were submitted to physicochemical, microbiological and sensory analyses on the1st, 10th and 21st days after production. Sugar inclusion did not affect the probiotic growth in fer-mented milks, but milk powder levels of 10–15% influenced positively the probiotic counts, whichwere above six log colony-forming units (cfu)/g. These findings can be useful for small dairy indus-tries that are interested in producing flavoured fermented milks without decreasing the viability ofprobiotic micro-organisms during its shelf life.

Keywords Fermented milk, Total solids, Probiotics, Quality.

INTRODUCTION

Dairy products, especially fermented milks, arethe most popular vehicles for delivery of probi-otics into the body due their good compatibility,their pleasant and attractive sensory profiles aswell as their high consumption around the world(Granato et al. 2010; Mohammadi and Mortaza-vian 2011). The ingestion of fermented milkscontaining probiotic bacteria has been proven toprovide several benefits to the human health(Wagar et al. 2009; De Vrese et al. 2011; Ejta-hed et al. 2011).The manufacture of probiotic fermented milks

is not an easy task; along the processing andstorage, probiotic bacteria are subjected to stresssuch as oxygen, acids, cold and osmotic stresses(Granato et al. 2010; Ahmadi et al. 2012; Cruzet al. 2012). The latter can be caused by highconcentrations of sugar added to the milk beforethe fermentation process, which results in a longfermentation time and also leads to the develop-ment of products with a low content of organicacids, due to a lower water activity (Shah andRavula 2000).

During the fermentation process, the degrada-tion of milk proteins plays an important role fordevelopment of textural and flavour characteris-tics of fermented milks (Alo�glu and €Oner 2011;Rodrigues et al. 2012). Probiotic bacteria havebeen shown to contribute to some extent toproteolysis in yoghurt (Ramchandran and Shah2010), and in this context, it is important toevaluate all the nitrogen fractions which areinvolved in the protein degradation along theprocessing of probiotic yoghurt. In addition,skim milk powder is widely used to fortify thesolid level in the milk base before processingand fermentation. Fortification of the proteincontent in the milk base improves the textureand enhances quality parameters of the product,as the nutritional and functional properties(Lucey 2004). However, there is little informa-tion on the evaluation of these parameters onthe survival of probiotic strains during theproduct’s storage (Mortazavian et al. 2010;Shafiee et al. 2010; Marafon et al. 2011).Therefore, the aim of the present study was toevaluate the viability of probiotic bacteria in fer-mented skim milk manufactured with different

*Author forcorrespondence. E-mail:[email protected]

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Vol 67, No 1 February 2014 International Journal of Dairy Technology 89

doi: 10.1111/1471-0307.12087

levels of milk powder and sugar during refrigerated storage.In addition, the physicochemical characteristics and sensoryevaluation were also performed.

MATERIAL AND METHODS

Experimental design and probiotic culturesThe experiment was composed of eight different fermentedmilks produced with ultra-high temperature (UHT) bovineskim milk (0.1% fat). A completely randomised design wasused with four levels of milk powder (0%, 5%, 10% and15%, w/v) and two levels of sugar (0% and 10%, w/v).A mixed culture (freeze-dried) containing Streptococcus

thermophilus, Bifidobacterium animalis subsp. lactis andLactobacillus acidophilus – DVS ABT-4 – was obtainedfrom Chr-Hansen and was dissolved aseptically in 1 L ofsterilised skim milk at 5 °C. Then, this content was distrib-uted in sterilised bottles and kept in a freezer at �18 °C.The bottles were only defrosted when the bacterial inoculumneeded to be prepared to produce the fermented milksamples (Thamer and Penna 2005).

Fermented milk productionUHT skim milk (5 L per treatment) was heated to 35 °C,and skim milk powder (La Serenissima, Braganc�a Paulista,Brazil) or sugar was added to achieve the specific levels asdescribed before. These ingredients were kept under con-stant and vigorous agitation. The mixture was heated up to85 °C and then kept in thermostatically controlled waterbath for 20 min. Then, the mixture was cooled to 42 °Cand inoculated (3% inoculum, v/v) with the mixed culture,followed by mixing and incubation at 42 °C, for approxi-mately 3 h until a pH of 4.50–4.65 was reached (Spreer1991). Then, the products were cooled rapidly to 20 °C andbottled in 1-L glass containers and kept in the refrigerator(5 °C). The experiment was replicated three times, once permonth, representing 24 batches of probiotic fermented milk,three within each treatment.The processed fermented milks were stored in a refrigera-

tor (5 °C) for 21 days, collected and submitted to micro-biological and physicochemical analyses, and sensoryevaluation on the 1st, 10th and 21st days of storage.

Analysis of fermented milksAnalysis of dry matter content, fat content and pH valuewas performed according to the procedures as described byInstituto Adolfo Lutz (1985). The total protein content wasdetermined by the Kjeldahl method according to the meth-odology described by the Association of Official AnalyticalChemists – AOAC (1995). The determination of nonproteinnitrogen (NPN) was performed after previous precipitationof the fermented milk proteins using a 15% trichloroaceticacid solution, followed by filtration and analyses were madeaccording to the Kjeldhal method (AOAC 1995). The non-

casein nitrogen (NCN) content was quantified according tothe procedures described by Lynch and Barbano (1998).The final result (NCN) was subtracted from the total nitro-gen (TN) content. This equation provides the final caseincontent present in the sample. The TN, NPN, NCN andcasein concentration were also determined on days 1, 10and 21 after production.Viable lactic acid bacteria (LAB) counts in fermented

skim milks were obtained by serial dilutions. For this pur-pose, a total of 10 mL of the sample was transferred to asterile Erlenmeyer flask containing 90 mL of sterile distilledwater. Then, this solution was homogenised and subsequentdilutions were prepared until a dilution of 10�6 wasreached, and samples were pour-plated. The fermented milkswere evaluated in duplicate. The S. thermophilus count wasdetermined using M 17 agar, followed by incubation(anaerobiosis) at 37 °C for 48 h (IDF 1997). The B. animal-is subsp. lactis count was determined using MRS-IM agaradded with glucose and dichloxallin, lithium chloride andcysteine chloride solutions, followed by incubation (anaero-biosis) at 37 °C for 72 h (Anon 1999). The L. acidophiluscount was determined using MRS-IM with maltose, fol-lowed by incubation (anaerobiosis) at 37 °C for 72 h (IDF1999).For the sensory evaluation of the fermented milks, the cri-

teria were considered and adjusted according to Spreer(1991). Overall impression (grade 0.5) consistency (grade1.0), odour (grade 0.5) and flavour (grade 2.0) wereassessed by 30 untrained panellists using a 9-point hedonicscale. The final score for each sample corresponded to themean score attributed by the panellists for each sensorycharacteristic. The samples were tested in their natural form,with no addition of other ingredients.

Statistical analysisThe results were submitted to one-way ANOVA according toprocedures established in the general linear model (SASInstitute 2004). The bacteria count values were converted toa log scale. The statistical analyses of the evaluated parame-ters, that involved repeated measurements in time, were exe-cuted using the procedures proc glm and proc mixed of theprogram SAS (SAS Institute 2004) being the differencestested by the Tukey test. All statements of significance werebased on the 0.05 level of probability.

RESULTS AND DISCUSSION

Table 1 shows the mean values obtained in the physico-chemical analyses of the experimental probiotic fermentedmilks evaluated on day 1 after production. Values of pHranged between 4.49 and 4.59 and did not vary among thefermented milks (P > 0.05), hence indicating that theacidification process occurred regardless the addition ofmilk powder or sugar, as observed in previous studies

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(Mortazavian et al. 2007; Almeida et al. 2008; Shafieeet al. 2010; Heydari et al. 2011). As expected, the additionof milk powder and sugar increased the total solids contentin the experimental fermented milks, the values for whichvaried from 8.41% to 24.15%. Coherently, higher concentra-tions of total protein and casein were also observed in thetreatments supplemented with 10% or 15% (w/v) skim milkpowder.During 21 days of storage, the total protein, casein, NNP

and NCN contents were similar among treatments(P > 0.05). Thus, the addition of different levels of sugar ormilk powder had no effect in the physicochemical character-istics of probiotic fermented milks. Similar results were alsoobtained by Oliveira and Damin (2003) and Marafon et al.(2011).

Microbiological evaluationThe microbiological analyses were performed separately forthe quantification of each micro-organism. The counts ofS. thermophilus, L. acidophilus and B. animalis subsp. lac-tis in each fermented milk are shown in Table 2. Strepto-coccus thermophilus counts ranged from 6.16 to 8.28 logcolony-forming units (cfu)/mL during 21 days of storage.

Higher counts (P < 0.05) were found in fermented milkswithout supplementation, or with 10% (w/v) of skim milkpowder alone or in combination with 10% sugar (w/v) ondays 1 and 10 after production. On day 21, all fermentedmilks had similar (P > 0.05) counts for S. thermophilus,except for the product containing only 10% sugar (w/v).The positive effect of milk powder on the counts of S. ther-mophilus may be attributed to the compact matrix and thebuffering capacity provided by the addition of skim milkpowder in the fermented milk matrix (Shafiee et al. 2010).When used in combination with 5% or 10% milk powder(w/v), sugar addition did not influence (P > 0.05) the countsof S. thermophilus in fermented milks. On the other hand,fermented milks containing 15% milk powder + 10% sugaror 10% sugar alone had lower counts of S. thermophilus.These results can be related to the high concentrations ofnutrients in those fermented milks, taking into account thathigh contents of milk powder and sugar (sucrose) maydecrease the water activity and negatively affect the viabilityof S. thermophilus.Lactobacillus acidophilus counts ranged from 6.22 to

8.26 log cfu/mL (Table 2), with increased values found inall fermented milks on day 21 of storage. Lower counts

Table 1 Physicochemical characteristics of experimental probiotic fermented milksa on day 1 after production

Milk Powder (%, w/v) Sugar (%, wv) pH Total solids (%) Protein (%) NNC (%) NNP (%) Casein (%)

0 0 4.53 8.41 3.15 0.59 0.081 2.415 0 4.52 12.97 4.43 0.88 0.102 3.3410 0 4.49 15.97 6.05 1.23 0.154 4.5215 0 4.59 18.66 6.72 0.79 0.166 5.720 10 4.57 15.58 2.90 0.50 0.071 2.295 10 4.50 19.28 4.12 0.82 0.096 3.1710 10 4.56 21.27 5.24 0.51 0.128 4.5515 10 4.54 24.15 6.81 1.58 0.146 4.81aFermented milk produced with skimmed milk (fat content: max. 0.1%).

Table 2 Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis viable counts (log cfu/g)

Milk Powder (%, w/v) Sugar (%, w/v)

Streptococcus thermophilus Lactobacillus acidophilusBifidobacterium animalissubsp. lactis

Day 1 Day 10 Day 21 Day 1 Day 10 Day 21 Day 1 Day 10 Day 21

0 0 8.23a 8.28a 6.44a 6.46a 6.47a 8.26a 6.08c 6.08bc 6.07c

5 0 8.11b 8.10b 6.44a 6.24b 6.25b 8.11b 6.07c 6.16b 6.14bc

10 0 8.22a 8.20ab 6.44a 6.46a 6.55a 8.27a 6.31ab 6.33ab 6.37b

15 0 8.12b 8.17ab 6.49a 6.52a 6.56a 8.17ab 6.51a 6.48a 6.56a

0 10 7.92c 7.92c 6.16b 6.24b 6.22b 7.88c 6.04c 6.03c 6.05c

5 10 8.18ab 8.12ab 6.47a 6.46a 6.52a 8.16ab 6.06c 6.05c 6.12bc

10 10 8.18ab 8.19ab 6.42a 6.35a 6.46a 8.19ab 6.24b 6.23b 6.25b

15 10 8.04c 8.03c 6,54a 6.52a 6.52a 8.01bc 6.58a 6.55a 6.61a

Means with different superscripts letters in the same column indicate the presence of statistical difference (P < 0.05) among treatments.

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(P < 0.05) were observed only in fermented milks contain-ing 5% milk powder (w/v) or 10% sugar (w/v) alone ondays 1, 10 and 21. Thus, higher total solids content did notinfluence significantly the counts of L. acidophilus in theexperimental fermented milks during storage. Tramer (1973)reported that L. acidophilus could substitute L. delbrueckiisubsp. bulgaricus in yoghurt. However, L. acidophilus pre-sents a low growing rate in milk, even when milk is supple-mented with nutrients, and this fact could explain the initiallow counts observed in fermented milks in the presentstudy. According to Collins and Aramaki (1980), the growthof L. acidophilus can be influenced by the acidity increase,mainly by the production of lactic, acetic and benzoic acidsduring storage. Lactic acid bacteria present in mixed cul-tures can be damaged due to the lactic acid produced byother bacteria present in the food, as well as by the increas-ing acidification in the product during storage (Shah 2000).In the present study, only fermented milks containing 5%milk powder (w/v) or 10% sugar (w/v) alone had lowercounts of L. acidophilus in all evaluations, although no vari-ation in the pH values was found in the fermented milksduring 21 days of storage.Bifidobacterium animalis subsp. lactis counts ranged from

6.03 to 6.61 log cfu/mL during storage (Table 2), with noremarkable variation in the values from day 1 to 21 of stor-age. Higher counts of B. animalis subsp. lactis were foundin fermented milks containing higher levels of total solids(P < 0.05), corroborating the data obtained by Zacarchencoand Massaguer-Roig (2004). Additionally, there was noeffect (P > 0.05) of sugar addition alone or in combinationwith skim milk powder on the counts of B. animalis subsp.lactis. In general, bifidobacteria can be better cultivated inartificial media than in milk. However, these media areexpensive for the bifidobacteria multiplication, and theexcessive growth of such bacteria may provide unpleasantflavours in the final product. Therefore, the improvement ofthe conditions for the growth of bifidobacteria in fermented

milk can be obtained by the addition of a nitrogen source asprovided by the skim milk powder in the present study, orsubstances that reduce the redox potential of the food matrix(Gomes and Malcata 1999).All the fermented milks can be considered as a suitable

food matrix for probiotic bacteria supplementation, as valuesabove 6–7 log cfu/g of probiotic bacteria are reported asadequate for providing positive effects on the health of con-sumers (Shah 2000), being in accordance with different reg-ulations worldwide. In Brazil, the legislation requires aminimum viable cell count of 6 log cfu/g of product, andthis information must be declared on the label (Brasil 2008).European countries and Canada require that a serving-sizeprobiotic product should contain at least 8 and 9 log cfu/gof probiotic bacteria, respectively (Canadian Food Inspec-tion Agency 2009; European Food Safety Authority 2010).

Sensory evaluationThe sensory evaluation of fermented milk covered fourparameters: overall impression, consistency, odour and fla-vour, which were evaluated on days 1, 10 and 21 after pro-duction. Table 3 shows the average notes attributed to thefermented milks by the panellists on days 1, 10 and 21 aftermanufacture.On day 1, fermented milks containing 10% sugar (w/v)

had higher grades (P < 0.05), hence indicating the relevanceof this ingredient in the production of fermented milks. Thisresult is in accordance with previous studies (Cruz et al.2011, 2012), although no differences (P > 0.05) were foundin the grades attributed to all fermented milks on day 10after storage. Coherently, the treatments with sugar additionwere evaluated as ‘moderately good’, whereas the fermentedmilks without sugar were evaluated as ‘moderately bad’ or‘indifferent’. On the 21st day, fermented milks containing10% of milk powder + 10% of sugar presented the highersensory score (P < 0.05), when compared with the othertreatments. Panellists characterised this fermented milk as

Table 3 Sensory analysis of probiotic fermented milk

Milk Powder (%, w/v) Sugar (%, w/v)

Sensory analysis

Day 1 Day 10 Day 21

0 0 5.7Ba 6.0Aa 4.2Cb

5 0 5.7Ba 6.2Aa 5.8Ba

10 0 4.7Bb 5.6Aa 5.5Ba

15 0 5.1Bb 6.1Aa 5.2Bb

0 10 6.3Aa 6.2Aa 5.5Ba

5 10 6.7Aa 6.5Aa 5.6Ba

10 10 6.9Aa 7.5Aa 7.0Aa

15 10 6.5Aa 6.7Aa 5.8Ba

Means with different uppercase letters in the same column indicate the presence of statistical difference (P < 0.05) among treatments.

Means with different lowercase letters in the same row indicate the presence of statistical difference (P < 0.05) among storage days.



‘good’, while the other treatments were characterised as‘indifferent’ or ‘moderately bad’.According to Almeida et al. (2008), several species or

subspecies of lactic acid bacteria may provide different sen-sory and physicochemical characteristics to the products.The final metabolic compounds of some cultures, either pureor mixed, may provide unpleasant sensory characteristics.Moreover, bifidobacteria are able to produce acetic and lac-tic acids in a proportion of 3:2 during fermentation; there-fore, the excessive growth of these bacteria can enhance theacetic flavour to the final products, which undermines thesensory acceptance (Cruz et al. 2010).

CONCLUSION

Results of this trial suggest that the addition of sugar andskim milk powder improved the quality parameters of fer-mented milks containing probiotic bacteria. No significanteffect of such ingredients was found on the physicochemicalparameters of fermented milks. However, high levels ofskim milk powder contributed to the growth of L. acidophi-lus and B. animalis subsp. lactis. From the sensory point ofview, the addition of sugar seems to be an important factorthat influences the acceptance of fermented milks. Addi-tional descriptive sensory analysis and the functionality ofthe fermented milks assessed in this study need to be per-formed to characterise more accurately such products.

ACKNOWLEDGEMENTS

The authors thank the Fundac�~ao de Amparo �a Pesquisa doEstado de S~ao Paulo (FAPESP, grant no. 2007/06353-1) forthe MSc. fellowship provided.

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