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LWT - Food Science and Technology 42 (2009) 672–678

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LWT - Food Science and Technology

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Research Note

Influence of total solids contents of milk whey on the acidifying profile andviability of various lactic acid bacteria

K.E. Almeida a, A.Y. Tamime b, M.N. Oliveira a,*

a Sao Paulo University, Department of Biochemical and Pharmaceutical Technology, Ave. Prof. Lineu Prestes, 580, 05508-900, Sao Paulo, Brazilb 24 Queens Terrace, Ayr KA7 1DX, Scotland, United Kingdom

a r t i c l e i n f o

Article history:Received 9 April 2007Received in revised form 7 February 2008Accepted 28 March 2008

Keywords:MilkWheyProbioticAcidification profile

* Corresponding author. Tel.: þ55 11 3091 3690.E-mail address: monolive@usp.br (M.N. Oliveira).

0023-6438/$34.00 � 2008 Swiss Society of Food Sciedoi:10.1016/j.lwt.2008.03.013

a b s t r a c t

The main objectives of the present study were (a) to study the effects of the different combinations ofLactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr),and Bifidobacterium animalis subsp. lactis (Bl) in co-culture with Streptococcus thermophilus (St) on therate of acid development in milk and milk-whey mixture, and (b) the effect of the level of the total solidsof the different bases on the acidification profile and viability of potential health-promoting micro-organisms. The co-culture of St-Lr showed the lowest values Vmax in all bases; while the co-culture St-Blhad high tVmax in milk and whey bases (12 and 10 g/100 g, respectively). Co-cultures St-La and St-Lbreached Vmax at pH 5.5, while St-Lr and St-Bl at pH 5.91. Fermentation time to reach pH 4.5 was longerwhen St-Lr co-culture was used, while St-Lb had the lowest value. All the products had slight de-velopment of acid during the storage period, and lowest values were observed when the St-Bl co-culturewas employed. Lb, Bl and St cultures had high counts at pH 4.5 in the three bases. The total solids affectedthe viability of Lb and La. The technological interest of these combinations is discussed in this article.

� 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Fermented lactic beverages are dairy products manufacturedusing milk and/or dairy derivatives with or without the addition ofother ingredients in which the lactic base represents almost 51%(w/w) of the total ingredients used (Brasil, 2005). Nevertheless, thestarter or probiotic bacteria should be abundant and alive in thefinal product (Mercosul, 1998). These products are characterized ofbeing refreshing having a smooth texture and low viscosity. Theconsumption of lactic beverages has increased in many countries(Nielsen, 2002; Tamime, 1997).

Whey-based lactic beverages represent an emerging segment ofnon-conventional dairy products that require sensory, physical, andchemical characterisation for quality control and product de-velopment (Gallardo-Escamilla, Nelly, & Delahunt, 2007). The use ofliquid or dried whey in lactic beverages is very common and, asa consequence, this method of processing may assist in the uti-lisation of whey, i.e. solution to dispose of high volume of wheygenerated by the cheese industry. In addition, the inclusion ofa high nutritive raw material in the beverage base rather than milkcan ultimately reduce the production cost. According to Drgalic,Tratnik, and Bo�zanic (2005), attention should be drawn to the

nce and Technology. Published by

natural whey, in a liquid form. Several lactic acid bacteria (LAB) andyeast could be employed in lactic beverages production. Hence,lactic beverages could be considered as an important vehicle for thedelivery of probiotic micro-organisms to the human gastro in-testine (Gardiner, Ross, Kelly, & Staton, 2002; Oliveira, Sodini,Remeuf & Corrieu, 2001).

In this way, several products are being developed using mix-tures of whey, milk, fruit juice, probiotic bacteria and/or yeast(Athanasiadis, Paraskevopoulou, Blekas, & Kiosseoglou, 2004;Djuric, Caric, Milanovic, Tekic, & Panic, 2004; Gallardo-Escamillaet al., 2007; Miglioranza et al., 2003; Suomalainen et al., 2006).However, little attention has been done to the kinetics parametersof milk-whey bases designated to whey-based fermented lacticbeverages. Quantifying the acidifying activity of LAB allows thecomparison of different combination of starter cultures in severalsubstrates in order to define the better process. The acidifying ac-tivity of LAB during the fermentation period could be describedthroughout pH curves; measuring pHs decrease in regular mini-mum time intervals, and maximum acidification rate (Vmax) couldbe calculated as dpH/dt. In addition, the time necessary to attainVmax and its corresponding pH could be obtained. According toPicque, Perret, Latrille, and Corrieu (1992), these are the parametersthat better describe the acidification kinetics. Studies of the acidi-fication kinetics of Streptococcus thermophilus and Lactobacillusdelbrueckii subsp. bulgaricus in milk and some probiotic microflorahad been reported by Beal, Louvet, and Corrieu (1989); Cachon,

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K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672–678 673

Jeanson, Aldarf, and Divies (2002); Chammas, Saliba, Corrieu, andBeal (2006); Kristo, Biliaderis, and Tzanetakis (2003); Lucas, Sodini,Monet, Jolivet, and Corrieu (2004); Oliveira and Damin (2003) andOliveira et al. (2001). However, to our knowledge, there are no datain literature concerning the acidification profile of probiotics inmilk-whey bases or in whey-based lactic beverages.

In a previous study (Almeida, Tamime, & Oliveira, 2008), it wasdemonstrated that the technological interest of using Minasfrescal cheese whey for the production of a probiotic lacticbeverage was feasible. In the present study, the influence of totalsolids contents of milk-whey bases on the acidifying profile ofL. delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La),Lactobacillus rhamnosus (Lr), and Bifidobacterium animalis subsp.lactis (Bl) was studied. These strains were used in co-culture withS. thermophilus (St) in milk-whey bases (10 and 8 g/100 g of totalsolids) at different pH levels at which the fermentation wasstopped. Milk (12 g/100 g of total solids) was used as control.Finally, the effect of total solids contents of the milk-whey baseson the viability of the probiotic was also examined.

2. Materials and methods

2.1. Materials

Commercial pasteurized whole milk (Paulista, Sao Paulo) wasused for the manufacture of Minas Frescal cheese according to themethod reported by Furtado and Neto (1994). The gross chemicalcomposition of whey was total solids (6.2� 0.026 g/100 g); protein(0.8� 0.015 g/100 g); fat (0.5� 0.115 g/100 g) and lactose (4.7�0.104 g/100 g). The whey was then pasteurized at 72 �C for 15 s ina plate heat exchanger (Laboratory Pasteurizer Armfield model FT-43, Ringewood, England) in the Laboratory of Food Engineering(Department of Chemical Engineering, Polytechnic School of SaoPaulo University), cooled to 4 �C in an ice bath, dispensed intoplastic bottles and frozen until required.

Freeze-dried starter cultures (Danisco, Sassenage, France) wereused for direct-to-vat inoculation (DVI), and starter organisms wereS. thermophilus TA040, L. delbrueckii subsp. bulgaricus LB340,L. acidophilus LAC4, L. rhamnosus LBA and B. animalis subsp. lactis BLO4. A pre-culture was prepared by weighting each culture in suf-ficient amounts to attain initial counts of 108 colony forming units(cfu) mL�1, and diluting in 50 mL skimmed milk, which had beensterilized at 121 �C for 10 min, and cooled to 5 �C. The pre-cultureswere warmed at 42 �C for 20 min before inoculation.

2.2. Experimental procedure

Thirty six trials were performed in which the studied variableswere (a) the different co-culture combinations (St-Lb, St-La, St-Lrand St-Bl), (b) the total solids of the milk (12 g/100 g) and milk-whey mixture (10 and 8 g/100 g), and (c) the pH values when thefermentation of the milk-whey bases was stopped (4.5, 5.0 and 5.5).

Based in the analyzed total solids contents of pasteurized milkand whey, milk-whey bases were prepared by blending parts ofmilk and pasteurized whey to achieve the desired levels of totalsolids content of the beverage bases. The Pearson’s Squaremethod was used to calculate the required components (Tamime& Robinson, 2007).

Flasks containing 250 mL of milk or milk-whey bases wereplaced in water bath at 42 �C. The warmed flasks were inoculatedat the fermentation temperature with 8.38 log10 cfu mL�1 of pre-culture of S. thermophilus, 8.11 log10 cfu mL�1 of L. bulgaricus,8.95 log10 cfu mL�1 of L. acidophilus, 8.15 log10 cfu mL�1 ofL. rhamnosus, and 8.81 log10 cfu mL�1 of B. animalis subsp. lactis ineach beverage base as described in Section 2.1. All the inoculatedbases were incubated at 42 �C in a water bath until the desired

pHs were reached, and each experiment was performed in tworeplicates. The fermentation profile was monitored by using theCinac system (Ysebaert, Frepillon, France) (Spinnler & Corrieu,1989), which allows continuous measurement and recording ofthe pH, and computes the acidification rate during the incubationperiod. Six kinetic parameters were considered: (a) Vmax

(maximum acidification rate, measured in pH units per min(upH.min�1), (b) tVmax (time to reach the maximum acidificationrate), (c) pH corresponding to Vmax and (d) tpH 4.5, tpH 5.0, andtpH 5.5 (time in hours to reach pH 4.5, 5.0, and 5.5, respectively).When the fermentation was stopped at the desired pHs, theproducts were transferred immediately to an ice bath. The fer-mented beverages were stored at 4 �C.

2.3. Chemical composition of milk-whey bases

Total solids content was determined by drying the samples at105 �C up to a constant weight (AOAC, 1995), protein content wasdetermined by micro Kjeldahl (AOAC, 1995), fat by Gerber method(Schmidt-Hebbel, 1956), and lactose by Eynon-Lane method (AOAC,1984). A digital potentiometer (Quimis, Diadema, Sao Paulo) wasused to measure the pH values. The post-acidification of the milk ormilk-whey bases was calculated by the difference of the pH value ofthe product after 24 h of storage and the pH of the productimmediately after fermentation. All the chemical analyses wereperformed in triplicate.

2.4. Microbiological analysis

Enumeration of the starter cultures was carried out after thefermented milk-whey bases were stored at 4 �C for 24 h, andeach sample was prepared according to the methods described bythe International Dairy Federation (IDF, 1996, 1997, 2003). Countsof S. thermophilus were enumerated on M17 agar (Merck,Darmstadt, Germany) by aerobic incubation at 37 �C for 48 h.Enumerations of lactobacilli species and bifidobacteria werecarried out on MRS agar (Merck, Darmstadt, Germany) adjustedto pH 5.4 with glacial acetic acid and by aerobic incubation at37 �C for 48 h for L. delbrueckii subsp. bulgaricus, and by anaerobicincubation at 37 �C for 72 h for L. acidophilus, L. rhamnosus andfor the bifidobacteria. The selectivity of the growth conditionswas confirmed by microscope appearance of the cells from singlecolonies.

2.5. Statistical analysis

Analysis of variance for multiple comparisons (ANOVA) usingStatistica 6.0, Statsoft (Tulsa, USA), was performed in order toconfirm statistical significance of differences among samples (P <

0.05). Mean values were compared using the Tukey test atP< 0.05.

3. Results and discussion

3.1. Chemical composition of milk-whey bases

The chemical composition (g/100 g) of the milk-whey basesis shown in Table 1, and the data fell within the following ranges:total solids (TS) 7.8–12 g/100 g, protein 1.4–2.9 g/100 g, and fat 1.2–3.6 g/100 g. These values were statistically different in all the bases,which was influenced by the amount of added whey. The milk-whey bases had similar amounts of lactose (w4.6 g/100 g), andSpadoti (1998) reported similar level of lactose in raw milk.However, the chemical composition of milk-whey bases was similarto those reported by Almeida, Bonassi, and Roça (2001).

Table 1Composition (g 100 g�1) of milk-whey basesa

Milk and milk-whey basesb

Total solids Protein Fat Lactose

12 12.0a (�0.347) 2.9a (�0.017) 3.6a (�0.153) 4.5a (�0.061)10 9.8b (�0.100) 2.1b (�0.022) 2.4b (�0.058) 4.5a (�0.012)8 7.8c (�0.038) 1.4c (�0.026) 1.2c (�0.058) 4.6a (�0.075)

Values in the same column with different letters are significantly different (Tukeytest at P< 0.05).

a Values are given as means with standard deviation in parenthesis.b 12, 10 and 8 are the predicted total solids content in the milk-whey bases.

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672–678674

3.2. Acidification profile

Vmax values ranged between 12.30 � 10�3 (St-Lr at pH 5.5) and16.35 � 10�3 upH.min�1 (St-La at pH 4.5) when milk (12 g/100 g)was used (Table 2). Co-culture St-La attained highest values, whichaveraged 16.08 � 10�3 upH.min�1. However, when St-Lr wasemployed, the lowest Vmax values were observed. These results canbe compared with those reported by Oliveira and Damin (2003)who investigated the kinetic parameters of the same strainsreported in present paper, L. acidophilus, L. delbrueckii subsp.bulgaricus and L. rhamnosus, in co-culture with S. thermophilus inwhole milk, and in milk supplemented with sucrose fermenteduntil pH 4.5. The same authors also reported the following Vmax

values 14.5 � 10�3, 7.7 � 10�3 and 12.8 � 10�3 upH.min�1 for co-cultures St-Lb, St-La and St-Lr, respectively. It is of interest to notethat to our knowledge there are no data available on the acidifyingprofile of St-Bl in milk or milk-whey bases.

In the bases containing 10 g/100 g, the co-culture St-Lr showed thelowest Vmax values irrespective of pH when the fermentation wasstopped (13.00�10�3, 12.65�10�3, and 12.60�10�3 upH.min�1)(Table 2). St-Lb and St-La had values that ranged between 14.60�10�3

and 15.75�10�3 upH.min�1 with no significant differences.According to the Vmax values in milk-whey base (8 g/100 g), the

co-cultures could be classified into three very distinct groups(Table 2). First, the least Vmax value was for St-Lr irrespective at whatpH the fermentation was stopped (12.40�10�3 upH.min�1 in aver-age), second, intermediate values for St-La (15.63�10�3 upH.min�1

in average), and third, high Vmax values for co-cultures St-Lb and St-Blat pH 4.5 (18.55�10�3 and 19.00�10�3 upH.min�1, respectively).

Co-cultures St-Lb and St-Bl showed an increase in Vmax as thetotal solids content was decreased, while St-La and St-Lr keptsimilar average values irrespective of which of the bases used. St-Lrwas characterized as slow culture when compared with the otherco-cultures in all bases.

Table 2Kinetic parameters of Lactobacillus delbrueckii subsp, bulgaricus (Lb), Lactobacillus acidophco-culture with Streptococcus thermophilus (St) in three different milk-whey basesa

Co-culture pH When fermentationwas stopped

Vmax (�10�3 upH.min�1) tV

12 g/100 ga 10 g/100 ga 8 g/100 ga 12

St-Lb 5.5 13.25ab 15.20ab 16.20ab 4.St-Lb 5.0 14.65bc 15.40ab 19.55b 4.St-Lb 4.5 13.75ab 14.60ab 18.55b 4.St-La 5.5 15.90c 15.75ab 16.15ab 3.St-La 5.0 16.00c 15.20ab 15.55ab 3.St-La 4.5 16.35c 14.70ab 15.20ab 3.St-Lr 5.5 12.30a 13.00ab 12.40a 3.St-Lr 5.0 13.10ab 12.65a 12.45a 3.St-Lr 4.5 13.00ab 12.60a 12.40a 2.St-Bl 5.5 13.20ab 16.55ab 16.90b 5.St-Bl 5.0 13.45ab 18.25b 17.60b 4.St-Bl 4.5 14.45bc 17.55ab 19.00b 4.

Vmax¼maximum acidification rate, tVmax¼ time to reach Vmax.Values in the same column with different letters are significantly different (Tukey test a

a 12, 10 and 8 g/100 g are the predicted total solids content in the milk-whey bases.

Values of Vmax found in this work were higher than those de-scribed by Chammas et al. (2006), who studied pure cultures ofS. thermophilus and L. delbrueckii subsp. bulgaricus isolated fromfermented milk known as ‘‘laban’’. According to the same authors,maximum acidification rates were 8.4�10�3 and 10.5�10�3

upH.min�1, respectively, for S. thermophilus and L. bulgaricus.The tVmax data shown in Table 2 varied from 2.86 to 5.23 h in

milk (12 g/100 g). It can be observed that co-culture St-Lr reachedVmax in less time 3.21, 3.51 and 2.86 h at pH 5.5, 5.0 and 4.5, re-spectively, followed by co-cultures St-La and St-Lb. Co-culture St-Blrequired longer tVmax. However, the tVmax values for co-cultures St-Lb (4.60 h) and St-La (3.66 h) were lower than those reported byOliveira and Damin (2003) in milk 12 g/100 g, but they were similarto the values obtained when the milk was supplemented with 8 gsucrose/100 g (4.7 and 3.8 h, respectively).

In milk-whey base (10 g/100 g), tVmax varied from 3.70 to 4.98 h(Table 2), where co-cultures St-La and St-Bl had similar values (i.e.on average 3.80 and 3.91 h, respectively). By contrast, St-Lb and St-Lr required longer times (w4.61 and w4.53 h, respectively). Theseresults showed different pattern than those observed in milk-wheybase (12 g/100 g) where St-Bl had the highest values and St-Lr thelowest values (Table 2).

When milk-whey bases (8 g/100 g) were fermented, all the co-cultures showed similar behavior for tVmax than in milk 12 g/100 g(Table 2). The tVmax values of co-cultures St-Lb and St-Bl werew4.17 h, while co-cultures St-La and St-Lr averaged 3.46 h. Never-theless, in 8 g/100 g milk-whey bases, tVmax was lower for allcultures when compared with those in 10 and 12 g/100 g milk-whey bases.

The pH corresponding to Vmax values can give important in-formation about LAB. There is no data reported of this parameterfor L. acidophilus, L. rhamosus and B. animalis subsp. lactis. In milk, itcan be observed that co-culture St-Lb reached Vmax at pHs 5.58,5.38 and 5.32, followed by St-La. These values were lower fromthose reported by Spinnler and Corrieu (1989) for S. thermophilusand L. delbrueckii subsp. bulgaricus in pure cultures. Co-culture St-Lrand St-Bl obtained Vmax at higher pHs (w5.87 and w6.01, re-spectively; see Table 2), and the same behavior/pattern was alsoobserved for all the different co-cultures grown in milk-whey base10 g/100 g).

When milk-whey bases (8 g/100 g) were fermented, St-Lbshowed a similar behavior as previously described, while St-La andSt-Bl obtained similar values at pH w5.77, indicating the possibilitythat the total solids have influenced this parameter.

Fermentation times (tpH) varied significantly (P� 0.05),which were influenced by the chemical composition of milk and

ilus (La), Lactobacillus rhamnosus (Lr), and Bifidobacterium animalis subsp. lactis (Bl) in

max (h) pH corresponding to Vmax

g/100 ga 10 g/100 ga 8 g/100 ga 12 g/100 ga 10 g/100 ga 8 g/100 ga

08cd 4.23bcd 4.06bc 5.58abc 5.96bc 5.56ab

37bc 4.63de 4.23c 5.38ab 5.52ab 5.27a

60de 4.98e 4.18c 5.32a 5.30a 5.30a

96bcd 3.82ab 3.06ab 5.57abc 5.53ab 5.90ab

76abc 3.70a 3.67abc 5.59abc 5.57ab 5.87ab

66abc 3.88ab 3.67abc 5.56abc 5.54ab 5.53ab

21ab 4.36cd 3.73abc 6.07d 6.05c 5.82ab

51abc 4.63de 3.73abc 5.84cd 5.80bc 5.86ab

86a 4.61de 2.90a 5.69bc 5.88bc 6.20b

23e 3.90abc 4.03bc 5.84cd 5.96bc 5.93ab

67de 4.00abc 4.20c 6.12d 5.91bc 5.73ab

73de 3.85ab 4.31c 6.09d 6.07c 5.63ab

t P< 0.05).

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672–678 675

milk-whey bases, and at what pH the fermentation was stopped. Ingeneral, the bases fermented by co-culture St-Lb were fastest toreach pH 4.5 (Fig. 1). Results from the fermentation of milk bya combination of the species S. thermophilus and L. bulgaricus,yogurt starter cultures, are known as protocooperation that isbeneficial for both species but is not indispensable for their growthor survival (Luquet & Corrieu, 2005).

In milk (12 g/100 g), the acidification times for co-cultures St-Lb,St-La, St-B1 and St-Lr to reach pH 4.5 were 6.50, 7.50, 11.30 and11.68 h, respectively (Fig. 1).

The acidification pattern of co-culture St-Lb in milk-whey base10 g/100 g was similar as in milk, and the acidifying tpH 4.5 was6.93 h. Fermentation time tpH 4.5 of St-La was statistically similar toSt-Bl (see Fig. 1). St-Lr acidified the same base in 12.07 h, albeithaving the same behavior as in milk. The tpH 4.5 in milk-whey base10 g/100 g was higher than in milk-whey base (12 g/100 g) whenco-cultures St-Lb, St-La and St-Lr were used. Even so, the tpH ofco-culture St-Bl was 2.4 times lower.

The acidification behavior of most co-cultures was faster inmilk-whey base (8 g/100 g), i.e. different to the other bases (seeFig. 1), but co-culture St-Lr required 14.02 h, which was the longestfermentation time observed in the present study. This slow acidi-fying performance of St-Lr has been observed in previous study inwhich liquid whey was employed as fermentation matrix. In thelatter case, St-Lr required 12.4 h to reach pH 4.5, even thoughpresenting high Vmax (w20�10�3 upH.min�1) (Almeida et al.,2008).

The co-cultures showed different acidification profiles in milk-whey mixtures. St-Lb and St-Bl had Vmax increased and tVmax, tpH 4.5

and tpH 5.5 decreased with the decrease in total solids contents. Lowvalues for tVmax and tpH 5.5 meant a high acidification activity for thestudied co-cultures. It could be argued, however, that the co-cultures had their acidifying capacity improved due to the lowertotal solids available. St-Bl showed high Vmax values in all bases,which characterise it as a fast acidifying co-culture as reported byXanthopoulos, Petridis, and Tzanetakis (2001). According to Kristoet al. (2003), lower total solids contents means lower buffering

12g/100 g

2

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H (h

)

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St-Lb St-LaSt-Lb St-La St-Lr St-Bl

a aab ab

bccd

de deef

f

gg

c

d

b

ab

a

ab

Fig. 1. Fermentation time (tpH) of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus atococcus thermophilus (St-Lb, St-La, St-Lr and St-B1, respectively) in milk-whey bases containidifferent; P� 0.05. (B pH 4.5, , pH 5.0, > pH 5.5).

capacity, which in turn means a higher decrease in pH for the sameamount of acid produced and vice versa.

St-La kept an average value of Vmax, tVmax and tpH 4.5 in milk-whey bases, but had the lower tpH 5.5 when compared with theother co-cultures. The co-culture St-Lr showed low acidificationability in all bases, which is characterized by lower Vmax andfermentation times until pH 4.5 above 11.5 h.

Nevertheless, the different co-cultures succeeded to decreasethe pH of the milk-whey in less than 6 h until reach pH 5.5.According to Cogan et al. (1997), micro-organisms with thesecharacteristics have good acid producing characteristics. However,Badis, Guetarni, Moussa Boudjema, Henni, and Kihal (2004)reported that the variations in the acidification activity of differentstrains of LAB are related to their specific aptitude to assimilate thenutritive compounds of the medium, which could explain thebehavior of co-culture St-Lr in milk-whey bases.

3.3. Post-acidification

All treatments showed post-fermentation acidification, whichvaried significantly (P� 0.05) (data not shown). Co-culture St-Lbexhibited the highest post-acidification in both milk and milk-wheybases; a similar behavior was suggested by Dave and Shah (1997).Co-culture St-La presented an intermediate post-fermentationacidification values in the different growth bases used but, when thefermentation was stopped at pH 5.0, these values were higher. Inmilk (12 g/100 g), co-culture St-Lr also gave high values for post-acidification, while co-culture St-Bl had lowest values, especiallywhen the fermentation was stopped at pH 4.5; similar results werealso observed in milk-whey bases 10 and 8 g/100 g.

3.4. Viable counts of starter cultures

In milk, counts of Lb, La and Lr were higher than 7.03 log10 cfumL�1, significantly different from Bl (i.e. averaged 8.79 log10 cfumL�1) (Fig. 2). Although the inoculation rate had similar viablenumber of cells, the counts of bifidobacteria were higher in all

/100 g 8g/100 g

St-Lb St-La St-Lr St-BlSt-Lr St-Bl

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cbc bc bc

cidophilus, Lactobacillus rhamnosus and Bifidobacterium lactis in co-culture with Strep-ng 12, 10 and 8 g/100 g total solids. Means (n¼ 2) with different letters are significantly

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ij

c

Fig. 2. Effect of milk-whey bases composition on the counts of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Bifido-bacterium animalis subsp. lactis (Bl), and Streptococcus thermophilus (St). Means (n¼ 2) with different letters are significantly different; P� 0.05. (a) Milk containing 12 g/100 g totalsolids; (b) milk-whey base containing 10 g/100 g total solids; (c) milk-whey base containing 8 g/100 g total solids; B St, , Lb, La, Lr or Bl).

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672–678 677

growth bases used. No significant difference in the counts ofL. delbrueckii subsp. bulgaricus and bifidobacteria were observeddespite the fact that the counts were higher when the fermentationwas stopped at pH 4.5. Counts of S. thermophilus were higher than8.5 log10 cfu mL�1.

In milk-whey base (10 g/100 g), the counts varied significantlyfrom 6.88 (for La at pH 5.5) to 8.91 log10 cfu mL�1 (for Bl at pH 4.5).Slightly lower counts for L. acidophilus were observed when grownin milk (12 g/100 g); the counts for L. rhamnosus were higherand reached w8.22 log10 cfu mL�1. Chiavari, Coloretti, Nanni,Sorrentino, and Grazia (2005) found similar counts of L. rhamnosusAT194 and L. rhamnosus CLT2/2 in fermented beverage usingdonkey’s milk.

In milk-whey (8 g/100 g), a slight decrease in the counts of La atpH 4.5 was observed, when compared to milk (12 g/100 g), whichwere significantly lower. These were the lowest counts observed inthe present study. The variations of the counts of L. acidophilus maysuggest that the chemical composition of the milk-whey basesinfluenced the growth of this organism; although mix containing8 g/100 g presented almost the same content of lactose, it has onlyhalf part of protein than in milk (12 g/100 g) (see Table 1).

A pattern of growth behavior was observed for cultures Lb andBl where counts were significantly higher when the fermentationwas stopped at pH 4.5 than at pH 5.5 in all bases; this may indicatethat longer fermentation time was required to increase the overallcounts of these micro-organisms.

The counts of S. thermophilus were higher in milk and milk-whey bases when fermentation was stopped at pH 4.5.

4. Conclusions

Co-culture combinations and total solids content of milk-wheybases affected the acidifying profile of the potential probiotic bac-teria, where St-Lr had low values of Vmax and tpH 4.5. St-Bl showedhigh Vmax values in all bases, which characterized it as a fast acid-ifying combination of strains. St-Lb and St-La reached Vmax at lowerpH than St-Lr and St-Bl. St-Lb had the lowest fermentation timewhen compared with the other co-culture combinations in thedifferent growth bases used. St-Lb and St-Bl had their acidifyingcapacity improved due to the lower total solids available. All fer-mented bases showed slight development of acid during the storageperiod, and lowest values were observed when the co-culture St-Blwas used. The different pH at which the fermentation was stoppedinfluenced the counts of B. animalis subsp. lactis and L. delbrueckiisubsp. bulgaricus, which had higher counts at pH 4.5. The counts ofL. rhamnosus were higher in bases containing 10 and 8 g/100 g totalsolids, but L. acidophilus had the lowest counts in these bases.

The results suggest that co-cultures St-Lb, St-La and St-Bl couldbe employed in milk-whey bases containing 8 and 10 g/100 g totalsolids; however, the counts of La were lower than 7 log10 cfu mL�1.Co-culture St-Lr was characterized as having a low acidifyingcapacity.

Acknowledgements

We wish to thank FAPESP (The State of Sao Paulo ResearchFoundation), CAPES and CNPq for financial support, and DANISCOfor providing the cultures.

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