LWT - Food Science and Technology 42 (2009) 672678

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Research Note

Inuence of total solids contents of milk whey on the acidifying prole and viability of various lactic acid bacteria
K.E. Almeida a, A.Y. Tamime b, M.N. Oliveira a, *
a b

o Paulo University, Department of Biochemical and Pharmaceutical Technology, Ave. Prof. Lineu Prestes, 580, 05508-900, Sa o Paulo, Brazil Sa 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 2007 Received in revised form 7 February 2008 Accepted 28 March 2008 Keywords: Milk Whey Probiotic Acidication prole

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 of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bidobacterium animalis subsp. lactis (Bl) in co-culture with Streptococcus thermophilus (St) on the rate of acid development in milk and milk-whey mixture, and (b) the effect of the level of the total solids of the different bases on the acidication prole and viability of potential health-promoting microorganisms. The co-culture of St-Lr showed the lowest values Vmax in all bases; while the co-culture St-Bl had high tVmax in milk and whey bases (12 and 10 g/100 g, respectively). Co-cultures St-La and St-Lb reached Vmax at pH 5.5, while St-Lr and St-Bl at pH 5.91. Fermentation time to reach pH 4.5 was longer when St-Lr co-culture was used, while St-Lb had the lowest value. All the products had slight development of acid during the storage period, and lowest values were observed when the St-Bl co-culture was employed. Lb, Bl and St cultures had high counts at pH 4.5 in the three bases. The total solids affected the 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 manufactured using milk and/or dairy derivatives with or without the addition of other ingredients in which the lactic base represents almost 51% (w/w) of the total ingredients used (Brasil, 2005). Nevertheless, the starter or probiotic bacteria should be abundant and alive in the nal product (Mercosul, 1998). These products are characterized of being refreshing having a smooth texture and low viscosity. The consumption of lactic beverages has increased in many countries (Nielsen, 2002; Tamime, 1997). Whey-based lactic beverages represent an emerging segment of non-conventional dairy products that require sensory, physical, and chemical characterisation for quality control and product development (Gallardo-Escamilla, Nelly, & Delahunt, 2007). The use of liquid or dried whey in lactic beverages is very common and, as a consequence, this method of processing may assist in the utilisation of whey, i.e. solution to dispose of high volume of whey generated by the cheese industry. In addition, the inclusion of a high nutritive raw material in the beverage base rather than milk , can ultimately reduce the production cost. According to Drgalic (2005), attention should be drawn to the Tratnik, and Bo zanic

* Corresponding author. Tel.: 55 11 3091 3690. E-mail address: monolive@usp.br (M.N. Oliveira).

natural whey, in a liquid form. Several lactic acid bacteria (LAB) and yeast could be employed in lactic beverages production. Hence, lactic beverages could be considered as an important vehicle for the delivery of probiotic micro-organisms to the human gastro intestine (Gardiner, Ross, Kelly, & Staton, 2002; Oliveira, Sodini, Remeuf & Corrieu, 2001). In this way, several products are being developed using mixtures of whey, milk, fruit juice, probiotic bacteria and/or yeast (Athanasiadis, Paraskevopoulou, Blekas, & Kiosseoglou, 2004; , Caric , Milanovic , Tekic , & Panic , 2004; Gallardo-Escamilla Djuric et al., 2007; Miglioranza et al., 2003; Suomalainen et al., 2006). However, little attention has been done to the kinetics parameters of milk-whey bases designated to whey-based fermented lactic beverages. Quantifying the acidifying activity of LAB allows the comparison of different combination of starter cultures in several substrates in order to dene the better process. The acidifying activity of LAB during the fermentation period could be described throughout pH curves; measuring pHs decrease in regular minimum time intervals, and maximum acidication rate (Vmax) could be calculated as dpH/dt. In addition, the time necessary to attain Vmax and its corresponding pH could be obtained. According to Picque, Perret, Latrille, and Corrieu (1992), these are the parameters that better describe the acidication kinetics. Studies of the acidication kinetics of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in milk and some probiotic microora al, Louvet, and Corrieu (1989); Cachon, had been reported by Be

0023-6438/$34.00 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.03.013

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672678

673

Jeanson, Aldarf, and Divies (2002); Chammas, Saliba, Corrieu, and al (2006); Kristo, Biliaderis, and Tzanetakis (2003); Lucas, Sodini, Be Monet, Jolivet, and Corrieu (2004); Oliveira and Damin (2003) and Oliveira et al. (2001). However, to our knowledge, there are no data in literature concerning the acidication prole of probiotics in milk-whey bases or in whey-based lactic beverages. In a previous study (Almeida, Tamime, & Oliveira, 2008), it was demonstrated that the technological interest of using Minas frescal cheese whey for the production of a probiotic lactic beverage was feasible. In the present study, the inuence of total solids contents of milk-whey bases on the acidifying prole of L. delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bidobacterium animalis subsp. lactis (Bl) was studied. These strains were used in co-culture with S. thermophilus (St) in milk-whey bases (10 and 8 g/100 g of total solids) at different pH levels at which the fermentation was stopped. Milk (12 g/100 g of total solids) was used as control. Finally, the effect of total solids contents of the milk-whey bases on the viability of the probiotic was also examined. 2. Materials and methods 2.1. Materials o Paulo) was Commercial pasteurized whole milk (Paulista, Sa used for the manufacture of Minas Frescal cheese according to the method reported by Furtado and Neto (1994). The gross chemical composition 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 in a plate heat exchanger (Laboratory Pasteurizer Armeld model FT43, Ringewood, England) in the Laboratory of Food Engineering o (Department of Chemical Engineering, Polytechnic School of Sa Paulo University), cooled to 4  C in an ice bath, dispensed into plastic bottles and frozen until required. Freeze-dried starter cultures (Danisco, Sassenage, France) were used for direct-to-vat inoculation (DVI), and starter organisms were S. thermophilus TA040, L. delbrueckii subsp. bulgaricus LB340, L. acidophilus LAC4, L. rhamnosus LBA and B. animalis subsp. lactis BL O4. A pre-culture was prepared by weighting each culture in sufcient amounts to attain initial counts of 108 colony forming units (cfu) mL1, and diluting in 50 mL skimmed milk, which had been sterilized at 121  C for 10 min, and cooled to 5  C. The pre-cultures were warmed at 42  C for 20 min before inoculation. 2.2. Experimental procedure Thirty six trials were performed in which the studied variables were (a) the different co-culture combinations (St-Lb, St-La, St-Lr and St-Bl), (b) the total solids of the milk (12 g/100 g) and milkwhey mixture (10 and 8 g/100 g), and (c) the pH values when the fermentation of the milk-whey bases was stopped (4.5, 5.0 and 5.5). Based in the analyzed total solids contents of pasteurized milk and whey, milk-whey bases were prepared by blending parts of milk and pasteurized whey to achieve the desired levels of total solids content of the beverage bases. The Pearsons Square method was used to calculate the required components (Tamime & Robinson, 2007). Flasks containing 250 mL of milk or milk-whey bases were placed in water bath at 42  C. The warmed asks were inoculated at the fermentation temperature with 8.38 log10 cfu mL1 of preculture of S. thermophilus, 8.11 log10 cfu mL1 of L. bulgaricus, 8.95 log10 cfu mL1 of L. acidophilus, 8.15 log10 cfu mL1 of L. rhamnosus, and 8.81 log10 cfu mL1 of B. animalis subsp. lactis in each beverage base as described in Section 2.1. All the inoculated bases were incubated at 42  C in a water bath until the desired

pHs were reached, and each experiment was performed in two replicates. The fermentation prole was monitored by using the pillon, France) (Spinnler & Corrieu, Cinac system (Ysebaert, Fre 1989), which allows continuous measurement and recording of the pH, and computes the acidication rate during the incubation period. Six kinetic parameters were considered: (a) Vmax (maximum acidication rate, measured in pH units per min (upH.min1), (b) tVmax (time to reach the maximum acidication rate), (c) pH corresponding to Vmax and (d) tpH 4.5, tpH 5.0, and tpH 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, the products were transferred immediately to an ice bath. The fermented beverages were stored at 4  C. 2.3. Chemical composition of milk-whey bases Total solids content was determined by drying the samples at 105  C up to a constant weight (AOAC, 1995), protein content was determined by micro Kjeldahl (AOAC, 1995), fat by Gerber method (Schmidt-Hebbel, 1956), and lactose by Eynon-Lane method (AOAC, o Paulo) was 1984). A digital potentiometer (Quimis, Diadema, Sa used to measure the pH values. The post-acidication of the milk or milk-whey bases was calculated by the difference of the pH value of the product after 24 h of storage and the pH of the product immediately after fermentation. All the chemical analyses were performed in triplicate. 2.4. Microbiological analysis Enumeration of the starter cultures was carried out after the fermented milk-whey bases were stored at 4  C for 24 h, and each sample was prepared according to the methods described by the International Dairy Federation (IDF, 1996, 1997, 2003). Counts of S. thermophilus were enumerated on M17 agar (Merck, Darmstadt, Germany) by aerobic incubation at 37  C for 48 h. Enumerations of lactobacilli species and bidobacteria were carried out on MRS agar (Merck, Darmstadt, Germany) adjusted to pH 5.4 with glacial acetic acid and by aerobic incubation at 37  C for 48 h for L. delbrueckii subsp. bulgaricus, and by anaerobic incubation at 37  C for 72 h for L. acidophilus, L. rhamnosus and for the bidobacteria. The selectivity of the growth conditions was conrmed by microscope appearance of the cells from single colonies. 2.5. Statistical analysis Analysis of variance for multiple comparisons (ANOVA) using Statistica 6.0, Statsoft (Tulsa, USA), was performed in order to conrm statistical signicance of differences among samples (P < 0.05). Mean values were compared using the Tukey test at P < 0.05. 3. Results and discussion 3.1. Chemical composition of milk-whey bases The chemical composition (g/100 g) of the milk-whey bases is shown in Table 1, and the data fell within the following ranges: total solids (TS) 7.812 g/100 g, protein 1.42.9 g/100 g, and fat 1.2 3.6 g/100 g. These values were statistically different in all the bases, which was inuenced by the amount of added whey. The milkwhey bases had similar amounts of lactose (w4.6 g/100 g), and Spadoti (1998) reported similar level of lactose in raw milk. However, the chemical composition of milk-whey bases was similar to those reported by Almeida, Bonassi, and Roa (2001).

674 Table 1 Composition (g 100 g1) of milk-whey basesa Milk and milkwhey basesb 12 10 8 Total solids 12.0a (0.347) 9.8b (0.100) 7.8c (0.038) Protein 2.9a (0.017) 2.1b (0.022) 1.4c (0.026)

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672678

Fat 3.6a (0.153) 2.4b (0.058) 1.2c (0.058)

Lactose 4.5a (0.061) 4.5a (0.012) 4.6a (0.075)

Values in the same column with different letters are signicantly different (Tukey test 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.

3.2. Acidication prole Vmax values ranged between 12.30 103 (St-Lr at pH 5.5) and 16.35 103 upH.min1 (St-La at pH 4.5) when milk (12 g/100 g) was used (Table 2). Co-culture St-La attained highest values, which averaged 16.08 103 upH.min1. However, when St-Lr was employed, the lowest Vmax values were observed. These results can be compared with those reported by Oliveira and Damin (2003) who investigated the kinetic parameters of the same strains reported in present paper, L. acidophilus, L. delbrueckii subsp. bulgaricus and L. rhamnosus, in co-culture with S. thermophilus in whole milk, and in milk supplemented with sucrose fermented until pH 4.5. The same authors also reported the following Vmax values 14.5 103, 7.7 103 and 12.8 103 upH.min1 for cocultures St-Lb, St-La and St-Lr, respectively. It is of interest to note that to our knowledge there are no data available on the acidifying prole of St-Bl in milk or milk-whey bases. In the bases containing 10 g/100 g, the co-culture St-Lr showed the lowest Vmax values irrespective of pH when the fermentation was stopped (13.00 103, 12.65 103, and 12.60 103 upH.min1) (Table 2). St-Lb and St-La had values that ranged between 14.60 103 and 15.75 103 upH.min1 with no signicant differences. According to the Vmax values in milk-whey base (8 g/100 g), the co-cultures could be classied into three very distinct groups (Table 2). First, the least Vmax value was for St-Lr irrespective at what pH the fermentation was stopped (12.40 103 upH.min1 in average), second, intermediate values for St-La (15.63 103 upH.min1 in average), and third, high Vmax values for co-cultures St-Lb and St-Bl at pH 4.5 (18.55 103 and 19.00 103 upH.min1, respectively). Co-cultures St-Lb and St-Bl showed an increase in Vmax as the total solids content was decreased, while St-La and St-Lr kept similar average values irrespective of which of the bases used. St-Lr was characterized as slow culture when compared with the other co-cultures in all bases.

Values of Vmax found in this work were higher than those described by Chammas et al. (2006), who studied pure cultures of S. thermophilus and L. delbrueckii subsp. bulgaricus isolated from fermented milk known as laban. According to the same authors, maximum acidication rates were 8.4 103 and 10.5 103 upH.min1, 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 reached Vmax in less time 3.21, 3.51 and 2.86 h at pH 5.5, 5.0 and 4.5, respectively, followed by co-cultures St-La and St-Lb. Co-culture St-Bl required longer tVmax. However, the tVmax values for co-cultures StLb (4.60 h) and St-La (3.66 h) were lower than those reported by Oliveira and Damin (2003) in milk 12 g/100 g, but they were similar to the values obtained when the milk was supplemented with 8 g sucrose/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 StLr required longer times (w4.61 and w4.53 h, respectively). These results showed different pattern than those observed in milk-whey base (12 g/100 g) where St-Bl had the highest values and St-Lr the lowest values (Table 2). When milk-whey bases (8 g/100 g) were fermented, all the cocultures 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 were w4.17 h, while co-cultures St-La and St-Lr averaged 3.46 h. Nevertheless, in 8 g/100 g milk-whey bases, tVmax was lower for all cultures when compared with those in 10 and 12 g/100 g milkwhey bases. The pH corresponding to Vmax values can give important information about LAB. There is no data reported of this parameter for L. acidophilus, L. rhamosus and B. animalis subsp. lactis. In milk, it can 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 from those reported by Spinnler and Corrieu (1989) for S. thermophilus and L. delbrueckii subsp. bulgaricus in pure cultures. Co-culture St-Lr and St-Bl obtained Vmax at higher pHs (w5.87 and w6.01, respectively; see Table 2), and the same behavior/pattern was also observed for all the different co-cultures grown in milk-whey base 10 g/100 g). When milk-whey bases (8 g/100 g) were fermented, St-Lb showed a similar behavior as previously described, while St-La and St-Bl obtained similar values at pH w5.77, indicating the possibility that the total solids have inuenced this parameter. Fermentation times (tpH) varied signicantly (P  0.05), which were inuenced by the chemical composition of milk and

Table 2 Kinetic parameters of Lactobacillus delbrueckii subsp, bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bidobacterium animalis subsp. lactis (Bl) in co-culture with Streptococcus thermophilus (St) in three different milk-whey basesa Co-culture pH When fermentation was stopped 5.5 5.0 4.5 5.5 5.0 4.5 5.5 5.0 4.5 5.5 5.0 4.5 Vmax (103 upH.min1) 12 g/100 g 13.25 14.65bc 13.75ab 15.90c 16.00c 16.35c 12.30a 13.10ab 13.00ab 13.20ab 13.45ab 14.45bc
ab a

tVmax (h)
a

pH corresponding to Vmax
a

10 g/100 g 15.20 15.40ab 14.60ab 15.75ab 15.20ab 14.70ab 13.00ab 12.65a 12.60a 16.55ab 18.25b 17.55ab
ab

8 g/100 g 16.20 19.55b 18.55b 16.15ab 15.55ab 15.20ab 12.40a 12.45a 12.40a 16.90b 17.60b 19.00b
ab

12 g/100 g 4.08 4.37bc 4.60de 3.96bcd 3.76abc 3.66abc 3.21ab 3.51abc 2.86a 5.23e 4.67de 4.73de
cd

10 g/100 g 4.23 4.63de 4.98e 3.82ab 3.70a 3.88ab 4.36cd 4.63de 4.61de 3.90abc 4.00abc 3.85ab
bcd

8 g/100 g 4.06 4.23c 4.18c 3.06ab 3.67abc 3.67abc 3.73abc 3.73abc 2.90a 4.03bc 4.20c 4.31c
bc

12 g/100 ga 5.58 5.38ab 5.32a 5.57abc 5.59abc 5.56abc 6.07d 5.84cd 5.69bc 5.84cd 6.12d 6.09d
abc

10 g/100 ga 5.96 5.52ab 5.30a 5.53ab 5.57ab 5.54ab 6.05c 5.80bc 5.88bc 5.96bc 5.91bc 6.07c
bc

8 g/100 ga 5.56ab 5.27a 5.30a 5.90ab 5.87ab 5.53ab 5.82ab 5.86ab 6.20b 5.93ab 5.73ab 5.63ab

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

Vmax maximum acidication rate, tVmax time to reach Vmax. Values in the same column with different letters are signicantly different (Tukey test at P < 0.05). a 12, 10 and 8 g/100 g are the predicted total solids content in the milk-whey bases.

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672678

675

milk-whey bases, and at what pH the fermentation was stopped. In general, the bases fermented by co-culture St-Lb were fastest to reach pH 4.5 (Fig. 1). Results from the fermentation of milk by a combination of the species S. thermophilus and L. bulgaricus, yogurt starter cultures, are known as protocooperation that is benecial for both species but is not indispensable for their growth or survival (Luquet & Corrieu, 2005). In milk (12 g/100 g), the acidication 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 and 11.68 h, respectively (Fig. 1). The acidication pattern of co-culture St-Lb in milk-whey base 10 g/100 g was similar as in milk, and the acidifying tpH 4.5 was 6.93 h. Fermentation time tpH 4.5 of St-La was statistically similar to St-Bl (see Fig. 1). St-Lr acidied the same base in 12.07 h, albeit having the same behavior as in milk. The tpH 4.5 in milk-whey base 10 g/100 g was higher than in milk-whey base (12 g/100 g) when co-cultures St-Lb, St-La and St-Lr were used. Even so, the tpH of co-culture St-Bl was 2.4 times lower. The acidication behavior of most co-cultures was faster in milk-whey base (8 g/100 g), i.e. different to the other bases (see Fig. 1), but co-culture St-Lr required 14.02 h, which was the longest fermentation time observed in the present study. This slow acidifying performance of St-Lr has been observed in previous study in which liquid whey was employed as fermentation matrix. In the latter case, St-Lr required 12.4 h to reach pH 4.5, even though presenting high Vmax (w20 103 upH.min1) (Almeida et al., 2008). The co-cultures showed different acidication proles in milkwhey 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. Low values for tVmax and tpH 5.5 meant a high acidication activity for the studied co-cultures. It could be argued, however, that the cocultures had their acidifying capacity improved due to the lower total solids available. St-Bl showed high Vmax values in all bases, which characterise it as a fast acidifying co-culture as reported by Xanthopoulos, Petridis, and Tzanetakis (2001). According to Kristo et al. (2003), lower total solids contents means lower buffering

capacity, which in turn means a higher decrease in pH for the same amount of acid produced and vice versa. St-La kept an average value of Vmax, tVmax and tpH 4.5 in milkwhey bases, but had the lower tpH 5.5 when compared with the other co-cultures. The co-culture St-Lr showed low acidication ability in all bases, which is characterized by lower Vmax and fermentation times until pH 4.5 above 11.5 h. Nevertheless, the different co-cultures succeeded to decrease the pH of the milk-whey in less than 6 h until reach pH 5.5. According to Cogan et al. (1997), micro-organisms with these characteristics have good acid producing characteristics. However, Badis, Guetarni, Moussa Boudjema, Henni, and Kihal (2004) reported that the variations in the acidication activity of different strains of LAB are related to their specic aptitude to assimilate the nutritive compounds of the medium, which could explain the behavior of co-culture St-Lr in milk-whey bases. 3.3. Post-acidication All treatments showed post-fermentation acidication, which varied signicantly (P  0.05) (data not shown). Co-culture St-Lb exhibited the highest post-acidication in both milk and milk-whey bases; a similar behavior was suggested by Dave and Shah (1997). Co-culture St-La presented an intermediate post-fermentation acidication values in the different growth bases used but, when the fermentation was stopped at pH 5.0, these values were higher. In milk (12 g/100 g), co-culture St-Lr also gave high values for postacidication, while co-culture St-Bl had lowest values, especially when the fermentation was stopped at pH 4.5; similar results were also 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 cfu mL1, signicantly different from Bl (i.e. averaged 8.79 log10 cfu mL1) (Fig. 2). Although the inoculation rate had similar viable number of cells, the counts of bidobacteria were higher in all

15 h 14 13 12 11 10 d d f de de bc ab a ab a ef cd 6 5 4 3 2 St-Lb St-La St-Lr St-Bl St-Lb St-La St-Lr St-Bl St-Lb St-La St-Lr St-Bl c e b ab a c bc bc a g f e d bc b g g e

tpH (h)

9 8 7

b ab ab a

12g/100 g

10g/100 g

8g/100 g

Fig. 1. Fermentation time (tpH) of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus and Bidobacterium lactis in co-culture with Streptococcus thermophilus (St-Lb, St-La, St-Lr and St-B1, respectively) in milk-whey bases containing 12, 10 and 8 g/100 g total solids. Means (n 2) with different letters are signicantly different; P  0.05. (B pH 4.5, , pH 5.0, > pH 5.5).

a
Counts of Viable Cells (log10 cfu/mL)

10.0

9.5 d d 9.0 d d d d d cd cd d cd d d cd 8.5 cd

8.0 b ab 7.5 ab 7.0 ab ab a ab ab ab

6.5

6.0

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

pH4.5

pH5.0

pH5.5

b
Counts of Viable Cells (log10 cfu/mL)

10.0

9.5 g 9.0 efg e d 8.5 c 8.0 b 7.5 a a 7.0 b a a efg g efg efg efg efg fg efg d

efg

ef

efg cd

6.5

6.0

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

pH4.5

pH5.0

pH5.5

c
Counts of Viable Cells (log10 cfu/mL)

10.0

9.5 hij hij j fghi ghij ghi ghi hij e d 8.0 d fghi ghi fgh fg e ij ef fg

9.0

8.5

7.5 ab 7.0 a a bc

6.5

6.0

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

St-Lb St-La St-Lr

St-Bl

pH4.5

pH5.0

pH5.5

Fig. 2. Effect of milk-whey bases composition on the counts of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Bidobacterium animalis subsp. lactis (Bl), and Streptococcus thermophilus (St). Means (n 2) with different letters are signicantly different; P  0.05. (a) Milk containing 12 g/100 g total solids; (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) 672678

677

growth bases used. No signicant difference in the counts of L. delbrueckii subsp. bulgaricus and bidobacteria were observed despite the fact that the counts were higher when the fermentation was stopped at pH 4.5. Counts of S. thermophilus were higher than 8.5 log10 cfu mL1. In milk-whey base (10 g/100 g), the counts varied signicantly from 6.88 (for La at pH 5.5) to 8.91 log10 cfu mL1 (for Bl at pH 4.5). Slightly lower counts for L. acidophilus were observed when grown in milk (12 g/100 g); the counts for L. rhamnosus were higher and reached w8.22 log10 cfu mL1. Chiavari, Coloretti, Nanni, Sorrentino, and Grazia (2005) found similar counts of L. rhamnosus AT194 and L. rhamnosus CLT2/2 in fermented beverage using donkeys milk. In milk-whey (8 g/100 g), a slight decrease in the counts of La at pH 4.5 was observed, when compared to milk (12 g/100 g), which were signicantly lower. These were the lowest counts observed in the present study. The variations of the counts of L. acidophilus may suggest that the chemical composition of the milk-whey bases inuenced the growth of this organism; although mix containing 8 g/100 g presented almost the same content of lactose, it has only half part of protein than in milk (12 g/100 g) (see Table 1). A pattern of growth behavior was observed for cultures Lb and Bl where counts were signicantly higher when the fermentation was stopped at pH 4.5 than at pH 5.5 in all bases; this may indicate that longer fermentation time was required to increase the overall counts of these micro-organisms. The counts of S. thermophilus were higher in milk and milkwhey bases when fermentation was stopped at pH 4.5. 4. Conclusions Co-culture combinations and total solids content of milk-whey bases affected the acidifying prole of the potential probiotic bacteria, where St-Lr had low values of Vmax and tpH 4.5. St-Bl showed high Vmax values in all bases, which characterized it as a fast acidifying combination of strains. St-Lb and St-La reached Vmax at lower pH than St-Lr and St-Bl. St-Lb had the lowest fermentation time when compared with the other co-culture combinations in the different growth bases used. St-Lb and St-Bl had their acidifying capacity improved due to the lower total solids available. All fermented bases showed slight development of acid during the storage period, and lowest values were observed when the co-culture St-Bl was used. The different pH at which the fermentation was stopped inuenced the counts of B. animalis subsp. lactis and L. delbrueckii subsp. bulgaricus, which had higher counts at pH 4.5. The counts of L. rhamnosus were higher in bases containing 10 and 8 g/100 g total solids, but L. acidophilus had the lowest counts in these bases. The results suggest that co-cultures St-Lb, St-La and St-Bl could be employed in milk-whey bases containing 8 and 10 g/100 g total solids; however, the counts of La were lower than 7 log10 cfu mL1. Co-culture St-Lr was characterized as having a low acidifying capacity. Acknowledgements o Paulo Research We wish to thank FAPESP (The State of Sa Foundation), CAPES and CNPq for nancial support, and DANISCO for providing the cultures. References
sticas f sicas e qu micas de Almeida, K. E., Bonassi, I. A., & Roa, R. O. (2001). Caracter cteas fermentadas preparadas com soro de queijo Minas Frescal. bebidas la ncia Tecnologia de Alimentos, 21, 187192. Cie Almeida, K. E., Tamime, A. Y., & Oliveira, M. N. (2008). Acidication rates of probiotic bacteria in Minas frescal cheese whey. LWT Food Science and Technology, 41, 311316.

AOAC. (1984). Ofcial methods of analysis (14 ed.). Arlington: Association of Ofcial Analytical Chemists. p. 1141. AOAC. (1995). Ofcial methods of analysis (16 ed.). Washington: Association of Ofcial Analytical Chemists. p. 1141. Athanasiadis, I., Paraskevopoulou, A., Blekas, G., & Kiosseoglou, V. (2004). Development of a novel whey beverage by fermentation with ker granules. Effect of various treatments. Biotechnology Progress, 20, 10911095. Badis, A., Guetarni, D., Moussa Boudjema, B., Henni, D. E., & Kihal, M. (2004). Identication and technological properties of lactic acid bacteria isolated from raw goat milk of four Algerian races. Food Microbiology, 21, 579588. al, C., Louvet, P., & Corrieu, G. (1989). Inuence of controlled pH and temperature Be on the growth and acidication of pure cultures of Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398. Applied Microbiology and Biotechnology, 32, 148154. rio da Agricultura, Pecua ria e Abastecimento. Legislaa o. SIBrasil. (2005). Ministe ` Legislaa o. Instrua o Normativa n.16, de 23 de SLEGIS: Sistema de Consulta a cnico de Identidade e Qualidade de agosto de 2005. Aprovar o Regulamento Te cteas. Dispon vel em: <http://extranet.agricultura.gov.br/sislegisBebidas La consulta/servlet/VisualizarAnexo?id3742>. Acesso em: 30 out. Cachon, R., Jeanson, S., Aldarf, M., & Divies, C. (2002). Characterisation of lactic starters based on acidication and reduction activities. Lait, 82, 281288. al, C. (2006). Characterisation of lactic Chammas, G. I., Saliba, R., Corrieu, G., & Be acid bacteria isolated from fermented milk laban. International Journal of Food Microbiology, 110, 5261. Chiavari, C., Coloretti, F., Nanni, M., Sorrentino, E., & Grazia, L. (2005). Use of donkeys milk for a fermented beverage with lactobacilli. Lait, 85, 481490. Cogan, T. M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, B., Cocconcelli, P. S., Fernandes, I., et al. (1997). Characterization of the lactic acid bacteria in artisanal dairy products. Journal of Dairy Research, 64, 409421. Dave, R. I., & Shah, N. P. (1997). Effect of level of starter culture on viability of yogurt and probiotic bacteria in yogurts. Food Australia, 4, 164168. , M., Milanovic , S., Tekic , M., & Panic , M. (2004). Development of , M., Caric Djuric whey based beverages. European Food Research Technology, 219, 321328. , I., Tratnik, L., & Bo , R. (2005). Growth and survival of probiotic bacteria Drgalic zanic in reconstituted whey. Lait, 85, 171179. cnico para Furtado, M. M., & Neto, J. P. M. (1994). Tecnologia de Queijos Manual Te o Industrial de Queijos. Dipemar. p. 115. Produa Gallardo-Escamilla, F. J., Nelly, A. L., & Delahunty, C. M. (2007). Mouthfeel and avour of fermented whey with added hydrocolloids. International Dairy Journal, 17, 308315. Gardiner, G. E., Ross, R. P., Kelly, P. M., & Staton, C. (2002). Microbiology of therapeutics milks. In R. K. Robinson (Ed.), Dairy microbiology handbook (3rd ed.). (pp. 431478) NY, USA: John Wiley & Sons Inc. IDF. (1996). Preparation of samples and dilutions for microbiological examination. Standard No. 122C. Brussels, Belgium: International Dairy Federation. IDF. (1997). Dairy starer cultures of lactic acid bacteria (LAB) Standard of identity. Standard No. 149A. Brussels, Belgium: International Dairy Federation. IDF. (2003). Yoghurt/enumeration of characteristic microorganisms Colony count technique at 37  C. Standard No. 117. Brussels, Belgium: International Dairy Federation. Kristo, E., Biliaderis, C. G., & Tzanetakis, N. (2003). Modelling of rheological, microbiological and acidication properties of a fermented milk product containing a probiotic strain of Lactobacillus paracasei. International Dairy Journal, 13, 517528. Lucas, A., Sodini, I., Monet, C., Jolivet, P., & Corrieu, G. (2004). Probiotic cell counts and acidication in fermented milks supplemented with milk protein hydrolysates. International Dairy Journal, 14, 4753. Luquet, F. M., & Corrieu, G. (2005). Lactic bacteria and probiotics. Secaucus: Lavosier. p. 34. o GMC 47/97: Leites fermentados. In: Mercosul. (1998). Leis e Decretos, etc. Resolua cteos e de Alimentos para Fins Especiais Diet, Light o de Produtos La Nova Legislaa es de Identidade e Qualidade. Sa o Paulo: Fonte e Enriquecidos. Padro es. Comunicao rdoba, G. M., Dichi, J. B., Cyrino, E. S., Miglioranza, L. H. S., Matsuo, T., Caballero-Co Oliveira, I. B. N., et al. (2003). Effect of long-term fortication of whey drink with ferrous bisglycinate on anemia prevalence in children and adolescents from deprived areas in Londrina, Parana, Brazil. Nutrition, 19(5), 419421. es Sobre o Crescimento de Alimentos e Bebidas. Os Nielsen, A. C. (2002). Informao Produtos mais quentes do mundo. <www.br.acnielsen.com> Assessed 28.03.07. lidos e da concentraa o de Oliveira, M. N., & Damin, M. R. (2003). Efeito do teor de so rias do iogurte e pro o, rmeza e viabilidade de bacte sacarose na acidicaa ticas em leite fermentado. Cie ncia e Tecnologia de Alimentos. Campinas, 23, bio 172176. Oliveira, M. N., Sodini, I., Remeuf, F., & Corrieu, G. (2001). Effect of milk supplementation and culture composition on acidication, textural properties and microbiological stability of fermented milks containing probiotic bacteria. International Dairy Journal, 11, 935942. risation et classication Picque, D., Perret, B., Latrille, E., & Corrieu, G. (1992). Caracte ries lactiques a ` partir de la mesure de leur cine tique dacidication. de bacte LWT Food Science and Technology, 25, 181186. Schmidt-Hebbel, H. (1956). Quimica y tecnologia de los alimentos. Santiago: Editorial Salesiana. do na fabricaa o de queijo mussarela. Spadoti L. M. (1998). Uso de leite reconstitu o Paulo, Dissertaa o de Mestrado], Piracicaba. p. 101. Diss. [Universidade de Sa Spinnler, H. E., & Corrieu, G. (1989). Automatic method to quantify starter activity based on pH measurement. Journal of Dairy Research, 56, 755764.

678

K.E. Almeida et al. / LWT - Food Science and Technology 42 (2009) 672678 Tamime A. Y., & Robinson R. K. (2007). Tamime and Robinsons Yoghurt Science and Technology (3rd ed.). Boca Raton: CRC Press. Xanthopoulos, V., Petridis, D., & Tzanetakis, N. (2001). Characterization and classication of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains isolated from traditional Greek yogurts. Journal of Food Science, 66(5), 747752.

Suomalainen, T., Lagstro, H., Matto, J., Saarela, M., Arvilommi, H., Laitinen, I., et al. (2006). Inuence of whey-based fruit juice containing Lactobacillus rhamnosus on intestinal well-being and humoral immune response in healthy adults. LWT Food Science and Technology, 39, 788795. cticas e probio ticas. In: Leites Fermentados Tamime, A. Y. (1997). Culturas starters la cteas: Tecnologia e Mercado. Campinas, Brazil: ITAL. e Bebidas La