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Arquivos do Instituto Biológico

Print version ISSN 0020-3653On-line version ISSN 1808-1657

Arq. Inst. Biol. vol.82  São Paulo  2015 

Scientific Communication

Interference of different storage temperatures in the dynamics of probiotic Bifidobacterium spp. and Streptococcus thermophilus starter cultures in fermented milk

Interferência de diferentes temperaturas de armazenamento na dinâmica de Bifidobacterium spp. probióticas e cultura starterStreptococcus thermophilus em leite fermentado

Rosângela Freitas 1  

Rodrigo Otávio Miranda 2  

Gabriel G. Netto 1  

Luís Augusto Nero 2   * 

Antônio Fernandes de Carvalho 1   * 

1Departamento de Tecnologia de Alimentos, Centro de Ciências Exatas; Universidade Federal de Viçosa (UFV) - Viçosa (MG), Brazil

2Departamento de Veterinária, Centro de Ciências Biológicas e da Saúde; UFV - Viçosa (MG), Brazil


The variations of temperature during the cold chain can impair the quality of live foods, such as fermented milks. Probiotic bacteria are commonly added to food to provide the consumer with beneficial effects. Nevertheless, the concentration of probiotic in the end products should be elevated to ensure functionality. The aim of this study was to evaluate the viability of probiotic strains of bifidobacteria and starter strain of Streptococcus thermophilus in fermented milks at storage temperatures of 4 and 10ºC, for a period of 28 days. Commercial cultures ofBifidobacterium spp. were added to milk fermented byStreptococcus thermophillus and stored for 28 days at 4 and 10ºC. During this period, bifidobacteria and S. thermophilluscultures were monitored to check their behavior in the evaluated storage conditions. Viable bifidobacteria and S. thermophillus counts showed no significant variation during storage at 4 and 10ºC (p < 0.05), indicating that both of these conditions are adequate for maintaining their initial concentrations. The results indicate that the storage conditions usually adopted in sale establishments of dairy products are suitable to maintain bifidobacteria and S. thermophillus cultures in fermented milk.

Key words: storage; probiotic; fermented milk; bifidobacteria


As variações de temperatura que ocorrem durante a cadeia refrigerada da produção leiteira pode interferir na qualidade de alimentos bioativos, como leites fermentados. Bactérias probióticas são usualmente adicionadas a alimentos, visando a oferecer ao consumidor efeitos benéficos. O objetivo deste trabalho foi avaliar a viabilidade de culturas probióticas de bifidobactérias e de cultura starter de Streptococcus thermophilus em leites fermentados, armazenados nas temperaturas de 4 e 10ºC por um período de 28 dias. Culturas comerciais de Bifidobacterium spp. foram adicionadas ao leite fermentado produzido com Streptococcus thermophillus e estocados por 28 dias a 4 e 10ºC. Nesse período, as culturas deBifidobacterium spp. e Streptococcus thermophillus foram monitoradas, com o objetivo de verificar seus comportamentos nas temperaturas de estocagem testadas. As contagens de ambos os micro-organismos não apresentaram variação significativa ao longo do período de estocagem a 4 e a 10ºC (p < 0,05), indicando que as duas temperaturas testadas podem oferecer condições adequadas de conservação desse produto. Os resultados obtidos indicaram que as condições de conservação de alimentos usualmente adotadas para produtos lácteos em estabelecimentos comerciais são adequadas para manter as contagens de bifidobactérias e Streptococcus thermophillus em leites fermentados.

Palavras-Chave: estocagem; probiótico; leite fermentado; bifidobactéria

These variations in storage temperature can determine a deleterious result in dairy products. These conditions enable the growth of distinct spoilage and pathogenic microorganisms that are able to jeopardize the quality and safety of such products (Ammor; Mayo, 2007). Such variation in storage temperature has a particular concern for dairy products that are considered as "live foods", such as fermented milks: the temperature variation while storing these products can affect the viability and the concentrations of the added starter and probiotic cultures (Shah, 2000).

According to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), probiotics are live organisms that, when administered at adequate amounts, have a beneficial effect on the health of the host. Once in the host's intestinal tract, these microorganisms cause changes in the intestinal microbiota in order to modulate the immune system, inhibit the adherence and multiplication of pathogens, and also prevent obesity and some types of cancer (Lourens-Hattingh; Viljoen, 2001). Ideally, probiotic cultures should present populations between 105 and 107 CFU.g-1during the entire shelf life of the food, so that it can assure the desired beneficial effects for the consumer after ingestion (Ammor; Mayo, 2007; Davidson et al., 2000).

The present study aimed to investigate how usual variation in the storage temperature can interfere in the populations of starter and probiotic cultures in a fermented milk, during its shelf-life.

Four commercial cultures of lyophilized Bifidobacterium spp. were used:B. animalis Bb 12 (B1, Chr. Hansen A/S, Hørsholm, Denmark),B. lactis SAB 440A (B2, Clerici-Sacco Group, Cadorago, Italy),B. lactis BI-07 (B3, Danisco A/S, Copenhagen, Denmark) andB. longum LGM P-17500 (B4, Centro Sperimentale del Latte, Zelo Buon Persico, Italy). An additional culture of S. thermophillus (ST 066, Clerici-Sacco Group) was used to produce fermented milk, previously added to 3 liters of milk and stored at -18ºC until it was ready to be used.

Pasteurized nonfat milk was heated at 90ºC for five minutes and cooled to 39ºC, and then fermented for 4 hours by the addition of ST 066 (Clerici-Sacco Group) in a concentration of about 107 colony forming units per g (CFU/g). After fermentation, B1, B2, B3 and B4 were added in concentrations of about 107 CFU/g, in batches separated from the fermented milk, and distributed in sterile 200 mL flasks. The fermented milk samples were produced in three repetitions and stored at 4 and 10ºC for 28 days.

Immediately after the fermented milk was bottled and at seven-day intervals, a sample of each fermented milk batch was submitted to microbiological analysis. Aliquots of the samples were submitted to ten-fold dilution using NaCl 8.5 g/L, being two dilutions selected and pour-plated in duplicate using NNLP (MRS broth [Oxoid Ltd., Basingstoke, England] added to nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) (Laroia; Martin, 1991; Shah, 2000) and M17 (Oxoid) culture media. NNLP plates were incubated at 37ºC for 48 hours under anaerobic conditions (GasPak EZ(tm) Gas Generating Container Systems, BD) to enumerate the Bifidobacterium spp., and M17 plates were incubated at 37ºC for 48 hours under aerobic conditions for S. thermophillus enumeration. After incubation, the colonies formed in both culture media were enumerated and the final results were expressed as CFU/g.

The obtained counts for bifidobacteria and S. thermophillus cultures were converted in log10, and compared considering different refrigeration temperatures and storage time, using the Analysis of Variance and the Tukey test (p < 0.05) to determine significant differences. All of the analyses were performed using STATISTICA Software 7.0 (StatSoft Inc., Tulsa, OK, USA).

The mean counts obtained for the different bifidobacteria and S. thermophillus cultures in fermented milk stored at 4 and 10ºC and in different storage periods are shown in Tables 1and 2, respectively. No significant differences were found between the different populations observed in the assessed incubation temperatures and periods, indicating that the populations of cultures used remained stable during the study.

Table 1. Mean counts of bifidobacteria cultures from fermented milk (prepared with Streptococcus thermophilus) stored at 4 and 10ºC for 28 days, obtained by NNLP culture media by conventional plating. 

Culture Storage temperature Days of Incubation Statistics
0 7 14 21 28
B1 4 6.20 6.38 6.16 6.21 6.32 F(4,10) = 0.08, p = 0.987
10 6,20 6,95 5,90 6,35 6,06 F(4,10) = 0.74, p = 0.588
F (1,4) = 0.00, p = 1.000 F(1,4) = 0.63, p = 0.473 F(1,4) = 0.49, p = 0.523 F(1,4) = 0.11, p = 0.759 F(1,4) = 0.14, p = 0.724
B2 4 5.70 5.56 4.99 5.05 5.28 F(4,10) = 0.85, p = 0.527
10 5.70 5.26 5.26 4.98 5.07 F(4,10) = 0.85, p = 0.526
F (1,4) = 0.00, p = 1.000 F(1,4) = 0.14, p = 0.734 F(1,4) = 0.20, p = 0.680 F(1,4) = 0.08, p = 0.792 F(1,4) = 0.98, p = 0.378
B3 4 6.87 6.84 6.86 6.75 6.42 F(4,10) = 0.23, p = 0.916
10 6.87 6.75 6.55 6.75 6.27 F(4,10) = 0.43, p = 0.786
F(1,4) = 0.00, p = 1.000 F(1,4) = 1.95, p = 0.235 F(1,4) = 5.88, p = 0.072 F(1,4) = 0.00, p = 0.996 F(1,4) = 0.01, p = 0.930
B4 4 7.09 6.90 6.93 6.61 7.35 F(4,10) = 1.85, p = 0.195
10 7.09 6.94 6.63 6.34 7.30 F(4,10) = 0.87, p = 0.514
F(1,4) = 0.00, p = 1.000 F(1,4) = 0.02, p = 0.905 F(1,4) = 0.31, p = 0.609 F(1,4) = 0.21, p = 0.673 F(1,4) = 0.01, p = 0.912
all 4 6.47 6.50 6.24 6.15 6.34 F(4,38) = 0.37, p = 0.829
10 6.47 6.47 6.09 6.21 6.16 F(4,38) = 0.49, p = 0.745
F(1,114) = 0.00, p = 1.000 F(1,114) = 0.00, p = 0.949 F(1,114) = 0.19, p = 0.671 F(1,114) = 0.02, p = 0.880 F(1,114) = 0.14, p = 0.711

*All statistics presented: Analysis of Variance (ANOVA). Mean counts with distinct lower case indicate significant differences in a same row (Tukey test, p < 0.05). Mean counts with distinct upper case indicate significant differences in the same column (Tukey test, p < 0.05); nc = no counts recorded; F = ANOVA test; df = degrees of freedom; p = level of significance.

Table 2. Mean counts of Streptococcus thermophilus from fermented milk (added to bifidobacteria cultures) stored at 4 and 10ºC for 28 days, obtained by M17 culture media by conventional plating. 

Added bifidobacteria Storage temperature Days of Incubation Statistics
0 7 14 21 28
B1 4 7.85 8.88 7.71 8.47 7.80 F(4,10) = 0.36, p = 0.831
10 7.85 9.03 8.92 8.42 7.90 F(4,10) = 1.36, p = 0.320
F(1,4) = 0.00, p = 1.000 F(1,4) = 0.02, p = 0.889 F(1,4) = 0.79, p = 0.425 F(1,4) = 0.01, p = 0.926 F(1,4) = 0.02, p = 0.889
B2 4 8.50 8.57 8.88 8.43 8.32 F(4,10) = 0.19, p = 0.940
10 8.50 8.55 8.85 9.16 8.62 F(4,10) = 0.52, p = 0.725
F(1,4) = 0.00, p = 1.000 F(1,4) = 0.09, p = 0.745 F(1,4) = 0.01, p = 0.937 F(1,4) = 1.10, p = 0.353 F(1,4) = 0.23, p = 0.657
B3 4 7.86 8.34 8.48 6.27 8.14 F(4,10) = 1.42, p = 0.298
10 7.86 8.72 7.65 7.08 7.72 F(4,10) = 0.79, p = 0.558
F(1,4) = 0.00, p = 1.000 F(1,4) = 0.10, p = 0.768 F(1,4) = 2.46, p = 0.192 F(1,4) = 3.20, p = 0.148 F(1,4) = 0.06, p = 0.816
B4 4 8.14 8.25 8.24 7.38 7.90 F(4,10) = 0.26, p = 0.896
10 8.14 8.32 8.43 7.18 8.75 F(4,10) = 0.89, p = 0.507
F(1,4) = 0.00, p = 1.000 F(1,4) = 0.00, p = 0.952 F(1,4) = 0.18, p = 0.692 F(1,4) = 0.05, p = 0.834 F(1,4) = 0.35, p = 0.594
all 4 8.07 8.51 8.33 7.64 8.06 F(4,38) = 0.99, p = 0.424
10 8.07 8.65 8.47 7.96 8.20 F(4,38) = 1.04, p = 0.395
F(1,114) = 0.00, p = 1.000 F(1,114) = 0.10, p = 0.758 F(1,114) = 0.12, p = 0.729 F(1,114) = 0.46, p = 0.502 F(1,114) = 0.07, p = 0.790

*All statistics presented: Analysis of Variance (ANOVA). Mean counts with distinct lower case indicate significant differences in a same row (Tukey test, p < 0.05). Mean counts with distinct upper case indicate significant differences in a same column (Tukey test, p < 0.05); nc = no counts recorded; F = ANOVA test; df = degrees of freedom; p = level of significance.

Considering that the initial populations of bifidobacteria cultures were about 107 CFU/g, typical of fermented milk with added probiotics (Zacarchenco; Massaguer-Roig, 2006), stocking at 4ºC allowed the concentration to be adequately preserved, and the same was true for the 10ºC temperature (Table 1). This temperature variation (4 to 10ºC) is usually observed in sale establishments, and, depending on the cultures that are present in different foods, it may lead to significant multiplication. Moreover, the 4ºC temperature is usually recommended to maintain microbial populations in milk and milk products to prevent their deterioration (Robinson, 2002). Nevertheless, Brazilian legislation allows fermented milk to be preserved at temperatures of 10ºC in sale establishments (Brasil, 2000). In a similar study, no significant changes were observed in the populations or in the culture viability of bifidobacteria added to yogurt (Vinderola et al., 2002) produced with different types of additives. In another study (Cruz et al., 2010), it was possible to observe that a commercial yogurt kept the count of bifidobacteria at 7 to 8 log for 84 days at 10ºC.

In relation to S. thermophillus, some types of fermented milk with specific added cultures of bifidobacteria ranged during different storage periods and temperatures, but at no significant level (Table 2). Oliveira et al.(2002) described a similar behavior for S. thermophilluscultures in fermented dairy products containing probiotic cultures, whereas significant interferences were observed only in products fermented by Lactobacillusafter storage at 4ºC for 21 days.

The viability of bifidobacteria cultures in fermented products during storage may be influenced by certain factors, such as post-acidification, oxygen content and presence of antimicrobial compounds (Shah, 2001). Considering that only the S. thermophillus culture was used for fermentation, and that its population remained stable during the entire storage period and at the different temperatures under assay (Table 2), acidity was most likely controlled, therefore, it did not interfere with the viability of the added probiotic culture (Table 1). By using a type of cheese with S. thermophilus as starter culture to deliver probiotic strains,Bergamini et al. (2010)showed that the B. lactis population maintained more than 107 CFU.g-1 throughout the 60 days of ripening at 12ºC, even if with some statistical difference.

The results obtained lead to the conclusion that the storage conditions assessed, usually observed in dairy industries and sale establishments, allowed the adequate preservation of populations of commercial bifidobacteria cultures added to fermented milk produced with S. thermophillus.



Luís A. Nero and Antônio F. Carvalho are supported by CNPq and FAPEMIG. Rodrigo O. Miranda, Gabriel Gama Netto, and Rosângela Freitas are supported by CAPES. The authors also thank Chr. Hansen A/S, Clerici-Sacco Group, Danisco A/S, and Centro Sperimentale del Latte, for providing the bifidobacteria and the S. thermophilus commercial cultures.


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Received: January 25, 2013; Accepted: March 12, 2014

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