The Impact of Selected Factors on the Survival Rate of Probiotic Bacteria in Natural Bio-yoghurts Made from Cow's Milk

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(1)Zeszyty Uniwersytet Ekonomiczny w Krakowie Naukowe Towaroznawstwo. 874 Kraków 2011. Wanda Kudełka. Department of Food Commodity Science. The Impact of Selected Factors on the Survival Rate of Probiotic Bacteria in Natural Bio-yoghurts Made from Cow’s Milk 1. Introduction According to many authors (Dave & Shah 1997; Holzapfel et al. 1998; Lankaputhra & Shah 1995; Lankaputhra, Shah & Britz 1996), numerous factors affect the survival rate of probiotic bacteria, including the presence of hydrogen peroxide, oxygen content, concentration of acetic and lactic acid, the type of starter cultures applied, the type of raw material & production process progress, and product storage conditions. Pursuant to the IDF/FIL and FAO/WHO standards, the count of living probiotic bacteria exhibiting documented probiotic features, which must be present in fermented milk drinks, should not fall below 106 cfu/ml at any point before the product’s expiry date. However, in order to achieve a probiotic effect, people should consume at least 106 to 109 living probiotic bacteria per day (Donnet-Hughes et al. 1999; Kleessen, Bezirtzoglou & Matto 2000). Numerous studies have confirmed the benefits of probiotic micro-flora on human health (Defecińska & Libudzisz 2000; Dunne, Murphy & Flynn 1999; Isolauri 2001; Isolauri et al. 2000; Ouwehand, Salminen & Isolauri 2002; Ouwehand & Salminen 1998; Pessi et al. 2000). Additionally, the presence of beneficial micro-flora enhances the sensory attractiveness of milk drinks fermented during production and storage (Lamoureux, Roy & Gauthier 2002; Shin et al. 2000)..

(2) 80. Wanda Kudełka. The objective of the study described in this paper was to determine the survival rate of probiotic bacteria in natural bio-yoghurts depending on the applied pasteurisation variant of raw material and packaging type.. 2. Materials and Methods The bio-yoghurts investigated were manufactured from cow’s milk using a container method. The milk being processed was centrifuged and normalised to a fat level of 2% and then pasteurised at a temperature of 95°C for 5 min and at 90°C for 10 min, then cooled to 40°C. The cooled milk was inoculated with DVS ABT1 inoculants (0.2% addition), a product of the Chr. Hansen Company. The inoculants applied contained the following 3 bacterial strains, all of which exhibited documented probiotic features: Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium bifidum. The bio-yoghurts were thermostated (controlled) in containers at a temperature of 40°C (+/–1°C) until they reached a pH level of 4.7. They were then cooled again to a temperature below 20°C, and poured into 4 different packagings made of polypropylene (PP), polystyrene (PS), polyethylene (PEHD), and glass (G). They were subsequently stored for 21 days at temperatures ranging from 2 to 5°C. The presence of probiotic bacteria in bio‑yoghurt was determined 12 h (T0) after the bio-yoghurts were manufactured, and on the 7th (T1), 14th (T2), and 21st (T3) days of storage. The quantitative determination of probiotic bacteria was carried out as indicated in the relevant instructions. An average laboratory sample was prepared from one batch. To achieve a homogenous consistency of the material analysed, each of the samples examined was thoroughly shaken prior to being opened. From a properly shaken sample, 1 ml was taken and placed in a test-tube, which was centrifuged for 1 minute in a “Wortex” centrifuge to obtain a homogenous suspension. Next, suitable selective culturing substrates (Lactobacillus acidophilus – an MRS substrate Oxoid Co.; Streptococcus thermophilus, a Mueller-Hinton substrate; and Bifidobacterium – from Agar BL) were inoculated with 1 ml of each individual dilution (dilutions ranged from 10 –1 to 10 –10). The laboratory tiles were then incubated at a temperature of 37ºC, under anaerobic conditions, for 72 hours. After incubation, the initial densities of bacteria from each of the three substrates were counted (Dave & Shah 1996; Lankaputhra & Shah 1996; Tharmaraj & Shah 2003). Further, the bacteria grown on the culturing substrata were identified according to genus. API 50 CHL test was used to identify Lactobacillus acidophilus and API 20A was used to identify Bifidobacterium. A special test to detect a fructose-6-phosphate phosphoketolase (F6PPK) enzyme was applied, and API STREP test to find Streptococcus thermophilus..

(3) The Impact of Selected Factors on the Survival…. 81. The computation was performed using procedures from the Statistica 7.0 PL computer software package. The hypotheses on the impact of pasteurisation temperature and type of packaging were verified using a two-factor analysis of variance. Null hypotheses on the non-occurrence of any significant impact of a given factor (pasteurisation temperature, type of packaging) and on the lack of significant interaction between factors were rejected in the case of the calculated value of the F characteristic, which was higher than the boundary value at a significance level of α = 0.05. The individual quality parameter levels of cow’s milk bio-yoghurts were determined at four points: T0, T1, T2, and T3, and then compared.. 3. Results and Discussion The effect of the temperature of pasteurisation used to manufacture bio‑yoghurts from cow’s milk was analysed. It was found that the temperature had a statistically significant impact on the count of Str. thermophilus and B. bifidum in the bio-yoghurt 12 hours after it was manufactured (Tab. 1). However, the type of packaging did not affect the parameters analysed (Tab. 1) because the milk drink was produced using a container method and simultaneously poured into all types of packagings, so the contact time between the product and the packaging was too short. Table 1. Results of the Variance Analysis – Impact of Pasteurisation Temperature and Type of Packaging at a T0 Time (12 Hours after Manufacturing) on the Bacteria Count in Bio‑yoghurts of Cow’s Milk Bacteria L. acidophilus B. bifidum. Str. thermophilus. Impact of. pasteurisation temperature. type of packaging. 14.792*. 0.476. 0.040. 0.459. 4.351*. 1.031. [pasteur. temp.] × [pack.] 0.561. 1.443. 0.378. The * symbol means that the calculated value of F characteristic, is higher than the boundary value (α = 0.05).. Source: the author’s own research.. Increasing the pasteurisation temperature by 5ºC caused a statistically significant differentiation in the amount of B. bifidum and Str. thermophilus bacteria (Fig. 1 and 2). The incubation of these bacteria multiplied more in the bio-yoghurt produced from cow’s milk pasteurised at a temperature of 95ºC for 5 minutes and, consequently, 12 hours after manufacturing, there was a greater amount. This.

(4) Wanda Kudełka. 82. B. bifidum [log cfu/ml]. 8,0 7,5 7,0. 6,5 6,0 5,5 5,0. 4,5 4,0. 90. Pasteurisation temperature [°C] PP. PS. PE. 95. G. Fig. 1. Impact of Pasteurisation Temperature on the Count of B. bifidum Cells in Cow’s Milk Bio-yoghurt in Different Types of Packaging 12 Hours after It Was Manufactured. Str. thermophilus [log cfu/ml]. Source: the author’s own research. 9,6. 9,4. 9,2 9,0. 8,8 8,6. 8,4. 8,2 8,0. 90. Pasteurisation temperature [°C] PP. PS. PE. 95. G. Fig. 2. Impact of Pasteurisation Temperature on the Number of Str. thermophilus Cells in Cow’s Milk Bio-yoghurt in Different Types of Packaging 12 Hours after It Was Manufactured Source: the author’s own research.. speaks to the value of operating at a higher temperature, which causes proteins to denature faster and low-molecular compounds to release, among other things, nitrogen compounds such as peptides, free amino acids, amino sugars, and lactulose, which is produced by lactose. These compounds stimulate the development of.

(5) The Impact of Selected Factors on the Survival…. 83. micro-flora added to the yoghurt (the micro-flora utilise the compounds to grow) (Wiatr-Szczepanik & Libudzisz 1997; Ziajka & Dzwolak 1997; Żbikowski 1997). After 7 days of storage, the packaging still played no role in determining the survival rate of probiotic bacteria, but the pasteurisation variant of raw material continued to have a statistically significant effect on the multiplication power, though only on the B. bifidum bacteria (Tab. 2). Table 2. Results of the Variance Analysis – Impact of Pasteurisation Temperature and Type of Packaging at a T1 Time (7 Days after Manufacturing) on the Survival Rate of Probiotic Bacteria in Cow’s Milk Bio-yoghurt Bacteria L. acidophilus. Impact of. pasteurisation temperature. type of packaging. 4.553*. 0.405. 0.414. B. bifidum. 1.776. 0.838. Str. thermophilus. [pasteur. temp.] × [pack.] 1.755. 0.973. 1.118. 0.602. The * symbol means that the calculated value of F characteristic, is higher than the boundary value (α = 0.05).. Source: the author’s own research.. B. bifidum [log cfu/ml]. 8,0 7,5 7,0. 6,5 6,0 5,5 5,0. 4,5 4,0. 90. Pasteurisation temperature [°C] PP. PS. PE. 95. G. Fig. 3. Impact of Pasteurisation Temperature on the Count of B. bifidum Cells in Cow’s Milk Bio-yoghurt in Different Types of Packages 7 Days after It Was Manufactured. Source: the author’s own research.. Following the above storage period, a differentiation was observed in the count of the B. bifidum genus which depended on the pasteurisation temperature of raw material, again demonstrating the virtue of operating at a raised temperature.

(6) Wanda Kudełka. 84. (Fig. 3); the differentiation resulting from the type of packaging was statistically insignificant. After 14 days of storage, the pasteurisation temperature of cow’s milk had a statistically significant effect on the B. bifidum count in bio-yoghurt irrespective of the type of packaging (Tab. 3). Those bacteria multiplied more and the quantity was higher in the bio-yoghurt produced using the pasteurisation variant of 95ºC/5 minutes (Fig. 4). This can be attributed to the bacteria having quicker access to components they utilise to grow (the low-molecular compounds, which are essential to the survival of the bacteria, release quicker) (Wiatr-Szczepanik & Libudzisz 1997; Ziajka & Dzwolak 1997; Żbikowski 1997). Table 3. Results of the Variance Analysis – Impact of Pasteurisation Temperature and Type of Packaging at a T2 Time (14 Days after Manufacturing) on the Survival Rate of Probiotic Bacteria in Cow’s Milk Bio-yoghurt Bacteria L. acidophilus. Impact of. pasteurisation temperature. type of packaging. 11.849*. 0.348. 0.388. B. bifidum. 1.293. 0.748. Str. thermophilus. [pasteur. temp.] × [pack.] 0.197. 0.635. 0.175. 0.110. The * symbol means that the calculated value of the F characteristic is higher than the boundary value (α = 0.05). Source: the author’s own research.. B. bifidum [log cfu/ml]. 8,0 7,5 7,0. 6,5 6,0 5,5 5,0. 4,5 4,0. 90. Pasteurisation temperature [°C] PP. PS. PE. 95. G. Fig. 4. Impact of the Pasteurisation Temperature on the Count of B. bifidum Cells in Cow’s Milk Bio-yoghurt in Different Types of Packaging 14 Days after It Was Manufactured Source: the author’s own research..

(7) The Impact of Selected Factors on the Survival…. 85. Table 4. Results of the Variance Analysis – Impact of Pasteurisation Temperature and Type of Packaging at a T3 Time (21 Days after Manufacturing) on the Survival Rate of Probiotic Bacteria in Cow’s Milk Bio-yoghurt Bacteria L. acidophilus B. bifidum. Str. thermophilus. pasteurisation temperature 0.658 1.393 0.016. Impact of type of packaging 1.520. 0.389. 0.325. [pasteur. temp.] × [pack.] 0.774. 1.079 0.653. The * symbol means that the calculated value of F characteristic, is higher than the boundary value (α = 0.05).. Source: the author’s own research.. On the last day of the storage of the bio-yoghurts, no statistically significant effect of the pasteurisation temperature or of the type of packaging was observed on the probiotic bacteria count in the drinks studied (Tab. 4).. 4. Conclusion The pasteurisation temperature had a statistically significant effect on the count of both the Str. thermophilous and the B. bifidum populations only 12 hours after the drinks were manufactured. However, the effect lasted only until the fourteenth day of storage (on the last day of storage, the effect was statistically insignificant). A higher pasteurisation temperature of raw material had a favourable effect on the B. bifidum population as there was more of it in the bio-yoghurts studied. Thus, it is recommended a higher pasteurisation temperature be applied during the production process so as to ensure a higher count of B. bifidum bacteria during storage. Irrespective of the applied pasteurisation temperature of raw material, the population of the L. acidophilus and Str. thermophilus bacteria remained at a level of above 106 cfu/ml, which is the therapeutic minimum of the drinks investigated. Conversely, the survival rate of B. bifidum was always below this value regardless of the pasteurisation temperature applied. Over the 21-day storage period, the type of packaging used did not affect the count of bacteria present in the drinks investigated..

(8) 86. Wanda Kudełka. Bibliography Dave R.I., Shah N.P. (1996), Evaluation of Media for Selective Enumeration of Streptococcus Thermophillus, Lactobacillus Delbrueckii Subsp. Bulgaricus, Lactobacillus Acidophilus and Bifidobacteria, “Journal of Dairy Science”, No 79. Dave R.I., Shah N.P. (1997), Viability of Yoghurt and Probiotic Bacteria in Yoghurts Made from Commercial Starter Cultures, “International Dairy Journal”, No 7. Defecińska A., Libudzisz Z. (2000), Bakterie fermentacji mlekowej – wpływ na funkcje życiowe człowieka, “Przegląd Mleczarski”, No 8. Donnet-Hughes A., Rochat F., Serrant P., Aseschlimann J.M., Schiffrin E.J. (1999), Modulation of Nonspecific Mechanisms of Defense by Lactic Acid Bacteria: Effective Dose, “Journal of Dairy Science”, No 82. Dunne C., Murphy L., Flynn S. (1999), Probiotics: from Myth to Realisty. Demonstration of Functionality in Animal Models and in Human Clinical Trials, “Antonie van Leeuwenhoek”, No 76. Holzapfel W.H., Haberer P., Snel J., Schillinger U., Huits in’t Veld J. (1998), Overview of Gut Flora and Probiotics, “International Journal Food Microbiology”, No 41. Isolauri E. (2001), Probiotics in Human Disease, “American Journal of Clinical Nutrition”, No 73. Isolauri E., Arvola T., Sütas Y., Salminen S. (2000), Probiotics in the Management of Atopic Eczema, “Clinical Experimental Allergy”, No 30. Kleessen B., Bezirtzoglou E., Matto J. (2000), Culture-based Knowledge on Biodiversity, Development and Stability of Human Gastrointestinal Microflora, “Microbial Ecology in Health and Disease”, No 12, suppl. 2. Lamoureux L., Roy D., Gauthier S.F. (2002), Production of Oligosaccharides in Yogurt Containing Bifidobacteria and Yogurt Cultures, “Journal of Dairy Science”, No 85. Lankaputhra W.E.V., Shah N.P. (1995), Survival of Lactobacillus Acidophilus and Bifidobacterium Spp. in the Presence of Acid and Bile Salts, “Cultured Dairy Products Journal”, No 30. Lankaputhra W.E.V., Shah N.P. (1996), A Simple Method for Selective Enumeration of Lactobacillus Acidophilus in Yoghurt Supplemented with L. Acidophilus and Bifidobacterium Spp., “Milchwissenschaft”, No 51. Lankaputhra W.E.V., Shah N.P., Britz M.L. (1996), Survival of Bifidobacteria During Refrigerated Storage in the Presence of Acid and Hydrogen Peroxide, “Milchwissenschaft”, No 51. Ouwehand A.C., Salminen S.J. (1998), The Health Effects of Cultured Milk Products with Viable and Nonviable Bacteria, “International Dairy Journal”, No 8. Ouwehand A.C., Salminen S.J., Isolauri E. (2002), Probiotics: An Overview of Beneficial Effects, “Antonie van Leeuwenhoek”, No 82. Pessi T., Sütas Y., Hurme M., Isolauri E. (2000), Interleukin-10 Generation in Atopic Children Following Oral Lactobacillus Rhamnosus GG, “Clinical Experimental Allergy”, No 30. Shin H.S., Lee J.H., Pestka J.J., Ustunol Z. (2000), Viability of Bifidobacteria in Commercial Dairy Products During Refrigerated Storage, “Journal Food Protection”, No 63. Tharmaraj N., Shah N.P. (2003), Selective Enumeration of Lactobacillus Delbrueckii Ssp. Bulgaricus, Streptococcus Thermophilus, Lactobacillus Acidophilus, Bifidobacteria,.

(9) The Impact of Selected Factors on the Survival…. 87. Lactobacillus Casei, Lactobacillus Rhamnosus and Propionibacteria, “Journal of Dairy Science”, No 86. Wiatr-Szczepanik A., Libudzisz Z. (1997), Porównanie wzrostu i aktywności kwaszącej szczepów Lb. acidophilus w mleku kozim i krowim, “Przegląd Mleczarski”, No 6. Ziajka S., Dzwolak W. (1997), Próba określenia właściwości fermentacyjnych i proteolitycznych bifidobakterii, “Przegląd Mleczarski”, No 3. Żbikowski Z. (1997), Nowoczesne trendy w technologii produkcji jogurtu, “Przegląd Mleczarski”, No 3.. Wpływ wybranych czynników na przeżywalność bakterii probiotycznych w biojogurtach naturalnych z mleka krowiego Celem badań było określenie przeżywalności bakterii probiotycznych w biojogurtach naturalnych z mleka krowiego w zależności od zastosowanego wariantu pasteryzacji surowca i rodzaju opakowania. Według zaleceń FAO/WHO przeżywalność bakterii probiotycznych w ostatnim dniu przydatności nie może być niższa niż 106 jtk/ml. Taka liczba bakterii jest minimum terapeutycznym bionapojów. Rodzaj zastosowanego opakowania biojogurtów naturalnych nie wpływał statystycznie istotnie na liczbę i przeżywalność bakterii probiotycznych. Natomiast temperatura pasteryzacji miała statystycznie istotny wpływ, ale jedynie na przeżywalność bakterii B. bifidum – zastosowanie wyższej temperatury sprzyjało wzrostowi populacji tych bakterii. W odniesieniu do liczby Str. thermophilus wpływ ten zauważono jedynie 12 godzin po wyprodukowaniu..

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