The sterilization conditions between experiment and dairy industrial level are different and concern about influence on product’s qualities. In this study, the fermentation properties of Streptococcus thermophilus , alone and in combination with Lactobacillus delbrueckii subsp. bulgaricus , were evaluated in skim milk that had been subjected to distinct sterilization conditions. Growth, organic acid generation, and EPS production were determined using pasteurized or autoclaved milk. When S. thermophilus was cultivated in pasteurized skim milk, the growth was strain-dependent. On the other hand, growth of S. thermophilus was accelerated in autoclaved milk. Exocellular polysaccharide (EPS) production by L. bulgaricus was not affected by the combination of S. thermophilus strains. Thus, we observed that yogurt fermented by L. bulgaricus was minimally affected by the combination of S. thermophilus strains; growth of L. bulgaricus was maintained under the constant environment. These results should facilitate the development of fermented milk produced from L. bulgaricus in the dairy industry.
The international standards of the WHO/FAO define yogurt as milk fermented with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and production of yogurt depends on a symbiosis established between the two species [
Both S. thermophilus and L. bulgaricus are able to produce lactic acid by homo-fermentation of carbohydrates. However, it has been found that Streptococcus genus possesses Pyruvate Formate-Lyase (PFL) and is able to produce formic acid in glycolysis. Namely, S. thermophilus can’t produce just lactic acid, or formic acid in aerobic condition. The organic acid profile of bacteria has been shown to change according to metabolic activity [
We have been investigating the use of probiotic strain L. bulgaricus OLL1073R-1 as a commercial yogurt starter. This strain produces exocellular polysaccharide (EPS), and the physiology of host immunological response has been elucidated [
Streptococcus thermophilus NIAI510 was obtained from National Institute of Animal Industry. Streptococcus thermophilus ME-551 and Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 were obtained from Meiji Co., Ltd. (Tokyo, Japan). The strains were propagated twice in autoclaved (115˚C, 15 min) 10% (w/v) skim milk. The cultures were inoculated into pasteurized (63˚C, 30 min) or autoclaved 10% skim milk and incubated at 37˚C for 24 hr.
The strains were propagated twice in autoclaved 10% skim milk and inoculated at 1% (v/v) to pasteurized or autoclaved skim milk. The tubes were incubated at 37˚C. After incubation for 3, 6, 9, 12, or 24 hr, cultures were examined for pH and total cell numbers. The pH was recorded with a pH meter (pH/ION meter F-24, HORIBA, Tokyo, Japan). Total cell numbers were determined using optical densities (ODs) at 600 nm. In the case of skim milk culture, ODs were determined after mixing one volume of culture with nine volumes of a solution composed of 0.2% (w/v) NaOH and 0.2% sodium EDTA according to the method of Sander et al. (2010). ODs were monitored using a UV-mini1240 spectrophotometer (Shimazu Co., Kyoto, Japan).
After 24 hr of incubation, analyze of organic acids in the culture was outsourced to the Kyoto Institute of Nutrition and Pathology Inc. (Kyoto, Japan). Levels of succinic acid, lactic acid, formic acid, and acetic acid were determined by ion-exclusion chromatography using HPLC. The amounts of each organic acid were estimated by comparing retention times and peak areas to those obtained using standards.
The concentrations of EPS in the cultures were determined as described elsewhere [
The strains, which had been cultivated in autoclaved skim milk, were inoculated at 2% (v/v) into pasteurized or autoclaved skim milk. The tubes were incubated for 24 hr at 37˚C. The samples then were serially diluted ten-fold using sterile physiological saline (0.85% NaCl (w/v)) to quantify the cell numbers of both strains. Diluted solutions (100 μl) of the co-cultures were spread on MRS agar and M17 agar plates for detection of L. bulgaricus and S. thermophilus, respectively. The plates were incubated at 37˚C for 48 hr under anaerobic condition with Anaero Pack (Sugiyama-Gen, Co., Ltd., Tokyo, Japan) and the colony forming units (CFU/ml) were enumerated.
Statistical significance in differences was determined using the Student’s t-test. The test was performed as two- tailed and P value was considered statistically significant difference less than 0.05.
We determined acidification and turbidity in skim milk of S. thermophilus strains in order to understand their fermentation pattern in substrates subjected to different sterilization conditions. The growth of the two S. thermophilus strains differed in skim milk sterilized by pasteurization, and the difference was starting at 6 hr after inoculation (
It is well known that formic acid generated by S. thermophilus is essential for the growth of L. bulgaricus. We detected organic acids generated by fermentation (
To investigate the influence of mixed culture with S. thermophilus and L. bulgaricus in skim milk treated under different conditions, i.e. pasteurized or autoclaved sterilization, viable cell numbers were counted (
Succinic acid | Lactic acid | Formic acid | Acetic acid | |
---|---|---|---|---|
Pasteurized skim milk (63˚C, 30 min) | ||||
Control | 0.085 | 0.079 | 0.014 | 0.000 |
S. thermophilus NIAI510 | 0.038 | 64.511 | 0.201 | 0.278 |
S. thermophilus ME-551 | 0.089 | 40.420 | 0.253 | 0.363 |
L. bulgaricus OLL1073R-1 | 0.981 | 63.263 | 0.000 | 1.974 |
L. bulgaricus OLL1073R-1 + S. thermophilus NIAI510 | 0.853 | 78.752 | 0.000 | 1.691 |
L. bulgaricus OLL1073R-1 + S. thermophilus ME-551 | 0.651 | 82.391 | 0.000 | 1.795 |
Autoclaved skim milk (115˚C, 15 min) | ||||
Control | 0.088 | 0.000 | 2.101 | 0.142 |
S. thermophilus NIAI510 | 0.037 | 64.553 | 1.247 | 0.319 |
S. thermophilus ME-551 | 0.126 | 55.124 | 3.083 | 0.856 |
L. bulgaricus OLL1073R-1 | 0.445 | 77.593 | 0.597 | 1.744 |
L. bulgaricus OLL1073R-1 + S. thermophilus NIAI510 | 0.697 | 83.162 | 0.316 | 1.870 |
L. bulgaricus OLL1073R-1 + S. thermophilus ME-551 | 0.568 | 81.998 | 0.549 | 1.841 |
The values are showed the concentration of each organic acid as mM.
Pasteurized skim milk (63˚C, 30 min) (CFU/ml) | Autoclaved skim milk (115˚C, 15 min) (CFU/ml) | |
---|---|---|
OLL1073R-1 | 9.87 × 108 | 4.20 × 109 |
OLL1073R-1 + ME-551 | ||
OLL1073R-1 | 4.04 × 109 | 4.03 × 109 |
ME-551 | 7.93 × 107 | <107 |
OLL1073R-1 + NIAI510 | ||
OLL1073R-1 | 2.10 × 109 | 6.27 × 109 |
NIAI510 | 1.82 × 108 | <107 |
The data shows the mean of three plates.
After co-cultivation for 24 hr in pasteurized milk, cell proliferation of L. bulgaricus was stimulated and L. bulgaricus cell numbers increased (compared to mono-culture) four- or two-fold upon co-culturing with S. thermophilus ME-551 or NIAI510, respectively. When L. bulgaricus was cultivated with S. thermophilus in autoclaved milk, the L. bulgaricus cell numbers also were enhanced. However, under these conditions, no S. thermophilus colonies were detected by replica plating, meaning that cell numbers fell below 107 CFU/ml (the limit of detection in our assay).
L. bulgaricus OLL1073R-1 is a starter used for the manufacture of yogurt and is hypothesized to confer benefits on the host by producing EPS. To investigate influence of the medium sterilization and co-culturing on EPS production by L. bulgaricus OLL1073R-1, the amount of EPS generated by L. bulgaricus OLL1073R-1 was determined on skim milk subjected to different sterilization conditions and co-cultivated with S. thermophilus (
The sterilization condition of milk can differ greatly between experimental and manufacturing environments. Industrial sterilization has been designed to preclude the denaturation of milk proteins as much as possible. On the other hand, milk-based medium in laboratory usually is sterilized by autoclaving at 110˚C - 115˚C for 10 to 15 min. The pressure on these conditions were about 1.3 atm (110˚C) and 1.6 atm (115˚C), it have been considered that milk components after autoclaving are degraded by both temperature and pressure. Thus, laboratory- derived experimental data may not be directly applicable for manufacturing purposes. In the present study, we investigated the difference between pasteurized and autoclaved skim milk as a medium for growth of yogurt starter cultures. In our hands, two different S. thermophilus strains yielded similar growth patterns when cultured in autoclaved skim milk, whereas the two strains yielded distinct growth patterns when cultured in pasteurized skim milk. And our tests revealed that the levels of lactic and formic acids in skim milk were higher following autoclaving (113.3˚C, 10 min); previous work demonstrated that propionic, pyruvic, butyric, and acetic acids also were increased following autoclaving [
S. thermophilus and L. bulgaricus are widely used as LAB in the dairy industry, and these species are known to form a symbiosis. To elucidate the cooperative growth of these species, we investigated the fermentation properties of S. thermophilus strains, focusing on the generation of organic acids. We observed that two separate S. thermophilus strains (ME-551 and NIAI510) generated 0.2 - 0.25 mM formic acid and 0.3 - 0.4 mM acetic acid in the course of 24 hr of growth in pasteurized milk; neither of the tested strains produced detectable levels of succinic acid in cultures growing on pasteurized milk. In autoclaved skim milk, ME-551 was able to produce organic acids to higher levels than was NIAI510. In a previous report, Streptococcus mutans was shown to produce higher levels of formic acid and lower levels of lactic acid under anaerobic conditions [
Qualitative analysis of organic acids in L. bulgaricus OLL1073R-1 mono-culture and in co-culture with S. thermophilus also was performed. Succinic and acetic acid levels were higher in the mono-culture of L. bulgaricus OLL1073R-1 on pasteurized milk compared to that on autoclaved milk; in particular, the concentration of succinic acid was remarkably increased on pasteurized milk. It has been recognized that L. bulgaricus is one kind of homofermentative lactic acid bacteria, in which only lactic acid is generated from pyruvic acid on glycolysis. On the other hand, hetero-fermentation is able to produce acetic acid and ethanol other than lactic acid via hexose monophosphate pathway. Especially, acetic acid is generated from acetyl-phosphate. In Lactobacillus plantarum, succinic acid is generated from phosphoenol pyruvic acid, pyruvic acid, and acetyl CoA via the TCA cycle, especially under anaerobic condition [
Furthermore, L. bulgaricus cell numbers exceeded those of S. thermophilus after co-cultivation for 24 hr. Previous reports indicated that the viable cell numbers of S. thermophilus in yogurt were higher than those of L. bulgaricus [
In addition, we investigated the effect of co-culturing with S. thermophilus on EPS production by L. bulgaricus OLL1073R-1. We showed that EPS production was influenced by the sterilization condition used, and did not differ for the two different S. thermophilus strains used for co-cultivation. In our previous study, we demonstrated that L. bulgaricus OLL1073R-1 cultivated in formate-containing medium produced EPS at levels four- fold greater than those of a control [
Thus, we have demonstrated that the process used for sterilization of milk for yogurt manufacture has large effects on the growth of the starter cultures, the level of functional EPS production, and the required time of fermentation. These data will facilitate improved economics for the dairy food industry.
The influences of different sterilization conditions of milk against dairy products have not been investigated. The present study demonstrated the growth of S. thermophilus and L. bulgaricus, and EPS production by L. bulgaricus, when grown on milk subjected to different sterilization processes. We showed that the growth and organic acid generation or consumption by both strains varied according to sterilization conditions. In addition, the yield of EPS produced from L. bulgaricus was influenced by sterilization condition rather than the specific identity of the co-cultured S. thermophilus strain. Our results are expected to provide fundamental data for use by the dairy industry.
This work was supported by a grant from the Food Science Institute Foundation (Ryoshokukenkyukai) to Prof. Saito.
JunkoNishimura,SeiyaMakino,KatsunoriKimura,EmikoIsogai,TadaoSaito, (2015) Influence of Different Sterilization Conditions on the Growth and Exopolysaccharide of Streptococcus thermophilus and Co-Cultivation with Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1. Advances in Microbiology,05,760-767. doi: 10.4236/aim.2015.511080