Evaluation of the yield,molar mass of exopolysaccharides,and rheological properties of gels formed during fermentation of milk by Streptococcus thermophilus strains St-143 and ST-10255y

The yield and chemical structures of exopolysaccharides (EPS) produced by many strains of Streptococcus thermophilus have been characterized. However, the kinetics (or production profile) for EPS during milk fermentation is not clear. In this study, we investigated whether any differences existed in the yield and molar mass of EPS when milk was fermented at the same acidification rate by 2 strains of S. thermophilus (St-143 and ST-10255y). The type of EPS produced by these 2 strains is different. Milk samples were analyzed for EPS concentration every 30 min during a fermentation period of 270 min (final pH 4.5) by using a modified quantification method, which was faster and validated for its recovery of added EPS. Rheological properties of milks during fermentation were also analyzed using small-strain dynamic oscillatory rheology. For the determination of molar mass, EPS extracts were isolated by ultrafiltration of whey obtained during fermentation of milk to pH values 5.2, 4.9, 4.7, and 4.5, and molar mass was analyzed using size-exclusion chromatography–multi-angle laser light scattering. During fermentation, both strains appeared to start producing significant amounts of EPS after about ～150 min, which corresponded to pH ～5.3, which was close to the point of gelation. During the remainder of the fermentation process (150–270 min), the EPS concentration from strains St-143 and ST-10255y significantly increased from 30 to 72 mg/L and from 26 to 56 mg/L, respectively. The quantity of EPS recovered by our modified method was estimated to represent ～60% of the total EPS added to milk. The molar mass of EPS produced by both strains appeared to slightly decrease during fermentation. At pH 5.2, EPS from St-143 and ST-10255y had molar masses of 2.9 × 10⁶ and 1.4 × 10⁶ g/mol, respectively, which decreased to 1.6 × 10⁶ and 0.8 × 10⁶ g/mol, respectively, when the pH of milk was 4.5. Distinct differences were apparent in the rheological properties of gels fermented by the 2 strains. At the end of fermentation, St-143 fermented milk had weaker gels with storage modulus (G′) value at pH 4.6 of 26 Pa, whereas gels made with ST-10255y were stiffer with a G′ value at pH 4.6 of 82 Pa. For St-143 gels, maximum loss tangent (LT_max) values were higher (0.50) and occurred earlier (at a higher pH value) than the LT_max values (0.46) for gels from ST-10255y strain. Because the fermentation conditions were identical for both strains, the observed changes in rheological properties could be due to the differences in chemical structures and molar mass of the EPS produced by these 2 S. thermophilus strains.     

Isolation and identification of high viscosity-producing lactic acid bacteria from a traditional fermented milk in Xinjiang and its role in fermentation process

Forty-four lactic acid bacteria (LAB) isolated from traditional Sayram ropy fermented milk (SRFM) in southern Xinjiang of China. Further two strains were selected based on their high viscosity-producing activity. Based on the API 50 CHL strip and 16S rDNA sequence analysis, strain MB 2-1 was a Gram-positive, rod-shaped Lactobacillus helveticus and strain MB 5-1 was identified as Streptococcus thermophilus. Both the two LAB strains were grown in the milk fermentations for pure and mixed cultures and were evaluated for their growth, acidification properties, EPS production, and ability to increase the apparent viscosity of fermented milk, respectively. L. helveticus MB 2-1 displayed a high increasing in viable cell counts and the acidifying capacity in pure cultures, whereas the relatively high EPS production and viscosity-producing capacity detected in pure culture with S. thermophilus MB 5-1 as starter culture. In addition, the mixed culture of the two strains showed a higher cell growth, EPS production, and high viscosity-producing capacity at 37?°C. Values of apparent viscosity were 4.03- and 2.41-fold higher in mixed culture than for pure cultures of L. helveticus MB 2-1 and S. thermophilus MB 5-1, respectively. There was a positive correlation between the viscosity and high molecular weight EPS production with pure and mixed cultures. Our data showed two strains combination, with high viscosity-producing and acidifying capacity, can be used in mixed cultures for the manufacture of fermented milk with improved functional properties.     

Altered nucleotide sugar metabolism in Streptococcus thermophilus interferes with nitrogen metabolism

Exopolysaccharide (EPS)-producing Streptococcus thermophilus strains have attracted interest recently, since the EPSs act as natural viscosifiers and texture enhancers of fermented foods. We have previously reported that the low level of EPS production by S. thermophilus LY03 could be improved by altering the activities of enzymes in the central carbon metabolism involved in the nucleotide sugar metabolism. In this study, we observed a reduced growth in milk for the strains with increased UDP-glucose pyrophosphorylase (GalU) activity together with either enhanced phosphoglucomutase activity, and/or enhanced activity of the Leloir enzymes. Rapid growth of these mutants in milk could be restored by the addition of four specific amino acids, i.e. Glu, His, Met, and Val. This amino acid requirement was confirmed in a defined medium. Furthermore, the 31P NMR spectra showed higher levels of the GalU reactants pyrophosphate (PPi) and UDP-glucose in the engineered strain, TMB 6013, compared to the parent strain, LY03. These products plus Glu and the GalU reactant UTP are known to be involved in the nitrogen regulatory system in many bacteria. Thus, these results suggest that the reaction catalyzed by GalU is connected to the nitrogen demand of these engineered strains.     

High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts

Streptococcus thermophilus is a commonly used starter bacterium in dairy industry. It reduces the pH of milk rapidly and equilibrates the medium for the growth of Lactobacillus delbrueckii subsp. bulgaricus during yoghurt fermentation. Efforts to increase the diversity of artisanal yoghurt starters are not only important to bring new strains with novel and desirable characteristics, but also for the preservation of natural diversity which diminishes with the overuse and spread of industrial starters to natural resources. In the present study, 14 artisanal yoghurt samples were processed for the isolation of promising strains for yoghurt starter culture production and 66 strains were subsequently characterized. They were all identified as S. thermophilus using species-specific PCR and 16S rRNA gene sequencing. Genotypic diversity at the strain level was investigated by pulsed field gel electrophoresis (PFGE), and 22 homology groups were obtained. Further phenotypic characterization unearthed a significant phenotypic heterogeneity within homology groups, mostly with atypical novel character. Only 7 out of 66 strains showed S. thermophilus type-strain like phenotypic traits. Majority of the isolates were determined to be protease positive and fast milk acidifier to be used as yoghurt starter culture.     

Effect of exopolysaccharide-producing starter cultures and post-fermentation mechanical treatment on textural properties and microstructure of low fat yoghurt

The microstructure and texture of yoghurts produced by four different exopolysaccharide (EPS)-producing starter cultures and mechanically treated post-fermentation at four levels of intensity (applied back-pressure) were studied. Two Lactobacillus delbrueckii ssp. bulgaricus (LB) strains were used in combination with two Streptococcus thermophilus (ST) strains and yoghurts were formulated by pairwise combining one LB and one ST strain. The choice of ST strain was the major determinant for the rheological properties of the yoghurts, since one of the ST strains conferred a ropy texture and resulted in yoghurts with decreased water holding capacity and an open microstructure. In addition, one of the LB strains used produced both aggregated and threadlike EPS and improved water holding. When combined with an ST strain that produced neglible amounts of EPS this LB strain resulted in yoghurt where a moderate mechanical treatment post-fermentation was able to further improve the water holding capacity.     

Gelation and resistance to shearing of fermented milk: Role of exopolysaccharides

Milk was fermented with the exopolysaccharide-producing (EPS⁺) strains Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and with the non-EPS-producing strain (EPS⁻) L. lactis subsp. cremoris. The kinetics of gelation and the behaviour of set fermented milk during and after shearing were studied using rheometry and confocal scanning laser microscopy. The time of gelation of milk depended on the kinetics of acidification of strains whereas the pH of gelation depended mostly on the presence of exopolysaccharides (EPS). In set fermented milk with EPS⁺ strains, bacteria were observed in protein-free areas likely filled with EPS. Phase-separated EPS and caseins contributed to induce the gelation of fermented milk at pH 5.6. The high resistance to shearing of milk fermented with the EPS⁺ strain L. lactis subsp. cremoris might be due to the negative charge of the exopolysaccharide allowing an attractive interaction with caseins.     

Comparison of volatile metabolic profiles in fermented milk of Streptococcus thermophilus during the postripening period at different incubation temperatures

Streptococcus thermophilus has been extensively applied in fermented milk. This study used gas chromatography-ion mobility spectroscopy to determine and evaluate the volatile metabolites in raw milk, milk fermented at 37°C, and milk fermented at 42°C. Ten discriminatory volatile metabolites were identified at different incubation temperatures: acetone, 2-heptanone, 2-pentanone, 2-hexanone, butanal, hexanal, ethyl acetate, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropanoic acid, indicating that fermentation temperature affected the spectrum of volatiles in milk fermented by different strains of S. thermophilus. Specifically, fermentation at 37°C led to accumulation of short-chain fatty acids, whereas fermentation at 42°C enriched ketones and other flavor substances in the fermented milk, enhancing the flavor of the product. This work examined the differences between the volatile metabolites produced by different S. thermophilus strains fermented at different temperatures to evaluate the effect of temperature on the metabolic pathways.     

Development of a dairy-based exopolysaccharide bioingredient

An exopolysaccharide (EPS)-producing Streptococcus thermophilus was used to develop an “all-dairy” ingredient with increased EPS content and greater functionality for dairy applications such as Mozzarella cheese. In laboratory-scale trials, milk protein hydrolysate (MPH), whey protein isolate (WPI) and whey protein hydrolysate (WPH) in reconstituted low-heat skim milk powder (LHSMP) were examined as additional dairy protein sources to increase the production of EPS. WPH supplementation resulted in the greatest EPS production. In batch fermentation, a medium based on LHSMP and WPH, combined with optimum conditions of temperature and pH, was used to obtain maximum production of EPS from S. thermophilus. EPS production was growth associated. The fermented biomass was harvested at the end of the exponential phase and freeze-dried. The reduced viable cell count and the retention of ropiness of the powder from the drying process enabled a higher level of EPS inoculation in a preliminary Mozzarella cheese manufacturing trial.     

Effect of fermentation temperature on the properties of exopolysaccharides and the acid gelation behavior for milk fermented by Streptococcus thermophilus strains DGCC7785 and St-143

We investigated the effect of fermenting milk with 2 strains (DGCC7785 and St-143) of Streptococcus thermophilus, which are known to produce different types of exopolysaccharide (EPS) structures. The yields and physical properties of these ropy EPS were monitored during the fermentation of milk at different temperatures. We wanted to understand how these types of EPS properties affected yogurt gelation. Reconstituted skim milk was fermented at 33, 39, or 45°C until pH values reached 5.2, 4.9, 4.7, and 4.5. Molar mass of ropy EPS samples was determined using size exclusion chromatography coupled with multiangle laser light scattering. Rheological properties of fermented milk gels were analyzed using small-strain dynamic oscillatory measurements. In both strains, concentrations of ropy EPS increased during fermentation and at all temperatures. Fermentation times, by both strains, were shortest at 45°C and longest at 33°C. For both strains, molar mass of ropy EPS ranged from 2 to 4 × 10⁶ g/mol during fermentation. A major proteinaceous contaminant that was co-isolated with the ropy EPS fraction by our isolation method was identified as a milk-derived phosphoglycoprotein PP3. Increase in fermentation temperature from 33 to 45°C significantly decreased the storage modulus values (from 170 to 41 Pa) for milk gelled by strain DGCC7785, whereas the gels made with St-143 had very low storage modulus values (11–17 Pa) regardless of fermentation temperatures. For both strains, the values of maximum loss tangent in the milk gels increased with fermentation temperature; the maximum loss tangent occurred at higher pH values when milk was fermented by strain DGCC7785. The specific type of EPS produced appeared to be responsible for the differences in yogurt texture rather than the concentration or molar mass of the EPS.     

Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-γ production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.     

Effect of mono or co-culture of EPS-producing Streptococcus thermophilus strains on the formation of acid milk gel and the appearance of texture defects

One acidifying (ST1) and two texturing strains (ST2 and ST3) of Streptococcus thermophilus were used as pure or co-cultures to identify and understand their effects on the structuring of acid milk gels and on the appearance of texture defects, i.e., syneresis and graininess. Symbiosis between specific texturing and acidifying strains reduced acidification time and increased exocellular polysaccharide (EPS) content. The texturing strain could simultaneously produce low and high molar mass EPS and their distribution in mass and/or in number were influenced by the proportion of acidifying to texturing strain used. The results of this study suggest that the high molar mass EPS contributes to acid gel firmness, but less so compared with the acidification rate. The ability of strain ST3 to prevent texture defects, specifically graininess, did not depend on the acidification kinetics or final EPS content, but rather on the structural properties of EPS and/or the bacterial chain morphology.     

Streptococcus thermophilus strains: Multifunctional lactic acid bacteria

Streptococcus thermophilus has “Generally Recognized as Safe” status and is extensively used for the manufacture of several important fermented dairy foods, including yoghurt and some cheese varieties (Swiss, Limburger, Brick). S. thermophilus also has a number of functional activities such as production of extracellular polysaccharides, bacteriocins and vitamins. In addition, it also has potential as a probiotic, as demonstrated by various health effects, transient survival, and moderate adherence in the gastrointestinal tract. Therefore, the natural diversity among S. thermophilus strains with respect to their capacity to produce different metabolites has the potential to be exploited beyond fermentation for lactic acid production.     

Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS⁺Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS⁺ strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS⁻) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.     

The effect of heteropolysaccharide‐producing strains of Streptococcus thermophilus on the texture and organoleptic properties of low‐fat yoghurt

Heteropolysaccharide‐producing Streptococcus thermophilus strains (LY03, ST 111, or 5077) were compared with nonropy S. thermophilus strains (NR or 5137), in respect of texture development of low‐fat yoghurt produced at 42°C. Whether milk solids were added or not, lower syneresis and improved texture and sensory properties were obtained in yoghurts produced with S. thermophilus LY03 or S. thermophilus 5077 compared with nonropy strains. Texture effects, lower syneresis and improved sensory properties because of the use S. thermophilus ST 111 were only noticeable in the presence of milk solids. Milk solids had no significant effect on the textural parameters of nonropy strains.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.     

Identification and characterization of lactic acid bacteria isolated from traditional cone yoghurt

Cone yoghurt is a yoghurt variety produced by adding only pine cones to milk without culture in a limited area of Turkey. The present study was conducted to identify and characterize lactic flora in traditional cone yoghurt. Morphological, cultural, physiological, biochemical, and genotypic characteristics were employed to identify lactic acid bacteria isolates from cones and cone yoghurts. Streptococcus salivarius subsp. thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) were obtained from both cones and yoghurts. Among the isolates, L. plantarum was frequently isolated except for these two bacteria (S. thermophilus and L. bulgaricus). The results indicate that the cone yoghurt has a mixed microflora contrary to the yoghurt produced by the addition of a starter culture and S. thermophilus, and L. bulgaricus in cone yoghurt originates from the pine cones.