The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH₃ and CO₂ that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH₃ or CO₂ supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO₂-responsive or CO₂-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO₂ for the fast- and slow-acidifying Strep. thermophilus and for the CO₂-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO₂ on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.     

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.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

Fortification of milk with plant extracts modifies the acidification and reducing capacities of yoghurt bacteria

The acidification and reducing capacities of yoghurt bacteria were evaluated in different plant extract‐enriched milk samples. The milk samples enriched with thyme and grape seed extracts exhibited the highest values of acidification capacity for Lactobacillus delbrueckii subsp. bulgaricus (LB) (0.0065 pH unit/min) and Streptococcus thermophilus (ST) (0.0068 pH unit/min). The highest values of reducing capacity were observed in thyme (?0.98 mV/min), grape (?1.92 mV/min) and green tea (?0.75 mV/min)‐enriched samples for LB, ST and mixed culture of LB + ST, respectively. The fortification of yoghurt with plant extracts modified the acidification and reducing activities of starters, thus changing the fermentation time and quality attributes of the product.     

Microbial and physiochemical changes in yoghurts containing different Lactobacillus delbrueckii subsp. bulgaricus strains in association with Lactobacillus plantarum as an adjunct culture

Two strains of slow and normal acid‐producing Lactobacillus delbrueckii subsp. bulgaricus, mixed separately with Streptococcus thermophilus, were used in the preparation of yoghurt with or without Lactobacillus plantarum A7. Viable cell counts of lactic bacteria, pH, lactic acid and acetic acid concentrations, soluble nitrogen content, spontaneous syneresis, viscosity and firmness were studied at selected points during the refrigerated storage period. Results showed that survival of L. plantarum A7 in yoghurt was independent of the acidification capability of L. delbrueckii subsp. bulgaricus strains which had already been characterised in their pure culture. Thus, in the preparation of probiotic yoghurt, using slow acid‐producing L. delbrueckii subsp. bulgaricus strain in combination with nondairy lactobacilli postfermentation acidification is not restricted but instability in other physicochemical properties of the yoghurt may be expected.     

Lactobacillus Gasseri LGZ 1029 in yogurt: rheological behaviour and volatile compound composition

Yogurt preserves and enhances nutritional value of milk. In this study, we have compared several strains to determine the physicochemical, sensory, rheological and aroma characteristics of different yogurts. We used Lactobacillus gasseri LGZ 1029 (LG), commercial probiotic L. rhamnosus (LGG) and traditional fermentation strains Streptococcus thermophilus and L. bulgaricus (SL). Results showed that the flavour and texture characteristics of mixed-strain yogurts were obviously better than in single-strain yogurts. Addition of LG increased pseudoplastic behaviour, as shown by Herschel–Bulkley model analysis of rheological behaviour. The LG + SL group also had both the highest viscosity consistency index and thickening ability. In addition, a total of 57 volatile compounds were detected in yogurts and the fermentation with the addition of LG was mainly affected by ketones. Our study suggested that a yogurt with new attributes can be produced by using LGZ 1029.     

Effect of high hydrostatic pressure treatment on the viability and acidification ability of lactic acid bacteria

The effect of high pressures of 100–450 MPa combined with temperatures of 20–40 °C on Lactobacillus delbrueckii subsp. bulgaricus ACA-DC0105, Streptococcus thermophilus ACA-DC0022 and Lactococcus lactis ACA-DC0049 cell viability and acid production ability was studied. The rates of decrease in pH and cell viability were estimated for all the process combinations studied. The viability and acidification ability of the cells depended on the process conditions. More intense process conditions resulted in a lower number of viable cells and simultaneous reduction of lactic acid production correlated to lower rates of decrease of pH. Lb. bulgaricus appeared to be the microorganism most resistant to pressure, while Lc. lactis the most sensitive. Similar behaviour was observed for the acidification ability of these microorganisms, with Lc. lactis being least able to decrease the pH value of cheese. The HP-treated strains could be used as adjunct starters for cheese production.     

RECENT DEVELOPMENTS IN YOGHURT STARTERS: A NOTE ON THE SUPPRESSION OF LACTOBACILLUS BULGARICUS IN MEDIA CONTAINING β-GLYCEROPHOSPHATE AND APPLICATION OF SUCH MEDIA TO SELECTIVE ISOLATION OF STREPTOCOCCUS THERMOPHILUS FROM YOGHURT*

A medium devised primarily for lactic streptococci and their phages but also recommended for Lactobacillus bulgaricus was found to be inhibitory to many strains of L. bulgaricus. The inhibition was attributed to the buffering agent, β-glycerophosphate, in the medium and could also be demonstrated when this buffer was incorporated in other media. Since the glycerophosphate containing medium supported good growth of Streptococcus thermophilus it was applied successfully to selectively isolate this organism from yoghurt.     

Characteristics of the Bulgarian yoghurt lactic acid bacteria-metabolism of lactose

The investigation was made with more than 200 selected strains of lactic acid bacteria isolated from home-made yoghurt produced in the high-mountains regions of Bulgaria. 94% of them (LBD strains) were attributed to L. bulgaricus spicies and only 6% (KBL strains) were attributed to the species 5. thermophilus. The results showed that the LBD strains utilize glucose completely. The LBL strains partially utilize galactose and most (or all) glucose derived from lactose hydrolysis. The starters for Bulgarian yoghurt contain selected strains of L bulgaricus and S. thermophilus, and the starters for Yana yoghurt contain selected strains L. helveticus and S. thermophilus. So the symbiotic starters used for production of Bulgarian yoghurt, as well as those used for the production of Yana yoghurt, guarantee the obtaining of a product which contains over 80% L(+) lactate, and in some cases over 93% L(+) lactate. According to the references of WHO these kinds of yoghurt could be used in child's nutrition.     

Survival of Lactic Acid Bacteria and Their Lactases under Acidic Conditions

Effects of sonication, survival, and β-galactosidase activity of four lactic cultures were investigated in pH 1.5-3.5 range. Lactobacillus delbruekii subsp. bulgaricus and Streptococcus thermophilus exhibited the highest β-galactosidase activity in skim milk and broth systems, respectively. The β-galactosidases from L. delbruekii subsp. bulgaricus, S. thermophilus, and L. acidophilus showed optimum activity in the neutral pH range and 55°C. Viable count of all four cultures decreased most rapidly at pH 1.5, but L. acidophilus and L. delbruekii subsp. bulgaricus survived better than the other organisms. The decrease of enzyme activity of unsonicated cultures with pH was slight, especially at pH 3.5. However, acidification of sonicated cultures to pH 3.5 or lower resulted in rapid and permanent loss of enzyme activity.     

Probiotic strains: survival under simulated gastrointestinal conditions,in vitro adhesion to Caco-2 cells and effect on cytokine secretion

This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of Water Activity of Milk upon Growth and Acid Production by Mixed Cultures of Streptococcus thermophilus and Lactobacillus bulgaricus

Acid production and growth rates were determined in pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus and cocci/ bacilli ratios in mixed cultures of both species, using milk with modified a_w as culture medium. Maximal growth rates were always at a_w lower than 0.992 for all strains whcn a_w of milk was adjusted with glycerol, while maximal acid production rates were at a_w 0.992-0.983. Glucose had an inhibitory effect additional to that caused by lowering a_w. The cocci/bacilli ratio in mixed cultures increased as a_w decreased in most cases, because of the higher susceptibility of L. bulgaricus to a_w decrease. The behavior of a mixed culture could not always be predicted from data of the component strains in a pure culture, showing some kind of symbiotic compatibility between species.     

Relationship between Lactobacillus bulgaricus and Streptococcus thermophilus under whey conditions: Focus on amino acid formation

In this study, we performed microarray experiments using Lactobacillus delbrueckii subsp. bulgaricus 2038 cultured with or without Streptococcus thermophilus in whey medium to investigate nitrogen utilisation of these two strains, since Lb. bulgaricus 2038 use whey as their nitrogen before casein. For the utilisation of environmental free amino acids and peptides in co-culture, Lb. bulgaricus 2038 imported the types of peptides necessary for growth more effectively compared with monoculture because S. thermophilus primarily preferred different types of peptides than Lb. bulgaricus, but has lost the ability to prioritise the use of free branched-chain amino acids. Considering the ratio of nitrogen versus carbon being lower during co-culture, Lb. bulgaricus 2038 was induced to stop converting aspartate into carbon-skeleton intermediates which is catalysed by phosphoenolpyruvate carboxykinase. Despite mutualism, Lb. bulgaricus underwent competitive co-evolution with S. thermophilus to optimise nitrogen utilisation during co-cultivation.     

Proteolytic profiles of yogurt and probiotic bacteria

Nine strains of Streptococcus thermophilus, 6 strains of Lactobacillus delbrueckii ssp. bulgaricus, 14 strains of Lactobacillus acidophilus and 13 strains of Bifidobacterium spp. were screened for proteolytic, amino-, di-, tri- and endopeptidase activity by using the o-pthaldialdehyde-based spectrophotometric assay. Strains showing the highest and lowest proteolytic activity were further studied for their peptidase activities at the extracellular and intracellular levels. The amounts of free amino groups released by S. thermophilus, L. delbrueckii ssp. bulgaricus and L. acidophilus strains were higher than that by Bifidobacterium strains. Aminopeptidase activity was detected for all bacterial strains both at the extracellular and intracellular levels. The specific activity towards the six substrates studied was higher at the intracellular level for all strains. High dipeptidase activity was demonstrated by all bacterial strains for L. delbrueckii ssp. bulgaricus, L. acidophilus, and Bifidobacterium spp. whereas S. thermophilus had greater dipeptidase activity at the extracellular level. All bacterial cultures tested were able to hydrolyse large biologically active peptides, bradykinin, Ala–Ala–Ala–Ala–Ala and the tripeptide substrate Gly–Ala–Tyr at both the extracellular and intracellular levels. However, with the substrates ending with a C-terminal of phenylalanine, the hydrolysis only occurred at the intracellular level.     

Potential of LAB starter culture isolated from Chinese traditional fermented foods for yoghurt production

A total of 32 indigenous lactic acid bacteria (LAB) isolates were previously obtained from 7 samples of traditional dairy products in specific ecological niches throughout the northwestern area of China. Among the 32 isolates, 9 strains of LAB were selected for their potential in fermented dairy products and identified by 16S rDNA sequence analysis. In this study, these 9 isolates were combined each other and 8 combinations were selected according to their fermentation characteristics in yoghurt production. The effects of these 8 combinations on acidification, post acidification, texture and sensory quality of yoghurt were analysed. Results indicated that Streptococcus thermophilus SP1.1 combined with Lactobacillus delbrueckii ssp. bulgaricus 3 4.5 was the best combination and better than the commercial starter culture in terms of yoghurt fermentation time and sensory quality, suggesting that the combination has potential as a commercial starter to improve the quality of yoghurt.     

Physico-chemical,microbiological, and sensory characteristics of yogurt as affected by various ingredients

l-Glutamine, quercetin, slippery elm bark, marshmallow root, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate have been reported to help treat leaky gut. The purpose of this research was to explore the impact of these functional ingredients on the physico-chemical, microbiological, and sensory properties of yogurt. The milk from same source was equally divided into 9 pails and the 8 ingredients were randomly assigned to the 8 pails. The control had no ingredient. Milk was fermented to yogurt. The pH, titratable acidity, syneresis, viscosity, color (L*, a*, b*, C*, and h*), Streptococcus thermophilus counts, and Lactobacillus delbrueckii spp. bulgaricus counts of yogurts were determined on d 1, 7, 14, 21, 28, 35, and 42, whereas coliform counts, yeast and mold counts, and rheological characteristics were determined on d 1 and 42. The sensory study was performed on d 3 and particle size of the functional ingredients (powder form) was also determined. When compared with control, the incorporation of slippery elm bark into yogurts led to less syneresis. l-Glutamine increased pH and n′ values (relaxation exponent derived from G′) and lowered titratable acidity values. N-Acetyl-d-glucosamine incorporation resulted in higher n′ and lower titratable acidity values, whereas maitake mushroom led to lower n′ values. Incorporating quercetin increased the growth of L. bulgaricus. Adding maitake mushrooms increased the growth of S. thermophilus but lowered apparent viscosity values, whereas quercetin decreased its S. thermophilus counts. Quercetin decreased L* and a* values but increased b* values, and maitake mushroom increased a* values. Thixotropic behavior increased with the addition of licorice root and quercetin. Adding slippery elm bark, N-acetyl-d-glucosamine, licorice root, maitake mushrooms, and zinc orotate into yogurt did not affect the sensory properties, whereas yogurts with quercetin had the lowest sensory scores. Overall, most of these ingredients did not cause major changes to yogurt properties.     

Lactase Activity of Cultured and Acidified Dairy Products

A method was standardized for determining lactase activity in cultured and acidified dairy products such as cottage cheese, sour cream, and yogurt. Cottage cheese and sour cream prepared by both the cultured and acidified processes and yogurt prepared by the direct acidification process did not possess lactase activity. However, cultured yogurt possessed considerable enzyme activity mainly due to lactase as an endoenzyme in the yogurt culture (Lactobacillus bulgaricus and Streptococcus thermophilus). Enzyme in yogurt increased with time of incubation, reaching a maximum of 8 orthonitrophenol β-d-galactopyranoside units per gram of yogurt in 4 h. S. thermophilus contained approximately three times more lactase than did L. bulgaricus. Also, an in vitro digestion process appeared to enhance the release of lactase from the composite yogurt culture. It was felt that cultured yogurt would be beneficial to individuals suffering from lactose intolerance not only because of reduced lactose but also because of lactase.     

Effect of papain‐hydrolysed casein peptides on the fermentation kinetics,microbiological survival and physicochemical properties of yoghurt

The objective of this study was to determine the effect of different molecular mass papain‐hydrolysed casein peptides (PCP) on the growth of lactic acid bacteria (LAB) (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in 1:1 ratio) and on the fermentation kinetics, microbiological survival and physicochemical properties in yoghurt. The fraction of PCP4 (<3 kDa) exhibited the highest growth‐promoting activity on the strains. The value of T_pH4.5 (time necessary to reach pH 4.5) of yoghurt fortified with PCP4 reduced from 5.21 to 4.29 h, and the total counts of LAB present in the final products increased from 8.29 to 8.42 log CFU mL^?1 with the addition of PCP4 at nitrogen replaced ratio of 3% (w/w). Moreover, the yoghurt exhibited lower postacidification power, higher viscosity and sensory evaluation scores than did the control product during storage period.