Biochemistry,genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review

Many strains of Streptococcus thermophilus synthesize extracellular polysaccharides. These molecules may be produced as capsules that are tightly associated with the cell, or they may be liberated into the medium as a loose slime (i.e., "ropy" polysaccharide). Although the presence of exopolysaccharide does not confer any obvious advantage to growth or survival of S. thermophilus in milk, in situ production by this species or other dairy lactic acid bacteria typically imparts a desirable "ropy" or viscous texture to fermented milk products. Recent work has also shown that exopolysaccharide-producing S. thermophilus can enhance the functional properties of Mozzarella cheese, but they are not phage-proof. As our understanding of the genetics, physiology, and functionality of bacterial exopolysaccharides continues to improve, novel applications for polysaccharides and polysaccharide-producing cultures are likely to emerge inside and outside the dairy industry. This article provides an overview of biochemistry, genetics, and applications of exopolysaccharide production in S. thermophilus.     

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.     

Hyaluronic acid production by recombinant Streptococcus thermophilus

Generally recognized as safe, Streptococcus thermophilus was transformed using a plasmid expressing endogenous hyaluronic acid (HA) synthase genes. A single expression of hyaluronic acid synthase (hasA), uridine diphosphate-glucose dehydrogenase gene (hasB), or pyrophosphorylase gene (glmU) and double expression of hasA and hasB were attempted. A streptococcus-Escherichia coli shuttle vector, pBE31, was successfully transfected in S. thermophilus. The single expression of hasA or hasB allowed S. thermophilus to produce about 0.5-1.0 g/l HA. The strains coexpressing of hasA and hasB showed a markedly increased HA production (1.2g/l) which was six-fold increase compared with the wild-type strain. The maximum cell concentration and specific growth rate of each recombinant strain were lower than those of the wild-type strain; however, the specific production rate was more than 100-fold higher. Galactose concentration decreased in the coexpressing strain after depletion of lactose. The bacterial metabolism would be altered in order to achieve a higher production by changing the intracellular metabolism. The average molecular weight of HA (1.0 × 10(6) Da) was not affected by the expression of hasA and hasB. HA produced from recombinant strain could be an alternative material for medical, cosmetic and food utilization instead of HA from conventional pathogenic streptococci.     

Effect of the metabolism of urea on the acidifying activity of Streptococcus thermophilus

One of the main functions of Streptococcus thermophilus strains used in the dairy industry is the production of lactic acid. In cheese and fermented milk manufacturing processes, the pH evolution kinetics must be reproducible in order to ensure the good quality of the final products. The objective of the present study was to investigate the effect of the metabolism of urea on the acidifying activity of fast- and slow-acidifying strains of S. thermophilus. Milk treatment with a purified urease and utilization of the urease inhibitor flurofamide revealed that urea metabolism by S. thermophilus influences the pH evolution kinetics through 2 distinct means. First, ammonia production from urea tends to increase the pH. This effect is greater when lactic acid concentration is low due to a lower buffering capacity of milk. Second, urea metabolism also modifies growth and lactic acid production by S. thermophilus. Depending on the strains and the growth stage of the cultures, consumption of urea induces either a faster or a slower pH decrease. For the slow-acidifying strain RD678, suppression of urea metabolism by adding flurofamide decreased the time necessary to reach pH 6 by 195 min. This effect was less pronounced for the 2 fast-acidifying strains RD674 and RD677. These results show that urea metabolism may have a considerable influence on the acidifying properties of S. thermophilus strains.     

Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275

Effects of pH, temperature, supplementation with whey protein concentrate (WPC), and non-EPS culture on the exopolysaccharide (EPS) production by Streptococcus thermophilus 1275 were studied. The organism was grown in 10% reconstituted skim milk (RSM) in a Biostat B fermenter for 24 h at various pH (4.5, 5.5 and 6.5) and temperatures (30, 37, 40, and 42 degrees C), and supplementation with WPC 392, and non-EPS producing S. thermophilus 1303 and the amount of EPS produced were determined. Bacterial counts were enumerated and the concentrations of lactic acid, lactose, glucose, and galactose were also determined. A maximum of 406 mg/L of EPS was produced in RSM at 37 degrees C after 24 h of fermentation at pH 4.08 when the pH was not controlled. A pH of 5.5 and temperature of 40 degrees C were found to be optimal for EPS production by S. thermophilus 1275, yielding 458 mg/L. The EPS production increased when RSM was supplemented with WPC 392. At optimum pH and at 37 degrees C with WPC supplementation, the level of EPS increased to 1029 mg/L. Co-culturing S. thermophilus 1275 with non-EPS S. thermophilus 1303 increased EPS production at 37 degrees C and pH 5.5 to 832 mg/L. High temperature (42 degrees C) reduced the amount of EPS production, and EPS production ceased at pH 4.5 when maintained constantly at this pH. The level of lactose utilization and lactic acid production depended on growth conditions of the organism. No glucose was detected, while galactose was found to accumulate in the medium.     

Selection and properties of Streptococcus thermophilus mutants deficient in urease

Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey.     

Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina

Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.     

Urease production by Streptococcus thermophilus

In order to identify potential alternative sources of urease for the removal of urea from alcoholic beverages, 205 strains of lactic acid bacteria belonging to 27 different species were screened for urease production. Only Streptococcus thermophilus produced urease. Cell permeabilization with toluene allowed to increase activity significantly. Optimal pH for urease activity in whole and permeabilized cells and of cell free extracts differed slightly, but was in the range 6.0-7.0. Significant activity was retained at pH 3.0 and 8.0, and, for cell free extracts, at pH 4.0 in the presence of ethanol. Urease production was evaluated in fermentations with pH control (5.25-6.5) and without pH control. Very little urease was produced in absence of urea, which at 5g/l slowed growth significantly in fermentations without pH control, but prevented a decrease in pH below 5.1 and resulted in higher final biomass. Optimal pH for growth was between 6.0 and 6.5 but specific urease activity was higher for fermentations at low pH at the beginning of the exponential phase. However, a higher total urease activity was obtained at pH 6.0 and 6.5 because of higher biomass. Potential technological applications of urease production by S. thermophilus are discussed.     

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.     

嗜热链球菌胞外多糖生物合成的研究进展

嗜热链球菌是一种公认的安全性益生菌,作为重要的食品发酵剂,广泛应用在发酵乳和奶酪等食品工业生产中,在发酵过程中起着重要的作用,日益成为食品科学研究的热点之一。由于与其他微生物菌株相比安全性高,目前在医药保健等行业中也有着广泛的应用。在嗜热链球菌代谢产物研究中,胞外多糖(exopolysaccharide,EPS)因独特的理化特性和生理功能而备受研究者青睐。嗜热链球菌来源的EPS不但具有降低胆固醇、抗肿瘤和促进肠道粘附等生理功能,而且还能够作为食品添加剂和稳定剂显著改善食品物性。本文重点介绍了嗜热链球菌EPS的生物合成、EPS合成基因簇及其代谢调控,期望为嗜热链球菌EPS生物合成的深入研究提供参考。     

嗜热链球菌胞外多糖的结构生物合成与遗传调控

介绍了嗜热链球菌胞外多糖的组成结构、生物合成、代谢途径和针对途径中关键酶的遗传调控,为胞外多糖研究提供参考.     

Effect of exopolysaccharide-producing strains of Streptococcus thermophilus on technological attributes of fat-free lassi

Sixty-four exopolysaccharide-producing thermophilic lactic acid bacteria (LAB) were isolated from traditionally made Indian fermented milk products. On the basis of morphological and biochemical tests, these isolates were identified as the species of Lactobacillus, Streptococcus, and Enterococcus genera. Initial screening for technological attributes revealed that Streptococcus thermophilus IG16 was a promising isolate, and produced both capsular and ropy polysaccharides at the concentration of 211 mg/L. Exopolysaccharide produced by IG16 was a heteropolysaccharide containing rhamnose and galactose in a ratio of 5.3:1 and had a molecular weight of 3.3 × 10⁴ Da. Use of IG16 as a starter culture controlled whey separation and improved viscosity, flavor, consistency, and color and appearance of lassi. Use of IG16 resulted in lassi having optimal acidity, less syneresis, high viscosity, and better scores for flavor, consistency, and color and appearance.     

Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes

The aim of this work was to use fluorescently labeled lectins to develop a convenient and reliable method to determine the relative abundance of capsular polysaccharides (CPS) at the surface of Streptococcus thermophilus MR-1C cells. Fluorescein isothiocyanate-labeled peanut agglutinin isolated from Arachis hypogaea was found to interact specifically with the CPS of Strep. thermophilus MR-1C. This labeled lectin was then used as an effective probe to detect and quantify CPS. A fluorescence-based lectin-binding assay was successfully applied to follow the accumulation of CPS during the growth of Strep. thermophilus MR-1C in milk and in M17 broth supplemented with lactose. Our results showed that in both media, CPS production by Strep. thermophilus MR-1C began during the exponential phase of growth and continued for several hours after the culture reached the stationary growth phase.     

Viability of Lactobacillus acidophilus La-5 added solely or in co-culture with a yoghurt starter culture and implications on physico-chemical and related properties of Minas fresh cheese during storage

The effect of a probiotic culture of Lactobacillus acidophilus (La-5), added solely or in co-culture with a starter culture of Streptococcus thermophilus, on texture, proteolysis and related properties of Minas fresh cheese during storage at 5 °C was investigated. Three cheese-making trials were prepared and produced with no addition of cultures (T1 - control), supplemented with La-5 (T2), and with La-5 + S. thermophilus (T3). Viable counts of La-5 remained above 6.00 log cfu g⁻¹ during the whole storage for T2, reaching 7.00 log cfu g⁻¹ on the 14th day. For T3, the counts of La-5 remained above 6.00 log cfu g⁻¹ after 7 days of storage. Due to the presence of S. thermophilus, T3 presented the highest proteolytic index increase and titratable acidity values. Nevertheless, these results and S. thermophilus addition had no influence on viability of La-5 which presented satisfactory populations for a probiotic food. Moreover, the use of a yoghurt culture for the production of Minas fresh cheese T3 supplemented with La-5 resulted in a good quality product, with a small rate of post-acidification, indicating that traditional yoghurt culture could be employed in co-culture with La-5 to improve the quality of this cheese.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Use of an exopolysaccharide-producing strain of Streptococcus thermophilus in the manufacture of Mexican Panela cheese

An exopolysaccharide (EPS) producing strain of Streptococcus thermophilus was evaluated for the production of Panela cheese using two total solids milk (TSM) concentrations (12.5 and 17.5 g/100 mL). This ropy strain increased cheese yield; nevertheless, with 12.5 TSM the increment was higher than with 17.5 TSM. Analysis of cheese composition showed that with 12.5 TSM, the ropy strain increased moisture, but did not change the fat or non fat solids on dry weight basis (dwb), suggesting that the increment of the yield is only due to water retention. In 17.5 TSM cheeses the ropy strain caused an increase in the moisture and fat (dwb), suggesting that besides water retention, fat also contributed to the yield. The difference in yield increment could be explained by cheese composition: higher fat content creates a more hydrophobic environment, which would expel more water than the cheese with lower fat content. Electron microscopy showed EPS attached to the protein matrix of the cheeses. In 17.5 TSM cheeses EPS was observed around the milk fat globules (MFG), confirming that higher TSM causes EPS to bind the MFG besides binding the protein matrix, retaining fat within the cheese. Sensory evaluation demonstrated that ropy cheeses were softer and creamier.     

西藏灵菇中产胞外多糖嗜热链球菌的分离筛选及其发酵性能测定

从西藏灵菇中分离筛选、鉴定高产胞外多糖的乳酸菌菌株,并对其发酵性能和流变学参数进行测试。筛选的9株菌,菌种鉴定结果均为嗜热链球菌,其中菌株KC1、KC2、KC6、KC17产胞外多糖,菌株KC5、KC7、KC15、KC16、KC22不产胞外多糖。产胞外多糖菌株发酵乳发酵性能、黏度和黏附性指标均明显高于不产胞外多糖的嗜热链球菌,进一步证实了嗜热链球菌产生的胞外多糖可以赋予发酵乳良好的质地。     

Structural characterization of the exopolysaccharide produced by Streptococcus thermophilus 05-34 and its in situ application in yogurt

An exopolysaccharide (EPS) producing strain was isolated from Tibetan kefir grains in China, which was identified by 16S rDNA tests and designated as Streptococcus thermophilus 05-34. The high-performance liquid chromatography analysis showed that it was composed of galactose and glucose in a molar ratio of 1.0:0.8 with a molecular mass of 2.5 × 10(4) Da. EPS was further revealed to have α-d-glucose, α-d-galactose, β-d-glucose, and β-d-galactose by Fourier transform infrared spectroscopy combined with 1D (1) H nuclear magnetic resonance spectroscopy. The length of EPS ranged from 10 to 100 nm and the maximal height of lumps was 2.5 nm through atomic force micrograph analysis. Furthermore, yogurt fermented with EPS-producing S. thermophilus 05-34 exhibited lower susceptibility to whey separation, higher viscosity, and sensory scores than those made with non-EPS-producing strain in yogurt production. These results suggested that EPS-producing Streptococcus thermophilus 05-34 provided a potential application in the fermented dairy industry.     

Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk

The duration of infection and pulsed-field gel electrophoresis (PFGE) types of bovine intramammary Streptococcus uberis isolates were examined. Milk samples were collected in duplicate from all 4 glands of 503 cows from 5 herds within 1 to 3 d of parturition and from 113 cows with clinical mastitis in the same herds throughout lactation. Glands from which S. uberis was isolated were resampled at 28-d intervals.The prevalence of S. uberis was 12% for cows around parturition, and the median duration of infection was 16 d. Cows >2 yr old had a longer duration of infection than 2 yr old cows, and duration varied among herds. A total of 173 different PFGE types were identified from a total of 234 S. uberis isolates. Each farm had a unique set of PFGE types. Only 3 PFGE types were common to each of 3 pairs of cows, and these occurred on the same farm. Where S. uberis was isolated on more than one occasion from a gland, only 55% of the PFGE types were the same across time. For cows with multiple glands infected, only one-half (9 of 18) had the same PFGE type in more than one gland. No predominant PFGE type was identified in any herd.It is concluded that there was wide heterogeneity of PFGE types, that the environment rather than other cows was the likely source of S. uberis infections, and that glands may be infected with multiple S. uberis PFGE types over a lactation.