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.     

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.     

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.     

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.     

Galactose metabolism and capsule formation in a recombinant strain of Streptococcus thermophilus with a galactose-fermenting phenotype

The capsule-producing, galactose-negative Streptococcus thermophilus MR-1C strain was first transformed with a low-copy plasmid containing a functional galK gene from Streptococcus salivarius to generate a recombinant galactose-fermenting Strep. thermophilus strain named MR-AAC. Then, we compared the functional properties of Strep. thermophilus MR-AAC with those of the parent MR-1C strain when used as starter for fermented products and cheese. In lactose-supplemented laboratory medium, MR-AAC metabolized galactose, but only when the amount of lactose was less than 0.1% (wt/vol). After 7 h of fermentation, the medium was almost depleted of galactose. The parent strain, MR-1C, showed the same pattern, except that the concentration of galactose decreased by only 25% during the same period. It was found that, during milk fermentation and Mozzarella cheese production, the galactose-fermenting phenotype was not expressed by MR-AAC and this strain expelled galactose into the medium at a level similar to the parent MR-1C strain. In milk and in lactose-supplemented medium, capsular exopolysaccharide production occurred mainly during the late exponential phase and the stationary growth phase with similar kinetics between MR-1C and MR-AAC.     

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.     

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.     

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.     

Production of hyaluronic acid from Streptococcus zooepidemicus MTCC 3523 and its wound healing activity

Exopolysaccharide produced and purified from Streptococcus zooepidemicus MTCC 3523 was identified as hyaluronic acid (HA) based on IR and NMR spectroscopy while its Mw was found to be 5.38 × 10(5)Da. HA produced passed bacterial endotoxin test and showed significant wound healing activity in Wistar rats on 12th and 16th day.     

Influence of hyaluronidase addition on the production of hyaluronic acid by batch culture of Streptococcus zooepidemicus

Enhancement of hyaluronic acid (HA) production by Streptococcus zooepidemicus through increasing oxygen transfer rate via degradation of HA by hyaluronidase was investigated. Dissolved oxygen (DO) level became a limiting factor for HA production during 8–16 h, and thus hyaluronidase (0.05, 0.10, 0.15, 0.20, 0.25 g/l) was added at 8 h to degrade HA. Oxygen transfer rate coefficient and DO level during 8–16 h increased with increased hyaluronidase concentration. Compared to 5.0 ± 0.1 g/l of the control without hyaluronidase addition, HA production was increased from 5.0 ± 0.1 g/l to 6.0 ± 0.1 g/l when hyaluronidase concentration was 0.15 g/l. Further increase of hyaluronidase concentration had no effect on HA production. The molecular weight of HA decreased with the increased hyaluronidase concentration and decreased to 21 kDa when hyaluronidase concentration was 0.25 g/l from 1300 kDa of the control. The prepared low molecular weight HA (LMW-HA) could function as potential anti-angiogenic substances, antiviral and anti-tumor agents to possibly be used as functional food ingredients.     

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.     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain

Efficient L-lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida utilis strain expressing an L-lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased L-lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).     

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.     

Biosynthesis of high molecular weight hyaluronic acid by Streptococcus zooepidemicus using oxygen vector and optimum impeller tip speed

The potential use of n-dodecane and n-hexadecane as oxygen vectors for enhancing hyaluronic acid (HA) biosynthesis by Streptococcus zooepidemicus ATCC 39920 was investigated using a 2-L stirred-tank bioreactor equipped with helical ribbon or Rushton turbine impellers. The volumetric fraction of the oxygen vector influenced the gas-liquid volumetric oxygen transfer coefficient (K(L)a) positively. Batch HA fermentation with 1% (v/v) n-dodecane or 0.5% (v/v) n-hexadecane addition was carried out at different impeller tip speeds. Even though cell growth was lower in the fermentation with oxygen vector addition, the HA productivity and molecular weight were higher when compared to the fermentation without oxygen vector at low impeller tip speed. The highest HA concentration (4.25 gHA/l) and molecular weight (1.54 × 10(7) Da) were obtained when 0.5% (v/v) n-hexadecane and 0.785 m/s impeller tip speed of helical ribbon were used.     

Efficient production of L-(+)-lactic acid from raw starch by Streptococcus bovis 148

Streptococcus bovis 148 was found to produce L-(+)-lactic acid directly from soluble and raw starch substrates at pH 6.0. Productivity was highest at 37 degrees C, with 14.7 g/l lactic acid produced from 20 g/l raw starch. The yield and optical purity of L-lactic acid were 0.88 and 95.6%, respectively.     

Production of trehalose by intramolecular transglucosylation of maltose catalysed by a new enzyme from Thermus thermophilus HB-8

Thermus thermophilus HB-8 is a source of trehalose synthase (GTase), which catalyses conversion of maltose into trehalose. Specific activity of maltose transglucosylation by cell-free extracts of the bacteria was about 0.1 U mg⁻¹ protein and precipitation at 28% saturation of ammonium sulphate caused 3.5-fold enzyme purification. The optimum temperature for conversion of maltose into trehalose was 65 °C with about 27% of maximum activity at 85 °C. The highest GTase productivity was achieved at cultivation temperature over 60 °C and at NaCl concentration range of 0.1–0.5% (w/v). However, larger concentrations of sodium chloride in the growth media caused a remarkable decrease of GTase synthesis. The results, of ammonium sulphate fractionation and activity towards maltotriose (0.028 U mg⁻¹), maltotetraose (0.16 U mg⁻¹) and GlcαpNp (0.27 U mg⁻¹), show that trehalose synthase and α-glucosidase activities reside in separate protein fractions of cell-free extracts from T. thermophilus cells.