Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus,Streptococcus thermophilus,Lactobacillus acidophilus,bifidobacteria, Lactobacillus casei,Lactobacillus rhamnosus,and propionibacteria

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.     

Production of the exopolysaccharides by Streptococcus thermophilus: effect of growth conditions on fermentation kinetics and intrinsic viscosity

Production of exopolysaccharides (EPS) by a commercial Streptococcus thermophilus strain was evaluated at different growth conditions [temperature (32-45 degrees C), carbon source and initial nitrogen (N) content]. Lactose from deproteinized whey and sucrose allowed to obtain EPS yields higher than 1200 mg/mM of the consumed carbon source. Intrinsic viscosity of the EPS was significantly reduced by ionic strength indicating a polyelectrolyte behavior. Growth conditions used for the production of the EPS had a significant effect (p<0.05) on the intrinsic viscosity. This was attributed to the effect of growth conditions on the molecular properties of the EPS [stiffness and molecular weight (MW)]. High MW EPS were produced when the bacteria grew at a high specific growth rate; however MW of the EPS and specific growth rate were not linearly associated. In the lactose fermentations carried out at different temperatures specific EPS synthesis rate was positive and linearly associated with the specific lactose consumption rate (R2=0.967) and specific galactose production rate (R2=0.967). Critical coil overlap parameter, [eta]C*, for the EPS produced in the lactose fermentations carried out at 43 and 45 degrees C was determined to be approximately 7.6, and their critical overlap concentrations (C*) were 0.45 and 0.87 g/dL, respectively.     

Influence of temperature on associative growth of Streptococcus thermophilus and Lactobacillus bulgaricus

Compatibility of Streptococcus thermophilus and Lactobacillus bulgaricus during associative growth as dependent on optimum growth temperature was determined. Optimum growth temperatures for 9 strains of S. thermophilus and 10 strains of L. bulgaricus ranged from 35 to 42 degrees C for S. thermophilus and 43 to 46 degrees C for L. bulgaricus. Streptococcus thermophilus and L. bulgaricus strains exhibiting similar to divergent optimum growth temperatures were combined (1:1 vol/vol) and incubated in milk at 37, 42, and 45 degrees C until pH 4.2 was reached. Initial and postincubation cell numbers were determined by plate count method. Streptococcus thermophilus strains reached greater cell numbers than L. bulgaricus at 37, 42, and 45 degrees C in 93.3% of the mixed culture trials. Average rod-coccus ratios obtained at 37, 42, and 45 degrees C were 1:2.2, 1:8, and 1:2.4, respectively. Optimum growth temperatures had no influence on growth of L. bulgaricus and S. thermophilus in mixed culture. Rather, temperature appeared to influence compatibility by determining the concentration or type of stimulatory factor(s) produced by L. bulgaricus. All strains of S. thermophilus exhibited an uncoupling of growth from acid production. Optimum temperature for acid production ranged from 2 to 8 degrees C above optimum growth temperature. These findings warrant consideration in the manufacture of yogurt and other fermented milk products.     

Growth and exopolysaccharide yield of Lactobacillus delbrueckii ssp. bulgaricus DSM 20081 in batch and continuous bioreactor experiments at constant pH

Some Lactobacillus delbrueckii ssp. bulgaricus strains are able to synthesize exopolysaccharides (EPS) and are therefore highly important for the dairy industry as starter cultures. The aim of this study was to investigate the nutritional requirements for growth and EPS production of Lactobacillus delbrueckii ssp. bulgaricus DSM 20081. A medium was developed from a semi-defined medium (SDM) in which glucose was replaced by lactose and different combinations of supplements (nucleobases, vitamins, salts, sodium formate and orotic acid) were added. Constant pH batch fermentation with the modified medium resulted in an EPS yield of approximately 210 mg glucose equivalents per liter medium. This was a 10-fold increase over flask cultivation of this strain in SDM. Although not affecting cell growth, the mixture of salts enhanced the EPS synthesis. Whereas EPS production was approximately 12 mg/g dry biomass without salt supplementation, a significantly higher yield (approximately 20 mg/g dry biomass) was observed after adding the salt mixture. In continuous fermentation, a maximal EPS concentration was obtained at a dilution rate of 0.31/h (80 mg EPS/L), which corresponded to a specific EPS production of 49 mg/g dry biomass.     

嗜热链球菌KLDS3.1012胞外多糖合成途径的基因组学及表型特征分析

在分子水平上挖掘嗜热链球菌KLDS3.1012合成胞外多糖的途径并且通过表型特征进行验证。首先,基于Illumina HiSeq与Illumina MiSeq测序平台对菌株KLDS3.1012进行基因组测序并构建基因组图谱;随后,从糖代谢、糖核苷酸合成、胞外多糖基因簇等方面进行生物信息学分析;最后,利用API 50CH测定菌株KLDS3.1012的糖发酵情况及采用高效离子交换色谱法测定胞外多糖的单糖组成。生物信息学分析结果表明菌株KLDS3.1012具有半乳糖、葡萄糖、果糖、甘露糖、乳糖和蔗糖转运系统和4 种糖核苷酸（UDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖和UDP-N-乙酰葡糖胺）合成的相关基因及1 个胞外多糖合成基因簇。表型特征分析结果表明菌株KLDS3.1012可以利用以上6 种碳源并能形成由鼠李糖、半乳糖和葡萄糖组成的胞外多糖。本研究为分析该菌株合成胞外多糖的遗传基础与胞外多糖结构的关系提供理论依据,并对将其开发为发酵剂具有一定指导意义。     

Proteolytic profiles and angiotensin-I converting enzyme and alpha-glucosidase inhibitory activities of selected lactic acid bacteria

ABSTRACT:  This study was conducted to examine the growth, proteolytic profiles as well as angiotensin-I converting enzyme (ACE) and α-glucosidase (α-glu) inhibitory potentials of selected strains of lactic acid bacteria (LAB). Two strains each of yogurt bacteria ( Streptococcus thermophilus —1275 and 285, and Lactobacillus delbrueckii ssp. bulgaricus —1092 and 1368), and probiotics ( L. acidophilus —4461 and 33200, and L. casei —2607 and 15286, and 1 strain of Bifidobacterium longum 5022), were cultivated in reconstituted skim milk (RSM) at 37 °C and their proteolytic profiles and ACE as well as α-glu inhibitory activities were determined. Among all the strains of lactic acid bacteria studied, yogurt bacteria grew very well, with the exception of L. delbrueckii ssp. bulgaricus 1368 which showed a slower growth during the initial 3 h of incubation. The growth pattern corresponded well with the decrease in pH for the organisms. All the organisms showed an increase in proteolysis with time. The variations in proteolytic capabilities translated into corresponding variations in ACE inhibitory potential of these organisms. Bifidobacterium longum 5022 showed the highest ACE inhibitory potential followed by L. delbrueckii ssp. bulgaricus 1368, L. casei 15286, S. thermophilus 1275, and L. acidophilus 4461. Organisms with high intracellular enzymatic activities grew well. Also, aminopeptidases of strains of L. acidophilus 4461 and S. thermophilus 1275 that could better utilize proline containing substrates showed enhanced ACE inhibitory potential. Lactic acid bacteria possessed the ability to inhibit α-glu activity, which endowed them an antidiabetic property as well.     

Low-fat mozzarella as influenced by microbial exopolysaccharides, preacidification, and whey protein concentrate

Low-fat Mozzarella cheeses containing 6% fat were made by preacidification of milk, preacidification combined with exopolysaccharide- (EPS-) producing starter, used independently or as a coculture with non-EPS starter, and preacidification combined with whey protein concentrate (WPC) and EPS. The impact of these treatments on moisture retention, changes in texture profile analysis, cheese melt, stretch, and on pizza bake performance were investigated over 45 d of storage at 4°C. Preacidified cheeses without EPS (control) had the lowest moisture content (53.75%). These cheeses were hardest and exhibited greatest springiness and chewiness. The meltability and stretchability of these cheeses increased most during the first 28 d of storage. The moisture content in cheeses increased to 55.08, 54.79, and 55.82% with EPS starter (containing 41.18 mg/g of EPS), coculturing (containing 28.61 mg/g of EPS), and WPC (containing 44.23 mg/g of EPS), respectively. Exopolysaccharide reduced hardness, springiness, and chewiness of low-fat cheeses made with preacidified milk in general and such cheeses exhibited an increase in cohesiveness and meltability. Although stretch distance was similar in all cheeses, those containing EPS were softer than the control. Cocultured cheeses exhibited the greatest meltability. Cheeses containing WPC were softest in general; however, hardness remained unchanged over 45 d. Cheeses made with WPC had the least increase in meltability over time. Incorporation of WPC did not reduce surface scorching or increase shred fusion of cheese shreds during pizza baking; however, there was an improvement in these properties between d 7 and 45. Coating of the cheese shreds with oil was necessary for adequate browning, melt, and flow characteristics in all cheese types.     

Production of an exopolysaccharide-containing whey protein concentrate by fermentation of whey

Using whey as a fermentation medium presents the opportunity to create value-added products. Conditions were developed to partially hydrolyze whey proteins and then ferment partially hydrolyzed whey with Lactobacillus delbrueckii ssp. bulgaricus RR (RR; an EPS-producing bacterium). In preliminary experiments, pasteurized Cheddar cheese whey was treated with Flavourzyme to partially hydrolyze the protein (2 to 13% hydrolyzed). Fermentation (2 L, 38 degrees C, pH 5.0) with RR resulted in EPS levels ranging from 95 to 110 mg of EPS per liter of hydrolyzed whey. There were no significant differences in the amount of EPS produced during fermentations of whey hydrolyzed to varying degrees. Since a high level of hydrolysis was not necessary for increased EPS production, a low level of hydrolysis (2 to 4%) was selected for future work. In scale up experiments, whey was separated and pasteurized, then treated with Flavourzyme to hydrolyze 2 to 4% of the protein. Following protease inactivation, 60 L of partially hydrolyzed whey was fermented at 38 degrees C and pH 5.0. After fermentation, the broth was pasteurized, and bacterial cells were removed using a Sharples continuous centrifuge. The whey was then ultrafiltered and diafiltered to remove lactose and salts, freeze-dried, and milled to a powder. Unfermented hydrolyzed and unhydrolyzed whey controls were processed in the same manner. The EPS-WPC ingredients contained approximately 72% protein and 6% EPS, but they exhibited low protein solubility (65%, pH 7.0; 58%, pH 3.0).     

Effect of carbon dioxide under high pressure on the survival of cheese starter cultures

A new processing method that rapidly forms curds and whey from milk has the potential to improve cheesemaking procedures if cheese starter cultures can tolerate the processing conditions. The survival of Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. lactis, or Streptococcus thermophilus through this new process was evaluated. Inoculated milk containing 0, 1, or 3.25% fat or Lactobacillus MRS broth or tryptone yeast lactose broth (depending on microorganism used) was sparged with CO2 to a pressure of 5.52 MPa and held for 5 min at 38 degrees C. Broth contained 7.93 to 8.78 log CFU/ ml before processing and 7.84 to 8.66 log CFU/ml afterward. Before processing, milk inoculated with L bulgaricus, L. lactis, or S. thermophilus contained 6.81, 7.35, or 6.75 log CFU/ml, respectively. After processing, the curds contained 5.68, 7.32, or 6.50 log CFU/g, and the whey had 5.05, 6.43, or 6.14 log CFU/ml, respectively. After processing, the pHs of control samples were lower by 0.41 units in broth, 0.53 units in whey, and 0.89 units in curd. The pH of the processed inoculated samples decreased by 0.3 to 0.53 units in broth, 0.32 to 0.37 units in whey, and 0.93 to 0.98 units in the curd. Storing curds containing L. lactis at 30 degrees C or control curds and curds with L. bulgaricus or S. thermophilus at 37 degrees C for an additional 48 h resulted in pHs of 5.22, 5.41, 4.53, or 4.99, respectively. This study showed that milk inoculated with cheese starter cultures and treated with CO2 under high pressure to precipitate casein-produced curds that contained sufficient numbers of viable starter culture to produce lactic acid, thereby decreasing the pH.     

Effect of Flavourzyme® on Angiotensin‐Converting Enzyme Inhibitory Peptides Formed in Skim Milk and Whey Protein Concentrate during Fermentation by Lactobacillus helveticus

Angiotensin‐converting enzyme inhibitory (ACE‐I) activity as affected by Lactobacillus helveticus strains (881315, 881188, 880474, and 880953), and supplementation with a proteolytic enzyme was studied. Reconstituted skim milk (12% RSM) or whey protein concentrate (4% WPC), with and without Flavourzyme^® (0.14% w/w), were fermented with 4 different L. helveticus strains at 37 °C for 0, 4, 8, and 12 h. Proteolytic and in vitro ACE‐I activities, and growth were significantly affected (P < 0.05) by strains, media, and with enzyme supplementation. RSM supported higher growth and produced higher proteolysis and ACE‐I compared to WPC without enzyme supplementation. The strains L. helveticus 881315 and 881188 were able to increase ACE‐I to >80% after 8 h of fermentation when combined with Flavourzyme^® in RSM compared to the same strains without enzyme supplementation. Supplementation of media by Flavourzyme^® was beneficial in increasing ACE‐I peptides in both media. The best media to release more ACE‐I peptides was RSM with enzyme supplementation. The L. helveticus 881315 outperformed all strains as indicated by highest proteolytic and ACE‐I activities.     

Evolution of carbohydrate fraction in carbonated fermented milks as affected by beta-galactosidase activity of starter strains

The influence of carbonation on the evolution of lactose, galactose and glucose in fermented milks with added probiotic bacteria (Lactobacillus casei, Lactobacillus acidophilus and/or Bifidobacterium bifidum) was evaluated and related to beta-galactosidase activity of starter strains. During incubation and first days of refrigeration, lactose hydrolysis resulting in the liberation of galactose and glucose occurred in CT (Streptococcus thermophilus/Lb. casei), AT (Str. thermophilus/Lb. acidophilus) and ABT fermented milks (Str. thermophilus/Lb. acidophilus/Bifid. bifidum). Levels of galactose were higher than those of glucose and could be related to the preferential consumption of glucose by actively growing bacteria. Through the incubation, lactose and monosaccharide levels were not affected by milk carbonation. However, during refrigerated storage the presence of this gas was associated with slightly lower content of lactose and higher levels of galactose and glucose in AT and ABT products but not in CT fermented milks. Through the refrigeration galactose was moderately utilised by Lb. acidophilus in AT products whereas the presence of Bifid. bifidum seems to prevent the consumption of this sugar in ABT fermented milks. Glucose remained constant, with minor variations in CT products but a continuous increase of this sugar occurred in carbonated AT and ABT fermented milks during storage. Beta-galactosidase activity displayed by Str. thermophilus strains was similar at pH 6.5 (initial pH of non-carbonated samples) and pH 6.3 (initial pH of carbonated samples) whereas Lb. acidophilus LaA3 showed greater beta-galactosidase activity at pH 6.3 than at higher pH values. Thus, the enhanced metabolic activity of Lb. acidophilus caused by the low initial pH of carbonated milk also promoted higher cellular beta-galactosidase activity that could have released greater amounts of galactose and glucose from lactose in AT and ABT fermented milks through the refrigerated period. In CT fermented milks, similar beta-galactosidase activity levels of Str. thermophilus at pH 6.5 and 6.3 together with the absence of beta-galactosidase activity in Lb. casei could explain the lack of differences on glucose and galactose content between carbonated and non-carbonated samples.     

Short communication: impact of pH and temperature on the acidifying activity of Carnobacterium maltaromaticum

The acidifying activity of Carnobacterium maltaromaticum LMA28, a strain isolated from French soft cheese, was studied in trypticase soy broth with yeast extract (TSB-YE) medium and in milk. In TSB-YE supplemented with lactose, glucose, or galactose, lactose and glucose were metabolized with a maximum growth rate of 0.32 h⁻¹ and galactose was not metabolized. During hydrolysis of lactose, the galactose moiety was not excreted. The major product was l(+) lactic acid, with no significant difference in the lactic acid yield. Glucose was not completely metabolized because cell growth stopped when pH values reached an average of 5.0. In sterilized UHT milk, the addition of 1 g/L of YE enhanced its coagulation. Compared with commercial starter lactic acid bacteria such as Lactococcus lactis DSMZ 20481 or Streptococcus thermophilus INRA 302, Carnobacterium maltaromaticum LMA 28 was shown to be a slow acidifying strain. However, in spite of this weak acidifying ability, C. maltaromaticum LMA 28 can sustain low pH values in coculture with Lc. lactis DSMZ 20481 or S. thermophilus INRA 302. The individual and interactive effects of initial pH values (5.2 to 8.0) and incubation temperatures (23 to 37°C) on acidifying activity were studied by response surface methodology. The 3 strains displayed different behaviors depending on pH and temperature. The psychrotrophic lactic acid strain C. maltaromaticum LMA 28 was able to grow at alkaline pH values and during storage conditions. It could be used as a potential ripening flora in soft cheese.     

Optimization of conditions for galactooligosaccharide synthesis during lactose hydrolysis by β-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP G)

A study on optimisation of the conditions for galactooligosaccharide (GOS) formation during lactose hydrolysis, produced by Lactozym 3000 L HP G, was carried out. The synthesis was performed during times up to 300 min at 40, 50 and 60 °C, pH 5.5, 6.5 and 7.5, lactose concentration 150, 250 and 350 mg/mL and enzyme concentration 3, 6 and 9 U/mL. The product mixtures were analysed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). During the hydrolysis of lactose, besides glucose and galactose, galactobiose, allolactose and 6′ galactosyl lactose were also formed as a result of transgalactosylation catalysed by the enzyme. The effect of the reaction conditions was different in the formation of di- and the trisaccharide. Thus, the optimal conditions for galactobiose and allolactose synthesis were 50 °C, pH 6.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 300 min, whereas the best reaction conditions for 6′ galactosyl lactose production were 40 °C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 120 min. These results show the possibility to obtain reaction mixtures with Lactozym 3000 L HP G, with different composition, depending on the assayed conditions.     

Changes in galactose and lactic acid content of sweet whey during storage

Whey is often stored or transported for a period of time prior to processing. During this time period, galactose and lactic acid concentrations may accumulate, reducing the quality of spray-dried whey powders in regard to stickiness and agglomeration. This study surveyed industry samples of Cheddar and mozzarella cheese whey streams to determine how galactose and lactic acid concentrations changed with storage at appropriate (4 degrees C) and abuse (37.8 degrees C) temperatures. Samples stored at 4 degrees C did not exhibit significant increases in levels of lactic acid or galactose. Mozzarella whey accumulated the greatest amount of galactose and lactic acid with storage at 37.8 degrees C. Whey samples derived from cheese made from single strains of starter culture were also evaluated to determine each culture's contribution to galactose and lactic acid production. Starter cultures evaluated included Streptococcus salivarius ssp. thermophilus. Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. cremoris, and Lactococcus lactis ssp. lactis. Whey derived from L. helveticus accumulated a significantly greater amount of lactic acid upon storage at 37.8 degrees C as compared with the other cultures. Galactose accumulation was significantly decreased in whey from L. lactis ssp. lactis stored at 37.8 degrees C in comparison with the other cultures. Results from this study indicate that proper storage conditions (4 degrees C) for whey prevent accumulation of galactose and lactic acid while the extent of accumulation during storage at 37.8 degrees C varies depending on the culture(s) used in cheese production.     

Exopolysaccharide production from whey lactose by fermentation with Lactobacillus delbrueckii ssp. bulgaricus

Ropy Lactobacillus delbrueckii ssp. bulgaricus (strain RR) was used for production of exopolysaccharide in sweet whey and simulated whey permeate (SWP) supplemented with combinations of lactose, KH₂PO₄, NH₄Cl, casamino acids, and mineral salts. Media were incubated at 32, 37, and 44°C for 72h. Periodic adjustment of pH to ～6.2 increased viscosity and lactose utilization, and the free galactose and lactic acid in the media. The effect of pH adjustment was greater than that of supplementation with nutrients or minerals. Fermentation of supplemented SWP generally produced lower viscosities than did fermentation of supplemented sweet whey. After 24h fermentation, viscosity decreased in pH adjusted media. Viscosity of media was highest when incubation was at 32°C and lowest with incubation at 44°C.     

Effects of exopolysaccharide-producing strains of Streptococcus thermophilus on technological and rheological properties of set-type yoghurt

This study investigated the effects of two Streptococcus thermophilus strains, ST 285 and ST 1275, on selected technological and rheological characteristics of set-type yoghurt. The strains were selected for their capability to produce distinctly different exopolysaccharides (EPS) and were thus coded as capsular (ST 285) or ropy-capsular (ST 1275). The culture performance and physico-chemical properties of yoghurt were assessed in relation to different fermentation temperatures (30, 37 or 42 °C) and prolonged storage (up to 30 days) at low temperature (4 °C). ST 1275 showed faster growth and acidification rates, resulting in yoghurt with lower syneresis and higher-flow behaviour index, than ST 285. EPS production appeared to be growth associated with the maximum given at growth temperatures of 37 and 42 °C for ST 285 and ST 1275, respectively; however, EPS concentration declined considerably during storage. Prolonged cold storage increased several rheological characteristics of yoghurt including G′, consistency index and hysteresis loop area. A weak correlation between EPS concentration and textural properties of yoghurt was observed.     

Sources, properties and suitability of new thermostable enzymes in food processing

Investigations concerning recombinant a-amylases from Pyrococcus woesei and thermostable a-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant ss-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are also presented. The activity of this enzyme in a wide pH range of 4.3-6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant a-amylase and ss-galactosidase was facilitated by cloning and expression of genes from Pyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained a-amylase has maximal activity at pH 5.6 and 93 degrees C. The half-life of this preparation (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. An advantageous attribute of recombinant a -amylase is independence of its activity and stability on calcium salt. a-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8-6.9. Thus, this enzyme can be used together with recombinant a-amylase.     

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.     

Influence of starter culture type and incubation temperatures on rheology and microstructure of low fat set yoghurt

The effects of different cultures and incubation temperatures on the physical properties of low fat yoghurts were investigated. The samples were incubated with exopolysaccharide (EPS)-producing and non-EPS-producing cultures at 37, 42 and 45°C. All measured parameters except firmness were influenced by culture type and incubation temperature. Firmness, G' and G" were maximised at 42°C for both cultures. Increased incubation temperature and EPS culture led to a higher water-holding capacity but lower syneresis, G' and G". The EPS treatment incubated at 37°C showed even lower syneresis than non-EPS treatments incubated at higher temperatures.     

Fermentation of reconstituted skim milk supplemented with soy protein isolate by probiotic organisms

ABSTRACT:  Utilization of lactose and production of organic acid were determined in reconstituted skim milk (RSM) and RSM supplemented with soy protein isolate (SPI) (RSMS) by 6 probiotic organisms, including L. acidophilus 4461, L. acidophilus 4962 , L. casei 290, L. casei 2607, B. animalis subsp. lactis bb12, and B. longum 20099. The viable counts of probiotic organisms of RSM and RSMS were enumerated and pH measured during fermentation. Our results showed that 3% to 10% more lactose was utilized by all the 6 probiotic microorganisms from RSMS than RSM. All 6 probiotic organisms produced significantly more acetic acid in RSMS than RSM. However, the viable microbial populations in RSMS were lower than those in RSM due to lower pH of the former. It appears that addition of SPI enhanced lactose utilization and acetic acid production but slightly reduced the lactic acid production and the growth of probiotic microorganisms.