Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and finds applications even in non-dairy foods and in therapeutics. Box-Behnken model of response surface methodology (RSM) was employed to formulate the production medium for exopolysaccharide (EPS). FT-IR spectral analysis of the purified EPS from Lactobacillus plantarum MTCC 9510 revealed prominent characteristic groups corresponding to polyhydric alcohols. The degradation temperature (Td) of the polysaccharide was found to be 260°C with the help of thermo gravimetric analysis (TGA). Structure elucidation of the EPS showed that it consists of a trisaccharide repeating unit of α-d-glucose, β-d-glucose and α-d-mannose.     

Binding Interactions Between α-glucans from Lactobacillus reuteri and Milk Proteins Characterised by Surface Plasmon Resonance

Interactions between milk proteins and ??-glucans at pH 4.0?C5.5 were investigated by use of surface plasmon resonance. The ??-glucans were synthesised with glucansucrase enzymes from Lactobacillus reuteri strains ATCC-55730, 180, ML1 and 121. Variations in the molecular characteristics of the ??-glucans, such as molecular weight, linkage type and degree of branching, influenced the interactions with native and denatured ??-lactoglobulin and ??-casein. The highest overall binding levels were reached with ??-(1,4) compared to ??-(1,3) linked glucans. Glucans with many ??-(1,6) linkages demonstrated the highest binding levels to ??-casein, whereas the interaction with native ??-lactoglobulin was suppressed by ??-(1,6) linkages. Glucans with a higher degree of branching generally displayed lower protein binding levels whereas a higher molecular weight resulted in increased binding to ??-casein. The interactions with ??-casein were not pH dependent, whereas binding to denatured ??-lactoglobulin was highest at pH 4.0 and binding to native ??-lactoglobulin was optimal at pH 4.5?C5.0. This study shows that molecular weight, linkage type and degree of branching of ??-glucans highly influence the binding interactions with milk proteins.     

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ_1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ_1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, α_S- and β-casein. Likewise, incubating Aβ_1–40 with αB-crystallin inhibited both Aβ_1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.     

Constitutive expression,purification and characterization of bovine prochymosin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> GS115

Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn²⁺, Fe³⁺, Mg²⁺ and Na⁺, but inhibited by K⁺, Co²⁺, Zn²⁺, Ni²⁺, and to a lesser extent by Cu²⁺. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.     

In vitro fermentation of prebiotics by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> CFR 2194: selectivity,viability and effect of metabolites on β-glucuronidase activity

Prebiotic Fructooligosaccharides (FOS) escape metabolism in upper GI tract undergo microbial metabolism in colon and thereby influence the nature, type and number of intestinal microbiota to improve host’s health. The present study focuses on the ability of Lactobacillus plantarum CFR 2194 to utilize FOS as a selective carbon and energy source. The effect of fermentative metabolites of L. plantarum on the β-glucuronidase was also investigated. A total of 16 strains of lactobacilli were assessed for their ability to ferment oligosaccharides. L. plantarum CFR 2194, an isolate from kanjika was found to utilize FOS effectively. Lactic acid was the main metabolic end product, followed by acetic acid, butyric acid, formic acid and ethanol. The inhibitory effects of these metabolites have been confirmed through the reduction of β-glucuronidase activity. L. plantarum when co-cultured with β-glucuronidase producing E. coli, in a basal media containing FOS as an energy source, could inhibit the growth of the pathogen during the course of fermentation. The results showed that L. plantarum CFR 2194 has the ability to utilize the prebiotic FOS as a selective carbon and energy source. The organism could inhibit the growth of the pathogen which produces β-glucuronidase and lowered its activity by the metabolites of FOS which indicates the probable use of L. plantarum through dietary intervention in combating colon carcinogenesis.     

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature

Aims: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α‐linolenic acid (α‐LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. Methods and Results: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1–60%). CLA conversion, occurring in three strains, was lower (2–5%). The LAI gene sequences of the ten LAI‐positive strains shared 75–99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6·2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5·5 (30°C) or 37°C (pH 6·2), LA was not converted and α‐LNA only slightly converted. Conclusions: LAB show strain‐dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. Significance and Impact of the Study: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.     

Partial characterization and dynamics of synthesis of high molecular mass exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

Exopolysaccharide (EPS) preparations from Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) strains LBB.B26 and LBB.B332 and Streptococcus thermophilus strains LBB.T54 and LBB.T6V were characterized using ion-exchange chromatography and gel filtration. All four preparations contained a neutral EPS with molecular mass in the range of 1.3−1.6 × 10⁶ Da (HMM-EPS). The EPS preparations from the two L. bulgaricus strains also contained an acidic low molecular mass EPS fraction (LMM-EPS) comprising from 10% to 34% of the total EPS yield. HMM-EPS preparations were subjected to High Pressure Liquid Chromatography (HPLC) analysis of monomer sugars after complete hydrolysis. Glucose, galactose and/or rhamnose in different ratios proved to be the principal sugars building the HMM-EPS from all four strains. The chemical composition of HMM-EPS was strictly strain-specific. The LMM-EPS contained galactose. The viscosifying properties of the four different HMM-EPS varied greatly with intrinsic viscosity in the range from 0.26 (strain B26) to 2.38 (strain T6V). For 24 h the two L. bulgaricus strains accumulated more HMM-EPS in milk (>70 mg l⁻¹) than S. thermophilus strains T54 and T6V (<30 mg l⁻¹), but maximal yields were reached earlier with cocci (8 h) than with rods (16–24 h). The contribution of HMM-EPS production to increased viscosity of fermented milk was demonstrated for all of the tested strains grown as monocultures or as mixed yogurt starters compared to non-EPS producing S. thermophilus LBB.A and poor EPS-producer L. bulgaricus LBB.B5. The extent of increased viscosity was strongly dependent on the nature of the produced HMM-EPS, rather than simply on polymer yield.     

Porcineβ-lactoglobulin A and C

Summary The occurrence of the dominant ‘whey’ protein in samples of milk from 1180 sows is examined. It exhibits genetic polymorphism with some unusual features. Although immunologically different from bovine β-lactoglobulin, it is shown by chemical studies of the isolated protein to be a β-lactoglobulin. Two homozygous genetic variants, designated porcine β-lactoglobulin A and C, are isolated and their amino acid compositions and peptide maps compared. It is shown that the C variant has +1 His, −1 Gln, and +1 Asp, −1 Glu, with respect to the A variant. These variants, containingca. 162 residues per molecule, are considered in relationship to porcine β-lactoglobulins isolated by other workers. The sequence of the first 50 residues is determined and compared with sequence of the bovine protein. The sequences ofca. 70% of the remaining residues is proposed on the basis of the composition of tryptic peptides and assumed homology.     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

Variation in specificity of the PrtP extracellular proteinases in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis>

Comparison of cell-wall-bound extracellular proteinases (CEPs) from Lactobacillus paracasei (LBP) ssp. paracasei natural isolates BGHN14, BGAR75 and BGAR76 with Lactococcus lactis (LCL) ssp. cremoris Wg2, in their action on α_S1-, β- and κ-casein was done. The CEPs of LBP strains were able to degrade α_S1- and β-caseins and their caseinolytic specificity depended on the type of buffer used. These CEPs, compared with LCL Wg2, differ in four amino acid residues in small segments predicted to be involved in substrate binding. The most striking features of this comparison are the presence of Ala instead of Ser³²⁹ and the presence of Thr instead of Asn²⁵⁶ and Ala²⁹⁹, in the subtilisin-like region of the CEP in LBP natural isolates. Additional conservative amino acid substitution Leu to Ile³⁶⁴ was found.     

Differential Roles of the Two-Component Peptides of Lactocin 705 in Antimicrobial Activity

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705α and Lac705β. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705α/Lac705β peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705β peptide alone. Furthermore, Lac705β increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705α protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Δψ and ΔpH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705α peptide is responsible for receptor recognition, and the Lac705β peptide is the active component on the cell membrane of CRL691 cells. Received: 12 April 2002 / Accepted: 24 May 2002     

Exopolysaccharide production by Lactobacillus casei under controlled pH

Summary The exopolysaccharide (EPS) production and growth characteristics of Lactobacillus casei CRL 87 under pH control were studied. Maximum polymer synthesis (488 mg/l) and cell viability (2.4×10¹⁰ cfu/ml) occurred when L. casei was cultured at a constant pH of 6.0 and 30°C for 24 h. However, the optimum specific EPS production (3.9×10^-5 g EPS/g cell dry weigt) and EPS yield (4.3%) were found at a pH of 4.0.     

Heat-Induced Whey Protein Gels: Effects of pH and the Addition of Sodium Caseinate

The effects of pH (6.7 or 5.8), protein concentration and the heat treatment conditions (70 or 90 °C) on the physical properties of heat-induced milk protein gels were studied using uniaxial compression, scanning electron microscopy, differential scanning calorimetry, and water-holding capacity measurements. The systems were formed from whey protein isolate (10–15% w/v) with (5% w/v) or without the addition of caseinate. The reduction in pH from 6.7 to 5.8 increased the denaturation temperature of the whey proteins, which directly affected the gel structure and mechanical properties. Due to this increase in the denaturation temperature of the β-lactoglobulin and α-lactalbumin, a heat treatment of 70 °C/30 min did not provide sufficient protein unfolding to form self-supporting gels. However, the presence of 5% (w/v) sodium caseinate decreased the whey protein thermo stability and was essential for the formation of self-supporting gels at pH 6.7 with heat treatment at 70 °C/30 min. The gels formed at pH 6.7 showed a fine-stranded structure, with great rigidity and deformability as compared to those formed at pH 5.8. The latter had a particulate structure and exuded water, which did not occur with the gels formed at pH 6.7. The addition of sodium caseinate led to less porous networks with increased gel deformability and strength but decreased water exudation. The same tendencies were observed with increasing whey protein concentration.     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Cloning and characterization of the lactate dehydrogenase genes from<Emphasis Type="Italic">Lactobacillus</Emphasis> sp. RKY2

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (IdhL andIdhD) encoding the L-(+) and D-(−) lactate dehydrogenases (L-LDH and D-LDH) were cloned fromLactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames ofIdhL for andIdhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(−)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes ofLactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.     

Measuring and modelling the glucose-fermenting activity of Lactobacillus plantarum

Summary The pH decrease in a phosphate buffer due to fermentation of glucose to lactic acid by non-growing Lactobacillus plantarum cells has been studied. The method used offers a quick and reproducible way of measuring the glucose-fermenting activity of L. plantarum. The maximum observed velocity of pH decrease is linear with the biomass concentration and is defined as the activity of the cell suspension. With L. plantarum, recalculation of this arbitrary unit (pH·min^–1 per gram dry weight) to a conceivable unit of lactic acid production rate (mol·min^–1 per gram dry weight) is possible. This recalculation is based on the titration theory of a weak base with a weak acid. The same theory together with the lactic acid production kinetics of L. plantarum is applied to model the entire pH-time curve.Offprint requests to: L. C. Lievense     

Use of virginiamycin to control the growth of lactic acid bacteria during alcohol fermentation

The antibiotic virginiamycin was investigated for its effects on growth and lactic acid production by seven strains of lactobacilli during the alcoholic fermentation of wheat mash by yeast. The lowest concentration of virginiamycin tested (0.5 mg Lactrol ^TMkg⁻¹ mash), was effective against most of the lactic acid bacteria under study, but Lactobacillus plantarum was not significantly inhibited at this concentration. The use of virginiamycin prevented or reduced potential yield losses of up to 11% of the produced ethanol due to the growth and metabolism of lactobacilli. However, when the same concentration of virginiamycin was added to mash not inoculated with yeast, Lactobacillus rhamnosus and L. paracasei grew after an extensive lag of 48 h and L. plantarum grew after a similar lag even in the presence of 2 mg virginiamycin kg⁻¹ mash. Results showed a variation in sensitivity to virginiamycin between the different strains tested and also a possible reduction in effectiveness of virginiamycin over prolonged incubation in wheat mash, especially in the absence of yeast. Received 05 August 1996/ Accepted in revised form 18 December 1996     

A κ-carrageenase from a newly isolated pseudoalteromonas-like bacterium, WZUC10

A bacterial strain able to produce κ-carrageenase, designated WZUC10, was isolated from a live specimen of the red alga Plocamium telfainae collected in the East China Sea. The phylogenetic evidence and phenotypic features indicate that this strain belongs to the genus Pseudoalteromonas. WZUC10 requires NaCl for growth and κ-carrageenan to induce κ-carrageenase synthesis; galactose and lactose do not induce it. The optimal growth temperature is 23∼27°C. The secreted enzyme, which has a molecular mass of 45 kDa, breaks down κ-carrageenan into κ-neocarratetraose sulfate and larger oligosaccharides with a repeating β-D-Galp4S-(1→4)-α-D-AnGalp structure, but cannot degrade κ-neocarratetraose sulfate or κ-neocarrahexaose sulfate into κ-neocarrabiose sulfate. The enzyme retains 90% of its activity after 2 h at 40°C and is completely inactivated after 7.5 min at 70°C. The enzyme’s optimal temperature is 30°C and its optimal pH is 7.5. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics, with the Michaelis constant (K _m) and the turnover number (k) being 0.015 mM and 125 s⁻¹, respectively. WZUC10 produces 50 U/mL κ-carrageenase after cultivation at 25°C for 35 h on a medium containing 80 g/L glucose, 5 g/L corn steep liquor, 3 g/L κ-carrageenan, and 15 g/L NaCl. κ-Neocarratetraose sulfate was prepared simply with precipitation by ethanol:water (5:1, v/v).     

β-Galactosidase production by the psychrotolerant yeast Guehomyces pullulans 17-1 isolated from sea sediment in Antarctica and lactose hydrolysis

A psychrotolerant yeast Guehomyces pullulans 17-1 was isolated from sea sediment in Antarctica. It was found that it could yield both extracellular and cell-bound β-galactosidase. After optimization of the medium and cultivation conditions, it was found that the yeast strain produced over 17.2 U mL⁻¹ of β-galactosidase activity within 120 h at the flask level while the yeast strain produced over 25.3 U mL⁻¹ of β-galactosidase activity within 144 h during the 2-L fermentation. This is the highest β-galactosidase activity produced by the wild type yeast strains reported so far. However, the optimal pH and temperature for the crude β-galactosidase were 4.0 and 50 °C, respectively. Lactose could be converted into glucose and galactose and a large amount of reducing sugar could be released from milk under catalysis of the yeast culture.