Identification of Lactobacillus curvatus TMW 1.624 dextransucrase and comparative characterization with Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 dextransucrases

Recently, it was affirmed that the exopolysaccharides (EPSs) of Lactobacillus curvatus TMW 1.624, Lactobacillus reuteri TMW 1.106 and Lactobacillus animalis TMW 1.971 improve the quality of gluten-free breads and that they can be produced in situ to levels enabling baking applications. In this study we provide insight into the molecular and biochemical background of EPS production of these three strains. EPS formation strongly correlated with growth and took place during the exponential phase. Gtf genes were heterologously expressed, purified and their enzymatic properties as well as the structures of the EPSs formed were compared. Structural comparison of EPS formed by heterologously expressed glucosyltransferases (Gtfs) and of those formed by the wildtype lactobacilli confirmed that the respective genes/enzymes were identified and examined. The glucan formed by L. animalis Gtf was identified as a linear low molecular weight dextran. Optimal enzymatic conditions were pH 4.4 and 45 °C for the L. reuteri Gtf and pH 4.4 and 31 °C for L. curvatus Gtf. The Gtf from L. animalis had an optimal pH of 5.8 and displayed more than 50% of activity over a broad temperature profile (22–59 °C). The three Gtfs were stimulated by various mono- and divalent metal ions, dextran, as well as levan to different extents.     

Effect of structurally different microbial homoexopolysaccharides on the quality of gluten-free bread

The effect of structurally different bacterial homoexopolysaccharides on gluten-free bread quality and their properties to act as hydrocolloids was investigated. Furthermore, exopolysaccharides (EPS) were analyzed structurally by asymmetrical flow field flow fractionation and methylation analysis. Breads were made of buckwheat and rice flour with EPS of Lactobacillus (L.) curvatus TMW 1.624, L. reuteri TMW 1.106, L. animalis TMW 1.971, and L. sanfranciscensis TMW 1.392 or hydroxypropylmethylcellulose (HPMC) at 1?% w/w flour base. Water-holding capacity, specific volume, crumb analysis, baking loss, moisture content, and crumb hardness were determined. Only HPMC and the glucan of L. curvatus TMW 1.624 retained water, and all supplements increased the specific volume. Furthermore, crumb hardness was decreased by additives to different extents. The moisture content, baking loss, and crumb firmness were improved most by dextran of L. curvatus TMW 1.624. Structure analysis of EPS revealed that L. animalis TMW 1.971 produces a fructan and a glucan and that the dextran of L. curvatus TMW 1.624 had highest molecular weight of analyzed EPS, ranging from 118 to 242 MDa. A methylation analysis demonstrated differences in branching. Dextran of L. reuteri TMW 1.106 is branched in position 4 (18?C19?%), whereas dextran of L. curvatus TMW 1.624 is branched in position 3 (8?C9?%). Overall, this study gives insight into structure function relations of different EPS. A structure function relation is suggested in which high weight average molar mass (Mw) and branching at position 3 of the glucose monomer foster a compact conformation of the molecule, which enables an increased water-binding capacity and promotes superior (structural) effects in gluten-free breads. The dextran of L. curvatus TMW 1.624 was the most promising candidate for applications in gluten-free bread quality improvements as it retains its size distribution and root mean square even with increasing Mw and forms an increasingly compact molecule.     

Optimization of exopolysaccharide yields in sourdoughs fermented by lactobacilli

In this study, the yields of exopolysaccharides (EPS) produced in situ during sourdough fermentations with Lactobacillus reuteri TMW 1.106 synthesizing glucan from sucrose were investigated under variation of the fermentation parameters dough yield (DY), pH, sucrose content and fermentation substrate. The obtained amounts of EPS after 1 day of fermentation were higher in softer (DY 500) than in firmer (DY 220) doughs. With the regulation of the pH to a constant value of 4.7, the optimum for EPS synthesis in liquid medium, the EPS production in dough also increased. The EPS yield could further be improved by additional sucrose fed-batch during fermentation. Fermentations with wheat flours, a rye-wheat mixture and rye bran with 10% sucrose as fermentation substrate showed, that the use of rye bran is a promising tool to get high dextran formation through L. reuteri even in the first 8 h of fermentation. Further, alternative production of oligosaccharides and organic acids from sucrose was investigated. Lactobacillus reuteri synthesized high amounts of acetic acid leading to low fermentation quotient values. In wheat doughs, the formation of maltooligosaccharides was observed. Confirmatory experiments with fructan producing Lactobacillus sanfranciscensis TMW 1.392 revealed the same trends with a few distinct differences, indicating that this approach is transferable to other EPS types and producers.     

Identification and characterization of a glucan-producing enzyme from Lactobacillus hilgardii TMW 1.828 involved in granule formation of water kefir

Water kefir is a home made fermented beverage based on a sucrose solution with fruit extracts. The inoculum of such fermentations consists of macroscopic granula containing lactic and acetic acid bacteria, and yeasts, which are embedded in an exopolysaccharide (EPS) matrix. In this work, a strain of Lactobacillus hilgardii producing large amounts of the granule-forming dextran could be isolated. The glycosyltransferans (Gtf) commonly called glucansucrase responsible for the production of this dextran was purified from L. hilgardii. Characteristic enzyme kinetic data were obtained. Optimum activity was observed between pH 4.3 and 4.6 and temperatures between 40 °C and 45 °C. A Michaelis–Menten kinetic could be fit to the experimental data and a K_M of 0.0385 M was calculated. The corresponding gtf gene was identified and characterized. It encodes a 1448 amino acid protein with higher homologies to Gtfs of Lactobacillus parabuchneri, Lactobacillus sakei and Lactobacillus fermentum followed by lower homologies to Lactobacillus reuteri Gtfs. By knockout experiments the role of this gene in granule dextran production was demonstrated.     

Effects of inulin,fructooligosaccharides/glucose and pH on the shape of the death kinetic of Lactobacillus reuteri DSM 20016

The viability of Lactobacillus reuteri, Lb. delbrueckii subsp. bulgaricus and Lb. paracasei was assessed in a laboratory medium containing inulin (5 g L^?1) or glucose + FOS (fructooligosaccharides) (2.5 + 2.5 g L^?1). Data were modelled through the Weibull equation pinpointing that prebiotics determined a decrease in the shape parameter. In the 2nd step, L. reuteri was used as the test microorganism; pH, storage temperature and the amounts of FOS + glucose and inulin were combined through a 4‐variable/5‐level central composite design. Temperature, pH and glucose + FOS affected the microbiological shelf life; inulin was not significant. The maximum value of shelf life was found at pH 8.5 and with 5.0 g L^?1 of glucose + FOS. The combination of pH and prebiotics affected the shape parameter with a shift from a convex to a concave trend and vice versa: their effect was different depending on the use of a single prebiotic or a mix.     

Antagonistic effects of Lactobacillus reuteri against Escherichia coli O157:H7 in white-brined cheese under different storage conditions

This study aimed to investigate the survival of the foodborne pathogen Escherichia coli O157:H7 in white-brined cheeses as influenced by the presence of Lactobacillus reuteri. The white cheeses were made from pasteurized bovine milk inoculated with E. coli O157:H7 (cocktail of 3 strains) to achieve ～5 log₁₀ cfu/g with absence or presence of Lb. reuteri (～6 log₁₀ cfu/g). Cheese samples were brined in 10% or 15% NaCl solution and stored at 10°C and 25°C for 28 d. The white-brined cheeses were assessed for salt content, pH, water activity (A_w), and numbers of E. coli O157:H7, Lb. reuteri, nonstarter lactic acid bacteria (NSLAB), yeasts, and molds. Results showed that E. coli O157:H7 survived in cheese stored in both brine solutions at 10°C and 25°C regardless of the presence of Lb. reuteri. A substantial reduction was observed in cheese stored in 10% NaCl brine at 25°C, followed by cheese stored in 15% NaCl brine at 10°C by 2.64 and 2.16 log₁₀ cfu/g, respectively, in the presence of Lb. reuteri and by 1.02 and 1.87 log₁₀ cfu/g, respectively, in the absence of Lb. reuteri under the same conditions. The pathogen in brine solutions survived but at a lower rate. Furthermore, the growth of Lb. reuteri and NSLAB were enhanced or slightly decreased in cheese and brine by 28 d, respectively. The salt concentrations of cheese ranged from 4 to 6% and 5 to 7% (wt/wt), during 28-d ripening in 10 and 15% brine, respectively. Values of pH and A_w slightly increased at d 1 after exposure to brine and reached 4.69 to 6.08 and 0.91 to 0.95, respectively, in all treatments. Therefore, the addition of Lb. reuteri can be used as a biopreservation method to inhibit the survival of E. coli O157:H7 in white-brined cheese when combined with the appropriate temperature, NaCl level, and storage time.     

Optimal conditions for the production of exopolysaccharide by Pediococcus acidilactici and impact of the bacterium,its EPS and dextran on the quality of Kariesh cheese

The effect of some growth conditions on the production of exopolysaccharide (EPS) by Pediococcus acidilactici was studied. Furthermore, the impact of utilisation of the isolated EPS, dextran and P. acidilactici on some properties of Kariesh cheese was investigated. The maximum EPS production by P. acidilactici was obtained after 10 h of incubation at 37°C in MRS medium with an initial pH of 7. Kariesh cheese manufactured with dextran or P. acidilactici exhibited the highest (P ≤ 0.05) moisture content and the lowest hardness values. Protein, fat, ash, total bacterial and yeast and mould counts were not significantly affected by the applied treatments. However, the body and texture of Kariesh cheese were significantly improved.     

Characterization of Lactobacillus isolates from infant faeces as dietary adjuncts

Lactobacillus paracasei subsp. paracasei (six strains), Lb. rhamnosus (six strains), Lb. acidophilus (two strains), Lb. gasseri (three strains) and Lb. reuteri (three strains) isolates from new-born infants were tested for their ability to grow and metabolize in milk and to resist specific conditions of the gastrointestinal tract. Many of the tested strains had desirable properties concerning their ability to withstand adverse conditions of the gastrointestinal tract, and produce carbonyl compounds. In general, strains of Lb. paracasei subsp.paracasei and Lb. rhamnosus were more resistant to low pH of the stomach than all the other strains. On the other hand, Lb. gasseri and Lb. reuteri strains did not acidify milk. Thus, Lb. paracasei subsp.paracasei , Lb. acidophilus and Lb. rhamnosus strains could be preferably used as starters to produce fermented milks with possibly interesting organoleptic properties, as well as dietary and possible therapeutic importance.     

Exopolysaccharides produced by Lactobacillus sp.: Biosynthesis and applications

Lactobacillus sp. synthesize exopolysaccharides (EPS), including both homo- and heteropolysaccharides, which play an important role in the production of fermented foods, and especially in the dairy industry, improving the gustatory and rheological properties of the finished products. These polymers are generated by starter cultures in situ in fermented foods, and so they are treated as natural thickening agents. As some Lactobacillus strains are generally recognized as safe and have been shown to exhibit probiotic activity, EPS from those bacteria can be used as functional food ingredients, conferring both health and economic benefits to the consumers. However, their industrial applications are hindered by the low yield of EPS from Lactobacillus and high costs of their purification. This review focuses on the latest reports concerning the biosynthesis and properties of Lactobacillus EPS.     

Performance of <Emphasis Type="Italic">Lactobacillus sanfranciscensis</Emphasis> TMW 1.392 and its levansucrase deletion mutant in wheat dough and comparison of their impact on bread quality

The performance of Lactobacillus sanfranciscensis TMW 1.392 and its levansucrase deletion mutant TMW 1.392 Δlev in wheat dough was compared. The effects of both strains on dough and bread characteristics were determined in order to find benchmarks for in situ production of exopolysaccharides (EPS). Growth and acidification were lower in doughs prepared with the Δlev mutant than in those employing the wild type. Extensogram resistance of the dough was reduced and extensibility increased with the addition of L. sanfranciscensis levan. Added EPS positively influenced water absorption, bread volume and firming of the crumb. In situ production of EPS was not sufficient to achieve the same positive effects of EPS, as they partially overlapped with effects resulting from enhanced acidification. Control doughs were made to separate effects of predough, EPS and different metabolism/acidification. High acetic acid levels decreased extensibility and volume. High lactic acid levels negatively influenced crumb hardness and firming kinetics. The use of knock out mutants proved helpful to judge overall performance of a strain, although the interpretation of specific effects must consider all changes in its metabolism.     

Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX

The putative protective role of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11, and its non-EPS-producing isogenic strain NB1, was tested upon HT29-MTX monolayers challenged with seven opportunistic pathogens. The probiotic strain Lactobacillus rhamnosus LMG18243 (GG) was used as a reference bacterium. Tested lactobacilli were able to efficiently reduce the attachment to HT29-MTX of most pathogens. Lb. paraplantarum NB1 and Lb. rhamnosus GG were more efficient reducing the adhesion of Clostridium difficile or Yersinia enterocolitica than Lb. paraplantarum BGCG11, while strain BGCG11 reduced, to a greater extent, the adhesion of Escherichia coli and Listeria monocytogenes. The detachment and cell lysis of HT29-MTX monolayers in the presence of pathogens alone and co-incubated with lactobacilli or purified EPS was followed. L. monocytogenes induced the strongest cell detachment among the seven tested pathogens and this effect was prevented by addition of purified EPS-CG11. The results suggest that this EPS could be an effective macromolecule in protection of HT29-MTX cells from the pathogen-induced lysis. Regarding innate intestinal barrier, the presence of C. difficile induced the highest IL-8 production in HT29-MTX cells and this capability was reinforced by the co-incubation with Lb. paraplantarum NB1 and Lb. rhamnosus GG. However, the increase in IL-8 production was not noticed when C. difficile was co-incubated with EPS-producing Lb. paraplantarum BGCG11 strain or its purified EPS-CG11 polymer, thus indicating that the polymer could hinder the contact of bacteria with the intestinal epithelium. The measurement of mucus secreted by HT29-MTX and the expression of muc1, muc2, muc3B and muc5AC genes in the presence of pathogens and lactobacilli suggested that all lactobacilli strains are weak “co-adjuvants” helping some pathogens to slightly increase the secretion of mucus by HT29-MTX, while purified EPS-CG11 did not induce mucus secretion. Taking altogether, Lb. paraplantarum BGCG11 could act towards the reinforcement of the innate mucosal barrier through the synthesis of a physical-protective EPS layer which could make difficult the contact of the pathogens with the epithelial cells.     

Effect of temperature on growth and metabolism of probiotic bacteria in milk

The growth and metabolism of six probiotic strains with documented health effects were studied in ultra-high temperature (UHT) treated milk supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose at different temperatures. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lb. johnsonii LA1, Lb. rhamnosus GG, Lb. reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 48 h at 20, 30, 37 and 45 °C and the samples were analysed for pH, log cfu mL⁻¹, volatile compounds, organic acids and carbon dioxide. All six probiotic strains showed very different profiles of metabolites during fermentation, however, the two Lb. acidophilus strains were the most alike. All strains, except Lb. reuteri SD 2112, showed viable cell numbers above 6.5 log cfu mL⁻¹ after 48 h fermentation at 30, 37 and 45 °C. The probiotic strains produced different amounts of metabolic products according to temperature and fermentation time illustrating the importance of controlling these parameters.     

Identification and Partial Characterization of a Bacteriocin‐Like Inhibitory Substance (BLIS) from Lb. Bulgaricus K41 Isolated from Indigenous Yogurts

Forty‐two strains of Lactobacillus bulgaricus isolated from locally made yogurts were examined and compared for bacteriocin producing ability using spot on lawn assay which improved by taking photo and image processing. Lb. bulgaricus K41 exhibited the highest inhibition level against indicators. K41 Bacteriocin‐like inhibitory substance is sensitive to proteolytic enzymes (proteinase K, pepsin, and trypsin) but α‐amylase makes slight reduction in its activity and it is resistant to lipase. This antibacterial peptide is extremely heat‐stable (121 °C for 15 min) and remains active over a wide pH range (pH = 2 to 10); also nonionic detergents (Tween‐20, Tween‐80, and Triton X100) showed no effect on its activity. The inhibitory spectrum is against Gram‐positive bacteria (except Staphylococcus aureus) with extremely antilisterial activity and it is almost ineffective against Gram‐negative bacteria. The mode of its action was identified as bactericidal against Listeria monocytogenes. The properties of K41 bacteriocin‐like inhibitory substance add to its safety as a biopreservative produced by a generally recognized as safe (GRAS) bacterium suggesting it can be used in hurdle technology for ready‐to‐eat foods as one of the main sources of Listeria contaminations.     

Influences of pH and electrolyte on the rheological properties of aqueous solution of exopolysaccharide secreted by a deep-sea mesophilic bacterium

The effects of pH and electrolytes (NaCl and CaCl₂) on the viscosity, dynamic modulus (storage modulus G′ and loss modulus G″) and thixotropy of aqueous solution of new exopolysaccharide (SM-A87 EPS) secreted by a deep-sea mesophilic bacterium Wangia profunda SM-A87 were investigated. It was found that at pH 3–12 and the electrolyte concentration of 0.1–80 g/L, the SM-A87 EPS solution showed very weakly pH-dependent and salt-dependent rheological properties and dominant elastic behavior. These properties were considered to arise from the hyper-branched structure of the SM-A87 EPS molecules through the comparison of rheological properties of SM-A87 EPS solutions with those of partially hydrolyzed polyacrylamide ones at different electrolyte concentrations. Such good pH stability and salt resistance of the SM-A87 EPS solution indicate that SM-A87 EPS has great potential for application as food additive, thickener, stabilizer and enhanced oil recovery system. In the studied conditions, 2 g/L SM-A87 EPS solution showed positive thixotropy. The relative thixotropic recovery decreased with pH increasing and the electrolyte concentration decreasing.     

The influence of exopolysaccharide-producing lactic acid bacteria on reconstructed ham

The influence of in-situ-formed exopolysaccharides (EPS) by lactic acid bacteria (LAB; Lactobacillus sakeiTMW 1.411 and Lactobacillus plantarumTMW 1.1478) on the yield and texture of reconstructed ham was investigated. No differences in yield (P > 0.05) were observed but weight loss during storage was significantly higher (P < 0.05) for hams produced with L. sakei 1.411. Furthermore, the produced EPS decreased the hardness (P < 0.05; 50.22 N for control compared to 44.81 N) but did not influence the cohesiveness (P > 0.05) of hams. Products with L. plantarum 1.1478 showed no significant differences in comparison to the control. This could be attributed to the lower amount of EPS formed during ripening. L. sakei 1.411 produced 194.49 ± 5.34 mg kg⁻¹ EPS, whereas L. plantarum 1.1478 formed 60.26 ± 2.96 mg kg⁻¹ EPS. Thus, the use of the examined LAB is not recommended in reconstructed ham, since they have no or even a negative influence on the product quality.     

Relationship between gastrointestinal tolerance and exopolysaccharide production of propionibacteria strains under different pH and bile conditions

In this study, we report the effects of exposure to simulated gastric and intestinal juices on the survival of six strains of dairy propionibacteria. Gastrointestinal tolerance of propionibacteria strains was highly variable, depending on the strain and pH (P < 0.05). Exopolysaccharides (EPS) production of six propionibacteria strains under different pH conditions and bile concentrations was also studied. A positive correlation was obtained between the EPS production quantity of the strains and tolerance to simulated gastric juices at pH 2.0, 3.0 (P < 0.05). This investigation showed that high EPS production may be important in the selection of probiotic strains for resistance to upper gastrointestinal system conditions.     

Potential probiotic characterization of Lactobacillus reuteri from traditional Chinese highland barley wine and application for room-temperature-storage drinkable yogurt

The aim of this study was to select probiotic strains that could be used in drinkable yogurt to yield viable cells following storage at room temperature (RT). The uniquely high altitude conditions in Tibet and the alcoholic environment of certain products, such as the highland barley wine homemade in Tibet, may induce unusual characteristics of microbial strains. A total of 27 lactic acid bacteria were isolated from homemade highland barley wines. One strain, Lactobacillus reuteri WHH1689, demonstrated no ability for lactose utilization, exhibited a high survival rate during storage at RT in drinkable yogurts, and produced very weak post-acidification. This strain showed great resistance to conditions simulating the gastrointestinal tract, including strong adherence to HT-29 cells and inhibitory activity against Escherichia coli, Shigella flexneri, Salmonella paratyphi β, and Staphylococcus aureus. A dextran sulfate sodium (DSS)–induced mouse model was used to evaluate the in vivo influence of Lb. reuteri WHH1689 on the intestinal flora and showed that strain WHH1689 increased viable counts of bifidobacteria in feces of mice. The probiotic strain selected in this study—with its high survival at RT and lack of serious post-acidification problems—may provide significant improvements for dairy industry products by extending the storage time of dairy products with living cells.     

Bistage control of pH for improving exopolysaccharide production from mycelia of Ganoderma lucidum in an air-lift fermentor

It was found that pH control definitely affects mycelial cell growth and exopolysaccharide (EPS) production of the mycelial cultivation of Ganoderma lucidum. Compared to the case of uncontrolled pH cultivation, a culture system whose pH was kept constant at 3 and 6 exhibited improved mycelial cell growth and EPS production, respectively. The bistage pH control technique, that is, shifting the pH from 3 to 6 at the initial phase of the exponential growth, is introduced to improve cell growth and EPS production. This technique can greatly increase EPS production to 20.1 g/l from 4.1 g/l in the case of uncontrolled pH cultivation, without adverse effects on cell growth as in the case of constant maintenance of a high pH. It was also proved that bistage pH control retained the desirable morphologies of the mycelia during cultivation and resulted in low viscosity and yield stress of the culture broth. It will be useful for the application of the culture process to mycelial growth in a large-scale fermentor.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Angiotensin I converting enzyme (ACE) inhibitory activity and antihypertensive effect of fermented milk

Milk was fermented with a total of 25 lactic acid bacteria to assay in vitro inhibitory activity towards angiotensin I converting enzyme (ACE). The tested strains belonged to Lactobacillus acidophilus, Lactobacillus casei, Lacobacillus helveticus, Lactobacillus jensenii, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis ssp. lactis, Lactococcus. raffinolactis and Leuconostoc mesenteroides ssp. cremoris. The ACE inhibitory potencies of theses strains varied and seven of them showing the highest ACE inhibitory activity were selected for further studies. The development of ACE inhibitory activity during fermentation correlated with degree of hydrolysis. Modification of fermentation conditions or pH control did not affect the ACE inhibitory activity. ACE inhibitory compounds from Lb. jensenii fermented milk were isolated by reversed phase HPLC and identified by MS-analysis and amino acid sequencing. The active compounds were peptides from β-casein. The milk fermented with Lb. jensenii caused a transient reduction of blood pressure in spontaneously hypertensive rats.