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.     

Influence of pH on the Formation of Glucan by Lactobacillus reuteri TMW 1.106 Exerting a Protective Function Against Extreme pH Values

Lactobacillus reuteri TMW 1.106, a dominant type II sourdough bacterium, produces glucan from sucrose in vitro and in situ. Exopolysaccharides positively affect the texture and mouth feel of foods and their in situ production in fermented foods could be an alternative to the addition of hydrocolloids from plants or non-GRAS microorganisms. The aim of this study was to elucidate a probable function of the EPS for the bacterium. Lb. reuteri TMW 1.106 harbors two glucosyltransferases, Gtf106A and Gtf106B and produces a dextran. Gtf106B exhibited hydrolysis but no transferase activity. Enzymatic production of dextran with the heterologously expressed, N-terminally truncated ΔN Gtf106A was highest at a pH of 4.0, whereas dextran formation in pH static fermentations was optimal between pH 4.7 and 5.4. The dextran synthesised at these pH values had the highest molecular mass (1.2 × 10⁷) and 15% α–(1–4) linkages. A protective effect of this EPS on Lb. reuteri TMW 1.106 against low pH, explaining the low pH-production maximum, could be demonstrated through the delay of cell death.     

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.     

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non-selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 °C or 42 °C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37 °C or 42 °C, aerobiosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37 °C or 42 °C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 °C or 42 °C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 °C, anaerobiosis, L. acidophilus: LC medium, 42 °C, aerobiosis or anaerobiosis and L. casei: LP-MRS, 42 °C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P ≤ 0.05).     

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.     

Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough

Cooperative metabolism of lactobacilli in silage fermentation converts lactate to propionate. This study aimed to determine whether propionate production by Lactobacillus buchneri and Lactobacillus diolivorans can be applied for bread preservation. Propionate formation was observed in cofermentation with L. buchneri and L. diolivorans in modified MRS broth as well as sourdough with low, medium and high ash contents. 48 mM of propionate was formed in sourdough with medium ash content, but only 9 and 28 mM propionate were formed in sourdoughs prepared from white wheat flour or whole wheat flour, respectively. Acetate levels were comparable in all three sourdoughs and ranged from 160 to 175 mM. Sourdough fermented with L. buchneri and L. diolivorans was used in breadmaking and its effect on fungal spoilage was compared to traditional sourdough or propionate addition to straight doughs. Bread slices were inoculated with Aspergillus clavatus, Cladosporium spp., Mortierella spp. or Penicillium roquefortii. The use of 20% experimental sourdough inhibited growth of three of the four moulds for more than 12 days. The use of 10% experimental sourdough deferred growth of two moulds by one day. Bread from traditional sourdough with added acetate had less effect in inhibiting mould growth.     

Survival of Lactobacillus acidophilus and Bifidobacterium animalis ssp. lactis in stirred fruit yogurts

The effect of commercial fruit preparations (mango, mixed berry, passion fruit and strawberry) on the viability of probiotic bacteria, Lactobacillus acidophilus LAFTI^® L10 and Bifidobacterium animalis ssp. lactis LAFTI^® B94 in stirred yogurts during storage (35 days) at refrigerated temperature (4 °C) was evaluated. The results showed that addition of either 5 or 10 g/100 g fruit preparations had no significant (p>0.05) effect on the viability of the two probiotic strains except on L. acidophilus LAFTI L10 yogurt with 10 g/100 g passion fruit or mixed berry. After the addition of fruit preparation, 96% of the yogurts incorporated with fruit preparation did not exhibit a greater loss in the viability of probiotic bacteria compared to plain yogurt during the storage period. A correlation between the post-storage pH in yogurts and the survival of probiotic bacteria was observed. All the yogurts, however, contained the recommended levels of (10⁶-10⁷ cfu/g) probiotic bacteria at the end of 35-day shelf life.     

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 °C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10⁶ cfu g⁻¹). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10 days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an “acid” off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.     

Changes in microbiological, biochemical and physico-chemical properties of Nham inoculated with different inoculum levels of Lactobacillus curvatus

The effect of inoculum level of Lactobacillus curvatus on Nham fermentation was studied. Nham inoculated with L. curvatus at 10⁴ (LC104) and 10⁶ cfu/g (LC106) exhibited a higher rate of fermentation than naturally fermented Nham (control) as indicated by greater rate of pH drop, lactic acid production, and changes in protein compositions. Based on the pH, the fermentation was completed within 72, 48 and 36 h for control, LC104, and LC106, respectively. Higher extent of proteolysis and lipolysis were observed during fermentation of Nham inoculated with starter bacteria (P<0.05). Due to higher acid production rate and extent, texture development of inoculated Nham was more rapid. Inoculated Nham exhibited higher TPA hardness and adhesiveness but lower fracturability than naturally fermented Nham (P<0.05). In terms of acceptability, control and LC104 had higher ratings on the overall liking than LC106 (P<0.05). However, LC104 was better accepted than control in terms of flavour, sourness, saltiness, and texture. Unusual smell was detected only in Nham inoculated at 10⁶ cfu/g. Based on physico-chemical properties and consumer acceptability, L. curvatus is a potential starter for Nham fermentation. However, inoculation of L. curvatus at high level may cause off-flavour in the product.     

Selenium enriched green tea increase stability of Lactobacillus casei and Lactobacillus plantarum in chitosan coated alginate microcapsules during exposure to simulated gastrointestinal and refrigerated conditions

The effects of selenium enriched green tea (SGT; 85.8–96 mg/kg) in different concentrations of 1 g and 2 g/100 mL, on the in vitro exposure to simulated gastrointestinal juice and refrigerated storage of encapsulated Lactobacillus casei and Lactobacillus plantarum were investigated in chitosan coated alginate beads. The encapsulation yield of viable cells in chitosan coated alginate beads with and without SGT was not significantly different (P < 0.05). These results together with the study about the survival of probiotic bacteria in microspheres with SGT during storage at 4 °C, demonstrated significantly higher number (P < 0.05) of survival bacteria in microcapsules with SGT 2 g/100 mL. Microencapsulated L. casei and L. plantarum with SGT 1 g and 2 g/100 mL were resistant to simulated gastric conditions (pH 2.0, 2 h) and bile solution (3 g/100 mL, 2 h) resulting in significantly (P < 0.05) improved survival when compared with microencapsulation without SGT addition.     

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)₅-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.     

The substitution of a traditional starter culture in mutton fermented sausages by Lactobacillus acidophilus and Bifidobacterium animalis

Common starter cultures used in fermented mutton sausages were substituted by probiotic strains of Lactobacillus acidophilus CCDM 476 and Bifidobacterium animalis 241a. Technological properties of the traditional and the probiotic sausages were compared. The potential probiotic effect was evaluated by enumeration of bifidobacteria and lactobacilli in stool samples of 15 volunteers before and after a 14-day consumption period. The numbers of lactobacilli (10⁷ cfu/g) and bifidobacteria (10³ cfu/g) in the final product did not affect the technological properties. The use of L. acidophilus as a starter culture was found more beneficial than the use of B. animalis. Even after 60 days of storage, high counts of L. acidophilus (10⁶ cfu/g) were detected; on the other hand, the counts of B. animalis were under the detection limit. Regarding sensory properties, the probiotic products showed better texture, and, curiously, a reduction of the typical smell of mutton. The numbers of lactobacilli in stool samples increased significantly after the consumption of the probiotic sausages.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

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.     

Aglycone production by Lactobacillus rhamnosus CRL981 during soymilk fermentation

Lactobacillus rhamnosus CRL981 showed the highest levels of β-glucosidase and was selected to characterize this enzyme system, among 63 strains of different Lactobacillus species. The maximum activity was obtained at pH 6.4 and 42 °C. The enzyme showed weak resistance to thermal inactivation maintaining only 20% of the initial activity when it was exposed at 50 °C for 5 min. It also, showed stability when stored at 4 °C for 60 days. Afterwards, L. rhamnosus was evaluated for hydrolysis of isoflavones to aglycones, cell population, residual sugars and organic acid produced during fermentation on soymilk (37 °C for 24 h). Higher viable counts were obtained after 12 h of fermentation (8.85 log CFU ml⁻¹) followed by a drop of pH and an increase of acidity during fermentation due the production of organic acids. L. rhamnosus CRL981 was able to proliferate in soymilk and produce a high β-glucosidase activity achieving a complete hydrolysis of glucoside isoflavones after 12 h of fermentation. The present study indicates that L. rhamnosus CRL981 could be used in the development of different aglycone-rich functional soy beverages.     

Isolation and characterization of acid-sensitive Lactobacillus plantarum with application as starter culture for Nham production

The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants. The growth of three representative mutants was characterized in MRS broth, which revealed that their cell numbers and acid production were lower than that of the wild-type. The H⁺-ATPase activities of the three mutants were found significantly lower than that of the wild-type under either neutral or acidic conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days) and after an additional 4 days of storage at 30 °C. These results indicate that the use of acid-sensitive L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of Nham at ambient temperature.     

Probiotic beverage from cashew apple juice fermented with Lactobacillus casei

This study optimized the conditions of Lactobacillus casei NRRL B-442 cultivation in cashew apple juice, as well as, determined the proper inoculum amount and fermentation time. Moreover, it was investigated the survivability ability of L. casei in cashew apple juice during refrigerated storage (4 °C) for 42 days. The optimum conditions for probiotic cashew apple juice production were initial pH 6.4, fermentation temperature of 30 °C, inoculation level of 7.48 Log CFU/mL (L. casei) and 16 h of fermentation process. It was observed that the L. casei grew during the refrigerated storage. Viable cell counts were higher than 8.00 Log CFU/mL throughout the storage period (42 days). The values of lightness, yellowness and total color change increased and the values of redness reduced along the fermentation and refrigerated storage periods. The fermented juice with L. casei is a good and healthy alternative functional food containing probiotics. Cashew apple juice showed to be as efficient as dairy products for L. casei growth.     

Microbial transformation of tectoridin from Pueraria flos by Lactobacillus and bifidobacteria

Tectoridin could be hydrolyzed to tectorigenin by β-glucosidase-producing intestinal bacteria. In this study, nine strains of Lactobacillus and bifidobacteria were screened for high levels of β-glucosidase activity. We investigated their ability to transform tectoridin from Pueraria flos to tectorigenin. Lactobacillus reuteri DSM20016 showed the highest cell-envelope associated β-glucosidase activity, whereas the intracellular β-glucosidase activity from Bifidobacterium adolescentis ATCC15703 was higher than the other screened bacterial strains. L. reuteri DSM20016, Lactobacillus rhamnosus GGB41031 and B. adolescentis ATCC15703 showed high bioconversion rate of tectoridin. L. reuteri DSM20016 showed the highest bioconversion efficiency of tectoridin, 100% tectoridin was hydrolyzed and there was an approximate 185-fold increase in the concentration of tectorigenin after 24 h. The present study suggests that L. reuteri DSM20016, L. rhamnosus GGB41031 and B. adolescentis ATCC15703 have great potential for converting tectoridin from Pueraria flos to more bioactive tectorigenin.