Persistence and ß-glucan formation of beer-spoiling lactic acid bacteria in wheat and rye sourdoughs.

Some beverage-spoiling lactic acid bacteria (LAB) produce capsular ß-glucans from UDP-glucose, which is accompanied by cell network formation causing viscosity increases of liquids. This feature of certain LAB is feared in breweries but could be useful for structural and nutritional improvement of baked goods, provided that these LAB are suited for the manufacture of sourdoughs. The aim of this study was to investigate the persistence and ß-glucan formation of the brewery isolates Levilactobacillus (L.) brevis TMW 1.2112 and Pediococcus (P.) claussenii TMW 2.340 in wheat and rye sourdoughs. Both the wild-type strains and the respective ß-glucan-deficient mutants were dominant in wheat and rye sourdoughs and acidified them to characteristic pH ranges. The formation of ß-glucan capsules during sourdough fermentations was stable in L. brevis TMW 1.2112 in contrast to P. claussenii TMW 2.340. Wheat sourdoughs fermented with the ß-glucan producing L. brevis TMW 1.2112 cells were significantly more viscous than doughs fermented by the P. claussenii TMW 2.340 cells and the applied mutant strains. In conclusion, L. brevis TMW 1.2112 and P. claussenii TMW 2.340 were suited and persistent wheat and rye sourdough starters, while the in situ ß-glucan formation in sourdoughs was hardly detectable in case of P. claussenii.

In Situ ß-Glucan Fortification of Cereal-Based Matrices by Pediococcus parvulus 2.6: Technological Aspects and Prebiotic Potential.

Bacterial exopolysaccharides produced by lactic acid bacteria are of increasing interest in the food industry, since they might enhance the technological and functional properties of some edible matrices. In this work, Pediococcus parvulus 2.6, which produces an O2-substituted (1,3)-ß-d-glucan exopolysaccharide only synthesised by bacteria, was proposed as a starter culture for the production of three cereal-based fermented foods. The obtained fermented matrices were naturally bio-fortified in microbial ß-glucans, and used to investigate the prebiotic potential of the bacterial exopolysaccharide by analysing the impact on the survival of a probiotic Lactobacillus plantarum strain under starvation and gastrointestinal simulated conditions. All of the assays were performed by using as control of the P. parvulus 2.6's performance, the isogenic ß-glucan non-producing 2.6NR strain. Our results showed a differential capability of P. parvulus to ferment the cereal flours. During the fermentation step, the ß-glucans produced were specifically quantified and their concentration correlated with an increased viscosity of the products. The survival of the model probiotic L. plantarum WCFS1 was improved by the presence of the bacterial ß-glucans in oat and rice fermented foods under starvation conditions. The probiotic bacteria showed a significantly higher viability when submitted to a simulated intestinal stress in the oat matrix fermented by the 2.6 strain. Therefore, the cereal flours were a suitable substrate for in situ bio-fortification with the bacterial ß-glucan, and these matrices could be used as carriers to enhance the beneficial properties of probiotic bacteria.

Effect of temperature (5-25°C) on epiphytic lactic acid bacteria populations and fermentation of whole-plant corn silage.

AIMS: The objective of this study was to investigate the effect of temperature (5-25°C) on epiphytic lactic acid bacteria (LAB) populations during 60 days of fermentation of whole-plant corn silage. METHODS AND RESULTS: Vacuum bag mini-silos of chopped whole-plant corn were incubated at five different temperatures (5, 10, 15, 20 and 25°C), according to a completely randomized design with four repetitions. The silos were opened and sampled on day 0, 1, 2, 3, 7, 28 and 60. At 20 and 25°C, Lactobacillus plantarum- and Pediococcus  pentosaceus-related operational taxonomic units (OTU) dominated the fermentation within 1 day. After 7 days, the OTU related to the heterofermentative species Lactobacillus buchneri began to appear and it eventually dominated silages incubated at these temperatures. Population dynamic of LAB at 5 and 10°C was different. At these temperatures, Leuconostoc citreum OTU was identified at the beginning of the fermentation. Thereafter, Lactobacillus sakei- and Lactobacillus curvatus-related OTU appeared and quickly prevailed. Corn silage at 15°C acted as a transition between 20-25°C and 5-10°C, in terms of LAB diversity and succession. CONCLUSION: The conditions of silage incubation temperature affect species diversity of LAB population with notable difference along the temperature gradient. Colder temperature conditions (5 and 10°C) have led to the identification of LAB species never observed in corn silage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the impact of temperature gradient on the diversity and some important population shift of lactic acid bacteria communities during fermentation of corn silage.

Phenolic acid degradation potential and growth behavior of lactic acid bacteria in sunflower substrates.

Sunflower flour provides a high content of protein with a well-balanced amino acid composition and is therefore regarded as an attractive source for protein. The use for human nutrition is hindered by phenolic compounds, mainly chlorogenic acid, which can lead under specific circumstances to undesirable discolorations. In this study, growth behavior and degradation ability of chlorogenic acid of four lactic acid bacteria were explored. Data suggested that significant higher fermentation performances on sunflower flour as compared to sunflower protein concentrate were reached by Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus gasseri and Bifidobacterium animalis subsp. lactis. In fermentation with the latter two strains reduced amounts of chlorogenic acid were observed in sunflower flour (-11.4% and -19.8%, respectively), which were more pronounced in the protein concentrate (-50.7% and -95.6%, respectively). High tolerances against chlorogenic acid and the cleavage product quinic acid with a minimum inhibitory concentration (MIC) of ≥20.48 mg/ml after 48 h were recorded for all strains except Bifidobacterium animalis subsp. lactis, which was more sensitive. The second cleavage compound, caffeic acid revealed a higher antimicrobial potential with MIC values of 0.64-5.12 mg/ml. In this proof of concept study, degradation versus inhibitory effect suggest the existence of basic mechanisms of interaction between phenolic acids in sunflower and lactic acid bacteria and a feasible way to reduce the chlorogenic acid content, which may help to avoid undesired color changes.

Analysis of cell growth dynamics of Pediococcus acidilactici in the presence of inulin in an optimized microenvironment.

The present investigation deals with the optimization of cell growth rate of the candidate probiotic Pediococcus acidilactici in the presence of the specific prebiotic inulin. Three independent variables viz. concentration of inulin, concentration of glucose and pH have been selected for optimization study using response surface methodology. Theoretical analysis indicates that the maximum cell growth rate occurs at pH 7, 20 g/dm(3) concentration of inulin and 20 g/dm(3) concentration of glucose. Validation of these values has been done through a set of programmed experiments. Studies on cell dynamics in the presence of different concentrations of inulin have also been carried out to identify any limitation on the initial inulin concentration. Results clearly indicate that cell growth is enhanced with the increase in inulin concentration. However, there is a critical value of the prebiotic concentration (20 g/dm(3) inulin) beyond which the cell growth is inhibited. A summative type growth model has been proposed to explain the growth behaviour of P. acidilactici in the presence of the dual substrate, i.e. glucose and inulin. While growth on glucose follows Monod model, Haldane-type substrate-inhibited growth model holds good for growth on inulin. Intrinsic kinetic parameters for all the model equations have been determined experimentally.

Effect of yogurt containing deep sea water on health-related serum parameters and intestinal microbiota in mice.

Deep sea water (DSW) has health benefits and is widely used as food supplement; however, its effect in fermented products has not been explored. Here, we investigated the effect of DSW-containing yogurt on health-related serum parameters and intestinal microbiota in mice. Animals were assigned to 3 feeding groups, which received water (control), normal yogurt (N-yogurt), or DSW-containing yogurt (DSW-yogurt) with a basal diet. Mice were killed at wk 4 or 8 of feeding and analyzed for serum parameters and microbial population in the small intestine. Both yogurt groups demonstrated increased populations of intestinal lactic acid bacteria compared with the control group. The activity of serum aspartate aminotransferase and alanine aminotransferase was markedly decreased in the DSW-yogurt and N-yogurt groups, and triglyceride level tended to be lower in the DSW-yogurt group compared with that in the control mice. Furthermore, the DSW-yogurt group showed a more significant decrease in the ratio of total cholesterol to high-density lipoprotein-cholesterol than did the N-yogurt group. These findings suggest that DSW supplementation of yogurt can increase its beneficial effects on lipid metabolism.

Effect of microbial inoculants on the quality and aerobic stability of bermudagrass round-bale haylage.

The objective of this study was to compare the efficacy of using 4 commercially available microbial inoculants to improve the fermentation and aerobic stability of bermudagrass haylage. We hypothesized that the microbial inoculants would increase the fermentation and aerobic stability of the haylages. Bermudagrass (4-wk regrowth) was harvested and treated with (1) deionized water (control); (2) Buchneri 500 (B500; Lallemand Animal Nutrition, Milwaukee, WI) containing 1×10(5) of Pediococcus pentosaceus and 4×10(5) of Lactobacillus buchneri 40788; (3) Biotal Plus II (BPII; Lallemand Animal Nutrition) containing 1.2×10(5) of P. pentosaceus and Propionibacteria freudenreichii; (4) Silage Inoculant II (SI; AgriKing Inc., Fulton, IL) containing 1×10(5) of Lactobacillus plantarum and P. pentosaceus; and (5) Silo King (SK; AgriKing Inc.), containing 1×10(5) of L. plantarum, Enterococcus faecium, and P. pentosaceus, respectively. Forty round bales (8 per treatment; 441±26kg; 1.2×1.2 m diameter) were made and each was wrapped with 7 layers of plastic. Twenty bales were stored for 112 d and the remaining 20 were stored for 30 d and sampled by coring after intermediary storage periods of 0, 3, 7, and 30 d. The pH of control and inoculated haylages sampled on d 3 did not differ. However, B500 and BPII had lower pH (5.77±0.04 vs. 6.16±0.04; 5.06±0.13 vs. 5.52±0.13) than other treatments by d 7 and 30, respectively. At final bale opening on d 112, all treatments had lower pH than the control haylage (4.77±0.07 vs. 5.37±0.07). The B500, BPII, and SI haylages had greater lactic acid and lactic-to-acetic acid ratios than SK and control haylages. No differences were detected in neutral detergent fiber digestibility, dry matter losses, dry matter, lactic and acetic acid concentrations, and yeast and coliform counts. The SK haylage had lower clostridia counts compared with the control (1.19±0.23 vs. 1.99±0.23 cfu/g). Treatments B500, BPII, SI, and SK tended to reduce mold counts and they improved aerobic stability by 236, 197, 188, and 95%, respectively, compared with the control (276±22 vs. 99±22h).

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BACKGROUND: Species of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3. RESULTS: Antimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity. CONCLUSION: To our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

In situ production of human ß defensin-3 in lager yeasts provides bactericidal activity against beer-spoiling bacteria under fermentation conditions.

AIMS: To examine the use of a natural antimicrobial peptide, human ß-defensin-3 (HBD3), as a means of preventing spoilage from bacterial contamination in brewery fermentations and in bottled beer. METHODS AND RESULTS: A chemically synthesised HBD3 peptide was tested for bactericidal activity against common Gram-positive and Gram-negative beer-spoiling bacteria, including species of Lactobacillus, Pediococcus and Pectinatus. The peptide was effective at the µmol l(-1) range in vitro, reducing bacterial counts by 95%. A gene construct encoding a secretable form of HBD3 was integrated into the genome of the lager yeast Saccharomyces pastorianus strain CMBS-33. The integrated gene was expressed under fermentation conditions and was secreted from the cell into the medium, but a significant amount remains associated with yeast cell surface. We demonstrate that under pilot-scale fermentation conditions, secreted HBD3 possesses bactericidal activity against beer-spoiling bacteria. Furthermore, when added to bottled beer, a synthetic form of HBD3 reduces the growth of beer-spoiling bacteria. CONCLUSIONS: Defensins provide prophylactic protection against beer-spoiling bacteria under brewing conditions and also in bottled beer. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have direct application to the brewing industry where beer spoilage due to bacterial contamination continues to be a major problem in breweries around the world.

Administration of Lactobacillus salivarius LI01 or Pediococcus pentosaceus LI05 improves acute liver injury induced by D-galactosamine in rats.

This work investigated the effect of the intragastric administration of five lactic acid bacteria from healthy people on acute liver failure in rats. Sprague-Dawley rats were given intragastric supplements of Lactobacillus salivarius LI01, Lactobacillus salivarius LI02, Lactobacillus paracasei LI03, Lactobacillus plantarum LI04, or Pediococcus pentosaceus LI05 for 8 days. Acute liver injury was induced on the eighth day by intraperitoneal injection of 1.1 g/kg body weight D-galactosamine (D-GalN). After 24 h, samples were collected to determine the level of liver enzymes, liver function, histology of the terminal ileum and liver, serum levels of inflammatory cytokines, bacterial translocation, and composition of the gut microbiome. The results indicated that pretreatment with L. salivarius LI01 or P. pentosaceus LI05 significantly reduced elevated alanine aminotransferase and aspartate aminotransferase levels, prevented the increase in total bilirubin, reduced the histological abnormalities of both the liver and the terminal ileum, decreased bacterial translocation, increased the serum level of interleukin 10 and/or interferon-γ, and resulted in a cecal microbiome that differed from that of the liver injury control. Pretreatment with L. plantarum LI04 or L. salivarius LI02 demonstrated no significant effects during this process, and pretreatment with L. paracasei LI03 aggravated liver injury. To the best of our knowledge, the effects of the three species-L. paracasei, L. salivarius, and P. pentosaceus-on D-GalN-induced liver injury have not been previously studied. The excellent characteristics of L. salivarius LI01 and P. pentosaceus LI05 enable them to serve as potential probiotics in the prevention or treatment of acute liver failure.

Biotyping of cultivable lactic acid bacteria isolated from donkey milk.

UNLABELLED: The diversity of lactic acid bacteria (LAB) species in donkey's milk was analysed by culture-dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heat-treated at 63°C for 10 min before incubation at 37°C (heat-treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkey's milk have been discussed below. SIGNIFICANCE AND IMPACT OF THE STUDY: There is increased interest in using donkey's milk as a source of human nutrition. The large amounts of antimicrobial components and defence factors present in donkey's milk provide protection from microbial infections and distinguish donkey's milk from the milks of other mammals. However, the microbiota in donkey's milk has so far been poorly characterized, specifically with regard to the lactic acid bacteria (LAB). This study has identified cultivable, acidifying and thermoduric LAB that could be used to develop starter cultures. This is the first study to investigate the culturable LAB microbiota present in donkey's milk.

Effect of regrowth interval and a microbial inoculant on the fermentation profile and dry matter recovery of guinea grass silages.

The objectives of this study were to characterize and quantify the microbial populations in guinea grass (Panicum maximum Jacq. cultivar Mombasa) harvested at different regrowth intervals (35, 45, 55, and 65 d). The chemical composition and fermentation profile of silages (after 60 d) with or without the addition of a microbial inoculant were also analyzed. Before ensiling, samples of the plants were used for the isolation and identification of lactic acid bacteria (LAB) in the epiphytic microbiota. A 4 × 2 factorial arrangement of treatments (4 regrowth intervals × with/without inoculant) was used in a completely randomized design with 3 replications. Based on the morphological and biochemical characteristics and the carbohydrate fermentation profile, Lactobacillus plantarum was found to be the predominant specie of LAB in guinea grass forage. Linear increases were detected in the dry matter (DM) content and concentrations of neutral detergent fiber, acid detergent fiber, acid detergent insoluble nitrogen, and DM recovery as well as linear reductions in the concentrations of crude protein and NH3-N with regrowth interval. Additionally, linear reductions for gas and effluent losses in silages were detected with increasing regrowth interval. These results demonstrate that guinea grass plants harvested after 55 d of regrowth contain a LAB population sufficiently large to ensure good fermentation and increase the DM recovery. The use of microbial inoculant further enhanced the fermentation of guinea grass at all stages of regrowth by improving the DM recovery.

Yerba mate enhances probiotic bacteria growth in vitro but as a feed additive does not reduce Salmonella Enteritidis colonization in vivo.

Yerba mate (Ilex paraguariensis) is a tea known to have beneficial effects on human health and antimicrobial activity against some foodborne pathogens. Thus, the application of yerba mate as a feed additive for broiler chickens to reduce Salmonella colonization was evaluated. The first in vitro evaluation was conducted by suspending Salmonella Enteritidis and lactic acid bacteria (LAB) in yerba mate extract. The in vivo evaluations were conducted using preventative and horizontal transmission experiments. In all experiments, day-of-hatch chicks were treated with one of the following 1) no treatment (control); 2) ground yerba mate in feed; 3) probiotic treatment (Lactobacillus acidophilus and Pediococcus; 9:1 administered once on day of hatch by gavage); or 4) both yerba mate and probiotic treatments. At d 3, all chicks were challenged with Salmonella Enteritidis (preventative experiment) or 5 of 20 chicks (horizontal transmission experiment). At d 10, all birds were euthanized, weighed, and cecal contents enumerated for Salmonella. For the in vitro evaluation, antimicrobial activity was observed against Salmonella and the same treatment enhanced growth of LAB. For in vivo evaluations, none of the yerba mate treatments significantly reduced Salmonella Enteritidis colonization, whereas the probiotic treatment significantly reduced Salmonella colonization in the horizontal transmission experiment. Yerba mate decreased chicken BW and decreased the performance of the probiotic treatment when used in combination. In conclusion, yerba mate had antimicrobial activity against foodborne pathogens and enhanced the growth of LAB in vitro, but in vivo yerba mate did not decrease Salmonella Enteritidis colonization.

Growth, acid production and bacteriocin production by probiotic candidates under simulated colonic conditions.

AIMS: The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA-1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine. METHODS AND RESULTS: The three bacteriocin-producing strains were grown in Macfarlane broth and in De Man-Rogosa-Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co-culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant. CONCLUSIONS: The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.

Development of a carbohydrate-supplemented semidefined medium for the semiselective cultivation of Lactobacillus spp.

The macronutrient and micronutrient compositions of traditional media used to cultivate Lactic Acid Bacteria (LAB) are largely undefined, which precludes their use in many metabolic bioassays. In order to address this deficiency, we developed MS: a carbohydrate-supplemented semidefined medium with low-background coloration. MS was designed to support the semiselective cultivation of a wide range of fastidious species belonging to the Lactobacillus clade of the LAB. When supplemented with 100 mM D-glucose, the MS medium stimulated the proliferation of 21 strains of LAB, including Pediococcus spp. and Lactobacillus spp. The MS medium supported biomass accumulation comparable with MRS, an undefined medium routinely used for the cultivation of lactobacilli. Interestingly, however, the novel MS medium exhibited greater semiselectivity against non-LAB than MRS. Together, these results suggest that MS is an acceptable alternative to MRS for use in metabolic and phenotypic bioassays that use a colorimetric reporter system or would benefit from a semidefined nutrient composition.

Complete genome sequence of the beer spoilage organism Pediococcus claussenii ATCC BAA-344T.

Pediococcus claussenii is a common brewery contaminant. We have sequenced the chromosome and plasmids of the type strain P. claussenii ATCC BAA-344. A ropy variant was chosen for sequencing to obtain genetic information related to growth in beer, as well as exopolysaccharide and possibly biofilm formation by this organism.

The synthesis of active and stable diaminopimelate analogues of the lantibiotic peptide lactocin S.

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.

RT-qPCR analysis of putative beer-spoilage gene expression during growth of Lactobacillus brevis BSO 464 and Pediococcus claussenii ATCC BAA-344(T) in beer.

Lactic acid bacteria (LAB) contamination of beer presents a continual economic threat to brewers. Interestingly, only certain isolates of LAB can grow in the hostile beer environment (e.g., as studied here, Lactobacillus brevis BSO 464 (Lb464) and a non-ropy isolate of Pediococcus claussenii ATCC BAA-344(T) (Pc344NR)), indicating that significant genetic specialization is required. The genes hitA, horA, horB, horC, and bsrA, which have been proposed to confer beer-spoiling ability to an organism, are suspected of counteracting the antimicrobial effects of hops. However, these genes are not present in the same combination (if at all) across beer-spoiling organisms. As such, we sought to investigate the extent to which these genes participate during Lb464 and Pc344NR mid-logarithmic growth in beer through reverse transcription quantitative PCR analysis. We first determined the optimal reference gene set needed for data normalization and, for each bacterium, established that two genes were needed for accurate assessment of gene expression. Following this, we found that horA expression was induced for Pc344NR, but not for Lb464, during growth in beer. Instead, horC expression was dramatically increased in Lb464 when growing in beer, whereas no change was detected for the other putative beer-spoilage-related genes. This indicates that HorC may be one of the principle mediators enabling growth of Lb464 in beer, whereas in Pc344NR, this may be attributable to HorA. These findings not only reveal that Lb464 and Pc344NR are unique in their beer-specific genetic expression profile but also indicate that a range of genetic specialization exists among beer-spoilage bacteria.

Development of a novel probiotic delivery system based on microencapsulation with protectants.

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host's gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 µm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 µm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F1F0-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 µm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 µm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.

Beta-1,3-glucanase from Delftia tsuruhatensis strain MV01 and its potential application in vinification.

During vinification microbial activities can spoil wine quality. As the wine-related lactic acid bacterium Pediococcus parvulus is able to produce slimes consisting of a ß-1,3-glucan, must and wine filtration can be difficult or impossible. In addition, the metabolic activities of several wild-type yeasts can also negatively affect wine quality. Therefore, there is a need for measures to degrade the exopolysaccharide from Pediococcus parvulus and to inhibit the growth of certain yeasts. We examined an extracellular ß-1,3-glucanase from Delftia tsuruhatensis strain MV01 with regard to its ability to hydrolyze both polymers, the ß-1,3-glucan from Pediococcus and that from yeast cell walls. The 29-kDa glycolytic enzyme was purified to homogeneity. It exhibited an optimal activity at 50°C and pH 4.0. The sequencing of the N terminus revealed significant similarities to ß-1,3-glucanases from different bacteria. In addition, the investigations indicated that this hydrolytic enzyme is still active under wine-relevant parameters such as elevated ethanol, sulfite, and phenol concentrations as well as at low pH values. Therefore, the characterized enzyme seems to be a useful tool to prevent slime production and undesirable yeast growth during vinification.