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γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10 g/L to 50 g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03 g/mL glucose and 0.4 g/mL sodium glutamate for 72 h, the GABA yield reached 239 g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields. [ABSTRACT FROM AUTHOR]

We evaluated the ability of an inoculant containing a combination of Lactobacillus hilgardii and Lactobacillus buchneri to modify the microbiome and improve the aerobic stability of whole-plant corn silage after various lengths of ensiling. Chopped whole-plant corn at about 33% dry matter (DM) was uninoculated (CTR) or inoculated with L. hilgardii CNCM I-4785 and L. buchneri NCIMB 40788 at 200,000 cfu/g of fresh forage weight each (combined application rate of 400,000 cfu of lactic acid bacteria/g of fresh forage weight; LHLB), L. buchneri NCIMB 40788 at 400,000 cfu/g of fresh forage weight and Pediococcus pentosaceus NCIMB 12455 at 100,000 cfu/g of fresh forage weight, used as a positive control (LB500) , L. hilgardii CNCM I-4785 at the application rate used in the LHLB formulation of 200,000 cfu/g of fresh forage weight (LH), or L. buchneri NCIMB 40788 at the application rate used in the LHLB formulation of 200,000 cfu/g of fresh forage weight (LB). Silos were opened after 34 and 99 d of ensiling and analyzed for nutrient composition, fermentation profile, microbiome, and aerobic stability. After 34 d of ensiling, the inoculated silages had greater numbers of culturable lactic acid bacteria, a bacterial community less rich and diverse, greater relative abundance of Lactobacillus , lower relative abundance of Klebsiella , and a greater concentration of propionic acid than uninoculated silages. Inoculation decreased the ratio of lactic acid to acetic acid, except for LB alone. Treatment LHLB resulted in silage with a greater concentration of 1,2-propanediol than LB500 and was the only treatment to have a lower relative abundance of Saccharomycetes compared with uninoculated silage. Treatments LHLB and LB500 improved the aerobic stability compared with CTR, but the individual LH and LB treatments applied at a low dose did not. Whereas LB500 was stable 34 h longer than CTR, LHLB was stable 91 h longer. After 99 d of ensiling, all inoculated silages had markedly greater aerobic stability than uninoculated silage and were stable for more than 360 h. The inoculant containing a combination of L. hilgardii and L. buchneri markedly improved the aerobic stability of corn silage after a relatively short period of ensiling, and such improvements were greater than the ones obtained from inoculation with the combination of L. buchneri and P. pentosaceus. Inoculating with the combination of L. hilgardii and L. buchneri may be helpful to producers that must feed silage shortly after ensiling. [ABSTRACT FROM AUTHOR]

A meta-analysis of 158 peer-reviewed articles was conducted to examine effects of inoculation with Lactobacillus buchneri (LB)-based inoculants (LBB) that did or did not include homolactic or obligate heterolactic bacteria on silage fermentation and aerobic stability. A complementary meta-analysis of 12 articles examined LBB inoculation effects on dairy cow performance. Raw mean differences between inoculant and control treatment means weighted by inverse variance were compared with a hierarchical effects model that included robust variance estimation. Meta-regression and subgrouping analysis were used to identify effects of covariates including forage type, application rate (≤104, 105, 106, or ≥ 107 cfu/g as fed), bacteria type (LB vs. LB plus other bacteria), enzyme inclusion, ensiling duration, and silo type (laboratory or farm scale). Inoculation with LBB increased acetate (62%), 1, 2 propanediol (364%) and propionate (30%) concentration and aerobic stability (73.8%) and reduced lactate concentration (7.2%), yeast counts (7-fold) and mold counts (3-fold). Feeding inoculated silage did not affect milk yield, dry matter intake, and feed efficiency in lactating dairy cows. However, forage type, inoculant composition, and dose effects on silage quality measures were evident. Inoculation with LBB increased aerobic stability of all silages except tropical grasses. Adding obligate homolactic or facultative heterolactic bacteria to LB prevented the small increase in DM losses caused by LB alone. The 105 and 106 cfu/g rates were most effective at minimizing DM losses while aerobic stability was only increased with 105, 106, and ≥ 107 cfu/g rates. Inoculation with LBB increased acetate concentration, reduced yeast counts and improved aerobic stability but did not improve dairy cow performance. [ABSTRACT FROM AUTHOR]

Aims: To evaluate the capacity of Lactobacillus hilgardii and Lactobacillus buchneri on modifying the bacterial community and improving fermentation and aerobic stability of high‐moisture corn (HMC). Methods and Results: High‐moisture corn was untreated (CTR), treated with L. hilgardii (LH) or L. buchneri (LB) at 600 000 CFU per gram fresh weight, or with L. hilgardii and L. buchneri at 300 000 CFU per gram fresh weight each (LHLB), and stored for 10, 30 or 92 days. Compared to CTR, inoculated silages had higher Lactobacillaceae relative abundance, lower yeasts numbers and higher aerobic stability. Treatment with LHLB resulted in a higher acetic acid concentration than LH and higher 1,2 propanediol concentration than LB, such differences were numerically greater at 10 and 30 days but statistically greater at 92 days. At 10 days, all inoculated silages were more stable than CTR, but LHLB was even more stable than LB or LH. Conclusions: The combination of L. hilgardii and L. buchneri had a synergistic effect on yeast inhibition, leading to greater improvements in aerobic stability as early as 10 days after ensiling. Significance and Impact of the Study: Lactobacillus hilgardii, especially in combination with L. buchneri, can improve the aerobic stability of HMC after a very short period of ensiling. [ABSTRACT FROM AUTHOR]

Elevated levels of biogenic amines (BAs) in fermented food can have negative effects on both the flavor and health. Mining enzymes that degrade BAs is an effective strategy for controlling their content. The study screened a strain of Lactobacillus hilgardii 1614 from fermented food system that can degrade BAs. The multiple copper oxidase genes LHMCO 1614 were successfully mined after the whole genome protein sequences of homologous strains were clustered and followed by homology modeling. The enzyme molecules can interact with BAs to stabilize composite structures for catalytic degradation, as shown by molecular docking results. Ingeniously, the kinetic data showed that purified LHMCO 1614 was less sensitive to the substrate inhibition of tyramine and phenylethylamine. The degradation rates of tyramine and phenylethylamine in huangjiu (18% vol) after adding LHMCO 1614 were 41.35 and 40.21%, respectively. Furthermore, LHMCO 1614 demonstrated universality in degrading tyramine and phenylethylamine present in other fermented foods as well. HS-SPME-GC-MS analysis revealed that, except for aldehydes, the addition of enzyme treatment did not significantly alter the levels of major flavor compounds in enzymatically treated fermented foods ( p > 0.05). This study presents an enzymatic approach for regulating tyramine and phenylethylamine levels in fermented foods with potential applications both targeted and universal.

The environment temperature and its effect on the temperature of silage is very important for the fermentation and subsequent quality of a silage. Obligate heterofermentative lactic acid bacteria (LAB) inocula, because of their ability to inhibit yeasts, have been developed to prevent the aerobic deterioration of silages. The temperature during silage conservation may also play an important role in the fermentation profile of silages. This study has evaluated the effect of temperature, during the conservation of whole crop corn silage, untreated or treated with different LAB inocula, on the fermentation profile and on the aerobic stability of the silage. Corn was harvested at 42% dry matter and either not treated (control) or treated with Lactobacillus buchneri NCIMB 40788 (LB) at 300,000 cfu/g fresh matter (FM); Lactobacillus hilgardii CNCM I-4785 at 150,000 cfu/g FM (LH 150); L. hilgardii CNCM I-4785 at 300,000 cfu/g FM (LH 300); or LB+LH at 150,000 cfu/g FM each. In an attempt to experimentally simulate temperature fluctuations in the mass or at the periphery of a silage bunker, corn was conserved in laboratory silos at a constant temperature (20 ± 1°C; MASS) or at lower and variable outdoor temperatures (PERIPH; ranging from 0.5 to 19°C), and the silos were opened after 15, 30, and 100 d of conservation. Lactic acid, acetic acid, and ethanol contents increased in all the silages over the conservation period. The lactic acid content was higher (+10%) in the silages kept at a constant temperature than those conserved at the lower and variable outdoor temperatures. The acetic acid was higher in the treated silages than in the control ones conserved at a constant temperature for 100 d. Moreover, 1,2-propanediol was only detected in the treated silages after at least 30 d at a constant temperature, whereas only traces were detected in the LB+LH treatment for the other temperature conditions. The yeast count decreased during conservation at a slower rate in PERIPH than in MASS and on average reached 2.96 and 4.71 log cfu/g for MASS and PERIPH, respectively, after 100 d of conservation. The highest aerobic stability values were observed for LH 300 (191 h) in the MASS silage after 100 d of conservation, whereas the highest aerobic stability was observed in LB+LH (150 h) in the PERIPH silages. After 7 d of air exposure, a pH higher than 4.5 and a higher yeast than 8.0 log cfu/g were detected in all the silages opened after 15 and 30 d of conservation. A pH value close to that of silo opening was detected in the LB, LH 150 , and LH 300 silages conserved under MASS conditions after 100 d, whereas LB+LH was the most effective under PERIPH conditions. The temperature and its fluctuation during conservation of silage in laboratory silos influenced the fermentation, which in turn had an effect on the quality of silage and on the extent of the effect of LAB inocula. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of inoculation of whole crop corn silage with a mixture of heterofermentative lactic acid bacteria (LAB) composed of Lactobacillus hilgardii and Lactobacillus buchneri on ensiling, aerobic stability, ruminal fermentation, total tract nutrient digestibility, and growth performance of beef cattle. Uninoculated control corn silage (CON) and silage inoculated with 3.0 × 105 cfu g−1 of LAB containing 1.5 × 105 cfu g−1 of L. hilgardii CNCM I-4785 and 1.5 × 105 cfu g−1 of L. buchneri NCIMB 40788 (INOC) were ensiled in silo bags. The pH did not differ (P > 0.05) between the two silages during ensiling but was greater (P < 0.001) for CON than INOC after 14 d of aerobic exposure (AE). Neutral detergent insoluble crude protein (NDICP) content (% of DM and % of CP basis) of terminal INOC silage was greater (P ≤ 0.05) than that of CON. In terminal silage, concentrations of total VFA and acetate were greater (P < 0.001), while water-soluble carbohydrates were lower (P < 0.001) for INOC than CON. Yeast and mold counts were lower for INOC than CON (P ≤ 0.001) in both terminal and aerobically exposed silages. The stability of INOC was greater (P < 0.001) than that of CON after 14 d of AE. Ruminal fermentation parameters and DMI did not differ (P > 0.05) between heifers fed the two silages, while there was a tendency (P ≤ 0.07) for lower CP and starch digestibility for heifers fed INOC than CON. Total nitrogen (N) intake and N retention were lower (P ≤ 0.04) for heifers fed INOC than CON. Dry matter intake as a percentage of BW was lower (P < 0.04) and there was a tendency for improved feed efficieny (G:F; P = 0.07) in steers fed INOC vs. CON silage. The NEm and NEg contents were greater for INOC than CON diets. Results indicate that inoculation with a mixture of L. hilgardii and L. buchneri improved the aerobic stability of corn silage. Improvements in G:F of growing steers fed INOC silage even though the total tract digestibility of CP and starch tended to be lower for heifers fed INOC are likely because the difference in BW and growth requirements of these animals impacted the growth performance and nutrient utilization and a greater proportion of NDICP in INOC than CON. [ABSTRACT FROM AUTHOR]

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BACKGROUND Lactic acid bacteria inocula have been developed over the years to improve the aerobic stability of silages. The aims of the study were to evaluate the effect of various conservation periods and the use of Lactobacillus hilgardii inoculum on aerobic stability, fermentative profile and microbial population of corn and sorghum silages. Trials were carried out on two corns and one sorghum crops. The crops were untreated or treated with L. buchneri (LB, application rate 300 000 cfu g−1 FM), L. hilgardii (LH, application rate 300 000 cfu g−1 FM) and a combination (LB + LH, application rate 150 000 cfu g−1 FM each). Silos were opened after 15, 30, 100 and 250 days of conservation, and the silages were analysed for fermentative profile, microbial count and aerobic stability. RESULTS: During conservation, the inocula influenced the fermentation profile. The use of LH increased the aerobic stability at 15 and 30 days in one out of three trials, while after 100 and 250 days, the presence of LB alone or with LH led to greater stability. In all the trials, the acetic acid content increased, the yeast count decreased and the aerobic stability increased during the conservation period. CONCLUSIONS: This study has shown that a long period in complete anaerobiosis reduced yeast count and improved aerobic stability in all silages. The addition of LB was confirmed to be a good option for increasing aerobic stability of silages, whereas the effect of LH alone or in combination with LB on aerobic stability was not consistent between trials. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The ensiling of sugarcane results in high dry-matter ( DM) loss, but the addition of glycerine may compensate for the loss during ensiling. Methanol is the most undesirable contaminant of crude glycerine destined for animal feeding. The aim of this study was to evaluate the ability of the yeast strain Pichia methanolica NCYC 1381 to reduce the methanol concentration in sugarcane silage inoculated with Lactobacillus hilgardii CCMA 0170 + glycerine. A randomized design consisted of four dose rates of glycerine (0, 4, 8 and 12% of fresh forage), three periods of silage fermentation (11, 34 and 68 d) and three combinations of microbial additives [ L. hilgardii ( LH), L. hilgardii plus P. methanolica ( LH + PM) and without microbial additive ( WI)]. The DM of the fresh sugarcane was 275 g kg−1. The linear reduction in neutral detergent fibre caused by glycerine inclusion was probably due to a dilution effect. The LH treatment increased the concentrations of the succinic, acetic and propionic acids, and 1,2-propanediol, and reduced the yeast population. The LH + PM treatment increased DM loss of sugarcane silage with 12% glycerine and L. hilgardii CCMA 0170 (6·1 log cfu g−1 of FM) reduced the DM loss when compared to the silage without additives. Under the conditions of the experiment, the P. methanolica treatment did not reduce the methanol concentration in silage. [ABSTRACT FROM AUTHOR]

[Display omitted] • Obligately heterofermentative Lactobacillus promoted the organic acid production. • Citric acid addition into inoculants improved the acetic acid concentration. • The combined treatment achieved the highest yield of organic acids. • The highest organic acid production was 225.41 g/kg dry matter. • The combined treatment stimulated obligately heterofermentative microbes. The effects of Lactobacillus buchneri , Lactobacillus hilgardii and citric acid on organic acid production, substrate consumption, protein degradation and microbial community were investigated in this study. The results indicated that combined inoculants induced a significant increase in levels of lactic acid (43 g/kg dry matter), acetic acid (14 g/kg dry matter), butyric acid (5 g/kg dry matter), total organic acid (60 g/kg dry matter) and ammonia nitrogen (20 g/kg total nitrogen). Furthermore, citric acid addition into the combined inoculants caused a significant increase in levels of acetic acid (12 g/kg dry matter), water-soluble carbohydrate (12 g/kg dry matter) and a reduction in ammonia nitrogen formation (22 g/kg total nitrogen). Microbiologically, combining inoculants and citric acid enriched Lactobacillus buchneri and Lactobacillus hilgardii and upregulated the functional pathways related to acid production and resistance. Collectively, combining citric acid and heterofermentative inoculants was beneficial to recycle Chinese cabbage waste in producing organic acids. [ABSTRACT FROM AUTHOR]

Water kefir is a slightly alcoholic, lactic and acetic beverage fermented by yeasts, lactic acid bacteria and acetic acid bacteria that are associated with the polysaccharide of the water kefir grains. In this study, the three main metabolic products of microorganisms were evaluated during a traditional 192-h water kefir fermentation and also after inoculating the microorganisms in fresh medium or sterilised broth from different fermentation stages. The first process to occur was alcoholic fermentation, carried out in particular by Saccharomyces cerevisiae. After 24 h, lactic and acetic acid accumulation was generated by Lactobacillus hilgardii and Acetobacter tropicalis. By the end of fermentation, ethanol had been almost entirely consumed and oxidised to acetic acid, possibly by a dissimilatory route of Acetobacter species. An original hypothetical diagram is proposed for the carbon flux from sucrose, and the metabolic role of the main yeasts and bacteria is assigned for the distinct stages of water kefir fermentation. [ABSTRACT FROM AUTHOR]

Aerobic stability is an important feature in the evaluation of silages. The aims were to investigate the chemical and microbiological changes that occur in sugar-cane ( Saccharum spp.) silage after aerobic exposure, to identify the major species of yeasts associated with the aerobic deterioration process and to select lactic acid bacteria ( LAB) strains that can improve the aerobic stability of this silage. Fourteen wild LAB strains belonging to Lactobacillus plantarum, L. brevis and L. hilgardii were evaluated using experimental silos. Silage samples were collected at 0, 96 and 216 h after aerobic exposure to determinate the DM, WSC, p H, products of fermentation, to evaluate the silage temperatures and to identify yeast species associated with the aerobic deterioration of silage. The strains tested were able to modify the fermentative and chemical parameters and the diversity of yeasts species of silage after aerobic exposure. There was no association between the facultative or obligatory heterofermentative fermentation patterns and the increased aerobic stability of silage. Aerobic stability of sugar-cane silages was associated with high acetic acid and 1,2-propanediol concentrations. L. hilgardii UFLA SIL51 and UFLA SIL52 strains promoted an increase in aerobic stability of silage. [ABSTRACT FROM AUTHOR]

During sugar cane ensilage an intense growth of yeasts can result in high dry matter (DM) loss and reduction in the quality of the feed. The aim of this study was to evaluate the fermentation profile of sugar cane ( Saccharum spp.) silage inoculated with new strains of lactic acid bacteria (LAB) screened for this forage silage. Fourteen wild LAB strains were evaluated, biochemically (API 50 CHL, BioMérieux) characterized, and identified by sequencing of 16S rDNA. The wild isolates were identified as Lactobacillus plantarum , Lactobacillus brevis , and Lactobacillus hilgardii . Different fermentation profiles were observed among strains of the same species. The silages inoculated with L. plantarum species showed the highest yeast population (5.97 log CFU/g silage), ethanol concentration (137 g kg/silage) and DM loss (20.6%) (P<0.01), therefore, they were not beneficial for sugar cane silage. The silages inoculated with L. brevis UFLA SIL17 and UFLA SIL24 and L. hilgardii UFLA SIL51 and UFLA SIL52 strains showed smaller DM loss (12.2%) and NDF-NDF content (573 g/kg silage) (P<0.01). When compared with these inoculated silages, the silages inoculated with L. hilgardii UFLA SIL51 and UFLA SIL52 strains resulted in 57% and 94% more acetic acid and 1,2-propanediol respectively. Obligatory heterofermentative strains showed better silage quality. L. hilgardii (UFLA SIL51 and UFLA SIL52) strains show promise for use in sugar cane silage. [ABSTRACT FROM AUTHOR]

Histidine decarboxylase activity was investigated in 21 strains of lactic acid bacteria isolated from Argentinian wines. This activity is not widely distributed between them, and occurs significatively only in some strains of Lactobacillus hilgardii. L.hilgardii 5w was selected for the study on the basis of its constitutive expression. Glucose is necessary for histidine transport. Maximum activity is observed at 37°C and pH 4.0. Enzyme activity is inhibited by SO2 and ethanol at concentrations usually found in wine. L-matic acid and citric acid act as stimulators of the activity.

The effect of organic acids on the growth of Leuconostoc oenos and Lactobacillus hilgardii strains was investigated, and the interaction between organic acids, ethanol and inoculum level was determined. The four selected strains of Leuconostoc oenos : X2L, E2, ST2 and L2 and the two selected strains of Lactobacillus hilgardii : 7j and X1B are resistent to 10 p. cent ethanol, a typical concentration for a table wine. The effect of the additives depends on the strain, except for dodecanoic acid which produces a growth inhibition in the six strains. Dodecanoic acid is an effective inhibitor when added to grape juice and might be used as a substitute of SO2, or rather in association with SO2.

Sugar cane (Saccharum spp.) is a forage crop widely used in animal feed because of its high dry matter (DM) production (25 to 40 t/ha) and high energy concentration. The ensiling of sugar cane often incurs problems with the growth of yeasts, which leads to high losses of DM throughout the fermentative process. The selection of specific inoculants for sugar cane silage can improve the quality of the silage. The present study aimed to select strains of lactic acid bacteria (LAB) isolated from sugar cane silage and to assess their effects when used as additives on the same type of silage. The LAB strains were inoculated into sugar cane broth to evaluate their production of metabolites. The selected strains produced higher concentrations of acetic and propionic acids and resulted in better silage characteristics, such as low yeast population, lower ethanol content, and lesser DM loss. These data confirmed that facultative heterofermentative strains are not good candidates for sugar cane silage inoculation and may even worsen the quality of the silage fermentation by increasing DM losses throughout the process. Lactobacillus hilgardii strains UFLA SIL51 and UFLA SIL52 resulted in silage with the best characteristics in relation to DM loss, low ethanol content, higher LAB population, and low butyric acid content. Strains UFLA SIL51 and SIL52 are recommended as starter cultures for sugar cane silage. [ABSTRACT FROM AUTHOR]

Aim To clone and characterize two related intracellular esterases from Oenococcus oeni and Lactobacillus hilgardii under wine-like conditions. Methods and Results The published genome sequences for O. oeni and Lact. hilgardii were used to identify, clone and purify putative esterase genes from these species designated EstCOo8 and EstC34, respectively. Both esterases are members of family V of lipolytic enzymes. However, EstC34 contains an SGSLG nucleophilic elbow structural motif instead of the usual GGSLG motif which is conserved in other lactic acid bacteria. Both esterases exhibited greatest specificity for C2-C4 pNP-linked substrates and retained activity under wine-like conditions. EstCOo8 had an optimum temperature, pH, and ethanol concentration of 40°C, 5·5 and 6% (v/v), respectively. Whereas EstC34 had an optimum temperature, pH and ethanol concentration of 50°C, 5·0 and 10% (v/v), respectively. Conclusions Both esterases were stable and retained activity under conditions that would be encountered in wine. They have the potential to reduce short-chain ethyl esters such as ethyl acetate. Significance and Impact of the Study This study provides information that might help improve the performance of LAB during malolactic fermentation in wine in the future, either by strain selection, optimization or direct enzyme addition. [ABSTRACT FROM AUTHOR]

Abstract: 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 0.0385M 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. [Copyright &y& Elsevier]

Abstract: The aim of this work was to investigate the effect of phenolic acids on cell membrane permeability of lactic acid bacteria from wine. Several phenolic acids were tested for their effects on the cell membrane of Oenococcus oeni and Lactobacillus hilgardii by measuring potassium and phosphate efflux, proton influx and by assessing culture viability employing a fluorescence technique based on membrane integrity. The experimental results indicate that hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids) induce greater ion leakages and higher proton influx than hydroxybenzoic acids (p-hydroxibenzoic, protocatechuic, gallic, vanillic, and syringic acids). Among the hydroxycinnamic acids, p-coumaric acid showed the strongest effect. Moreover, the exposure of cells to phenolic acids caused a significant decrease in cell culture viability, as measured by the fluorescence assay, in both tested strains. The results agree with previous results obtained in growth experiments with the same strains. Generally, phenolic acids increased the cell membrane permeability in lactic acid bacteria from wine. The different effects of phenolic acids on membrane permeability could be related to differences in their structure and lipophilic character. [Copyright &y& Elsevier]

Abstract: The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain. [Copyright &y& Elsevier]

Abstract: The influence of phenolic compounds on the growth of Lactobacillus hilgardii X1B and putrescine formation was assayed at concentrations normally present in wine. Agmatine degradation increased growth and survival of the microorganism and the alkalinity of the media. Bacterial growth was stimulated by phenolic compounds, except for gallic acid and quercetin. Putrescine formation from agmatine diminished in the presence of protocatechuic, vanillic and caffeic acids, and the flavonoids catechin and rutin. The concentration of phenolic compounds decreased after five days of incubation of L hilgardii X1B, except for gallic acid and quercetin. The results indicate that phenolic compounds, besides their already known beneficial properties to human health, seem to be a natural way of diminishing putrescine formation. [Copyright &y& Elsevier]

The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%( w/ v) PEG 4000, 75 m M sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%( v/ v) 2-methyl-2,4-pentanediol, 7.5%( w/ v) PEG 4000, 100 m M HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å3 Da−1, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code ) as the search model. [ABSTRACT FROM AUTHOR]

Abstract: Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated. [Copyright &y& Elsevier]

Aims: A contribution towards the elucidation of the mechanisms of tannins on bacteria growth inhibition, with particular focus on the interaction between tannins and bacterial proteins. Methods and results: The interaction between tannic acid (TA) and Lactobacillus hilgardii, a wine spoilage bacterium, was investigated by a combination of physiologic and proteomic approaches. Growing tests were performed on medium supplemented with TA at concentrations ranging from 100 to 1000 mg l−1 demonstrating the inhibitory effect of TA on the growth rate. Total proteins extracted from cells unexposed and exposed to TA were then analysed by 2D-electrophoresis and significant quantitative variations with a marked decrease of protein intensity upon TA exposure were observed. Most of the proteins, identified by ESI tandem Mass Spectrometry, were metabolic enzymes of different pathways, located in cytoplasm and membrane. Conclusions: The effects of TA on cells are deduced by the involvement of metabolic enzymes, and functional proteins on the tannin–protein interaction. These results might be related to the altered functions of the cell metabolism. Significance and impact of the study: The possible role of tannins in the inhibition of the bacterial survival and growth in a natural environment such as wine. A similar approach could be applied for evaluating the effects of tannins on food borne and pathogenic bacteria. [ABSTRACT FROM AUTHOR]

• Sugarcane tops ensiled with LH and homofermentative LP were analyzed. • LH and LHLP addition improved the aerobic stability of sugarcane tops silage. • LH and LHLP stabled pH value and organic acid contents after aerobic exposure. • Combined addition of LH and LP showed least DM loss after 5 days of aerobic exposure. • L. hilgardii inhibited spoilage microorganisms: A. pasteurianus and K. humilis. This study investigated the effects of Lactobacillus hilgardii (LH), alone or in combination with Lactobacillus plantarum (LP), on the aerobic stability, fermentation quality and dynamics of the bacterial and fungal communities of sugarcane top silage. Results demonstrated that LH and LHLP (LH combined with LP) improved the aerobic stability of sugarcane top silages. As the exposure time increased, the pH values and the contents of lactic acid, acetic acid, as well as propionic acid remained stable in silage treated with LH and LHLP. The abundance of L. hilgardii was enriched and the undesirable microorganisms, such as Acetobacter pasteurianus , Paenibacillus amylolyticus and yeasts like Kazachstania humilis , were suppressed in silages treated with LH and LHLP. In conclusion, LH-treated silage, whether with LP or not, positively impacted the fungal and bacterial microbes. This improved the quality of fermentation, the aerobic stability, and reduced aerobic spoilage in sugarcane top silage. [ABSTRACT FROM AUTHOR]

The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X1B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.