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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. more...

BackgroundIn recent years, researchers are interested in the discovery of active compounds from traditional remedies and natural sources, as they reveal higher therapeutic efficacies and improved toxicological profiles. Among the various traditional treatments that have been widely studied and explored for their potential therapeutic benefits, kefir, a fermented beverage, demonstrates a broad spectrum of pharmacological properties, including antioxidant, anti-inflammation, and healing activities. These health-promoting properties of kefir vary among the kefir cultures found at the different part of the world as different media and culture conditions are used for kefir maintenance and fermentation.MethodsThis study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays.ResultsThe 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir wasLactobacillus hilgardiifollowed byLactobacillus harbinensis,Acetobacter lovaniensis,Lactobacillus satsumensis,Acetobacter tropicalis,Lactobacillus zeae, andOenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples.ConclusionsThis study will help to intensify the knowledge on the water kefir microbial composition, available phytochemicals and its toxicological and antioxidant effects on BALB/c mice since there are very limited studies on the water kefir grain fermented in Malaysia. more...

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. more...

Our objective was to determine the effects of two strains of obligate heterofermentative bacteria, alone or in combination, on the fermentation profile, gas production kinetics, chemical composition, and aerobic stability of sugarcane silage. A plot of sugarcane was manually harvested, mechanically chopped and treated with: distilled water (5 mL kg–1; Control), Lentilactobacillus hilgardii CNCM I-4785 [3 × 105 colony-forming units (cfu) g–1; LH], Lentilactobacillus buchneri NCIMB 40788 (3 × 105 cfu g–1; LB), and LH+LB (1.5 × 105 cfu g–1 of each strain). Treated forages were packed into 1.96-L gas-tight silos (0.40 porosity) and stored at 25 ± 1.5°C for 70 days (4 replicates per treatment). All heterolactic inoculants were effective to increase acetic acid concentration and inhibit yeast metabolism, as treated silages had lower formation of ethanol, ethyl esters and gas during fermentation. Lower fungal development spared soluble carbohydrates, consequently resulting in silages with higher in vitro digestibility. Nevertheless, L. buchneri was the most effective strain to extend the aerobic stability of sugarcane silage (based on both temperature and pH rise). The use of L. buchneri alone or in combination with L. hilgardii, applied at 3 × 105 cfu g–1, is a feasible strategy to inhibit yeast metabolism and increase the nutritional quality of sugarcane silage. more...

Eleven series of water kefir fermentation processes differing in the presence of oxygen and the type and concentration of inoculum and substrate, were followed as a function of time to quantify the impact of these parameters on the kinetics of this process via a modeling approach. Increasing concentrations of the water kefir grain inoculum increased the water kefir fermentation rate, so that the metabolic activity during water kefir fermentation was mainly associated with the grains. Water kefir liquor could also be used as an alternative means of inoculation, but the resulting fermentation process progressed slower than the one inoculated with water kefir grains, and the production of water kefir grain mass was absent. Substitution of sucrose with glucose and/or fructose reduced the water kefir grain growth, whereby glucose was fermented faster than fructose. Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei), Lentilactobacillus hilgardii (formerly known as Lactobacillus hilgardii), Liquorilactobacillus nagelii (formerly known as Lactobacillus nagelii), Saccharomyces cerevisiae, and Dekkera bruxellensis were the main microorganisms present. Acetic acid bacteria were present in low abundances under anaerobic conditions and only proliferated under aerobic conditions. Visualization of the water kefir grains through scanning electron microscopy revealed that the majority of the microorganisms was attached onto their surface. Lactic acid bacteria and yeasts were predominantly associated with the grains, whereas acetic acid bacteria were predominantly associated with the liquor. more...

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 (LH150); L. hilgardii CNCM I-4785 at 300,000 cfu/g FM (LH300); 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 LH300 (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, LH150, and LH300 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. more...

Aerobic deterioration of silage following feeding out is responsible for the deterioration of its quality. Inoculation of silage with lactic acid bacteria is one strategy to limit these effects. A trial was performed using whole‐plant corn ensiled in bag silo, and forage was inoculated with Lentilactobacillus buchneri NCIMB 40788 (Lactobacillus buchneri) and Lentilactobacillus hilgardii CNCM‐I‐4785 (Lactobacillus hilgardii) or not (Control silage). After 159 days of fermentation, the silos were opened and the silage was sampled at 24‐h intervals during a 10‐day aerobic stability assay to measure pH, the fermentation profile, mycotoxins, and microbial and fungal populations. In inoculated silage, lactic acid concentrations and pH remained stable during the aerobic phase and higher microorganism alpha‐diversity was observed. Treated silage was characterized by a high abundance of Saccharomycetes and maintenance of Lactobacillus throughout the aerobic stability assay. The high aerobic stability of the inoculated silage contrasted with the decrease in lactic acid contents and the increase in pH observed in the Control silage, concomitantly with an increase in lactate‐assimilating yeast (Pichia and Issatchenkia), and in Acetobacter and Paenibacillus OTUs. Remarkably, Penicillium and roquefortine C were detected in this silage by day 8 following exposure to air. Our study highlighted the fact that the use of L. buchneri with L. hilgardii modified the consequences of exposure to air by maintaining higher microbial diversity, avoiding the dominance of a few bacteria, and preventing fungi from having a detrimental effect on silage quality., Aerobic deterioration of silage following feeding out is responsible for important quality losses on farms. Inoculation with a combination of Lentilactobacillus buchneri and Lentilactobacillus hilgardii modified the consequence of exposure to air and maintained higher microbial diversity, avoiding spoiling microorganisms detrimental effect on the silage. more...

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. more...

Background: It has been proven that probiotic Lactobacillus bacteria have inhibitory effects on human cancer cell lines. The aim of this study is to isolate and characterize the antioxidant probiotic Lactobacillus and determine the possible anticancer activities of the selected strain. Methods: One of the Lactobacillus strain isolated from camel doogh sample showed the high antioxidant activity by using of different methods such as resistance to hydrogen peroxide, hydroxyl radical and superoxide anions. The antioxidant strain was characterized by sequencing of 16S rRNA V2-V3 regions and the 16S-23S intergenic spacer (ITS). The methanol extract of this strain supernatant was fractionated using thin layer chromatography (TLC) and antioxidant activity of fractions was detected by 0.1% of DPPH through TLC-DPPH bioautography. In vitro anticancer activity of each fraction was investigated by using MTT and flow cytometry methods. Results: According to the phylogenetic results, the antioxidant Lactobacillus strain was closely related to Lactobacillus hilgardii strain E91 (Accession No. EF536365). After fractionation and anti-proliferation assessments of Lactobacillus hilgardii strain AG12a extracellular materials, one of the antioxidant fraction (F4) showed maximum DPPH radical scavenging activity (IC50 of 535.27 μg/mL). MTT assay of the F4 fraction demonstrated cytotoxic activity against Caco-2 with the IC50 value of 299.05 μg/mL. The cell death activity of the fraction was confirmed by flow cytometry with 30.925. Conclusions: In this study, the anticancer and apoptotic properties of Lactobacillus hilgardii against Caco-2 cell line was reported for the first time. The isolated bioactive fraction from the extracellular methanol extract needs to be further investigated in human studies of cancer therapy. more...

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. Fil: Nair, Jayakrishnan. Lethbridge Research Centre; Canadá Fil: Huaxin, Niu. Inner Mongolia University for Nationals; China Fil: Andrada, Lidia Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Yang, Hee-Eun. Lethbridge Research Centre; Canadá Fil: Chevaux, Eric. Lallemand SAS; Francia Fil: Drouin, Pascal. Lallemand Specialties Inc; Estados Unidos Fil: McAllister, Tim A.. Lethbridge Research Centre; Canadá Fil: Wang, Yuxi. Lethbridge Research Centre; Canadá more...

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. more...

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. more...

Selected ion flow tube-mass spectrometry (SIFT-MS) is a direct-injection mass spectrometric technique that has been introduced recently into the field of food and flavor analysis. It also shows potential for use in the monitoring of food fermentations. Therefore, this study aimed at the online monitoring of different volatile compounds produced during starter culture-initiated liquid sourdough fermentations by SIFT-MS, for which a new workflow was developed.; Results: The online monitoring of the volatile sample compounds acetoin and ethyl acetate, diacetyl, and ethanol was made possible during the production of sourdoughs obtained through fermentation with several interesting strains belonging to the species Lactobacillus crustorum, Lactobacillus fermentum, Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus sakei, and Gluconobacter oxydans. Acetoin and ethyl acetate could not be distinguished based solely on SIFT-MS data. Diacetyl production was monitored in the case of Lb. crustorum LMG 23699 as a starter culture strain, thereby making the distinction between those volatiles produced in sourdough without extra ingredients added or after the addition of citrate or malate.; Conclusion: Starter culture-initiated liquid sourdough fermentations were monitored successfully. The volatile compound production of the different starter culture strains tested reflected differences in their metabolism and/or competitiveness in a sourdough matrix. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry. more...

The wild Lactobacillus hilgardii (CCMA 0170) strain isolated from sugarcane silage showed good results as an inoculant in this silage, so the aim of this study was to evaluate its effect on corn silage. After harvested, three replicates for each fermentation time of corn silage: 19, 60 and 103 days were prepared with L. hilgardii or uninoculated (control silage). Experimental silos were opened and their chemical composition was determined. The silages opened after 103 days were also assessed for aerobic stability and microbial population after aerobic exposure. The new inoculant reduced the dry matter losses and conserved more water-soluble carbohydrates. The inoculated silage showed higher lactic acid bacteria populations and lower yeasts and filamentous fungi than the control silage. The concentration of lactic and acetic acid and 1,2-propanediol increased in inoculated silage. The population of filamentous fungi and yeasts after aerobic exposure in the inoculated silage was less than the minimum detectable ( more...

Certain acetic and lactic acid bacteria are major causes of quality defects in musts and wines, giving rise to defects such as a “vinegary,” “sharp, like nail polish-remover” taste or preventing alcoholic and/or malolactic fermentation. Sulfur dioxide is the major tool currently used in the control of these bacteria in wine. The aim of this work was to isolate bacteriophages from musts and wine of different grape varieties that were able to eliminate lactic and acetic acid bacteria spoilages at the laboratory scale. Musts obtained from grape-berries of Vitis vinifera cv. Chardonnay and Moscatel and a red wine made with V. vinifera cv. Tintilla de Rota were used to isolate bacteriophages. Bacteriophages were obtained from each of the musts and the wine and belonged to the order Caudovirals and the family Tectivirals. They were isolated by classical virology methods and identified by electron microscopy. The host bacteria used in the study were lactic acid bacteria of the species Lactobacillus hilgardii, Lactobacillus plantarum, and Oenococcus oeni and the acetic bacteria Acetobacter aceti. A comparative study was performed by adding phage titrations and SO2 to musts and wines, which had been previously inoculated with bacteria, to study the effectiveness of bacteriophages against bacteria. The comparative study showed that some bacteriophages were as effective as sulfur dioxide at low concentrations. more...

Eight water kefir fermentation series differing in buffer capacity and calcium concentration of the water used for fermentation were studied during eight backslopping steps. High buffer capacities resulted in high pH values and high calcium concentrations resulted in low pH values at the end of each backslopping step. When the water buffer capacity and/or calcium concentration were below certain minima, the water kefir grain growth decreased gradually over multiple backsloppings. High water buffer capacities resulted in high concentrations of residual total carbohydrate concentrations and low metabolite concentrations. Further, high water buffer capacities resulted in high ratios of lactic acid bacteria to yeasts, which was reflected in high molar ratios of the concentrations of lactic acid to ethanol and acetic acid to ethanol. The most prevalent microorganisms of the water kefir grain inoculum and grains of all fermentation series at the end of the eighth backslopping step were Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus paracasei, Bifidobacterium aquikefiri, Saccharomyces cerevisiae, and Dekkera bruxellensis. These microbial communities were influenced by the water buffer capacity and had an impact on the substrate consumption and metabolite production during water kefir fermentation. more...

Lactic acid bacteria (LAB) used as silage additives have been shown to improve several fermentation parameters, including aerobic stability. Inoculation with a combination of Lactobacillus buchneri NCIMB40788 and Lactobacillus hilgardii CNCM-I-4785, contributes to an increase in aerobic stability, compared to each strain inoculated independently. To understand the mode of action of the combination on the LAB community, a fermentation-kinetic study was performed on corn. Four treatments, Control, Lb. buchneri, Lb. hilgardii, and a combination of the two strains, were fermented 1, 2, 4, 8, 16, 32, and 64 days. Corn silage inoculated by both strains had a lactate:acetate ratio of 0.59 after 64 days and a higher concentration of lactate than Lb. buchneri. Analysis of the microbiota by 16S and ITS amplicon metasequencing demonstrated that inoculation led to lower bacterial diversity after 1 day, from 129.4 down to 40.7 observed operational taxonomic units (OTUs). Leuconostocaceae represented the dominant population by day 1, with 48.1%. Lactobacillaceae dominated the succession by day 4, with 21.9%. After 32 days, inoculation by both strains had the lowest bacterial alpha diversity level, with 29.0 observed OTUs, compared to 61.3 for the Control. These results confirm the increased fermentation efficiency when the two Lactobacillus strains are co-inoculated, which also led to a specific yeast OTUs diversity profile, with Hannaella as the main OTU. more...

Aims To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process. Methods and results Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile, and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations, and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these microorganisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii, and Saccharomyces cerevisiae were always present and may be the key microorganisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of microorganisms, fast fermentation, and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria. Conclusions The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key microorganisms was always present during fermentation. Significance and Impact of the Study This study allows a rational selection of a water kefir grain inoculum. This article is protected by copyright. All rights reserved. more...

The objectives of the present study were to characterize the biofilm microbiota of 11 different farms (from A to K), producing PDO Ragusano cheese, and to investigate on its ability to generate volatile organic compounds (VOCs) in milk samples inoculated with biofilm and incubated under Ragusano cheese making conditions. The biofilms were subjected to plate counting and PCR/T/DGGE analysis and the VOCs generated in incubated milk samples were evaluated through SmartNose, GC/O, and GC/MS. Streptococcus thermophilus was the dominant species both in biofilms and in incubated milks. Lactobacillus, Lactococcus, Enterococcus and Leuconostoc were also identified. Low levels of Pseudomonas spp. and yeasts counts were detected, whereas coliforms, Listeria monocytogenes and Salmonella spp., were never found. SmartNose and GC/O analyses were able to differentiate incubated milk samples on the basis of the odour compounds, highlighting that samples E and F overlapped and sample C was clearly separated from the others. These results complied with those acquired by GC/MS analysis, that detected in total 20 VOCs. Principal component analysis showed positive correlations (r > 0.6; P < 0.05) between some lactic acid bacteria (LAB) and VOCs: such as Enterococcus hirae with alcohols, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii with aldehydes, and Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus hilgardii with ketones. This work demonstrates that biofilm represents an excellent source of LAB biodiversity, which contribute to generate VOCs during the production of PDO Ragusano cheese. more...

A poorly performing industrial water kefir production process consisting of a first fermentation process, a rest period at low temperature, and a second fermentation process was characterized to elucidate the causes of its low water kefir grain growth and instability. The frozen-stored water kefir grain inoculum was thawed and reactivated during three consecutive prefermentations before the water kefir production process was started. Freezing and thawing damaged the water kefir grains irreversibly, as their structure did not restore during the prefermentations nor the production process. The viable counts of the lactic acid bacteria and yeasts on the water kefir grains and in the liquors were as expected, whereas those of the acetic acid bacteria were high, due to the aerobic fermentation conditions. Nevertheless, the fermentations progressed slowly, which was caused by excessive substrate concentrations resulting in a high osmotic stress. Lactobacillus nagelii, Lactobacillus paracasei, Lactobacillus hilgardii, Leuconostoc mesenteroides, Bifidobacterium aquikefiri, Gluconobacter roseus/oxydans, Gluconobacter cerinus, Saccharomyces cerevisiae, and Zygotorulaspora florentina were the most prevalent microorganisms. Lb. hilgardii, the microorganism thought to be responsible for water kefir grain growth, was not found culture-dependently, which could explain the low water kefir grain growth of this industrial process. more...

The effects of wine phenolic compounds (PCs), hydroxybenzoic acids, hydroxycinnamic acids, and a flavanol, on the growth of Lactobacillus hilgardii and Pediococcus pentosaceus, both isolated from Argentine red wine, were assayed in synthetic winelike medium for the first time. The greatest inhibition of L. hilgardii 6F, L. hilgardii X1B, and P. pentosaceus 12p occurred with 400 mg/L of either trans-caffeic or trans-p-coumaric acid and 96 hr of incubation. P. pentosaceus was the most sensitive to PCs. A structure-antibacterial activity relationship study of the PCs revealed that trans-caffeic and trans-p-coumaric acid have similar electron distributions with interatomic distances of 8 Å between the catechol hydroxyls and the carboxyl group. These minimum structural requirements (pharmacophore pattern) present in the molecule of the phenolic compounds are necessary to produce antibacterial activity against wine spoilage lactic acid bacteria. Fil: Stivala, Maria Gilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Villecco, Margarita Beatriz. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Enriz, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina Fil: Aredes Fernández, Pedro Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina more...

Summary 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. more...

Grapevine shoots, known to be a source of bioactive stilbenes, are considered one of the most important and abundant waste products from the vineyard. The objective of this study was to obtain a pure stilbene extract from grapevine shoots and test its properties, mainly antimicrobial ones, as a preservative in wine. A new extract with a purity of 99% in stilbenes was obtained by centrifugal partition chromatography. The extract was characterized by HPLC-MS, the main compounds being E-e-viniferin (70%) and E-resveratrol (18%). The stability conditions for the extract solution were low temperature (4 °C) and darkness for a maximum of 21 days. Data regarding to the stability of E-ϵ-viniferin, ω-viniferin and r-viniferin are reported first. The antimicrobial activity of the extract was higher against yeast (Brettanomyces bruxellensis, Zygosaccharomyces bailli, Haseniaspora uvarum, Candida zemplinina) than against bacteria (Lactobacillus hilgardii, Oenococcus oeni, Pediococcus pentasaceus). The best antimicrobial activity was found on Brettanomyces bruxellensis and Zygossaccharomyces bailli. The aromatic characterization of the extract allows us to establish that it does not affect the sensory properties of wine, and thus wine quality is not compromised with the addition of the extract. All of the above allow us to propose ST99 as a promising alternative to SO2 in wines, including rose and white wines. more...

Water kefir is a fruity, sour, slightly alcoholic and carbonated beverage, which is made by fermentation of an aqueous sucrose solution in the presence of dried figs and water kefir grains. These polysaccharide grains contain lactic acid bacteria (LAB), yeasts, and sometimes bifidobacteria and/or acetic acid bacteria, which consume sucrose to produce exopolysaccharides, lactic acid, acetic acid, ethanol, and carbon dioxide. Shotgun metagenomic sequencing was used to examine the microbial species diversity present at two time points during water kefir fermentation in detail, both in the water kefir liquor and on the water kefir grains, hence representing four samples. Lactobacillus harbinensis, Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus paracasei, and a Lactobacillus species similar to Lactobacillus hordei/mali were present in the water kefir examined, along with Bifidobacterium aquikefiri and two yeast species, namely Saccharomyces cerevisiae and Dekkera bruxellensis. In addition, evidence for a novel Oenococcus species related to Oenococcus oeni and Oenococcus kitaharae was found. Its genome was derived from the metagenome and made available under the name of Candidatus Oenococcus aquikefiri. Through functional analysis of the four metagenomic data sets, it was possible to link the production of lactic acid, acetic acid, ethanol, and carbon dioxide to subgroups of the microbial species found. In particular, the production of mannitol from fructose was linked to L. hilgardii, Candidatus O. aquikefiri, and B. aquikefiri, whereas glycerol production was associated with S. cerevisiae. Also, there were indications of cross-feeding, for instance in the case of amino acid supply. Few bacterial species could synthesize a limited number of cofactors, making them reliant on the figs or S. cerevisiae. The LAB species in turn were found to be capable of contributing to water kefir grain growth, as dextransucrase-encoding genes were attributed to L. hilgardii, L. hordei/mali, and Candidatus O. aquikefiri. more...

Lactic acid bacteria isolated from wine fermentations, particularly from the malolactic fermentation, and belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus paracasei and Lactococcus lactis species were tested for their effectiveness in inhibiting the development of different microorganisms. The different strains showed, to varying degrees, an antagonistic effect against bacteria of the genera Bacillus and Staphylococcus. The specificity of the species L. hilgardii that inhibits only strains of the genus Bacillus is remarkable, on the other hand, L. plantarum was more effective against the strains of the genus Staphylococcus. The greatest effectiveness, considering both the degree of inhibition and the number of inhibited species, was presented by strains of L. lactis and L. paracasei. Seven strains belonging to the species that showed a more broad-spectrum activity, L. paracasei and L. plantarum, were also tested for their ability to inhibit the growth of fungi. All of them showed, in different degree (55&ndash, 76%), activity against Fusarium oxysporum. Finally, the ability of the L. paracasei LPAUV12 and L. plantarum LPLUV10 strains was evaluated to protect Lycopersicon esculentum plants against the fungus F. oxysporum and promote its growth. Strain LPLUV10, showed capacity to significantly inhibit the harmful effect of F. oxysporum in tomato plants as well as to significantly stimulate their growth. more...

Acetic and some lactic acid bacteria are the main reason for the loss of quality of musts and wines, giving rise to defects such as “vinegary”, “chopped” or preventing alcoholic and / or malolactic fermentation. The solution to this problem consists in the application of authorized bactericidal agents, such as sulfurous. The aim of this work is to isolate bacteriophages from musts and wines of different grape varieties, able to eliminate lactic and acetic acid bacteria spoilages. Musts obtained from grape-berries of Vitis vinifera cv. Chardonnay and Moscatel and a red wine made with the Tintilla de Rota variety were used to isolate bacteriophages. It were isolated by classical virology methods and identified by electron microscopy. Host bacteria used in the study were the lactic acid bacteria of the species Lactobacillus hilgardii, L. plantarum and Oenococcus oeni and the acetic bacteria Acetobacter aceti. A comparative study was performed on musts and wines, previously inoculated with bacteria, by phage titration and SO2 addition to study the effectiveness of bacteriophages against bacteria. Bacteriophages were obtained from all musts and wine, belonging to the order of Caudovirals and Tectivirals. The comparative study showed that a cocktail of bacteriophages at low concentration is as effective as sulfur. more...

While lactic acid bacteria are widespread in various ecological niches with the ex cess of nutrients and extensive use in food industry, many aspects of the nitrogen metabolism of remain unexplored. The most preferred nitrogen sources for most bacteria are glutamine and ammonium ions. In bacterial cells an enzyme glutamine synthetase synthetizes glutamine from glutamate and ammonium ion. While L. hilgardii LMG 7934 genome carries two genes encoding glutamine synthetases with 55% of mutual identity of amino acid sequences, which protein plays the major role is still unclear. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. In this study we report the cloning of both genes encoding glutamine synthetases from L. hilgardii LMG 7934. more...

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed was asked to deliver a scientific opinion on the safety and efficacy of a strain of Lactobacillus hilgardii and of Lactobacillus buchneri when used as a technological additive intended to improve ensiling at a proposed application rate of 3.0 x 108 colony forming units (CFU)/kg fresh material. The two bacterial species are considered by EFSA to be suitable for the qualified presumption of safety approach to safety assessment. In previous opinions the identity of the strains has been clearly established and no antibiotic resistance of concern was detected. Therefore, the use of the strains as a silage additive is considered safe for livestock species, for consumers of products from animals fed the treated silage and for the environment. In the absence of data, no conclusion can be drawn on the skin and eye irritancy or skin sensitisation of the additive. The additive should be considered a potential respiratory sensitiser. In a previous application, three studies involving both strains used in combination at the same concentration were made with samples of whole crop maize with varied dry matter content. The same studies have been submitted in this application. The Panel reiterates its previous conclusions that Lactobacillus hilgardii CNCM I‐4785 and Lactobacillus buchneri NCIMB 40788/CNCM I‐4323 at 1.5 × 108 CFU of each active agent per kg of fresh forage, equivalent to 3 x 108 CFU total lactobacilli/kg of forage significantly improve the aerobic stability of the silage. more...

Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains. more...

This work evaluated the effect of lysozyme on lactobacilli isolated from Port wine. Bacterial growth experiments were conducted in MRS/TJ medium and inactivation studies were performed in phosphate buffer (KH2PO4), distilled water and wine supplemented with different concentrations of lysozyme. The response of bacteria to lysozyme was found to be highly strain dependent. Some strains of Lactobacillus hilgardii together with Lactobacillus collinoides and Lactobacillus fructivorans were found to be resistant to concentrations of lysozyme as high as 2000 mg/L. It was observed that among the L. hilgardii taxon the resistant strains possess an S-layer coat. Apparently, the strains of L. collinoides and L. fructivorans studied are also S-layer producers as suggested by the total protein profile obtained by SDS-PAGE. Thus, the hypothetical protective role of the S-layer against the action of lysozyme was investigated. From the various treatments used to remove the protein from the surface of the cells, the one employing LiCl (5 M) was the most effective. LiCl pre-treated cells exposed to lysozyme (2000 mg/L) in KH2PO4 buffer maintained its resistance. However, when cells were suspended in distilled water an increased sensitivity to lysozyme was observed. Moreover, it was found that the addition of ethanol (20% v/v) to the suspension medium (distilled water) triggered a strong inactivation effect especially on cells previously treated with LiCl (reduction of >6 CFU log cycles). The results suggest that the S-layer exerts a protective effect against lysozyme and that the cell suspension medium influences the bacteriolysis efficiency. It was also noted that ethanol enhances the inactivation effect of lysozyme. more...

The microbial basis of acidification process during spontaneous cheese whey wastewater fermentation was decrypted by implementing both culture-dependent and culture-independent techniques. Lac tobacillus and Bifidobacterium were the predominant taxa among the microbiota growing on MRS (deMan, Rogosa, and Sharpe), while Kazachstania unispora and Dekkera anomala yeast species were also isolated. Almost all Lactobacillus isolates were heterofermentative that could ferment glucose and lactose, with most of them being related to Lactobacillus hilgardii (99.0–100 % similarity). By employing fluorescence techniques, the dominance of long crescent-shaped bacteria in the acidogenic sludge was observed. Temperature gradient gel electrophoresis (TGGE), clone library, and next-generation sequencing techniques revealed the dominance of Selenomonas lacticifex. Based on Illumina data, Selenomonas in the continuous stirred-tank reactor (CSTR) represented 70.13 ± 4.64 % of the bacterial reads, while other Veillonellaceae taxa (Megasphaera and Pectinatus) represented a notable proportion (6.54 %). Prevotella was only detected by Illumina sequencing as an important constituent of the microbial population (14.97 ± 1.71 %). Budding yeasts represented 97 % of the fungal population in the CSTR, with Yarrowia strains representing 88.85 ± 5.52 % of the fungal reads. Spontaneous cheese whey acidification can favor the dominance of rumen bacteria and here was driven by the rarely reported S. lacticifex-type fermentation, which should be taken into consideration during evaluation of acidogenesis in process simulation and modelling. Moreover, the important nervonic acid content detected indicates that acidogenic sludge can be used as a source for the production of high value-added biomedical substrates. more...

Water kefir is a beverage fermented by a microbial consortium captured in kefir grains. The kefir grains matrix is composed of polysaccharide, primarily dextran, which is produced by members of the microbial consortium. In this study, we have isolated lactic acid bacteria (LAB) from non-commercial water kefir grains (from Belgrade, Serbia) and screened for dextran production. Among twelve LAB isolates three produced slime colonies on modified MRS (mMRS) agar containing sucrose instead of glucose and were presumed to produce dextran. Three LAB were identified, based on morphological, physiological and biochemical characteristics and 16S rRNA sequencing, as Leuconostoc mesenteroides (strains T1 and T3) and Lactobacillus hilgardii (strain T5). The isolated strains were able to synthesize a substantial amount of dextran in mMRS broth containing 5% sucrose. Maximal yields (11.56, 18.00 and 18.46 g/l) were obtained after 16, 20 and 32 h for T1, T3 and T5, respectively. Optimal temperature for dextran production was 23oC for two Leuconostoc mesenteroides strains and 30 °C for Lactobacillus hilgardii strain. The produced dextrans were identified based on paper chromatography, while the main structure characteristics of purified dextran were observed by FT-IR spectroscopy. Our study shows that water kefir grains are a natural source of potent dextran producing LAB. Vodeni kefir je napitak koji nastaje fermentacionom aktivnošću bakterija mlečno-kiselinskog vrenja, kvasca i sirćetnih bakterija. Ova kompleksna smeša mikroorganizama smeštena je unutar polisaharidnog matriksa - kefirnog zrna. Glavnu šećernu komponentu matriksa čini dekstran koji sintetišu bakterije mlečno-kiselinskog vrenja. Dekstran je homo polisaharid velike molekulske mase čija je monomerna jedinica glukoza. Zahvaljujući dobroj rastvorljivosti i stabilnosti, dekstran ima široku primenu u različitim granama industrije, kao što su medicina, farmacija, prehrambena, tekstilna i hemijska industrija. Nerastvorni dekstran može da posluži kao nosač za imobilizaciju biomolekula. Imajući u vidu veliku primenjivost dekstrana, cilj ovog rada je bio izolovati bakterije mlečno-kiselinskog vrenja iz zrna vodenog kefira koje produkuju ovaj egzopolisaharid sa visokim prinosima. U radu je prikazana izolacija i karakterizacija tri selektovana soja i optimizacija uslova za produkciju dekstrana. Na osnovu morfoloških, fizioloških i biohemijskih osobina i 16S rRNK sekvenciranja utvrđeno je da su izolati T1 i T3 sojevi vrste Leuconostoc mesenteroides, dok je izolat T5 identifikovan kao Lactobacillus hilgardii. Za produkciju dekstrana korišćena je modifikovana MRS podloga sa 5% saharoze, kao jedinim izvorom ugljenika. Maksimalni prinosi dekstrana (11,56, 18,00 i 18,46 g/l) su dobijeni nakon 16, 20 i 32 h fermentacije, za T1, T3 i T5, redom. Optimalne temperature za produkciju dekstrana su 23 °C za dva Leuconostoc mesenteroides soja, a za Lactobacillus hilgardii 30 °C. Uzorci sintetisanih polisaharida su identifikovani kao dekstrani na osnovu papirne hromatografije, dok su glavne strukturne karakteristike prečišćenih dekstrana utvrđene na osnovu FTIR spektroskopije. . more...

Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts.Pediococcus pentosaceusandLactobacillus graminiswere the most abundant LAB on grapes, whileLactobacillus plantarumdominated in fermenting musts from both regions. No particular structure ofLactobacillus plantarumpopulations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally ofOenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associatedLactobacillus hilgardiistrains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine. more...

Eight water kefir fermentation series differing in the presence of oxygen, the nutrient concentration, and the nutrient source were studied during eight consecutive backslopping steps. The presence of oxygen allowed the proliferation of acetic acid bacteria, resulting in high concentrations of acetic acid, and decreased the relative abundance of Bifidobacterium aquikefiri. Low nutrient concentrations resulted in slow water kefir fermentation and high pH values, which allowed the growth of Comamonas testosteroni/thiooxydans. Further, low nutrient concentrations favored the growth of Lactobacillus hilgardii and Dekkera bruxellensis, whereas high nutrient concentrations favored the growth of Lactobacillus nagelii and Saccharomyces cerevisiae. Dried figs, dried apricots, and raisins resulted in stable water kefir fermentation. Water kefir fermentation with dried apricots resulted in the highest pH and water kefir grain growth, whereas that with raisins resulted in the lowest pH and water kefir grain growth. Further, water kefir fermentation with raisins resembled fermentations with low nutrient concentrations, that with dried apricots resembled fermentations with normal nutrient concentrations, and that with fresh figs or a mixture of yeast extract and peptone resembled fermentations with high nutrient concentrations. more...

Fortified wines contain a high level of unfermented sugars and are prone to spoilage by alcohol-tolerant lactic acid bacteria. A total of 62 strains were isolated from various production stages of one of the more popular fortified wines produced in South Africa. The strains were identified by using numerical analysis of total soluble cell protein patterns and 16S rRNA sequence analyses. The species most frequently isolated were Lactobacillus vermiforme (24 strains) and Lactobacillus casei subsp. casei (32 strains). Twenty-four of the strains of L. vermiforme, three strains of Lactobacillus buchneri, one strain of Lactobacillus plantarum and two strains of L. casei subsp. casei were isolated from spoiled fortified wine which contained 22% (vol/vol) ethanol. The majority of strains of L. casei subsp. casei (25 of the 32) and two strains of Lactobacillus zeae were isolated from wine before submerged fermentation. Five strains of L. casei subsp. casei were isolated from wine undergoing submerged fermentation, with an alcohol content of 11.92% (vol/vol). No strain was isolated from unbottled wine which underwent the complete fermentation process and with an alcohol content of 17.20% (vol/vol). Three distinct phenotypic groups of L. vermiforme were identified at r ≥ 0. 70, separate from Lactobacillus brevis,' L. buchneri and Lactobacillus hilgardii. Three phenotypic clusters have been identified for L. casei subsp. casei. This is the first report of the presence of L. vermiforme, L. zeae, L. casei subsp. casei and L. plantarum in fortified wines. more...

The present study aimed to explore a novel attribute of the bacterium Lactobacillus hilgardii IITRKH159, isolated from Sapodilla, which was found to produce Bacterial cellulose. The compositional analysis of BC was carried out by HPLC system using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone showed that it consisted of glucan. The Physiochemical study of BC showed that it had bacterial nano-cellulose like structure, immensely pure, highly crystalline (90.25%) and significantly enriched with Iα form of cellulose. Additionally, FE-SEM and TEM of the BC revealed the morphological similarities with bacterial nano-cellulose due to the presence of a reticulated structure consisting of nano-sized fibrils with an average width of 45 nm. The effect of different carbon sources was analyzed to evaluate BC production and demonstrated the production of 7.23 ± 0.59 g L−1 BC with conversions yield (0.30 g BC/g sugar) and productivity (0.45 ± 0.15 g L−1 d−1) in a modified Y-medium after 16 days of cultivation. more...

Silage making has become a significant method of forage conservation worldwide. To determine how tomato pomace (TP) may be used effectively as animal feed, it was ensilaged for 90 days and microbiology counts, fermentation characteristics and chemical composition of tomato pomace silage (TPS) were evaluated at the 30th, 60th, and 90th days, respectively. In addition, 103 lactic acid bacteria were isolated from TPS. Based on the phenotypic and chemotaxonomic characteristics, 16S rDNA sequence and carbohydrate fermentation tests, the isolates were identified as 17 species namely: Lactobacillus coryniformis subsp. torquens (0.97%), Lactobacillus pontis (0.97%), Lactobacillus hilgardii (0.97%), Lactobacillus pantheris (0.97%), Lactobacillus amylovorus (1.9%), Lactobacillus panis (1.9%), Lactobacillus vaginalis (1.9%), Lactobacillus rapi (1.9%), Lactobacillus buchneri (2.9%), Lactobacillus parafarraginis (2.9%), Lactobacillus helveticus (3.9%), Lactobacillus camelliae (3.9%), Lactobacillus fermentum (5.8%), Lactobacillus manihotivorans (6.8%), Lactobacillus plantarum (10.7%), Lactobacillus harbinensis (16.5%) and Lactobacillus paracasei subsp. paracasei (35.0%). This study has shown that TP can be well preserved for 90 days by ensilaging and that TPS is not only rich in essential nutrients, but that physiological and biochemical properties of the isolates could provide a platform for future design of lactic acid bacteria (LAB) inoculants aimed at improving the fermentation quality of silage. more...

Molecular identification of microorganisms associated with submerged cassava fermentation was carried out and isolates of lactic acid bacteria (LAB) were examined for antimicrobial activity, including ability to produce antimicrobial peptides as a first step to define starter cultures for controlled cassava fermentations. A total of 75 isolates, including 41 LAB, 31 aerobic bacteria (AB) and three anaerobic bacteria were isolated from unfermented and fermenting cassava roots, cassava leaves and fermented cassava dough and identified by a combination of phenotypic tests and sequencing of 16S rRNA, rpoA, rpoB and pheS genes. Microbial diversity at interspecies and intraspecies level was screened by, respectively, PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR) and repetitive sequence based PCR (rep-PCR). Antimicrobial activity of LAB cultures and supernatants against indicator bacteria; Escherichia coli, Salmonella enterica serotype Typhimurium (S. Typhimurium), Bacillus cereus and Staphylococcus aureus was studied using agar diffusion tests. Furthermore, inactivation of indicator bacteria was investigated in both liquid medium and during controlled cassava fermentation. Results revealed a diversity of bacterial genera, species and subspecies associated with submerged cassava fermentation. DNA sequencing enabled identification of LAB isolates as Lactobacillus plantarum, Weissella confusa, Weissella paramesenteroides, Lactobacillus rhamnosus, Lactobacillus hilgardii, Lactobacillus paracasei, Leuconostoc mesenteroides, Enterococcus faecium, Enterococcus casseliflavus, and Pediococcus acidilactici. Lactobacillus spp. were the predominant LAB and were present in all cassava samples studied. Aerobic bacteria were predominantly Bacillus spp., including Bacillus subtilis, Bacillus amyloliquefaciens and B. cereus. Other species identified included Staphylococcus pasteuri and Clostridium beijerinkii. Cells, supernatants and cell free supernatants (CFS) of selected LAB isolates were able to inhibit both Gram positive and Gram negative pathogenic bacteria. LAB isolates inactivated all indicator organisms during controlled cassava fermentations, with a 4–6 log reduction after 48 h fermentation. The antimicrobial effect of the LAB was attributed to acid production. more...

Evolution of lactic acid bacteria (LAB) from two wineries located in Rioja Alavesa region has been analyzed during the whole winemaking process. Special attention was focused on determining their biogenic amine (BA) forming potential. From 295 isolates, 36 different genotypic profiles were identified, from which 17 belonged to Oenococccus oeni. Although LAB species evolution differed in both wineries during winemaking process, O. oeni became the predominant species in both wineries once malolactic fermentation (MLF) began. Each winery showed an exclusive ecosystem with own microbiota, as low coincident O. oeni strains were identified. Both phenotypical and molecular methods revealed the low incidence of BA producing LAB strains, as exclusively Lactobacillus hilgardii strains were positive for putrescine production. Other bacteria genus rarely found in wine environment such as Staphylococcus and Paenibacillus were also identified. Indeed, this is the first work in which those species have been reported as BA producers in wine. In addition, BA concentration was also monitored. Both wineries showed low concentrations for each BA, far from limits recommended for safety and quality principles. These results emphasize the potential application of these autochthonous strains in order to minimize BA formation during the whole winemaking process. more...

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, pH, 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. more...

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. more...

Oenococcus oeni is the best malolactic bacterium adapted to low pH and the high SO2 and ethanol concentrations in wine. Leuconostoc mesenteroides and Leuconostoc paramesenteroides (now classified as Weissella paramesenteroides) have also been isolated from wine. Pediococcus damnosus is not often found in wine and is considered a contaminant of high pH wines. Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus have occasionally been isolated from wines. Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus hilgardii (previously Lactobacillus vermiforme), Lactobacillus fructivorans (previously Lactobacillus trichoides and Lactobacillus heterohiochii) and Lactobacillus fermentum have been isolated from most wines. Lactobacillus hilgardii and L. fructivorans are resistant to high acid and alcohol and have been isolated from spoiled fortified wines. Lactobacillus vini, Lactobacillus lindneri, Lactobacillus nagelii and Lactobacillus kunkeei have been described more recently. The latter two species are known to cause sluggish or stuck alcoholic fermentations in wine. Although Lactobacillus collinoides and Lactobacillus mali (previously Lactobacillus yamanashiensis) decarboxylate L-malic acid, they are more often found in cider and fruit juices. Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus diolivorans, Lactobacillus jensenii and Lactobacillus paracasei are seldomly isolated from wines. Some strains of Lactobacillus casei may be closer related to Lactobacillus paracasei or a distant relative, Lactobacillus zeae. Oenococcus kitaharae, isolated from compost is genetically closely related to Oenococcus oeni, but does not decarboxylate malate, prefers higher growth pH and is phenotypically well distinguished from O. oeni. This review summarises the current taxonomic status of malolactic bacteria and lists key phenotypic characteristics that may be used to identify the species. more...

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production. more...

In order to analyze the genes related to histamine production in halophilic lactic acid bacteria, 16 strains of histamine-producing bacteria were isolated from three fermented seafoods produced in the Hokuriku region of Japan. Phenotypic and 16S rRNA gene sequence analyses identified all of the strains as those of Tetragenococcus muriaticus. Pyruvoyl-dependent histidine decarboxylase gene (hdcA) was determined from all strains using the PCR method with an hdcA-specific detection primer set. Genetic analyses (Southern blot and restriction fragment length polymorphism analysis) of hdcA and genes related to histamine production (the hdc cluster) confirmed that all of the strains harbored 21–23 kbp plasmids encoding a single copy of hdcA. The four representative strains were selected based on isolation source and genetic analysis, and subsequently full sequences of plasmids harbored in these strains were determined. hdc cluster sequences from the plasmids showed very high similarity (>99 %) to known hdc clusters of T. halophilus, Lactobacillus hilgardii 0006, and other lactic acid bacteria. The structures of the plasmids, the replication region, the hdc cluster, and the plasmid maintenance system were conserved between the plasmids present in new isolates and the T. halophilus strains. These results indicate that plasmids encoding hdcA are widely distributed among T. halophilus and T. muriaticus and function in both species. more...

Aims To investigate the impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and results Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO(2) at pH 3.50 and 3.70. Low concentrations of acetaldehyde- and pyruvic acid-bound SO(2) were inhibitory to the growth of all bacteria although acetaldehyde-bound SO(2) was generally more inhibitory than pyruvic acid-bound SO(2). Inhibition was greater at pH 3.50 than 3.70, and Lact. hilgardii was the most sensitive to acetaldehyde-bound SO(2), while O. oeni was the most sensitive to pyruvic acid-bound SO(2). Degradation of SO(2)-bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO(2)-bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3.70. Degradation of SO(2)-bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO(2). Conclusions Acetaldehyde- and pyruvic acid-bound SO(2) were inhibitory to wine LAB growth at concentrations as low as 5 mg l(-1). Despite this inhibition, all wine LAB degraded SO(2)-bound acetaldehyde and pyruvic acid suggesting that bound SO(2) may have a bacteriostatic rather than bacteriocidal action. Significance and impact of the study Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus. more...

The application of high hydrostatic pressure (HHP) to inactivate microorganisms in a grape must (crushed grapes), coupled with autoclavable microscale fermentors, was investigated as a potential tool for studying the impact of yeast on red wine aroma and flavor. A Pinot noir grape must was inoculated with Saccharomyces cerevisiae, Brettanomyces bruxellensis, Kluveromyces thermotolerans, Lactobacillus hilgardii, Oenococcus oeni, and Acetobacter aceti at ~1 × 105 cfu/mL and subjected to HHP treatment for 10 min at 551 MPa (5510 bar). After HHP treatment no viable cells were detected in the grape must. Autoclavable microscale fermentors were then used to conduct replicate fermentations of HHP-treated and untreated Pinot noir grapes producing sufficient wine for chemical and sensory analysis. No differences were observed in fermentation rate between treatments, and variability between replicates was very low. No significant differences in color or hue were observed, but wine produced from HHP-treated grapes contained higher total phenolics. Sensory analysis of the wines by a trained panel revealed that other than a slight increase in overall fruit aroma there were no significant differences between any aroma and flavor descriptors. Results suggest that HHP processing of grapes, in conjunction with autoclavable microscale fermentors, could be used to conduct experimental red wine fermentations without the influence of native yeast and bacteria present on grapes and fermentation equipment. more...

Five strains (Ryu1-2T, Gon2-9, Ryu4-3, Nog8-1 and Aza1-1) of lactic acid bacteria were isolated from flowers in mountainous areas in Japan, Oze National Park, Iizuna mountain and the Nikko area. The five isolates were found to share almost identical (99.6–100 % similar) 16S rRNA gene sequences and were therefore deemed to belong to the same species. These isolates exhibited low levels of 16S rRNA gene sequence similarity to known lactic acid bacteria; the closest recognized relatives to strain Ryu1-2T were the type strains of Lactobacillus hilgardii (92.8 % similarity), Lactobacillus kefiri (92.7 %), Lactobacillus composti (92.6 %) and Lactobacillus buchneri (92.4 %). Comparative analyses of rpoA and pheS gene sequences demonstrated that the novel isolates did not show significant relationships to other Lactobacillus species. The strains were Gram-stain-positive, catalase-negative and homofermentative. The isolates utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose and fructose. On the basis of phenotypic characteristics and phylogenetic data, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus floricola sp. nov. is proposed. The type strain is Ryu1-2T ( = NRIC 0774T = JCM 16512T = DSM 23037T). more...

International audience; A collection of 810 lactic acid bacteria (LAB) strains isolated from wine and cider was screened for potential biogenic amine (BA) producers by combining molecular and phenotypic approaches. A newly developed multiplex PCR method allowed for the simultaneous detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via either ornithine decarboxylase, odc, or agmatine deiminase, agdi) while TLC and HPLC analysis allowed for BA-production determination. One hundred and fifty-eight LAB strains were monitored by the molecular/phenotypic double approach and revealed a good correlation between genotypic and phenotypic data. Eighteen per cent of the tested strains were positive for at least one BA target gene with up to three detected simultaneously, in particular amongst Lactobacillus brevis and Lactobacillus hilgardii isolates for the tyrdc and agdi genes. The most frequent gene corresponded to the agdi gene detected in 112 strains (14% of all LAB strains) of 10 different LAB species. The tyrdc gene was detected in 67 strains represented by 7 different LAB species (8% overall), especially those isolated from wine. Lower levels of hdc+ (2% of strains) and especially odc+ (0.5% of strains) strains were observed. Interestingly, species that have never been described to carry BA-producing pathway genes were identified in this study. Furthermore, only one cadaverine-producer was detected and corresponded to Lactobacillus 30a, a collection strain not found in fermented beverages, although cadaverine is commonly detected in wines. more...

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. more...

The aim of this work was to determine the ability of six yeast and two bacterial species associated with wine spoilage to form biofilms in mono- or co-culture using the Calgary Biofilm Device (CBD). Moreover, the efficacy of several disinfectants was evaluated against these spoilage microorganisms, both in the planktonic and the biofilm states. Results showed that Dekkera bruxellensis, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Schizosaccharomyces pombe and Acetobacter aceti formed biofilms both in wine and in synthetic medium. Zygosaccharomyces bailii formed biofilm only in wine and Pichia guilliermondii and Lactobacillus hilgardii formed biofilms only in synthetic medium. In wine, D. bruxellensis presented the same biofilm population when grown in pure culture or in mixed culture with acetic acid bacteria. There was a 3-log increase in biofilm formed by A. aceti in mixed culture with L. hilgardii. Alkaline chlorine-based disinfectant was the most effective in decontaminating spoilage yeast and bacteria both in planktonic and biofilm tests. Sodium hydroxide-based detergents and peracetic-based disinfectant were also efficient against suspended cells, but at least 10-fold more concentrated solutions were needed to remove the biofilms. Furthermore, the results showed that, except for the neutral detergent VK10, the tested agents were actually effective when used under the conditions recommended by manufacturers. In any case, biofilms showed greater tolerance to biocides when compared to the same microorganisms in the planktonic state. To our knowledge, this is the first study in which the CBD is used to assess the ability of wine spoilage microorganisms to form biofilms and their susceptibilities to disinfectant agents. more...