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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.

Lactic acid bacteria are widespread in various ecological niches with the excess of nutrients and have reduced capabilities to adapt to starvation. Among more than 280 Lactobacillus species known to the date, only five, including Lactobacillus hilgardii, carry in their genome the gene encoding for PII-like protein, one of the central regulators of cellular metabolism generally responding to energy- and carbon–nitrogen status in many free-living Bacteria, Archaea and in plant chloroplasts. In contrast to the classical PII encoding genes, in L. hilgardii genome the gene for PII homologue is located within the potABCD operon, encoding the ABC transporter for polyamines. Based on the unique genetic context and low sequence identity with genes of any other so-far characterized PII subfamilies, we termed this gene potN (Pot-protein, Nucleotide-binding). The second specific feature of L. hilgardii genome is that many genes encoding the proteins with similar function are present in two copies, while with low mutual identity. Thus, L. hilgardii LMG 7934 genome carries two genes of glutamine synthetase with 55% identity. One gene is located within classical glnRA operon with the gene of GlnR-like transcriptional regulator, while the second is monocistronic. Together with the relative large genome of L. hilgardii as compared to other Lactobacilli (2.771.862 bp vs ~ 2.2 Mbp in median), these data suggest significant re-arrangements of the genome and a wider range of adaptive capabilities of L. hilgardii in comparison to other bacteria of the genus Lactobacillus.

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.

The production of mead holds great value for the Polish liquor industry, which is why the bacterium that spoils mead has become an object of concern and scientific interest. This article describes, for the first time, Lactobacillus hilgardii FLUB newly isolated from mead, as a mead spoilage bacteria. Whole genome sequencing of L. hilgardii FLUB revealed a 3 Mbp chromosome and five plasmids, which is the largest reported genome of this species. An extensive phylogenetic analysis and digital DNA-DNA hybridization confirmed the membership of the strain in the L. hilgardii species. The genome of L. hilgardii FLUB encodes 3043 genes, 2871 of which are protein coding sequences, 79 code for RNA, and 93 are pseudogenes. L. hilgardii FLUB possesses three clustered regularly interspaced short palindromic repeats (CRISPR), eight genomic islands (44,155 bp to 6345 bp), and three (two intact and one incomplete) prophage regions. For the first time, the characteristics of the genome of this species were described and a pangenomic analysis was performed. The concept of the pangenome was used not only to establish the genetic repertoire of this species, but primarily to highlight the unique characteristics of L. hilgardii FLUB. The core of the genome of L. hilgardii is centered around genes related to the storage and processing of genetic information, as well as to carbohydrate and amino acid metabolism. Strains with such a genetic constitution can effectively adapt to environmental changes. L. hilgardii FLUB is distinguished by an extensive cluster of metabolic genes, arsenic detoxification genes, and unique surface layer proteins. Variants of MRS broth with ethanol (10–20%), glucose (2–25%), and fructose (2–24%) were prepared to test the strain’s growth preferences using Bioscreen C and the PYTHON script. L. hilgardii FLUB was found to be more resistant than a reference strain to high concentrations of alcohol (18%) and sugars (25%). It exhibited greater preference for fructose than glucose, which suggests it has a fructophilic nature. Comparative genomic analysis supported by experimental research imitating the conditions of alcoholic beverages confirmed the niche specialization of L. hilgardii FLUB to the mead environment.

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.

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.

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.

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.

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.

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

Highlights • Conventional pasteurization drastically decreases Lactobacillus sp. • Thermosonication (TS) for less 9 min is sufficient for processing pulque. • Physicochemical properties of pulque were not modified by the use of TS. • Thermosonication extending shelf life of pulque up to 24 days. • TS is a viable technology for processing pulque., The aim of this study was to evaluate thermosonication as an alternative method for the pasteurization of pulque in order to improve its shelf life and retain its quality parameters. Thermosonication was carried out at 50 °C using amplitudes of 75% (for 6 and for 9 min), 85% (for 4 and for 6 min), and 95% (for 3 and for 5 min). These were the optimal conditions found for processing pulque by thermosonication. Physicochemical (acidity, color, alcohol content, and sensory analysis) and microbiological (lactic acid bacteria and yeasts) parameters were determined during 30 days for storage at 4 ± 1 °C. Conventional pasteurization (63 °C, 30 min) and raw pulque were used as controls. According to the results, the shelf life of pulque was extended up to 24 days storage at 4 °C. After this time, the quality of beverage decreased, due that the microbial load increases. Thermosonication treatments at 75% and 85% showed a higher content of LAB (6.58–6.77 log CFU/mL) and yeasts (7.08–7.27 log CFU/mL) than conventional pasteurization (3.64 log CFU/mL of LAB and 3.97 log CFU/mL of yeasts) at 24 days of storage. Raw pulque demonstrated up to 7.77 log CFU/mL of yeasts and 7.51 log CFU/mL of LAB. Pulque processed by thermosonication exhibited greater lightness, sensory acceptance, a maximal acidity of 0.83 g/lactic acid, and an alcohol content of 4.48–4.95% v/v. The thermosonication process preserves sensory and physicochemical properties better than conventional pasteurization. Lactic acid bacteria such as Lactobacillus kefiri, Lactobacillus acidophilus, and Lactobacillus hilgardii and yeasts such as Saccharomyces cereviasiae were identified in thermosonicated pulque.

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.

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.

A novel nucleoside 2'-deoxyribosyltransferase (LhNDT) from Lactobacillus hilgardii ZJS01 was cloned and expressed in Escherichia coli BL21 (DE3). The amino acid sequence of LhNDT displayed the highest identity of 26% to other reported NDTs. LhNDT was a monomer as revealed by gel filtration chromatography with or without addition of Ca2+ in elution buffer. This enzyme showed a high activity and stability over a broad pH range and was sensitive to temperature. With the addition of 15mM Ca2+, the thermostability of LhNDT was greatly promoted by 63 times and 46 times at 30°C and 35°C, respectively. With the use of 2'-deoxyuridine as sugar donor at an economic molar ratio of 1:1, 5-aza-2'-deoxycytidine (46%) and 5-fluoro-2'- deoxycytidine (58%) were efficiently synthesized from 5-azacytosine and 5-fluorocytosine, respectively, by LhNDT for 2h.

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 (

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.

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.

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.

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.

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.

Lactic acid bacteria (LAB) are Gram-positive, non-spore forming, facultative anaerobic or microaerophilic bacteria living in various nutrients-rich ecological niches and are widely used for dairy food and silage production as well as probiotics for the Human and animals. While bacteria can assimilate various nitrogen-containing compounds, the glutamine and ammonium are the most preferred nitrogen sources since they could be directly involved into the nitrogen metabolism of the cell. In bacterial cells, the glutamine can be synthetized from glutamate and ammonium ions by the metalloenzyme glutamine synthetase (GS). In contrast to other bacteria which generally have one gene encoding for the glutamine synthetase, two genes encoding proteins with 53% mutual identity and predicted glutamine synthetase activity were found in the genome of Lactobacillus hilgardii LMG 7934. One gene (glnA_2) is located in the glnRA operon with the transcriptional factor GlnR gene (glnR) similarly to GS genes from other bacteria. The second GS gene (glnA_1) is monocistronic. While the biosynthetic activity glutamine synthetases could be detected in L. hilgardii cells, which protein plays the major role is still unclear.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.