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

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.

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.

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

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.

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.

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X1B. Glucose and L-malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors. Copyright © 2012 Society of Chemical Industry

The effects of pH, temperature and initial yeast:bacteria ratio on Lactobacillus hilgardii and Saccharomyces florentinus cultivated either in pure or mixed culture were studied. Quadratic polynomial as a function of factors was proposed to express the lactic acid production at different sampling times, and the percentage increase in lactic acid production by Lact. hilgardii in mixed culture compared with pure culture. Temperature was the factor which had the main influence on lactic acid production in mixed culture, whereas stimulation of bacteria depended greatly on pH value. In the range 0.1-20%, the initial yeast-bacteria ratio had no effect on these responses, but presence of the yeast was absolutely necessary to obtain high production of lactic acid. Optimum culture conditions were determined to maximize these characteristics.

Growth, sugar and organic acid metabolism of Lactobacillus hilgardii X 1 B and Leuconostoc oenos X 2 L isolated from Argentinian wine were examined in pure and mixed cultures. In mixed culture Leuc. oenos X 2 L did not grow, no viable cells were detected after 24 h, but the consumption of glucose and fructose by Lact. hilgardii was higher. An increase of mannitol and acetic acid production was detected during the early stages of growth. Over 12 h, higher levels of D(-) lactic acid and slightly increased levels of L(+) lactic acid were observed. Citric and malic acid were simultaneously metabolized, but a slight increase in citric acid consumption was observed in mixed culture

Eighty‐two ethanol‐tolerant (≥ 15% v/v) isolates of Lactobacillus were obtained from Douro fortified wines and associated winery equipment. The diversity and specific identity of these isolates was studied with computer‐assisted comparison of the whole‐cell, SDS‐solubilized protein profiles (as generated by SDS‐PAGE); with this method four species were identified. The majority of isolates (89%) were identified as Lact. hilgardii, and three differentiable electrophoretic groups of this species were evident. The identification was confirmed by DNA‐DNA dot‐blot hybridization studies. Also identified were one strain each of Lact. fructivorans, Lact. collinoides and Lact. mali.

Stimulation of the growth of Lactobacillus hilgardii and production of lactic acid in the presence of Saccharomyces florentinus have already been observed in a poor glucose medium. In this study we showed that CO2, pyruvate, propionate, acetate and succinate excreted by the yeast were responsible for this phenomenon, whereas ethanol, fumarate and a cocktail of vitamins had no effect. Fermentation by the yeast did not enrich the total nitrogen or free amino acid content of the medium. The use of proteases confirmed that it was improbable that any nitrogenous compound could be responsible for the bacterial activation. Between values of 0.1 and 5%, the initial yeast: bacteria ratio had no effect on the stimulation of Lact. hilgardii, even though yeast fermentation was drastically affected by a low initial value. The interactions differed greatly according to the sugar source. With monosaccharides, stimulation began after 48 h of culture, and was higher on glucose than on fructose. With sucrose, stimulation appeared at the beginning of fermentation. Different mechanisms seemed to be involved.

Different techniques were tested for studying the synergism between the micro-organisms of sugary kefir grains. Agar cultures in Petri dishes did not give reproducible results. In sequential cultures, i.e. growing one organism, sterile filtering and then inoculating the other, 10 of 18 selected lactic acid bacteria/yeast pairs revealed stimulation of bacterial growth in a poor glucose medium. In mixed culture, Saccharomyces florentinus supported better survival of Lactobacillus hilgardii and a significant increase in lactic acid production; at the same time, the growth and alcoholic fermentation of S. florentinus were drastically reduced. The inter-relationships between these two strains were the same when immobilized in calcium alginate beads, even though total metabolite production was always lower than with free cells. The stimulation of Lact. hilgardii by Candida lambica in sequential culture was not confirmed in mixed culture, where the two organisms grew as in pure culture, and bacterial growth and lactic acid production were inhibited in the immobilized system.

Nineteen atypical heterofermentative strains of Lactobacillus spp. isolated from South African red wines were identified with the API 50 CHL system and by computerized comparison of their total soluble cell protein patterns. Eleven strains were identified as Lactobacillus hilgardii and eight strains as L. brevis. Three strains of L. hilgardii and three strains of L. brevis produced small amounts of H2S in peptone water supplemented with 001%l-cysteine. All strains produced mannitol from fructose. Proteolytic activity was detected in all except two strains of L. hilgardii. Strains capable of digesting gelatine, hydrolized casein, but not vice versa. The excessive production of sulphur-containing compounds, mannitol and acetaldehyde by lactic acid bacteria could have serious quality implications for the wine industry. Three strains of L. hilgardii and all strains of L. brevis decarboxylated l-malic acid to l-lactic acid.

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.

SUMMARY: An improved growth medium for lactobacilli is described. It supports good growth of lactobacilli generally and also is particularly useful for a number of fastidious strains which grow only poorly in other general media. In addition, tomato juice, a highly variable material, is not required. In a slightly modified form, it can also be used as a basal medium for fermentation tests.

Growth of two heterofermentative lactic acid bacteria (Lactobacillus hilgardii and Leuconostoc mesenteroides) isolated from wine, was inhibited in a medium to which sulphurous acid and an excess of acetaldehyde had been added. It was found that the bacteria rapidly attacked the aldehyde and liberated sufficient free sulphur dioxide to prevent further growth. A homofermentative bacterium (Lactobacillus arabinosus) consumed much less aldehyde than the two heterofermentative organisms, but nevertheless failed to grow in the presence of 100 p.p.m. of bound sulphur dioxide. The significance of these results in relation to the malo-lactic fermentation in wines is discussed.