Heterofermentative Lactobacilli Research Articles

Lactobacilli - Probiotic as well as an Uncommon Pathogen – Current Status Regarding the “Two Sides of the Same Coin:” An Overview

Abstract Lactobacilli are nonsporing, lactic acid producing, anaerobic bacilli, classified under the phylum Firmicutes that form part of the normal flora in specific areas of the human body inclusive of the oral cavity, gastrointestinal tract, and the urogenital system. This genus, for instance contains Lactobacillus acidophilus, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, and others, when they are involved in the breakdown of glucose, they release lactic acid. It plays a major role in the production of fermented foods and in addition, it can serve as a probiotic which can be good to the host in that it can help in digestion, synthesis of vitamins, and can prevent colonization by pathogens. Lactobacilli are in association, although, in a commensal manner in large numbers during puberty and pregnancy and are also acidity maintainers, thereby preventing the pathogens to occupy the vaginal tract. They have also been declared to influence antiviral functions together with the suppression of virulence factors of pathogens based on recent studies. However, some categories of strains have been associated with life-threatening conditions such as endocarditis, meningitis, liver abscess, and bacteremia, particularly in immunocompromised clients. Regarding Lactobacilli, speciation has grown with methods such as MALDI-TOF, as it is faster and accurate than biochemical assays. Because their fermentative metabolism differs, they are grouped into the obligate homofermentative, facultative heterofermentative, and the obligate heterofermentative lactobacilli that assists in their identification and functionality. Lactobacilli being the probiotics are known to be safe and effective as endorsed by such bodies as the US Food and Drug Administration and European Food Safety Authority. Prebiotics are most effective in preterm infants and neonates where they help in strengthening the Gut barrier and Immunity. Thus, two probiotic strains like L. rhamnosus, and L. acidophilus have shown some positive impacts against different infections such as Helicobacter pylori, Clostridium difficile-associated diarrhea, and periodontitis. Nevertheless, one cannot disregard the possible pathogenicity of Lactobacilli as a number of strains are known to produce enzymes and organic acids which are hazardous to the human body. These examples of invasive infections stemming from strict regard for the use of probiotics only serve to enforce the prospect of heedful application of probiotics, especially to the vulnerable groups of people. Furthermore, diseases such as vaginal lactobacillosis are created to focus on the need for moderation in the consumption of probiotics. In summary, it can be seen that both, the roles and effects of Lactobacilli are twofold, as positive components with great potential for benefiting health as well as negative aspects that need further assessment.

Conversion of Phytochemicals by Lactobacilli: (Phospho)-β-glucosidases Are Specific for Glucosylated Phytochemicals Rather than Disaccharides.

Food-fermenting lactobacilli convert glycosylated phytochemicals to glycosyl hydrolases and thereby alter their biological activity. This study aimed to investigate the microbial transformation of β-glucosides of phytochemicals in comparison with utilization of cellobiose. Four homofermentative and four heterofermentative lactobacilli were selected to represent the metabolic diversity of Lactobacillaceae. The genomes of Lactobacillus crispatus, Companilactobacillus paralimentarius, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum encoded for 8 to 22 enzymes, predominantly phospho-β-glucosidases, with predicted activity on β-glucosides. Levilactobacillus hammesii and Furfurilactobacillus milii encoded for 3 β-glucosidases, Furfurilactobacillus rossiae for one, and Fructilactobacillus sanfranciscensis for none. The hydrolysis of amygdalin, esculin, salicin, glucosides of quercetin and genistein, and ginsenosides demonstrated that several strains hydrolyzed β-glucosides of phytochemicals but not cellobiose. Taken together, several of the carbohydrate-active enzymes of food-fermenting lactobacilli are specific for glycosides of phytochemicals.

Comparative genomic analysis of Periweissella and the characterization of novel motile species.

The genus Periweissella was proposed as a novel genus in the Lactobacillaceae in 2022. However, the phylogenetic relationship between Periweissella and other heterofermentative lactobacilli, and the genetic and physiological properties of this genus remain unclear. This study aimed to determine the phylogenetic relationship between Periweissella and the two closest genera, Weissella and Furfurilactobacillus, by the phylogenetic analysis and calculation of (core gene) pairwise average amino acid identity. Targeted genomic analysis showed that fructose bisphosphate aldolase was only present in the genome of Pw. cryptocerci. Mannitol dehydrogenase was found in genomes of Pw. beninensis, Pw. fabaria, and Pw. fabalis. Untargeted genomic analysis identified the presence of flagellar genes in Periweissella but not in other closely related genera. Phenotypes related to carbohydrate fermentation and motility matched the genotypes. Motility genes were organized in a single operon and the proteins shared a high amino acid similarity in the genus Periweissella. The relatively low similarity of motility operons between Periweissella and other motile lactobacilli indicated the acquisition of motility by the ancestral species. Our findings facilitate the phylogenetic, genetic, and phenotypic understanding of the genus Periweissella.ImportanceThe genus Periweissella is a heterofermentative genus in the Lactobacillaceae which includes predominantly isolates from cocoa fermentations in tropical climates. Despite the relevance of the genus in food fermentations, genetic and physiological properties of the genus are poorly characterized and genome sequences became available only after 2020. This study characterized strains of the genus by functional genomic analysis, and by determination of metabolic and physiological traits. Phylogenetic analysis revealed that Periweissella is the evolutionary link between rod-shaped heterofermentative lactobacilli and the coccoid Leuconostoc clade with the genera Weissella and Furfurilactobacillus as closest relatives. Periweissella is the only heterofermentative genus in the Lactobacillaceae which comprises predominantly motile strains. The genomic, physiological, and metabolic characterization of Periweissella may facilitate the potential use of strains of the genus as starter culture in traditional or novel food fermentations.

The contribution of epiphytic microbiota in oat and Italian ryegrass to silage fermentation products and bacterial community structure of whole-crop maize

BackgroundThe purpose of this research is to study the contribution of epiphytic microbiota in fresh oat (OT), Italian ryegrass (IR) and whole-crop maize (MZ) to silage fermentation products and bacterial community structure of MZ. After γ-ray irradiation, the sterile MZ was treated via microbiota transplantation method: (1) sterile deionized water (STMZ); (2) microbiota epiphytic on MZ (MZMZ); (3) microbiota epiphytic on OT (MZOT); (4) microbiota epiphytic on IR (MZIR). Triplicate silos of each treatment were tested after 1, 3, 7, 15, 30 and 60 days of ensiling.ResultsMZMZ had higher (P < 0.05) lactic acid contents, and lower (P < 0.05) ammonia nitrogen and ethanol contents than MZIR and MZOT on day 60. The relative abundance of Lactobacillus in MZMZ decreased from 84.0% on day 3 to 44.7% on day 60. MZMZ had higher (P < 0.05) abundances of ‘Nucleotide metabolism’, ‘Replication and repair’ and ‘Membrane transport’, and lower (P < 0.05) abundance of ‘Amino acid metabolism’ than MZOT and MZIR on day 3.ConclusionsThe silage fermentation products of MZ were highly affected by the activity and compositions of epiphytic microbiota. The Enterobacteriaceae, Hafnia-Obesumbacterium, hetero-fermentative and acid-resistant Lactobacillus took primary responsibility for the high dry matter loss and ethanol contents and low lactic acid contents in MZ silage.Graphical

Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

BIOTECHNOLOGICAL APPROACHES TO THE CREATION OF NEW FERMENTED DAIRY PRODUCTS

The industrial production of balanced and healthy food has always been one of the most important tasks facing humanity. To solve this problem, research is relevant in the field of creating functional milk-based synbiotic products, which, along with traditional lactic acid microorganisms, include strains of probiotic bacteria with a proven specific positive effect on the macroorganism, and prebiotic substances that increase the selectivity of the benefits of the "useful" microflora of the gastrointestinal tract-intestinal tract and its biological activity.Along with lactic acid bacteria used as starters in fermented milk products, there is a population of predominantly adventitious heterofermentative lactobacilli that can have a positive effect on dairy products.This article describes the use of facultatively and obligately heterofermentative species such as Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus fermentum 14. The general characteristics of these species are described, as well as the desired properties (probiotic activity and taste formation) that affect the characteristics of dairy products.The general characteristics of these species are described along with the desired (probiotic activity and taste formation) properties affecting the characteristics of dairy products.Samples of yogurt based on probiotic starter cultures enriched with plant fillers were studied. The technological process of production was carried out in the traditional way. The dynamics of acid accumulation was studied, titratable and active acidity were determined. As a result of the study, it was revealed that the combination of fermented milk products with plant fillers slightly accelerates the fermentation process.

Homo- and heterofermentative lactobacilli are distinctly affected by furanic compounds.

Second generation (2G) ethanol is produced using lignocellulosic biomass. However, the pre-treatment processes generate a variety of molecules (furanic compounds, phenolic compounds, and organic acids) that act as inhibitors of microbial metabolism, and thus, reduce the efficiency of the fermentation step in this process. In this context, the present study aimed to investigate the effect of furanic compounds on the physiology of lactic acid bacteria (LAB) strains that are potential contaminants in ethanol production. Homofermentative and heterofermentative strains of laboratory LAB, and isolated from first generation ethanol fermentation, were used. LAB strains were challenged to grow in the presence of furfural and 5-hydroxymethylfurfural (HMF). We determined that the effect of HMF and furfural on the growth rate of LAB is dependent on the metabolic type, and the growth kinetics in the presence of these compounds is enhanced for heterofermentative LAB, whereas they are inhibitory to homofermentative LAB. Sugar consumption and product formation were also enhanced in the presence of furanic compounds for heterofermentative LAB, who displayed effective depletion kinetics when compared to the homofermentative LAB. Homo- and heterofermentative LAB are affected differently by furanic compounds, in a way that the latter type is more resistant to the toxic effects of these inhibitors. This knowledge is important to understand the potential effects of bacterial contamination in 2G bioprocesses.

Fermentative products and bacterial community structure of C4 forage silage in response to epiphytic microbiota from C3 forages.

The observation that temperate C3 and tropical C4 forage silages easily produce large amounts of ethanol or acetic acid has puzzled researchers for many years. Hence, this study aimed to assess the effects of epiphytic microbiota from C3 forages (Italian ryegrass and oat) on fermentative products and bacterial community structure in C4 forage (sorghum) silage. Through microbiota transplantation and γ-ray irradiation sterilization, the irradiated sorghum was treated: i) sterile distilled water (STSG); ii) epiphytic microbiota from sorghum (SGSG); iii) epiphytic microbiota from Italian ryegrass (SGIR); iv) epiphytic microbiota from oat (SGOT). After 60 days, all the treated groups had high lactic acid (>63.0 g/kg dry matter [DM]) contents and low pH values (<3.70), acetic acid (<14.0 g/kg DM) and ammonia nitrogen (<80.0 g/kg total nitrogen) contents. Notably, SGIR (59.8 g/kg DM) and SGOT (77.6 g/kg DM) had significantly (p<0.05) higher ethanol concentrations than SGSG (14.2 g/kg DM) on day 60. After 60 days, Lactobacillus were predominant genus in three treated groups. Higher proportions of Chishuiella (12.9%) and Chryseobacterium (7.33%) were first found in silages. The ethanol contents had a positive correlation (p<0.05) with the abundances of Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium. The epiphytic bacteria on raw materials played important roles in influencing the silage fermentation products between temperate C3 and tropical C4 forages. The quantity and activity of hetero-fermentative Lactobacillus, Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium may be the key factors for the higher ethanol contents and DM loss in silages.

Effects of ensiling conditions on pyrrolizidine alkaloid degradation in silages mixed with two different Senecio spp.

ABSTRACT Pyrrolizidine alkaloid (PA) producing plants like Senecio jacobaea or Senecio vernalis are undesirable in fields for forage production, since PA are toxic to animals and humans. Previous studies have shown that ensiling can decrease the PA content in forages; however, no direct comparison of diverse PA from different Senecio spp. under various ensiling conditions has been made. Therefore, it was hypothesised that individual PA might react differently to ensiling, and silage inoculation with Lactobacillus will affect PA degradation because of a quick drop in pH, contrastingly to poor silage qualities resulting from contamination with soil. Laboratory scale grass silages were prepared in a multifactorial design with two levels of dry matter contents, four ensiling treatments and two storage durations (10 and 90 d). For each combination, four replicates were prepared individually. Ensiling treatments were (1) 10 ml water per kg fresh matter as control (CON), (2) 10 ml heterofermentative Lactobacillus buchneri strain LN4637 at 3 · 105 cfu/kg fresh matter plus 25 g molasses/kg fresh matter (LBHE), (3) 10 ml homofermentative lactobacilli at 3 · 105 cfu/kg fresh matter plus 25 g molasses/kg fresh matter (LBHO) and (4) 10 g soil/kg fresh matter (SOIL). Treatments affected formation of fermentation acids. Acetic acid was highest with treatment LBHE, and butyric acid was highest with treatment SOIL. All ensiling treatments effectively reduced total PA content by degrading the PA N-oxide (PANO) fraction. In parallel, though, the fraction of the tertiary base forms increased by around one-tenth of the original PANO content. Contents of jaconine and senkirkine were higher after ensiling than before, with regards to the sum of PA and PANO for jaconine, indicating higher stability or new formation through degradation of other PA. Overall, ensiling offers opportunities to decrease the PA-PANO content in feed and therefore lowers the risk of intoxication by Senecio in livestock.

Assessment of the microbiological origin of blowing defects in Grana Padano Protected Designation of Origin cheese

Recognized worldwide for its history, flavor, and high nutritional quality, Grana Padano (GP) is one of the most traditional Italian raw-milk, hard-cooked, long-ripened cheese. Throughout GP manufacturing, some well-known and undesired bacterial species, such as clostridia, can proliferate and lead to spoilage defects that mischaracterize the final product; however, little is known about the development of late-blowing defects in hard cheese samples without clostridia. Therefore, in this study we aimed to use metataxonomic analysis to identify bacterial taxa associated with the development of late-blowing defect in GP samples. Furthermore, the presence of several heterofermentative lactobacilli species in defective zones were verified by primer-specific PCR assay. Considering α- and β-diversity analyses, no statistically significant differences were detected between cheese samples with and without blowing defect. Following taxonomic assignment, Lactobacillus and Streptococcus were the dominant genera, whereas clostridia-related taxa were not detected in any of the 20 analyzed samples. Using EdgeR, the genera Propionibacterium and Acinetobacter were found to be prevalently more abundant in samples categorized as having "big regular holes." In samples with "small regular holes," multiplex PCR amplification revealed differences in terms of Lactobacillus population composition, mainly obligate homofermentative lactobacilli, between defective and non-defective zones of the same cheese wheel. This study demonstrated that GP samples with blowing defects not caused by clostridial development share similar biodiversity indices with GP collected from control zones, but an imbalance of obligate homofermentative lactobacilli was noticed between samples, which requires further analysis to better comprehend the exact mechanism involved in this process.

Biotechnology of Newly Created Bacterial Composition for Siloing Based on Lactic Acid Bacteria

Peculiarities of high-quality silage production are the use of biological products based on lactic acid bacteria. The composition of such starters varies greatly according to the use of bacterial cultures, so among the starters available on the market, the range of their effectiveness is also different. It is very common to use a one-sided approach to the choice of bacterial components, which in combination with imperfect production technology have low preservative activity. The study of combined preparations, which combine homo- and heterofermentative types of lactic acid fermentation, allows to stabilize the preservative properties throughout the ensiling time, and increase the aerobic stability of the silage after access of oxygen. Aim. Development of biotechnology of bacterial preparation for corn ensiling, optimization of cultivation conditions of newly created bacterial composition, and selection of cryoprotectants for its lyophilization. Methods. The combined preparation was created on the basis of heterofermentative strain Lactobacillus buchneri 3806 combining it in two- and three-strain compositions with other representatives of lactic acid bacteria, which are characterized by obligate homofermentative and facultative heterofermentative types of metabolism. Optimization of the environment and technological parameters was carried out using a central-compositional plan, further statistical analysis of the obtained data and determination of optimal values of input parameters according to the created mathematical model of optical density response. The effectiveness of the selected protective media was tested for the survival of bacteria after lyophilization. Results. The most effective bacterial composition was found during experiments: L. buchneri 3806, Enterococcus faecium C-8-12, L. plantarum 3216. The effectiveness of the obtained composition was tested by laboratory silage of corn. Tests of the drug based on the selected bacterial composition showed an improvement in the chemical composition of the silage compared to the untreated control and treated only with monoculture L. buchneri 3806, namely: there was a decrease in dry matter loss by 2.21% and 2.04%, 22 due to the increase of lactic acid content, and increase of aerobic stability of silage – 341 h against 57 h of the control sample, and 313 h in case of using monoculture. For the obtained bacterial composition, the culture medium of the following composition was optimized: base (hydrolyzed milk with the addition of the following components: monosubstituted potassium phosphate – 2 g/L; 5-aqueous manganese sulfate – 0.05 g/L; 7-aqueous magnesium sulfate – 0.2 g/L; twin-80 – 1.0 g/L); glucose – 19.7 g/L; yeast extract – 7.8 g/L; corn extract – 23.6 g/L; peptone – 9.1 g/L; sodium citrate – 6.6 g/L; sodium acetate – 3,4 g/L. Cultivation of the bacterial composition on an optimized medium made it possible to obtain the maximum biomass yield, at which the optical density was 2.01 units, which is almost twice as much as the value obtained by culturing the same composition in MRS medium. The optimal technological parameters of culturing the bacterial composition were established, namely the best growth was observed at a temperature of 36.4±0.4°C with constant maintenance of the pH value in the culture medium at the level of 6.5±0.1 units. In addition, the optimal composition of the protective medium containing sodium citrate, sucrose and agar was selected, and ensures the survival rate of lactic acid bacteria 98.4% after lyophilization. Conclusions. The newly formed bacterial composition can be used for the production of preparations for corn silage, and tested on other raw materials, in particular on some perennial legumes (alfalfa, clover), and the conditions of its production can be used to scale the technology.

Silage fermentation characteristics and microbial diversity of alfalfa (Medicago sativa L.) in response to exogenous microbiota from temperate grasses.

The objective of this study was to explore the microbiological factors that cause the difference in silage fermentation characteristics between grass and legume. Specifically, the effects of epiphytic microbiota from alfalfa, oat and Italian ryegrass on ensiling characteristics and microbial community of alfalfa were assessed. By γ-ray irradiation sterilization and microbiota transplantation technology, the sterile alfalfa was inoculated as follows: (i) aseptic water (STAL); (ii) epiphytic bacteria from alfalfa (ALAL); (iii) epiphytic bacteria from oat (ALOT); (iv) epiphytic bacteria from Italian ryegrass (ALIR). Alfalfa at the initial flowering stage was ensiled in laboratory-scale silos for 1, 3, 7, 14, 30 and 60 days. Compared with ALAL and ALIR, higher lactic acid contents and ratio of lactic acid to acetic acid, and lower acetic acid, propionic acid, ethanol and ammonia nitrogen contents were observed in ALOT after 60 days of fermentation. In each treated group, Lactobacillus was the most dominant genus after 60 days of ensiling. Relatively higher abundance of Weissella, Hafnia-Obesumbacterium, Enterobacteriaceae or hetero-fermentative Lactobacillus was found in ALAL and ALIR after 60 days. Co-occurrence network analysis proved Pediococcus and Lactococcus were pivotal in deciding the fermentation pattern of alfalfa silage. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was inhibited by the epiphytic microbiota from oat. Overall, ALOT showed a homo-fermentative process, whereas ALAL and ALIR exhibited a hetero-fermentative pattern. Furthermore, the exogenous microorganisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.

Fermentation profile and microbial diversity of temperate grass silage inoculated with epiphytic microbiota from tropical grasses.

This study was designed to evaluate the effects of epiphytic microbiota from Italian ryegrass (IRIR), Napier grass (IRNP) and Sudan grass (IRSD) on ensiling characteristics and microbial community of Italian ryegrass silage. Each treatment was prepared in triplicate and ensiled in plastic bag silos for 1, 3, 7, 14, 30 and 60days. The γ-ray irradiation sterilization method, microbiota transplantation and next generation sequencing technology were used. Results indicated that significantly (P < 0.05) higher ratio of lactic acid to acetic acid, and lower acetic acid and ammonia nitrogen contents were observed in IRNP than IRIR and IRSD after 60days of ensiling. Lactobacillus was the most predominant in each treatment at the late stage of fermentation. Lactococcus was eventually replaced by Lactobacillus in IRSD, whereas higher abundance of Lactococcus was continuously found in IRNP. Co-occurrence network analysis demonstrated Lactococcus was pivotal in determining the silage fermentation pattern of Italian ryegrass. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was enhanced by the epiphytic microbiota from Italian ryegrass and Sudan grass, while the carbohydrate metabolism was accelerated by the epiphytic microbiota from Napier grass. Overall, IRNP had a homo-fermentative process, whereas IRIR and IRSD possessed a hetero-fermentative pattern. The Lactococcus and heterofermentative Lactobacillus were mainly responsible for this. It also confirmed that the exogenous microorganisms that promote the carbohydrate metabolism and inhibit the metabolism of amino acids could be a good potential source to improve the silage quality of temperate grass.

Exploring the ensiling characteristics and bacterial community of red clover inoculated with the epiphytic bacteria from temperate gramineous grasses.

To explore the microbiological factors that cause the difference in silage fermentation characteristics between temperate gramineous (Italian ryegrass and oat) grass and legume forage. Through γ-ray irradiation sterilization and bacterial transplantation technology, the sterilized red clover was inoculated as follows: (i) aseptic water (STRC); (ii) epiphytic bacteria on red clover (RCRC); (iii) epiphytic bacteria on oat (RCOT); and (iv) epiphytic bacteria on Italian ryegrass (RCIR). Red clover was ensiled in laboratory-scale silos made of vacuum-packed plastic bags for 1, 3, 7, 14, 30 and 60days. STRC remained unfermented state based on similar chemical components with fresh red clover. Compared with RCRC and RCIR, higher lactic acid content and ratio of lactic acid to acetic acid, and lower pH, acetic acid and ammonia nitrogen contents were observed in RCOT after 60days of ensiling. Using next-generation sequencing, higher abundances of Methylobacterium and Sphingomonas were observed in the epiphytic bacteria on red clover. Pediococcus was dominant in RCRC after 3days of ensiling. Lactobacillus was the most predominant in each group after 60days of fermentation. RCRC and RCIR had a more hetero-fermentative process, while RCOT possessed a more homo-fermentative pathway. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was accelerated by the epiphytic bacteria from red clover. Inoculating the epiphytic bacteria from oat could alter the fermentation characteristics and bacterial compositions of red clover silage. The relative abundance and activity of Methylobacterium, Sphingomonas, Enterobacteriaceae and hetero-fermentative Lactobacillus in red clover silage could be changed by the epiphytic bacteria on temperate gramineous grass. The exogenous micro-organisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.

Gluconate metabolism and gas production by Paucilactobacillus wasatchensis WDC04

Paucilactobacillus wasatchensis, a nonstarter lactic acid bacteria, can cause late gas production and splits and cracks in aging cheese when it metabolizes 6-carbon substrates, particularly galactose, to a 5-carbon sugar, resulting in the release of CO2. Previous studies have not explained late gas production in aging cheese when no galactose is present. Based on the genome sequence of Pa. wasatchensis WDC04, genes for potential metabolic pathways were mapped using knowledgebase predictive biology software. This metabolic modeling predicted Pa. wasatchensis WDC04 could metabolize gluconate. Gluconate contains 6 carbons, and Pa. wasatchensis WDC04 contains genes to convert it to 6-P-gluconate and then to ribulose-5-P by using 6-phosphogluconate dehydrogenase in a decarboxylating step, producing CO2 during its metabolism. The goal of this study was to determine if sodium gluconate, often added to cheese to reduce calcium lactate crystal formation, could be metabolized by Pa. wasatchensis WDC04, resulting in gas production. Carbohydrate-restricted DeMan, Rogosa, and Sharpe broth was mixed with varying ratios of ribose, sodium gluconate, or d-galactose (total added substrate content of 1% wt/vol). Oxyrase (Oxyrase Inc.; 1.8% vol/vol) was also used to mimic the anaerobic environment of cheese aging in selected tubes. Tubes were inoculated with a 4-d culture of Pa. wasatchensis WDCO4, and results were recorded over 8 d. When inoculated into carbohydrate-restricted DeMan, Rogosa, and Sharpe broth containing only sodium gluconate as the added substrate, Pa. wasatchensis WDC04 grew, confirming gluconate utilization. Of the 10 ratios used, Pa. wasatchensis WDC04 produced gas in 6 scenarios, with the most gas production resulting from the ratio of 100% sodium gluconate with no added ribose or galactose. It was confirmed that obligately heterofermentative nonstarter lactobacilli such as Pa. wasatchensis WDC04 can utilize sodium gluconate to produce CO2 gas. Addition of sodium gluconate to cheese thus becomes another risk factor for unwanted gas production and formation of slits and cracks.

Galactose-positive adjunct cultures prevent gas formation by Paucilactobacillus wasatchensis WDC04 in a model gas production test

Gas production by obligatory heterofermentative lactic acid bacteria such as Paucilactobacillus wasatchensis is a sporadic problem in Cheddar cheese and results in undesired slits and cracks in the cheese. Growth of Pa. wasatchensis is not rapid, which makes investigations of gas production difficult to consistently execute. A primary objective of this study was to develop a model gas production test that could be used to investigate the effect of galactose and ribose utilization on gas production by Pa. wasatchensis and determine whether galactose-fermenting adjunct cultures could prevent gas formation. Paucilactobacillus wasatchensis WDC04 was inoculated at 101 to 106 cfu/mL into carbohydrate-restricted MRS broth containing different ribose and galactose levels and incubated for up to 21 d at 23°C. Gas production in the broth was detected using a Durham tube inverted on a 6-cm-long capillary tube; cells were enumerated at 4, 8, and 12 d; and residual galactose was also measured. Gas production was sporadic except for when 105 cfu/mL of Pa. wasatchensis WDC04 was inoculated into broth containing 0.3% ribose and 0.7% galactose. In those tubes, gas production was consistently observed after 8-d incubation, by which time galactose levels had decreased to 0.15%. Co-inoculation of Pa. wasatchensis WDC04 with as few as 103 cfu/mL of a lactose-negative galactose-positive adjunct culture (Pediococcus acidilactici 23F, Lacticaseibacillus paracasei UW4, or Lactobacillus helveticus 7995) resulted in galactose depletion by d 4 and no observable gas production by d 12. With less galactose available to the slower-growing Pa. wasatchensis WDC04, its growth was limited to 108 cfu/mL when any of the adjunct cultures was co-inoculated, compared with 109 cfu/mL when grown on its own. We concluded that galactose-fermenting adjunct cultures have potential for preventing unwanted gas production in cheese by competition for resources and especially by removing the 6-carbon galactose before it can be utilized for energy by an obligatory heterofermentative lactobacilli such as Pa. wasatchensis and produce carbon dioxide.

Characterization of γ-glutamyl cysteine ligases from Limosilactobacillus reuteri producing kokumi-active γ-glutamyl dipeptides.

γ-Glutamyl cysteine ligases (Gcls) catalyze the first step of glutathione synthesis in prokaryotes and many eukaryotes. This study aimed to determine the biochemical properties of three different Gcls from strains of Limosilactobacillus reuteri that accumulate γ-glutamyl dipeptides. Gcl1, Gcl2, and Gcl3 were heterologously expressed in Escherichia coli and purified by affinity chromatography. Gcl1, Gcl2, and Gcl2 exhibited biochemical with respect to the requirement for metal ions, the optimum pH and temperature of activity, and the kinetic constants for the substrates cysteine and glutamate. The substrate specificities of the three Gcls to 14 amino acids were assessed by liquid chromatography-mass spectrometry. All three Gcls converted ala, met, glu, and gln into the corresponding γ-glutamyl dipeptides. None of the three were active with val, asp, and his. Gcl1 and Gcl3 but not Gcl2 formed γ-glu-leu, γ-glu-ile, and γ-glu-phe; Gcl3 exhibited stronger activity with gly, pro, and asp when compared to Gcl2. Phylogenetic analysis of Gcl and the Gcl-domain of GshAB in lactobacilli demonstrated that most of Gcls were present in heterofermentative lactobacilli, while GshAB was identified predominantly in homofermentative lactobacilli. This distribution suggests a different ecological role of the enzyme in homofermentative and heterofermentative lactobacilli. In conclusion, three Gcls exhibited similar biochemical properties but differed with respect to their substrate specificity and thus the synthesis of kokumi-active γ-glutamyl dipeptides. KEY POINTS: • Strains of Limosilactobacillus reuteri encode for up to 3 glutamyl cysteine ligases. • Gcl1, Gcl2, and Gcl3 of Lm. reuteri differ in their substrate specificity. • Gcl1 and Gcl3 produce kokumi-active dipeptides.

The microbiology of Kasseri cheese during the maturation

The microbiology of Kasseri cheese, a Protected Designation of Origin (PDO) cheese of pasta-filata type was studied in order to identify the dominant species and strains that may contribute to the maturation process. Chemical composition and microbiological quality of Kasseri cheese samples from two different dairies during the maturation was studied at 0, 7, 25, 60 and 90 days of the maturation. Lactic acid bacteria and Enterococcus spp. were found to be the dominant microflora in fresh cheese. P. pentosaceous and P. acidilactici, E. hirae, E. faecium, E. durans and E. gallinarium, together with facultatively heterofermentative lactobacilli were found to be the dominant microflora. Since these strains are dominating the microflora of Kasseri cheese during maturation, the enzymic system need to be further studied in order to select the proper strains for adjunct culture in Kasseri cheese.

Effects of homo- and hetero-fermentative lactic acid bacteria on the quality and aerobic stability of corn silage

This study aimed to investigate the effects of homo/heterofermentative lactic acid bacteria (LAB) on the quality of corn silage and its rumen digestibility. Maize (Zea mays strain Xingsiyu No. 10), at the early dough stage, was harvested, chopped, ensiled in vacuum bags, and divided into four groups: (1) control (without added bacteria); (2) with the homofermentative LAB Lactobacillus plantarum and Pediococcus pentosaceus (1:1 mixture, 1 × 105 cfu·g−1); (3) with the heterofermentative LAB Lactobacillus buchneri (1 × 105 cfu·g−1); and (4) with the homo/heterofermentative LAB L. plantarum, P. pentosaceus, and L. buchneri (1:1:1 mixture). At 2, 8, 15, 45, and 60 d of ensiling, nutritional quality, fermentation characteristics, and microbial content were assessed. After 60 d, aerobic stability and rumen digestibility tests were performed. The addition of homo- and (or) hetero-fermentative LAB significantly improved the nutritional quality, fermentation characteristics, and microbial content. Addition of the heterofermenter L. buchneri, with or without the homofermenters L. plantarum and P. pentosaceus, improved aerobic stability. The different LAB inoculants increased the degradation rates of dry matter, acid detergent fiber, and neutral detergent fiber of the corn silage by sheep ruminants. Analysis indicated that L. buchneri yielded the best corn silage.

Microbial Succession and Identification of Effective Indigenous Pectinolytic Yeasts From Orange Juice Processing Wastewater

Although the management of citrus residues is of concern, their valorization is a challenging alternative. In this work, raw orange juice processing wastewater was spontaneously fermented to determine the succession in microbial communities and identify new indigenous microbial strains of high pectinolytic activity. Pectinolytic strains were isolated using citrus pectin as growth substrate. A shift in fungal population observed during spontaneous fermentation, where the abundances of Hanseniaspora, Cystofilobasidium and Meyerozyma were significantly increased, whereas Pichia fermentans followed by Saccharomyces spp. became the predominant taxa in the fermented wastewater. A significant reduction in the relative abundance of homofermentative aerococci and an increase in the relative abundance of heterofermentative leuconostocs and lactobacilli occurred during fermentation, while enterococci were predominant throughout the fermentation process. Regarding ecological indices, opposite changes took place in the bacterial compared to fungal community structure, but in a less pronounced degree. Examination of beta-diversity revealed the key role of Pichia species in driving fungal community structure during fermentation. P. fermentans, the predominant taxon in the fermented wastewater, was found to be the most effective pectinolytic microorganism. Pectin hydrolysis, carbohydrate fermentation to ethanol, lactic acid and acetate, and citric acid fermentation were the most important factors, influencing microbial community structure.