DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations.
ABSTRACT: Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.
Project description:Northeastern Chinese sauerkraut is a well-known traditional fermented vegetable in China. Incomplete identification of the microorganisms' (bacteria in spontaneous fermentation) diversity and accumulation of nitrite make it difficult to normalize the fermentation process and product qualities of northeastern Chinese sauerkraut. Conventional culturing and polymerase chain reaction-denaturing gradient gel electrophoresis methods were combined to describe microbial structure and diversity. Lactobacillus, Leuconostoc, Enterobacter, Accumulibacter, Thermotoga, Pseudomonas, Clostridium, Rahnella and Citrobacter were predominant microorganisms in different fermentation periods. The pH value and nitrite concentration presented a certain relevance to the amount of lactic acid bacteria. Lactobacillus and Leuconostoc had the ability to decrease nitrite by inhibiting nitrate-reducing bacteria such as Enterobacter. Therefore, Northeastern Chinese sauerkraut should not be eaten until 4 weeks of fermentation for the safety and quality of fermented foods. Northeastern Chinese sauerkraut is rich in lactic acid bacteria, which demonstrate its ability as an excellent probiotic for applications in functional foods.
Project description:BACKGROUND:Panax ginseng is a physiologically active plant widely used in traditional medicine that is characterized by the presence of ginsenosides. Rb1, a major ginsenoside, is used as the starting material for producing ginsenoside derivatives with enhanced pharmaceutical potentials through chemical, enzymatic, or microbial transformation. METHODS:To investigate the bioconversion of ginsenoside Rb1, we prepared kimchi originated bacterial strains Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49 and analyzed bioconversion products using LC-MS/MS mass spectrometer. RESULTS:L. mesenteroides WiKim19 and Pediococcus pentosaceus WiKim20 converted ginsenoside Rb1 into the ginsenoside Rg3 approximately five times more than Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49. L mesenteroides WIKim19 showed positive correlation with ?-glucosidase activity and higher transformation ability of ginsenoside Rb1 into Rg3 than the other strains whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 even with lack of ?-glucosidase activity but have the highest acidity among the five lactic acid bacteria (LAB). CONCLUSION:Ginsenoside Rg5 concentration of five LABs have ranged from ?2.6 ?g/mL to 6.5 ?g/mL and increased in accordance with the incubation periods. Our results indicate that the enzymatic activity along with acidic condition contribute to the production of minor ginsenoside from lactic acid bacteria.
Project description:Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.
Project description:Kimchi fermentation depends on diverse lactic acid bacteria, which convert raw materials into numerous metabolites that contribute to the taste of food. Amino acids and saccharides are important primary metabolites. Arginine is nearly exhausted during kimchi fermentation, whereas the concentrations of other amino acids are reported not to increase or decrease dramatically. These phenomena could imply that arginine is an important nutritional component among the amino acids during kimchi fermentation. In this study, we investigated the arginine-catabolism pathway of seven lactic acid bacteria isolated from kimchi and evaluated the products of arginine catabolism (citrulline and ornithine) associated with the bacteria. The arginine content dramatically decreased in cultures of Lactobacillus brevis and Weissella confusa from 300 ?g/mL of arginine to 0.14 ± 0.19 and 1.3 ± 0.01 ?g/mL, respectively, after 6 h of cultivation. Citrulline and ornithine production by L. brevis and W. confusa showed a pattern that was consistent with arginine catabolism. Interestingly, Pediococcus pentosaceus, Lactobacillus plantarum, Leuconostoc mesenteroides, and Leuconostoc lactis did not show increased citrulline levels after arginine was added. The ornithine contents were higher in all bacteria except for L. lactis after adding arginine to the culture. These results were consistent with the absence of the arginine deiminase gene among the lactic acid bacteria. Arginine consumption and ornithine production were monitored and compared with lactic acid bacteria by metagenomics analysis, which showed that the increment of ornithine production correlated positively with lactic acid bacteria growth.
Project description:Sauerkraut, one of the most popular traditional fermented vegetable foods in northern China, has been widely consumed for thousands of years. In this study, the physicochemical characteristics, microbial composition and succession, and metabolome profile were elucidated during the fermentation of traditional northeast sauerkraut sampled from different households. The microbial community structure as determined by high-throughput sequencing (HTS) technology demonstrated that Firmicutes and Proteobacteria were the predominant phyla and Weissella was the most abundant genus in all samples. Except for Weissella, higher relative abundance of Clostridium was observed in #1 sauerkraut, Clostridium and Enterobacter in #2 sauerkraut, and Lactobacillus in #3 sauerkraut, respectively. Meanwhile, Principal component analysis (PCA) revealed significant variances in the volatilome profile among different homemade sauerkraut. Acids and lactones were dominant in the #1 sauerkraut. The #2 sauerkraut had significantly higher contents of alcohols, aldehydes, esters, sulfides, and free amino acids (FAAs). In comparison, higher contents of terpenes and nitriles were found in the #3 sauerkraut. Furthermore, the potential correlations between the microbiota and volatilome profile were explored based on Spearman's correlation analysis. Positive correlations were found between Clostridium, Enterobacter, Lactobacillus, Leuconostoc, Weissella and most volatile compounds. Pseudomonas, Chloroplast, Rhizobium, Aureimonas, and Sphingomonas were negatively correlated with volatile compounds in sauerkraut. This study provided a comprehensive picture of the dynamics of microbiota and metabolites profile during the fermentation of different homemade northeast sauerkraut. The elucidation of correlation between microbiota and volatile compounds is helpful for guiding future improvement of the fermentation process and manufacturing high-quality sauerkraut.
Project description:A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.
Project description:Feed deficiency during the long cold period of a year is one of the major problems that the traditional year-round animal grazing system has faced on the Qinghai-Tibetan plateau (QTP) since ancient time. Therefore, ensiling the grasses from grassland could be a desirable feeding regime to preserve high quality forage and to alleviate the seasonal unbalanced feed supply problem on this plateau. The present study was designed to investigate dynamics of bacterial community and natural fermentation quality of ensiled Elymus nutans collected from grasslands in four different areas with different elevations [Tianzhu County (TZ), 2965 m; Golog Prefecture (GL), 3763 m; Damxung County (DX), 4228 m, and Nagqu Prefecture (NQ), 4752 m] on the QTP. The bacterial community was characterized by using the PacBio single molecule with real-time sequencing technology (SMRT). The harvested fresh E. nutans grasses were ensiled in vacuum-sealed polyethylene bags for 14, 30, 60, and 90 days. Obvious differences in the epiphytic bacterial community of the fresh E. nutans samples from the four areas were observed, which resulted in various bacterial community dynamics and fermentation qualities of ensiled E. nutans. Higher fermentation quality was observed in silage samples from Nagqu than in those from the other areas (P < 0.05). Lactic acid bacteria (LAB) involved in fermentation of E. nutans from low altitude areas consisted of Pediococcus pentosaceus, Lactobacillus sp., Leuconostoc mesenteroides, and Lactobacillus coryniformis, whereas major LAB species involved in the fermentation of E. nutans silage from high altitudes included L. mesenteroides, Lactobacillus brevis, and Lactobacillus sp. Correlation analysis between bacterial composition and fermentation quality of E. nutans silages made from the four different areas in the QTP indicated that the LAB species responsible for silage fermentation in different areas were totally different, which was mainly due to the different epiphytic bacterial compositions in fresh E. nutans before ensiling. The present results provide important information on revealing the bacterial communities and fermentation quality of ensiled E. nutans, and on future screening of LAB isolates for making high quality silage in order to alleviate feed shortage of the traditional year-round grazing system on the QTP.
Project description:To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations.
Project description:Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.
Project description:Consumer demand for natural pathogen-control agents for substitution of synthetic food preservatives and traditional antibiotics is increasing. This study aimed to reveal the distribution of lactic acid bacteria (LAB) in raw camel milk and to characterize their antimicrobial traits. The genetic identification by 16S rRNA sequencing of 58 LAB isolates showed the predominance of Enterococcus (24.2%), Lactococcus (22.4%) and Pediococcus (20.7%) genera in raw camel milk. These genera exhibited inhibitory activity against a broad spectrum of Gram-positive and Gram-negative bacteria including multidrug-resistant Salmonella. Among these LAB, two isolates-identified as Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22-were selected for their strong bacteriocinogenic anti-listerial activity estimated at 1600 and 800 AU/mL, respectively. The bacteriocins produced were partially purified by ammonium sulphate precipitation and gel filtration and then biochemically characterized. The proteinaceous nature of bacteriocins was confirmed by the susceptibility to enzymes. These bacteriocins showed significant technological characteristics such as heat-resistance, and stability over a wide range of pH (2.0-10.0). In conclusion, these results indicated that Pediococcus pentosaceus CM16 and Lactobacillus brevis CM22 could be useful as potential probiotics. Moreover, their partially purified bacteriocins may play an important role as food preservatives and feed additives. To our knowledge, this is the first report describing the distribution of LAB population in raw camel milk and the characterization of their bacteriocins from the Arabian Peninsula of western Asia.