Genomic and metabolomic analysis of Latilactobacillus sakei DCF0720 for black soybean yogurt fermentation.

Lactic acid bacteria are commonly used in plant-based fermentation to reduce off-flavor and improve sensory characteristics. However, there have been few studies on Latilactobacillus sakei for plant-based yogurt fermentation and, particularly, its metabolic features at the genomic level remain unclear. This study aims to analyze the fermentation characteristics of the L. sakei DCF0720 strain and compare genetics and metabolic relations. For this, DCF0720 was used to ferment the black soybean milk and conduct the physicochemical analysis and sensory test. The genomic and metabolic analyses were performed by complete genome sequencing and 500 MHz 1H NMR, respectively. As a result, DCF0720 exhibited enhanced fermentation performance and sensory evaluations at 37 °C compared to 30 °C, which is generally recognized as the optimal growth temperature for most L. sakei strains. It also produced flavor enhancing volatile compounds such as acetoin and hydroxyacetone, possessing all three key genes for acetoin biosynthesis. DCF0720 lacks 2,3-butanediol dehydrogenase, which leads to the inhibition of acetoin production. DCF0720 possesses a complete pathway to utilize primary black soybean carbon sources such as sucrose, raffinose, and stachyose. DCF0720 also possesses genes for the GH28 family, including the key enzymes in the hydrolysis of pectin substances, which means eliminating the main soybean nonstarch polysaccharides. This study demonstrates that DCF0720 is a suitable starter for plant-based yogurt fermentation, providing a better understanding of fermentation conditions with genetic and metabolic features for black soybean yogurt. Various carbon source utilization abilities with depth metabolic pathway analysis provide that DCF0720 can be employed to develop enhanced starter cultures for black soybean yogurt and diverse plant-based yogurts.

Transcriptome Analysis Reveals the Role of Sucrose in the Production of Latilactobacillus sakei L3 Exopolysaccharide.

Latilactobacillus (L.) sakei is a species of lactic acid bacteria (LAB) mostly studied according to its application in food fermentation. Previously, L. sakei L3 was isolated by our laboratory and possessed the capability of high exopolysaccharide (EPS) yield during sucrose-added fermentation. However, the understanding of sucrose promoting EPS production is still limited. Here, we analyzed the growth characteristics of L. sakei L3 and alterations of its transcriptional profiles during sucrose-added fermentation. The results showed that L. sakei L3 could survive between pH 4.0 and pH 9.0, tolerant to NaCl (<10%, w/v) and urea (<6%, w/v). Meanwhile, transcriptomic analysis showed that a total of 426 differentially expressed genes and eight non-coding RNAs were identified. Genes associated with sucrose metabolism were significantly induced, so L. sakei L3 increased the utilization of sucrose to produce EPS, while genes related to uridine monophosphate (UMP), fatty acids and folate synthetic pathways were significantly inhibited, indicating that L. sakei L3 decreased self-growth, substance and energy metabolism to satisfy EPS production. Overall, transcriptome analysis provided valuable insights into the mechanisms by which L. sakei L3 utilizes sucrose for EPS biosynthesis. The study provided a theoretical foundation for the further application of functional EPS in the food industry.

Genomic and metabolic features of the Lactobacillus sakei JD10 revealed potential probiotic traits.

Lactic acid bacteria that inhabit in the lung play important roles in maintaining the microbiome balance by interacting with the host immune system. Numerous metabolites (e.g., short chain fatty acids, bacteriocins, and hydrogen peroxide) produced by Lactobacillus sakei possess a special inhibitory spectrum against invading pathogens. In this research, the whole genome of L. sakei JD10 strain isolated from the porcine lung was sequenced and investigated. The whole size of the L. sakei JD10 chromosome was 1,989,921 bp, which encoded a total of 1951 predicted genes. Genome analyses revealed that many genes encoded carbohydrate-active enzymes (CAZymes) were predicted, which were responsible for the carbohydrate degradation and short chain fatty acids production. The metabolic profiles of short chain fatty acids in the L. sakei JD10 culture medium were measured by GC/TOFMS, and their regulatory effects on bacterial phagocytosis of RAW264.7 cells were also determined. The bacteriocin-producing genes of the L. sakei JD10 genome were also predicted, and a bacteriocin gene encoding carnocin was characterized and its molecular structure was analyzed. Two CRISPR-Cas system related genes were identified from the L. sakei JD10 genome, revealed that precise and efficient genome editing technologies could be applied for genetic engineering-manipulation. In all, investigation on the genomic features and metabolic features of L. sakei JD10 showed the potential probiotic traits to fight against pathogenic infection and regulate the host immune function.

Characterization of a new bacteriocin-like inhibitory peptide produced by Lactobacillus sakei B-RKM 0559.

The biopreservation strategy allows extending the shelf life and food safety through the use of indigenous or controlled microbiota and their antimicrobial compounds. The aim of this work was to characterize an inhibitory substance with bacteriocin-like activity (Sak-59) produced by the potentially probiotic L. sakei strain from artisanal traditional Kazakh horse meat product Kazy. The maximum production of Sak-59 occurred at the stationary phase of the L. sakei growth. Sak-59 showed inhibitory activity against gram-positive meat spoilage bacteria strains of Listeria monocytogenes, Staphylococcus aureus, and pathogenic gram-negative bacteria strains of Serratia marcescens and Escherichia coli, but not against the tested Lactobacilli strains. Sak-59 activity, as measured by diffusion assay in agar wells, was completely suppressed after treatment with proteolytic enzymes and remained stable after treatment with α-amylase and lipase, indicating that Sak-59 is a peptide and most likely not glycosylated or lipidated. It was concluded that Sak-59 is a potential new bacteriocin with a characteristic activity spectrum, which can be useful in the food and feed industries.

Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis.

Latilactobacillus sakei group comprises four closely related species, making it difficult to accurately distinguish them with standard markers such as the 16S rRNA gene. The objective of our study was to mine novel markers for PCR detection and discrimination of L. sakei group species and L. sakei subspecies by comparative pan-genomic analysis. A total of 63 genome sequences of L. sakei group species consisted of 119,899 coding genes, yielding 5741 pan-genomes, 831 core-genomes, 3347 accessory-genomes, and 1563 unique-genomes. The accessory-genome was compared to extract unique candidate genes common only to genomes of the same species. The candidate genes were then aligned with the other bacterial genomes to select marker genes present in all genomes of a given species, but not in the genomes of other species. We identified the arginine/ornithine antiporter, putative cell surface protein precursor, sodium:solute symporter, PRD domain protein, PTS sugar transporter subunit IIC, and phosphoenolpyruvate-dependent sugar phosphotransferase system EIIC as marker genes for L. sakei, L. sakei subsp. sakei, L. sakei subsp. carnosus, L. curvatus, L. graminis, and L. fuchuensis, respectively. Primer pairs were designed for each marker and showed 100% specificity for 48 lactic acid bacterial reference strains. The PCR method developed in this study was used to evaluate 106 strains isolated from fermented foods to demonstrate that the marker genes provided a viable alternative to the 16S rRNA gene. We also applied the method to the monitoring of kimchi samples to quantify L. sakei group species or subspecies. Our PCR method based on novel markers can rapidly identify L. sakei group with high accuracy and high throughput.

High-Fat-Diet-Induced Oxidative Stress Linked to the Increased Colonization of Lactobacillus sakei in an Obese Population.

Obesity is a major public health problem related to various chronic health conditions. Lactobacillus species has been reported in obese individuals; however, its role is unknown. We compared the abundance and composition of Lactobacillus species by analyzing feces from 64 healthy control subjects and 88 obese subjects. We isolated one Lactobacillus strain from the feces of a subject with obesity and further analyzed its genetic and molecular features. We found that an increased abundance and higher prevalence of Lactobacillus sakei distinguished the fecal microbiota of the obese group from that of healthy subjects and that it was related to the increased levels of reactive oxygen species (ROS) induced by higher fat intake. The L. sakei ob4.1 strain, isolated from the feces of a subject with obesity, showed high catalase activity, which was regulated by oxidative stress at the gene transcription level. L. sakei ob4.1 maintained colon epithelial cell adhesion ability under ROS stimulation, and treatment with saturated fatty acid increased colon epithelial ROS levels in a dose-dependent manner; however, L. sakei ob4.1 did not change the level of fat-induced colon epithelial ROS. Exposing mice to a high-fat diet revealed that high-fat-diet-induced colon ROS was associated with the increased colonization of L. sakei ob4.1 through catalase activity. Four-week supplementation with this strain in mice fed a high-fat diet did not change their body weights or ROS levels. A high-fat diet induces changes in the colon environment by increasing ROS levels, which provides a colonization benefit to an L. sakei strain with high catalase activity. IMPORTANCELactobacillus provides many health benefits; its various species are widely used as probiotics. However, an increased abundance of Lactobacillus has been reported in obesity, and the role of Lactobacillus strains in obesity remains unknown. We found a high abundance of the Lactobacillus sakei species in a group of obese subjects and examined its relationship with a high-fat diet and reactive oxygen species (ROS) in the feces. To find the underlying mechanism, we analyzed and characterized an L. sakei strain isolated from a severely obese individual. We found that higher gut oxidative stress could link high-fat-diet-induced obesity and L. sakei. This translational research identifies the roles of the host gut environment in the colonization and survival of L. sakei.

The Complete Genome of Probiotic Lactobacillus sakei Derived from Plateau Yak Feces.

Probiotic bacteria are receiving increased attention due to the potential benefits to their hosts. Plateau yaks have resistance against diseases and stress, which is potentially related to their inner probiotics. To uncover the potential functional genes of yak probiotics, we sequenced the whole genome of Lactobacillus sakei (L. sakei). The results showed that the genome length of L. sakei was 1.99 Mbp, with 1943 protein coding genes (21 rRNA, 65 tRNA, and 1 tmRNA). There were three plasmids found in this bacteria, with 88 protein coding genes. EggNOG annotation uncovered that the L. sakei genes were found to belong to J (translation, ribosomal structure, and biogenesis), L (replication, recombination, and repair), G (carbohydrate transport and metabolism), and K (transcription). GO annotation showed that most of the L. sakei genes were related to cellular processes, metabolic processes, biological regulation, localization, response to stimulus, and organization or biogenesis of cellular components. CAZy annotation found that there were 123 CAZys in the L. sakei genome, with glycosyl transferases and glycoside hydrolases. Our results revealed the genome characteristics of L. sakei, which may give insight into the future employment of this probiotic bacterium for its functional benefits.

Heme Uptake in Lactobacillus sakei Evidenced by a New Energy Coupling Factor (ECF)-Like Transport System.

Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [lsa1836-1840]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δlsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems.IMPORTANCELactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system.

Genomic and metabolic features of Lactobacillus sakei as revealed by its pan-genome and the metatranscriptome of kimchi fermentation.

The genomic and metabolic features of Lactobacillus sakei were investigated using its pan-genome and by analyzing the metatranscriptome of kimchi fermentation. In the genome-based relatedness analysis, the strains were divided into the Lb. sakei ssp. sakei and Lb. sakei ssp. carnosus lineage groups. Genomic and metabolic pathway analysis revealed that all Lb. sakei strains have the capability of producing d/l-lactate, ethanol, acetate, CO2, formate, l-malate, diacetyl, acetoin, and 2,3-butanediol from d-glucose, d-fructose, d-galactose, sucrose, d-lactose, l-arabinose, cellobiose, d-mannose, d-gluconate, and d-ribose through homolactic and heterolactic fermentation, whereas their capability of d-maltose, d-xylose, l-xylulose, d-galacturonate, and d-glucuronate metabolism is strain-specific. All strains carry genes for the biosynthesis of folate and thiamine, whereas genes for biogenic amine and toxin production, hemolysis, and antibiotic resistance were not identified. The metatranscriptomic analysis showed that the expression of Lb. sakei transcripts involved in carbohydrate metabolism increased as kimchi fermentation progressed, suggesting that Lb. sakei is more competitive during late fermentation stage. Homolactic fermentation pathway was highly expressed and generally constant during kimchi fermentation, whereas expression of heterolactic fermentation pathway increased gradually as fermentation progressed. l-Lactate dehydrogenase was more highly expressed than d-lactate dehydrogenase, suggesting that l-lactate is the major lactate metabolized by Lb. sakei.

Comparative genomics of Lactobacillus sakei supports the development of starter strain combinations.

Strains of Lactobacillus sakei can be isolated from a variety of sources including meat, fermented sausages, sake, sourdough, sauerkraut or kimchi. Selected strains are widely used as starter cultures for sausage fermentation. Recently we have demonstrated that control about the lactic microbiota in fermenting sausages is achieved rather by pairs or strain sets than by single strains. In this work we characterized the pan genome of L. sakei to enable exploitation of the genomic diversity of L. sakei for the establishment of assertive starter strain sets. We have established the full genome sequences of nine L. sakei strains from different sources of isolation and included in the analysis the genome of L. sakei 23K. Comparative genomics revealed an accessory genome comprising about 50% of the pan genome and different lineages of strains with no relation to their source of isolation. Group and strain specific differences could be found, which namely referred to agmatine and citrate metabolism. The presence of genes encoding metabolic pathways for fructose, sucrose and trehalose as well as gluconate in all strains suggests a general adaptation to plant/sugary environments and a life in communities with other genera. Analysis of the plasmidome did not reveal any specific mechanisms of adaptation to a habitat. The predicted differences of metabolic settings enable prediction of partner strains, which can occupy the meat environment to a large extent and establish competitive exclusion of autochthonous microbiota. This may assist the development of a new generation of meat starter cultures containing L. sakei strains.

Characterization and distribution of CRISPR-Cas systems in Lactobacillus sakei.

Clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) structures, known as prokaryotes 'immune system', have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR-Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR-Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR-Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.

Robust Domination of Lactobacillus sakei in Microbiota During Traditional Japanese Sake Starter Yamahai-Moto Fermentation and the Accompanying Changes in Metabolites.

The successful production of sake (Japanese rice wine) is brought about by drastic changes in microbial flora and chemical components during fermentation. In the traditional manufacturing process of sake starter (yamahai-moto), spontaneous growth of lactic acid bacteria suppresses inappropriate microorganisms and prepares the optimum environment for the alcohol fermentative yeast. In this study, we analyzed the changes in bacterial flora and chemical components of yamahai-moto. High-throughput next-generation sequencing (NGS) of the 16S ribosomal RNA gene V4 region revealed that various kinds of bacteria, including nitrate-reducing bacteria, existed in the early fermentation stage; however, Lactobacillus sakei then increased drastically to become dominant in the middle stage. Interestingly, this result was different from that obtained in the previous year at the same manufacturer; the early-stage major bacterium was Lactobacillus acidipiscis. Lactic acid, glucose, isomaltose, and total free amino acids increased throughout the fermentation process, which was attributable to the metabolism of L. sakei and the koji mold. It is noteworthy that significant ornithine accumulation and arginine consumption were observed from the middle to late stages. Thirty-eight percent of the L. sakei isolates from yamahai-moto exhibited significant ornithine production, indicating that the arginine deiminase pathway of L. sakei was working to survive the extremely low pH environment of the moto after the middle stage. This is the first report that includes concurrent analyses of the NGS-based bacterial flora and chemical components of yamahai-moto, providing further knowledge to help understand and improve the process of sake brewing.

Integrated phenotypic-genotypic approach to understand the influence of ultrasound on metabolic response of Lactobacillus sakei.

The lethal effects of soundwaves on a range of microorganisms have been known for almost a century whereas, the use of ultrasound to promote or control their activity is much more recent. Moreover, the fundamental molecular mechanism influencing the behaviour of microorganisms subjected to ultrasonic waves is not well established. In this study, we investigated the influence of ultrasonic frequencies of 20, 45, 130 and 950 kHz on growth kinetics of Lactobacillus sakei. A significant increase in the growth rate of L. sakei was observed following ultrasound treatment at 20 kHz despite the treatment yielding a significant reduction of ca. 3 log cfu/mL in cells count. Scanning electron microscopy showed that ultrasound caused significant changes on the cell surface of L. sakei culture with the formation of pores "sonoporation". Phenotypic microarrays showed that all ultrasound treated L. sakei after exposure to various carbon, nitrogen, phosphorus and sulphur sources had significant variations in nutrient utilisation. Integration of this phenotypic data with the genome of L. sakei revealed that various metabolic pathways were being influenced by the ultrasound treatments. Results presented in this study showed that the physiological response of L. sakei in response to US is frequency dependent and that it can influence metabolic pathways. Hence, ultrasound treatments can be employed to modulate microbial activity for specialised applications.

Developing novel species-specific DNA markers for PCR-based species identification of the Lactobacillus sakei group.

Identification of members of the Lactobacillus sakei group (LSG) by common phenotypic and genotypic methods is generally inadequate and time-consuming. The objective of this study was to develop novel species-specific primers based on sequence-characterized amplified region (SCAR) markers using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Three species-specific fragments were gel-purified, cloned and sequenced after preliminary screening of 80 random primers. Accordingly, three pairs of primers Lcur-F/R, Lgram-F/R and Lsakei-F/R were designed based on single species-specific bands (281, 278 and 472 bp) that were obtained from Lactobacillus curvatus, Lactobacillus graminis and L. sakei, respectively. The specificities of these primer pairs were confirmed in 21 LSG strains and 31 nontarget Lactobacillus strains. In addition, the detection limits for each primer pair were approx. 105 , 104 and 106 cells per gram of meat samples spiked with L. curvatus, L. graminis and L. sakei, respectively. In conclusion, we have successfully developed a rapid, accurate and effective PCR-based method for identification of species in the LSG. SIGNIFICANCE AND IMPACT OF THE STUDY: Neither phenotypic nor the most commonly used genotypic method (16S rRNA gene sequencing) provides sufficient resolution for accurate identification of the Lactobacillus sakei group. A sequence-characterized amplified region method developed in this study provides a rapid, cost-effective way to detect the member of the L. sakei group in meat sample.

Effects of glucose availability in Lactobacillus sakei; metabolic change and regulation of the proteome and transcriptome.

Effects of glucose availability were investigated in Lactobacillus sakei strains 23K and LS25 cultivated in anaerobic, glucose-limited chemostats set at high (D = 0.357 h-1) and low (D = 0.045 h-1) dilution rates. We observed for both strains a shift from homolactic towards more mixed acid fermentation when comparing high to low growth rates. However, this change was more pronounced for LS25 than for 23K, where dominating products were lactate>formate>acetate≥ethanol at both conditions. A multivariate approach was used for analyzing proteome and transcriptome data from the bacterial cultures, where the predictive power of the omics data was used for identifying features that can explain the differences in the end-product profiles. We show that the different degree of response to the same energy restriction revealed interesting strain specific regulation. An elevated formate production level during slow growth, more for LS25 than for 23K, was clearly reflected in correlating pyruvate formate lyase expression. With stronger effect for LS25, differential expression of the Rex transcriptional regulator and NADH oxidase, a target of Rex, indicated that maintainance of the cell redox balance, in terms of the NADH/NAD+ ratio, may be a key process during the metabolic change. The results provide a better understanding of different strategies that cells may deploy in response to changes in substrate availability.

Proteomic and genetics insights on the response of the bacteriocinogenic Lactobacillus sakei CRL1862 during biofilm formation on stainless steel surface at 10°C.

Some lactic acid bacteria have the ability to form biofilms on food-industry surfaces and this property could be used to control food pathogens colonization. Lactobacillus sakei CR1862 was selected considering its bacteriocinogenic nature and ability to adhere to abiotic surfaces at low temperatures. In this study, the proteome of L. sakei CRL1862 grown either under biofilm on stainless steel surface and planktonic modes of growth at 10°C, was investigated. Using two-dimensional gel electrophoresis, 29 out of 43 statistically significant spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten proteins resulted up-regulated whereas 16 were down-regulated during biofilm formation. Differentially expressed proteins were found to belong to carbohydrate, nucleotide, aminoacid and lipid metabolisms as well as translation, peptide hydrolysis, cell envelope/cell wall biosynthesis, adaption to atypical conditions and protein secretion. Some proteins related to carbohydrate and nucleotide metabolisms, translation and peptide degradation were overexpressed whereas those associated to stress conditions were synthesized in lower amounts. It seems that conditions for biofilm development would not imply a stressful environment for L. sakei CRL1862 cells, directing its growth strategy towards glycolytic flux regulation and reinforcing protein synthesis. In addition, L. sakei CRL1862 showed to harbor nine out of ten assayed genes involved in biofilm formation and protein anchoring. By applying qRT-PCR analysis, four of these genes showed to be up regulated, srtA2 being the most remarkable. The results of this study contribute to the knowledge of the physiology of L. sakei CRL1862 growing in biofilm on a characteristic food contact surface. The use of this strain as green biocide preventing L. monocytogenes post-processing contamination on industrial surfaces may be considered.

Metabolite profile of koji amazake and its lactic acid fermentation product by Lactobacillus sakei UONUMA.

The koji amazake is a traditional sweet Japanese beverage. It has been consumed for over a thousand years in Japan; nonetheless, little is yet known of the ingredients in koji amazake. Therefore, this study aimed to analyze the metabolites of koji amazake using a metabolomics approach. Additionally, we reformed the flavor of koji amazake by lactic acid fermentation (LAF-amazake) using Lactobacillus sakei UONUMA, which was isolated from snow caverns. The purpose of this article is to identify the ingredients in these beverages. In LAF-amazake and koji amazake, sugars, amino acids, organic acids, and vitamin B complex were determined in the two beverages, and over 300 compounds were detected in total. Thirteen saccharides were identified including two unknown trisaccharides, and there were no differences in these between the two beverages. In LAF-amazake, lactic acid, vitamin B2 (riboflavin), B3 (nicotinic acid and nicotinamide), and B6 (pyridoxine) were significantly increased as compared to koji amazake, whereas malate and glutamine decreased. These results suggested that LAF, malolactic fermentation, and glutamine deamidation occurred simultaneously in LAF-amazake. L. sakei UONUMA strains produced these vitamins. Moreover, it was surprising that acetylcholine, a well-known neurotransmitter, was newly generated in LAF-amazake. Here, we have succeeded in reforming the flavor of koji amazake and obtained these metabolic data on the two beverages. The present study could provide useful basic information for promoting functional analyses of koji amazake and LAF-amazake for human health.

Isolation and identification of a novel bacterium, Lactobacillus sakei subsp. dgh strain 5, and optimization of growth condition for highest antagonistic activity.

In the present study, we isolated Lactobacillus sakei strain DGH5 from raw beef meat. This bacterium plays an inhibitory effect against food-spoiling bacteria and food-borne pathogens, including Listeria monocytogenes, a gram-positive and pathogenic bacterium. Lactobacillus sakei strain DGH5 was identified through both phenotypical and biochemical tests accompanied with 16S rRNA sequence analysis. Among all the sources of carbon, nitrogen and phosphorous forms, we selected the most potent compounds to optimize the condition for the highest antagonistic activity. Among the sugars, polygalacturonic acid demonstrated to improve the antagonistic activity. Ammonium nitrate demonstrated to be suitable nitrogen sources. Amongst phosphorous sources, disodium hydrogen phosphate had the greatest antagonistic effect. According to Taguchi's orthogonal array, temperature, disodium hydrogen phosphate and soy Peptone had significant effect on antagonistic activity. Furthermore, mean comparisons showed that the optimum conditions achieved at pH 6.0, 25 °C temperature, 1.5% (w/v) Na2HPO4 and 0.5% (w/v) peptone.

Detection of different microenvironments and Lactobacillus sakei biotypes in Ventricina, a traditional fermented sausage from central Italy.

The present study evaluated the physico-chemical and microbiological features of Ventricina, considering for the first time the presence of different compartments deriving from the technology of production. In fact meat pieces (pork muscle and fat cut into cubes of about 10-20cm3), mixed with other ingredients and then stuffed into pig bladder, are still distinguishable at the end of the ripening. They appear delimited on the outside by the casing and inside by thin layers consisting of spices (mainly red pepper powder), salt and meat juices. Our results showed that the exterior (portion of the product in contact with the casing), the interstice (area between the different cubes of meat or fat) and the heart (the inner portion of meat cubes) had distinctive values of pH and aw, and a typical microbial progression, so that they can be considered as different ecological niches, here called microenvironments. The study of lactic acid bacteria population, performed with PCR-DGGE and sequence analysis targeting the V1-V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Lactobacillus sakei, Lb. plantarum, Weissella hellenica and Leuconostoc mesenteroides. The RAPD-PCR analysis performed on Lb. sakei, recognised as the predominant species, allowed the differentiation into three biotypes, with that characterised by the highest acidifying and proteolytic activities and the highest ability to grow in the presence of sodium chloride prevailing. This leading biotype, detectable in the interstice during the entire ripening period, was isolated in the microenvironments exterior and heart starting from the 30th d of ripening, and it was the sole biotype present at the end of the ripening. The analysis of microenvironments through the scanning electron microscopy (SEM) evidenced the presence of micro-channels, which could favour the microbial flow from the interstice to the exterior and the heart. Moreover, the SEM analysis allowed the detection of biofilms, recognised as responsible for the correct colonisation of the different meat niches.