Probiogenomic In-Silico Analysis and Safety Assessment of Lactiplantibacillus plantarum DJF10 Strain Isolated from Korean Raw Milk.

The whole genome sequence of Lactiplantibacillus plantarum DJF10, isolated from Korean raw milk, is reported, along with its genomic analysis of probiotics and safety features. The genome consists of 29 contigs with a total length of 3,385,113 bp and a GC content of 44.3%. The average nucleotide identity and whole genome phylogenetic analysis showed the strain belongs to Lactiplantibacillus plantarum with 99% identity. Genome annotation using Prokka predicted a total of 3235 genes, including 3168 protein-coding sequences (CDS), 59 tRNAs, 7 rRNAs and 1 tmRNA. The functional annotation results by EggNOG and KEGG showed a high number of genes associated with genetic information and processing, transport and metabolism, suggesting the strain's ability to adapt to several environments. Various genes conferring probiotic characteristics, including genes related to stress adaptation to the gastrointestinal tract, biosynthesis of vitamins, cell adhesion and production of bacteriocins, were identified. The CAZyme analysis detected 98 genes distributed under five CAZymes classes. In addition, several genes encoding carbohydrate transport and metabolism were identified. The genome also revealed the presence of insertion sequences, genomic islands, phage regions, CRISPR-cas regions, and the absence of virulence and toxin genes. However, the presence of hemolysin and antibiotic-resistance-related genes detected in the KEGG search needs further experimental validation to confirm the safety of the strain. The presence of two bacteriocin clusters, sactipeptide and plantaricin J, as detected by the BAGEL 4 webserver, confer the higher antimicrobial potential of DJF10. Altogether, the analyses in this study performed highlight this strain's functional characteristics. However, further in vitro and in vivo studies are required on the safety assurance and potential application of L. plantarum DJF10 as a probiotic agent.

Bacterial diversity of traditional fermented food, Idli by high thorough-put sequencing.

The bacterial composition of naturally fermented Indian food, Idli was studied by high-throughput Illumina amplicon sequencing at different taxonomic levels. Metagenomic investigation revealed fold change with respect to some of the phylotypes in 06th to 12th h of fermentation, suggesting the synergistic mode of nutrition. After 12th h fermentation, bacterial populations were stabilized towards 15th h fermentation. The bacterial phyla found as Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and others in various proportions with respective to fermentation time. Among these Firmicutes and Proteobacteria were the predominant bacterial associates in this product. Initially at 0th h time interval Firmicutes (7%) and Proteobacteria (93%) were present adequately in the product which has been changed to Firmicutes (68%), Proteobacteria (31%) at the end of the fermentation (15th h). Phylum Firmicutes represented various major genus such as Lactococcus, Weissella, Lactobacillus, Enterococcus, Bacillus and Macrococcus whereas Proteobacteria revealed the presence of Enterobacter, Erwinia, Serratia, Pseudoalteromonas, Vibrio and Klebsiella genus. Co-occurrence and Co-exclusion network were developed to ensure the positive and negative association in the eubacterial genus detected in entire batter fermentation event. Some genera like Weissella, Lactococcus and Enterococcus are showing increase in abundances in auxiliary succession events can be established for starter culture development. Supplementary material: The online version contains supplementary material available at 10.1007/s13197-022-05421-4.

Oxidative stress alleviating potential of galactan exopolysaccharide from Weissella confusa KR780676 in yeast model system.

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.

Rapid Discrimination and Authentication of Korean Farmstead Mozzarella Cheese through MALDI-TOF and Multivariate Statistical Analysis.

Geographical origin and authenticity are the two crucial factors that propel overall cheese perception in terms of quality and price; therefore, they are of great importance to consumers and commercial cheese producers. Herein, we demonstrate a rapid, accurate method for discrimination of domestic and import mozzarella cheeses in the Republic of Korea by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The protein profiles' data aided by multivariate statistical analysis successfully differentiated farmstead and import mozzarella cheeses according to their geographical location of origin. A similar investigation within domestic samples (farmsteads/companies) also showed clear discrimination regarding the producer. Using the biomarker discovery tool, we identified seven distinct proteins, of which two (m/z 7407.8 and 11,416.6) were specific in farmstead cheeses, acting as potential markers to ensure authentication and traceability. The outcome of this study can be a good resource in building a database for Korean domestic cheeses. This study also emphasizes the combined utility of MALDI-TOF MS and multivariate analysis in preventing fraudulent practices, thereby ensuring market protection for Korean farmstead cheeses.

Quantitative Analysis of Biogenic Amines in Different Cheese Varieties Obtained from the Korean Domestic and Retail Markets.

To evaluate the safety and risk assessment of cheese consumption in the Republic of Korea, sixty cheese samples purchased from the farmstead and retails markets (imported) were analyzed for their biogenic amine (BA) contents. The BA profiles and quantities of eight amines (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine) were determined using high-performance liquid chromatography (HPLC). Spermine was the only amine detectable in all the samples. The BAs of fresh cheeses from both farmstead and retail markets were mostly undetectable, and comparatively at lower levels (<125 mg/kg) than ripened samples. Putrescine was undetectable in all the domestic ripened cheeses. The sum of BA levels in the imported ripened cheeses of Pecorino Romano (1889.75 mg/kg) and Grana Padano (1237.80 mg/kg) exceeds >1000 mg/kg, of which histamine accounts nearly 86 and 77% of the total levels, respectively. The tolerable limits of the potential toxic amines, histamine and tyramine surpassed in four and three imported ripened samples, respectively. Furthermore, the presence of potentiators (putrescine and cadaverine) together in samples even with a lower level of toxic amines alarms the risk in consumption. Therefore, adoption of strict hygienic practices during the entire chain of cheese production, along with obligatory monitoring and regulation of BA in cheeses seems to be mandatory to ensure the safety of the consumers.

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities.

In this study, the 1H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that 1H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.

Report on aflatoxin-binding activity of galactan exopolysaccharide produced by Weissella confusa KR780676.

Galactan exopolysaccharide (EPS) produced by Weissella confusa KR780676 isolated from an Indian traditional fermented food has been reported earlier. In this manuscript, we have studied aflatoxin-binding ability of this galactan EPS. Aflatoxin B1 (AFB1) binding ability of galactan EPS was observed in an increasing trend with increasing EPS concentration (20-100 mg/mL). At lower concentrations (< 20 mg/mL) of EPS, the binding activity was undetectable, while notable binding was seen from 30 mg/mL. Enhanced AFB1 binding (32.40%) was recorded at 50 mg/mL of EPS and it increased gradually up to 34.79% at 100 mg/mL concentrations of EPS. The intensity of bands in high-performance thin-layer chromatography (HPTLC) analysis confirms the AFB1 binding efficiency of galactan EPS, which shows its potential application for removal of toxins in food and feed industry. Galactan EPS binding activity to AFB1 is further studied with particle size analysis (PSA). This is the first study reporting the aflatoxin-binding activity of any kind of EPS from lactic acid bacteria.

Characterisation of fungal contamination sources for use in quality management of cheese production farms in Korea.

OBJECTIVE: This study was conducted to determine the composition and diversity of the fungal flora at various control points in cheese ripening rooms of 10 dairy farms from six different provinces in the Republic of Korea. METHODS: Floor, wall, cheese board, room air, cheese rind and core were sampled from cheese ripening rooms of ten different dairy farms. The molds were enumerated using YM petrifilm, while isolation was done on yeast extract glucose chloramphenicol agar plates. Morphologically distinct isolates were identified using sequencing of internal transcribed spacer region. RESULTS: The fungal counts in 8 out of 10 dairy farms were out of acceptable range, as per hazard analysis critical control point regulation. A total of 986 fungal isolates identified and assigned to the phyla Ascomycota (14 genera) and Basidiomycota (3 genera). Of these Penicillium, Aspergillus, and Cladosporium were the most diverse and predominant. The cheese ripening rooms was overrepresented in 9 farms by Penicillium (76%), while Aspergillusin a single farm. Among 39 species, the prominent members were Penicillium commune, P. oxalicum, P. echinulatum, and Aspergillus versicolor. Most of the mold species detected on surfaces were the same found in the indoor air of cheese ripening rooms. CONCLUSION: The environment of cheese ripening rooms persuades a favourable niche for mold growth. The fungal diversity in the dairy farms were greatly influenced by several factors (exterior atmosphere, working personnel etc.,) and their proportion varied from one to another. Proper management of hygienic and production practices and air filtration system would be effective to eradicate contamination in cheese processing industries.

Evaluation of functional properties of potential probiotic isolates from fermented brine pickle.

Fermented foods have been consumed for centuries in various parts of the world and are known to be rich resources of functionally important microorganisms. This study documents the antioxidative, anticancer and enzyme-inhibiting properties of potential probiotic Bacillus strains isolated from fermented brine mango pickle. Antioxidant activity was determined through in-vitro assays namely, DPPH•, ABTS•+, hydroxyl radical scavenging ability, reducing activity, superoxide anion scavenging ability, linoleic acid and plasma lipid peroxidation ability. Both intact cells (IC) and intracellular cell-free extracts (CFE) from most of the strains exhibited prominent antioxidant activity. Likewise, CFE and intracellular cell-free supernatants (CFS) exhibited potential inhibitory activities towards α-amylase, α-glucosidase and tyrosinase. Interestingly, CFS and crude ethyl acetate extracts of PUFSTP35 (Bacillus licheniformis KT921419) displayed strong anticancer activity against HT-29 colon cancer cell line. Hence, these probiotic strains have been showed to exhibit unique functional properties and could be further commercially exploited.

Safety and Stability Assessment of Potential Probiotic Strains from Fermented Mango Brine Pickle.

Fermented foods are known to be rich source of valuable nutrients and probiotics. Previously, our study reported the isolation and characterization of eight potential probiotic strains from traditional mango brine pickle, which has been conventionally consumed for ages in raw form in Southern India. The present study reports on the safety assessment of these strains for the selection of prospective probiotic candidates. Hydrogen peroxide production, histidine decarboxylase activity (production of histamine), DNase activity, and presence of the virulence factor genes (assessed by PCR) were carried out to evaluate its safety. Bacillus licheniformis KT921419 and B. methylotrophicus KT921422 was found to show no adverse safety characteristics. These two strains were further assessed for their ability to survive in the native substrate (mango brine pickle) as single and mixed inoculums. Above strains maintained significant viability in mango brine pickle for a period of 6 months during storage at the room temperature. Results clearly proved the safety and stability of two of the potential probiotic strains, which can be further utilized in food applications under harsh conditions of high salt, low pH, and room temperature making these strains unique.

Effect of γ-irradiation on physico-chemical and antioxidant properties of galactan exopolysaccharide from Weissella confusa KR780676.

Exopolysaccharide (EPS) produced by Weissella confusa KR780676 from an Indian fermented food (Idli) has been characterized as galactan in earlier report. In this study, the galactan EPS was irradiated at different doses (0, 4, 8, and 16 kGy) using 60Co as γ-source and effect of γ-irradiation on physico-chemical and antioxidant potential of galactan EPS was studied. Color and flow properties showed moderate change after the γ-irradiation treatment. Particle size analysis and scanning electron microscopy exhibited significant breakdown of the EPS particles into smaller average hydrodynamic diameter size from 495.54 to 198.44 nm with increasing irradiation doses. Overall, pH showed decrease, while the water activity showed an increasing trend with the increasing irradiation dose. No significant effect was observed in the functional groups of EPS as revealed in the FT-IR spectroscopic analysis. After the treatment, semi-crystalline nature of the EPS changed to amorphous as shown in XRD analysis due to the breakdown. Antioxidant activities of galactan EPS increased marginally after the treatment. However, hydroxyl radical scavenging activity increased significantly from 48 to 67%. Changes in physico-chemical and antioxidant properties of galactan EPS with γ-irradiation treatment altered the functional properties of EPS positively, making it more suitable for wider application in the food industry.

Production, partial characterization and antioxidant properties of exopolysaccharide α-d-glucan produced by Leuconostoc lactis KC117496 isolated from an idli batter.

Exopolysaccharide (EPS) produced from Leuconostoc lactis KC117496 isolated from the naturally fermented idli batter has been reported earlier. Here, study aimed to optimize the carbon source to enhance the yield of EPS production, partial characterization and antioxidant activity of α-d-glucan EPS. Among different disaccharides (sucrose, maltose, lactose) and monosaccharides (glucose, galactose and fructose), combination of sucrose and glucose at 2% showed highest EPS production of 4.55 g/L in MRS medium. The molecular weight was identified as ~ 4.428 × 103 kDa with MALDI-TOF mass spectroscopy. 1D and 2D NMR results exhibited the presence of α-1-6 and α-1-3 linked glucose revealed EPS as a glucan. Antioxidant properties of glucan (500 µg/mL) revealed the significant oxidation alleviation potential such as DPPH (74%) and Hydroxyl radical activity (97.8%), whereas metal chelating activity (70%) was lower as compare to control standard. These characteristics of glucan EPS reveals its potential application in food and pharmaceutical industry.

Bioprospecting thermophilic glycosyl hydrolases, from hot springs of Himachal Pradesh, for biomass valorization.

The harnessing of biocatalysts from extreme environment hot spring niche for biomass conversion is significant and promising owing to the special characteristics of extremozymes attributed by intriguing biogeochemistry and extreme conditions of these environments. Hence, in the present study 38 bacterial isolates obtained from hot springs of Manikaran (~ 95 °C), Kalath (~ 50 °C) and Vasist (~ 65 °C) of Himachal Pradesh were screened for glycosyl hydrolases by in situ enrichment technique using lignocellulosic biomass (LCB). Based on their hydrolytic potential 5 isolates were selected and they were Bacillus tequilensis (VCB1, VCB2 and VSDB4), and B. licheniformis (KBFB2 and KBFB3). Cellulolytic activity assayed by growth under submerged fermentation showed that B. tequilensis VCB1 had maximum FPA activity (3.38 IU ml-1) in 48 h, while B. licheniformis KBFB3 excelled for endoglucanase (EGA of 4.81 IU ml-1 in 24 h) and cellobiase (0.71 IU ml-1 in 48 h) activities. Among all the thermophilic biocatalysts evaluated, highest exoglucanase (0.06 IU ml-1) activity was observed in B. tequilensis VSDB4 while endoglucanase of B. licheniformis KBFB3 showed optimum specific activity at pH 7 and 70 °C. Further, the presence of celS, celB and xlnB genes in the isolates suggest their possible role in biomass conversion. Protein profiling by SDS-PAGE analysis revealed that cellulase isoforms migrated with molecular masses of 75 kDa. The endoglucanase activity of promising strain B. licheniformis KBFB3 was enhanced in the presence of Ca2+, mercaptoethanol and sodium hypochlorite whereas moderately inhibited by Cu2+, Zn2+, urea, SDS and H2O2. The results of this study indicate scope for the possible development of novel biocatalysts with multifunctional thermostable glycosyl hydrolases from hot springs for efficient hydrolysis of the complex lignocellulosic biomass into simple sugars and other derived bioproducts leading to biomass valorization.

High Level Secretion of Laccase (LccH) from a Newly Isolated White-Rot Basidiomycete, Hexagonia hirta MSF2.

Newer and novel laccases attract considerable attention due to its promising and valuable multiple applications in biotech industry. This present investigation documents, for the first time, on high level extracellular secretion of laccase (LccH) in newly isolated wood-degrading basidiomycete Hexagonia hirta MSF2. LccH was optimally active at 40°C in citrate phosphate buffer with a pH of 3.4. Optimized Cu(2+) in glucose yeast extract (GY) medium enhanced the LccH production by H. hirta to 1944.44 U.ml(-1). A further increment in LccH activity of 5671.30 U.ml(-1) was achieved by the addition of a phenolic inducer, 2,5 Xylidine. Zymogram and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of LccH revealed that LccH is a monomer with a molecular mass of 66 kDa. MALDI-TOF-MS based peptide mass fingerprinting and comparative modeling of the amino acid sequence of LccH showed that it was closer to Trametes sp. AH28-2 (PDB: 3KW7) with 48% identity, 95% coverage, 0.011 alignment score and RMSD of 0.497Å. Crude LccH delignified lignocellulosic biomass such as wood and corncob, to a level of 28.6 and 16.5%, respectively. Such high level secretion, thermal and solvent stability of LccH make H. hirta a potential candidate not only for LccH production and biodelignification but also generation of lignin derived aromatic feed stock chemicals for industrial and environmental applications.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater.

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.

Aerobic cyanide degradation by bacterial isolates from cassava factory wastewater

Ten bacterial strains that utilize cyanide (CN) as a nitrogen source were isolated from cassava factory wastewater after enrichment in a liquid media containing sodium cyanide (1 mM) and glucose (0.2% w/v). The strains could tolerate and grow in cyanide concentrations of up to 5 mM. Increased cyanide levels in the media caused an extension of lag phase in the bacterial growth indicating that they need some period of acclimatisation. The rate of cyanide removal by the strains depends on the initial cyanide and glucose concentrations. When initial cyanide and glucose concentrations were increased up to 5 mM, cyanide removal rate increased up to 63 and 61 per cent by Bacillus pumilus and Pseudomonas putida. Metabolic products such as ammonia and formate were detected in culture supernatants, suggesting a direct hydrolytic pathway without an intermediate formamide. The study clearly demonstrates the potential of aerobic treatment with cyanide degrading bacteria for cyanide removal in cassava factory wastewaters.