Karthiyaini Damodharan

Streptomyces sp. strain PGPA39 alleviates salt stress and promotes growth of ‘Micro Tom’ tomato plants

To identify an actinobacterial strain that can promote growth and alleviate salinity stress in tomato plants.

Genetic and functional characterization of culturable plant-beneficial actinobacteria associated with yam rhizosphere

Actinobacteria were isolated from the rhizosphere of yam plants from agricultural fields from Yeoju, South Korea and analyzed for their genetic and plant‐beneficial functional diversity. A total of 29 highly occurring actinobacterial isolates from the yam rhizosphere were screened for various plant‐beneficial traits such as antimicrobial activity on fungi and bacteria; biocontrol traits such as production of siderophore, protease, chitinase, endo‐cellulase, and β‐glucanase. The isolates were also screened for plant growth‐promoting (PGP) traits such as auxin production, phosphate solubilization, 1‐aminocyclopropane‐1‐carboxylic acid (ACC) deaminase activity, and in vitro Arabidopsis growth promotion. 16S rDNA sequence‐based phylogenetic analysis was carried out on the actinobacterial isolates to determine their genetic relatedness to known actinobacteria. BOX‐PCR analysis revealed high genetic diversity among the isolates. Several isolates were identified to belong to the genus Streptomyces and a few to Kitasatospora. The actinobacterial strains exhibited high diversity in their functionality and were identified as novel and promising candidates for future development into biocontrol and PGP agents.

Preliminary probiotic and technological characterization of Pediococcus pentosaceus strain KID7 and in vivo assessment of its cholesterol-lowering activity

The study was aimed to characterize the probiotic properties of a Pediococcus pentosaceus strain, KID7, by in vitro and in vivo studies. The strain possessed tolerance to oro-gastrointestinal transit, adherence to the Caco-2 cell line, and antimicrobial activity. KID7 exhibited bile salt hydrolase activity and cholesterol-lowering activity, in vitro. In vivo cholesterol-lowering activity of KID7 was studied using atherogenic diet-fed hypercholesterolemic mice. The experimental animals (C57BL/6J mice) were divided into 4 groups viz., normal diet-fed group (NCD), atherogenic diet-fed group (HCD), atherogenic diet- and KID7-fed group (HCD-KID7), and atherogenic diet- and Lactobacillus acidophilus ATCC 43121-fed group (HCD-L.ac) as positive control. Serum total cholesterol (T-CHO) level was significantly decreased by 19.8% in the HCD-KID7 group (P < 0.05), but not in the HCD-L.ac group compared with the HCD group. LDL cholesterol levels in both HCD-KID7 and HCD-L.ac groups were decreased by 35.5 and 38.7%, respectively, compared with HCD group (both, P < 0.05). Glutamyl pyruvic transaminase (GPT) level was significantly lower in the HCD-KID7 and HCD-L.ac groups compared to HCD group and was equivalent to that of the NCD group. Liver T-CHO levels in the HCD-KID7 group were reduced significantly compared with the HCD group (P < 0.05) but not in the HCD-L.ac group. Analysis of expression of genes associated with lipid metabolism in liver showed that low-density lipoprotein receptor (LDLR), cholesterol-7α-hydroxylase (CYP7A1) and apolipoprotein E (APOE) mRNA expression was significantly increase in the HCD-KID7 group compared to the HCD group. Furthermore, KID7 exhibited desired viability under freeze-drying and subsequent storage conditions with a combination of skim milk and galactomannan. P. pentosaceus KID7 could be a potential probiotic strain, which can be used to develop cholesterol-lowering functional food after appropriate human clinical trials.

In vitro probiotic characterization of Lactobacillus strains from fermented radish and their anti-adherence activity against enteric pathogens

In this study, we evaluated the probiotic properties of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus fermentum strains isolated from fermented radish. All the strains survived the simulated oro-gastrointestinal transit condition and showed significantly higher adherence to Caco-2 cells compared with the probiotic strain Lactobacillus rhamnosus GG. The strains showed broad-spectrum antimicrobial activity, autoaggregation, and coaggregation capacity with pathogens. Furthermore, the Lactobacillus strains inhibited the adherence of Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Salmonella choleraesuis to the Caco-2 cell line. The strains possessed bile salt hydrolase activity and their cholesterol-lowering activity in vitro was above 50% in the presence of bile. Strains of L. plantarum and L. pentosus possessed the plantaricin-encoding plnEF gene. In addition, the Lactobacillus strains maintained about 80% cell viability after freeze-drying in the presence of a combination of 5% skim milk and 5% maltodextrin as cryoprotectant, and 70% recovery of cell viability was observed in the absence of any cryoprotectant.

Functional Probiotic Characterization and In Vivo Cholesterol-Lowering Activity of Lactobacillus helveticus Isolated from Fermented Cow Milk.

We characterized the probiotic properties of Lactobacillus helveticus strains KII13 and KHI1 isolated from fermented cow milk by in vitro and in vivo studies. The strains exhibited tolerance to simulated orogastrointestinal condition, adherence to Caco-2 cells, and antimicrobial activity. Both L. helveticus strains produced bioactive tripeptides, isoleucylprolyl-proline and valyl-prolyl-proline, during fermentation of milk. KII13 showed higher in vitro cholesterol-lowering activity (47%) compared with KHI1 (28%) and L. helveticus ATCC 15009 (22%), and hence, it was selected for in vivo study of cholesterol-lowering activity in atherogenic diet-fed hypercholesterolemic mice. For the study, mice were divided into four groups (viz., normal diet control group, atherogenic diet control group (HCD), KII13- atherogenic diet group (HCD-KII13), and Lactobacillus acidophilus ATCC 43121-atherogenic diet group (HCD-L.ac) as positive control). The serum total cholesterol level was significantly decreased by 8.6% and 7.78% in the HCD-KII13 and HCD-L.ac groups (p < 0.05), respectively, compared with the HCD group. Low-density lipoprotein cholesterol levels in both HCD-KII13 and HCD-L.ac groups were decreased by 13% and 11%, respectively, compared with the HCD group (both, p < 0.05). Analysis of cholesterol metabolism-related gene expression in mice liver showed increased expression of LDLR and SREBF2 genes in mice fed with KII13. By comparing all the results, we conclude that L. helveticus KII13 could be used as a potential probiotic strain to produce antihypertensive peptides and reduce serum cholesterol.

Enrichment of ginsenoside Rd in Panax ginseng extract with combination of enzyme treatment and high hydrostatic pressure

Ginsenoside Rd, a minor ginseng saponin, has several pharmacological activities. Traditionally, saponins are extracted using organic solvents or hot water extraction. However, both of these methods have disadvantages such as formation of artefacts and compound decomposition. Additionally, the use of organic solvents for extraction is hazardous to the environment. Therefore, we aimed to produce ginsenoside Rd without using organic solvents or hot extraction. We developed a simultaneous extraction and transformation process for higher yields of ginsenoside Rd using a combination of high hydrostatic pressure (HHP) and enzymes. Several commercial glycosidases in various combinations were studied for the enrichment of ginsenoside Rd from major ginsenosides by enzymatic transformation and HHP. We found that treatment with a combination of cellulase (2 U/mL), cellobiase (4 U/mL) and HHP of 100 Mpa at pH 4.8 and 45°C for 24 h resulted in a ginsenoside Rd content of 3.47 ± 0.35 mg/g of fresh ginseng. This yield is 2.1-fold higher than that of the corresponding enzyme treatment at atmospheric pressure (AP, 0.1 Mpa) at pH 4.8, 45°C and for 24 h. This simultaneous extraction and transformation process can be used for the preparation of Rd enriched ginseng beverage without using hazardous organic solvents.

Co-encapsulation of lactic acid bacteria and prebiotic with alginate-fenugreek gum-locust bean gum matrix: Viability of encapsulated bacteria under simulated gastrointestinal condition and during storage time

The aim of this study was to enhance the viability of probiotic strains Pediococcus pentosaceus KID7, Lactobacillus plantarum KII2, Lactobacillus fermentum KLAB6 and Lactobacillus helveticus KII13 in gastrointestinal transit, freeze-drying condition and during storage time by microencapsulation using a combination of alginate, fenugreek gum and locust bean gum. The microcapsules were prepared using various ratio of alginate to fenugreek gum to locust bean gum and tested for its dissolution in colonic fluid. The combination that efficiently dissolved in colonic fluid was selected for co-encapsulation of the probiotic strains and prebiotics to produce synbiotic microcapsules. Further, we observed that the bacteria encapsulated with alginate-fenugreek gum-locust bean gum (AFL) matrix tolerated gastrointestinal condition efficiently compared to non-encapsulated bacteria. The encapsulated bacterial cells retained higher viability than non-encapsulated cells during freeze-drying condition and subsequent storage for 3 months at 4°C. These results show the utility of AFL matrix in microencapsulation of probiotics for use in food industry.

Fermentative transformation of ginsenoside Rb1 from Panax ginseng C. A. Meyer to Rg3 and Rh2 by Lactobacillus paracasei subsp. tolerans MJM60396

Lactic acid bacteria (LAB) were screened for ginsenoside transforming activity using crude ginseng extract. Thin-layer chromatography analysis of fermented ginseng extract showed that LAB strain MJM60396 possessed higher ginsenoside transformation ability than other strains. It converted major ginsenosides into minor ginsenosides such as Rg3 and Rh2. MJM60396 also showed high β-glucosidase activity. Strain MJM60396 was identified as Lactobacillus paracasei subsp. tolerans based on 16S rRNA gene sequence. To delineate the pathway involved in the production of the minor ginsenosides Rg3 and Rh2, strain MJM60396 was incubated with pure ginsenoside Rb1. HPLC analysis revealed the appearance of Rg3 and Rh2 peak from the incubation mixture containing Rb1 and strain MJM60396. Furthermore, β-glucosidase enzyme was prepared from strain MJM60396. To achieve its maximum activity, we optimized the pH and temperature conditions. Cell-free β-glucosidase enzyme hydrolyzed ginsenoside Rb1 through the following pathway: ginsenoside Rb1 → Rd → Rg3 → Rh2. This is the first report on the transformation of ginsenosides Rb1 to Rg3 and Rh2 by a Lac. paracasei subsp. tolerans strain. Our results indicate that Lac. paracasei subsp. tolerans MJM60396 has the potential to be used for preparing ginsenosides Rg3 and Rh2 as nutraceuticals.

Functional Characterization of an Exopolysaccharide Produced by Bacillus sonorensis MJM60135 Isolated from Ganjang

The present study focused on the production, characterization, and in vitro prebiotic evaluation of an exopolysaccharides (EPS) from Bacillus sonorensis MJM60135 isolated from ganjang (fermented soy sauce). Strain MJM60135 showed the highest production (8.4 ± 0.8 g/l) of EPSs compared with other isolates that were screened for EPS production based on ropy culture morphology. Furthermore, MJM60135 was cultured in 5 L of medium and the EPS was extracted by ethanol precipitation. The emulsification activity of the EPS was higher in toluene than in o-xylene. Fourier transform infrared spectroscopy analysis showed the presence of hydroxyl and carboxyl groups and glycosidic linkages. The isolated EPS contained mannose and glucose, as observed by thin-layer chromatography analysis of the EPS hydrolysate. Lactic acid bacteria (LAB) and pathogenic E. coli K99 and Salmonella enterica serovar Typhimurium were tested for their growth utilizing the EPS from B. sonorensis MJM60135 as the sole carbon source for its possible use as a prebiotic. All the tested LAB exhibited growth in the EPS-supplied medium compared with glucose as carbon source, whereas the pathogenic strains did not grow in the EPS-supplied medium. These findings indicate that the EPS from B. sonorensis MJM60135 has potential application in the bioremediation of hydrocarbons and could also be used as a prebiotic.

Streptomyces sp. strain SK68, isolated from peanut rhizosphere, promotes growth and alleviates salt stress in tomato (Solanum lycopersicum cv. Micro-Tom)

A novel actinobacterium, strain SK68, was isolated from the rhizosphere of peanut plant and its salinity stress alleviation ability was studied using tomato (Solanum lycopersicum cv. Micro-Tom) plants. Based on 16S rDNA based phylogenetic analysis, strain SK68 has been identified as a Streptomyces sp. Strain SK68 had branched substrate mycelium bearing smooth surfaced spores and the spore colour is brownish grey on ISP4 medium. It exhibited enzyme activities such as xylanase, cellulase, amylase, and pectinase and degraded hypoxanthine, casein, and L-tyrosine. The strain SK68 differed in its banding pattern in BOX-PCR and RAPD fingerprinting compared to the closely matching type strains Streptomyces erythrochromogenes NBRC 3304T (AB184746), S. flavotricini NBRC 12770T (AB184132), S. racemochromogenes NBRC 12906T (AB184235), and S. polychromogenes NBRC 13072T (NR041109). Strain SK68 was evaluated for its salinity stress-alleviating activity in tomato plants with 180 mmol/L NaCl under gnotobiotic condition. A significant increase in plant biomass was observed in strain SK68-inoculated tomato plants under salt stress compared to control and salt-stressed non-inoculated plants.