Diapolycopenedioic-acid-diglucosyl ester and keto-myxocoxanthin glucoside ester: Novel carotenoids derived from Exiguobacterium acetylicum S01 and evaluation of their anticancer and anti-inflammatory activities.

Inflammation is pivotal for the development of gastrointestinal cancer and linked to poor survival and limited therapeutic options. In this study, six structurally different carotenoids were isolated and identified from the methanolic extract of Exiguobacterium acetylicum S01 namely lycopene (Car-I), diapolycopenedioic-acid-diglucosyl-ester (Car-II), ß-carotene (Car-III), zeaxanthin (Car-IV), astaxanthin (Car-V), and keto-myxocoxanthin glucoside-ester (Car-VI). Further, their anti-cancer, anti-inflammatory, and antioxidant potentials were evaluated. The MTT assay was used to determine the effect of carotenoids on viability of colorectal cancer (HT-29) as well as peripheral blood mononuclear cells (PBMCs). Results revealed that all the six carotenoids were demonstrated a significant inhibition of HT-29 cells viability in a dose-dependent manner whereas there was no cytotoxic effect in PBMCs. The study also recorded that six carotenoids considerably inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-α), and lipid peroxidation in PBMCs. Moreover, antioxidant potentials of Car-II and Car-VI were significantly (p = 0.001) higher than ascorbic acid as determined by a 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. Therefore, our results ascertained the role of carotenoids derived from E. acetylicum S01 in developing potential therapeutic agents for inflammation-associated cancer.

Modulation of innate immunity, expression of cytokine genes and disease resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus) by dietary supplementation with Exiguobacterium acetylicum S01.

Probiotic strains play an increasing role in the production of healthy animals used as a food source. Elucidating the mechanisms of action that allow probiotic-driven immunomodulation may facilitate different applications such as the prevention of infectious diseases in food organisms. This study elucidates the probiotic effects of Exiguobacterium acetylicum S01 on the growth, haematological profile, innate immune capacity, expression of cytokine genes, and resistance to diseases of Carassius auratus caused by Aeromonas hydrophila infection. Three fish groups were fed with the following diets containing different doses of E. acetylicum S01 (CFU g-1): basal diet 0 (BD, without probiotic), 2.5 × 107 (DI) and 2.7 × 109 (DII)-CFU g-1 for 4 weeks. After 4 weeks, the fish were injected intraperitoneally with A. hydrophila and the percentage of survival was recorded over 21 days of post-challenge. Results revealed that dietary supplementation of E. acetylicum S01 significantly (P < 0.05) enhanced the growth, haematological profile and cellular immune responses including respiratory burst, phagocytic activities and antimicrobial enzymes (myeloperoxidase and lysozyme) and total immunoglobulin levels were improved by probiotic feeding at both occasions. Comparatively, expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGFß) was up-regulated in kidney, head-kidney and spleen. Moreover, fish fed with diet DII had a significantly higher (P < 0.05) survival rate (73.2%) after challenging. The survival rate was only 33.2% of the BD group against A. hydrophila infection. Our results revealed that E. acetylicum S01 delivered probiotic in feed exerts its influence on growth performance and provides disease resistance by stimulating the immune system at the cellular and molecular levels in C. auratus.

Gene cloning and enzymatic characterization of alkali-tolerant type I pullulanase from Exiguobacterium acetylicum.

UNLABELLED: A pullulanase gene (Pul3YH5) of 2568 bp, which encodes a protein containing 855 amino acid residues, was cloned from the alkaliphilic bacterium Exiguobacterium acetylicum YH5. The pullulanase (Pul3YH5) contains the YNWGYDP motif of type I pullulanase as well as four conserved glycoside hydrolase sequences of the GH13 (α-amylase) family. When the pullulanase gene was cloned and expressed in Escherichia coli BL21 (DE3) plysS, the recombinant pullulanase had a molecular mass of ˜100·0 kDa. It was optimally active at 50°C and pH 6·0, alkali-tolerant and displayed excellent stability (>93·0%) over a broad pH range (4·0-10·0) when incubated for 30 min without substrate. The enzyme activity of Pul3YH5 was significantly enhanced in the presence of Co(2+) , Fe(2+) and Mn(2+) and was inhibited by Cu(2+) , SDS, ß-mercaptoethanol and EDTA. The enzyme displayed the highest specificity for pullulan (Km  = 0·12 ± 0·02 mg ml(-1) ), followed by soluble starch (Km  = 0·69 ± 0·04 mg ml(-1) ). Substrate hydrolysis demonstrated that pullulanase from E. acetylicum liberated maltotriose from pullulan, although hydrolytic activity was also detected with soluble starch, amylopectin, ß-limited dextrin and glycogen. These enzymatic properties indicate that Pul3YH5 is alkali-tolerant pullulanase and that Pul3YH5 could be useful in the detergent industry. SIGNIFICANCE AND IMPACT OF THE STUDY: Pullulanases have great potential in various industries, ranging from food (high fructose and glucose syrups) to washing detergent industries. In this study, the gene encoding the novel pullulanase from E. acetylicum YH5 was cloned and sequenced, then expressed in E. coli. The properties of the recombinant enzyme in E. coli were also determined. The pullulanase from E. acetylicum YH5 is alkaline tolerant and has a high optimum temperature, making it a candidate for applications in the detergent industry.

Effects of Dietary Multi-Strain Probiotics on Growth Performance, Antioxidant Status, Immune Response, and Intestinal Microbiota of Hybrid Groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂).

This study aims to examine the effects of the mixture of Bacillus cereus G1-11 and Exiguobacterium acetylicum G1-33, isolated from the gut of hybrid groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), on the host. The hybrid groupers were divided into a control (C, without any probiotics), B. cereus (BC, 1010 cfu/g), E. acetylicum (EA, 108 cfu/g), compound (mix, a 1:1 mixture of B. cereus and E. acetylicum), and positive reference group (P, Lactobacillus acidophilus, 5 × 108 cfu/L). Each group had four replicates, with 30 fish per replicate (53.30 ± 0.50 g), and were fed for 60 days. The results showed that adding probiotics to the feed significantly improved the weight gain, weight growth rate, specific growth rate, and digestive enzyme activities of hybrid groupers compared to the C group. The compound group was the most significant. In addition, composite probiotics added to feed significantly upregulated the expression levels of several growth-related genes in the liver and muscles. The activities of alkaline phosphatase, catalase, glutathione peroxidase, glutathione transferase, lysozyme, and total antioxidant capacity in the serum and liver were significantly influenced through mixed probiotic feeding. Moreover, the expression levels of several immune-related genes in the liver, spleen, and head kidney were significantly enhanced by adding single and mixed probiotics to feed, with the synergy of mixed probiotics being the best. An analysis of the gut microbiota showed that adding composite bacteria enhanced the richness and diversity of the gut microbiota, significantly increasing the relative abundance of potential probiotics (Cetobacterium and Microbacterium) while decreasing the presence of potential pathogens (Mycoplasma). Overall, our findings highlighted the efficacy of mixed probiotics (B. cereus and E. acetylicum) in enhancing growth performance, nutritional value of hybrid grouper feed, antioxidant capacity, immune response, and intestinal health, in finding the best combination of functional feed additives.

Growth promotion of wheat seedlings by Exiguobacterium acetylicum 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas.

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 µg of P ml(-1) day(-1)), and produced IAA (14.9 µg ml(-1) day(-1)) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.

ß-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization.

The main goal of this work was to evaluate the performance of ß-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-ß-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-ß-d-glucopyranoside, butyl- and pentyl-ß-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 ß-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary ß-galactosidase is used) can be avoided.