Regulatory T cells (Tregs

Inflammation of the gastrointestinal tract is associated with reactive oxygen species (ROS) genesis. Alleviation of oxidative stress is achieved by using antioxidants and probiotics. Present study investigates a synergistic effect of the probiotic Escherichia coli CFR 16 containing Vitreoscilla haemoglobin gene (vgb), green fluorescent protein (gfp) gene and pyrroloquinoline quinone (pqq) gene cluster on oxidative stress induced by 1,2-dimethylhydrazine (DMH). Adult virgin Charles foster male rats (3-4 months) weighing 200-250 g were administered with DMH (25 mg/kg body weight, s.c.) twice a week for eight consecutive weeks. Rats receiving only DMH dose showed increased lipid peroxidation in liver and intestinal tissues with reduced activity of antioxidant enzymes, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Oral dose of E. coli CFR 16::vgb-gfp harbouring pqq gene cluster increased rat faecal PQQ concentration by twofold, reduced lipid peroxidation and retained SOD, CAT and GPx activities close to normal levels in liver and colonic tissues following DMH treatment. In addition, significant protection was found in colonic histological sections of these rat groups. This study demonstrates a protective efficacy in the following order: E. coli CFR 16 < E. coli CFR 16::vgb-gfp < vitamin C = PQQ < E. coli CFR 16::vgb-gfp (pqq).

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The present study describes the beneficial effects of potential probiotic E. coli 16 (pUC8:16gfp) expressing Vitreoscilla hemoglobin (vgb) gene, associated with bacterial respiration under microaerobic condition, on gastrointestinal (GI) colonization and its antioxidant activity on carbon tetrachloride (CCl4) induced toxicity in Charles Foster rats. In vitro, catalase activity in E. coli 16 (pUC8:16gfp) was 1.8 times higher compared to E. coli 16 (pUC-gfp) control. In vivo, E. coli 16 (pUC8:16gfp) not only was recovered in the fecal matter after 70 days of oral administration but also retained antibacterial activities, whereas E. coli 16 (pUC-gfp) was not detected. Oral administration of 200 and 500 μL/kg body weight of CCl4 to rats at weekly interval resulted in elevated serum glutamyl pyruvate transaminase (SGPT) and serum glutamyl oxalacetate transaminase (SGOT) levels compared to controls. Rats prefed with E. coli 16 (pUC8:16gfp) demonstrated near to normal levels for SGPT and SGOT, whereas the liver homogenate catalase activity was significantly increased compared to CCl4 treated rats. Thus, pUC8:16gfp plasmid encoding vgb improved the growth and GI tract colonization of E. coli 16. In addition, it also enhanced catalase activity in rats harboring E. coli 16 (pUC8:16gfp), thereby preventing the absorption of CCl4 to GI tract.

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Escherichia coli 16 has potential probiotic properties including antimicrobial activity due to extracellular secretion of colicins E1/1a1b. Inulosucrase (InuJ) enzyme catalyses the polymerization of a fructose moiety of sucrose leading to the formation of fructooligosaccharides. The present investigation compared the activity of InuJ enzymes cloned into pMAL-p2ΔlacI Q a deletion vector and transformed into E. coli 16 and standard strain that is, E. coli BL21. Specific activities of InuJ enzyme were estimated in supernatant, periplasm and lysate. Specific activities of InuJ activity in cell lysate were similar in E. coli 16 and E. coli BL21 without induction of tac promoter with isopropyl thio-β-Dgalactoside (IPTG). InuJ activity is mainly present in the periplasm of E. coli BL21 whereas in E. coli 16, most of the activity is in the supernatant. Superantant of E. coli 16 strain also showed good antibacterial activity due to colicin E1/Ia1b. Colicin E1/1a1b transport system could allow extracellular secretion of InuJ proteins in probiotic E. coli 16.Key words: Colicin, extracellular, E. coli, fructooligosaccharide, inulosucrase, prebiotic, probiotic. INTRODUCTIONPrebiotics are a category of nutraceutical product that has the ability to promote the growth of specific beneficial gut bacteria (Kelly, 2008). In 2007, Roberfroid defined prebiotics as "a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gut microflora that confers health benefit". Fructooligosaccharides (FOS) has been used as prebiotic and is considered as a functional food ingredient (Cherbut, 2002;Fanaro et al., 2005;Bouhnik et al., 2006;Roberfroid, 2007;Paineau et al., 2008). FOS is the common name for fructose oligomers that are mainly composed of 1-kestose (GFS2), 2-nystose (GF3) among others in which fructose units are bound at the β-2, 1 position of sucrose through the transfructosylating enzymes such as glucosyltransferases, fructosyltransferases and inulosucrase (Yun et al., 1996). Inulosucrase has been previously shown to be involved in the synthesis of FOS (Van Hijum et al., 2006).Escherichia coli, a Gram negative bacterium is widely used as a host strain for recombinant protein production in industry. Some natural E. coli strains secrete protein extracellulally but their mechanisms of secretion are not clearly understood, nor are they widely exploited for recombinant protein production and metabolic engineering (Ni and Chen, 2009). In some case, recombinant proteins directed to the periplasm were found in the medium but the process is not known (Choi and Lee, 2004;Mergulhao et al., 2005). Sommer et al. (2010) constructed a plasmid which contains bacteriocin release proteins (BRP) that allow secretion of recombinant protein from the periplasm into the culture medium (Sommer et al., 2010). It was known that BRP or lysis proteins are responsible for the release of colicins such as A, E1, E2, K, N, U, and Y (Cascales et al., 2007;Singh et al., 2012). Previously, we had reported E. coli ...

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