Study Of Proboitic Properties Of Photosynthetic Bacteria

Background:Many species of bacteria have evolved and adapted to live and grow in the human gastrointestinal tract(GIT ) and perform a bio-film barrier of mucosa. Whereas the human GIT always results from endogenous and exogenous oxidative damage unceasingly, which brings oxidative damage to the mucosa. There have been some researches on microorganisms' resistance to oxidative stress. It has been shown that some lactobacilli possess antioxidative activity, and are able to ecrease the risk of accumulation of reactive oxygen species(ROS) during the ingestion of food. Lactic acid bacteria are able to degrade the superoxide anion and hydrogen peroxide. It is reported that Rhodobacter sphaeroides has antioxdative ability in vivo and in vitro, but in vivo the function position of Rhodobacter sphaeroides is still unknown and potential mechanisms of antioxidation of Rhodobacter sphaeroides remain to be done. In addition, whether other species of photosynthetic bacteria(PSB) possess antioxidative properties is unclear. There is no report on the protection of GIT mucosa against from oxidative damage by PSB possing antioxidative ability.PSB acting as probiotics has been introduced into health food in some foreign countries such as Japan and Amerian and England and so on , but PSB hasn't belonged to the approved list in health food at home nowadays. So far, there is no systematic report on probiotic properties of PSB.Objective:This thesis aimed to study the safety, functionary and technological characteristics of 8 PSB strains tentatively, to identify the strains systematically, and to provide with data of probiotic properties of PSB to exploit health food and medicine of PSB.Methods:1. Morphologic and cultural and bio-chemical characteristics along with their sequences of 16S rDNA were analyzed to identify the 8 strains. 2. The susceptibility of 8 strains to antibiotics scrips was done in vitro. The safety of strain 2c was assessed systematically, which included its acute and toxic experiment, its effects on the structure of villus and cells ofileum, its effects on the haematologic parameters reflecting the function of kidney and liver. In addition whether BT occurred was studied. Which were to determine the safety of PSB strains. 3. The functionary properties of PSB strains were investigated followed by: (1) Experiments which aimed to forecast the survival and persistence of PSB in vivo were performed. The tolerance of strains under condition of pH2.5 and 0.5%/0.9% ox bile salt as well as the survival in the simulated gastric juice (pH2.0, 3.0, 4.0) and small intestinal juice containing or uncontaining 0.45% ox bile salt were assessed. Besides, the inhibition to 4 kinds of common enteropathogenic bacteria, the adhesion to enterocyte-like cells HT-29 were carried out. An enrichment PSB experiment during and after the rats ingested PSB were done too.(2) The resistance to 0.8mmol/L H2O2 and superoxide anions produced by 10mmol/L paraquat and hydroxyl radicals out from Fenton system were executed to evaluate the tolerance of PSB to ROS in vitro. (3) The antioxidative performance of PSB in vitro were done using the following methods: first, samples of cultures, cells and their crusher by sonification respectively were prepared; second, commercial kits were used to determine the content of T-SOD, GSH, MDA in samples along with the eliminating ability of samples to superoxide anions and hydroxyl radicals in vitro. (4) An acute water-immersion restraint stress model in rats was constructed to discuss the protection GIT mucosa against from damage induced by oxidative stress by PSB. The damge degree of GIT mucosa was evaluated through macrography and micrography of HE stained pafaffin slice under light-microscope. The parameters (the content of GSH, GSH-Px, SOD, CAT and MDA along with the ability of eliminating superoxide anion and hydroxyl radicals) which reflecting the redox status of the GIT mucosa were determined. The grinded liquid of spleen and MLN were cultured on LB and BHI media aerobicly and anaerobicly respectively for 3 days to find whether BT occurred. (5) A plate count experiment using PSB selective media was done to know the viability of strain 2c in different position of GIT during various periodes after rats ingested 2c once(10 CFU/mL). 4. The culture media, culture condition and product line of manufacturing on scale for 8 strains of PSB were investigated. In addition, the stability of the cultures produced on scale during the period of storage and transportation was researched and the subcultured generations of the strains were studied.Results:1. 8 PSB strains growed better anaerobicly in light than aerobicly in dark. All the strainsproduced red pigments and carotenoides and CoQ10 under phototrophic conditions. Optimal growth occurred at 30℃~40℃ and at pH7.0~pH8.0. Based on the morphologic and cultural and bio-chemical properties, strain Ga. Il and WS8N were identified as Rhodobacter sphaeroides; strain 2c, 2C and 13 ing as Rhodopseudomonas palustris; strain MT as Rhodobacter capsulatus; strain rub as Rhodospirillum rubrum. The accession numbers in GenBank of the sequences of 16S rDNA of the strains were followed by Ga(DQ001152), rub(DQ001159), 2c(AY834756), 2C(DQ001155), S8N(DQ001157), Il(DQ001153), MT(DQ001154, 13ing(DQ001158). The Blast and phylogenetic tree analysis showed that the sequenses' similarity between Il and the same species strain whose accession number was AL162757 wasn't high(e.g. 86%), while total different genus and species strains (such as strain Ga, rub, WS8N, 13ing, MT and some Alcaligenes) spread faraway though their sequences' similarity was as high as 98%/99%. 2. 8 PSB strains showed susceptibility to most of antibiotics scrips in vitro. Strain 2c hadn't acute toxicity, when it was orally administered to mice with dose of 5×1011CFU/Kg bt. 2c didn't produce endogenous toxin in blood, it neither translocated itself, nor caused other bacterium in intestinal tract translocate; it had no adverse effects on the function of liver and kidney, when 2c was orally administered to rats. 3. Results of functionary characters of the strains follwed by: (1) 8 PSB strains survived well under condition of pH2.5(P>0.05). The viablility of the strains wasn't affected significantly under the treatment of 0.5%/0.9% ox bile (P>0.05) except for strain Ga(P<0.05) and Il(P<0.01). The low pH value (such as pH<2.0 ) of gastric juice reduced the survival of PSB in vivo significantly (P<0.01/P<0.001); pH4.0 of the gastric juice didn't affect the survival of the PSB(P>0.05); When pH>3.0, the survival of PSB in vivo wasn't affected(P>0.05) except for rub and Ga(P<0.05). PSB strains didn't inhibit the 4 kinds of common enteropathogenic bacteria in vitro. PSB strains had some adhesion ability onto HT-29 cells in vitro, but the ablility was not good. PSB could stayed in vivo for 3 days at least after it was ingested for several times. (2) 8 PSB strains showed good resistance to o.8mmol/L H2O2, superoxide anion produced by 10mmol/L paraquat (P>0.05). Strain Ga, 2c, WS8N and 13ing showed good tolerance to hydroxyl radicals out from Fenton system(P>0.05), while strain MT, rub, Il and 2C hadn't good resistance to hydroxyl radicals(P<0.05). (3) 8 PSB strains had the ability to eliminate superoixide anion and hydroxyl radicals in vitro, among them strain 2c had the strongest ability.(4) The results of macrograph and mcirograph underlight microscope and the items imaging the redox status of GIT mucosa of model control and normal control did show that damage of the former was severer than that of the latter. So the water-immersion restraint oxidative stress model was recognized successful. PSB could improve the GIT mucosa's damage induced by oxidative stress partially, the effects of the cultures was better than that of the cultures crusher by sonification. (5) When rats ingested strain 2c(109CFU/mL) once, the viability of 2c in stomach after 3h was 1.0×101CFU/g stomach, till 24h the viability was zero; while in small intestine and colon the viability of 2c was 1.2×102. 1.0×l02(CFU/g tissue) respectively after 3h; After 24h, the viability was 1.0×101. 1.1 × 102 respectively; Till 48h only tens of viability was found in the intestinal tract; After 72h no viability was found in vivo of rats. 4. 8 PSB strains subcultured stablely and could be cultured on scale . The product line was simple and could be manipulated easily. Conclusion:1. This thesis studied PSB probiotic properties tentatively and found that PSB was safe, possessing antioxidative capacity, able to be produced on scale and the product line was simple and could be manipulated easily, which provided with probiotic properties data to exploit PSB health food and medicine.2. The identification of the 8 PSB strains and analysis their sequences of 16S rDNA and gaining their accession numbers in GenBank afforded systematical background data to identify PSB and performed bio-marker and technological marker to protect intellectual property of the strains .3. Antioxidation experiments were done. Experiments in vitro demonstrated that 4 species of PSB such as Rhodobacter sphaeroides , Rhodobacter capsulatus , Rhodopseudomonas palustris and Rhodospirilium rubrum had antioxidative ability in vitro. The acute water-immersion restraint oxidative stress model in rats showed PSB improved the damage of GIT mucosa induced by oxidative stress partially, which afford function orientation to exploit health food and medicine of PSB.4. Among the 8 PSB strains, 2c had clear background and good probiotic properties, acting as probiotic, 2c was worth of extensive study.