Industrial Producing Condition Of Selenium-enriched Probiotics And Its Inhibitory Activity Against Pathogenic Escherichia Coli

Selenium(Se) is an essential micro-mineral element which is relatively deficient in most area of the world.Its functions include regulating metabolism,improving immunity,enhancing reproductive performance,preventing cancer,resisting free radicals,postponing aging,detoxification from some heavy metals and preventing some local epidemic diseases.In most of countries,supplementation with Se in humans and animal diets is a fine routine step on human and animal health.While supplementation with sodium selenite as inorganic Se in diet has been limited in some countries because inorganic form has disadvantages of lower incorporation,higher toxicity and potential environment pollution compared with organic form.Hence, there is an increasing interest in the use of organic Se in diets.Probiotic could enhance the organism health and growth by promoting intestinal flora.Se-enriched probiotics,which combines the virtues of probiotics with the virtues of organic Se, can exert dual effects of organic Se and probiotics at the same time.The aim of the present study was to find a substitute media and optimize the culture conditions to achieve industrial production.In further,effect and mechanism on antibacterial activity of the Se-enriched probiotics against E.coli was evaluate in vitro and vivo,which was to find a new and no-polluted alternative to drug to prevent the diarrhea caused by E.coli in future.Experiment 1.The study was to evaluate whether the four strains of probiotic could live together without inter-inhibition and find applicable media for its industrial producing.Four stains of probiotic were cultured singly and four strains mix to count the number of each strain.Medias(flour and soybean power,wort and YEPD) used to produce bacteria were studied to compare the bacterial growth condition which was to utilize for industrial production.In Further,Medias composed of different ratio of flour and soybean powder were researched though bacterial growth conditions to make sure the suitable ratio to be used.Orthogonal test was done to optimize different levels of nitrogen,phosphorus,kalium and magnesium which were to supply the needy minerals to probiotic.Results indicated that the four strains of bacteria could live together.Probiotic can grow better in flour media than that in any other one.The suitable ingredient of medium were 8%flour powder and 2%soybean powder. Probiotic could grow better when 0.5%ammonium sulfate,0.1%potassium dihydrogen phosphate and 0.01%bitter salt were added into the primitive media.Experiment 2.The study was to evaluate the effect of temperature,culture durations, inorganic selenium concentration,inoculum sizes and the initial pH on probiotics production and organic Se conversion.Single-factor tests and orthogonal test were done to optimize the cultural conditions on selenium-enriched probiotics to explore the conditions for industrial:produce.It was concluded that four kinds of probiotic were fermented in flour medium,with 5%inoculum sizes and 10mg/mL inorganic-selenium concentration,cultured for 36h at 34℃and the initial pH of the medium was 6.2.Under the conditions,93.82%inorganic-selenium was converted into organic-selenium and the number of four kinds of probiotic was reached 10⁸-10⁹ CFU·mL^-1.Experiment 3.The purpose is to research the process of producing Se-enriched probiotics and its stabilization in storage.The difference on the growth of probiotic and conversion of organic Se between different culture apparatus:fermentation tank and conical flask under certain conditions was researched.When mixed the absorbent with selenium enriched probiotics even,the product was processed after drying.Varies on growth of probiotic and organic Se conversion were estimated when the product was stored.The result showed that there is no marked difference on two indexes when culture apparatus changed between fermentation tank and conical flask.Total Se content in product reached 50 ppm,while organic Se concentration reached 46.32μg·g^-1.Conversion of organic Se was about 92.64%.Population of each probiotic strain was 5.6×10⁸,1.0×10¹⁰,2.9×10⁹ and 5.8×10⁸CFU·g^-1 respectively.The content of organic selenium remained unchanged in storage.Population of Cu reduced to 5.1×10⁵ CFU·g^-1when storage duration was up to one month.Population of La, LGG,St reduced to 1.5×10⁹,2.1×10⁷,6.8×10⁷ CFU·g^-1respectively when duration reached three months.Experiment 4.The study was to research the effecet and mechanism of inhibitaory action of Se-enriched probiotics against pathogenic Escherichia Coli K88 in vitro. Four kinds of probiotic werecultured with K88 cultured singly or four-strain mixed. The difference of inhibitory ability between Se-enriched probiotic and four-strain mixed were detected.Fermented filtrated liquor was gotten to research the antibacterial activity using Oxford plate assay system.Two kinds of fermentation were produced from diverse media whose inhibitory activity was compared. Fermented filtrated liquor was treated with Lactate dehydrogenase,acid,alkali and heat to determine the inhibitory activity.The results indicated that the four kinds of probiotic owned the antagonistic properties.There was no difference on whether probiotc enriched inorganic Se and inhibitory activity of diverse fermentation derive from different media.Escherichia Coli K88 was inhibited by fermented filtrated liquor me.tabolized by three kinds of probiotic.Antibacterial component owned heat-stability advantage.The antagonistic abilities were reduced greatly after treated with Lactate dehydrogenase or alkali while the abilities were raised after treated with lactic acid.It was concluded that Escherichia Coli K88 can be antagonized by the bacterium from nutrition competition,the organic-acids especially lactic acid and bacteriocin produced.Experiment 5.The purpose was to research the effect and potential inhibitory action of selenium-enriched probiotics against the pathogenic Escherichia Coli K88.96 male Kuming mice were divided into control,probiotics sodium selenite,organic-selenium with prebiotics,selenium-enriched probiotics or drug groups(n=16) in the experiment. All the mice had been fed for 30 days."Productive performance,selenium concentration in blood and skeletal muscle,anti-oxidation status and resident bacterial flora of each treatment were studied to analysis the difference after fed for 15 and 30 days.Pathogenic E.coli was irrigated into gaster to inspect the mortality in the 30th days.The survived were sacrificed to detect other items.Population of E.coli was enumerated in the caecal feces;pH of different segment of intestine and Spleen and thymus indexes were monitored.Results indicated that anti-oxidation status and blood Se content of mice with Se-enriched probiotics supplemented were enhanced higher than that of others after fed for 15 days;the normal bacteria flora of intestine was improved;number of resident flora(Lactobacillus,Saccharomyce) was markedly more than that of others(P＜0.05).Skeletal selenium content of the mice with inorganic or organic selenium supplementation was higher than those without supplementary and lower than those of mice with Se-enriched probiotic(P＜0.05).The body weight of mice with Se-enriched probiotics supplemented was more increased than those of others.The mortalities of the mice and No.of E.coli in the caecal feces were lowest with selenium-enriched probiotic supplementation.In conclusion,growth performance,anti-oxidation status and immunity were boosted for mice with Se-enriched probiotic;pathogenic E.coli was antagonized efficiently in vivo.