Screened Out And Cultivated Antagonism Strain: Bacillus Licheniforms L₁ And Elementary Discussed Its Applications

There were rich vitamin C and inorganic salt in greenstuffs and fruits, the value of nourishment was quit high, but greenstuffs and fruits were eroded by pathogenic organism after picking, down to blet and deteriorated. Nowadays, the important methods that could restrain blet and deteriorated after picking were chemical prevention and cure, but because of toxin side-effect of some chemistry medicament , even polluted environment, and the questions that the retained quantity of chemical agents and their toxin side-effect were concerned by humans, so it was a hotpot that searched new and safe methods of controlling blet after picking in the study of greenstuffs and fruits store; the effect of application of probiotic in feed industry lied on two aspects: validity of strains and live colony quantity. Screened out high effect strains was a prophase basic work, seek new availability strains, constructed astronomical resource storeroom of probiotic in feed industry, and then benefited farming industry. Just about basing on these advisement, in the experiment first screened out high effect antagonism strains from nature colony, and reviewed their quality, in order to offering science thereunder for their using antisepsis industry of greenstuffs and fruits and feed additive industry.Five strains which have stronger antagonism were screened out in this experiment, according to observing characters of colony and shape of them with Gram stain test, the result of sixteen item test of physiological and biochemical characters, consulted appraisal manual of Bacillus, confirmed that L1was Bacillus licheniformis, L2,L4 and L5 were Bacilli cereus B. L3was Brevibacilli breris B.The antagonism collection of illustrative platesatlas of three strains were mensurated ,the characters of physics and chemistry such as depression titre,thermostability,acid-base stability, pankrin K stability, and so on. The results show: L1 and L5 have the wider antagonism collection of illustrative platesatlas, L1 has the strongest antagonism. The depression titre of fermentation filtrate of strain L1 to Staphylococcus aureus was 2000IU/mL, to Bacillius subtilis was 1500 IU/mL, and to Escherichia coli was 1500IU/mL;the fermentation filtrate of strain L1 had the strongest thermostability:when placed it at 30℃, it had the biggest antagonism, when placed it at 37℃one hour, the depression reactivity only decreased six percent, when placed it at 60℃one hour ,the depression reactivity decreased twenty-nine percent, when placed it at 100℃thirty minutes, the depression reactivity decreased forty-five percent, when placed it one hour, the depression reactivity decreased forty-eight percent, when placed it at 121℃thirty minutes ,the depression reactivity decreased fifty-five percent, placed one hour, the depression reactivity only keep forty percent; the fermentation filtrate of strain L3 had the worst thermostability:when placed it at 121℃one hour ,the depression reactivity was closed to nothing. the fermentation filtrate of strain L1 had the strongest acid-base stability: when the pH was 7.0 it reached the biggest depression reactivity, when the pH was 3.0,it can keep about seventy-one percent depression reactivity, when the pH was 11.0,the depression reactivity decreased thirty-three percent; the fermentation filtrate of strain L3 had the worst acid-base stability: when the pH was 11.0,it can retain only thirty percent depression reactivity. When acceded pankrin K to the fermentation filtrate of these three strains ,the depression reactivity of the fermentation filtrate swelled at different degree, thereinto, the depression reactivity of L1 swelled most.At the same time, obtained the antibacterial protein of fermentation filtrate of strain L1 by using ammonium sulfate precipitation method. The synergistic building effect of strains L1 and L5 was reviewed in this experiment, the result showed: when mixed the inoculum of two strains ,the mixed inoculum could boost up the antagonism to the indicating bacterium, when mixed the inoculum of two strains in the light of the proportion three to one ,would obtain the most repress bacterium diameter, and the titre could advance 96%; when mixed the filtrated inoculum of two strains ,the mixed filtrated inoculum could boost up the antagonism to the indicating bacterium, when mixed the filtrated inoculum of two strains in the light of the proportion three to one ,would obtain the most repress bacterium diameter, and the titre could advance 89.1%.In order to realize the industrialization production of the Bacillus licheniformis L1, investigated the growth condition of the strain, the raised condition of liquid state of the strain, preliminary studied the raised condition of solid state of the strain; contemporary, preliminary discussed the application in practice.At first, reviewed the optimum growth conditions of the Bacillus licheniformis L1, the conclusion was: the optimum growth temperature was 35℃,the optimum culture medium was Beef extract peptone culture medium which added 0.05% lysine; the optimum original pH was 7.0,the optimum rotate speed of the shaker was 150 rings every minute, because strain L1 was aerobic bacteria ,so used less volume filled in the shake flask test, in this experiment, used the volume filled was added fifty milliliter inoculum into 500 milliliter delta-flask, the seed growth curve was 18 to 21 hours.The influence factors of liquid state fermentation of Bacillus licheniformis L1 was investigated through single-factor test .The main factor include: temperature,initial pH and rotate speed. The optimum fermentation conditions were:temperature 35℃, initial pH 7.0, 150 r/min. The orthogonal experiment was used to optimize the medium, in the optimum condition, the bacterium concentration to be increased from 5.10×1010cfu/mL to 4.93×1010cfu/mLThe appropriate solid state culture medium of Bacillus licheniformis L1 was obtained through the orthogonal experiment, the culture medium was: every kilogram solid materiel contained 600 gram rice chaff, 400 gram bran, 6 gram brown sugar, the moisture was 60%.when used the liquid germ inoculation, the inoculate quantity was 10%,after cultivating 48 hours, the concentration of thalli was 7.15×1010CFU/g, the concentration of endospore was 5. 75×1010CFU/g,the ratio of deformation of endospore was upwards 80%.The conclusion of beastie experiments were: Bacillus licheniformis L1 has obvious effect on boosting use ratio of feed, accommodating colony balance of animal endogastric and enteric, avoiding diarrhea and so on; the experiment of fruits antisepsis showed: the fruits that were dealt with the broth of strain L1,the preservative schedule time would prolong three to five days.