| Koumiss is a traditional beverage that can nourish vessel, sooth mood, and promote digestion. It had supportive therapeutic effects on cardiovascular disease, digestive disorders, tuberculosis, diabetes and diarrhea. Yeasts are main microorganism in Koumiss, they can produce antibacterial substance in metabolism, such as organic acids and killer toxin. But, there have no comprehensive reports about using yeasts metabolites from Koumiss on animal. So, the study isolated and screened yeasts in Koumiss which had better antibacterial effects on pathogenic Escherichia coli (E. coli) O8 from cow feces and produced more antibacterial substance (killer toxins), purified and cultured these yeasts and extracted yeasts metabolites. Antibacterial effects of yeasts metabolites on E. coli O8 in vitro were determined and their antibacterial effect mechanism were analyzed, meantime, their antibacterial effects on mice challenged with E. coli O8 in vivo were determined. The study will provide fundamental basis for utilizing yeasts metabolites from Koumiss.Experiment 1. Isolation, Screening, and identification of yeasts in Koumiss which produce antibacterial substances. After isolated and purified, yeasts morphology were observed by staining methods, and they were identified by biochemical methods, the more killer toxins producing yeasts were screened by oxford cup method, then, these yeasts were identified by 26 S rDNA amplification. Results:the yeasts isolates in Koumiss were milky white, and the diameters of the colonies ranged from 0.2 mm to 1 mm. They were radial with pseudo or true mycelia. The 5 strains of yeasts isolates were 2 Kluyveromyces,1 Saccharomyces,1 Candida, 1 Leucosporidium identified by biochemical methods. Two yeasts had better antibacterial effects on E. coli O8 were identified by 26S rDNA amplification, they were Kluyveromyces marxianus and Saccharomyces cerevisiae.Experiment 2. Extraction of yeasts metabolites from Koumiss and determination of their main antibacterial components. Yeasts metabolites were extracted by ethyl acetate, their organic acids were determined by HPLC, and killer toxins were determined by kit. Results:4 yeasts metabolites of Kluyveromyces marxianus and Saccharomyces cerevisiae were obtained, they were K. marxianus pH 2, K. marxianus pH 8, S. cerevisiae pH 2, and S. cerevisiae pH 8 (named as K2, K8, S2, S8). The main antibacterial components of them were organic acids and killer toxins. The main organic acids of them were propionic acid, lactic acid, and ascorbic acid. The contents of killer toxins of them were range form 68.31 to 74.99 mg/100 g. The 4 yeasts metabolites had the characteristic of heat-stable and negative correlated with temperature, and pH-tolerant.Experiment 3. Antibacterial effect of yeasts metabolites from Koumiss in vitro, and their antibacterial spectrum. The minimum inhibition concentration (MIC) and the minimum bactericidal concentration (MBC) of 4 yeasts metabolites of Kluyveromyces marxianus and Saccharomyces cerevisiae on E. coli O8 were determined by broth dilution method. The effects of 4 yeasts metabolites on the growth curve of E. coli O8 were determined by turbidimetry. The effects of 4 yeasts metabolites on the cell surface hydrophobicity of the E. coli O8 were determined using the microbial adhesion to solvents method, and the effects of 4 yeasts metabolites on the cell membrane permeation of E. coli O8were determined by ONPG method. Moreover, the effects of 4 yeasts metabolites on the conductivity, cell wall, and large molecules DNA and RNA of E. coli O8were determined by conventional methods. Antibacterial spectrum of 4 yeasts metabolites on common pathogenic bacteria were analyzed. Results:The MICs of K2, K8, S2 and S8 were 0.0250,0.1000,0.025 0,0.025 0 g/mL, respectively. The MBCs of K2, K8, S2 and S8 were 0.100 0,0.200 0,0.1000 and 0.200 0 g/mL, respectively. The growth curve of E. coli O8 was S-shape, the cell surface characteristic of E. coli O8 was basic and relative hydrophilic. Four yeasts metabolites could inhibited obviously the growth of E. coli O8, increased its cell surface hydrophobicity and promoted its cell membrane permeation, destroyed its cell membrane and cell wall, leaked its cytoplasm, increased the alkline phosphatase (AKP) and conductivity of E. coli O8 solution. Four yeasts metabolites had wide antibacterial spectrum.Experiment 4. Antibacterial effects of yeasts metabolites from Koumiss in vivo.1.The pathogenic rule of E. coli O8 on mice.I selected 144 Kunming strain mice and divided them into 18 groups,9 groups were the control groups,9 groups were the experiment groups, the detecting time were 0,2,4,8,12,24,36,48,72 h. Mice in the control groups were injected sterile PBS and collected small intestine samples at different time. Mice in the experiment groups were injected 50% minimum lethal dose (MLD) E. coli O8. Clinical symptoms were observed and small intestine samples were collected. Paraffin section were prepared by traditional method and pathological section were observed after HE and PAS stainings. Results:Compared with the control groups, regular changes were appeared from 0 h to 72 h in the experiment groups, they revealed that the length of villus, length of villus/depth of crypt (V/C) value, the thickness of muscularis, and the numbers of intraepithelial lymphocytes and goblet cell were decreased at the beginning, then increased. However, the depth of crypt was increased at the beginning, then decreased. The most serious time was 36 h after mice were injected E. coli O8, and the mice recovered gradually after 72 h.2. Screening for the best dose of yeasts metabolites from Koumiss on mice challenged with E. coli O8. I selected 150 Kunming strain mice and divided them into 15 groups, contained the control group, the negative control group, the positive control group, the high, middle, and low doses of K2, K8, S2 and S8. Mice in the control group and the negative control group were administered with sterile PBS by gavage for 7 d. Mice in the positive control group were administered with 0.13 g/kg-bw Ciprofloxacin (CPFX). Mice in the high, middle, and low doses groups of K2, K8, S2, and S8 were administered with 10 000,5 000,2 500 mg/kg-bw K2, K8, S2, and S8, respectively. Besides the control group, mice in other groups were injected with E. coli O8 at the 4th day. Results:mice in the low dose of K2, the middle dose of K8, the low dose of S2, and the low dose of S8 had higher survival rates than the other groups, it indicated that the low dose of yeasts metabolites (2 500 mg/kg-bw) was the best dose by gavage.3. Antibacterial effect of yeasts metabolites from Koumiss on mice challenged with E. coli O8. I selected 464 Kunming strain mice and divided them into 8 groups, contained the control group, the negative control group, the positive control group, the Mongolian compound group (25 g/kg·bw), K2, K8, S2 and S8 groups (250 0 mg/kg·bw). The treatments were as before. Blood lymphocyte subgroups were analyzed by flow cytometry. Cytokines and enzyme in serum were measured by enzyme-linked immunosorbent assay (ELISA). The thymus index and spleen index were weighed and calculated. NK cell activities were measured by cell-cultured method. Caecal microflora were calculated by spread plate method. Phagocytosis of O8 were measured by carbon clearance method, and survival rate of mice were detected. Results:after challenged with E. coli O8, every detected indexes were changed in different degrees of mice in the negative control group, it resulted in caecal microflora disorder, immune function reduced, and the ability of resistance to diseases decreased of mice. Four yeasts metabolites groups, the positive group, and the Mongolian compound group could maintain normal caecal microflora, enhance immune function, increase survival rate, and cure diseases of mice.In summarize, yeasts metabolites of Kluyveromyces marxianus and Saccharomyces cerevisiae from Koumiss had antibacterial effects on pathogenic E. coli O8, they can effectively enhance mice immune function and the ability of resistance to diseases after mice challenged with pathogenic E. coli O8. |
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