优良益生菌筛选及利用菌糠生产益生菌制剂研究

发布时间：2018-04-14 06:17

本文选题：益生菌制剂 + 菌糠　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：随着抗生素的耐药性和抗生素残留问题日益严重,研究和开发可替代抗生素的绿色饲料添加剂产品已成为世界性的研究课题。微生物制剂具有补充、调整和维持动物肠道微生态平衡,达到防病治病,促进健康和提高生产性能的作用。近年来我国食用菌技术发展迅速,逐步形成规模化和产业化生产,收菌后剩余底料菌糠的数量也急剧增加,菌糠内富含菌丝体且蕴含着丰富的营养物质,合理利用食用菌菌糠成为食用菌行业需要解决的问题。而利用菌糠生产益生菌制剂为其提供了重要途径。本研究以本实验室多年分离并保存的乳酸菌为出发菌株,从生长性能、产酸性能和耐受性等方面出发,筛选优良乳酸菌;并进一步研究筛选获得的乳酸菌的生长性能、产酸规律等生物学特性。以实验室保存和购买的芽孢杆菌为出发菌株,从生长规律、耐受性和产酶能力等方面出发,筛选获得优良芽孢杆菌;并对其进行鉴定和生物学特性研究。利用菌糠混合培养芽孢杆菌、乳酸菌和酵母菌,采用实验室小型罐式发酵,优化菌糠发酵培养基和发酵条件,筛选出最佳菌株配伍,并进行验证试验。主要研究结果如下:(1)实验共筛选获得3株优良乳酸菌,分别为植物乳杆菌R02(Lactobacillus plantarum)、粪肠球菌R07(Enterococcus faecalis)和戊糖片球菌R09(Pediococcus pentosaceus)。其中菌株R09生长最快,产酸能力最强,乳酸占总酸含量最高,达95.45%,可耐受0.12 g/mL NaCl;菌株R02对温度的耐受性最强,可耐受55℃高温;菌株R07对强酸强碱环境有较强的耐受能力,在pH为3.0-9.5的条件下均可生长。在药敏试验中,菌株R02和R07对抗菌药物表现出较强的耐受性,而R09表现出极强的耐药性。(2)筛选获得3株优良芽孢杆菌,分别为解淀粉芽孢杆菌Ba1(Bacillus amyloliquefaciens);蜡样芽孢杆菌Bc1(Bacillus cereus);枯草芽孢杆菌Bs1(Bacillus subtilis)。筛选结果表明,菌株Bs1和Bc1对高温、高盐条件有较好的耐受能力,3株菌对酸碱均有较强的耐受范围;且3株菌都具有良好的产酶活性;生长曲线测定结果表明,菌株Bc1、Ba1和Bs1在稳定期活菌含量较高,均超过1010cfu/mL,其中菌株Bc1在稳定期的活菌含量最高,达1014 cfu/mL。菌株Bc1、Ba1和Bs1的皿内拮抗试验结果表明菌株Ba1和Bs1对链球菌产生了较强的抑制作用。3株菌均具有较高的SOD酶活力。菌株Bc1对抗菌药物有较强的耐受性,菌株Ba1和Bs1对大多数抗菌药物都很敏感。(3)以乳酸菌R09和芽孢杆菌Bc1为出发菌株,通过L_9(3~4)正交试验获得菌糠发酵的最适培养基配方:菌糠22.5 g,麸皮5.2 g,油渣2.5 g,尿素1.2 g。在此条件下乳酸菌R09的活菌数可达2.5×10~9cfu/g,芽孢杆菌Bc1的活菌数可达3.16×10~(10)cfu/g。通过乳酸菌、芽孢杆菌和酵母菌混菌发酵,获得两组最优组合:处理3Bc1+1314+R02+R09和处理5 Ba1+1314+R02+R09。并得出处理3 Bc1+1314+R02+R09发酵产物的pH值显著(p0.05)低于其它菌株组合,发酵产物中的粗蛋白含量和还原糖含量均显著(p0.05)高于其它菌株组合,与对照组相比,粗蛋白含量增加了13.4%,还原糖含量增加了108.5%。通过L_9(3~4)正交试验获得菌糠发酵的最适发酵条件,因此通过综合分析,菌糠发酵最适发酵条件为:温度30℃,初始pH值4.5,料水比2:2.7,培养时间96 h。在此条件下乳酸菌、芽孢杆菌和酵母菌的活菌数分别可达2.0×10~9 cfu/g,8.71×10~(10) cfu/g,1.95×10~8cfu/g。(4)经验证,两种菌株配伍的乳酸菌和芽孢杆菌活菌数都达到1010 cfu/g,各菌株活菌含量均较高,其中菌株配伍Bc1+1314+R02+R09中乳酸菌活菌数达4.27×10~(10) cfu/g,芽孢杆菌活菌数为8.24×10~(10)cfu/g,酵母菌为7.08×10~9cfu/g;Ba1+1314+R02+R09中乳酸菌活菌数达1.58×10~(10) cfu/g,芽孢杆菌活菌数为5.75×10~(10) cfu/g,酵母菌为2.69×10~8cfu/g。
[Abstract]:With the problem of antibiotic residue and antibiotic resistance is becoming increasingly serious, green feed additive products research and development of alternative antibiotics has become a worldwide research topic. Microbial has added, adjust and maintain the animal intestinal micro ecological balance, achieve the treatment and prevention of diseases, health promotion and improve production performance. In recent years China's edible fungus the rapid development of technology, gradually formed the scale and industrial production, fermentaing number of WMLE remaining bottom material is increased rapidly, WMLE rich mycelium and contains rich nutrients, rational use of edible fungus chaff become edible fungus industry need to solve the problem. And the use of WMLE probiotics production it provides an important way. In this study, lactic acid bacteria in our laboratory for many years separated and preserved as the starting strain, the growth performance, acid production and other aspects of tolerance And screening of lactic acid bacteria; and further study on the growth performance of the lactic acid bacteria strains were obtained, the biological characteristics of acid production law. Kept in laboratory and purchase of Bacillus strain from growth, tolerance and enzyme production ability and other aspects, to obtain excellent Bacillus screening; and identification and biological research the characteristics of the WMLE. Using the mixed culture of Bacillus, lactic acid bacteria and yeast, the laboratory tank fermentation medium and Fermentation Conditions Optimization of mushroom bran fermentation, screened the best strain compatibility test. The main results are as follows: (1) were screened 3 strains of lactic acid bacteria, respectively. For Lactobacillus plantarum R02 (Lactobacillus plantarum), R07 (Enterococcus faecalis) Enterococcus and Pediococcus pentosaceus R09 (Pediococcus pentosaceus). The strain R09 grew fastest, strongest acid producing ability of lactic acid. The total acid content was the highest, up to 95.45%, can withstand 0.12 g/mL NaCl; strain R02 had the strongest tolerance to temperature, can withstand the high temperature of 55 DEG C; strain R07 had strong tolerance to acid and alkali environment in pH under the condition of 3.0-9.5 can grow. In the drug sensitivity test, the strains of R02 and R07 on antibacterial the drug showed strong tolerance, while R09 showed strong drug resistance. (2) screened 3 strains of Bacillus were excellent, Bacillus amyloliquefaciens Ba1 (Bacillus amyloliquefaciens); Bacillus cereus Bc1 (Bacillus cereus); Bacillus subtilis Bs1 (Bacillus subtilis). The results showed that the strains of Bs1 and Bc1 on the high temperature, high salt conditions have better resistance, 3 strains had strong tolerance to pH range; and all of the 3 strains have good enzyme activity; the growth curve showed that strains Bc1, Ba1 and Bs1 were higher in the stable period of live bacteria, More than 1010cfu/mL, the strain of Bc1 in the stable period of live bacteria content is the highest, up to 1014 cfu/mL. strains Bc1, Ba1 and Bs1 test results of antagonistic activity of SOD enzyme showed that dish strains Ba1 and Bs1 had a strong inhibitory effect on Streptococcus strains of.3 were high. The strain Bc1 has strong resistance to antimicrobial agents the strains Ba1 and Bs1 are sensitive to most antimicrobial agents. (3) the R09 of lactic acid bacteria and Bacillus Bc1 strain by L_9 (3~4) orthogonal test, the optimum medium formula of WMLE Junkang fermentation: 22.5 g, 5.2 g and 2.5 g wheat bran, oil residue, live lactic acid bacteria R09 1.2 g. urea under this condition number is 2.5 * 10~9cfu/g, living bacterium Bacillus Bc1 number is 3.16 * 10~ (10) cfu/g. by lactic acid bacteria, fermentation by mixed strains bacillus and yeast, obtained two groups: optimal combination treatments of 3Bc1+1314+R02+R09 and Ba1+1314+R02+R09. and 5 The treatment of 3 Bc1+1314+R02+R09 fermentation product of pH was significantly lower than the combination of other strains (P0.05), crude protein content of fermentation product and reducing sugar content were significantly higher than other strains (P0.05) combination, compared with the control group, the crude protein content increased by 13.4%, reducing sugar content increased by 108.5%. L_9 (3~4) orthogonal test of mushroom bran fermentation the fermentation conditions, therefore, through comprehensive analysis, WMLE optimum fermentation conditions of fermentation were 30 degrees Celsius temperature, the initial pH value of 4.5, the ratio of material to water 2:2.7, culture time 96 h. under these conditions, the number of viable lactic acid bacteria, Bacillus bacteria and yeast are up to 2 * 10~9 cfu/g 10~ (10), 8.71 * cfu/g, 1.95 * 10~8cfu/g. (4) verified, two strains of lactic acid bacteria and Bacillus and living bacteria number reached 1010 cfu/g, the strains of live bacteria had high content of Bc1+1314+ R02+R09 in which lactic acid bacteria and bacteria living bacteria number up to 4.27 x 1 0~ (10) cfu/g, the number of viable bacilli was 8.24 * 10~ (10) cfu/g, yeast was 7.08 * 10~9cfu/g, the number of viable lactic acid bacteria in Ba1+1314+R02+R09 reached 1.58 * 10~ (10) cfu/g, the number of viable bacilli was 5.75 * 10~ (10) cfu/g, yeast was 2.69 * X.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816

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