耐酸性反刍兽月形单胞菌构建及对瘤胃微生物发酵的影响
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摘要
本研究无菌采取健康奶牛瘤胃液,按照乳酸杆菌分离鉴定的方法步骤,利用乳酸杆菌分离培养基SL培养基,共筛选出8株瘤胃内耐酸的乳酸杆菌,通过形态学特征及生物化学反应特性分析,并对其进行16S rRNA基因扩增、测序,经BLAST与GenBank数据库进行同源性分析对其进行鉴定,通过耐酸性试验,最终获得一株能在pH值3.24条件下生存的耐酸能力强的乳酸杆菌-粘膜乳杆菌。
对本实验室保存的反刍兽月形单胞菌K6及分离鉴定的粘膜乳杆菌lm4208进行了菌株生长曲线绘制,确定了粘膜乳杆菌lm4208及反刍兽月形单胞菌K6的对数生长期,粘膜乳杆菌lm4208对数生长期为4~12h,反刍兽月形单胞菌K6对数生长期为8~16h。
采用L9(34)正交实验法确定了粘膜乳杆菌lm4208及反刍兽月形单胞菌K6原生质体制备及再生的最佳条件。
确定了粘膜乳杆菌lm4208原生质体的热灭活条件:56℃下灭活60min,反刍月形单胞菌K6的最低生长pH值为5.4。
采用化学融合法即在融合剂PEG作用下将反刍兽月形单胞菌K6和灭活的粘膜乳杆菌lm4208原生质体进行了融合,在再生培养基上获得4株融合子,经传代最终获得一株遗传稳定的融合子F-K6-lm4208。
对融合子F-K6-lm4208进行了形态学、生理生化性质及DNA分子量的鉴定,证明其是双亲产生的融合子,酸耐受试验表明融合子能在pH值5.1条件下存活。
取健康奶牛瘤胃液,分别加入反刍兽月形单胞菌原始菌K6和耐酸工程菌F-K6-lm4208,并设立对照组,在体外模拟急性瘤胃酸中毒,于培养0、2、4、6、8、10、12、14、16、18h,检测培养液中pH、乳酸、乙酸、丙酸、丁酸含量,研究其对瘤胃微生物发酵的影响。结果表明工程菌组培养液pH值在培养第4h之后达到最低点然后显著升高(p<0.01),培养液中乳酸没有蓄积。添加原始菌和工程菌都能影响瘤胃微生物的发酵,影响乙酸和丙酸的生成比例,使发酵倾向于生成丙酸,但工程菌影响的程度要大于原始菌。
Based on the isolation program of lactobacillus and using the SL culture mediums which were specific for isolating acid-tolerant lactobacillus from ruminal fluids.Eight acid-tolerant lactobacilli(named 1,2,3,4,5,6,7,8) were isolated from ruminal fulid and the morphological and biochemical characteristics were observe and analyzed.The consensus primers were designed according to the conservative region of bacterial 16S rRNA and the 16S rRNA genes were cloned from their genome.The 16S rRNA genes of the eight strains were amplified by polymerase chain reaction (PCR) and sequenced. The sequence obtained was 98% identical to those of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus brevis, Lactobacillus mucosae in GenBank.The strains 1,2,3,4,5,6,7,8 were identified through morphology and biochemical characteristics and 16S rRNA gene sequence as Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus casei,Lactobacillus curvatus, Lactobacillus brevis, Lactobacillus mucosae, Lactobacillus mucosae,respectively.
The growth curve of Selenomonas ruminantium K6 and Lactobacillus mucosae lm4208 were drawn and the logarithmic phase of them are definited. The lm4208’s logarithmic phase is 4h to 12h and the K6’s is 8h-16h.
To establish the optimum conditions of the protoplast preparation and regeneration of the lactobacillus mucosae lm4208 and Selenomonas ruminantium K6,the L9(34)orthogonal experiments were used.The results showd that the optimized conditions of the strain of lm4208 as follows: 10h incubation was for protoplast release,100μg/mL lysing enzyme for protoplast preparation, 37℃and 60min. for the release of protoplast.And the ratios of protoplast preparation and regeneration were 98.3% and 24%,respectively.The optimum conditions of K6 were as follows: 30min of treating time, 600μg/mL lysozyme and 12h incubation time.Under the optimum conditions,the ratios of formation and regeneration protoplast were 94% and 17.2%,resperctively.
The thermal inactivating conditions of strains lm4208 were determined by survival rate.In 56℃,the strain lm4208 treated for 60min were optimal,and the lowest pH of K6 to grow is 5.4.
Following all conditions were optimized, preparation,fusion and regeneration of protoplast were done and four recombinant strains were selected according to the rate of survival strains at the pH5.4 in the regenerating medium.After the future generation,the fusant F-K6-lm4208 was gained at last.
The fusant F-K6-lm4208 can’t make gelatin liquefy,but can hydrogenize the nitrate,and ferment maltose, xylose, glucose, fructose, cellobiose, lactose, glucopyranoside, sorbose and mannose,but can’t ferment rhamnose,aesculin and salicin.The biochemical and physiological characteristics of the fusants F-K6-lm4208 are similar to Selenomonas ruminantium K6. The molecular weight of the fusants was larger than their parents. According to the morphological,the biochemical and physiological characteristics,the molecular weight and the acid resistant experiment, the fusants were identified as acid-tolerant fusants and can survive at pH5.1.
The influence of the liminary strain K6 and the engineering bacteria F-K6-lm4208 on in vitro rumen fermentation was studied using co-incubating the strains with mixed rumen micro-organisms of dairy cows as incoculums. Culture fluid and ruminal fluid was sampled for analysis of pH and Volatile Fatty Acid (VFA) at 0、2、4、6、8、10、12、14、16、18h.
The results showed that the pH in engineering bacteria group was the lowest after 4h incubation then stepped up significantly.The lactic acid was not accumulated and at the 18h ,its concentration was 0.53 mmol/L.Compared to control group at 18h,the concentration of VFAs in the liminary bacteria group and the engineering bacteria group increased by 24% and 105%,the ratio of acetic acid decreased by 12% and 16%, the ratio of propanoic acid increased by 22% and 35%,respectly.And the ratio of acetic acid to propanoic acid decreased 0.42and 0.57,respectly.This demonstrate that the liminary strains K6 and its engineering bacteria both can have effect on the fermentation and the ratio of acetic acid to propanoic acid,and can make the fermentation prone to propionic acid production but the effects of the engineering bacteria were more marked higher than the liminary strains K6.
对本实验室保存的反刍兽月形单胞菌K6及分离鉴定的粘膜乳杆菌lm4208进行了菌株生长曲线绘制,确定了粘膜乳杆菌lm4208及反刍兽月形单胞菌K6的对数生长期,粘膜乳杆菌lm4208对数生长期为4~12h,反刍兽月形单胞菌K6对数生长期为8~16h。
采用L9(34)正交实验法确定了粘膜乳杆菌lm4208及反刍兽月形单胞菌K6原生质体制备及再生的最佳条件。
确定了粘膜乳杆菌lm4208原生质体的热灭活条件:56℃下灭活60min,反刍月形单胞菌K6的最低生长pH值为5.4。
采用化学融合法即在融合剂PEG作用下将反刍兽月形单胞菌K6和灭活的粘膜乳杆菌lm4208原生质体进行了融合,在再生培养基上获得4株融合子,经传代最终获得一株遗传稳定的融合子F-K6-lm4208。
对融合子F-K6-lm4208进行了形态学、生理生化性质及DNA分子量的鉴定,证明其是双亲产生的融合子,酸耐受试验表明融合子能在pH值5.1条件下存活。
取健康奶牛瘤胃液,分别加入反刍兽月形单胞菌原始菌K6和耐酸工程菌F-K6-lm4208,并设立对照组,在体外模拟急性瘤胃酸中毒,于培养0、2、4、6、8、10、12、14、16、18h,检测培养液中pH、乳酸、乙酸、丙酸、丁酸含量,研究其对瘤胃微生物发酵的影响。结果表明工程菌组培养液pH值在培养第4h之后达到最低点然后显著升高(p<0.01),培养液中乳酸没有蓄积。添加原始菌和工程菌都能影响瘤胃微生物的发酵,影响乙酸和丙酸的生成比例,使发酵倾向于生成丙酸,但工程菌影响的程度要大于原始菌。
Based on the isolation program of lactobacillus and using the SL culture mediums which were specific for isolating acid-tolerant lactobacillus from ruminal fluids.Eight acid-tolerant lactobacilli(named 1,2,3,4,5,6,7,8) were isolated from ruminal fulid and the morphological and biochemical characteristics were observe and analyzed.The consensus primers were designed according to the conservative region of bacterial 16S rRNA and the 16S rRNA genes were cloned from their genome.The 16S rRNA genes of the eight strains were amplified by polymerase chain reaction (PCR) and sequenced. The sequence obtained was 98% identical to those of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus brevis, Lactobacillus mucosae in GenBank.The strains 1,2,3,4,5,6,7,8 were identified through morphology and biochemical characteristics and 16S rRNA gene sequence as Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus casei,Lactobacillus curvatus, Lactobacillus brevis, Lactobacillus mucosae, Lactobacillus mucosae,respectively.
The growth curve of Selenomonas ruminantium K6 and Lactobacillus mucosae lm4208 were drawn and the logarithmic phase of them are definited. The lm4208’s logarithmic phase is 4h to 12h and the K6’s is 8h-16h.
To establish the optimum conditions of the protoplast preparation and regeneration of the lactobacillus mucosae lm4208 and Selenomonas ruminantium K6,the L9(34)orthogonal experiments were used.The results showd that the optimized conditions of the strain of lm4208 as follows: 10h incubation was for protoplast release,100μg/mL lysing enzyme for protoplast preparation, 37℃and 60min. for the release of protoplast.And the ratios of protoplast preparation and regeneration were 98.3% and 24%,respectively.The optimum conditions of K6 were as follows: 30min of treating time, 600μg/mL lysozyme and 12h incubation time.Under the optimum conditions,the ratios of formation and regeneration protoplast were 94% and 17.2%,resperctively.
The thermal inactivating conditions of strains lm4208 were determined by survival rate.In 56℃,the strain lm4208 treated for 60min were optimal,and the lowest pH of K6 to grow is 5.4.
Following all conditions were optimized, preparation,fusion and regeneration of protoplast were done and four recombinant strains were selected according to the rate of survival strains at the pH5.4 in the regenerating medium.After the future generation,the fusant F-K6-lm4208 was gained at last.
The fusant F-K6-lm4208 can’t make gelatin liquefy,but can hydrogenize the nitrate,and ferment maltose, xylose, glucose, fructose, cellobiose, lactose, glucopyranoside, sorbose and mannose,but can’t ferment rhamnose,aesculin and salicin.The biochemical and physiological characteristics of the fusants F-K6-lm4208 are similar to Selenomonas ruminantium K6. The molecular weight of the fusants was larger than their parents. According to the morphological,the biochemical and physiological characteristics,the molecular weight and the acid resistant experiment, the fusants were identified as acid-tolerant fusants and can survive at pH5.1.
The influence of the liminary strain K6 and the engineering bacteria F-K6-lm4208 on in vitro rumen fermentation was studied using co-incubating the strains with mixed rumen micro-organisms of dairy cows as incoculums. Culture fluid and ruminal fluid was sampled for analysis of pH and Volatile Fatty Acid (VFA) at 0、2、4、6、8、10、12、14、16、18h.
The results showed that the pH in engineering bacteria group was the lowest after 4h incubation then stepped up significantly.The lactic acid was not accumulated and at the 18h ,its concentration was 0.53 mmol/L.Compared to control group at 18h,the concentration of VFAs in the liminary bacteria group and the engineering bacteria group increased by 24% and 105%,the ratio of acetic acid decreased by 12% and 16%, the ratio of propanoic acid increased by 22% and 35%,respectly.And the ratio of acetic acid to propanoic acid decreased 0.42and 0.57,respectly.This demonstrate that the liminary strains K6 and its engineering bacteria both can have effect on the fermentation and the ratio of acetic acid to propanoic acid,and can make the fermentation prone to propionic acid production but the effects of the engineering bacteria were more marked higher than the liminary strains K6.
引文
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