摘要
本研究采用无菌采取健康奶牛瘤胃液,按照厌氧菌分离鉴定的方法,运用厌氧菌专用培养基CDC厌氧血琼脂和丙酸杆菌属特异性培养基SLB琼脂,筛选出12株瘤胃厌氧菌,编号分别为5、6、10、11、13、16、17、22、26、27、30、32,并进行形态学观察及生化分析。
根据GenBank中细菌16S rRNA序列保守区设计通用引物,从基因组DNA中分别克隆出12株瘤胃厌氧菌16S rRNA基因。经过序列测定,将所得序列经BLAST对GenBank数据库进行同源性分析,同时结合形态学及生物化学反应鉴定结果证实,所分离菌株分别为:蜡样芽孢杆菌(Bacillus cereus)、草乳杆菌(Lactobacillus graminis)、弯曲乳杆菌(Lactobacillus curvatus)、吉氏库特菌(Kurthia gibsonii)、粪肠球菌(Enterococcus faecalis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、牛链球菌(Streptococcus bovis)、志贺氏菌(Shigellae)、痤疮丙酸杆菌(Propionibacterium acnes)、氧化木糖无色杆菌(Achromobacter xylosoxidans)、夏威夷肠球菌(Enterococcus hawaiiensis)、产粘棒状杆菌(Corynebacterium vitarumen)。其中编号26的痤疮丙酸杆菌为瘤胃内生成丙酸的优势菌之一。
为了阐明痤疮丙酸杆菌的体外发酵特性,取健康羊瘤胃液,加入痤疮丙酸杆菌菌液(浓度为2.7×10~8个/mL)及其作用底物乳酸(浓度为2g/L),进行连续培养,于培养0、2、4、6、8、10、12、24、36、48h,分别检测培养液的pH及乙酸、丙酸、丁酸、乳酸含量。体外培养试验结果显示:随着培养时间的延长,培养液pH在培养到12h降至最低值,为6.13;乙酸、丙酸、丁酸浓度(mmol/L)均在培养到12h升至最大值,分别为39.86、28.57、33.91;乙酸/丙酸值在培养到12h降至1.40的最低值;乳酸浓度则一直呈下降趋势。
为了阐明痤疮丙酸杆菌在瘤胃内的发酵特性,取造瘘羊6只,分为实验组和对照组,每组3只。实验组羊经瘤胃瘘管注入痤疮丙酸杆菌培养液100mL(浓度为2.7×10~8个/mL),对照组注入等量生理盐水,于培养0、2、4、6、8、10、12、24h,采集瘤胃液检测pH、乙酸、丙酸、丁酸、乳酸含量。体内试验结果显示:在培养12h时,对照组和实验组的pH均降至最低,分别为6.16和6.02,并且实验组显著地低于对照组(p<0.05);对照组和实验组的乙酸和丙酸及丁酸的浓度(mmol/L)均升至最高,分别为59.25、69.13和23.27、33.36及10.76、15.86,并且实验组三项指标均显著地高于对照组(p<0.05);对照组和实验组的乳酸浓度(mmol/L)、乙酸/丙酸值均呈下降趋势,但是实验组两项指标下降幅度显著地大于对照组(p<0.05)。
本试验痤疮丙酸杆菌体内外发酵试验证明痤疮丙酸杆菌能利用乳酸生成挥发性脂肪酸(VFA),使pH降低,提高发酵生成丙酸的量,降低了乙酸/丙酸的值,发酵类型倾向于丙酸型。
Twelve strains of the anaerobic bacteria, which named as 5, 6, 10, 11, 13, 16, 17, 22, 26, 27, 30 and 32, respectively, were isolated from rumen fluid of health dairy cows by CDC Blood Agar Base and Sodium Lactate Broth (SLB) which are specific to anaerobes and Propionibacterium respectively , and the morphological and biochemical character of the bacteria were analyzed for each of them . The 16S ribosomal RNA (16S rRNA) genes of them were amplified by polymerase chain reaction (PCR) and sequenced. The strains of 5, 6, 10, 11, 13, 16, 17, 22, 26, 27, 30 and 32 were identified as Bacillus cereus, Lactobacillus graminis, Lactobacillus curvatus, Kurthia gibsonii, Enterococcus faecalis, Bacillus amyloliquefaciens, Streptococcus bovis, Shigellae, Propionibacterium acnes, Achromobacter xylosoxidans, Enterococcus hawaiiensis, Corynebacterium vitarumen, respectively through morphology, biochemical characteristics and 16S rRNA gene sequence.
Propionibacterium acnes is an anaerobic bacterium known as the ruminal predominant lactate-fermenting species, and can ferment lactic acid to acetic acid and propionic acid via Embden-Meyerhof-Parnas pathway (EMP) and hexose monophosphate pathway (HMP).
To understand fermentation characteristics of Propionibacterium acnes in vitro, in vitro rumen fluid from health sheep, which added into lactic acid (2g/L), were cultured for 48h with Propionibacterium acnes(2.7×108/mL). Rumen fluid was sampled for analysis of pH and Volatile Fatty Acid (VFA, acetic acid, propionic acid, butyric acid) and lactic acid, at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, and 48h after being cultured. The results shown: with prolongation of the culture time, at 12h of culture the pH of the rumen fluids decreased to the lowest, which is 6.13. The concentrations of acetic acid, propionic acid and butyric acid increased to the highest at same time, which were 39.86、28.57、33.91 respectively. The ratio of C2/C3 decreased to the lowest at same time. The concentrations of lactic acid decreased contimuously.
To understand fermentation characteristics of Propionibacterium acnes in vivo, six sheep were medially individed into two groups. One group was infused into 100 mL culture fluid of Propionibacterium acnes (2.7×108/mL) via the rumen fistulae, and another group was infused into the same dose of normal sodium. Rumen fluid was sampled for analysis of pH and Volatile Fatty Acid(VFA) at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h after being cultured. The results shown: pH of rumen fluids were significantly (P<0.05) lower in the experimental groups than control groups in each time point, and at 12h of culture two groups decreased to the lowest, which were 6.02 and 6.15 respectively. The significant increase (P<0.05) of acetic acid, propionic acid, and butyric acid content was observed in rumen fluids of experimental group, at 12h of culture increasec to the highest in two groups, which were 59.24, 69.13 for acetic acid, 21.27, 33.36 for propionic acid, and 10.76, 15.86 for butric acid respectively. The lactic acid content and the ratio of C2/C3 were significantly (P<0.05) lower in the experimental group as compared with the control group.
Conclusion obtained that Propionibacterium acnes could utilize lactate to generate propionic acid mainly, and belong to propionic acid producing strain.
引文
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