Isolation Of Twelve Strains Of Rumen Bacteria And Effect Of Propionibacterium Acnes On The Fermentation Of Rumen Microorganism

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.