Screening And Identification Of Conjugated Linoleic Acid Producing Ruman Bacteria

In this research, rumen bacteria were isolated from the rumen contents of cattles and sheeps by Hungate anaerobic method. Then using the ultraviolet spectroscopy and gas chromatography, strains producing cis-9, trans11-CLA and trans10, cis12-CLA were screened. These strains were classified according to morphological observation,16S rDNA sequence analyzing, fatty acid composition analyzing, physiological and biochemical characteristics assaying results. The research results are as follows:(1) Nine rumen samples were collected from the breeding experiment base of China Agricultural University and the Erdos of Inner Mongolia.58 rumen bacterias were isolated from these rumen samples by the Hungate anaerobic operation method, and purified by rolling tube cultivation. Among these 58 strains, most of their colonies were white, only a few were yellow. All the colonies were small, the diameters ranged from 1 to 5 mm. Colonies of most strains were round and with smooth edge, but small parts of them were irregular.(2) By ultraviolet spectroscopy screening,8 strains which have the ability to produce conjugated linoleic acid were screened from the 58 isolates. Among them, four strains had an absorption peak at 233nm, three strains at 232nm, while another one at 234nm. The OD of these strains at the absorption peak was between 0.4 and 0.9, and the highest OD was 0.8086 for strain 7512.(3) Gas chromatography was use to screen the strains with ability to produce cis9, trans 11-CLA and trans 10, cis12-CLA from the 8 strains. The results showed that all the strains had the capacity to produce cis9, trans 11-CLA and trans 10, cis12-CLA. The strain 111 had the highest yield of conjugated linoleic acid, and its yield of c9, t11-CLA and t10, c12-CLA reached to 141.7μg/mL and 127.5μg/mL, respectively.(4). colonial morphology characteristics of the 8 strains were observed in the MRS solid medium, then stained by Gram staining method, and their cells morphological characteristics were observed by electron microscopy scanning. The results showed that these strains were Gram-positive, and the colonies were white. The main shape of colonies was round, small parts were irregular. Most of the colonies had an orderly and smooth edge, while there were a few wavy. All of the colonies diameters were between 1-5mm. All the strains were spherical, and cell size was between 0.5 to 1.0μm.(5). According to the 16S rDNA sequence analyzing, fatty acid composition analyzing, physiological and biochemical characteristics assaying results, the 8 conjugated linoleic acid producing ruman bacterias are preliminary identified as genus Streptococcus infantarius.