Extraction And Immunity Activity Of Peptidoglycan In Lactobacillus Acidophilus Cell Wall

As an important member of lactic acid bacteria, Lactobacillus acidophilus can inhibit in intestinal tract. When comes to substantial amounts, Lactobacillus acidophilus can improve or regulate the balance of intestinal tract flora, strengthen body immunity and restrain the formation of tumor and etc. Peptidoglycan which is also called mucopetide, glycopeptide and murein is a special component in prokaryotic organism cell wall. Peptidoglycan becomes the ideal target for recognizing immune system of eukaryotes as there is no Peptidoglycan in eucytes. Researches indicated that there was a lot of Peptidoglycan recognizing molecules in advanced eukaryotes, while these active molecules induced the releasing of immunal regulating materials by different signal transmitting ways. Thus immune system was stimulated to perform immune functions. Peptidoglycan in the cell wall of Bifdobacterium had already been extracted successfully and was proved to be a new nonspecific immunity enhancer while reports related to peptidoglycan in cell wall of Lactobacillus acidophilus was scarce, so it is of great importance to study the process of extraction and purification of peptidoglycan in cell wall of Lactobacillus acidophilus we have got and combines it to pharmacology assay to development of medicine.Lactobacillus acidophilus of short trophophase and large yield La2 was scanned and the enrichment medium for La2 was optimized in the early stage of this paper, then peptidoglycan was extracted and purified by ultrasonic wave method, lysozyme method and trichloroacetic method, anti-tumor and immunity regulation effect were tested by pharmacology assay after the study and development to probitics immunity adjuvant. The content and results of the paper were as follows.1. La2 was scanned as a strain of Lactobacillus acidophilus of short trophophase and large yield from lot of Lactobacillus acidophilus strains and enriched medium was optimized through optimizing basic medium and enrichment factors by single factor and orthogonal test. Growth curve with optimized enriched medium were determined by following the change of pH and OD₆₀₀. According to the growth curve, the terminal time was at 12h. La2 got to 3.8×10⁹cfu/mL in this culture for 12h at 37℃when OD600 was 2.167while pH value was 4.01.2. Peptidoglycan in La2 cell wall was extracted and purified by ultrasonic wave method, lysozyme method and trichloroacetic method. The optimal conditions for ultrasonic wave were ultrasonic power 400W, ultrasonic time 30min, ultrasonic temperature 40℃, yield rate of peptidoglycan was 10.2%; The optimal conditions for lysozyme method were lysozyme 4mg/mL, temperature 55℃, time 5h, pH value 7, yield rate of peptidoglycan was 10%; Yield rate of peptidoglycan by trichloroacetic method was 15.8%. So trichloroacetic method was selected to extract peptidoglycan in La2 cell wall through multiple comparisons.3. Peptidoglycan in La2 cell wall was identified. Components and content of amino acid was determined by HPLC method that Asp, Glu, Gly, Lys were the amino acid molecular connected peptidoglycan of La2 and N-acetylmuramic acid; DNS method was used to analysis the concentration of aminohexose, results showed that the concentration of aminohexose was 2.831mmol/g; Content of phosphorus which was 0.0319mg/g was determined by phosphorus content analysis; SDS-PAGE electrophoresis result indicated that the molecular weight was around 30kD.4. The effect of peptidoglycan extracted from La2 cell wall on S-180 tumor in S-180 tumor mice model was tested. Results showed that peptidoglycan(80mg/mL)(anti-tumor ratio 54.5% ) could extremely significantly restrained the growth of S-180 tumor in S-180 tumor mice model (P≤0.01) , what more, peptidoglycan(80mg/mL)could extremely significantly increased the spleen index and adrenal gland index (P≤0.01) . As there was no significantly effect on the immune activity of peptidoglycan in LactobaciUus acidophilus cell wall obtained by TCA method, this method could be used to exact active peptidoglycan in Lactobacillus acidophilus cell wall.