Isolation Of Cell Wall Polysaccharides From Probiotics And Studies On Their Bioactivities

Probiotics are able to live in the intestine of animals. They can enhance intestinal environment and develop immunity, and they are totally benefit to the health of the host. The two most common probiotics are Lactobacillus and Bifidobacterium, while probiotics also include Bacillus, bacteria, yeast, some photosynthetic bacteria and a few kinds of Escherichia coli.Many functional of probiotics are related to the outer structure. For a bacteria cell,the cell wall is the most direct part contacted with the outside world, and the cell wall plays a quiet important role. The cell wall of probiotics is composed of a plurality of peptidoglycan, which looks like a sealed cystic structure, and with special components such as acid, polysaccharide and related proteins in it. There are many specific receptors on the surface of the cell wall, which affect the relationship between the bacteria and the host or phages. Such the functionals as enhancing immunity and inhibiting tumor growth have a great relationship with it.CWPS (cell wall polysaccharides) include the polysaccharide from the probiotic cell wall structure and a small amount of polysaccharides that are not easily removed.Peptidoglycan is the main component of CWPS. Peptidoglycan is one of the most abundant microbial conserved molecules in the cell, which plays an important role in the relationship between probiotics and host.In this study, five kinds of probiotics (Lactobacillus rhamnosus BMBL0606, L.reuteri BMBL0612, L. acidophilus BMBL0618, Bifidobacterium Lactis BMBL0624,B. animal BMBL0630) were selected as raw materials, respectively to optimize the extraction process of cell wall polysaccharides. According to the production of optimized process, the total sugar content, fat content, protein content, lysozyme,morphology, heavy metal content and yield were analyzed. MTT method was also used to investigate the inhibitory effect of five kinds of probiotic CWPS on the growth of cancer cells. The experimental results were as follows:1 The extracting process of CWPSs from probiotics was optimized. After degreased by five different organic solvents (chloroform methanol mixed solution, ethyl ether,petroleum ether, acetone, cyclohexane), the content of remained fat was detected by the method of vanillin and the best organic solvent could be chosen. Six different proteases (trypsin, acid protease. alkaline protease, neutral protease, papain.bromelain) were used and the content of amino nitrogen from the samples determined the best one. The results showed that: 1) The degreasing ability of chloroform methanol mixed solution to all the five probiotics was the strongest out of the five different organic solvents. 2) In the protein removal process, for L. acidophilus and B.lactis, the acid protease was the best protease out of other proteases. For L. rhamnosus,alkaline protease was the best protease out of other proteases. For L. reuteri and B.animal, neutral protease was the best protease out of other proteases. The final CWPS product was milky white, cottony like powder and the yield was between 0.150 g/L and 0.225 g/L liquid bacteria.2 The compositions of CWPSs from probiotics were analysted. In the finished CWPS products, total sugar content was between 94.70 ?g/mg and 161.36 ?g/mg. The fat content was between 18.53 ?g/mg and 28.57 ?g/mg. The protein content was between 135.00 ?g/mg and 179.29 ?g/mg. The peptidoglycan content was between 76.5% and 83.0%. ICP-AES detection of Pb, Hg, As, Cr, Cd, Cu, Fe, Zn and Mn showed that, contents of the harmful heavy element like Pb, Hg, As, Cr, Cd, Cu, and Mn were either very low or not found. Fe got the highest content between 0.0204 g/mg and 0.0416 g/mg, followed by the content of Zn between 0.0152 g/mg and 0.0240 g/mg. Each kind of heavy metals did not exceed the national standard of healthy food.3 Biological activities of CWPSs were studied. MTT experiments were carried out to study the inhibitory effect of probiotic CWPS on HepG2 hepatoma cells and BGC-823 gastric cancer cells. The results showed that 1600 g/ml B. lactis CWPS had the greatest inhibitory effect on the HepG2 hepatoma carcinoma cells, and reached the maximum inhibition rate of up to 36.9%. For BGC-823 gastric cancer cells, 1600 g/ml of L. acidophilus CWPS is for the greatest effect of the inhibition rate of up to 72,reached a maximum of 34.7%. By constructing the mouse S180 tumor model, we found that the inhibition rate of B. lactis CWPS in mice was 42.2%.In summary, in this study, based on five kinds of probiotic cell wall polysaccharides,the extraction process of degreasing and protein removal method was optimized to enhance the quality of the finished product. The components of CWPS obtained from the optimized extraction process were analyzed, which provided a reference for its future application in food industry. The results of MTT showed that the five kinds of probiotic cell wall polysaccharides got good antitumor effect in vitro, and the cell wall polysaccharides of B. lactis also showed good anti-tumor effect in mice. This will provide an important theoretical basis for the future development of probiotic cell wall polysaccharides as a new type of functional food.