| Conjugated linoleic acid(CLA)has physiological functions such as inhibiting tumour,anti-atherosclerosis,weight losing,lowering insulin resistance and blood sugar,anti-oxidation and lowering blood pressure.CLA mainly obtained from ruminant meat,milk and its products,but the content is very low.Breast cancer is a hazard of women's health and being the disease of highest morbidity and mortality.CLA showed inhibitory effect on breast tumour in vitro and in vivo.Existing conventional methods for producing CLA include chemical methods and biological methods.The CLA products prepared by chemical method are complex in composition,it is difficult in separation and purification,and the safety of some isomers is unknown while CLA obtained by biotechnology with low conversion rate.Based on this,a strain producing t10,c12-CLA was screened from the rumen,and the conversion yield of t10,c12-CLA was improved by optimizing the permeabilization and immobilization conditions,and its inhibitory on mammary tumours in rats induced by DMBA was studied,the main experimental results are as follows:(1)A strain was screened from the rumen to produce t10,c12-CLA at 39°C for 24 h in medium with emulsified linoleic acid(LA)as substrate,and the conversion rate was 31.2%.The strain was named Propionibacterium acidipropionici Lx1 by physiological and biochemical experiments and 16 S rDNA identification,the GenBank accession number is MK050541.(2)The permeabilization conditions of the strain were optimized for t10,c12-CLA production.Optimization of single factor condition showed that the CTAB concentration was 1.5%(w/v),the permeabilization temperature was 37°C,permeabilization time was 60 min,pH was 5.8;the optimum conditions by response surface methodology were as follows: CTAB concentration of 1.9%(w/v),permeabilization time of 56.5 min,temperature of 31°C,pH of 5.8,with 1.0 g/L emulsified LA as the substrate,the yield of t10,c12-CLA was 741.3 mg/L after biotransformation for 12 h in PBS buffer at 39?.The membrane structure of permeabilized cells was changed,and the permeability was increased while the overall structure remains intact,could be used for biotransformation.(3)The immobilization conditions were optimized for t10,c12-CLA production,too.The single factor condition of immobilization were 20.0 g/L sodium alginate and 30.0 g/L calcium chloride,the immobilization time was 60 min and the cellmass concentration was tripled;the immobilization conditions optimized by response surface methodology showed that the sodium alginate concentration was 25.0 g/L,calcium chloride was 40.0 g/L,immobilized for 52.0 min,and biotransformed for 12 h with 1.0 g/L emulsified LA as substrate in PBS at 39?,the yield of t10,c12-CLA was 716.3 mg/L.The biotransformation ability of the immobilized cells was determined,after 48 h of biotransformation under optimized immobilization conditions,the yield of t10,c12-CLA reached 851.7 mg/L,and the bioconversion rate was 85.2%.The immobilized cells were able to be used repeatedly for 6 times in PBS.(4)Five-week-old SD rats received 50 mg/kg of DMBA once a week for three weeks,after 76 days,all rats were tumourigenic,and the tumour formation rate was 100%.Rats were given 0.5 g/kg t10,c12-CLA every two days at the first time of DMBA for twelves weeks.Results showed that t10,c12-CLA treatment significantly prolonged the tumour latency period,but there were no significant differences in tumour weight and volume at advanced stage.HE staining and immunohistochemical analysis of tumour tissues also showed that t10,c12-CLA had inhibitory effects on the deterioration of mammary tumour induced by DMBA in SD rats. |
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