| Conjugated Linoleic Acid is a kind of natural active substance which exists in the milk and meat products of ruminant, with a series of physiological functions such as reducing weight and fat, lower cholesterol, prevent atherosclerosis and enhance immunity. It is the collectively referred of a series of octadecadienoic acid isomer mixture, these isomers containing conjugated double bonds, and have different spatial and location configuration which c9,t11-CLA and t10,c12-CLA have stronger physiological activity. But the naturally occurring CLA content is extremely low and can not meet people’s needs, therefore, to explore bioconversion method to generate CLA is imperatively. At present, microbial conversion synthesis method in resting cells is concerned because of its simple operation, less synthetic product impurities and high conversion rate. But the immobilized resting cells while retaining these characteristics,it also has enhanced stability,can be reused, ease to separate and other characteristics.Currently, mostly research used castor oil, linoleic acid, soybean oil and other expen- sive materials as substrates to synthesis CLA, where higher concentration of linoleic acid also inhibits the growth of cells.In our country, the yield of rapeseed oil is very high and ranks first in all countries. In the grease processing process will produce a large number of by-products, rapeseed oil sediment is one of them, it remains not only a small amount of unsaturated fatty acids, but also contain 15-20%of linoleic acid. Therefore, using rapeseed oil sediment as the substrate to synthesis CLA, not only reduce production costs, but also to protect the environment from pollution waste.This paper choosen lactic acid bacteria 3-9 which screening from kimchi for producing bacterium, first carried on the 16 srDNA molecular biology identification,the results showed that the bacteria is Lactobacillus plantarum,was named as Lactobacillus plantarum 3-9.At first,the paper compared the ability which Aspergillus niger, Pocine pancreas,Mucor javanicus three lipase hydrolyzed rapeseed oil sediment to produce free fatty acids.And determined the ability of Mucor javanicus lipase hydrolyzed rapeseed oil sediment is stronger, the hydrolysis rate was 15.02 ±0.32% which was 1.46 times thatof Pocine pancreas,1.14 times that of Aspergillus niger. Then through the single factor experiment, optimizing the adding quantity of the Mucor javanicus lipase, buffer pH,enzymolysis temperature, time and oscillation speed, and further adopt orthogonal test design to study the process parameters of the first four single factor. Determining the optimum reaction condition of Mucor javanicus lipase hydrolysis grease is: the adding quantity of Mucor javanicus lipase was 250 U/mL, pH6.5 citric acid sodium citrate buffer, at 150 r/min and 40 ℃ enzymolysis 60 min. The hydrolysis rate of enzyme hydrolysis grease reached the highest,was 26.38±0.64%.Then,compared the ability of Lactobacillus plantarum3-9, Lactobacillus plantarum CGMCC1.557 and Lactobacillus acidophilus CGMCC1.1854 converted rapeseed oil sediment into CLA under the same transformation medium system and condition.Among them,the yield of CLA was the highest which converted by Lactobacillus plantarum 3-9,was 135.39±3.75μg/m L which was 1.33 times that of Lactobacillus plantarum CGMCC1.557,1.23 times that of Lactobacillus acidophilus CGMCC1.1854.In order to prove the Lactobacillus plantarum3-9 had ability to convert rapeseed oil sediment into CLA,compared the CLA content in the Lactobacillus plantarum3-9 Conversion liquid and rapeseed oil sediment.The CLA content in the Conversion liquid was significantly higher than the original CLA content in the rapeseed oil sediment.Proved that the bacteria had ability to convert rapeseed oil sediment into CLA.And then the conditions of Lactobacillus plantarum 3-9producing linoleic acid isomerase were studied,when the linoleic acid emulsion concentration in the medium was 0.3%,the initial pH was 6.5, cultivated 20 h at 30℃,Lactobacillus plantarum3-9 had the strongest transformation ability and the yield of CLA could reach 167.68±1.12μg/mL.Finally,optimized process conditions of immobilized Lactobacillus plantarum3-9 convert rapeseed oil sediment into CLA. At first, investigating the adsorption immobilization materials and conditions of Lactobacillus plantarum3-9,determining the optimum immobilization material was 1-3mm ceramic particle, immobilization time was 1h, immobilization rate of cells could reach 75%. The immobilized cells were placed into rapeseed oil sediment conversion liquid which was hydrolyzed bymucor javanicus lipase, the yield of CLA was195.83 ±3.35μg/mL.It increased 1.16 times compared with the resting cells.Then the main influence factors including transformation based liquid medium,temperature, time, pH, concentration of cells and oscillation speed of adsorbing immobilized Lactobacillus plantarum3-9 convert rapeseed oil sediment were optimized. When pH7.5 phosphate buffer as transformation based liquid medium, the cell concentration was 0.6 g/mL, at 35 ℃ and 120r/min transformation 17 h, the yield of CLA could reach 274.31 ±3.21μg/mL, the conversion rate was 18.39%. At the same time, using the method of temperature shock treatment cells, to achieve the purpose of increase cell permeability, the result showed that the yield of CLA could reach 288.73±1.37μg/mL after cells were handled which was1.06 times that of cells without treatment. Then, using the adsorption immobilized Lactobacillus plantarum3-9 consecutive batches to convert rapeseed oil sediment into CLA.The result showed that the Immobilized cells ceramic particles could convert 16 batches continuously, the highest yield could reach 302.81± 6.02μg/mL, the conversion rate was 20.42% and the yield of CLA was higher than 200μg/ mL among the 10 batches. |
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