| Various fatty acids with conjugated double bonds(—C=C—C=C—)occur in nature. For example, edible fats derived from ruminant animals (cattle, sheep, etc), such as milk fat and tallow, contain conjugated linoleic acid (CLA), it is a generic term for a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. Generally considered that a total of16kinds of isomers of conjugated linoleic acid, so its type is very rich, in humans and animals, the most prevalent type is that c9,t11-CLA, t10,c12-CLA and c11,t13-CLA, c9,t11-CLA is considered to be the most relevant and most physiological activity isomer with human and animal nutrition.Conjugated linoleic acid is found as one of the most important functional lipid in recent years, medical studies have shown that some isomers have biological activity, including anticarcenogenic activity, antiatherogenic activity, antioxidation activity, cholesterol-lowering, prevention and treatment of diabetes, the ability to reduce the catabolic effects of immune stimulation, to reduce body fat, to regulate blood sugar, to enhance immunity, to increase bone density and improve sleep function. So these conjugated fatty acids have attracted widespread attention from the international medical community and nutritionist.In recent years, it is of concern for many researchers to product CLA by resting cells of microorganisms. For the reasons, the microbial cell transformation technique is simple, interfering substances is very rare in the reaction system liquid, contamination can hardly occur, it is more conducive to downstream purification and detection, etc. but it is an important research direction to look for a cheaper conversion substrate at the present time. Colza oil is much cheaper than linoleic acid, which contains15-20%linoleic acid, its sources is very wide, linoleic acid can be converted into high value CLA by linoleic acid isomerase of lactic acid bacteria. Jiangxi Province is one of the main producing areas of the national colza, colza planting area is more than800million acres, the total production of colza is nearly100million tons, colza oil is about29million tons. Colza oil is one of China’s major edible oil, waste cooking oil is also a good source of linoleic acid, rational use of waste cooking oil can not only brings economic benefits, but also prevents the spread of waste oil, ptotects environment.In the study, we convert the rich linoleic acid in colza oil into CLA by the Lactobacillus acidophilus CGMCC1.1854resting cells. Our overall objective:to a wide sources and cheap colza oil as raw material, product a variety of physiological activity, high value-added functional fatty acid(CLA). Production process of CLA is improved by immobilization technology innovation, improve the conversion generation rate, enhance the CLA product quality, reduce production costs, meet the growing market demand, reduce environment pollution caused by traditional fermentation production, achieve a high efficiency, high quality, low consumption and pollution-free production.When colza oil concentration was7.5mg/ml and the amount of lipase was2mg/30ml, we determined the optimal reaction medium, medium concentration, pH, temperature, time and cell concentration through the single factor experiment, the optimum conversion liquid medium was0.1mol/L citric acid-sodium citrate buffer, we obtained the optimum pH, the temperature, the conversion time and the cell concentration. At the same time, it was studied that the different metal ions effected on linoleic acid isomerase activity,1mmol/L Ca2+and Cu2+played a weak role in promoting linoleic acid isomerase activity, increased0.35%and0.62%respectively compared with the control group,2mmol/L Zn2+and Ca2+played a aidant role in promoting linoleic acid isomerase activity, increased2.40%and1.75%respectively compared with the control group. On this basis, using Box-Benhnken central composite design and response surface method, we optimized and analyzed the technological design consisted of time, pH, temperature and cell concentration, When colza oil concentration was7.5mg/ml and the amount of lipase was2mg/30ml, the optimal conversion process was time25h, pH6.5, temperature36℃, cell concentration40mg/ml, reaction medium was the0.1mol/L citric acid-sodium citrate buffer solution, the optimized CLA yield could reach230.12±7.52μg/ml.We studied effect of different permeabilizing treatments on the yield of CLA and determined that the biotin was the most suitable permeabilizing treatment. Biotin was not only a good anti-oxidant and easy to operate, can also increase the cell permeability. the optimum adding amount is4mg/30ml, CLA yield could reach256.25μg/ml.Using centrifugal and adsorption immobilization technology, the continual convertion of lactobacillus cell for CLA kept yeild at more than115μg/ml in10batches. The centrifugal and adsorption immobilization technique solved the problems that substances exchange and cumbersome operation of embedding immobilized technology, at the same time, overcomed disadvantages of adsorption immobilization technique (a small amount of adsorption and cell shedding), achieved high density and multi-batch conversion purpose.Finally, we determined that the reaction mixture contained c9t11, t10c12, cc and tt CLA isomers by gas chromatography, the most biological activity isomers accounted for53.49%of the total CLA. |
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