| Conjugated linoleic acid is a series of the mixture which (conjugated linolenic acid, LA) contains the location (8,10-,9,11-,10,12-,11,13-) and geometry (cis, cis; cis, trans; trans, cis; trans, trans) isomers of the conjugated double bonds of linoleic acid, t is a new healthy fatty acids which has lots of physiological functions, for example:anti-cancer, anti-tumor, improving immunity, reducing fatty deposits.Among the isomers of CLA,9c,11t-CLA and10t,12c-CLA have strong physiological activity. Natural CLA is found mainly in meat and dairy products of ruminant and the content is small. Therefore, using bio-transformation method is a good way to produce CLA.Lactobacillus helveticus L7, which was isolated and kept in our Lab, can efficiently produce9c,11t-CLA. The bacterial cell was permeabilized to improve the permeability of the cell membrane, and then immobilized with the optimal conditions. The CLA-converting activity, physical and chemical properties of the immobilized cell in bioconversion of CLA were investigated. The main results was as follows:1. The fermentation conditions of Lactobacillus helveticus L7for9c,11t-CLA production was optimized. The optimal bacterial cell concentration is about5×1010CFU/mL, and the optimum LA concentration was1mg/mL. The yield of9c,11t-CLA increased quickly after12h, and reached the maximum after36h.2. L. helveticus L7cell was permeabilized by16alkyl trimethyl ammonium bromide (CTAB), ether, ethanol, toluene and lysozyme, respectively. The permeability feasibility and effect were compared. It was indicates that20%of the ethanol had the higher permeating effect on the cell, and9c,11t-CLA production of permeabilized cell could reach more than468μg/mL after30min at room temperature. The crude enzyme was prepared from the cell broken by using the ultrasonic cell disruption method. The crude enzyme had poor operational stability, and the enzyme activity dropped to about35%to the initial activity, when kept at4℃after120h. On the contrary, permeabilized cell had a better stability than crude enzyme.3. The conditions of immobilization of permeable L. helveticus L7cell have been optimized by using response surface methodology. The optimum conditions for preparation of immobilized bell is:sodium alginate,1.78%; calcium chloride,3.13%; time,60.30min. fermenting by immobilized bell made under the conditions, the highest accumulation of9c,11t-CLA was561μg/mL. Immobilized cell displayed good bioconverting properties. The activity of the immobilized cell didn't change significantly after reuse for five times, and9c,11t-CLA yielding remained in549μg/mL. The activity had not significantly decline in a4℃storage after120h, and remained above90%of the initial activity, which showed that immobilized bell had good storage stability. Through an environmental scanning electron microscope (ESEM), the internal and surface structure of immobilized bead were observed, and it was found suitable to the mass transfer. The sodium alginate immobilized cell had a good performance in improving the cell's recycling rate, reducing the9c,11t-CLA production costs, and improving economic efficiency of enterprises. |
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