Study On The Technology Of Biotransformation Of ?-decalactone By Yeast MF-Y11 From Ricinoleic Acid

The natural?-decalactone?GDL?prepared by biotransformation has an attractive peach aroma in low content,and it is a very important lactone substance,which has been recognized as a safe food and drug additive.The controlled release of the concentration of ricinoleic acid in the biotransformation medium,the strong feedback inhibition effect of the high concentration product on the yeast,and the toxic effect of the substrate are the bottlenecks restricting the industrialization of GDL.In this study,active dry yeast and the natural ricinoleic acid was used to conduct research on the biotransformation of GDL.Firstly,23 kinds of yeasts were screened to determine a strain of MF-Y11 with high GDL production capacity.The transformation conditions of the shake flask were optimized as follows:shaking speed 250 r/min,temperature 30°C,the initial pH of the medium 6.23,and the substrate concentration 40 g/L.When ricinoleic acid was 150 g/L,the amount of cellular protein exuded out of the yeast nearly doubled,and the growth of yeast was also severely inhibited.Secondly,the mechanism of converting calcium ricinoleate into GDL by yeast was studied.The effects of calcium ricinoleate and ricinoleic acid on the production of GDL were compared.Experiments showed that calcium ricinoleate was first acidified to ricinoleic acid in the buffer medium,and then GDL was transformed by yeast from ricinoleic acid.3.3g/L,7g/L and 20g/L calcium ricinoleate were added to the buffer medium of pH=5.It was found that calcium ricinoleate could quickly acidified into ricinoleic acid in 6-9h,and the acidification rate was over 80%.The GDL was produced in calcium ricinoleate system during48-60h with no difference in ricinoleic acid system.When calcium ricinoleate was used as the substrate,the yield of GDL was higher than that of ricinoleic acid system.Furthermore,the calcium ion was proved to be an important factor to promote the production of GDL,and the optimum concentration was 0.6g/L.Thirdly,in order to increase the probability of the contact between the ricinoleic acid and the yeast MF-Y11 and improve the mass transfer efficiency of substrate,two methods were used including the addition of polar organic solvents and modified vermiculite.The experiments showed that the addition of acetone contributes to the yield of GDL,and the highest GDL yield was 5.32g/L when the optimal amount of acetone was 1%?v/v?,which was32.34%higher than the control.Modified vermiculite was added to the medium as a substrate dispersed release material.The results of scanning electron microscopy,thermogravimetry and fourier transform infrared spectroscopy showed that modified vermiculite had good adsorption and embedding with ricinoleic acid.For 60h in the biotransformation,the yield of GDL was increased by 33.55%to 6.21 g/L compared to the control in this delivery system.Fourthly,to alleviate the toxicity of substrates and products to yeast cells,a preliminary study of immobilized cell techniques was performed.The method for immobilizing cells was as follows:0.3 g yeast powder was added to the complex solution with 20 g/L sodium alginate and 20 g/L attapulgite clay,when they were mixed uniformly,15 g/L CaCl₂ solution was dropped in for 2 hours.It was proved that cell immobilization could maintain cell viability effectively.Results showed that addition of 1g/LCaCl₂ to the fermentation broth could enhance the mechanical strength of the gel balls,and it could be recycled 5 times without significant damage to sphere.The total yield of GDL was the highest at 5.44 g/L for 5 cycles.It was explored whether temperature and embedded substrate could improve mass transfer efficiency of substrate.The experimental results showed that:?1?30?was the best temperature for the production of GDL;?2?After the substrate was embedded in the gel balls,the total GDL production was lower than the control,and extracellular GDL was mostly present in the gel balls,accounting for about 74.7%of the total GDL in the entire system.Fifthly,experiments have shown that the feedback inhibition effect on the yeast is existed when the concentration of GDL is high.In the liquid-liquid two-phase system,GDL was transferred to organic phase to relieve this feedback inhibition effect.It was determined that DC345?cyclopentasiloxane and cyclohexasiloxane?was the best organic solvent which had small damage to cells.When 30%?v/v?DC345 was added,the highest GDL yield in shake flasks was 5.35 g/L,which was 61.88%higher than the control.The result showed that DC345 was not easily emulsified under mechanical shear,and the demulsification rate reached 65.22%under centrifugation.The highest GDL yield could reach 7.08g/L,which was68.17%higher than the control.