Study On The Formation Of Emulsification And Process Optimization Of The Preparation Of Natural Perfume γ-Decalactone Via Biotransformation

Gamma-decalactone（GDL）is a five-membered lactone with peach fragrance,which is widely used in food,beverages and cosmetics.Biotransformation recently has become a hot topic due to its good aroma and quality of GDL,and environmental protection.Ricinoleic acid（RA）usually served as the substrate and natural yeast was utilized as the strain.However,it is difficult to industrialize GDL via biotransformation.Aiming at increasing GDL yield,four aspects were investigated,including revealing the mechanism of emulsification formation,improving substrate dispersion,alleviating product feedback inhibition and process optimization.The main conclusions were as follows:Firstly,the mechanism of emulsification during biotransformation were revealed.The appearance,RA droplet size,cell concentration and protein content of the biotransformation system were analyzed via appearance-process analysis.The emulsifier contained in yeast powder,metabolites secreted by yeast cells（such as lipopeptide）,Pickering effect of yeast cells and stirring shear force were the comprehensive causes of emulsification.RA droplet size reached 1353.13±54.06 nm at 12 h of emulsification proto-phase,and then stabilized to less than 1μm.Emulsification promoted substrate dispersion,changed oil-water ratio and increased GDL concentration before 48 h.However,local GDL concentration was too high after 48 h emulsification leading to the inhibitions of product and cell growth.The biological process analysis of lipopeptides produced during biotransformation was conducted,and 106 proteins were found.The proteins,involved in ribosome related proteins,β-oxidation process,stress response and cell wall protection,were detected.Lipopeptide was the stress result of yeast cells responding to lactones,oxygen and nutrient changes during emulsification.Secondly,substrate was dispersed to improve its uptake.The substrate was adsorbed and embedded with halloysite,clay,130NP silica and 130H silica.The best adsorption and embedding rate of 130H was 70.94±0.27%,and the other materials were between 40-50%.FTIR,TG and SEM showed that there was intermolecular hydrogen bonding between the adsorbed material and the substrate,and the materials became the immobilized carrier of yeast.The changes of p H,cell concentration,RA and GDL concentration during biotransformation were analyzed.The p H did not change significantly,but the cell vitality in the medium with clay was decreased due to the mechanical force formed by agitation.The concentration of GDL after adsorption-embedding continued to increase at 12-36 h,and its generation rate was higher than control group.Among them,the highest conversion rate in the medium with halloysite at36 h reached 88.99%,and the highest lactone concentration in the medium with 130H reached2.79 g/L,which increased by 29.77%.With the help of materials,the biotransformation time shortened,the energy consumption reduced,and the product yield increased.Thirdly,in situ product removal was utilized to alleviate product feedback inhibition.In situ product removal with resin showed that the best addition of DA-201 resin was 0.3 g/30m L,the extraction rate of GDL was higher than 90%,and the inhibition of product feedback was alleviated.The DA-201 resin combined with HPD-400 could improve cell viability and reduce protein overflow.But GDL production was not significantly changed.Liquid-liquid extraction showed that after GDL extraction with Sera Sense-SF-DDM,cetearyl ethylhexanoate and ERCAREL G20,the residue of aqueous lactone was less and the cell viability was higher.When they were mixed with equal mass,10%addition had better extraction effect,less addition and increased cell viability.It alleviated product feedback inhibition and increased GDL concentration,and continuous in situ product removal was more effective than one time,alleviating cellular protein overflow and enhancing resistance to adversity.Finally,biological transformation process was optimized.The extraction process was optimized,the best extraction addition of n-butanol and n-hexane on substrate and product were both equal volumes.High temperature and pressure,and Tween 80 emulsification could improve the extraction effect.The GDL production medium was optimized using Plackett-Burman and Box-Behnken designs.Growth of S.cerevisiae was inhibited when GDL concentrations exceeded 2.5 g/L.The optimal formula（g/L）was determined:K₂HPO₄ 7,glycerin 5,Mg SO₄·7H₂O 7,L-carnitine 0.1,RA 25,Tween 80 3.25 g/L RA was the best choice due to the high conversion rate with relatively low cost.Mixed solvent could slow down substrate and product feedback inhibition,promote cell proliferation,and increase the GDL continuously.At 72 h,GDL concentration reached at 3.71 g/L increased by 43.53%.200 rpm was the best speed for GDL production,and the reduction of speed to 150 rpm at 36 h was beneficial to accumulate GDL,which increased by 10.64%.The above results provide a theoretical basis for the industrialization of biotransformation to prepare GDL.In future,the stress response of yeast can be further analyzed through metabolic pathways.Milder adsorbent materials can be used to achieve the dual functions of substrate dispersion and cell immobilization.For in situ product removal,mathematical model should be combined to develop supporting equipment with temperature,p H,dissolved oxygen,carbon dioxide and bacterial concentration.Finally,the adsorption embedding system,in situ product removal and process control were combined to increase the product yield.