Study On The Deacidification Of High-acid Oils And Fats By Lipase SMG1-F278N

High-acid oils and fats face many difficulties during their processing,utilization,and storage owing to the high content of free fatty acids.The traditional alkali refining is not applicable for the deacidification of high-acid oils and fats,because a large amount of soap is produced.The produced soap causes serious loss of neutral oil and needs a large amount of water to wash,and thereby produces a large amount of waste water.Removal of free fatty acid through physical distillation needs high energy input and less phosphorus content in raw materials.Most importantly,it is not applicable for the deacidification of high-acid oils and fats that rich in n-3 polyunsaturated fatty acids.Comparatively,enzymatic deacidification of highacid oils and fats has drawn considerable attention owing to its mild reaction conditions,high efficiency,and environmental friendliness.The utilization of enzymatic deacidification instead of traditional alkali refining and physical distillation for deacidification can achieve technological upgrading,energy conservation,and emission reduction,and increase the overall utilization of raw materials.However,all the existing enzymatic deacidification approaches face many problems,such as lack of catalysts,poor deacidification efficiency,side-reaction occurred,and lack of industrial application prospects.Therefore,the development of special enzyme preparations for highly efficient deacidification of high-acid oils and fats and try to establish a truly feasible process for industrial deacidification of high-acid oils and fats are of great significance.Therefore,the present study first screened the existing lipases,and lipases SMG1 with high esterification capability and good specificity towards long-chain and ultra long-chain fatty acids was screened for the succeeding studies.Subsequently,based on the resolved crystal structure and the clarified activation mechanism of lipase SMG1,rational modification was performed and lipase SMG1-F278 N with 1.94-fold increased esterification activity was obtained.Interestingly,it was found that lipase SMG1-F278 N still has good specificity towards long-chain and ultra long-chain fatty acids.Finally,lipase SMG1-F278 N was immobilized and the obtained immobilized lipase SMG1-F278 N showed excellent storage stability.Subsequently,through the screening of acyl acceptors,optimization of acyl acceptor addition methods,screening of reaction mediums,optimization of reaction medium addition amounts,and investigation of the effects of reaction parameters on the deacidification,a novel and highly efficient deacidification process for high-acid rice bran oil was established.After 6 h of reaction,the content of free fatty acids in high-acid rice bran oil decreased from 25.14% to 0.05%.After purification,the obtained final product consisted of 97.76% triacylglycerols,2.16% diacylglycerols,and 0.08% fatty acids and both the acid and peroxide value of the final product reached the grade one standard of edible oil.Interestingly,after enzymatic deacidification and low temperature molecular distillation for purification,the oryzanol content in rice bran oil increased from 18.6 g/kg to 27.8 g/kg.The accumulation fold of oryzanol reached 1.5 which was the highest value reported thus far.Afterwards,to investigate the feasibility and application potential of the developed novel enzymatic deacidification process,we developed a biorefining process for high-acid rice bran oil through combining enzymatic degumming with enzymatic deacidification.The developed biorefining process was employed for biorefining high-acid rice bran oil and the effect of each refining process on the retention of oryzanol and phytosterol was investigated.The acid and peroxide value of the final product reached the grade one standard of edible oil and the retention rate of oryzanol and phytosterol reached 71.67% and 59.2%,respectively,achieving moderate refining of high-acid rice bran oil.Although the established highly efficient deacidification process showed great potential in deacidification of high-acid rice bran oil that rich in long-chain fatty acids,its potential in deacidification of high-acid marine fish oils that rich in EPA and DHA is still unknown as the employed enzyme showed different specificities towards different fatty acids during deacidification.Thus,we further investigated the application potential of the established deacidification process above,and an efficent deacidification process for the deacidification of high-acid squid visceral oil was established.After 36 h of deacidification,the fatty acid content in high-acid squid visceral oil decreased from 13.84% to 0.06%.The deacidified high-acid squid visceral oil was further purified by molecular distillation and the obtained final product contained 99.41% triacylglycerols which contained 36.34% n-3 polyunsaturated fatty acids.Concurrently,the recovered by-product of fatty acid ethyl esters contained 46.29% n-3 polyunsaturated fatty acids and was further concentrated.After concentration,the n-3 polyunsaturated fatty acid content was elevated to 89.26%.The obtained n-3 polyunsaturated fatty acids-rich ethyl esters were used as substrates for the synthesis of n-3 polyunsaturated fatty acids-rich triacylglycerols through Novozym 435-catalyzed transesterification combining with immobilized lipase SMG1-F278N-catalyzed ethanolysis or Novozym 435-catalyzed esterification combining with immobilized lipase SMG1-F278N-catalyzed hydrolysis.Finally,the obtained final product contained more than 98.5% triacylglycerols and the obtained triacylglycerols contained more than 88.44% n-3 polyunsaturated unsaturated fatty acids.Through this process,the deacidified by-product achieved high-value utilization.High-acid waste cooking oils are common high-acid industrial raw oils.This study continued to investigate the application potential of immobilized lipase SMG1-F278 N in the deacidification of high-acid waste cooking oils using methanol as an acyl acceptor.Under the optimized conditions,the fatty acid content in high-acid waste cooking oils decreased from 28.69% to 0.05%.The immobilized lipase SMG1-F278 N showed excellent operational stability during deacidification of high-acid waste cooking oils and could be used for 20 consecutive batches without significant loss in activity.Subsequently,the obtained deacidied product was further transesterified by sodium hydroxide for the production of fatty acid ethyl esters and 98.24% fatty acid methyl esters were obtained after 20 min of reaction.This study extends the application area of the novel deacidification approach.In summary,in this study,a highly efficient deacidification enzyme preparation for highacid oils and fats was obtained through screening,rational design,and immobilization.Besides,this study established efficient enzymatic deacidification processes for high-acid oils and fats that are derived from different sources.The fatty acid content of the deacidified product could decrease to less than 0.06%.Moreover,based on the established enzymatic deacidification process for the deacidification of high-acid oils and fats,a biorefining process for high-acid rice bran oil,a high-value utilization process for high-acid squid visceral oil,as well as a novel twostep process for the production of biodiesel from high-acid waste cooking oils were developed.Overall,the established enzymatic deacidification processes allow mild reaction conditions,high catalytic efficiency,and the acid value of the deacidified products meets the grade one standard of edible oil.Most importantly,the established enzymatic deacidification approaches have great potential for industrial application.