Study On Mechanism Of Flavor Esters Formation By Microorganisms During The Fermentation Of Suanyu

Biological fermentation not only increase the utilization rate,improve the texture and colour and lustre,prolong the storage period,also can produce unique ester aroma and dispel the earthy aroma,which has become a hot research topic in the field of freshwater fish processing in recent years.Rich esters were dectected in Suanyu added with autochthonous mixed starter cultures.However,the mechanism of microbial aroma production and its synthetic pathway were not clear.Effects of autochthonous starter cultures on the overall aroma,the relationship between ester compounds and each strain,biosynthesis pathway of flavor acetate ester compounds,presence and influencing factors of microbial esterase activity were studied to elaborate the flavor formation mechanism.This study has important academic significance to help understand the mechanism of microbial aroma producing and how to improve and enhance the microbial aroma producing ability through the metabolic regulation,but also provides theoretical guidance for application of fermentation flavoured technology in the freshwater fish processing industry.Effects of autochthonous starter cultures on the volatile flavor compounds of Suanyu were studied.Physicochemical properties and volatile flavor compounds of Suanyu with a single strain(S1: Lp-120,S2: Sx-135,S3: Sc-31;fermentation for 6 weeks)and without inoculation(NS1: fermentation for 6 weeks,NS2: fermentation for 9 weeks,NS3: fermentation for 13 weeks)during the fermentation were determinated,combined with odor activity value and statistical means,to explore the influence of different strains on the overall flavor of Suanyu,and to reveal the relationship between different flavor esters with each strain.Volatiles were extracted by headspace-solid phase micro extraction(SPME)and analyzed by gas chromatographic-mass spectrometry technology(GC-MS).The results showed that a total of 88 volatile compounds were identified in the inoculation group s(S1,S2 and S3)and control groups(NS1,NS2 and NS3),and 46,57,53,57,60,59 types were from S1,S2,S3,NS1,NS2,and NS3,respectively.Esters and alcohols were the main components of volatiles,accounting for over 50 percentage points in all samples.The highest content of esters(3034.54 ?g / kg)was observed in S1 inoculated with L.plantarum 120,while the highest content of alcohols(2164.53 ?g / kg)and ketones(379.98 ?g / kg)were detected in S3 inoculated with S.cerevisiae 31.The content of acids and aldehydes were lower in inoculated samples.Ethyl acetate,isoamyl acetate,1-octen-3-ol and aliphatic aldehydes(C5-C9)with the highest OAVs and ROC were the major contributors to the overall aroma of Sunayu and furans(2-pentylfuran)followed.Principal component analysis(PCA)revealed that the volatile composition was primarily influenced by the inoculation of starter cultures.L.plantarum 120 and S.xylosus 135 could accelerate fermentation.Effects of autochthonous starter cultures on flavor ester compounds of Suanyu were studied.Microbial colony counts and flavor ester compounds of Suanyu with or without a single strain(S1: Lp-120,S2: Sx-135,S3: Sc-31)during the fermentation were determinated,compared to flavor ester compounds of Suanyu inoculated with mixed starter culture(MS:Lp-120/ Sx-135/ Sc-31),to reveal the relationship between flavor esters and each strain.The results showed that esters of Suanyu just contained acetate esters and ethyl esters.Ethyl acetate and ethyl palmitate,only found in Suanyu before fermentation,had different degrees of increase in content after fermentation,while other esters disappeared only after fermentation.At the end of fermentation,the contents of esters in five groups of Suanyu(NS,S1,S2,S3 and MS)were 307.78 ?g / kg,874.12 ?g / kg,472.07 ?g / kg,495.01?g / kg and 5284.63 ?g / kg,respectively.The types of esters detected in five samples were 7,8,10,12 and 11,respectively.The content and types of esters in all inoculated samples were higher than those in the sample without inoculation.Principal component analysis(PCA)revealed that L.plantarum 120 was related to the formation of acetate esters composed of C2-C6 aliphatic alcohols and 2-phenylethyl acetate,whilst S.xylosus 135 and S.cerevisiae 31 was related to the formation of ethyl esters composed of C6-C16 fatty acids.The release of free fatty acids by microbial lipolysis was investigated.The changes of free fatty acids in Suanyu during the fermentation were analyzed,and a hydrolysis system contained fish oil and a hydrolysis system contained long chain fatty acids(myristic acid C14:0,palmitic acid C16:0 and oleic acid C18:2,respectively)were established to investigate the ability of different strains to release medium-chain and short-chain fatty acids by lipid degradation.The results showed that the free fatty acids in Suanyu during the fermentation increased firstly and then decreased,and reached the maximum value of 375.70 mg / 100 g fish in medium stage.Fish oil hydrolysis system experiment displayed that the FFA contents of systems added with S.xylosus 135 and S.cerevisiae 31 were significantly higher than the control group,whilst no significant difference exist between the system added with L.plantarum 120 and the control group(P > 0.05),and the carbon number of released FFA was greater than 12.These suggested that Sx-135 and Sc-31 could release long chain fatty acids via lipid hydrolysis.Myristic acid and palmitic acid hydrolysis experiments showed that a large number of short-chain and medium-chain fatty acids were detected in all inoculated systems,including acetic acid,pentanoic acid,hexanoic acid,octanoic acid,heptanoic acid,azelaic acid,capric acid and lauric acid,indicating that saturated long-chain fatty acids can be transformed into in the medium chain and short-chain fatty acids under the action of microorganisms.Moreover,linoleic acid hydrolysis experiment showed that no medium-chain and short-chain fatty acids were detected in all systerms,indicating unsaturated long chain fatty acids cannot be transformed into short-chain fatty acids by the strains.Therefore,the microbial lipid metabolism in Suanyu is that firstly long-chain free fatty acids were released via lipid hydrolysis,and then long-chain free fatty acids were transformed into medium-chain and short-chain fatty acids.Acetate esters biosynthesis pathways by strains and the influences of alcohol and fatty acid structure on ester synthesis were studied.The biosynthesis of acetate esters by strains was determined by the reaction systems: acetic acid and ethanol/isoamyl alcohol(esterification),glyceryl triacetate/acetyl-CoA and ethanol / isoamyl alcohol(alcoholysis).And the effects of fatty acid with different chain lengths on the synthesis of ethyl esters and the effects of alcohol with different chain lengths on the synthesis of acetate esters were investigated.The results showed that the biosynthesis pathway of L.plantarum 120 was esterification and alcoholysis,while the biosynthesis pathway of S.xylosus 135 and S.cerevisiae 31 was esterification.The ester-synthesizing activity of L.plantarum 120 via alcoholysis was higher than that via esterification at high p H value,while ester-synthesizing activity of each strain via esterification was higher than that via alcoholysis.Moreover,the ester-synthesis activity of L.plantarum 120 was higher than that of Sx-135 and Sc-31.At the range of carbon number from 2-6,microbial ester-synthesis activity increased with the increase of aliphatic alcohol carbon number,whilst microbial ester-synthesis activity decreased with the increase of fatty acid carbon number(C > 8).The effects of factors on esterolytic activities of the strains were studied.Hydrolysis activities of esterase from intracellular and extracellular fractions of the strains using different pNP-esters as substrate and at different values of p H ? Aw and temperatures were experimentally determined,and effect of inoculation on Suanyu esterase activities was also conducted.The results showed that all microflora exhibited activity in both intracellular and extracellular fractions against pNP-acetate,pNP-butyrate and pNP-octanoate,also highest activity was observed in the intracellular fraction with pNP-octanoate.Esterase activities of all strains were positively inhibited by p H and Aw,with exception of L.plantarum 120 exhibiting higher activity in intracellular fraction with decreasing Aw.However L.plantarum 120 and S.xylosus 135 were negatively affected by temperature,while a positive influence was observed in intracellular faction of S.cerevisiae 31.This paper defined the characteristic flavor compounds of Suanyu and the relationship between each strain and esters,confirmed that each strain was capable of releaing free fatty acids by lipolysis,illustrated the biosynthetic pathyway of flavour acetate esters by each strain and the influence of alcohols and fatty acids chain lengths on microbial ester-synthesis,and factors affecting esterolytic activity of each strain,preliminarily revealed the mechanism of esters formation by microorganisms in Suanyu.