Study On The Formation Mechanism Of Key Off-flavor Compounds And The Effect Of Processing On Soymilk Flavor

Soymilk has become the most familiar breakfast food for residents because of its nutrition,high quality and low price.Meanwhile,it can also be further processed to make soybean products.However,the beany flavor of traditional soymilk restricts its application in new plant-based products.Most of the volatile compounds are formed in pulping processing.Similar to the reaction of all plant cells when subjected to external environmental stress,complex chemical and biochemical changes also occur in the soybean processings,involving different reaction types(enzymatic,non-enzymatic),enzyme types(hydrolase,oxidase,lyase),precursors(lipid,protein,carbohydrates)and reaction kinetics.At present,the formation mechanism of volatile compounds in soybean processing is still poorly understood.In this paper,the formation mechanism of key off-flavor compounds in soymilk and the effect of processings on soymilk flavor were studied in order to provide theoretical basis for the improvement of new processing technologies and high-quality soybean-based plant protein products.The main research contents and results are as follows:Firstly,the formation mechanism of C6 aldehydes(hexanal and(E)-2-hexenal),the representative beany flavor compounds,was investigated.It was found that PLA₂was another key enzyme besides LOX and HPL from the experiments of addition of enzyme inhibitors.The changes of fatty acid contents in lipid species of cotyledon and hypocotyl before and after soaking and grinding were analyzed by TLC combined with UPLC Q–TOF/MS.It was found that LA and LEA at sn-2 position in PLPC,SLPC,PLn PC and SLn PC were the main precursor lipids for C6 aldehyde.The relationship between different reaction behaviors of soybean during soaking and grinding and the distribution of LOX at the subcellular level was investigated.It was found that only membrane-LOX worked during soaking and the consumed LA or LEA were completely converted to hexanal or(E)-2-hexenal.However,cytoplasm-LOX was also involved in grinding and the substrate-product conversion rate is only about 60%.The content changes of total hydroperoxides,PCOOH,FAOOH and C6aldehyde with time were studied.It was found that LOX reaction occurred almost instantaneously and ended within 10 s,PLA₂ and HPL catalyzed reaction ended within 60 s,and the hydrolysis of PCOOH by PLA₂ was the rate-limiting step of C6 aldehyde formation.The results of enzymatic kinetics showed that LOX had the highest catalytic efficiency,K_catwas 8.70 s^-1,and the catalytic efficiency of PLA₂ and HPL had little difference.Subsequently,the related lipid free radicals in soymilk were further studied.Five lipid free radical adducts were found in soymilk,which were DMPO-X·,DMPO-L·,DMPO-R·,DMPO-LOO·and DMPO-RO·.The effects of soaking soaking,temperature and p H value on lipid free radicals were investigated.The total amounts of spins increased as soaking temperature increased from 4 to 50°?and soaking p H increased from 3 to 9.Prolonged soaking time at 50°?resulted in a higher signal intensity of DMPO-R·than that of DMPO-LOO·.The changes of lipid free radicals in simulation systems of LOX-deficient soybean and of LA–LOX–soybean exudates were investigated.The correlation between lipid free radicals and LOX was found and there was a positive correlation between loss of endogenous substances and lipid free radicals during soybean soaking.Then,the formation mechanism of(E)-2-heptenal during soybean soaking was studied.Through changing soaking conditions,it was found that(E)-2-heptenal formed through non-enzymatic reaction and was independent of LOX and HPL activities,but related to the concentration of Fe²⁺and chelating agent.In dry matter,the content of(E)-2-heptenal in hypocotyls was much higher than that in cotyledons.A phospholipid hydroperoxide was purified from hypocotyls by silica gel chromatography and amino propyl chromatography and was identified by HRMS and NMR as 1-palmitoyl-2-(12-hydroperoxadioyl)-sn-glycerol-3-phosphatidylethanolamine(12-PEOOH).The effects of p H and Fe²⁺concentration on the degradation of 12-PEOOH were investigated.The results showed that at p H 5.8,the degradation rate of 12-PEOOH was linearly positively correlated with the concentration of Fe²⁺,but the molar conversion of(E)-2-heptenal was 74%and remained unchanged,independent of the concentration of Fe²⁺.The formation of LOO·and R·radicals showed similar p H and Fe²⁺concentration dependence to that of(E)-2-heptenal.The formation mechanism of two C8 alcohols,1-octen-3-ol and 3-octanol,during soybean soaking was studied.It was found that C8 alcohols were not from the lipids the precursors and were independent of LOX pathway enzymes.Studies have shown that more than 90%of C8 alcohols were derived from soybean hypocotyl.1-Octen-3-yl-?-primeveroside was purified from hypocotyls by macroporous adsorption resin chromatography,reversed-phase chromatography and preparative reversed-phase high performance liquid chromatography.Meanwhile,a newly 3-octanyl-?-primeveroside was discovered and purified.NMR,HRMS and GC–MS were used to confirm the structures of two precursors of C8 alcohols as 1-octen-3-yl 6-O-?-D-xylopyranosyl-?-D-glucopyranoside and 3-octanyl 6-O-?-D-xylopyranosyl-?-D-glucopyranoside,respectively.A glycosidase was existed in soybean hypocotyls and its optimal temperature and p H were 50°?and 5.5,respectively.The hydrolysis activity of glycosidase was 5?11 times that of almond?-glucosidase.The non-enzymatic hydrolysis kinetics of C8 alcohol-?-primeveroside was investigated.It was found that non-enzymatic hydrolysis kinetics conforms to the first-order kinetic model.At p H 4.0,the E_a of 1-octen-3-yl-?-primeveroside and 3-octanyl-?-primeveroside were 30.41 k J/mol and 38.08 k J/mol,respectively.Finally,the effects of dry dehulled hot grinding,wet hot grinding and anaerobic grinding on off-flavors,hydroperoxides and free radicals of soymilk were investigated.The results showed that C8 alcohols and(E)-2-heptenal could be removed by dry processing,but lipids in dehulled soybean were prefer to be oxidized during storage.The contents of1-octen-3-ol in wet and anaerobic processings was higher.Soaking in alkaline p H and increasing washing times of soaked soybean were beneficial to remove 1-octene-3-ol.All three methods could significantly inhibit the LOX reaction The results of gas chromatography-olfaction-mass spectrometry conbined with sensory evaluation showed that the flavor profile of dry soymilk was fatty,grass and mushroom flavor,the flavor profile of wet soymilk was mainly grass and mushroom flavor and the flavor profile of anaerobic soymilk were fruity and mushroom flavor.In summary,flavors of soymilk prepared by dry and anaerobic pulping processings were better.