Study On The Influence Of Trans-2-nonenal On Beer Freshness And Regulative Mechanism

Freshness is one of the most important indexes to evaluate beer drinkability and indicate beer quality.There is a big gap between the freshness of Chinese beer and that of the international brands.This is mainly due to the lack of core technology for aging flavor control in domestic beer industry.Beer is a complicate fermentation product with various type,which makes it difficult to illustrate the aging mechanism,to control the aging,and therefore to keep the freshness.In this study,the key aging substance that impacts the freshness of Chinese light Lager beer was revealed as trans-2-nonenal（T2N）.And the T2N formation pathway was analyzed with enzymology,isotope labeling and metabolomics.In addition,the key factors that influence T2N formation were determined.Finally,a set of freshness control techniques were established and applied in industrial production.The main results were as follows:A total of 270 beer samples of different domestic brands were gathered from the market for freshness analysis using a combination of headspace solid-phase microextraction and gas chromatography mass spectrometry（HS-SPME-GC-MS）flavor detection technology and the national professional judges.It was found that cardboard flavor mainly contributed to the caused aging flavor defect of Chinese light Lager beer.The lipid oxidation product,T2N,was the key aging substance.Furthermore,the synergistic effects of T2N and other aging flavor substances,such as 3-methional,?-nonanolactone,3-methyl butylaldehyde,could aggravate the aging degree of beer.When stored under harsh conditions such as high temperature and bump or for a long period,the T2N concentration in beer significantly increased,which would cause aging and poor freshness of beer.A control standard of T2N concentration of beer(<0.15?g?L^-1)was therefore established using stepwise linear regression to guarantee beer freshness.The lipoxygenase（LOX）inhibitor could influence the T2N concentration in wort to some extent,depending on the malt variety used.The total reduction rate of aging precursors in wort was 31%,22%and 9%,when Copeland,Gairdner,and Ganpi 4#malt was used,respectively.It was therefore speculated that non-enzymatic oxidation pathway played a dominant role in generating T2N.In addition,beer was brewed using the LOX-less malt and common Copeland and Gairdner malt,respectively.Compared with the control beer,the beer brewed with LOX-less malt contained less T2N when stored in cool environment for a short period.However,as storage temperature and period arose,the concentrations of T2N and other aging aldehydes in beer brewed with the LOX-less malt were higher than the control beers.The results suggested that either the enzymatic or non-enzymatic oxidation pathway could play a major role on T2N formation in different storage periods of beer.To illustrate the T2N generating pathway,linoleic acid labeled with ¹³C isotope was used during mashing process and the metabolic flux was traced with targeted metabolomics.A new metabolic pathway was found（linoleic acid---cis-9-hexadecenal---decanol---T2N）using high-performance liquid chromatography-tandem quadrupole time of fligh mass spectrometry（HPLC-QTOF-MS）,which supplemented and improved the T2N formation mechanism.The metabolic groups of malts with different LOX activities were compared with untargeted metabolomics.The compounds with significant differences which may be involved in T2N generation were chemically identified,including tyrosine and valine.The solution model was therefore constructed to confirm the addition reaction of T2N and amino acid to form aging precursor.Furthermore,the effects of mashing and boiling process on T2N and amino acid addition reaction was qualitatively and quantitatively analyzed,which laid a theoretical foundation for process control.The LOX activities of 181 malts that produced with 13 barley varieties by 34 malt factories were analyzed.The results showed that the LOX activities were significantly different among malts.And to a certain extent,LOX activity could influence malt quality,beer T2N concentration and freshness.Compared with malt LOX activity,malt nonenal poteintial（NP）showed higher correlation with T2N concentration and freshness scores.Malt LOX activity and NP value could be used as the indicators to evaluate malt freshness and predict beer freshness.As monophenols were found to be capable to inhibit malt LOX activity,whereas metal ions could promote LOX activity,the lipid oxidation impacting indexes were increased.With the 11 impacting indexes,a mathematical model for evaluating malt lipid oxidation degree were established using principal component analysis,providing the theory basis for brewmasters to screen malt with low lipid oxidation capability.The key factors that influence malt LOX vitality and the concentrations of T2N and other aging precursors during malting and mashing process were studied.New approaches to control LOX activity in malting process and T2N concentration in mashing process were developed.The malt LOX activity was incorporated into the quality evaluation system.And the lipid oxidation control system from raw materials to the sales terminal was constructed and applied to industrial production.The malt LOX activity decreased by 52%,the T2N concentration in beer decreased by 48%on average,freshness score increased by 19%and the fresh-keeping period increased from 90 days to 180 days,which greatly enhanced beer quality.