Mechanism Of Methanol Production And Regulation In Apple Wine

Apple materials are rich in pectin,and pectin can generate methanol under the action of pectinases endogenously existed in apple juice and inoculated yeast during fermentation,which affects the apple wine safety and quality.Therefore,it is meaningful to further decrease the methanol production and improve the wine quality by exploring effective methods and deep understanding the mechanism of methanol production.A large part of fresh apples is stored in cold storage for a long period to keep them available for further utilization,but apple storage period affects the fruits quality and thus influences the wine quality.Here,the fresh Fuji and Ralls Janet apples were stored for five months,the changes of apple texture properties,pectin structure and pectinase activity during apple storage were tracked to identify the factors affecting the production of methanol in apple wine.Subsequently,the relationship between pectinase activity and methanol production was illustrated by comparing the activities of pectin methylesterase（PME）,pectin lyase（PL）and polygalacturonase（PG）during spontaneous fermentation,inoculated fermentation and inoculated fermentation combined with Ca Cl₂treatment.Furthermore,bentonite,Ca Cl₂ and the combination of bentonite and Ca Cl₂ were applied to treat apple juice to explore a method that could decrease methanol production effectively.Finally,the pectin in bentonite and Ca Cl₂-treated apple wine was separated and purified to analyze the pectin structure and explore the mechanism of bentonite and Ca Cl₂affecting the methanol and pectin content in apple wine,thus revealing the effect of pectin structure on the methanol production.The main results are as follows:（1）With the extension of apple storage period,the water-soluble pectin content increased and its structure changed,and the methanol content in apple wine increased generally.Apple Ralls Janet softened by one-month storage,exhibiting the increasing water-soluble pectin by692.44 mg/L in juice and methanol by 82.51 mg/L in wine.However,apple Fuji maintained firm during the five-month storage,demonstrating the increasing water-soluble pectin by124.42 mg/L in juice and methanol by 4.11 mg/L in wine.The effect of apple storage period on methanol production was different between Fuji and Ralls Janet due to their different pectin structures.Triple-helix structure existed in pectin Fuji,but it disintegrated gradually during storage and disappeared by five-month storage.Un-helix structure existed in pectin Ralls Janet,which was beneficial to the action of pectin methyl esterase,and thus methanol increased dramatically.With the extension of Fuji and Ralls Janet storage period,their activities of PME,PL and PG increased firstly and then decreased,pectin molecular weights decreased,chains were cleaved,side chains were degraded,and the esterification degree was changed,which thus influenced the methanol production in apple wine.（2）Apple storage affected the organic acids and volatiles in apple wines by affecting the organic acids and volatiles in apple juice and yeast metabolism simultaneously.By 5-month storage of Fuji and Ralls Genet apples,L-malic acid in juices decreased by 2269.69 mg/L and1755.78 mg/L,respectively;succinic acids in juices decreased by 582.25 mg/L and 943.10mg/L respectively.Correspondingly,L-malic acid decreased by 665.73 mg/L and 987.45 mg/L in Fuji and Ralls Genet apple wine,respectively;succinic acids decreased by 256.85 mg/L and232.40 mg/L,respectively.The increased p H of apple juice during apple storage affected yeast metabolism,and succinic acid from fermentation was positively correlated with the p H of juices（r=0.709*）.By fermentation,citric acid increased by 1443.78 mg/L-2854.72 mg/L,and its proportion to total organic acids was positively correlated with the p H of wines（r=0.757**）.Six volatiles were generated in Ralls Genet,but only one volatile was generated in Fuji during storage.Isobutylacetate,isoamylacetate and hexylacetate were increased by 32.80,20.54 and7.99 times by 3-months storage.Meanwhile,isoamylol,1-hexanol and 6-methyl-5-hepten-2-ol in Ralls Genet juice rose up by 11.43,3.92 and 137.37 times during storage.Esters were mostly affected by storage of apple wines,its content decreased by 104.05 mg/L in Fuji apple wine and59.40 mg/L in Ralls Genet apple wine.The content of volatiles in apple wine was also affected by the p H of apple juice.Twelve volatiles in Ralls Genet apple wine were significantly correlated with the p H of apple juice,but only five volatiles in Fuji apple wine were significantly correlated with the p H of the juice.（3）The apple wine fermented with Ralls Genet apples has the risk of excessive methanol.The following experiment was carried out with Ralls Genet apples.The activities of PME,PL and PG,the content of methanol,pectin and the molecular weight of pectin among spontaneous fermentation,inoculated fermentation and inoculated fermentation combined with Ca Cl₂treatment were compared.It was found that the changes of pectinase activities,methanol production and pectin breaking were different due to the differing fermentation ways.The three fermentation methods strongly inhibited the activities of PME,PL and PG,the pectinases retained in spontaneous apple wine were the lowest（PME 3.41%、PL 9.46%、PG 9.17%）,and the methanol in spontaneous apple wine was the lowest（117.37mg/L）.However,the pectinases retained in Ca Cl₂-treated apple wine were the highest（PME 5.84%、PL 17.71%、PG 10.31%）and the methanol in Ca Cl₂-treated apple wine was the highest（137.87mg/L）.Spontaneous fermentation withstood a long lag period,then its activities of PME,PL and PG decreased significantly,the methanol content increased,the pectin content decreased,and the pectin chain broke from the third fermentation day.Spontaneous fermentation was conducive to the cleavage of pectin to produce oligo-galacturonic acid and free Gal A with Mp lower than3 k Da continuously,its further degradation led to the lowest pectin content in wine.The activities of PME,PL and PG decreased significantly,methanol content increased,pectin content decreased,and the pectin chain broke from the first fermentation day during inoculated fermentation.The Ca Cl₂ treatment promoted the methanol production and the cleavage of pectin chains via retaining the PME and PL activities.Because the pectin with Mp around 3.07k Da was retained by Ca Cl₂,thus the highest pectin content was found in wine CC0（160.69mg/L）,which was 95.47 mg/L higher than that in wine from inoculated fermentation,and107.03 mg/L higher than that in wine from spontaneous fermentation.（4）Yeast and lactic acid bacteria（LAB）existed in Ralls Genet apple juice natively.The LAB multiplied during spontaneous fermentation and reached 7.89 lg CFU/m L,and then triggered malolactic fermentation（MLF）,thus malic acid decreased by 3880.52 mg/L and lactic acid increased by 4787.55 mg/L.The four-day lag period in spontaneous fermentation led to the later production of acetaldehyde and higher alcohols and their lower content in apple wine.The inoculated yeast（Saccharomyces cerevisiae）inhibited LAB,but the Ca Cl₂ treatment promoted the multiplication of the yeast and LAB,especially Leuconostoc pseudomesenteroides.Yeasts were key to produce the citric acid,acetaldehyde and 3-methyl-1-butanol in apple wine;while bacteria were closely related to the formation of lactic acid,acetic acid and ethyl acetate.It suggested that the yeast and LAB in apple juice are key factors affecting the organic acid and volatiles in apple wine,and L.pseudomesenteroides can work as candidate to reduce the L-malic acid and citric acid in apple cider.（5）The bentonite treatment could effectively decrease the methanol content and improve the sensory characteristics of apple wine.Among them,inoculation immediately after bentonite treatment（B0）decreased 46.03%～79.20%of methanol.Inoculation after bentonite clarification for 24 h（B24）or treated with bentonite and Ca Cl₂（B&CC）also decreased the methanol content,but the reduction effect was not as good as that of B0.Ca Cl₂ treatment,especially fermented at 25 ^oC,elevated the methanol content and changed the profile of individual organic acids.The wine treated by B&CC0 at 25°C showed dramatic changes in individual organic acids.The content of L-malic acid and succinic acid was only 2.22%and6.29%of the control,respectively,while the lactic acid content was 17.72 times that of the control.It suggested that B0 was the most effective way to decrease the methanol content and maintain the wine quality.（6）The bentonite treatment decreased the pectin content and PME activity,affected the pectin structure,and thus decreased the methanol content in apple wine.The bentonite adsorbed pectin,and the pectin content decreased by 291.81 mg/L in apple wine.The bentonite also adsorbed protein,and the protein content decreased by 41%in apple wine and the PME activity decreased,which avoided the breakdown of pectin chain,led to 98.81%of pectin clustered in the peak molecular weight around 235.59 k Da.The long pectin chain and high branching extent affected the PME activity and decreased the methanol production.The bathochromic shift at1749 cm^-1 in FT-IR,increasedζ-potential,and the characteristic peaks of XRD appearing at20.8^o and 12.4^o in bentonite-treated pectin proved that the bentonite adsorbed pectin and affected the pectin structure.The pectin chains broke and mainly clustered to the peak molecular weight around 2.76 k Da and 3.21 k Da.The Ca Cl₂ treatment decreased RG-I region and reduced the steric hindrance of Ca²⁺crosslinking with pectin chain.In addition,the Ca²⁺promoted the dissociation of the esterified methoxy group and provided the action site for the cross-linking of Ca²⁺and pectin chains.The bathochromic shift at 1734 cm^-1 in FT-IR and the decrease ofζ-potential in Ca Cl₂-treated pectin proved the cross-linking of Ca²⁺and pectin chain.