低甲醇酿酒酿酒的诱变筛选与甘蔗酒的发酵

Research ArticleReceived:9July2014Revised:3September2014Accepted article published:10September2014Published online in Wiley Online Library: ()DOI10.1002/jsfa.6908Mutation breeding of Saccharomyces cerevisiae with lower methanol content and the effectsof pectinase,cellulase and glycine in sugar cane spiritsMing-Hua Liang,a,b Ying-Jie Liang,a Xiao-Na Wu,a Shi-Shui Zhou a*and Jian-Guo Jiang b*AbstractBACKGROUND:To decrease the methanol content of the sugar cane sprits,mutagenesis of ultraviolet(UV)coupled with diethyl sulfate(DES)was used to generate a mutant of Saccharomyces cerevisiae with lower methanol content.Meanwhile,the effects of the additions of pectinase,cellulase and glycine on the production of methanol in sugar cane spirits were evaluated. RESULTS:After mutagenesis of UV coupled with DES,a mutant S.cerevisiae DU9with low production of methanol (97.3±1.7mg/L)was selected,with a12.3%decrease of that of S.cerevisiae D4only with DES treatment,and with a27.8% reduction of that of the strain without any treatment.Pectinase and cellulase significantly increased the methanol levels of the sugar cane spirits.The results showed that there was linear relationship between glycine(concentration within0∼0.9g/L)and methanol in sugar cane sprits and the linear equation was y=104.7×−4.79with the conversion rate of glycine conversion to methanol as24.56%.CONCLUSION:Mutagenesis of UV coupled with DES is an efficient way to generate a mutant of S.cerevisiae with lower methanol content.Also,it is necessary to control the additions of pectinase,cellulase and glycine in the fermentation medium,and other unknown ways to generate methanol metabolic pathway in yeasts may need further study.©2014Society of Chemical IndustryKeywords:Saccharomyces cerevisiae;diethyl sulfate(DES);ultraviolet(UV);methanol;glycine;sugar cane spirits; INTRODUCTIONSugar cane spirits are defined as beverages with an alcohol contentof38–54%(v/v)at20∘C.Sugar cane spirit production involves theextraction of sugar cane juice,fermentation and distillation.Alongwith sugars such as sucrose,glucose and fructose,sugar canejuice also consists of minerals such as potassium and magnesium,amino acids(proline,aspartic acid,asparagine,serine,histidineand glycine are the most representative amino acids),1vitaminB 3,organic acids and small amounts of cellulose,hemicelluloseand pectin,2and may have immense health benefits for people suffering from a variety of conditions.Although sugar cane juice has high sugar content,it is good for diabetic patients because of to its low glycemic index.Methanol has been reported to be associated with harmful health effects such as headache,fatigue,nausea,visual impair-ment or complete blindness,3and its presence in alcoholic beverages is strictly controlled.Here we used butyl acetate as internal standard with the method of headspace gas chromatog-raphy(HS-GC)to determine the identity and concentration of methanol in sugar cane spirits.As the sugar cane spirits sample matrices are complex,the GC method cannot be applied directly. These sample matrix problems can be largely eliminated by the proposed HS sampling technique,coupled with analysis by GC.In addition,sample clean-up and pre-concentration are not necessary,and tedious and error-producing steps can be elimi-nated.Thus methanol in sugar cane spirits can be analyzed easily and quickly by HS-GC.4In sugar cane spirits,methanol is formed mainly from sugar cane bagasse particles,a type offiber that contains pectin and cellulose,which remains in the juice after filtration.5Moreover,the use of commercial macerating enzymes like pectinase and cellulase in winemaking is a common and well-known practice.These preparations degrade the structural ∗Correspondence to:Shi-Shui Zhou,School of Biological Science and Engineering,South China University of Technology,Guangzhou510006, China.E-mail:hgzhouss@;or Jian-Guo Jiang,College of Food Science and Engineering,South China University of Technology,Guangzhou 510640,China.E-mail:jgjiang@a School of Biological Science and Engineering,South China University of Tech-nology,Guangzhou510006,Chinab College of Food Science and Engineering,South China University of Technology,Guangzhou510640,ChinaJ Sci Food Agric(2014)©2014Society of Chemical Industry Ming-Hua Liang et al. polysaccharides of the cell walls,facilitating the release of pheno-lic compounds.Enzymatic treatment can also cause a significantincrease in the methanol concentration of wine.Several investiga-tors have reported the influence of pectinase enzyme treatmenton the methanol concentrations of wines.3,6,7As for glycine,thiscan be from sugar cane juice or yeast extract,which is one elementof the medium for Saccharomyces cerevisiae.Glycinefirst generatesmethylamine by glycine decarboxylase,then reacts with nitrousacid to produce methanol.The reactions are as follows:H 2N—CH2—COOH→CH3NH2+CO2;CH3NH2+HNO2→CH3OH+N2+H2OThe relationship between glycine content in the fermentation medium and the production of methanol in sugar cane spirits was first researched in this study.Mutation breeding is a common method to obtain high muta-tion efficiency.8–10Chemical mutagenesis is a useful tool to study the physiological processes in microorganisms.Diethyl sulfate (DES)is a powerful alkylating agent.Breaking of the DNA double helix,as result of the hydrolysis of alkylated phosphate groups, may cause chromosome breaks and lethality.11Additionally,ultra-violet(UV)light has strong genotoxic effects to produce DNA damage and induce mutations.In this study,a mutant of Saccha-romyces cerevisiae,which was obtained by DES and UV mutation, was used for fermentation of sugar cane spirits to decrease the methanol content.A sugar cane spirit was obtained by the dis-tillation of fermented sugar cane juice with wort as a source of supplementary substrate.Meanwhile,the effects of adding pecti-nase,cellulase and glycine on the production of methanol were evaluated.MATERIALS AND METHODSMaterialsSaccharomyces cerevisiae was kept in the labs of South China University of Technology.Methanol and ethanol are high-performance liquid chromatography(HPLC)grade.Butyl acetate is analytical grade.Dansyl chloride(DNS-Cl)and glycine were from Sigma(St Louis,MO,USA).The medium for S.cerevisiae cultivation was yeast extract peptone dextrose(YPD):2%glucose,1%yeast extract,2%peptone.Apparatus and operations of HS-GCHS-GC measurements were carried out with an automatic headspace sampler(DANI HS86.50,Italy)and a GC system(Agilent GC7890A,USA)equipped with aflame ionization detector(H2 30mL min−1;air350mL min−1),and a DB-FFAP capillary column (30m×0.53mm×1.00μm),operating with nitrogen carrier gas (flow rate=1.0mL min−1).Oven temperature was controlled with a temperature elevation program during analysis,which was initially set at35∘C for4min,elevated to180∘C at a rate of 20∘C min−1and maintained for5min.The temperatures at the detector and vaporizer were set at250∘C and150∘C,respectively, and50:1split ratio in split mode was used.HS operating condi-tions were as follows:25min of strong shaking for the sample equilibration at80∘C;sample loop temperature=85∘C;transfer line temperature=90∘C;vial pressurization time=12s;sample loopfill time=30s;and transfer time=6s.Analysis of methanol content in sugar cane spirits by HS-GC The methanol concentration of each wine sample was calculated by internal standard method with respect to the internal standard from response factor,so that HS-GC is qualitative(by the use of retention time)and quantitative(by the use of signal strength). The procedure to obtain a calibration curve was as follows:20% (v/v)ethanol solution was added to10mL,containing internal standard butyl acetate(300mg L−1)and different methanol con-centrations(0,100,200,400and800mg L−1),respectively.The response from the methanol peak was compared to the internal standard peak.Here RF is defined as the ratio of the peak area of the methanol to the peak area of internal standard.The results with RF on the y-axis and methanol concentration on the y-axis were then plotted,and these data werefitted to obtain a calibra-tion curve.The sample preparation and measurement procedures were as follows:10mL sugar cane spirit sample with300mg L−1 butyl acetate were added to20mL HS vials,which were then closed.The sample underwent an equilibration period to achieve vapor–liquid phase equilibrium.100μL of the HS vapor was put into the HS sampler for measurement.Mutation procedure of S.cerevisiae by DES and UVTo obtain strains with lower methanol content,the cell suspension of S.cerevisiae was treated with DES and UV irradiation.The treat-ment condition that produced a lethality of about80%was set as the mutation concentration.12For DES treatment,the suspension was treated with DES in six concentrations(0.05%,0.1%,0.2%, 0.3%,0.4%and0.5%)in phosphate buffer(pH7.2)for60min,and 25%sodium thiosulfate was used to terminate the procedure. Treated cells were suitably diluted by sterile water,and then the suspension was spread on to YPD agar plates and incubated at 28∘C for48h.For UV irradiation,the suspension was placed in sterile Petri dishes,stirred with a magnetic stirrer,and irradiated with UV light(250–280nm)15W at a distance of30cm for100, 120,140,160,240and480s.After irradiation,the plates were immediately kept away from light.The suspension was diluted appropriately and spread on to YPD agar plates,and incubated at 28∘C in darkness for48h.Lethality was computed as follows:Lethality(%)=(Number of strains before mutation−Number of strains after mutation)∕(Number of strains before mutation)×100Determination of glycine content in yeast extractA reversed-phase high-performance liquid chromatography (RP-HPLC)method with pre-column derivatization was developed for the determination of glycine in the yeast extract.13Dansyl chlo-ride(DNS-Cl)is a chromophoric reagent employed for the labeling of glycine.Glycine labeled with DNS-Cl is highly stable and shows maximum absorption at254nm with a detection time of8.9min. Glycine standard working solutions(1.5, 3.0, 4.5, 6.0,7.5and 9.0mg L−1)were used to obtain the working curve of glycine.An Agilent1100liquid chromatograph with a dual-solvent delivery system,auto sampler,column thermostat and multi-wavelength UV detector was used for the measurement.A reverse-phase C18column(Waters Sun Fire;Dp,5μm)was used for separation and the elution was performed with50mmol L−1 sodium acetate solution(pH7.2)and acetonitrile(75:25,v/v).The/jsfa©2014Society of Chemical Industry J Sci Food Agric(2014)Mutation breeding of Saccharomyces cerevisiae with lower methanol content flow rate was1.0mL min−1,the column temperature was30∘C and the detection wavelength was at254nm.Effect of pectinase on the production of methanol Pectinase(0.1,0.2,0.4,0.8and1.6mL;70U mL−1)was added to a250mLflask with mixed fermentation medium with sugar cane juice and wort(9:1,v/v),respectively,and incubated at50∘C for1h.After placing in an ice bath to terminate the reaction, the medium was pitched with S.cerevisiae(OD=1.5–1.8)and fermented at23∘C.Effect of cellulase on the production of methanolCellulase(0.2,0.4,0.8,1.6and3.2mL;30U mL−1)was added to a 250mLflask with mixed fermentation medium1with sugar cane juice and wort(9:1,v/v)and medium2withfiltered sugar cane juice and wort(9:1,v/v)(sugar cane juice wasfiltered three tofive times),respectively,and then incubated at65∘C for1h.After plac-ing in an ice bath to terminate the reaction,the medium was inoc-ulated with S.cerevisiae(OD=1.5–1.8)and fermented at23∘C. Relationship between glycine and methanol produced by S. cerevisiaeThe glycine content of the yeast extract was determined by RP-HPLC.Fermentation medium without yeast extract and fermen-tation medium with12.5g L−1of yeast extract were prepared.Then 0,0.3,0.6and0.9g L−1glycine were added to the fermentation media above,respectively.The medium was then inoculated with S.cerevisiae(4×107cfu mL−1)and fermented at23∘C for7days. Analytical procedures for sugar cane spiritsTotal soluble solids of sugar cane spirit samples were determined with the help of an ERMA hand refractometer having a range of 0–32Brix at20∘C.The alcoholic content in sugar cane spirits was estimated by portable alcohol meter.Statistical analysisValues were expressed as mean±SD of three parallel measure-ments.The significance of differences between groups was assessed by one-way analysis of variance(ANOVA).P<0.05indi-cated the presence of a statistically significant difference and P<0.01was considered highly significant.RESULTSDetermination of methanol content in sugar cane spiritsby HS-GCWith HS-GC,butyl acetate as internal standard and20%(v/v) ethanol solution as the solvent,we established an equation: Y=0.0003X+0.0042,R2=0.9993,in which Y represents RF and X represents the content of methanol.Here we give the HS-GC chromatogram of one sample of sugar cane spirits(Fig.1).For ethanol,the retention time was about4.7min;for methanol,the retention time was about3.8min;for internal standard butyl acetate,the retention time was about7.1min.Validation for the determination of methanol contentby HSGCThe repeatability of the HS-GC method was demonstrated by using the standard sample(methanol concentration=200mg L−1).A relative standard deviation(RSD)of measured methanol content is less than5.0%forfive times testing,indicating that the tech-nique of HS-GC to detect the methanol content has excellent repeatability and high precision and accuracy.Screening for S.cerevisiae with low methanol content by DES and UVEffects of mutagen dosage on lethality of S.cerevisiae are shown in Fig.2.Figure2(A)shows that the lethality increased with increas-ing DES concentration.The treatment condition that produced a lethality of about80%was set as the mutation concentration.12Figure1.HS gas chromatogram of one sample of sugar cane spirits.A,ethanol;B,methanol;C,butyl acetate.J Sci Food Agric(2014)©2014Society of Chemical Industry /jsfa Ming-Hua Liang et al.A B 0204060801000.10.20.30.40.50.6L e t h a l i t y (%)Concentration of DES (%)020*********80160240320400480560L e t h a l i t y (%)Irradiation time (s)Figure 2.Lethality curve during mutagenesis.(A)Lethality curve of S.cere-visiae under DES mutagenesis by treatment of different DES concentra-tions.0.4%DES (lethality 84.81%)was set as the mutation conditions for S.cerevisiae .Results are mean ±SD.(B)Lethality curve of S.cerevisiae under UV mutagenesis by treatment of different irradiation times.A UV irradia-tion time of 240s (lethality 85.92%)was set as the mutation conditions for S.cerevisiae .Results are mean ±SD.Thus 0.4%DES concentration was set as mutation conditions for S.cerevisiae .Figure 2(B)shows that the lethality increased with increasing UV irradiation time.The lethality reached 85.92%and 86.85%with a UV irradiation time of 240s and 480s,respectively.Therefore,a UV irradiation time of 240s was set as mutation con-ditions for S.cerevisiae .In conclusion,0.4%DES (lethality 84.81%)and a UV irradiation time of 240s (lethality 85.92%)were set as the mutation conditions for S.cerevisiae .After treatment by 0.4%DES for 60min,10mutants (D1–D10)were collected to use for fermentation of sugar cane spirits.The methanol contents of sugar cane spirits were detected and are shown in Fig.3(A).Methanol content from fermentation of mutant D4was the lowest (111.0±2.4mg L −1),which was a decrease of 17.7%compared with the strain without any treatment (134.8±2.8mg L −1).Mutant D4was then treated by UV for 240s,and 10mutants (DU1–DU10)were used to ferment sugar cane spirits.The variation of methanol content in the sugar cane spirits is shown in Fig.3(B).It was found that DU9could produce the low-est methanol (97.3±1.7mg L −1),with a 12.3%decrease of that of the strain D4only with DES treatment,and with a 27.8%reduction of that of the strain without any treatment.Effect of adding pectinase on the production of methanol The effect of pectinase addition on methanol level is shown in Fig.4(A);methanol content varied between 240and 300mg L −1.A B ****************50100150200250ControlD1D2D3D4D5D6D7D8D9D10Strains for testM e t h a n o l c o n t e n t (m g L –1)***********4080120160200D4DU1DU2DU3DU4DU5DU6DU7DU8DU9DU10M e t h a n o l c o n t e n t (m g L –1)Strains for testFigure 3.Screening for S.cerevisiae mutant with low methanol content by DES and UV.(A)Methanol content of sugar cane spirits by S.cere-visiae mutants with DES mutation.D4could produce the lowest methanol (111.0±2.4mg L −1).Results are mean ±SD.*P <0.05;**P <0.01,statisti-cally significant in comparison with control.(B)Methanol content of sugar cane spirits by S.cerevisiae mutants with DES and UV mutation.DU9could produce the lowest methanol (97.3±1.7mg L −1).Results are mean ±SD.*P <0.05;**P <0.01,statistically significant in comparison with the control (D4).Without pectinase treatment,the methanol content was 186.4mg L −1.It was shown that pectinase significantly increased the methanol levels of sugar cane spirits and methanol content increased with the dose of pectinase.When 0.8mL pectinase (70U mL −1)–corresponding to 56U pectinase –was added,pectic substances in sugar cane juice had been adequately broken down,and the content of methanol almost reached the maximum,with no subsequent significant change.Effect of cellulase on the production of methanolThe effect of cellulose addition on methanol level is shown in Fig.4(B);methanol content varied from 250to 410mg L −1.Without cellulase treatment,the methanol content was 206.3mg L −1.From these data it is seen that the use of cellulase significantly increased the methanol content of sugar cane spirits.When 0.8mL cellulase (30U mL)–corresponding to 24U cellulase –was added,cellu-lose in sugar cane juice had been adequately broken down,and the content of methanol almost reached the maximum,with no subsequent significant change.As cellulose does not dissolve in water at room temperature,filtered sugar cane juice is lacking in cellulose.Thus,from Table 1,it can be concluded that methanol content produced from cellulose reached 11.2mg L −1.Determination of glycine content in yeast extractWith RP-HPLC,we established an equation:y =1368.1x +22.269,R 2=0.9993,in which y represents the peak area of glycine and/jsfa ©2014Society of Chemical IndustryJ Sci Food Agric (2014)Mutation breeding of Saccharomyces cerevisiae with lower methanol content AB220240260280300020406080100120Dose of pectinase (U)M e t h a n o l c o n t e n t (m g L –1)260290320350380410020406080100Dose of cellulase (U)M e t h a n o l c o n t e n t (m g L –1)Figure 4.Effects of adding pectinase and cellulose in sugar cane spirits.(A)Effect of the addition of pectinase on the production of methanol.When 56U pectinase was added,the content of methanol was almost maximal with no subsequent significant change.Results are mean ±SD.(B)Effect of the addition of cellulase on the production of methanol.When 24U cellulase was added,the content of methanol was almost maximal with no subsequent significant change.Results are mean ±SD.Table 1.Methanol content produced by cellulose in sugar cane spiritsParameter Medium 1Sugar cane juice and wort Medium 2Filtered sugar cane juice and wort Methanol content produced from cellulose (mg L −1)Alcohol content (%V/V,20∘C)28.433.5–Methanol content (mg L −1)227.1215.911.2x represents glycine concentration.After calculation,the glycine content in 1g L −1yeast extract solution reached 6.85mg L −1.From the reactions above,glycine in 1g L −1yeast extract solution could produce 2.92mg L −1methanol in theory.Figure 5(for Fig.5B,only the factor of glycine concentra-tion was considered)shows that methanol content in sugar cane spirits increased with the increase of glycine content,and there was linear relationship between glycine (concentration within 0–0.9g L −1)and methanol in fermentation liquor and the linear equation was y =104.7x −4.79.According to the cal-culation method of the theoretical methanol content,there was a linear relationship between glycine (concentration within 0–0.9g L −1)and theoretical methanol content and the linear equation was y =426.28x −0.001.Thus the rate of glycine con-version to methanol was 104.7/426.28=24.56%.From Fig.5(A),while 18Brix sugar was used as fermentation medium,onlyABy = 63.733x + 24.22R 2 = 0.9811y = 104.9x + 52.57R 2 = 0.976902040608010012014016018000.10.20.30.40.50.60.70.80.9118 Brix sugar18 Brix sugar+12.5 g L –1 yeast extractGlycine concentration (g L –1)M e t h a n o l c o n c e n t r a t i o n (m g L –1)y = 63.733x -3.18R 2 = 0.9811y = 104.7x -4.79R 2 = 0.9751y = 426.28x -0.001R 2 = 1-5005010015020025030035040000.10.20.30.40.50.60.70.80.9118 Brix sugar18 Brix sugar+12.5g L –1 yeast extract Glycine to produce methanol in theoryGlycine concentration (g L –1)M e t h a n o l c o n c e n t r a t i o n (m g L –1)Figure 5.The influence of different glycine concentrations and fermenta-tion conditions on methanol yield.(A)The factors of glycine concentration,fermentation conditions,fermentation process and yeast metabolic path-way were considered.(B)Only the factor of glycine concentration was con-sidered.Other factors influencing the methanol yield were deducted.faint bubbles were generated in the fermentation process,which meant that it was difficult to ferment normally for yeast with only sugar as fermentation medium,and 27.4mg L −1methanol was produced without adding glycine.While 18Brix sugar and 12.5g L −1yeast extract was adopted as fermentation medium,S.cerevisiae was able to ferment normally and more methanol content (57.3mg L −1)was produced without adding extra glycine.DISCUSSIONThe main polysaccharides responsible for turbidity,viscosity and filter stoppages in sugar cane juice are pectins,glucans (the major component of cellulose)and,to a lesser extent,hemicellu-lose (mainly xylans).14In winemaking,commercial pectinase and cellulase preparations are used to improve juice yields by degrad-ing structural polysaccharides that interfere with juice extraction,the release of color and flavor compounds,with the clarification and filtration of wine,and reducing viscosity and turbidity.15–17However,the enzymatic treatment employed in the juice can cause a significant increase in the methanol concentrations of wines.Several investigators reported the influence of pectinase enzyme treatment on the methanol concentrations of wines.3,7,18In this study,it was also found that pectinase and celluloseJ Sci Food Agric (2014)©2014Society of Chemical Industry /jsfa Ming-Hua Liang et al.significantly increased the methanol levels of sugar cane spir-its(Fig.4).It has been reported that the high methanol content is ascribed to the use of pectinase,which is commonly used in mango wine production and is responsible for the splitting of pectic substances to galacturonic acid and methanol.19However, the higher methanol concentration in the present wines could be reduced significantly by further optimizing the treatment condi-tions of pectinase and cellulase.On the other hand,it was pointed out that many other fac-tors,such as oenological practices and the yeast strain used,can influence methanol production.15Yeast species are used in many industrial fermentation processes,including alcoholic beverage production.The quality of wine produced greatly depends on the yeast strain.20It was reported that methanol concentration var-ied between9.51%with S.cerevisiae var.ellipsoideus and14.93% with S.carlsbergensis.21In this study,a mutant of S.cerevisiae with lower methanol content was obtained by DES and UV mutation. The concentration of mutagen and the time of mutation are cru-cial to mutation efficiency.If the concentration is higher and the time is longer,the harm would be greater and the chance of screening would be lost.However,if the concentration is lower and the time is shorter,variability would be less and the chance of screening would be reduced.A concentration of mutagen with about80%lethality was suitable for selecting positive mutation strains.12In this study,for DES treatment,0.4%DES(lethality 84.81%)for60min was adopted for mutation breeding of low methanol-producing S.cerevisiae(Fig.2A).For UV irradiation,an irradiation time of240s(lethality85.92%)15W at a distance of 30cm was set as the mutation condition for S.cerevisiae(Fig.2B). It was reported that mutagenesis of UV coupled with DES was car-ried out because the combined mutation was more efficient.8,22,23 It was reported that Klebsiella oxytoca mutants were isolated from the wild type strain ME-303after mutagenesis with UV coupled with diethyl sulfate,and it was found that ME-UD-3produced 7.8%more2,3-butanediol than ME-303.8Here,a mutant S.cere-visiae DU9with low production of methanol(97.3mg L−1)was selected by mutation of DES and UV radiation(Fig.3B),with a 12.3%decrease of that of S.cerevisiae D4only with DES treat-ment,and with a27.8%reduction of that of the strain without any treatment.Amino acids are essential growth factors for proper implemen-tation and growth of yeasts during the course of alcoholic fer-mentation.Some biogenic amines are formed in variable amounts in wines by decarboxylation of their precursor amino acids due to the action of yeasts during alcoholic fermentation.24Esters, higher alcohols and volatile fatty acids are important contributors to the fermentation bouquet of wine.These compounds princi-pally arise as primary metabolites of yeast sugar and amino acid metabolism.Amino acids are precursors of volatile compounds (ethanol,ethyl acetate,acetic acid,higher alcohols and some of their acetates,methionol,isobutyric acid,ethyl butyrate,and hex-anoic and octanoic acids)and,consequently,wine composition is affected by the content of amino acids in the must.Further-more,the addition of amino acids can improve fermentation, as the amino acids can be incorporated directly into yeast pro-tein synthesis.25Methionol is correlated with methionine,histi-dine and glutamine.Acetic acid is correlated with proline,and the other aroma compounds(isoamyl alcohol,hexanoic and octanoic acids,ethyl butyrate,isobutyl acetate and isoamyl acetate)are mainly correlated with serine,threonine and glutamic acid.26It has been described that Monod-like equations are more adequate to describe the relationship between the amino acid content and the production of volatile compounds in simple microbiological systems.27In this study,the relationship between glycine content in the fermentation medium and the production of methanol wasfirst researched.Glycinefirst generates methylamine by glycine decar-boxylase,then reacts with nitrous acid to produce methanol.It was found that better growth,better reducing sugar consump-tion and higher fermentative efficiency occurred in the medium supplemented with12.5g L−1yeast extract compared with that without yeast extract.The inference is that it was difficult for S.cerevisiae to grow in the medium without yeast extract(or glycine),which meant that S.cerevisiae required yeast extract(or glycine)for normal growth.It was reported that the addition of a mixture of20amino acids greatly improved the fermentation effi-ciency of this yeast,increasing the consumption of reducing sugars and production of ethanol.28It was also shown that better growth, reducing sugar consumption and ethanol tolerance for Kloeckera africana cultures supplemented with yeast extract compared with those supplemented with ammonium sulfate.29When glycine was not added to the medium,methanol produced in the medium supplemented with12.5g L−1yeast extract was29.9mg L−1more than that produced in the medium without yeast extract(Fig.5A). With the same species and process conditions,methanol content in the two different media showed a difference of29.9mg L−1 after fermentation.This indicated that the content of methanol generation was directly related to the propagation and fermen-tation of yeasts,but the methanol production metabolic pathway was unclear.Methanol content in sugar cane spirits was increased with increased addition of glycine content,and there was a linear relationship between glycine(concentration within0–0.9g L−1) and methanol in the fermentation liquor,and the linear equation was y=104.7x−4.79with the conversion rate of glycine con-version to methanol as24.56%(Fig.5B).Thus it is necessary to control the addition of glycine in the fermentation medium and new ways to generate the methanol metabolic pathway in yeasts may need further study.It is important to note that methanol is an undesirable product in juice processing and winemaking and is a common problem in industrial beverage production. 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