马铃薯为原料茁霉多糖的发酵研究
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摘要
茁霉多糖(Pullulan)是由出芽短梗霉(Aureobasidium Pullulans)在发酵过程中产生的一种胞外多糖,具有易溶于水、不凝胶化、不老化、可任意加工成型和无任何毒性等优良特点,可以被广泛应用于医药制造、食品包装、水果和海产品保鲜、化妆品工业、烟草制造业和农业种子保护及工业废水处理等众多领域。
本文以马铃薯为原料,首先经中温α-淀粉酶液化,采用二次通用旋转组合试验的方法对不同的糖化工艺(SNase303糖化酶和β-淀粉酶)和处理方法(脱色脱盐和未脱色未脱盐)进行研究,制备出不同DE值的马铃薯水解液并以其作为诱变菌株出芽短梗霉D_1-11发酵的碳源;同时以多糖产量、生物量、色素产量等为测定指标,采用正交试验、均匀设计等方法,研究了不同培养条件(装液量、pH、接种量、温度)和不同营养条件(碳源、二价阳离子)对茁霉多糖发酵的影响,得到如下结果:
1.经单因素试验和正交试验确定了D_1-11的最适培养条件,最优培养条件为装液量25%,初始pH6.5,温度30℃,接种量8%,其中装液量对多糖产量的影响最大,pH和接种量对多糖产量的影响也比较大,只有温度的影响比较小,按此条件培养的茁霉多糖产量为19.71g/L。
2.中温α-淀粉酶液化马铃薯粉浆的工艺条件为:粉浆浓度25%,中温α-淀粉酶酶用量10u/g,液化时间50min。
3.单因素试验确定了符合二次回归通用旋转组合试验的糖化工艺参数,采用多元回归分析法得到二阶多项式回归方程式,其相关系数为0.9831,并且模型在a=0.05的水平上回归显著。响应面分析表明:在60℃,pH3.8-4.5下,改变β-淀粉酶酶用量(30-90u/g),酶解时间(3-5h)和液化液DE值(15%-19%),可以制备出20-60%之间任意DE值的淀粉糖浆,用于茁霉多糖发酵的碳源。影响糖化液DE值由大到小的因素为酶解时间＞β-淀粉酶用量＞液化液DE值。
4.通过单因素试验和二次回归通用旋转组合试验确定SNase303糖化酶糖化工艺参数,采用多元回归分析法得到二阶多项式回归方程式,其相关系数为0.9823,并且模型在a=0.05的水平上回归显著。响应面分析表明:在60℃,pH3.8-4.5下,改变液化液DE值(15-19%),SNase303糖化酶酶用量(150-250u/g),液化时间(3-5h),可以制备出20-60%之间任意DE值的淀粉糖浆,用于茁霉多糖发酵的碳源。影响糖化液DE值由大到小的因素为酶解时间＞液化液DE值＞SNase303糖化酶用量。
5.经单因素试验分别对碳源和二价阳离子进行考察,结果表明经β-淀粉酶制备的水解液较经SNase303糖化酶制备的水解液更适合做茁霉多糖的发酵碳源,且脱盐脱色处理会提高多糖的产率,DE值50%的β-淀粉酶糖化液经脱色脱盐处理,在80g/L时多糖产量最高为21.98g/L,CaCO_3在0.1g/L时多糖产量最高为21.11g/L。
6.通过均匀试验确定摇瓶培养最佳发酵工艺为CaCO_3 0.06g/L,碳源82.25g/L,装液量26.62%,接种量11.99%,pH 7.5,根据本工艺茁霉多糖产量可达最大产量28.90g/L;经变量分析可知碳源对多糖产量的影响最大。
Pullulan,an extracelluler polysaccharide produced by Aureobasidium Pullulans, in the process of fermentation.The pullulan is water-soluble,no-gelation,no-aging, without toxicity,easy machine-shaping and so on.It can be applied in many fields including pharmaceutical and cosmetic industry,food packaging,refreshing of fruits and seafood,tobacco manufacturing,protection of seeds and industrial sewage disposal etc.
This paper used potato as its raw material,the potato starch syrup of different DE values which liquefacted byα-amylase was used as the carbon source of the mutant strain Aureobasidium pullulans D1-11.The influence of different saccharification enzymes(SNase303 glucoamylase orβ-amylas) and treatment methods(decolor and desalt treatment) were studied by the rotational composite design of quadratic regression experiments.Polysaccharide yield,biomass,pigment content are the detection index in this study.There are three different experiment designs such as the orthogonal experiment design and the uniform design used to studied the effects of fermentation conditions and nutritional conditions on pullulan fermentation, fermentation conditions include liquid volume in flask,pH,inoculum size, fermentation temperature,nutritional conditions include the carbon source and divalent cation.The results showed that:
1.The optimal formulation was obtained by the single factor screening test and the L_9(3~4) orthogonal experiment design,that was 25%liquid volume in flask,pH6.5, fermentation temperature 30℃,fermented with 8%inoculum size,the proper order of influential factors is liquid volume in flask,temperature,the inoculum size,pH,the yeild of pullulan can reach 19.71g/L under this fermentation process.
2.The liquefication technology of potato syrup by medium temperatureα-amylase was obtained by single factor screening,that was 25%concentration of potato,10u/g dosage of medium temperatureα-amylase,50 min of liquefaction.
3.Based on the single factor screening test for the saccharification process,a second-order model was obtained with a correlation coefficient 0.9831(a＜0.05), through multiple regression analysis.Analysis of the response surface showed that dextrose equivalent(DE) 20-60%were prepared at 60℃,pH3.8-4.5 by altering the amount ofβ-amylase(30-90u/g),digested time(3-5h) and the dextrose equivalent (DE) of liquefication(15%-19%).The carbon resource was made from potato starch, which could meet standard requirement.The results had the capability of providing the basis for pullulan in the next step.The proper order of factors influent saccharification liquid was digested time,the amount ofβ-amylase,the dextrose equivalent(DE) of liquefication.
4.Based on the single factor screening test for the saccharification process,a second-order model was obtained with a correlation co-efficient 0.9823(a＜0.05) through multiple regression analysis.Analysis of the response surface showed that dextrose equivalent(DE) 20-60%were prepared at 60℃,pH3.8-4.5 by altering the amount of glucoamylase(150-250u/g),digested time(3-5h) and the dextrose equivalent(DE) of liquefication(15%-19%).The carbon resource was made from potato,which could meet standard requirement for pullulan.The proper order of factors influent saccharification liquid was digested time,the dextrose equivalent(DE) of liquefication,the amount of glucoamylase.
5.The single factor screening test including the carbon source and divalent cations,the syrup maded byβ-amylase and decolor and desalt treatment was superior to SNase303 glucoamylase.The best result was the potato syrup byβ-amylase in DE 50%80 g/L,CaCO_3 0.1 g/L,the yield of pullulan was 21.98 g/L,21.11 g/L.
6.The optimal formulation was obtained by the uniform design,that was:CaCO_3 0.06 g/L,β-amylase in DE 50%82.25g/L,26.62%liquid volume in flask,fermented with 11.99%inoculum size,pH 7.5,the production of pullulan can reach 28.90 g/L under this fermentation process;an analysis of variable was indicated that the carbon source was the most influential factor.
本文以马铃薯为原料,首先经中温α-淀粉酶液化,采用二次通用旋转组合试验的方法对不同的糖化工艺(SNase303糖化酶和β-淀粉酶)和处理方法(脱色脱盐和未脱色未脱盐)进行研究,制备出不同DE值的马铃薯水解液并以其作为诱变菌株出芽短梗霉D_1-11发酵的碳源;同时以多糖产量、生物量、色素产量等为测定指标,采用正交试验、均匀设计等方法,研究了不同培养条件(装液量、pH、接种量、温度)和不同营养条件(碳源、二价阳离子)对茁霉多糖发酵的影响,得到如下结果:
1.经单因素试验和正交试验确定了D_1-11的最适培养条件,最优培养条件为装液量25%,初始pH6.5,温度30℃,接种量8%,其中装液量对多糖产量的影响最大,pH和接种量对多糖产量的影响也比较大,只有温度的影响比较小,按此条件培养的茁霉多糖产量为19.71g/L。
2.中温α-淀粉酶液化马铃薯粉浆的工艺条件为:粉浆浓度25%,中温α-淀粉酶酶用量10u/g,液化时间50min。
3.单因素试验确定了符合二次回归通用旋转组合试验的糖化工艺参数,采用多元回归分析法得到二阶多项式回归方程式,其相关系数为0.9831,并且模型在a=0.05的水平上回归显著。响应面分析表明:在60℃,pH3.8-4.5下,改变β-淀粉酶酶用量(30-90u/g),酶解时间(3-5h)和液化液DE值(15%-19%),可以制备出20-60%之间任意DE值的淀粉糖浆,用于茁霉多糖发酵的碳源。影响糖化液DE值由大到小的因素为酶解时间＞β-淀粉酶用量＞液化液DE值。
4.通过单因素试验和二次回归通用旋转组合试验确定SNase303糖化酶糖化工艺参数,采用多元回归分析法得到二阶多项式回归方程式,其相关系数为0.9823,并且模型在a=0.05的水平上回归显著。响应面分析表明:在60℃,pH3.8-4.5下,改变液化液DE值(15-19%),SNase303糖化酶酶用量(150-250u/g),液化时间(3-5h),可以制备出20-60%之间任意DE值的淀粉糖浆,用于茁霉多糖发酵的碳源。影响糖化液DE值由大到小的因素为酶解时间＞液化液DE值＞SNase303糖化酶用量。
5.经单因素试验分别对碳源和二价阳离子进行考察,结果表明经β-淀粉酶制备的水解液较经SNase303糖化酶制备的水解液更适合做茁霉多糖的发酵碳源,且脱盐脱色处理会提高多糖的产率,DE值50%的β-淀粉酶糖化液经脱色脱盐处理,在80g/L时多糖产量最高为21.98g/L,CaCO_3在0.1g/L时多糖产量最高为21.11g/L。
6.通过均匀试验确定摇瓶培养最佳发酵工艺为CaCO_3 0.06g/L,碳源82.25g/L,装液量26.62%,接种量11.99%,pH 7.5,根据本工艺茁霉多糖产量可达最大产量28.90g/L;经变量分析可知碳源对多糖产量的影响最大。
Pullulan,an extracelluler polysaccharide produced by Aureobasidium Pullulans, in the process of fermentation.The pullulan is water-soluble,no-gelation,no-aging, without toxicity,easy machine-shaping and so on.It can be applied in many fields including pharmaceutical and cosmetic industry,food packaging,refreshing of fruits and seafood,tobacco manufacturing,protection of seeds and industrial sewage disposal etc.
This paper used potato as its raw material,the potato starch syrup of different DE values which liquefacted byα-amylase was used as the carbon source of the mutant strain Aureobasidium pullulans D1-11.The influence of different saccharification enzymes(SNase303 glucoamylase orβ-amylas) and treatment methods(decolor and desalt treatment) were studied by the rotational composite design of quadratic regression experiments.Polysaccharide yield,biomass,pigment content are the detection index in this study.There are three different experiment designs such as the orthogonal experiment design and the uniform design used to studied the effects of fermentation conditions and nutritional conditions on pullulan fermentation, fermentation conditions include liquid volume in flask,pH,inoculum size, fermentation temperature,nutritional conditions include the carbon source and divalent cation.The results showed that:
1.The optimal formulation was obtained by the single factor screening test and the L_9(3~4) orthogonal experiment design,that was 25%liquid volume in flask,pH6.5, fermentation temperature 30℃,fermented with 8%inoculum size,the proper order of influential factors is liquid volume in flask,temperature,the inoculum size,pH,the yeild of pullulan can reach 19.71g/L under this fermentation process.
2.The liquefication technology of potato syrup by medium temperatureα-amylase was obtained by single factor screening,that was 25%concentration of potato,10u/g dosage of medium temperatureα-amylase,50 min of liquefaction.
3.Based on the single factor screening test for the saccharification process,a second-order model was obtained with a correlation coefficient 0.9831(a＜0.05), through multiple regression analysis.Analysis of the response surface showed that dextrose equivalent(DE) 20-60%were prepared at 60℃,pH3.8-4.5 by altering the amount ofβ-amylase(30-90u/g),digested time(3-5h) and the dextrose equivalent (DE) of liquefication(15%-19%).The carbon resource was made from potato starch, which could meet standard requirement.The results had the capability of providing the basis for pullulan in the next step.The proper order of factors influent saccharification liquid was digested time,the amount ofβ-amylase,the dextrose equivalent(DE) of liquefication.
4.Based on the single factor screening test for the saccharification process,a second-order model was obtained with a correlation co-efficient 0.9823(a＜0.05) through multiple regression analysis.Analysis of the response surface showed that dextrose equivalent(DE) 20-60%were prepared at 60℃,pH3.8-4.5 by altering the amount of glucoamylase(150-250u/g),digested time(3-5h) and the dextrose equivalent(DE) of liquefication(15%-19%).The carbon resource was made from potato,which could meet standard requirement for pullulan.The proper order of factors influent saccharification liquid was digested time,the dextrose equivalent(DE) of liquefication,the amount of glucoamylase.
5.The single factor screening test including the carbon source and divalent cations,the syrup maded byβ-amylase and decolor and desalt treatment was superior to SNase303 glucoamylase.The best result was the potato syrup byβ-amylase in DE 50%80 g/L,CaCO_3 0.1 g/L,the yield of pullulan was 21.98 g/L,21.11 g/L.
6.The optimal formulation was obtained by the uniform design,that was:CaCO_3 0.06 g/L,β-amylase in DE 50%82.25g/L,26.62%liquid volume in flask,fermented with 11.99%inoculum size,pH 7.5,the production of pullulan can reach 28.90 g/L under this fermentation process;an analysis of variable was indicated that the carbon source was the most influential factor.
引文
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