黑曲霉液体发酵法产酶制备阿魏酸和低聚糖的研究
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摘要
本文采用黑曲霉液体发酵法生产含阿魏酸酯酶和阿拉伯木聚糖酶的混合酶制剂，使用该酶制剂酶解去淀粉麦麸制备低聚糖和反式阿魏酸，达到了综合利用工业副产品的目的。
采用不同温度和时间进行发酵试验，确定最佳培养条件为：在32℃下培养4天。使用Plackett—Burman和响应面中心点组合设计对可能影响产酶的8个培养基因素进行综合评估，得到3个显著性影响因素，并对其进行了优化，获得最佳发酵产酶培养基条件：麦麸10％，NH_4NO_3 0.28％，KH_2PO_4 0.2％，MgSO_4·7H_2O0.08％，NaNO_3 0.31％，培养基初始pH为5.5。在此基础上，使用5L发酵罐对酶制剂的生产进行了初步放大试验。
混合酶制剂中阿魏酸酯酶和阿拉伯木聚糖酶的最适反应pH和温度分别为4.4及40℃；外加金属离子(Mn~(2+)、Co~(2+))可在一定程度上提高阿拉伯木聚糖酶的活性。混合酶制剂酶解麦麸的最适工艺条件为：料液比16％(W／V)，在最适反应pH和最适反应温度下酶解4h。底物粉碎处理、高温、高压、微波等前处理措施能显著影响反式阿魏酸的产率。
实验对酶解产物的功能性进行了初步研究。结果表明在离体环境下，麦麸酶解产物可有效清除羟基自由基、超氧阴离子自由基以及DPPH，并能防止自由基引发的DNA损伤；其对油脂的抗氧化活性优于同浓度的阿魏酸溶液。
Mixed enzymes containing ferulic acid esterase and arabinoxylanase prepared from liquid culture of Aspergillus niger were used to hydrolyze destarched wheat bran to prepare trans-ferulic acid and oligosaccharide.
1 Optimization of culture conditions
Plackett-Burman design and response surface methodology were used to optimize fermentation technology for production of mixed-enzymes (ferulic acid esterase and arabinoxylanase) by Aspergillus niger in conic flask. The results showed that mixed-enzymes production was influenced by 3 factors of the tested 8 factors according to Plackett-Burman design; Two response surface models and response surface plots were obtained by using response surface methodology to optimize 3 significant factors. The optimal fermentation condition were: pH 5.5 and 10% wheat bran, 0.28% NH4NO3, 0.2% KH2PO4, 0.08% MgSO4 ?7H2O, 0.31% NaNO3. The fermentation time and temperature were 4d and 32℃.
2 Properties of ferulic acid esterase and arabinoxylanase
The optimal pH of the mixed-enzymes reaction and stability were at 4.4 and 5.0, respectively, and the optimal temperature of the mixed-enzymes reaction and stability
were at 50 ℃ and 35℃, respectively. Furthermore, the metal ions ( Mn2+ Co2+ ) showed significant effect on the activity of arabinoxylanase.
Inorganic ceramic membrane ultrafiltration with molecular weight cut-off of 20,000 and 100,000, respectively was also used to concentrate the enzymes, the results showed that the inorganic ceramic membrane with molecular weight cut-off of 20,000 could retain 80% mixed-enzymes when concentrated by 1.5 times.
3 Other influencing parameters for enzymatic hydrolysis
The effective factors on the hydrolysis of de-starched wheat bran, including enzymatic hydrolysis time, the concentration of enzymes, the ratio of substrate to mixed-enzymes, the substrate particle size and pre-treatment of the substrate were investigated. Results showed that activity of ferulic acid esterase was inhibited by high concentration of ferulic acid. In the range of particle size between 2-0.149mm, the smaller the particle size was, the less the ferulic acid and oligosaccharides was released. The high pressure (0.1 MPa), microware treatments of de-starched wheat bran showed significant effect on the production of ferulic acid.
4 Antioxidant activity of enzyme-released products from wheat bran
The antioxidant activity of enzyme-released products from wheat bran was investigated their protection against peroxidation of linoleic acid. The results showed that the product had higher antioxidant activity than ferulic acid at the same concentration. Moreover, the enzyme-released products could effectively scavenge free radicals and protect against DNA damage caused by hydroxyl free radical in vitro.
采用不同温度和时间进行发酵试验，确定最佳培养条件为：在32℃下培养4天。使用Plackett—Burman和响应面中心点组合设计对可能影响产酶的8个培养基因素进行综合评估，得到3个显著性影响因素，并对其进行了优化，获得最佳发酵产酶培养基条件：麦麸10％，NH_4NO_3 0.28％，KH_2PO_4 0.2％，MgSO_4·7H_2O0.08％，NaNO_3 0.31％，培养基初始pH为5.5。在此基础上，使用5L发酵罐对酶制剂的生产进行了初步放大试验。
混合酶制剂中阿魏酸酯酶和阿拉伯木聚糖酶的最适反应pH和温度分别为4.4及40℃；外加金属离子(Mn~(2+)、Co~(2+))可在一定程度上提高阿拉伯木聚糖酶的活性。混合酶制剂酶解麦麸的最适工艺条件为：料液比16％(W／V)，在最适反应pH和最适反应温度下酶解4h。底物粉碎处理、高温、高压、微波等前处理措施能显著影响反式阿魏酸的产率。
实验对酶解产物的功能性进行了初步研究。结果表明在离体环境下，麦麸酶解产物可有效清除羟基自由基、超氧阴离子自由基以及DPPH，并能防止自由基引发的DNA损伤；其对油脂的抗氧化活性优于同浓度的阿魏酸溶液。
Mixed enzymes containing ferulic acid esterase and arabinoxylanase prepared from liquid culture of Aspergillus niger were used to hydrolyze destarched wheat bran to prepare trans-ferulic acid and oligosaccharide.
1 Optimization of culture conditions
Plackett-Burman design and response surface methodology were used to optimize fermentation technology for production of mixed-enzymes (ferulic acid esterase and arabinoxylanase) by Aspergillus niger in conic flask. The results showed that mixed-enzymes production was influenced by 3 factors of the tested 8 factors according to Plackett-Burman design; Two response surface models and response surface plots were obtained by using response surface methodology to optimize 3 significant factors. The optimal fermentation condition were: pH 5.5 and 10% wheat bran, 0.28% NH4NO3, 0.2% KH2PO4, 0.08% MgSO4 ?7H2O, 0.31% NaNO3. The fermentation time and temperature were 4d and 32℃.
2 Properties of ferulic acid esterase and arabinoxylanase
The optimal pH of the mixed-enzymes reaction and stability were at 4.4 and 5.0, respectively, and the optimal temperature of the mixed-enzymes reaction and stability
were at 50 ℃ and 35℃, respectively. Furthermore, the metal ions ( Mn2+ Co2+ ) showed significant effect on the activity of arabinoxylanase.
Inorganic ceramic membrane ultrafiltration with molecular weight cut-off of 20,000 and 100,000, respectively was also used to concentrate the enzymes, the results showed that the inorganic ceramic membrane with molecular weight cut-off of 20,000 could retain 80% mixed-enzymes when concentrated by 1.5 times.
3 Other influencing parameters for enzymatic hydrolysis
The effective factors on the hydrolysis of de-starched wheat bran, including enzymatic hydrolysis time, the concentration of enzymes, the ratio of substrate to mixed-enzymes, the substrate particle size and pre-treatment of the substrate were investigated. Results showed that activity of ferulic acid esterase was inhibited by high concentration of ferulic acid. In the range of particle size between 2-0.149mm, the smaller the particle size was, the less the ferulic acid and oligosaccharides was released. The high pressure (0.1 MPa), microware treatments of de-starched wheat bran showed significant effect on the production of ferulic acid.
4 Antioxidant activity of enzyme-released products from wheat bran
The antioxidant activity of enzyme-released products from wheat bran was investigated their protection against peroxidation of linoleic acid. The results showed that the product had higher antioxidant activity than ferulic acid at the same concentration. Moreover, the enzyme-released products could effectively scavenge free radicals and protect against DNA damage caused by hydroxyl free radical in vitro.
引文
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95 Brézillon C, Kroon PA, Faulds CB, Brett GM, Williamson G. Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar beet pulp [J]. Biotechnology and Applied Biochemistry, 1996, 16: 371-376.
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97 Ralet MC, Faulds CB, Williamson G, Thibault JF. Feruloylated oligosaccharides from cell wall polysaccharides. Part Ⅲ. Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger [J]. Carbohydrate Research, 1994, 263: 257-269.
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