米渣霉菌发酵产物分离鉴定及抗氧化活性研究
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摘要
我国是稻谷的生产大国,年产量在2000亿公斤左右。我国的稻米除了供应人们的日常粮食需求外,很大一部分低值大米被用作淀粉糖生产和工业发酵,产生了大量的副产物。米渣是大米淀粉糖生产、有机酸发酵和味精生产的副产物,蛋白质含量高达40%以上,是非常理想的蛋白质资源。此外,米渣中还含有丰富的糊精和多聚糖,极有开发利用价值。本研究以米渣为原料,利用霉菌固态发酵,经超滤、凝胶层析和RP-HPLC进行分离纯化,获得高抗氧化活性产物,并进行结构鉴定。主要研究结果如下：
1首次选用米曲霉(Aspergillus oryzae)、根霉(Rhizopus oligosporrus)、毛霉(Mracemosus)、黑曲霉(Aspergillium niger)、青霉(Penicillium glaucum)分别对米渣进行固态发酵制备抗氧化产物。测定米渣发酵产物(RRFPs)的还原力,清除DPPH自由基、ABTS自由基、OH自由基以及抗脂质过氧化能力等体外抗氧化活性,并建立细胞氧化损伤模型,通过MTT比色法,考察各RRFPs的体内抗氧化活性,筛选出黑曲霉为最佳发酵菌种。对上述5种霉菌发酵产物进行成分分析,结果表明各发酵产物均具有较高的水解度,并结合米渣基本成分分析,初步推断米渣发酵产物的抗氧化性可能主要是后发酵过程中米渣蛋白水解产生的小分子肽表现出来的。
2首次以黑曲霉为发酵菌种,以成曲的蛋白酶活性和糖化酶活力为评价指标,通过单因素试验得出前发酵期的最佳工艺条件为：米渣含水量45%,发酵周期3天、发酵温度32℃。通过后发酵期的单因素试验证实发酵温度、发酵时间、加水量对黑曲霉发酵产物(RRFPsⅣ)抗氧化活性和水解度具有显著影响,适当的水解度下RRFPsⅣ具有较高的抗氧化活性。以DPPH·清除率为指标,采用Box-Beknken响应面对各因素水平进行模型拟合,其回归方程为：Y=53.62-0.82X1-0.85X2+0.40X3-2.55X12-2.86X22-1.55X32+0.99X1X2-0.71X1X3。经MINITAB16软件分析获得黑曲霉对米渣固态发酵制备抗氧化产物的最佳工艺条件为：温度40.4℃,时间5.5d,加水量83.4mL/10g米渣,RRFPsIV对DPPH·清除率Y值的预测为53.81%。验证试验获得的RRFPsIV对DPPH·清除率为53.56%,水解度为38.92%。
3研究了RRFPsⅣ抗氧化的稳定性以及与其它天然抗氧化剂的协同作用。结果表明,温度、酸碱度、胃蛋白酶水解对RRFPsⅣ抗氧化活性无显著影响,Cu2+、Fe3+对RRFPsⅣ抗氧化活性具有显著影响。RRFPsⅣ与VC、茶多酚复配,具有很好的抗氧化协同增效作用；与VE复配则无明显协同增效作用。4利用10KDa的超滤膜对RRFPsⅣ进行了膜分离,DPPH·(?)青除率检测结果表明：10KDa以下组分的DPPH·清除率为64.98±3.45%,高于10KDa以上组分的DPPH·清除率。利用紫外光谱检测10Kda以下组分,在210nm处有最大吸收峰。10KDa以下组分经SephadexG-25凝胶层析色谱分离成F1、F2和F3三个组分,其中F2组分的DPPH·清除率为74.71±2.66%。利用制备型RP-HPLC将F2组分分离成5个主要组分,其中F2d组分和F2e组分的DPPH·(?)青除率较高,分别达到77.37±1.01%和83.04±1.69%。采用分析型RP-HPLC进行纯度分析表明F2d组分和F2e组分为相对单一峰,峰面积分别为90.36%和91.69%。
5首次采用MicroTOF-QII串联质谱对分离纯化后的F2d组分和F2e组分进行分子量及结构式鉴定,得到分子量分别为1072.97Da和1130.5132Da的小分子多肽；从N端开始排序,其氨基酸序列分别为：Val-Ala-Glu-Glu-Glu-Leu-Gly-Gly-Asn-Arg；Leu-Asp-Pro-Glu-Gly-Thr-G1y-Thr-Phe-Pro-Pro
As a rice producing country, the output of rice is about200billion kg per year in China. Besides the supply of daily need for food, most of the low-value rice is used as material for starch-sugar production and industrial fermentation, which may leave a great deal of byproducts. Rice residue is a kind of byproducts produced in the processes of starch-sugar production, organic acid fermentation and monosodium glutamate production. Rice residue in which the content of protein is more than40%and rich in dextrine and polysaccharide, is an ideal protein resource and worthy of exploiting. Based on rice residue as raw material for the solid-state fermentation of fungi, product with high antioxidant activity was isolated and purified by ultrafiltration, gel chromatography and PR-HPLC, and the structure has been identified as well. The main results were as follows:
1It was the first time to ferment rice residue with strains of genus of Aspergillus oryzae, Rhizopus oligosporrus, M. racemosus, Aspergillium niger and Penicillium glaucum, on solid state to produce antioxidant product. Antioxidant activity in vivo has been determined by MTT assay. Based on the results of reducibility, clearance ability of DPPH·free radical, ABTS·free radical and OH·free radical and anti-lipid peroxidation ability of each product, Aspergillium niger has been considered the optimum strain for the fermentation. Analysis for the products fermented by the five strains above have exhibited that each product showed a high degree of hydrolysis, and that the inoxidability of fermented products of rice residue mainly depended on the behavior of micromolecular peptide by the hydrolyzation of protein during the post-fermentation has been primarily concluded, combining the analysis of basic ingredients of rice residue.
2Using the activity of protease and glucoamylase of koji as evaluation index, primary fermentation conditions have been concluded as follows:water content of rice residue45%,3d for the fermentation at32℃, according to single factor experiment. It has been confirmed that factors of fermentation temperature, fermentation time and water content significantly affect the inoxidability and degree of hydrolysis of RRFPsIV during the after fermentation, and higher antioxidant activity has been detected against RRFPsIV with proper degree of hydrolysis. Using the clearance of DPPH·as index, models for each factor level have been fitted according Box-Beknken response surface. The regression equation was as follow: Y=53.62-0.82X1-0.85X2+0.40X3-2.55X12-2.86X22-1.55X32+0.99X1X2-0.71X1X3According the analysis software of MINITAB16, optimum process for the
fermentation of rice residue has been concluded:temperature40.4℃, time5.5d, water content83.4mL/10g rice residue, and the predicted Y of clearance of RRFPsIV against DPPH-was53.56%, degree of hydrolysis38.92%.
3The antioxidative stability of RRFPsⅣ and its synergism with other natural antioxidants were studied. Results exhibited that no significant effect of inoxidability of RRFPsIV had been detected by the variation of temperature, pH value and pepsin hydrolysis, however, Cu2+and Fe3+did. Synergistic interaction has been detected when RRFPsIV was blended with Vc and tea polyphenol, but no Synergistic interaction could be detected when blended with VE.
4Barrier separation for the hydrolysate of RRFPsIV has been studied with a ultrafiltration membrane of10KDa intercepting, tests showed that clearance of constituent less than10KDa was64.98±3.45%against DPPH·, and it was higher than that above lOKDa. Maximum absorption peak was scaned at210nm towards constituent less than10KDa with UV spectrum. Three constituents named F1, F2and F3was isolated by SephadexG-25from constituent less than10KDa. Clearance of F2against DPPH·was74.71±2.66%. Five main constituents from F2have been isolated by preparative RP-HPLC, of which F2d and F2e have showed a high clearance against DPPH·of77.37±1.01%and83.04±1.69%respectively. It has been confirmed that F2d and F2e were both relative single peaks, and the peak area were90.36%and91.69%respectively according to the purity analysis of analytical RP-HPLC.
5It was the first time to identify the molecular weight and constitutional formula of F2d and F2e by MicroTOF-QII tandem mass spectrometry. And the molecular weight were1072.97Da and1130.5132Da respectively, their amino acid sequences have identified as follows (started from N terminal): Val-Ala-Glu-Glu-Glu-Leu-Gly-Gly-Asn-Arg Leu-Asp-Pro-Glu-Gly-Thr-Gly-Thr-Phe-Pro-Pro
1首次选用米曲霉(Aspergillus oryzae)、根霉(Rhizopus oligosporrus)、毛霉(Mracemosus)、黑曲霉(Aspergillium niger)、青霉(Penicillium glaucum)分别对米渣进行固态发酵制备抗氧化产物。测定米渣发酵产物(RRFPs)的还原力,清除DPPH自由基、ABTS自由基、OH自由基以及抗脂质过氧化能力等体外抗氧化活性,并建立细胞氧化损伤模型,通过MTT比色法,考察各RRFPs的体内抗氧化活性,筛选出黑曲霉为最佳发酵菌种。对上述5种霉菌发酵产物进行成分分析,结果表明各发酵产物均具有较高的水解度,并结合米渣基本成分分析,初步推断米渣发酵产物的抗氧化性可能主要是后发酵过程中米渣蛋白水解产生的小分子肽表现出来的。
2首次以黑曲霉为发酵菌种,以成曲的蛋白酶活性和糖化酶活力为评价指标,通过单因素试验得出前发酵期的最佳工艺条件为：米渣含水量45%,发酵周期3天、发酵温度32℃。通过后发酵期的单因素试验证实发酵温度、发酵时间、加水量对黑曲霉发酵产物(RRFPsⅣ)抗氧化活性和水解度具有显著影响,适当的水解度下RRFPsⅣ具有较高的抗氧化活性。以DPPH·清除率为指标,采用Box-Beknken响应面对各因素水平进行模型拟合,其回归方程为：Y=53.62-0.82X1-0.85X2+0.40X3-2.55X12-2.86X22-1.55X32+0.99X1X2-0.71X1X3。经MINITAB16软件分析获得黑曲霉对米渣固态发酵制备抗氧化产物的最佳工艺条件为：温度40.4℃,时间5.5d,加水量83.4mL/10g米渣,RRFPsIV对DPPH·清除率Y值的预测为53.81%。验证试验获得的RRFPsIV对DPPH·清除率为53.56%,水解度为38.92%。
3研究了RRFPsⅣ抗氧化的稳定性以及与其它天然抗氧化剂的协同作用。结果表明,温度、酸碱度、胃蛋白酶水解对RRFPsⅣ抗氧化活性无显著影响,Cu2+、Fe3+对RRFPsⅣ抗氧化活性具有显著影响。RRFPsⅣ与VC、茶多酚复配,具有很好的抗氧化协同增效作用；与VE复配则无明显协同增效作用。4利用10KDa的超滤膜对RRFPsⅣ进行了膜分离,DPPH·(?)青除率检测结果表明：10KDa以下组分的DPPH·清除率为64.98±3.45%,高于10KDa以上组分的DPPH·清除率。利用紫外光谱检测10Kda以下组分,在210nm处有最大吸收峰。10KDa以下组分经SephadexG-25凝胶层析色谱分离成F1、F2和F3三个组分,其中F2组分的DPPH·清除率为74.71±2.66%。利用制备型RP-HPLC将F2组分分离成5个主要组分,其中F2d组分和F2e组分的DPPH·(?)青除率较高,分别达到77.37±1.01%和83.04±1.69%。采用分析型RP-HPLC进行纯度分析表明F2d组分和F2e组分为相对单一峰,峰面积分别为90.36%和91.69%。
5首次采用MicroTOF-QII串联质谱对分离纯化后的F2d组分和F2e组分进行分子量及结构式鉴定,得到分子量分别为1072.97Da和1130.5132Da的小分子多肽；从N端开始排序,其氨基酸序列分别为：Val-Ala-Glu-Glu-Glu-Leu-Gly-Gly-Asn-Arg；Leu-Asp-Pro-Glu-Gly-Thr-G1y-Thr-Phe-Pro-Pro
As a rice producing country, the output of rice is about200billion kg per year in China. Besides the supply of daily need for food, most of the low-value rice is used as material for starch-sugar production and industrial fermentation, which may leave a great deal of byproducts. Rice residue is a kind of byproducts produced in the processes of starch-sugar production, organic acid fermentation and monosodium glutamate production. Rice residue in which the content of protein is more than40%and rich in dextrine and polysaccharide, is an ideal protein resource and worthy of exploiting. Based on rice residue as raw material for the solid-state fermentation of fungi, product with high antioxidant activity was isolated and purified by ultrafiltration, gel chromatography and PR-HPLC, and the structure has been identified as well. The main results were as follows:
1It was the first time to ferment rice residue with strains of genus of Aspergillus oryzae, Rhizopus oligosporrus, M. racemosus, Aspergillium niger and Penicillium glaucum, on solid state to produce antioxidant product. Antioxidant activity in vivo has been determined by MTT assay. Based on the results of reducibility, clearance ability of DPPH·free radical, ABTS·free radical and OH·free radical and anti-lipid peroxidation ability of each product, Aspergillium niger has been considered the optimum strain for the fermentation. Analysis for the products fermented by the five strains above have exhibited that each product showed a high degree of hydrolysis, and that the inoxidability of fermented products of rice residue mainly depended on the behavior of micromolecular peptide by the hydrolyzation of protein during the post-fermentation has been primarily concluded, combining the analysis of basic ingredients of rice residue.
2Using the activity of protease and glucoamylase of koji as evaluation index, primary fermentation conditions have been concluded as follows:water content of rice residue45%,3d for the fermentation at32℃, according to single factor experiment. It has been confirmed that factors of fermentation temperature, fermentation time and water content significantly affect the inoxidability and degree of hydrolysis of RRFPsIV during the after fermentation, and higher antioxidant activity has been detected against RRFPsIV with proper degree of hydrolysis. Using the clearance of DPPH·as index, models for each factor level have been fitted according Box-Beknken response surface. The regression equation was as follow: Y=53.62-0.82X1-0.85X2+0.40X3-2.55X12-2.86X22-1.55X32+0.99X1X2-0.71X1X3According the analysis software of MINITAB16, optimum process for the
fermentation of rice residue has been concluded:temperature40.4℃, time5.5d, water content83.4mL/10g rice residue, and the predicted Y of clearance of RRFPsIV against DPPH-was53.56%, degree of hydrolysis38.92%.
3The antioxidative stability of RRFPsⅣ and its synergism with other natural antioxidants were studied. Results exhibited that no significant effect of inoxidability of RRFPsIV had been detected by the variation of temperature, pH value and pepsin hydrolysis, however, Cu2+and Fe3+did. Synergistic interaction has been detected when RRFPsIV was blended with Vc and tea polyphenol, but no Synergistic interaction could be detected when blended with VE.
4Barrier separation for the hydrolysate of RRFPsIV has been studied with a ultrafiltration membrane of10KDa intercepting, tests showed that clearance of constituent less than10KDa was64.98±3.45%against DPPH·, and it was higher than that above lOKDa. Maximum absorption peak was scaned at210nm towards constituent less than10KDa with UV spectrum. Three constituents named F1, F2and F3was isolated by SephadexG-25from constituent less than10KDa. Clearance of F2against DPPH·was74.71±2.66%. Five main constituents from F2have been isolated by preparative RP-HPLC, of which F2d and F2e have showed a high clearance against DPPH·of77.37±1.01%and83.04±1.69%respectively. It has been confirmed that F2d and F2e were both relative single peaks, and the peak area were90.36%and91.69%respectively according to the purity analysis of analytical RP-HPLC.
5It was the first time to identify the molecular weight and constitutional formula of F2d and F2e by MicroTOF-QII tandem mass spectrometry. And the molecular weight were1072.97Da and1130.5132Da respectively, their amino acid sequences have identified as follows (started from N terminal): Val-Ala-Glu-Glu-Glu-Leu-Gly-Gly-Asn-Arg Leu-Asp-Pro-Glu-Gly-Thr-Gly-Thr-Phe-Pro-Pro
引文
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