豆渣曲制备工艺及其提取物抗氧化性研究
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摘要
豆渣是大豆制品加工中的主要副产物,占全豆干重的15%～20%,并含有丰富的蛋白质等生理活性物质。米曲霉是生产酱油等发酵的重要微生物,且具有多种酶系。本文研究以豆渣为基质,制备豆渣曲,并利用豆渣曲中多种酶水解曲料,以获得具有抗氧化的活性提取物,为揭示酱油的抗氧化性奠定基础。研究得到的以下主要结论:
1.优化了以豆渣和麸皮为基质的固态发酵条件:在30℃,麸皮豆渣比为1:9,培养时间为48h,接种量为0.15%,水分含量为50%水分,米曲霉在此条件下,生长较好,米曲霉蛋白酶活力达到3283U/g。同时考察了添加葡萄糖和硫酸铵可以提高豆渣曲的蛋白酶活力。
2.利用豆渣曲自身酶系水解曲料可获得具有较强抗氧化的提取物,添加纤维素酶和果胶酶可以有效提高抗氧化生理活性物质溶出。直接提取:提取时间为3h,提取温度:40℃,固液比为1:20,在此条件下,测得豆渣曲蛋白质溶出率为69%。分子量大多小于1000,大部分为二肽、三肽及氨基酸。外加纤维素酶提取条件为:加酶量为0.3g/L,提取温度为40℃,固液比为1:25,提取时间为4h。在此条件下,测得豆渣曲蛋白质溶出率为77.3%;外加果胶酶提取抗氧化液最优条件:固液比为1:20,提取温度为45℃,提取时间为4h,加酶量为1g/L。提取液的抗氧化活性分别为:0.20ml提取液清除DPPH能力为67.5%,30μl提取液清除ABTS+能力为72.1%。
3.外加纤维素酶和果胶酶,不同程度的提高曲料中小肽,多酚,多糖以及黄酮抗氧化物质溶出速率和溶出量,从而提高了提取液的抗氧化能力,其中果胶酶效果最好,制得提取液具有最高的抗氧化活性。当含有7.5mg/ml多肽,1.4mg/ml多糖,多酚含量为2.0 mg/ml,以及6.5μg/ml黄酮,具有清除ABTS+和DPPH·自由基的IC50分别是21.5μl和0.21ml,0.1ml的提取液就具有49.2%的羟基自由基清除能力,而邻苯三酚半数抑制率IC50为0.49ml,0.2ml其还原能力就很强,还原产物吸光值就达到0.723。
Soybean residue, the main by-product of soy products, is not only accounting for 15%~20% in dried soybean, but also rich in protein and physiological active elements. Aspergillus oryzae is a universal strain in traditional fermented industry, which contain complexed enzymes. Using soybean residue as raw material, Koji was produced by fermentation of Aspergillus oryzae. The complex enzymes from aspergillus oryzae were used to hydrolyze Koji, anti-oxidant elements was obtained. In this paper, using soybean residue as a medium, Koji is obtained ,and then hydrolyze the koji to obtain the anti-oxidant elements. The results concluded from experiments were as follows.
1. Optimized cultivation conditions for the production of enzyme by Aspergillus oryzae under solid-state fermentation were obtained. The results showed the optimum conditions as followed: at 30℃, wheat bran, bean dregs ratio 1:9, incubation time 48h, inoculum dosage 0.15%, moisture 50%. Under these conditions, the protease activity reached 3283U/g. In addition, Glucose and Ammonium Sulfate can increase the neutral protease activity of the Koji.
2. By the Koji autolysis, the extract with antioxidants was obtained. When temperature is 40℃, the solid-liquid ratio is 1:20, the hydrolysis time is 3h, the dissolution rate of protein was 69%. Polypeptides are mostly less than 1000 MW. They were tri-peptides, dipeptides and amino acids. The cellulase and the pectinnase were used to improve the antioxidant components. the dissolution rate of protein was 77.3%, at the dosage of cellulase: 0.3g/L, temperature: 40℃, the solid-liquid ratio of 1:25 for 3h; 0.2ml of the extract can scavenge 49.2% DPPH·free radical, 30μl can scavenge 72.1 % ABTS+, at amount of pectinnase added 1g / L, solid-liquid ratio of Koji to water 1:20, temperature 40℃,for 4h.
3. The added cellulase and pectinnase can increased the extract rate and content of antioxidant from the Koji, and the antioxidant activity of the extract improved. As pectinase was added, the antioxidant activities of extract are the best. The extract contained 7.5 mg/ml polypeptides, 1.4mg/ml polysaccharide, 2.0mg/ml polyphenol, 6.5μg/ml flavones,which has high antioxidant activities in different system, the IC50 were 21.5μl, 0.21ml and 0.49ml in ABTS+, DPPH·free radical and pyrogallic acid auto-oxidation system , respectively. 0.1ml of the extract can scavenged 49.2% OH·free radical, and the reduce power reach 0.732 with 0.2ml of the extract。
1.优化了以豆渣和麸皮为基质的固态发酵条件:在30℃,麸皮豆渣比为1:9,培养时间为48h,接种量为0.15%,水分含量为50%水分,米曲霉在此条件下,生长较好,米曲霉蛋白酶活力达到3283U/g。同时考察了添加葡萄糖和硫酸铵可以提高豆渣曲的蛋白酶活力。
2.利用豆渣曲自身酶系水解曲料可获得具有较强抗氧化的提取物,添加纤维素酶和果胶酶可以有效提高抗氧化生理活性物质溶出。直接提取:提取时间为3h,提取温度:40℃,固液比为1:20,在此条件下,测得豆渣曲蛋白质溶出率为69%。分子量大多小于1000,大部分为二肽、三肽及氨基酸。外加纤维素酶提取条件为:加酶量为0.3g/L,提取温度为40℃,固液比为1:25,提取时间为4h。在此条件下,测得豆渣曲蛋白质溶出率为77.3%;外加果胶酶提取抗氧化液最优条件:固液比为1:20,提取温度为45℃,提取时间为4h,加酶量为1g/L。提取液的抗氧化活性分别为:0.20ml提取液清除DPPH能力为67.5%,30μl提取液清除ABTS+能力为72.1%。
3.外加纤维素酶和果胶酶,不同程度的提高曲料中小肽,多酚,多糖以及黄酮抗氧化物质溶出速率和溶出量,从而提高了提取液的抗氧化能力,其中果胶酶效果最好,制得提取液具有最高的抗氧化活性。当含有7.5mg/ml多肽,1.4mg/ml多糖,多酚含量为2.0 mg/ml,以及6.5μg/ml黄酮,具有清除ABTS+和DPPH·自由基的IC50分别是21.5μl和0.21ml,0.1ml的提取液就具有49.2%的羟基自由基清除能力,而邻苯三酚半数抑制率IC50为0.49ml,0.2ml其还原能力就很强,还原产物吸光值就达到0.723。
Soybean residue, the main by-product of soy products, is not only accounting for 15%~20% in dried soybean, but also rich in protein and physiological active elements. Aspergillus oryzae is a universal strain in traditional fermented industry, which contain complexed enzymes. Using soybean residue as raw material, Koji was produced by fermentation of Aspergillus oryzae. The complex enzymes from aspergillus oryzae were used to hydrolyze Koji, anti-oxidant elements was obtained. In this paper, using soybean residue as a medium, Koji is obtained ,and then hydrolyze the koji to obtain the anti-oxidant elements. The results concluded from experiments were as follows.
1. Optimized cultivation conditions for the production of enzyme by Aspergillus oryzae under solid-state fermentation were obtained. The results showed the optimum conditions as followed: at 30℃, wheat bran, bean dregs ratio 1:9, incubation time 48h, inoculum dosage 0.15%, moisture 50%. Under these conditions, the protease activity reached 3283U/g. In addition, Glucose and Ammonium Sulfate can increase the neutral protease activity of the Koji.
2. By the Koji autolysis, the extract with antioxidants was obtained. When temperature is 40℃, the solid-liquid ratio is 1:20, the hydrolysis time is 3h, the dissolution rate of protein was 69%. Polypeptides are mostly less than 1000 MW. They were tri-peptides, dipeptides and amino acids. The cellulase and the pectinnase were used to improve the antioxidant components. the dissolution rate of protein was 77.3%, at the dosage of cellulase: 0.3g/L, temperature: 40℃, the solid-liquid ratio of 1:25 for 3h; 0.2ml of the extract can scavenge 49.2% DPPH·free radical, 30μl can scavenge 72.1 % ABTS+, at amount of pectinnase added 1g / L, solid-liquid ratio of Koji to water 1:20, temperature 40℃,for 4h.
3. The added cellulase and pectinnase can increased the extract rate and content of antioxidant from the Koji, and the antioxidant activity of the extract improved. As pectinase was added, the antioxidant activities of extract are the best. The extract contained 7.5 mg/ml polypeptides, 1.4mg/ml polysaccharide, 2.0mg/ml polyphenol, 6.5μg/ml flavones,which has high antioxidant activities in different system, the IC50 were 21.5μl, 0.21ml and 0.49ml in ABTS+, DPPH·free radical and pyrogallic acid auto-oxidation system , respectively. 0.1ml of the extract can scavenged 49.2% OH·free radical, and the reduce power reach 0.732 with 0.2ml of the extract。
引文
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[2][美]KESHUNLIU著,江连洲译.大豆化学加工工艺与应用[M].哈尔滨:黑龙江科学技术出社,2005.
[3]朱会霞,孙金旭.大豆豆渣膳食纤维的制备条件研究[J].中国酿造,2008,24:400-405.
[4]司方,王明力.酶法提取豆渣水溶性膳食纤维的研究.农产品加工,2009,3:108-110.
[5] E.M. Bainy, S.M. Tosh, M. Corredig et al. Varietal differences of carbohydrates in defatted soybean flour and soy protein isolate by-products [J]. Carbohydrate Polymers 2008, 72: 664–672
[6]朱运平,程永强,汪丽君,等.发酵豆渣的功能性及应用研究现状[J].食品科学,2004,29(5):475-478.
[7]王华博,程裕东,包海蓉.大豆副产品中功能性物质的研究与开发[J].食品科学,2005,26(9):562-566.
[8] Hartmann R, Meisel.H. Food-derived peptides with biological activity:From research to food applications[J]. Current Opinion in Biotechnology, 2007 18,163–169.
[9] Liu F,Ooi V E C., Chang S T. Free radical scavenging activities of mushroom polysaccharide extracts[J]. Life Sciences,1997,60( 10) :763- 771
[10]Yasser F.M. Kishk_, Hanan MA, Al- Sayed.Free- radical scavenging and antioxidative activities of some polysaccharides in emulsions[J].Article in press,2005,11:1- 6
[11] hinde, A. N., Malpathak, N., & Fulzele, D. P.. Studied enhancement strategies for phytoestrogens production in shake flasks by suspension culture of Psoraleacorylifolia[J]. Bioresource Technology, 2009,100, 1833–1839.
[12] Akitha Devi, M. K., Gondi, M., Sakthivelu G., Giridhar,P et al. Functional attributes of soybean seeds and products, with reference to isoflavone content and antioxidant activity. Food Chemistry, 2009,114: 771–776.
[13] Stefano Petti, Crispian Scully . Polyphenols, oral health and disease: Areview[J].journal of dentistry , 2009,37:413-423.
[14] MARK L D. Dietary fiber overview in: dietary fiber in health and disease, handbook of dietary fiber[M]. New York: Marcel Dekker Inc,2001.
[15]刘明,罗远栩,倪辉,等.固态发酵大豆制备的抗氧化肽的活性分析[J].大豆科学,2007,26:381-385.
[16] Faithong, N., et al. Chemical composition and antioxidative activity of Thai traditional fermented shrimp and krill products[J].Food Chemistry ,2009:1-8.
[17]Jiaoyan Ren, Mouming Zhao, John Shi et al. Purification and identification of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry[J]. Food Chemistry ,2008,108:727–736
[18] E.Aida Pen-Ramos, Youling L.Xiong. Whey and soy protein hydrolysates inhibit lipid oxidation in cooked pork patties. Meat Science,2003,64:259–263
[19] Anne Pihlanto,Sari Akkanen,Hannu J.Korhonen . ACE-inhibitory and antioxidant properties of potato (Solanum tuberosum). Food Chemistry 2008,109:104–112
[20]司方,王明力.大蒜多糖的酶法提取及其抗氧化性研究[J].农产品加工, 2009,3:108-110
[21]李光,张雅利,梁花香.豆渣异黄酮超声提取工艺的响应曲面法优化[J].食品科技,2008,2:144-148
[22] Chih-Hung Liang,Jia-Li Syu,Jeng-Leun Mau. Antioxidant properties of solid-state fermented adlay and rice by Phellinus linteus[J]. Food Chemistry, 2009,116:841–845
[23] Yu-Ling Lee,Joan-Hwa Yang. Antioxidant properties of water extracts from Monascusfermented soybeans[J]. Food Chemistry,2008,106:1128–1137
[24] Ana M. Romero,Mirtha M. Doval,Mario A. Sturla. Antioxidant properties of polyphenol-containing extract from soybean fermented with Saccharomyces cerevisiae[J]. Eur. J. Lipid Sci. Technol. 2004,106:424–431
[25] Masako MATSUO. Antioxidant Activity of Hydrophilic Compounds of Defatted Soybean Fermented with Neurospora intermedia (D-Ontjom)[J]. Food Sci. Technol. Res., 2002 ,8 (3): 235–238.
[26]李文林,黄凤洪.天然抗氧化剂研究现状[J].粮食与油脂,2003,110:10-13.
[27]张敬尧,张东杰.天然抗氧化剂在大豆油脂中抗氧化活性的研究[J].中国酿造,2009,3:24-26.
[28] Yu-Ping Sun, Cheng-Chun Chou, Roch-Chui Yu.Antioxidant activity of lactic-fermented Chinese cabbage[J].food chemistry,2009,115:912–917。
[29]张强,牟雪姣,周正义等.豆渣多糖的提取及其抗氧化活性研究[J].中国粮油学报,2007,5:49-52。
[30]吴丽萍,郑辉,薛喜文等.广昌白莲微生物发酵产物多肽的抗氧化活性研究[J].食品科学,2008,.09:365-369
[31] Sandhya, C., Sumantha, A., Szakacs, G., & Pandey, A.. Comparative evaluationof neutral protease production by Aspergillus oryzae in submerged and solid–state fermentation. Process Biochemistry[J], 2005,40: 2689–2694.
[32] M.J. García-Gómez, S. Huerta-Ochoa, O. Loera-Corral, L.A. Prado-Barragán. Advantages of a proteolytic extract by Aspergillus oryzae from fish flour over a commercial proteolytic preparation[J]. Food Chemistry,2009, (112): 604–608
[33] Dyah H. Wardhani, JoséA. Vázquez, Severino S. Pandiella. Optimisation of antioxidants extraction from soybeans fermented by Aspergillus oryzae[J]. Food Chemistry, 2010,118: 731–739
[34] Yu-Ping Sun, Cheng-Chun Chou, Roch-Chui Yu. Antioxidant activity of lactic-fermented Chinese cabbage. Food Chemistry, 2009,115 : 912–917
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