纳豆芽孢杆菌固体发酵低温豆粕的功能活性及机制研究
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摘要
低温豆粕是大豆经提取豆油后的湿粕经低温脱溶工艺得到的副产品,是一种蛋白变性低、质量高的植物蛋白资源。也是一种来源易得、价格便宜的大宗农副产品资源,而目前主要用作饲料使用,造成资源浪费。本文着眼于豆粕的深度开发,旨在为开发一种健康食品资源提供试验依据。
以纳豆芽孢杆菌发酵大豆而得的纳豆具有溶血栓、抗肿瘤、抗氧化、抗菌、预防骨质疏松症以及调节肠功能等许多作用。同时纳豆芽孢杆菌还是FDA规定的43种益生菌之一以及我国农业部公布的16种可直接饲喂的微生物之一。
本试验以纳豆芽孢杆菌作为发酵菌种,以低温豆粕为基质进行固体发酵,研究发酵豆粕提取物的抗氧化活性、抗氧化活性成分的分离以及发酵豆粕提取物对小鼠肠道菌群的调节作用。
主要研究内容及结论如下：
(1)对发酵豆粕提取物的营养成分进行了分析与评价。发酵豆粕提取物蛋白含量高、脂肪含量低,分别为：57.26%和0.86%。发酵豆粕提取物含有铁、镁等多种矿物质元素。豆粕发酵以后的氨基酸组成有一定的变化,但总氨基酸含量基本不变。与发酵豆粕比较,发酵豆粕提取物在天冬氨酸、谷氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、赖氨酸及脯氨酸含量方面占明显优势,总氨基酸含量提高3%,发酵豆粕提取物包含人体所必需的8种氨基酸,尤其亮氨酸、赖氨酸、色氨酸含量较高。
(2)对发酵豆粕提取物的体外抗氧化活性进行考察。采用相同条件下的60%的乙醇提取物进行试验。分别考察了超氧阴离子自由基及DPPH自由基清除活性,Fe3+还原力,对鼠肝匀浆脂质过氧化的抑制能力以及对小鼠红细胞溶血的抑制能力。结果表明：与豆粕提取物相比,发酵豆粕提取物具有更高的清除DPPH·和O2-·的活性、Fe3+还原能力、抑制脂质过氧化以及红细胞溶血的能力,其IC50分别为0.321mg/mL,7.404mg/mL,2.128mg/mL,0.737mg/mL和4.599mg/mL.
(3)对以D-半乳糖诱导的衰老模型小鼠的体内抗氧化活性进行研究。发酵豆粕提取物可以提高模型小鼠血清及肝组织中的SOD、 GSH-Px、CAT活性,减少MDA的生成；减少模型小鼠脑组织中的MAO活性、提高NOS、 Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,其抗氧化活性强于未发酵豆粕。
(4)为分离发酵豆粕提取物中的抗氧化活性物质,进行了豆粕发酵工艺及活性成分提取工艺的优化试验研究,固体发酵条件优化结果为：基质含水量为73%,接种量19%,发酵时间94.5h。在此基础上,通过单因素以及正交试验优化的提取条件,结果为：乙醇体积分数50%,液固比10：1,提取时间90min,提取3次。
(5)在抗氧化活性指导下,对发酵豆粕提取物进行了系统分离与柱色谱分离,得到一种抗氧化活性物质,高效液相色谱(HPLC)分析显示为单一峰,紫外扫描最大吸收波长为260nm,质谱检测分子量为270,经碳谱、氢谱及文献比对鉴定为染料木素,分子式为C15H10O5.建立了发酵豆粕提取物中染料木素含量测定的HPLC分析法,其具体色谱条件为：色谱柱：KrOmasil C18柱(250mm×4.6mm,5μm)；流动相：甲醇-水(60：40)；流速：0.8mL／min；检测波长：260nm；进样量：20μL。柱温：室温(25℃)。测定FSME中染料木素含量为545.73±8.14μg/g,即为0.055%；发酵豆粕中染料木素含量为0.024%,而测得的豆粕中染料木素含量为0.011%,低温豆粕经纳豆芽孢杆菌发酵后,染料木素含量提高了1.2倍。
(6)对健康小鼠及抗生素诱导的腹泻模型小鼠肠道菌群的调节作用进行研究,结果表明：发酵豆粕提取物能够显著促进健康小鼠及抗生素诱导的腹泻模型小鼠肠道中厌氧菌群的生长,抑制好氧菌群的增殖,其调节肠道菌群作用强于未发酵豆粕。发酵豆粕提取物有望成为一种益生元类物质应用于食品、药品等行业
Low-temperature soybean meal is a by-product of the oil extraction of soybean, which is obtained from the wet soybean meal by removing solvent at the condition of low temperature. It is a kind of high-quality protein plant resource and highly produced in China as a source of readily available and inexpensive agricultural resources, but it is mainly used for feed, which results in a waste of resources. It is necessary to develop a kind of healthy food resources in order to make full use of soybean meal.
Natto has many functions including thrombolyitc activity, antitumor, antioxidation, prevention of osteoporosis, antibiosis and regulation of intestinal function. Bacillus natto has been recognized as one of strains can be fed directly to animals by Ministry of Agriculture of China and FDA.
In the present work, Bacillus natto was employed to ferment low-temperature soymean meal and antioxidant activity of fermented soybean meal extract from fermented low-temperature soybean meal was investigated. The ingredients of antioxidant activity waere separated and regulation of intestinal flora in diarrhea mice induced by antibiotics was carried out.
The main contents and conclusions are as follows:
(1) Nutritional components were analyed and evaluated on FSME. The content of protein and fat in FSME was57.26%and0.86%, respectively. FSME contained iron, magnesium and other minerals. Compared with soybean meal, the content of some amino acid specicies of FSME were changed, but the total amino acid content remained unchanged. Compared with fermented soybean meal, FSME in aspartic acid, glutamic acid, isoleucine, leucine, tyrosine, phenylalanine, lysine and proline content was dominant; the total amino acid content increased3%. There were8essential amino acids in FSME, especially leucine, lysine, tryptophan content were high.
(2) The antioxidant activity of fermented low-temperature soybean meal in vitro was studied. Experiments were conducted using60%ethanol extracts prepared under the same conditions. The superoxide anion radical and DPPH radical scavenging activities, Fe3+reducing power, rat liver homogenate inhibitory ability of lipid peroxidation and red blood cells of hemolysis inhibitory ability were checked. The results shows that:compared with soybean meal extract, fermented low-temperature soybean meal extract(FSME) had strong scavenging activities of DPPH radical and superoxide anion radical, had strong Fe+reducing ability, inhibition of lipid peroxidation and red blood cells hemolysis, the IC50was0.321mg/mL,7.404rag/mL, 2.128mg/mL,0.737mg/mL,4.599mg/mL, respectively.
(3) The antioxidant activity in vivo was investigated by using the D-galactose induced aging mice as models. Fermented soybean meal extract had increased SOD, GSH-Px, CAT activity in the serum and liver tissue of aging mice, decreased the content of lipid peroxide malondialdehyde. Fermented soybean meal extract had reduced MAO activity in brain tissue of aging mice, increased NOS, Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity. The antioxidant activity of fermented soybean meal extract was better than that of soybean meal extract.
(4) In order to separate antioxidant activity ingredient of fermented soybean meal extract, process conditions are optimized. Solid fermentation optimization result is: matrix water content73%, inoculation19%, fermentation time94.5h. Based on these results, the single factor and orthogonal tests were performed to determine the optimal extraction conditions:volume fraction of ethanol50%, the liquid-solid ratio10:1, extracting time90min, extracted three times.
(5) The system separation and column chromatography was subjected to FSME, and an active substance was obtained and showed a single peak in HPLC. The maximum absorption wavelength was at260nra by ultraviolet scanning. Molecular weight of MS detection was270, and then genistein was identified by comparison of'H NMR and13C NMR spectra with literature data, formulas was C15H10O5.
The analytic method with HPLC has been established for quantitation of genistein content in FSME. The chromatography conditions were:chromatographic column: Kromasil C18column (250mm x4.6mm,5u.m); Mobile phase:methanol-water (60:40); Flow rate:0.8mL/min; Detected wavelength:260nm; sample amount:20μL. The column temperature:room temperature (25℃). Genistein content of FSME is545.73±8.14μg/g, is0.055%; Genistein content of fermented soybean meal is0.024%. Genistein content of soybean meal is0.011%, which shows that Genistein content is increased by1.2times when soybean meal is fermented by Bacillus natto.
(6) Regulation of intestinal flora in nomal mice and diarrhea mice induced by antibiotics was tested with FSME. It could significantly promote the growth of intestinal anaerobic bacteria; inhibit the proliferation of aerobic bacteria. The activity of FSME was better than non-fermented soybean meal. FSME was a potential substance, could be used as a prebiotics in Food or pharmaceutical industry.
以纳豆芽孢杆菌发酵大豆而得的纳豆具有溶血栓、抗肿瘤、抗氧化、抗菌、预防骨质疏松症以及调节肠功能等许多作用。同时纳豆芽孢杆菌还是FDA规定的43种益生菌之一以及我国农业部公布的16种可直接饲喂的微生物之一。
本试验以纳豆芽孢杆菌作为发酵菌种,以低温豆粕为基质进行固体发酵,研究发酵豆粕提取物的抗氧化活性、抗氧化活性成分的分离以及发酵豆粕提取物对小鼠肠道菌群的调节作用。
主要研究内容及结论如下：
(1)对发酵豆粕提取物的营养成分进行了分析与评价。发酵豆粕提取物蛋白含量高、脂肪含量低,分别为：57.26%和0.86%。发酵豆粕提取物含有铁、镁等多种矿物质元素。豆粕发酵以后的氨基酸组成有一定的变化,但总氨基酸含量基本不变。与发酵豆粕比较,发酵豆粕提取物在天冬氨酸、谷氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、赖氨酸及脯氨酸含量方面占明显优势,总氨基酸含量提高3%,发酵豆粕提取物包含人体所必需的8种氨基酸,尤其亮氨酸、赖氨酸、色氨酸含量较高。
(2)对发酵豆粕提取物的体外抗氧化活性进行考察。采用相同条件下的60%的乙醇提取物进行试验。分别考察了超氧阴离子自由基及DPPH自由基清除活性,Fe3+还原力,对鼠肝匀浆脂质过氧化的抑制能力以及对小鼠红细胞溶血的抑制能力。结果表明：与豆粕提取物相比,发酵豆粕提取物具有更高的清除DPPH·和O2-·的活性、Fe3+还原能力、抑制脂质过氧化以及红细胞溶血的能力,其IC50分别为0.321mg/mL,7.404mg/mL,2.128mg/mL,0.737mg/mL和4.599mg/mL.
(3)对以D-半乳糖诱导的衰老模型小鼠的体内抗氧化活性进行研究。发酵豆粕提取物可以提高模型小鼠血清及肝组织中的SOD、 GSH-Px、CAT活性,减少MDA的生成；减少模型小鼠脑组织中的MAO活性、提高NOS、 Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,其抗氧化活性强于未发酵豆粕。
(4)为分离发酵豆粕提取物中的抗氧化活性物质,进行了豆粕发酵工艺及活性成分提取工艺的优化试验研究,固体发酵条件优化结果为：基质含水量为73%,接种量19%,发酵时间94.5h。在此基础上,通过单因素以及正交试验优化的提取条件,结果为：乙醇体积分数50%,液固比10：1,提取时间90min,提取3次。
(5)在抗氧化活性指导下,对发酵豆粕提取物进行了系统分离与柱色谱分离,得到一种抗氧化活性物质,高效液相色谱(HPLC)分析显示为单一峰,紫外扫描最大吸收波长为260nm,质谱检测分子量为270,经碳谱、氢谱及文献比对鉴定为染料木素,分子式为C15H10O5.建立了发酵豆粕提取物中染料木素含量测定的HPLC分析法,其具体色谱条件为：色谱柱：KrOmasil C18柱(250mm×4.6mm,5μm)；流动相：甲醇-水(60：40)；流速：0.8mL／min；检测波长：260nm；进样量：20μL。柱温：室温(25℃)。测定FSME中染料木素含量为545.73±8.14μg/g,即为0.055%；发酵豆粕中染料木素含量为0.024%,而测得的豆粕中染料木素含量为0.011%,低温豆粕经纳豆芽孢杆菌发酵后,染料木素含量提高了1.2倍。
(6)对健康小鼠及抗生素诱导的腹泻模型小鼠肠道菌群的调节作用进行研究,结果表明：发酵豆粕提取物能够显著促进健康小鼠及抗生素诱导的腹泻模型小鼠肠道中厌氧菌群的生长,抑制好氧菌群的增殖,其调节肠道菌群作用强于未发酵豆粕。发酵豆粕提取物有望成为一种益生元类物质应用于食品、药品等行业
Low-temperature soybean meal is a by-product of the oil extraction of soybean, which is obtained from the wet soybean meal by removing solvent at the condition of low temperature. It is a kind of high-quality protein plant resource and highly produced in China as a source of readily available and inexpensive agricultural resources, but it is mainly used for feed, which results in a waste of resources. It is necessary to develop a kind of healthy food resources in order to make full use of soybean meal.
Natto has many functions including thrombolyitc activity, antitumor, antioxidation, prevention of osteoporosis, antibiosis and regulation of intestinal function. Bacillus natto has been recognized as one of strains can be fed directly to animals by Ministry of Agriculture of China and FDA.
In the present work, Bacillus natto was employed to ferment low-temperature soymean meal and antioxidant activity of fermented soybean meal extract from fermented low-temperature soybean meal was investigated. The ingredients of antioxidant activity waere separated and regulation of intestinal flora in diarrhea mice induced by antibiotics was carried out.
The main contents and conclusions are as follows:
(1) Nutritional components were analyed and evaluated on FSME. The content of protein and fat in FSME was57.26%and0.86%, respectively. FSME contained iron, magnesium and other minerals. Compared with soybean meal, the content of some amino acid specicies of FSME were changed, but the total amino acid content remained unchanged. Compared with fermented soybean meal, FSME in aspartic acid, glutamic acid, isoleucine, leucine, tyrosine, phenylalanine, lysine and proline content was dominant; the total amino acid content increased3%. There were8essential amino acids in FSME, especially leucine, lysine, tryptophan content were high.
(2) The antioxidant activity of fermented low-temperature soybean meal in vitro was studied. Experiments were conducted using60%ethanol extracts prepared under the same conditions. The superoxide anion radical and DPPH radical scavenging activities, Fe3+reducing power, rat liver homogenate inhibitory ability of lipid peroxidation and red blood cells of hemolysis inhibitory ability were checked. The results shows that:compared with soybean meal extract, fermented low-temperature soybean meal extract(FSME) had strong scavenging activities of DPPH radical and superoxide anion radical, had strong Fe+reducing ability, inhibition of lipid peroxidation and red blood cells hemolysis, the IC50was0.321mg/mL,7.404rag/mL, 2.128mg/mL,0.737mg/mL,4.599mg/mL, respectively.
(3) The antioxidant activity in vivo was investigated by using the D-galactose induced aging mice as models. Fermented soybean meal extract had increased SOD, GSH-Px, CAT activity in the serum and liver tissue of aging mice, decreased the content of lipid peroxide malondialdehyde. Fermented soybean meal extract had reduced MAO activity in brain tissue of aging mice, increased NOS, Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity. The antioxidant activity of fermented soybean meal extract was better than that of soybean meal extract.
(4) In order to separate antioxidant activity ingredient of fermented soybean meal extract, process conditions are optimized. Solid fermentation optimization result is: matrix water content73%, inoculation19%, fermentation time94.5h. Based on these results, the single factor and orthogonal tests were performed to determine the optimal extraction conditions:volume fraction of ethanol50%, the liquid-solid ratio10:1, extracting time90min, extracted three times.
(5) The system separation and column chromatography was subjected to FSME, and an active substance was obtained and showed a single peak in HPLC. The maximum absorption wavelength was at260nra by ultraviolet scanning. Molecular weight of MS detection was270, and then genistein was identified by comparison of'H NMR and13C NMR spectra with literature data, formulas was C15H10O5.
The analytic method with HPLC has been established for quantitation of genistein content in FSME. The chromatography conditions were:chromatographic column: Kromasil C18column (250mm x4.6mm,5u.m); Mobile phase:methanol-water (60:40); Flow rate:0.8mL/min; Detected wavelength:260nm; sample amount:20μL. The column temperature:room temperature (25℃). Genistein content of FSME is545.73±8.14μg/g, is0.055%; Genistein content of fermented soybean meal is0.024%. Genistein content of soybean meal is0.011%, which shows that Genistein content is increased by1.2times when soybean meal is fermented by Bacillus natto.
(6) Regulation of intestinal flora in nomal mice and diarrhea mice induced by antibiotics was tested with FSME. It could significantly promote the growth of intestinal anaerobic bacteria; inhibit the proliferation of aerobic bacteria. The activity of FSME was better than non-fermented soybean meal. FSME was a potential substance, could be used as a prebiotics in Food or pharmaceutical industry.
引文
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