乳酸菌发酵酶解豆粉及其生物活性研究
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摘要
大豆制品是人类重要的植物蛋白资源。乳酸菌发酵可显著改善大豆制品的营养价值,但目前国内外研究多集中在乳酸菌发酵对大豆制品营养成分和风味的改变上,很少研究报道乳酸菌发酵对大豆制品其它生物活性,如抗氧化和ACE抑制活性的影响。本论文首先选育了适合固态发酵豆粉的乳酸菌菌株,在此基础上,对乳酸菌固态发酵豆粉的条件,发酵过程中营养成分的变化,发酵对豆粉抗氧化和ACE抑制活性的影响,发酵产物中ACE抑制和抗氧化活性肽的制备与分离纯化,发酵豆粉中活性肽对生物大分子和离体培养小鼠胸腺细胞氧化损伤的抑制作用及其机制,以及乳酸菌发酵酶解豆粉缓解实验动物断奶应激进行了研究。
1.乳酸菌发酵酶解豆粉菌株选育及发酵条件优化研究
本文首先对适于固态发酵豆粉的乳酸菌菌株进行了选育,通过比较不同乳酸菌菌株在豆粉中生长和产酸性能、以及发酵产物中蛋白质水解度,选育出植物乳杆菌Lp6(L.plantarum Lp6)作为最终的发酵菌株,通过单因素和正交优化实验,得出Lp6固态发酵豆粉的最佳条件为接种量为0.2%,含水量为55%,酸性蛋白酶添加量为0.5%,自然pH值,37℃密闭发酵72 h。
2.乳酸菌发酵酶解豆粉过程中成分变化研究
以植物乳杆菌Lp6固态发酵酶解豆粉,对发酵过程中蛋白质等营养成分的变化进行了研究。乳酸菌发酵可显著提高豆粉中蛋白质的体外消化率,分子量分布分析表明,豆粉中蛋白质降解,生成小分子肽。同时,豆粉中游离氨基酸含量显著增加。发酵还可使豆粉中总糖含量减少,还原糖含量增加,水苏糖和棉籽糖等胀气因子含量显著下降。抗营养因子含量分析表明,乳酸菌发酵可显著降低豆粉中胰蛋白酶抑制因子、植酸和脲酶等抗营养因子含量。
3.乳酸菌发酵酶解豆粉中活性肽的制备
乳酸菌发酵酶解可显著增强豆粉的ACE抑制活性和DPPH、·OH自由基清除能力,但对其Fe~(2+)螯合能力影响不显著。发酵豆粉蛋白提取物经大孔吸附树脂除盐、除糖,中空纤维膜超滤处理后,其蛋白质含量提高,糖和灰分含量降低,其中分子量小于1500 Da的肽含量在90%以上,且ACE抑制和抗氧化活性显著增加。活性肽经消化酶和ACE处理后,仍然保持较强的ACE抑制活性,说明其主要含有抑制型的ACE抑制肽,且具有通过胃肠道进入血液循环的可能性,从而在体内发挥降血压作用。
4.发酵酶解豆粉中活性肽的抗氧化作用研究
乳酸菌发酵酶解豆粉中活性肽可抑制质粒pBR322 DNA中开环和链状DNA的生成,抑制牛血清白蛋白中羰基的产生,从而减轻氧化剂对其造成的氧化损伤。用乳酸菌发酵豆粉中活性肽处理小鼠胸腺细胞,可提高H_2O_2损伤细胞的活性,提高其中T-AOC、SOD、GSH-Px和CAT的活性,降低细胞中ROS和MDA的含量,提高细胞中GSH的含量和GSH/GSSG值,同时,发酵豆粉中活性肽下调细胞中p53 mRNA的表达水平,抑制p53基因介导的细胞调亡,减轻细胞的氧化损伤。
5.发酵酶解豆粉中活性肽的分离纯化和结构鉴定
本论文分离纯化得到了发酵豆粉中一种具有较强ACE抑制和抗氧化活性肽。样品经Sephadex G-15凝胶柱分离,对具OLC分离,借助MALDI-TOF-MS/MS和氨基酸组成分析确定活性峰7-Ⅱ-f-R2的氨基酸序列为VPDALR,其ACE抑制和·OH清除活性IC_(50)值分别为29.3μg/mL、83.2μg/mL。
6.乳酸菌发酵酶解豆粉缓解断奶应激研究
在断奶仔猪日粮中添加乳酸菌发酵酶解豆粉,显著增加了断奶仔猪ADG,并且改善了FCR,降低仔猪血浆中TG含量,增加HDL含量,但对CHOL和LDL影响不显著。同时,还能够提高断奶仔猪抗氧化酶活性,增强机体的抗氧化能力。仔猪肠道菌群分析表明,日粮中添加乳酸菌发酵酶解豆粉,可增加其肠道中乳酸杆菌的数量,降低大肠杆菌的数量,但对双歧杆菌的数量影响不显著。添加发酵豆粉,能增加仔猪肠道消化酶活性,降低肠道内容物pH值,增加其中挥发性脂肪酸的含量。小肠粘膜形态结构分析表明,日粮中添加发酵豆粉,可显著增加仔猪小肠绒毛高度,绒毛高度与隐窝深度比值也显著增加。上述结果表明,乳酸菌发酵酶解豆粉可有效缓解断奶对动物造成的应激。
Soybean is an important plant protein source for human nutrition.Lactic acid fermentation can highly improve the nutritive value of soybean products.However,most of studies until now have been investigated the effects of lactic acid fermentation on the components and flavor of soybean products.Little study has been done about the beneficial physiological effects of lactic acid fermented soybean products,such as antioxidant activity and ACE inhibition activity.In the present study,firstly,we selected a strain of lactic acid bacteria which is suitable for the solid fermentation of soybean flour.Second,we investigated the parameters of solid-state fermentation,the changes of components in soybean flour during the fermentation,the effect of lactic acid fermentation on the antioxidant and ACE inhibitory activity of soybean flour,the isolation and purification of bioactive peptides from the fermented soybean flour.Furthermore,the effect of lactic acid fermented soybean flour on the weaning transition of newly weaned piglets and its mechanism were investigated.
1.Selection of lactic acid bacteria strains for the fermentation of soybean flour and study of the parameters for fermentation
Firstly,in the present study,we compared the growth performance,titratabale acidity and the degree of protein hydrolysis of different strains of lactic acid bacteria in soybean flour. After that,we selected the Lactobacillus plantarum Lp6 as the best strain for the solid-state fermentation of soybean flour.After one factor and orthogonal design experiment,we obtained the best parameters of fermentation are 0.2%inoculum volume,55%of moisture contents,0.5%acid protease addition and natural pH.The mixture was then fermented at 37℃for 72 h.
2.Changes of components in soybean flour during the lactic acid fermentation and enzyme hydrolysis
Soybean flour was fermented by L.plantarum Lp6 and the changes of some components in soybean flour were investigated during the fermentation.Lactic acid fermentation can improve the in vitro digestibility of protein in soybean flour.The high molecule weight protein in soybean flour was degraded into small peptides.Lactic acid fermentation can also decrease the content of total sugars,increase the content of reductive sugars,at the same time, the content of stachyose and raffinose were also decreased.Furthermore,the content of antinutritive factors,such as trypsin inhibitor,phytate and urease were also degraded.
3.Extraction of bioactive peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
Lactic acid fermentation can improve the antioxidant and ACE inhibitory activity of soybean flour.The ACE inhibitory and·OH scavenging activities of the extract from fermented soybean flour was increased after sugars and salt removal by macroporous resinby and hollow fibre ultrafiltration with molecular weight cut-off 6000 Da,the molecule weight of 90%of peptides in the water extract after ultrafiltration was less than 1500 Da suggesting ultrafiltration is an effective technique to enrich for food protein-derived bioactive peptides. The 6000 Da permeates have higher contents of aromatic amino acid than the corresponding protein in soybean flour.The impact of digestive proteases on ACE inhibitory activity of the ACE inhibitory peptides was evaluated under simulated gastrointestinal digestion.Little change has been observed after simulated gastrointestinal digestion,indicated that the ACE inhibitory peptides may exert their ACE inhibitory activity in human body after administration.
4.Antioxidant activity of bioactive peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
The oxidative models of thymocytes induced by H_2O_2 was established and to study the protective effects of bioactive peptides from fermented soybean flour on thymocytes.The peptides could enhance the cell viability and activities of GSH-Px,SOD,A-SAC,and T-AOC in cells.The concentration of GSH and the ratio of GSH/GSSG were increased by peptide from fermented soybean flour.The peptides decreased the concentration of ROS and MDA in cells,it also raised the expression of p53 gene in cells.The peptides from fermented soybean flour could block the oxidative damage of thymocytes induced by H202.
5.Purification and identification of peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
In the present study,isolation and characterization of ACE inhibitory and antioxidant peptides derived from the fermented soybean flour was done in order to realize the relationship between the bioactive activity of peptides and its structure.A peptide 7-Ⅱ-f-R2 was isolated from fermented soybean flour by Sephadex G-15 column,SP-Sephadex c-15 column, two-step reverse-phase high performance liquid chromatography(RP-HPLC) and identified by matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF- MS/MS) and amino acid composition analysis.The amino acid sequence of peptide 7-Ⅱ-f-R2 is VPDALR,and its IC_(50) of ACE inhibitory and·OH scanvenging activity is 29.3μg/mL,83.2μg/mL,respectively.
6.Effect of fermented soybean flour on the weaning transition of newly weaned animals
The effect of lactic acid fermented soybean flour on the weaning transition of newly weaned piglets and its mechanism was investigated in the present study.Seventy-two piglets were allotted to three dietary treatments for 28 days.Aider the feeding trial,the piglets were slaughtered and samples of digesta and intestinal tissue were collected for analysis.The results showed the addition of fermented soybean flour in the diet of piglets improved the growth performance,decreased the diarrhea rate,increased the activity of antioxidant enzyme in the blood,increased the numbers of intestinal lactobacilli,decreased the numbers of intestinal enterobacteria and increased villus height and the villus height to crypt depth ratio at the small intestinal mucosa compared to the control group.In conclusion,lactic acid fermented soybean flour can be used as an excellent protein source for newly weaned animals.
1.乳酸菌发酵酶解豆粉菌株选育及发酵条件优化研究
本文首先对适于固态发酵豆粉的乳酸菌菌株进行了选育,通过比较不同乳酸菌菌株在豆粉中生长和产酸性能、以及发酵产物中蛋白质水解度,选育出植物乳杆菌Lp6(L.plantarum Lp6)作为最终的发酵菌株,通过单因素和正交优化实验,得出Lp6固态发酵豆粉的最佳条件为接种量为0.2%,含水量为55%,酸性蛋白酶添加量为0.5%,自然pH值,37℃密闭发酵72 h。
2.乳酸菌发酵酶解豆粉过程中成分变化研究
以植物乳杆菌Lp6固态发酵酶解豆粉,对发酵过程中蛋白质等营养成分的变化进行了研究。乳酸菌发酵可显著提高豆粉中蛋白质的体外消化率,分子量分布分析表明,豆粉中蛋白质降解,生成小分子肽。同时,豆粉中游离氨基酸含量显著增加。发酵还可使豆粉中总糖含量减少,还原糖含量增加,水苏糖和棉籽糖等胀气因子含量显著下降。抗营养因子含量分析表明,乳酸菌发酵可显著降低豆粉中胰蛋白酶抑制因子、植酸和脲酶等抗营养因子含量。
3.乳酸菌发酵酶解豆粉中活性肽的制备
乳酸菌发酵酶解可显著增强豆粉的ACE抑制活性和DPPH、·OH自由基清除能力,但对其Fe~(2+)螯合能力影响不显著。发酵豆粉蛋白提取物经大孔吸附树脂除盐、除糖,中空纤维膜超滤处理后,其蛋白质含量提高,糖和灰分含量降低,其中分子量小于1500 Da的肽含量在90%以上,且ACE抑制和抗氧化活性显著增加。活性肽经消化酶和ACE处理后,仍然保持较强的ACE抑制活性,说明其主要含有抑制型的ACE抑制肽,且具有通过胃肠道进入血液循环的可能性,从而在体内发挥降血压作用。
4.发酵酶解豆粉中活性肽的抗氧化作用研究
乳酸菌发酵酶解豆粉中活性肽可抑制质粒pBR322 DNA中开环和链状DNA的生成,抑制牛血清白蛋白中羰基的产生,从而减轻氧化剂对其造成的氧化损伤。用乳酸菌发酵豆粉中活性肽处理小鼠胸腺细胞,可提高H_2O_2损伤细胞的活性,提高其中T-AOC、SOD、GSH-Px和CAT的活性,降低细胞中ROS和MDA的含量,提高细胞中GSH的含量和GSH/GSSG值,同时,发酵豆粉中活性肽下调细胞中p53 mRNA的表达水平,抑制p53基因介导的细胞调亡,减轻细胞的氧化损伤。
5.发酵酶解豆粉中活性肽的分离纯化和结构鉴定
本论文分离纯化得到了发酵豆粉中一种具有较强ACE抑制和抗氧化活性肽。样品经Sephadex G-15凝胶柱分离,对具OLC分离,借助MALDI-TOF-MS/MS和氨基酸组成分析确定活性峰7-Ⅱ-f-R2的氨基酸序列为VPDALR,其ACE抑制和·OH清除活性IC_(50)值分别为29.3μg/mL、83.2μg/mL。
6.乳酸菌发酵酶解豆粉缓解断奶应激研究
在断奶仔猪日粮中添加乳酸菌发酵酶解豆粉,显著增加了断奶仔猪ADG,并且改善了FCR,降低仔猪血浆中TG含量,增加HDL含量,但对CHOL和LDL影响不显著。同时,还能够提高断奶仔猪抗氧化酶活性,增强机体的抗氧化能力。仔猪肠道菌群分析表明,日粮中添加乳酸菌发酵酶解豆粉,可增加其肠道中乳酸杆菌的数量,降低大肠杆菌的数量,但对双歧杆菌的数量影响不显著。添加发酵豆粉,能增加仔猪肠道消化酶活性,降低肠道内容物pH值,增加其中挥发性脂肪酸的含量。小肠粘膜形态结构分析表明,日粮中添加发酵豆粉,可显著增加仔猪小肠绒毛高度,绒毛高度与隐窝深度比值也显著增加。上述结果表明,乳酸菌发酵酶解豆粉可有效缓解断奶对动物造成的应激。
Soybean is an important plant protein source for human nutrition.Lactic acid fermentation can highly improve the nutritive value of soybean products.However,most of studies until now have been investigated the effects of lactic acid fermentation on the components and flavor of soybean products.Little study has been done about the beneficial physiological effects of lactic acid fermented soybean products,such as antioxidant activity and ACE inhibition activity.In the present study,firstly,we selected a strain of lactic acid bacteria which is suitable for the solid fermentation of soybean flour.Second,we investigated the parameters of solid-state fermentation,the changes of components in soybean flour during the fermentation,the effect of lactic acid fermentation on the antioxidant and ACE inhibitory activity of soybean flour,the isolation and purification of bioactive peptides from the fermented soybean flour.Furthermore,the effect of lactic acid fermented soybean flour on the weaning transition of newly weaned piglets and its mechanism were investigated.
1.Selection of lactic acid bacteria strains for the fermentation of soybean flour and study of the parameters for fermentation
Firstly,in the present study,we compared the growth performance,titratabale acidity and the degree of protein hydrolysis of different strains of lactic acid bacteria in soybean flour. After that,we selected the Lactobacillus plantarum Lp6 as the best strain for the solid-state fermentation of soybean flour.After one factor and orthogonal design experiment,we obtained the best parameters of fermentation are 0.2%inoculum volume,55%of moisture contents,0.5%acid protease addition and natural pH.The mixture was then fermented at 37℃for 72 h.
2.Changes of components in soybean flour during the lactic acid fermentation and enzyme hydrolysis
Soybean flour was fermented by L.plantarum Lp6 and the changes of some components in soybean flour were investigated during the fermentation.Lactic acid fermentation can improve the in vitro digestibility of protein in soybean flour.The high molecule weight protein in soybean flour was degraded into small peptides.Lactic acid fermentation can also decrease the content of total sugars,increase the content of reductive sugars,at the same time, the content of stachyose and raffinose were also decreased.Furthermore,the content of antinutritive factors,such as trypsin inhibitor,phytate and urease were also degraded.
3.Extraction of bioactive peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
Lactic acid fermentation can improve the antioxidant and ACE inhibitory activity of soybean flour.The ACE inhibitory and·OH scavenging activities of the extract from fermented soybean flour was increased after sugars and salt removal by macroporous resinby and hollow fibre ultrafiltration with molecular weight cut-off 6000 Da,the molecule weight of 90%of peptides in the water extract after ultrafiltration was less than 1500 Da suggesting ultrafiltration is an effective technique to enrich for food protein-derived bioactive peptides. The 6000 Da permeates have higher contents of aromatic amino acid than the corresponding protein in soybean flour.The impact of digestive proteases on ACE inhibitory activity of the ACE inhibitory peptides was evaluated under simulated gastrointestinal digestion.Little change has been observed after simulated gastrointestinal digestion,indicated that the ACE inhibitory peptides may exert their ACE inhibitory activity in human body after administration.
4.Antioxidant activity of bioactive peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
The oxidative models of thymocytes induced by H_2O_2 was established and to study the protective effects of bioactive peptides from fermented soybean flour on thymocytes.The peptides could enhance the cell viability and activities of GSH-Px,SOD,A-SAC,and T-AOC in cells.The concentration of GSH and the ratio of GSH/GSSG were increased by peptide from fermented soybean flour.The peptides decreased the concentration of ROS and MDA in cells,it also raised the expression of p53 gene in cells.The peptides from fermented soybean flour could block the oxidative damage of thymocytes induced by H202.
5.Purification and identification of peptides from soybean flour processed by lactic acid fermentation and enzyme hydrolysis
In the present study,isolation and characterization of ACE inhibitory and antioxidant peptides derived from the fermented soybean flour was done in order to realize the relationship between the bioactive activity of peptides and its structure.A peptide 7-Ⅱ-f-R2 was isolated from fermented soybean flour by Sephadex G-15 column,SP-Sephadex c-15 column, two-step reverse-phase high performance liquid chromatography(RP-HPLC) and identified by matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF- MS/MS) and amino acid composition analysis.The amino acid sequence of peptide 7-Ⅱ-f-R2 is VPDALR,and its IC_(50) of ACE inhibitory and·OH scanvenging activity is 29.3μg/mL,83.2μg/mL,respectively.
6.Effect of fermented soybean flour on the weaning transition of newly weaned animals
The effect of lactic acid fermented soybean flour on the weaning transition of newly weaned piglets and its mechanism was investigated in the present study.Seventy-two piglets were allotted to three dietary treatments for 28 days.Aider the feeding trial,the piglets were slaughtered and samples of digesta and intestinal tissue were collected for analysis.The results showed the addition of fermented soybean flour in the diet of piglets improved the growth performance,decreased the diarrhea rate,increased the activity of antioxidant enzyme in the blood,increased the numbers of intestinal lactobacilli,decreased the numbers of intestinal enterobacteria and increased villus height and the villus height to crypt depth ratio at the small intestinal mucosa compared to the control group.In conclusion,lactic acid fermented soybean flour can be used as an excellent protein source for newly weaned animals.
引文
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