大豆肽的分离纯化及应用研究
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摘要
大豆肽具有许多独特的理化特性与生物学活性,同时具有原料来源广泛、价廉,安全性好,无毒副作用,便于产业化生产的优点。本文以两种样品肽作为研究对象,分别对其理化性质,分离纯化方法以及具体的应用进行研究,为大豆肽产品的继续深入研究提供了试验基础。
分析测定两种样品肽的一般成分及理化性质,结果表明:大豆分离蛋白肽和豆粕肽的氨基酸组成基本一致,但蛋白肽含量相差较大;在较广泛的pH值条件下,前者保持更好的溶解状态。随着浓度的增大,两种样品肽的粘度均有增加,但浓度超过60%后,大豆分离蛋白肽保持良好的流动性,而豆粕肽的流动性降低;大豆分离蛋白肽具有更强的还原能力和抗氧化能力,但乳化性及其稳定性却不如豆粕肽。
大豆分离蛋白肽经凝胶过滤层析洗脱分离,选用Sephadex G-25为填料,最佳洗脱条件为:径高比1:35;上样体积3mL;上样浓度0.01g/mL;洗脱流速16mL/h;洗脱液为蒸馏水。高效凝胶渗透色谱(HPGPC)分析结果表明,各洗脱组分均为单一组成,分子量分别为:组分1:1860Da;组分2:1426Da;组分3:853Da;组分4:376Da。红外色谱分析表明:四个组分的峰形基本一致,在3049-3406cm~(-1)处和1690-1630cm~(-1)之间具有典型的酰胺吸收峰。
选用DA021-CⅡ型大孔吸附树脂作为填料洗脱分离豆粕肽,径高比1:30,样品肽液pH值3.0,浓度45mg/mL,上样量为40mL,吸附流速为0.5mL/min,解析流速为1.0mL/min,水解析体积为2BV,经梯度乙醇洗脱可以得到4个组分;继续用Sephadex G-25柱层析洗脱得6个组分,HPGPC测定其分子量分别为:aⅠ:1845Da、209Da;bⅠ:288Da;bⅡ:4019Da、402Da;cⅠ:2314Da、197Da;dⅠ:1318Da、180Da;dⅡ:2719Da、209Da。
采用响应面法(RSM)对大豆肽与钙的螯合工艺条件进行优化,最佳工艺参数为:大豆肽与氯化钙的混合比4.2:1(w/w),水浴时间21.3min,无水乙醇添加6倍,大豆肽-钙的得率可达65.48%,钙含量为7.74g/(100g大豆肽-钙产品)。红外色谱分析表明:在大豆肽与钙的螯合试验中,氨基与羧基均参与了螯合反应。
试验采用活菌计数法和光密度法分析两种样品肽对S.thermophilus生长的影响,结果表明:两种样品肽均促进了S.thermophilus的生长,但随着在培养基中添加量的增加,豆粕肽对S.thermophilus的促生长作用逐渐减弱,而大豆分离蛋白肽的促生长作用则增强。利用光密度法比较两种样品肽对B.infantis生长的影响,结果表明:添加较少量的豆粕肽对B.infantis的促生长作用很明显,但随着添加量的增加,促生长作用减弱;而大豆分离蛋白肽对B.infantis的促生长作用却随着添加量的增加平缓增强。
Soybean peptides have many distinct physical-chemical properties and biochemical activities, and also possess merits respectively in widespread source of raw materials, low price, great safety, no side effects, easy for industrial production. This article respectively studied the physical-chemical properties, methods for separation, purification and applications in some aspects, with two kinds of soybean peptides as the investigate objects, offered experimental foundation for further research of soybean peptides product.
Common compositions, physical-chemical properties of the two kinds of sample peptides were determined and analyzed, the results showed that: the two kinds of peptides generally owned the same amino acids compositions, but were quite different in protein content; soybean separation protein peptides kept better dissolved state in wide pH condition. Viscosity of the two kinds of sample peptides augmented with the concentration increasing, soybean separation protein peptides kept good fluidity when concentration overtopping 60%, but crude soybean peptides not; soybean separation protein peptides showed more powerful abilities both in deoxidation and antioxidation, but less in emulsibility and its stability.
Gel filtration chromatography (GFC) was used to elute and separate the soybean separation protein peptides with Sephadex G-25 as the loading material, the optimum elution requirements were: the ratio of diameter to height of the chromatographic column was 1:35; addition volume of sample was 3mL; sample concentration was 0.01 g/mL; elution flow rate was 16mL/h; elutriant was distilled water. The analysis result of HPGPC showed that : each elution composition was single component, molecular weights respectively were: component 1: 1860 Da; component 2: 1426Da; component 3: 853Da; component 4: 376Da. Infrared chromatographic analysis showed that: the peak forms of the four components were fundamentally coincident with each other, and showed typical amides absorption peaks between 3049-3406cm~(-1) and 1690-1630cm~(-1).
DA201-CⅡmacroporous adsorptive resins was used as the packing material to elute and separate crude soybean peptides, diameter to height of the column was 1:30, the pH of sample peptides solution was 3.0, the concentration was 45mg/mL, the amount of the active peptides solution was 40mL, the adsorption flow rate was 0.5mL/min, the resolution flow rate was 1.0mL/min, resolution volume of water was 2BV, 4 compositions was attained through gradient alcohol eluting; after being further eluted by gel chromatography Sephadex G-25, 6 compositions were attained, and the molecule weights determined by HPGPC were respectly: aⅠ: 1845Da, 209Da; bⅠ: 288Da; bⅡ: 4019Da, 402Da, 207Da; cⅠ: 2314Da, 197aD; dⅠ: 1318Da, 180Da; dⅡ: 2719Da, 209Da.
Response surface method (RSM) was used to was used to optimize the technology and conditions of soybean peptides chelating Calcium, the optimum process parameters were: the ratio of soybean peptides to CaCl_2 was 4.2:1 (w/w), water bath time was 22.3 min, the times of absolute alcohol volume to mixed liquor was 6, under which, the yield of soybean peptides-Calcium was 65.48%, and the content of calcium was up to 7.74g per 100g soybean peptides-Calcium. The result of infrared chromatographic analysis showed that: both the amino group and carboxyl group participated in the chelating reaction.
The experiment analyzed the effect of the two sample peptides to the growth of S. thermophilus by using methods of count plate and optical density, the result showed that: both the two sample peptides promoted the growth of S. Thermophilu, with the addition amounts increasing in the nutrient mediums, the growth promotion action made by crude soybean peptides tapered while soybean separation protein peptides enhanced. Optical density method was used to analyze the effect of the two sample peptides to the growth of B. infantis, the result showed that: smaller addition amount of crude soybean peptides made a obvious growth promotion action but tapered with the addition amount increasing, while the growth promotion action made by soybean separation protein peptides enhanced with the addition amount increasing.
分析测定两种样品肽的一般成分及理化性质,结果表明:大豆分离蛋白肽和豆粕肽的氨基酸组成基本一致,但蛋白肽含量相差较大;在较广泛的pH值条件下,前者保持更好的溶解状态。随着浓度的增大,两种样品肽的粘度均有增加,但浓度超过60%后,大豆分离蛋白肽保持良好的流动性,而豆粕肽的流动性降低;大豆分离蛋白肽具有更强的还原能力和抗氧化能力,但乳化性及其稳定性却不如豆粕肽。
大豆分离蛋白肽经凝胶过滤层析洗脱分离,选用Sephadex G-25为填料,最佳洗脱条件为:径高比1:35;上样体积3mL;上样浓度0.01g/mL;洗脱流速16mL/h;洗脱液为蒸馏水。高效凝胶渗透色谱(HPGPC)分析结果表明,各洗脱组分均为单一组成,分子量分别为:组分1:1860Da;组分2:1426Da;组分3:853Da;组分4:376Da。红外色谱分析表明:四个组分的峰形基本一致,在3049-3406cm~(-1)处和1690-1630cm~(-1)之间具有典型的酰胺吸收峰。
选用DA021-CⅡ型大孔吸附树脂作为填料洗脱分离豆粕肽,径高比1:30,样品肽液pH值3.0,浓度45mg/mL,上样量为40mL,吸附流速为0.5mL/min,解析流速为1.0mL/min,水解析体积为2BV,经梯度乙醇洗脱可以得到4个组分;继续用Sephadex G-25柱层析洗脱得6个组分,HPGPC测定其分子量分别为:aⅠ:1845Da、209Da;bⅠ:288Da;bⅡ:4019Da、402Da;cⅠ:2314Da、197Da;dⅠ:1318Da、180Da;dⅡ:2719Da、209Da。
采用响应面法(RSM)对大豆肽与钙的螯合工艺条件进行优化,最佳工艺参数为:大豆肽与氯化钙的混合比4.2:1(w/w),水浴时间21.3min,无水乙醇添加6倍,大豆肽-钙的得率可达65.48%,钙含量为7.74g/(100g大豆肽-钙产品)。红外色谱分析表明:在大豆肽与钙的螯合试验中,氨基与羧基均参与了螯合反应。
试验采用活菌计数法和光密度法分析两种样品肽对S.thermophilus生长的影响,结果表明:两种样品肽均促进了S.thermophilus的生长,但随着在培养基中添加量的增加,豆粕肽对S.thermophilus的促生长作用逐渐减弱,而大豆分离蛋白肽的促生长作用则增强。利用光密度法比较两种样品肽对B.infantis生长的影响,结果表明:添加较少量的豆粕肽对B.infantis的促生长作用很明显,但随着添加量的增加,促生长作用减弱;而大豆分离蛋白肽对B.infantis的促生长作用却随着添加量的增加平缓增强。
Soybean peptides have many distinct physical-chemical properties and biochemical activities, and also possess merits respectively in widespread source of raw materials, low price, great safety, no side effects, easy for industrial production. This article respectively studied the physical-chemical properties, methods for separation, purification and applications in some aspects, with two kinds of soybean peptides as the investigate objects, offered experimental foundation for further research of soybean peptides product.
Common compositions, physical-chemical properties of the two kinds of sample peptides were determined and analyzed, the results showed that: the two kinds of peptides generally owned the same amino acids compositions, but were quite different in protein content; soybean separation protein peptides kept better dissolved state in wide pH condition. Viscosity of the two kinds of sample peptides augmented with the concentration increasing, soybean separation protein peptides kept good fluidity when concentration overtopping 60%, but crude soybean peptides not; soybean separation protein peptides showed more powerful abilities both in deoxidation and antioxidation, but less in emulsibility and its stability.
Gel filtration chromatography (GFC) was used to elute and separate the soybean separation protein peptides with Sephadex G-25 as the loading material, the optimum elution requirements were: the ratio of diameter to height of the chromatographic column was 1:35; addition volume of sample was 3mL; sample concentration was 0.01 g/mL; elution flow rate was 16mL/h; elutriant was distilled water. The analysis result of HPGPC showed that : each elution composition was single component, molecular weights respectively were: component 1: 1860 Da; component 2: 1426Da; component 3: 853Da; component 4: 376Da. Infrared chromatographic analysis showed that: the peak forms of the four components were fundamentally coincident with each other, and showed typical amides absorption peaks between 3049-3406cm~(-1) and 1690-1630cm~(-1).
DA201-CⅡmacroporous adsorptive resins was used as the packing material to elute and separate crude soybean peptides, diameter to height of the column was 1:30, the pH of sample peptides solution was 3.0, the concentration was 45mg/mL, the amount of the active peptides solution was 40mL, the adsorption flow rate was 0.5mL/min, the resolution flow rate was 1.0mL/min, resolution volume of water was 2BV, 4 compositions was attained through gradient alcohol eluting; after being further eluted by gel chromatography Sephadex G-25, 6 compositions were attained, and the molecule weights determined by HPGPC were respectly: aⅠ: 1845Da, 209Da; bⅠ: 288Da; bⅡ: 4019Da, 402Da, 207Da; cⅠ: 2314Da, 197aD; dⅠ: 1318Da, 180Da; dⅡ: 2719Da, 209Da.
Response surface method (RSM) was used to was used to optimize the technology and conditions of soybean peptides chelating Calcium, the optimum process parameters were: the ratio of soybean peptides to CaCl_2 was 4.2:1 (w/w), water bath time was 22.3 min, the times of absolute alcohol volume to mixed liquor was 6, under which, the yield of soybean peptides-Calcium was 65.48%, and the content of calcium was up to 7.74g per 100g soybean peptides-Calcium. The result of infrared chromatographic analysis showed that: both the amino group and carboxyl group participated in the chelating reaction.
The experiment analyzed the effect of the two sample peptides to the growth of S. thermophilus by using methods of count plate and optical density, the result showed that: both the two sample peptides promoted the growth of S. Thermophilu, with the addition amounts increasing in the nutrient mediums, the growth promotion action made by crude soybean peptides tapered while soybean separation protein peptides enhanced. Optical density method was used to analyze the effect of the two sample peptides to the growth of B. infantis, the result showed that: smaller addition amount of crude soybean peptides made a obvious growth promotion action but tapered with the addition amount increasing, while the growth promotion action made by soybean separation protein peptides enhanced with the addition amount increasing.
引文
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