酶解与体外模拟胃肠消化对脱脂羊奶粉ACE抑制活性的影响

田维杰; 马海乐; 刘丹丹; 陆峰; 朱俊松

doi:10.13386/j.issn1002-0306.2022090216

酶解与体外模拟胃肠消化对脱脂羊奶粉ACE抑制活性的影响

doi: 10.13386/j.issn1002-0306.2022090216

田维杰^1,,
马海乐^{1, 2,*, ,},
刘丹丹¹,
陆峰¹,
朱俊松¹

1.
江苏大学食品与生物工程学院，江苏镇江 212013
2.
江苏大学食品物理加工研究院，江苏镇江 212013

基金项目: 国家“863”计划：高生物利用度蛋白制备关键技术研究与开发（2013AA102203）。

详细信息

作者简介:
田维杰（1996−），男，硕士研究生，研究方向：功能性多肽的制备与评价，E-mail：1093283637@qq.com

通讯作者:
马海乐（1963−），男，博士，教授，研究方向：食品物理加工技术及装备研发，E-mail：mhl@ujs.edu.cn

中图分类号: TS209
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出版历程
- 收稿日期: 2022-09-22
- 刊出日期: 2023-05-01

Effect of Enzymolysis and in Vitro Simulated Gastrointestinal Digestion on the ACE Inhibitory Activity of Defatted Goat Milk Powder

TIAN Weijie^1
,,
MA Haile^{1, 2,*
, ,},
LIU Dandan¹,
LU Feng¹,
ZHU Junsong¹

1.
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
2.
Institute of Food Physical Processing, Jiangsu University, Zhenjiang 212013, China

摘要

摘要: 本研究旨在探究商业蛋白酶酶解与体外模拟胃肠消化对高消化率的脱脂羊奶粉释放ACE抑制肽的影响，以评价脱脂羊奶粉酶解必要性及所需酶解程度。采用中性蛋白酶、碱性蛋白酶、复合蛋白酶和风味蛋白酶对脱脂后的圭山羊奶粉进行酶解反应，并对脱脂羊奶粉及其酶解物进行体外模拟胃肠消化，测定酶解液及模拟胃肠消化液的多肽含量、ACE抑制活性以及分子量分布。研究发现，羊奶粉经过体外模拟胃肠消化后，消化液的多肽含量与ACE抑制率分别达到20.81 mg/mL和62.55%；而经过碱性蛋白酶和风味蛋白酶的适度酶解，其模拟胃肠消化液多肽含量与ACE抑制活性反而下降；经过复合蛋白酶的适度酶解，其模拟胃肠消化液的多肽含量与ACE抑制活性小幅度提高，分别达到22.67 mg/mL和69.29%；经过中性蛋白酶的适度酶解，其模拟胃肠消化液多肽含量与ACE抑制活性均显著提高，分别达到23.76 mg/mL和81.10%。分子量分布结果显示，经过体外模拟胃肠消化后，<1000 Da的小分子肽由酶解液中的70.77%提高到90%。综上，圭山羊奶粉经过体内胃肠消化就可以释放出较多的ACE抑制肽，而经过中性蛋白酶酶解至水解度8%后，ACE抑制活性还可以再提升28.83%，因此在一定程度上具有进一步酶解的必要性。
- 羊奶蛋白 /
- 酶解 /
- 多肽 /
- ACE抑制活性 /
- 体外模拟胃肠消化
Abstract: This study aimed to investigate the effect of commercial protease enzymolysis and in vitro simulated gastrointestinal digestion on the release of ACE inhibitory peptides from the defatted goat milk powder with high digestibility, and to evaluate the necessity and the extent of enzymatic hydrolysis. Neutrase, alcalase, protamex, and flavourzyme were used for the enzymolysis of the defatted Kuishan goat milk powder. The goat milk and its enzymatic hydrolysates were further digested via simulated gastrointestinal digestion in vitro. The peptide content, ACE inhibitory activity and molecular weight distribution of the enzymatic hydrolysates and the simulated gastrointestinal digests were determined. Results showed that after the simulated gastrointestinal digestion in vitro, the peptide content and ACE inhibitory activity of the goat milk powder digests were 20.81 mg/mL and 62.55%, respectively. However, after moderate enzymatic hydrolysis by alcalase and flavourzyme, the peptide content and ACE inhibitory activity of simulated gastrointestinal digests decreased. After moderate enzymolysis by protamex, the peptide content and ACE inhibitory activity of simulated gastrointestinal digests were increased slightly to 22.67 mg/mL and 69.29%, respectively. After moderate enzymolysis by neutrase, the peptide content and ACE inhibitory activity of simulated gastrointestinal digests were significantly increased to 23.76 mg/mL and 81.10%, respectively. The molecular weight (MW) distribution results showed that after in vitro simulated gastrointestinal digestion, the amount of the small molecular peptides with MW<1000 Da increased from 70.77% to 90%. Above results indicate that Kuishan goat milk powder can exert ACE inhibitory activity after gastrointestinal digestion, and the ACE inhibitory activity can be further increased by 28.83% after neutral protease hydrolysis to degree of hydrolysis of 8%. Therefore, it was necessary to further enzymatic hydrolysis to a certain extent.
- goat milk protein /
- enzymatic hydrolysis /
- peptide /
- ACE inhibitory activity /
- in vitro simulated gastrointestinal digestion

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图 1 碱性蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的多肽含量

Figure 1. Peptide content of alcalase hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 2 中性蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的多肽含量

Figure 2. Peptide content of neutrase hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 3 复合蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的多肽含量

Figure 3. Peptide content of protamex hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 4 风味蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的多肽含量

Figure 4. Peptide content of flavourzyme hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 5 碱性蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的ACE抑制活性

Figure 5. ACE inhibitory activity of the alcalase hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 6 中性蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的ACE抑制活性

Figure 6. ACE inhibitory activity of the neutrase hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 7 复合蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的ACE抑制活性

Figure 7. ACE inhibitory activity of the protamex hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 8 风味蛋白酶酶解物、模拟胃消化产物和模拟胃肠消化产物的ACE抑制活性

Figure 8. ACE inhibitory activity of flavourzyme hydrolysates, simulated gastric digests and simulated gastrointestinal digests

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图 9 中性蛋白酶酶解物及胃肠模拟消化产物分子量色谱图

注：A为中性蛋白酶酶解物，B为酶解物的胃肠模拟消化产物。

Figure 9. Chromatogram of molecular weight distribution of the neutrase hydrolysates and its simulated gastrointestinal digests

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表 1 羊奶蛋白氨基酸评分

Table 1. Amino acid rating of goat milk protein

必需氨基酸	婴幼儿均值	2~5岁儿童	10~12岁儿童	成人
His	1.11	1.52	1.52	1.80
Ile	1.42	2.32	2.32	5.01
Leu	1.30	1.83	2.75	6.37
Lys	1.23	1.40	1.85	5.08
Met+Cys	0.73	1.23	1.40	1.81
Phe+Tyr	1.20	1.37	3.93	4.56
Thr	1.46	1.85	2.25	7.00
Val	1.23	1.94	2.71	5.21
Trp	/	/	/	/

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表 2 蛋白酶的酶活、最适作用条件及酶切位点

Table 2. Enzymatic activity, optimum reaction conditions and cleavage sites of the different proteases

蛋白酶	蛋白酶活力（U/g）	最适作用条件	主要酶切位点
中性蛋白酶	155153.80	pH7.0; T 50 °C	酶切位点较为广泛
碱性蛋白酶	220157.69	pH9.0; T 50 °C	疏水性氨基酸残基，如Ala和Leu
复合蛋白酶	184000.00	pH6.5; T 50 °C	酶切位点较为广泛
风味蛋白酶	26553.85	pH6.5; T 50 °C	酶切位点较为广泛
注：T 温度。

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表 3 羊奶粉基本成分

Table 3. Elementary components of goat milk powder

指标	水分	灰分	粗蛋白	脂肪	总糖
含量（%）	2.26±0.26	4.60±0.19	20.00±0.21	19.40±0.36	52.00±0.34

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表 4 羊奶蛋白的氨基酸组成及含量

Table 4. Amino acid composition and content of goat milk protein

氨基酸	含量（%）	氨基酸	含量（%）
Asp	7.96±0.11	Ile	6.51±0.10
Thr	6.30±0.09	Leu	12.11±0.06
Ser	4.88±0.01	Tyr	4.29±0.01
Glu	21.01±0.25	Phe	4.36±0.02
Gly	2.11±0.01	His	2.89±0.01
Ala	3.70±0.02	Lys	8.13±0.09
Val	6.77±0.06	Arg	3.96±0.03
Met	3.08±0.02	Pro	1.93±0.01

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表 5 羊奶蛋白必需氨基酸及氨基酸分布

Table 5. Amino acid distribution and essential amino acids of goat milk protein

	氨基酸种类	含量（%）
必需氨基酸	Lys、Trp、Phe、Met、Thr、Ile、Leu、Val	47.27
疏水性氨基酸	Ala、Phe、Ile、Leu、Met、Pro、Val、Trp	38.45
亲水性氨基酸	Ser、Thr、Tyr、Cys、Gly	17.58
酸性氨基酸	Glu、Asp	28.98
碱性氨基酸	Lys、Arg、His	14.98

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表 6 羊奶蛋白营养价值分析

Table 6. Nutritional value of goat milk protein

营养评价参数	评分
E/T（%）	47.27
PER I	4.75
PER II	4.58
BV	93.63

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表 7 中性蛋白酶酶解物及其胃肠模拟消化产物中肽的相对分子质量分布和其分布范围

Table 7. Molecular weight distribution of the neutrase hydrolysates and its simulated gastrointestinal digests

分子量范围（Da）	酶解液各组分含量（%）	胃肠模拟消化液各组分含量（%）
>10000	2.83	0.34
5000~10000	2.52	0.27
3000~5000	3.31	0.78
2000~3000	6.00	1.64
1000~2000	14.57	6.98
500~1000	19.45	16.96
180~500	36.37	46.43
<180	14.95	26.60

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