酶法制备大豆蛋白成骨活性肽

doi:10.3864/j.issn.0578-1752.2021.13.016

中国农业科学 ›› 2021, Vol. 54 ›› Issue (13): 2885-2894.doi: 10.3864/j.issn.0578-1752.2021.13.016

酶法制备大豆蛋白成骨活性肽

李宇¹(),汪芳¹,翁泽斌²,宋海昭¹(),沈新春¹()

¹南京财经大学食品科学与工程学院/江苏省现代粮食流通与安全协同创新中心/江苏高校粮油质量安全控制及深加工重点实验室,南京 210023
²南京中医药大学中医学院/中西医结合学院,南京 210000

收稿日期:2020-10-29 修回日期:2021-02-08 出版日期:2021-07-01 发布日期:2021-07-12
通讯作者: 宋海昭,沈新春
作者简介:李宇,Tel：18795845118;E-mail： 18795845118@163.com。
基金资助:
国家自然科学基金(21476103);国家自然科学基金(31800280);江苏省自然科学基金(BK20181415);江苏省高等学校自然科学基金(18KJB550006);江苏省研究生科研与实践创新计划(KYCX19_1403)

Preparation of Soybean Protein-Derived Pro-osteogenic Peptides via Enzymatic Hydrolysis

LI Yu¹(),WANG Fang¹,WENG ZeBin²,SONG HaiZhao¹(),SHEN XinChun¹()

¹College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Quality Control and Processing, Nanjing 210023
²College of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210000

Received:2020-10-29 Revised:2021-02-08 Online:2021-07-01 Published:2021-07-12
Contact: HaiZhao SONG,XinChun SHEN

摘要/Abstract

摘要：

【目的】探究从大豆分离蛋白双酶分步酶解产物中分离促成骨细胞增殖肽的工艺,为大豆产品的开发提供参考。【方法】以促成骨细胞增殖活性作为测定指标,选用木瓜蛋白酶酶解大豆分离蛋白（SPI）,采用MTT法检测不同浓度的木瓜蛋白酶酶解产物对成骨细胞的促增殖活性。采用pH-Stat法检测水解时间为1、2、3、4、5和6 h的木瓜蛋白酶酶解产物的水解度,并检测相应水解时间的酶解产物对成骨细胞的促增殖活性。然后分别采用截留分子量为30 kD和10 kD两种超滤膜对活性较高的酶解产物进行超滤,并测定各超滤组分对成骨细胞的促增殖活性。再将具有较高促成骨细胞增殖活性的超滤组分分别在0.5、1、1.5、2和2.5 h进行碱性蛋白酶酶解,并检测相应水解时间的酶解产物对成骨细胞的促增殖活性。然后选用交联葡聚糖（Sephadex G-15）对具有较高促成骨细胞增殖活性的酶解产物进行分离,并检测各分离组分对成骨细胞的促增殖活性。最后,对各分离组分进行氨基酸分析,并采用质谱（ESI-TOF MS/MS）对最高促成骨细胞增殖活性的分离组分进行结构鉴定,并筛选出具有较强促成骨细胞增殖的大豆活性肽。【结果】在浓度为200 μg·mL ^-1时,木瓜蛋白酶酶解物对成骨细胞的促增殖活性随着酶解时间的增加逐渐增强,当酶解时间为5 h时达到最高促增殖活性（（118.24±2.73）%）。木瓜蛋白酶酶解产物经超滤分离、碱性蛋白酶酶解和凝胶过滤色谱分离后,对各分离组分进行氨基酸分析并筛选得到对成骨细胞具有最强增殖活性的组分F3,该组分的成骨细胞增殖率为（125.80±2.94）%,且F3中丙氨酸、缬氨酸、异亮氨酸、亮氨酸、苯丙氨酸等疏水性氨基酸以及芳香族氨基酸和必需氨基酸的含量明显高于其他组分。进一步通过质谱鉴定出F3主要由10个肽段组成。其中,DAMDGWFRL、GQTPLFPR、ADFYNPK、KDWYDIK的疏水性氨基酸、芳香族氨基酸以及必需氨基酸占比较高。对上述4个肽段进行活性测定,发现GQTPLFPR的促成骨细胞增殖活性最强,在浓度为100 μmol·L^-1时,其成骨细胞增殖率为（129.11±3.12）%。【结论】采用双酶分步酶解结合超滤和凝胶过滤色谱等蛋白分离纯化技术,从大豆分离蛋白中分离纯化出了具有较强促成骨细胞增殖活性的多肽,为促成骨细胞增殖活性肽的制备和应用提供了技术参考。

关键词: 大豆分离蛋白, 成骨细胞增殖, 双酶分步酶解, 分离纯化, 活性肽

Abstract:

【Objective】 The aim of this study was to develop an effective method and to identify the active peptides and to provide reference for the development of soybean products, so as to promote the proliferation of osteoblasts from soybean protein isolated with the two-step enzymatic hydrolysis. 【Method】The proliferative activity of osteoblast was served as an indicator. Soybean protein isolate (SPI) was hydrolyzed by papain, and the effect of papain hydrolysates on osteoblast proliferation was detected by a MTT assay. The degree of hydrolysis of the papain hydrolysates was determined by the pH-Stat method after 1, 2, 3, 4, 5 and 6 h treatment, respectively. The effects of papain hydrolysates on pro-liferation of osteoblast were determined. Two kinds of ultrafiltration membrane with 30 kDa and 10 kDa cut-off were used to separate the fractions of papain hydrolysates, and their proliferation activities on osteoblast were determined. The enzymatic hydrolysates with the highest pro-proliferative activity of osteoblast were further hydrolyzed with alkaline. The effect of alkaline enzymatic hydrolysate on the proliferation of osteoblast was examined after 0.5, 1, 1.5, 2 and 2.5 h treatment, respectively. The Sephadex G-15 was used to isolate the enzymatic hydrolysates with high pro-proliferative activity of osteoblasts, and the proliferation activity of each component on osteoblasts was determined. Furthermore, the amino acids of the separated fractions were analyzed. The structure of the most active fraction was identified by ESI-TOF MS/MS, and the soybean protein derived from peptides with strong activity to promote osteoblast proliferation was identified. 【Result】 The proliferative rate of osteoblasts was (118.24±2.73)% by the treatment with enzymatic hydrolysis products of papain for 5 h at the concentration of 200 μg/ml. After the enzymatic hydrolysates were separated by ultrafiltration membrane, the active fraction was further hydrolyzed with alkaline protease. The final hydrolysates were separated with gel filtration chromatography (Sephadex G-15). A fraction of component with the strongest pro-proliferative activity on osteoblasts, F3, was identified, and the proliferative rate of osteoblasts treated with this faction was (125.80±2.94)%. Amino acid analysis showed that the contents of hydrophobic amino acids, such as alanine, valine, isoleucine, leucine, and phenylalanine, aromatic amino acids and essential amino acids in F3, were significantly higher than other fractions. In addition, mass spectrometry (ESI-TOF MS/MS) revolved that F3 was mainly composed of 10 small peptides, among which, DAMDGWFRL, GQTPLFPR, ADFYNPK and KDWYDIK had high contents of hydrophobic amino acids, aromatic amino acids and essential amino acids. Moreover, 100 μmol∙L ^-1 of GQTPLFPR had the strongest pro-proliferative activity on osteoblasts, with a proliferative rate of (129.11±3.12)%.【Conclusion】 Two-step enzymatic hydrolysis combined with ultrafiltration membrane and gel filtration chromatography could identify the most active pro-proliferative peptides from SPI, which provided a technical reference for the preparation of peptides with the bio-activity to stimulate the proliferation of osteoblasts.

Key words: soybean protein isolate, osteoblast proliferation, two-step enzymatic hydrolysis, separation and purification, active peptide

李宇,汪芳,翁泽斌,宋海昭,沈新春. 酶法制备大豆蛋白成骨活性肽[J]. 中国农业科学, 2021, 54(13): 2885-2894.

LI Yu,WANG Fang,WENG ZeBin,SONG HaiZhao,SHEN XinChun. Preparation of Soybean Protein-Derived Pro-osteogenic Peptides via Enzymatic Hydrolysis[J]. Scientia Agricultura Sinica, 2021, 54(13): 2885-2894.

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氨基酸名称 Amino acid name		氨基酸含量 Amino acid content (g/100 g)
氨基酸名称 Amino acid name		F1	F2	F3	F4
天冬氨酸 Aspartic acid	Asp	10.20±0.66d	8.37±0.68c	4.67±0.33a	6.27±0.36b
苏氨酸 Threonine	Thr	2.15±0.19a	3.69±0.23c	3.24±0.23b	2.88±0.28b
丝氨酸 Serine	Ser	2.66±0.23a	5.13±0.31b	2.52±0.17a	2.58±0.24a
谷氨酸 Glutamate	Glu	20.97±2.17d	14.78±0.92c	6.48±0.52a	12.06±0.69b
甘氨酸 Glycine	Gly	2.64±0.23a	3.61±0.23c	3.08±0.23b	3.45±0.33c
丙氨酸 Alanine	Ala	1.57±0.14a	4.58±0.28b	7.02±0.42d	5.43±0.52c
半胱氨酸 Cysteine	Cys	0.52±0.05d	0.00±0.00a	0.21±0.02c	0.00±0.00a
缬氨酸 Valine	Val	3.98±0.17c	3.59±0.28b	4.98±0.19d	2.51±0.24a
甲硫氨酸 Methionine	Met	0.76±0.07b	0.25±0.02a	1.88±0.14e	1.56±0.16d
异亮氨酸 Isoleucine	Ile	1.90±0.17c	3.91±0.23e	1.69±0.12b	0.86±0.09a
亮氨酸 Leucine	Leu	5.39±0.21b	6.49±0.45c	7.58±0.50d	3.61±0.35a
酪氨酸 Tyrosine	Tyr	6.43±0.43c	5.47±0.52b	13.57±0.45d	3.97±0.47a
苯丙氨 Phenylalanine	Phe	6.38±0.54b	5.79±0.54b	13.06±1.14c	3.41±0.68a
赖氨酸 Lysine	Lys	4.69±0.42c	4.14±0.24b	3.57±0.26a	10.34±0.55d
组氨酸 Histidine	His	1.83±0.16b	1.53±0.09a	3.60±0.26c	6.77±0.64d
精氨酸 Arginine	Arg	5.47±0.47b	4.65±0.28a	5.49±0.40b	7.38±0.71c
脯氨酸 Proline	Pro	4.30±0.38a	5.11±0.31b	4.66±0.33a	4.73±0.45ab
疏水性氨基酸 Hydrophobic amino acid	—	30.71±0.31b	35.19±1.13c	54.44±1.84d	26.08±1.02a
芳香族氨基酸 Aromatic amino acid	—	12.81±0.29c	11.26±0.52b	26.63±0.78d	7.38±0.54a
必需氨基酸 Essential amino acid	EAA	27.08±1.70a	29.39±2.10b	39.60±3.08c	31.94±2.51b

序列 Sequence	离子 Ion (m/z)	观察质量 Observed mass	蛋白质 Protein	补充 Accession
KDWYDIK	484.25 (2)	967.49	40S ribosomal protein S3a OS=Glycine max OX=3847 GN=100813273 PE=2 SV=1	C6TI64
DAMDGWFRL	563.76 (2)	1126.50	Truncated Kunitz trypsin inhibitor OS=Glycine max OX=3847 PE=4 SV=1	Q9ATY0
GQTPLFPR	458.26 (2)	915.50	Uncharacterized protein OS=Glycine max OX=3847 GN=100801918 PE=3 SV=1	A0A0R0JL19
ADFYNPK	427.71 (2)	854.40	Soybean glycinin A3-B4 subunit (Fragment) OS=Glycine max OX=3847 PE=2 SV=1	Q39858
DAMDGWFR	499.22 (2)	997.42	Truncated Kunitz trypsin inhibitor OS=Glycine max OX=3847 PE=4 SV=1	Q9ATY0
NNNPFK	367.19 (2)	733.36	Uncharacterized protein OS=Glycine max OX=3847 GN=GLYMA_03G163500 PE=3 SV=1	A0A0R0KKD6
SNDVYLPR	482.25 (2)	963.49	Seed linoleate 9S-lipoxygenase-3 OS=Glycine max OX=3847 GN=LOX1.3 PE=1 SV=1	P09186
GGQLAMINLESR	435.56 (3)	1304.66	Uncharacterized protein OS=Glycine max OX=3847 GN=100796275 PE=2 SV=1	C6TKL7
DAMDGWFR	507.21 (2)	1013.41	Truncated Kunitz trypsin inhibitor OS=Glycine max OX=3847 PE=4 SV=1	Q9ATY0
THINIVVIGHVDSGK	530.29 (3)	1588.88	Elongation factor 1-alpha OS=Glycine max OX=3847 GN=100785429 PE=3 SV=1	A0A0R0ESZ8

酶法制备大豆蛋白成骨活性肽

Preparation of Soybean Protein-Derived Pro-osteogenic Peptides via Enzymatic Hydrolysis

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