利用乳酸菌B2-1发酵乳清蛋白,分离纯化发酵产物以获得具有抗氧化及抗肿瘤活性的双功能活性肽组分。将乳酸菌B2-1接种于乳清蛋白-MRS培养基中,厌氧培养5周,对乳酸菌B2-1发酵乳清蛋白代谢产物采用大孔吸附树脂D101层析柱及葡聚糖凝胶G15层析柱进行分离纯化,并采用邻苯二甲醛(O-phthaldialdehyde,OPA)法、ABTS法、噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)法检测实验对分离组分进行跟踪监测,选出既具有抗氧化活性又具有抗肿瘤活性的多肽组分,利用SDS-PAGE观察其分子质量分布。通过乳酸菌B2-1发酵乳清蛋白的产物经分离纯化后获得了具有抗氧化及抗肿瘤活性的双功能活性肽组分F3-1,其ABTS阳离子自由基清除率为69.29%;当其质量浓度为8 g/L时,对人结肠癌LoVo细胞增殖的抑制率达到75.36%。可以利用乳酸菌B2-1发酵乳清蛋白得到具有抗氧化及抗肿瘤的双功能活性肽组分,为双功能活性肽的利用提供理论依据。
Using Lactobacillus B2-1 to ferment whey protein, the product was separated and purified to obtain biologically active peptides with antioxidant and antitumor activities. Whey protein was fermented by Lactobacillus B2-1 under anaerobic cultivation for five weeks, and the metabolites were purified by macroporous adsorption resin D101 column chromatography and Sephadex G15 gel column chromatography. The antioxidant activity of biologically active peptides was studied by examining their ability to scavenge ABTS radical. Its antitumor activity was studied by examining the survival of colon and oral cancer cells. Molecular weight distribution was observed by polyacrylamide gel electrophoresis. The results showed that the purified fraction F3-1 was a good component with antitumor and antioxidant activities. In vitro antioxidation experiments showed that F3-1 had the best ability to eliminate ABTS free radicals. When the concentration was 8 g/L, the inhibition rate of human colon cancer LoVo cell proliferation was 75.36%. Based on these results, the Lactobacillus B2-1 can be used to ferment whey protein to obtain the biologically active peptide components with antitumor and antioxidant functions.
[1] ARASU M V, AL-DHABI N A, REJINIEMON T S, et al.Identification and characterization of Lactobacillus brevis P68 with antifungal, antioxidant and probiotic functional properties[J].Indian Journal of Microbiology, 2015, 55(1):19-28.
[2] FUCHS-TARLOVSKY V.Role of antioxidants in cancer therapy[J].Nutrition, 2013, 29(1):15-21.
[3] ZENG Q, XIE L, ZHOU N, et al.Detection of PIK3CA mutations in plasma DNA of colorectal cancer patients by an ultra-sensitive PNA mediated PCR[J].Molecular Diagnosis & Therapy, 2017, 21(4):443-451.
[4] CHAI T T, LAW Y C, WONG F C, et al.Enzyme-assisted discovery of antioxidant peptides from edible marine invertebrates:A review[J].Marine Drugs, 2017, 15(2):42.
[5] HAN J M, LIM H N, JUNG H J.Hovenia dulcis Thunb and its active compound ampelopsin inhibit angiogenesis through suppression of VEGFR2 signaling and HIF-1α expression[J].Oncology Reports, 2017, 38(6):3 430-3 438.
[6] ROCHA G F, KISE F, ROSSO A M, et al.Potential antioxidant peptides produced from whey hydrolysis with an immobilized aspartic protease from Salpichroa origanifolia fruits[J].Food Chemistry, 2017, 237:350-355.
[7] 刘利锋. 乳清蛋白在运动营养中的应用[J].食品安全质量检测学报, 2020(21):7 740-7 745.
LIU L F.Application of whey protein in sports nutrition[J].Journal of Food Safety and Quality, 2020(21):7 740-7 745.
[8] 侯惠玲, 关宏, 郭皓, 等.脱盐乳清粉代谢产物的抗氧化和抗肿瘤活性初步研究[J].中国食品添加剂, 2016(4):104-109.
HOU H L, GUAN H, GUO H, et al.A preliminary study on antioxidant activity and antitumor activity of desalted whey powder metabolites[J].China Food Additives, 2016(4):104-109.
[9] 杨文钦, 关宏, 陈雪, 等.乳清蛋白肽美拉德反应产物的制备及其抗氧化特性的评价[J].中国食品添加剂, 2015(8):101-106.
YANG W Q, GUAN H, CHEN X, et al.Maillard reaction products from whey protein peptides and study on antioxidant activities[J].China Food Additives, 2015(8):101-106.
[10] WANG X Q, YU H H, XING R, et al.Characterization, preparation, and purification of marine bioactive peptides[J].Biomed Research International, 2017.DOI:10.1155/2017/9746720.
[11] DE CASTRO R J S, SATO H H.Biologically active peptides:Processes for their generation, purification and identification and applications as natural additives in the food and pharmaceutical industries[J].Food Research International, 2015, 74:185-198.
[12] OUDART J B, BRASSART-PASCO S, VAUTRIN A, et al.Plasmin releases the anti-tumor peptide from the NC1 domain of collagen XIX[J].Oncotarget, 2015, 6(6):3 656-3 668.
[13] ZHANG P C, CUI Y G, ANDERSON C F, et al.Peptide-based nanoprobes for molecular imaging and disease diagnostics[J].Chemical Society Reviews, 2018, 47(10):3 490-3 529.
[14] LIN J Z, ZOU Y X, CAO K L, et al.The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in Lactobacillus casei[J].Journal of Industrial Microbiology & Biotechnology, 2016, 43(5):703-711.
[15] NIELSEN P M, PETERSEN D, DAMBMANN C.Improved method for determining food protein degree of hydrolysis[J].Journal of Food Science, 2001, 66(5):642-646.
[16] GUO H, GUAN H, YANG W Q, et al.Pro-apoptotic and anti-proliferative effects of corn silk extract on human colon cancer cell lines[J].Oncology Letters, 2017, 13(2):973-978.
[17] 郭皓, 关宏, 侯惠玲, 等.玉米须提取液抗氧化功能特性的研究[J].中国临床药理学杂志, 2017, 33(3):258-261.
GUO H, GUAN H, HOU H L, et al.Study on antioxidative activity of the extract of corn silk[J].The Chinese Journal of Clinical Pharmacology, 2017, 33(3):258-261.
[18] DASKAYA-DIKMEN C, YUCETEPE A, KARBANCIOGLU-GULER F, et al. Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from plants[J]. Nutrients, 2017, 9(4):316.
[19] VALIYARI S, SALAMI M, MAHDIAN R, et al.IL-24 peptide, a human interleukin-24 isoform, induces mitochondrial-mediated apoptosis in human cancer cells[J].Cancer Chemotherapy and Pharmacology, 2017, 80(3):451-459.
[20] CARVALHO F, PRAZERES A R, RIVAS J.Cheese whey wastewater:Characterization and treatment[J].Science of the Total Environment, 2013, 445-446:385-396.
