不同热处理方式的α-乳白蛋白对HIEC增殖、周期及凋亡的影响

摘要/Abstract

摘要： 摘要：本文以经过巴氏杀菌方式（63℃加热30 min、72℃加热15 s、85℃加热15 s、95℃加热10 min）处理的α-乳白蛋白（α-Lactalbumin, α-LA）为研究对象，研究其对正常人肠道上皮细胞株（HIEC）增殖、周期及凋亡的影响，并比较不同热处理对HIEC作用效果的差异性。在通过圆二色谱法测定α-LA的二级结构、分析不同热处理方式对α-LA二级结构影响的基础上，模拟婴儿肠道条件对α-LA进行体外消化，采用CCK-8法和流式细胞术检测热处理消化后α-LA作用HIEC 48 h后，对细胞增殖、细胞周期以及细胞凋亡的影响。结果显示，经不同加热处理及未经热处理的消化后α-LA均对HIEC增殖具有促进作用，且在一定浓度范围内促进效果随浓度升高而加强，在浓度为0.10 mg/mL、72℃处理15 s时促进效果最为显著(P＜0.05)；经不同加热处理及未经热处理的消化后α-LA在浓度为0.10 mg/mL时，作用HIEC细胞48 h后，G0/G1期细胞比例显著降低，S期和G2M期细胞比例显著升高(P＜0.05)，细胞凋亡率显著降低(P＜0.05)，从而促进细胞增殖。因此表明，热处理α-LA可以显著促进HIEC增殖，其作用机制可能与分裂的细胞比例增加从而抑制细胞凋亡有关。α-LA应用于婴儿配方产品中或可提高婴儿免疫能力，推荐添加浓度为0.10 mg/mL，推荐热处理方式为72℃加热15 s，仍需进一步研究。

关键词: 关键词：α-乳白蛋白（α-Lactalbumin, α-LA）, 正常人肠道上皮细胞株（HIEC）, 二级结构, 肠道免疫

Abstract: Abstract: The effects of α-Lactalbumin(α-LA) treated by pasteurization (63℃/30 min, 72℃/15 s, 85℃/15 s, 95℃/10 min) on the proliferation, cycle and apoptosis of normal human intestinal epithelial cells HIEC were studied, and the differences of various sterilization conditions were compared and analyzed. The secondary structure of α-LA was determined by circular dichroism chromatography, and the effect of different sterilization conditions on the secondary structure of α-LA was analyzed. Then, α-LA was digested in vitro under simulated infant intestinal conditions and the effects of α-LA hydrolysate with different sterilization on the proliferation, cycle and apoptosis of HIEC cells at 48 h was detected by the method of the cell counting kit-8 (CCK-8) assay and flow cytometry. The results showed that after pasteurization and digestion, all of α-LAs could significantly promote proliferation of HIEC cells at 48h in a certain range, the promotion would be enhanced with the concentration increased. The most significant (P < 0.05) result was displayed on the condition of 72℃ for 15 s and the concentration of 0.10 mg/mL. At the concentration of 0.10 mg/mL, flow cytometry showed that pasteurized α-LA hydrolyzate reduced the percentage of HIEC cells in the G0/G1 phase, increased the percentage of HIEC cells in the G2/M and S phase, and delayed cell apoptosis as same as the untreated α-LA hydrolyzate. The results indicated that α-LA can significantly promote the proliferation of HIEC cells, and its mechanism may be related to the increase of the proportion of dividing cells and the inhibition of apoptosis. Applied α-LA to infant formulations might improve the immune ability of infants, which needs further research. The recommended heat treatment is 72℃ for 15 s at the concentration of 0.10 mg/mL.

Key words: Key words: α-Lactalbumin (α-LA), normal human intestinal epithelial cells (HIEC), secondary structure, intestinal immunity

中图分类号:

TS252.1

盛雪张居典李梦寒崔东影席恩泽许晓曦. 不同热处理方式的α-乳白蛋白对HIEC增殖、周期及凋亡的影响[J]. 食品科学, 0, (): 0-0.

Meng-Han LI XI EnZe Xiaoxi Xu. Effects of α-lactalbumin with different heat treatment methods on proliferation,cell cycle and apoptosis of HIEC cells[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

[1]仲玉梅. α-乳白蛋白在婴儿营养中的重要性[J].食品工业科技, 1992, 2(018):53-57
[2]Ruiz-Valdepe?as M V, Campuzano S, Torrente-Rodríguez R M, et al.Electrochemical magnetic beads-based immunosensing platform for the determination of α-lactalbumin in milk[J].Food Chemistry, 2016, 213(07):595-601
[3] Delavari B, Saboury A A, Atri M S, et al.Alpha-lactalbumin: A new carrier for vitamin D 3, food enrichment[J].Food Hydrocolloids, 2015, 45(017):124-131
[4]Crittenden RG, Bennett LE.Cow's milk allergy: a complex disorder[J].Journal of the American College of Nutrition, 2005, 24(6 Suppl):582-S
[5] Wada Y, Phinney B S, Weber D, et al.In vivo, digestomics of milk proteins in human milk and infant formula using a suckling rat pup model[J].Peptides, 2017, 88(012):18-31
[6]Ma J, Li Q, Li Y, et al.Expression of recombinant human α-lactalbumin in milk of transgenic cloned pigs Is sufficient to enhance intestinal growth and weight gain of suckling piglets[J].Gene, 2016, 584(1):7-16
[7]Ryan M P, Walsh G.The biotechnological potential of whey[J].Reviews in Environmental Science & Biotechnology, 2016, 15(3):1-20
[8] Br??Ck W M, Kelleher S L, Gibson G R, et al..rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli[J].J Pediatr Gastroenterol Nutr, 2003, 37(3):273-280
[9] Berkhout, B, Derksen, G.C.H., Back, N.K.T., Klaver, B., De Kruif, C.G., Visser, S..Structural and functional analysis of negatively charged milk proteins with anti-HIV activity[J].AIDS Research and Human Retroviruses, 1997, 13(13):1101-1107
[10]Hill D R, Newburg D S.Clinical applications of bioactive milk components[J].Nutrition Reviews, 2015, 73(7):463-476
[11]Ng T B, Cheung R C, Wong J H, et al.Antiviral activities of whey proteins[J].Applied Microbiology & Biotechnology, 2015, 99(17):6997-7008
[12] Gauthier, SF., Pouliot, Y., Saint-Sauveur, D..Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins[J].International Dairy Journal, 2006, 16(11):1315-1323
[13]Jakopovic′ K L, Baruk?ic′ I, Boz?Anic′ R.Physiological significance,structure and isolation of α-lactalbumin[J].Mljekarstvo, 2016, 66(1):3-11
[14] Nongonierma A B, Fitzgerald R J.Bioactive properties of milk proteins in humans: A review.[J].Peptides, 2015, 73(08):20-34
[15] 徐睿锶, 吴楠.婴幼儿配方奶粉生产技术的现状及展望[J].科技风, 2018, 20(7):244-245
[16]姜红, 李向红, 王薇,等.高乳清蛋白婴儿配方奶对新生儿生长发育及氨基酸代谢的影响[J].中华实用儿科临床杂志, 2012, 27(19):1482-1484
[17]Davis A，Harris B，Lien E，et al.Lactalbumin-rich infant formula fed to healthy term infants in a multicenter study: plasma essential amino acids and gastrointestinal tolerance[J].European Journal of Clinical Nutrition, 2007, 62(11):1294-1301
[18] 杨怀谷, 郑楠, 王加启.巴氏杀菌乳和超高温灭菌乳营养价值及卫生安全对比研究[J].中国乳业, 2016, 7(02):62-67
[19]杨晋辉, 李松励, 郑楠, 等.热处理对牛乳成分的影响以及热敏感指标的变化研究进展[J].食品科学, 2017, 38(7):302-308
[20]Crowley S V, Dowling A P, Caldeo V, et al.Impact of α-lactalbumin:β-lactoglobulin ratio on the heat stability of model infant milk formula protein systems[J].Food Chemistry, 2016, 194(07):184-190
[21]Nguyen T T P, Bhandari B, Cichero J, et al.Gastrointestinal digestion of dairy and soy proteins in infant formulas: An in vitro,study[J].Food Research International, 2015, 76(Pt 3):348-358
[22]冯炎雯, 李娜, 徐纪璇, et al..脂质体在婴儿体外胃肠道消化的膜结构稳定性 [J].食品科学, 2017, 38(13):60-65
[23]YWada, B. L?nnerdal.Bioactive peptides released from in vitro digestion ofhuman milk with or without pasteurization[J].Pediatric Research, 2015, 77(4):546-553
[24] Islam M A, Devle H, Comi I, et al.Ex vivo digestion of raw, pasteurised and homogenised milk - Effects on lipolysis and proteolysis[J].International Dairy Journal, 2017, 65(02):14-19
[25]Yee Hsiung Chen， Jen Tsi Yang，Kue Hung Chau.Determination of the helix and β form of proteins in aqueous solution by circular dichroism[J].Biochemistry, 1974, 13(16):3350-3359
[26]Sreerama H，Venyaminov S Y，Woody R W.Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis[J].Analytical Biochemistry, 2000, 287(2):243-251
[27]包永睿, 王帅, 孟宪生, 等.薏苡仁脂肪酸类成分对人肝癌细胞株-细胞周期及细胞凋亡的影响[J].中成药, 2014, 36(2):235-239
[28]等.Lactalbumin Hydrolysate Stimulates Glucagon-Like Peptide-2 Secretion and Small Intestinal Growth in Suckling Rats[J].Journal of Nutrition, 2009, 139(7):1322-1327
[29]Sternhagen, LG., Allen, J.C..Growth rates of a human colon adenocarcinoma cell line are regulated by the milk protein alphalactalbumin[J].Adv. Exp. Med. Biol, 2001, 5(01):115.-120
[30]Su S L, Kuo C D.Induction of cell death in RAW 2647 cells by alpha-lactalbumin.[J].Food & Chemical Toxicology An International Journal Published for the British Industrial Biological Research Association, 2008, 46(3):842-853