部分脱磷酸β-酪蛋白的体外消化特性

摘要/Abstract

摘要： 为明确部分脱磷酸β-酪蛋白（partially dephosphorylated β-casein, PDP-BCN）的消化特性及肽谱特征，本研究采用酶法脱磷酸技术制备了PDP-BCN，并通过动态光散射、激光共聚焦显微镜和液相串联质谱技术对其体外模拟胃肠消化特性及肽谱进行表征。结果表明，PDP-BCN在模拟婴幼儿胃液中形成的絮凝结构及粒径显著小于天然β-酪蛋白（β-casein, β-CN），更易消化成小颗粒；而且其在模拟婴幼儿胃肠消化过程中降解产生了880种肽段，远高于天然β-CN的533种，这种肽段差异在蛋白N端位置尤为显著。此外，仅由PDP-BCN降解产生的活性肽有8种，具有活性肽潜力的肽段有137种，相较于β-CN而言更多且涉及的活性范围更广。因此，在消化特性方面，PDP-BCN相较于天然β-CN具有明显优势。本研究可为PDP-BCN未来作为一种新型蛋白配料在婴幼儿配方奶粉中应用提供理论依据。

关键词: 脱磷酸β-酪蛋白, 体外消化, 粒径, 肽谱, 活性肽

Abstract: To elucidate the digestion characteristics and peptide profile of partially dephosphorylated β-casein (PDP-BCN), this study prepared PDP-BCN using enzymatic dephosphorylation technology and characterized its in vitro simulated gastrointestinal digestion properties and peptide profile via dynamic light scattering, confocal laser scanning microscopy, and liquid chromatography-tandem mass spectrometry. The results demonstrated that the flocculent structures and particle size formed by PDP-BCN in simulated infant gastric fluid were significantly smaller than those of native β-casein (β-CN), making it more easily digestible into smaller particles. Moreover, during simulated infant gastrointestinal digestion, PDP-BCN degradation generated 880 peptide fragments, far exceeding the 533 peptides produced by native β-CN. This difference in peptide fragments was particularly notable at the N-terminal region of the protein. Additionally, PDP-BCN degradation yielded 8 unique bioactive peptides and 137 peptides with potential bioactivity, which were more numerous and covered a broader range of activities compared to those derived from β-CN. Therefore, PDP-BCN exhibits significant advantages over native β-CN in terms of digestibility. This study provides a theoretical basis for the future application of PDP-BCN as a novel protein ingredient in infant formula.

Key words: Dephosphorylated β-casein, In vitro digestion, Particle size, Peptide mapping, Bioactive peptides

中图分类号:

TS252.1

宋思家林莹莹张雨凝郭慧媛. 部分脱磷酸β-酪蛋白的体外消化特性[J]. 食品科学.

Ying-Ying LIN. The in Vitro Digestion Characteristics of Partially Dephosphorylated Bovine β-casein[J]. FOOD SCIENCE.

参考文献

[1] ZHANG L, MA Y, HETTINGA K, et al. Suckling rat pup model: do caprine milk lactoferrin and immunoglobulins have different digestion and absorption properties from that of human and bovine species?[J]. Journal of Agricultural and Food Chemistry, 2025, 73(5): 3069-3079. DOI:10.1021/acs.jafc.4c10539.
[2] SONG S, LIN Y, XUE P, et al. Pilot-scale co-enrichment of beta-casein and whey protein from skim milk via ceramic membrane cold microfiltration[J]. Applied Food Research, 2025, 5, 100942. DOI:10.1016/j.afres.2025.100942.
[3] HOLGERSEN K, MUK T, GHISARI M, et al. Neonatal gut and immune responses to β-casein enriched formula in piglets[J]. The Journal of Nutrition, 2024, 154(7): 2143-2156. DOI:10.1016/j.tjnut.2024.04.036.
[4] SONG S, LIN Y, ZHANG Y, et al. The self-association properties of partially dephosphorylated bovine beta-casein[J]. Food Hydrocolloids, 2023, 134: 108019. DOI:10.1016/j.foodhyd.2022.108019.
[5] 王园园. 牛乳蛋白酶法脱磷酸化修饰及其消化性的研究 [D]. 无锡: 江南大学, 2015: 3-51.
[6] LIU D S, ZHANG J, WANG L L, et al. Membrane-based fractionation, enzymatic dephosphorylation, and gastrointestinal digestibility of beta-casein enriched serum protein ingredients [J]. Food Hydrocolloids, 2019, 88: 1-12. DOI:10.1016/j.foodhyd.2018.09.032.
[7] WAL J M. Structure and function of milk allergens [J]. Allergy, 2001, 56(s67): 35-38. DOI:10.1034/j.1398-9995.2001.00911.x.
[8] DOCENA G H, FERNANDEZ R, CHIRDO F G, et al. Identification of casein as the major allergenic and antigenic protein of cow's milk [J]. Allergy, 1996, 51(6): 412-416. DOI:10.1111/j.1398-9995.1996.tb00151.x.
[9] BAUM F, EBNER J, PISCHETSRIEDER M. Identification of multiphosphorylated peptides in milk [J]. Journal of Agricultural and Food Chemistry, 2013, 61(38): 9110-9117. DOI:10.1021/jf401865q.
[10] POTH A G, DEETH H C, ALEWOOD P F, et al. Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms [J]. Journal of Proteome Research, 2008, 7(11): 5017-5027. DOI:10.1021/pr800387s.
[11] LI-CHAN E, NAKAI S. Enzymic dephosphorylation of bovine casein to improve acid clotting properties and digestibility for infant formula [J]. Journal of Dairy Research, 1989, 56(3): 381-390. DOI:10.1017/S0022029900028843.
[12] LIU D S, WANG Y Y, YU Y, et al. Effects of enzymatic dephosphorylation on infant in vitro gastrointestinal digestibility of milk protein concentrate [J]. Food Chemistry, 2016, 197: 891-899. DOI:10.1016/j.foodchem.2015.11.074.
[13] VAN LIESHOUT G A A, LAMBERS T T, BRAGT M C E, et al. How processing may affect milk protein digestion and overall physiological outcomes: A systematic review [J]. Critical Reviews in Food Science and Nutrition, 2020, 60(14): 2422-2445. DOI:10.1080/10408398.2019.1646703.
[14] MCCARTHY N A, KELLY A L, O'MAHONY J A, et al. The physical characteristics and emulsification properties of partially dephosphorylated bovine beta-casein [J]. Food Chemistry, 2013, 138(2-3): 1304-1311. DOI:10.1016/j.foodchem.2012.11.080.
[15] LU Y, LI Y, LIN Y, et al. Effects of heat treatment and simulated digestion on the properties and osteogenic activity of bovine lactoferrin [J]. LWT-Food Science and Technology, 2022, 162: 113475. DOI:10.1016/j.lwt.2022.113475.
[16] DUPONT D, MANDALARI G, MOLLE D, et al. Comparative resistance of food proteins to adult and infant in vitro digestion models [J]. Molecular Nutrition & Food Research, 2010, 54(6): 767-780. DOI:10.1002/mnfr.200900142.
[17] MANGUY J, JEHL P, DILLON E T, et al. Peptigram: A web-based application for peptidomics data visualization [J]. Journal of Proteome Research, 2017, 16(2): 712-719. DOI:10.1021/acs.jproteome.6b00751.
[18] NIELSEN S D, BEVERLY R L, QU Y, et al. Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization [J]. Food Chemistry, 2017, 232: 673-682. DOI:10.1016/j.foodchem.2017.04.056.
[19] 胡锦华, 王园园, 刘大松, 等. 酶法脱磷酸化修饰酪蛋白酸钠及其体外模拟消化 [J]. 食品与发酵工业, 2016, 42(7): 35-39. DOI:10.13995/j.cnki.11-1802/ts.201607006.
[20] 李星. 热处理和贮藏期间乳蛋白的消化吸收机制研究 [D]. 哈尔滨: 哈尔滨工业大学, 2021: 67-128.
[21] SáNCHEZ-RIVERA L, MéNARD O, RECIO I, et al. Peptide mapping during dynamic gastric digestion of heated and unheated skimmed milk powder [J]. Food Research International, 2015, 77: 132-139. DOI:10.1016/j.foodres.2015.08.001.
[22] 于文皓. 人乳内源肽组成及其与泌乳期的相关性研究 [D]. 大连: 大连海洋大学, 2019: 13-21.
[23] ATAMER Z, POST A E, SCHUBERT T, et al. Bovine β-casein: Isolation, properties and functionality. A review [J]. International Dairy Journal, 2017, 66: 115-125. DOI:10.1016/j.idairyj.2016.11.010.
[24] CAO Y, MIAO J Y, LIU G, et al. Bioactive peptides isolated from casein phosphopeptides enhance calcium and magnesium uptake in Caco-2 cell monolayers [J]. Journal of Agricultural and Food Chemistry, 2017, 65(11): 2307-2314. DOI:10.1021/acs.jafc.6b05711.
[25] SEDAGHATI M, EZZATPANAH H, BOOJAR M M A, et al. Isolation and identification of some antibacterial peptides in the plasmin-digest of beta-casein [J]. LWT-Food Science and Technology, 2016, 68: 217-225. DOI:10.1016/j.lwt.2015.12.019.
[26] ADAMS C, SAWH F, GREEN-JOHNSON J M, et al. Characterization of casein-derived peptide bioactivity: Differential effects on angiotensin-converting enzyme inhibition and cytokine and nitric oxide production [J]. Journal of Dairy Science, 2020, 103(7): 5805-5815. DOI:10.3168/jds.2019-17976.
[27] TONOLO F, MORETTO L, FERRO S, et al. Insight into antioxidant properties of milk-derived bioactive peptides in vitro and in a cellular model [J]. Journal of Peptide Science, 2019, 25(5): e3162. DOI:10.1002/psc.3162.
[28] TONOLO F, FIORESE F, MORETTO L, et al. Identification of new peptides from fermented milk showing antioxidant properties: Mechanism of action [J]. Antioxidants, 2020, 9(2): 117. DOI:10.3390/antiox9020117.
[29] GOBBETTI M, FERRANTI P, SMACCHI E, et al. Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp bulgaricus SS1 and Lactococcus lactis subsp cremoris FT4 [J]. Applied and Environmental Microbiology, 2000, 66(9): 3898-3904. DOI:10.1128/AEM.66.9.3898-3904.2000.
[30] YAMAMOTO N, AKINO A, TAKANO T. Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus-helveticus CP790 [J]. Journal of Dairy Science, 1994, 77(4): 917-922. DOI:10.3168/jds.S0022-0302(94)77026-0.
[31] JIANG X X, PAN D D, ZHANG T, et al. Novel milk casein-derived peptides decrease cholesterol micellar solubility and cholesterol intestinal absorption in Caco-2 cells [J]. Journal of Dairy Science, 2020, 103(5): 3924-3936. DOI:10.3168/jds.2019-17586.
[32] RIVAL S G, BOERIU C G, WICHERS H J. Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition [J]. Journal of Agricultural and Food Chemistry, 2001, 49(1): 295-302. DOI:10.1021/jf0003911.