臺灣博碩士論文加值系統

　　本研究探討卵白水解胜肽之鈣螯合活性（calcium-chelating activity），以瞭解卵白胜肽是否能開發為鈣補充劑（calcium supplement）。凍乾卵白先以以三種商業酵素水解後，再以消化道酵素水解淂EHA、EHP與EHB水解物，結果顯示三水解物分子量都比45 kDa來的低，而EHP水解率最高（31.24 ％）。三者水解液經超過濾（UF, MWCO: 5 kDa）收集濾液得U-EHA、U-EHP與U-EHB超濾物（ultrafiltrate），發現U-EHP亦有最佳鈣螯合活性，鈣螯合率為7.75 calcium-mg/ protein-g。將分U-EHP依分子量區分介於3～5 kDa（U-EHP35）、3～1 kDa（U-EHP31）與1 kDa以下（U-EHP1）的三超濾物，探討三者之鈣螯合活性與in vitro促進鈣吸收的效應，結果顯示U-EHP35具有最高的鈣螯合活性（11.60 calcium-mg/ protein-g），與促進鈣吸收率（0.937 mmole calcium / protein-g）。U-EHP35經胺基酸分析，發現Asp和Glu兩者占全胺基酸近21 %，推測是螯合鈣的主要效應。U-EHP35進一步以膠體過濾層析之區分物U-EHP35-II的鈣螯合活性為12.8 calcium-mg/ protein-g。U-EHP35-II續以離子交換層析得活性較高的U-EHP35-II-2區分物，其再經RP-HPLC純化以瞭解U-EHP35-II-2胜肽胺基酸組成。經本研究成果顯示，U-EHP35具有發展成為鈣螯合胜肽（calcium-chelating peptide）作為鈣補充劑的潛力。

　　This study is to investigate egg white protein hydrolystes (EH) with calcium chelating activity. Egg white proteins were digested by three commercial enzymes, and then by gastrointestinal enzymes. The hydrolysates are EHA, EHP and EHB. Electrophoretic analysis indicated that the MWs of these hydrolysates are generally lower than the 45 kDa. Chemical analysis showed that EHP has the highest degree of hydrolysis 31.24 %. The main molecular weight of U-EHP (the highest calcium chelating activity among U-EHA, U-EHP and U-EHB) which was ultrafiltrated by UF membrane (MWCO: 5 kDa) ranges from 480~1422 Da. The calcium chelating activity of U-EHP is 7.75 calcium-μg/ protein-g. The U-EHP35 with MW ranging from 3 to 5 kDa separated from U-EHP, exhibited the highest chelating activity (11.60 calcium-mg/-protein-g) among three ultrafiltrated U-EHPs. Moreover, in vitro calcium absorption exhibited that the absorption of U-EHP35 is 0.937 mmole calcium / protein-g. Amino acid analysis of U-EHP35 showed that the concentrations of Asp and Glu are the highest (the two account for nearly 21% of all amino acids). It is assumed that Asp and Glu are the main effect on calcium chelating. Then gel filitration chromatography was employed for U-EHP35 purification. The fraction U-EHP35-II exhibited the highest calcium chelating activity (12.8 calcium-mg / protein-g). U-EHP35-II was further purified by ion-exchange chromatography and the fraction U-EHP35-II-2 was obtained and its amino acid composition was analyzed. Moreover, RP-HPLC was used for advance study. Calcium chelating peptide with calcium chelating activity have the potential to develop calcium supplements.

壹、前言 1
貳、文獻回顧 2
一、雞蛋特性與加工利用 2
（一） 雞卵卵白特性與研究探討 2
（二） 雞蛋之消費現況 10
二、蛋白質水解 14
（一） 蛋白酶（Protease） 15
（二）蛋白水解物的特性 19
（三）活性胜肽的應用 20
（四）雞蛋中卵白活性胜肽的應用 27
三、鈣質之介紹 29
（一）鈣質的重要性 29
（二） 鈣質的吸收與代謝 30
（三）鈣補充劑（Dietary calcium supplement） 34
四、鈣螯合胜肽之介紹 39
五、純化原理與應用 43
參、實驗材料與方法 49
一、實驗材料 49
（一） 實驗原料 49
（二）實驗藥品 49
（三）儀器與設備 53
二、實驗架構 55
三、實驗步驟 57
（一）蛋清水解物製備 本實驗係參考參考 57
（二） 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳與染色（SDS-PAGE） 58
（三）蛋白質定量（Bradford法） 60
（三）Amino group胺基濃度的測定（o-Phthalaldehyde呈色法） 61
（四）Amino group胺基濃度的測定 （TNBS：2,4,6-Trinitrobenzene sulfonic acid呈色法） 62
（五）水解率（Degree of Hydrolysis） 62
（六）胜肽分子量分布（High-performance liquid chromatography） 63
（七）固定化金屬親和層析（Immobilized-Metal Affinity Chromatography） 64
（八）篩選各分子量範圍的水解物胜肽 65
（九）鈣螯合胜肽製備 66
（十）鈣濃度檢測法 (o-Cresolphthalein Complexone 呈色法) 67
（十一）促進鈣吸收功能評估：體外消化透析法 68
（十二）可吸收胺基濃度測定 70
（十三）膠體管柱層析 (Gel filitration chromatography) 70
（十四）陰離子交換管柱層析（Amino acid composition analysis） 71
（十五）胺基酸組成分析（Amino acid composition analysis） 71
（十六）逆相高效能液相分析（RP-HPLC） 73
肆、結果與討論 74
一、 卵蛋白與卵蛋白水解物特性探討 74
（一） 蛋白質電泳分析 74
（二） 蛋白與水解物溶液中可溶性蛋白質含量（Bradford method測量） 76
（三） 水解效果的量測 78
3-1. o-Phthalaldehyde呈色法測定胺基（amino group）的濃度 78
3-2. TNBS呈色法測定胺基（Amino group） 80
3-3.水解率（Degree of Hydrolysis）的測定 83
二、製備蛋白及各水解物的超濾物（MWCO：5 kDa） 85
三、蛋白水解物的超濾物之分子量分布 87
四、超濾物之鈣螯合率的測定：o-Cresolphthalein Complexone 呈色法量測 90
五、U-EHP之親合性管柱層析分離與純化 93
六、不同分子量的U-EHP區分物之鈣螯合活性與促進鈣吸收率探討 100
七、U-EHP35之分離純化與鈣螯合活性與促進鈣吸收探討 105
八、U-EHP35-II之離子交換層析與鈣螯合活性 113
九、U-EHP35-II與U-EHP35-II-2之C18 RP-HPLC層析 116
十、胺基酸組成分析 119
伍、結論 123
陸、參考文獻 124
柒、附錄 136

A+醫學百科，http://cht.a-hospital.com/。
GE Healthcarem。http://www.gelifesciences.com/。
台灣Wiki，http://www.twwiki.com/wiki/。
雞蛋產業現況，2009．台灣畜產 財團法人中央畜產會，
http://www.naif.org.tw/industrialList.aspx?frontTitleMenuID=12&frontMenuID=13。
國人膳食營養素參考攝取量查詢(DRIs)，2011，行政院 衛生福利部食品藥物管理，https://consumer.fda.gov.tw/Pages/List.aspx?nodeID=636。
Ｄｒ．ボーンペップ，2011，ワキ製薬株式会社，http://www.a-kusuri.co.jp/news/2011/11/1123.html。
莊榮輝，酵素化學實驗，2005。JuangWeb 莊榮輝網頁系統。
蕭寧馨，2009。透視營養學二版。台北。藝軒圖書出版社。
陳建元，2014。最新食品化學。華格那企業。台中。
何國慶、丁立孝，2007。食品酵素學，五南圖書出版股份有限公司。
臺大營養師 編著，2000。《家庭營養師 : 臺大營養師教你如何吃出健康 / 15位臺大營養師執筆》第一版 p.216，天下生活出版股份有限公司，台北。
王增興，2010。日本雞蛋產業現況。國際農業科技新知 56期。台北。
黃仁治，2013。高醫醫訊月刊第三十三卷第一期，高醫藥劑部。
David L. Nelson，Michael M. Cox，2008，Lehninger Principles of biochemistry. New York. W.H.Freeman and company.
林敬彬，2014。水產品加工技術研究。國立高雄海洋科技大學。高雄。
劉雅欣，2014。魚卵水解胜肽純化與活性探討。國立高雄海洋科技大學。高雄。
廖桂玉，2012。虱目魚魚鱗膠原蛋白胜肽的製備與鈣結合能力探討，國立高雄海洋科技大學。高雄。
蘇芷瑩，2012。活性胜肽及熱休克蛋白轉基因番茄之研究。國立宜蘭大。宜蘭。
莫長原，2010。虱目魚魚肉水解液的製備及應用。國立高雄海洋科技大學。高雄。
Abdallah, F.B. and Chahine, J.M., 1999. Transferrins, the mechanism of iron release by ovotransferrin. Eur. J. Biochem. 263:912–920.
Aguilar, F.Autrup, H., Barlow, S.,. Castle, S., Crebelli, R., Dekant, W.,Engel, K.H., Gontard,N., Gott, D., Grilli, S., Gürtler, R., Larsen, J.C., Leclercq, C., Leblanc, J.C., Malcata, F.X., Mennes, W., Milana, M.R., Pratt, I., Rietjens, I., Tobback, P., Toldrá,F., 2008. Opinion on certain bisglycinates as sources of copper, zinc, calcium, magnesium and glycinate nicotinate as source of chromium in foods intended for the general population (including food supplements) and foods for particular nutritional uses. The EFSA Journal (2008) 718, 1-26
Allcroft, WM., 1964. Incubation and Hatchery Practice . London : Her Majesty’s Stationery Office
Allen, L. H., 1982. Calcium bioavailability and absorption: a review. Am J. Clin Nutr., 35, 783-808.
Aristotelis, T.H., Anthony, K.D.T. and Anne, J.M., 2011. A Study of the Enzymatic Hydrolysis of Fish Frames Using Model Systems. Food and Nutrition Sciences, 2011, 2, 575-585
Avioli , L.V., 2000. The osteoporotic syndrome.Academic press.San duego. USA.
Bouayed, J., Deußer, H., Hoffmann, L. and Bohn, T., 2012. Bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns. Food Chemistry 131 (2012) 1466–1472
Charoenphun, N., Cheirsilp, B., Sirinupong, N. and Youravong., W., 2013. Calcium-binding peptides derived from tilapia ( Oreochromis niloticus ) protein hydrolysate. European Food Research and Technology January 2013, Volume 236, Issue 1, pp 57-63
Chiang, W., Lee, M., Guo, W. and Tsai,T., 2006. Protein hydrolysates batch production with angiotensin I–Converting enzyme inhibitory activity from egg whites. J. Food Drug Anal. 14:385–390.
Choi, D.W., Lee, J.H., Chun, H.H. and Song, K.B., 2012. Isolation of a Calcium-binding Peptide from Bovine Serum Protein Hydrolysates.Food Sci. Biotechnol. 21(6): 1663-1667 (2012) DOI 10.1007/s10068-012-0221-z
Daengprok, W., Garnjanagoonchorn, W., Onanong- Naivikul, Pornsinlpatip, P., Issigonis, K. and Mine. Y., 2003. Chicken Eggshell Matrix Proteins Enhance Calcium Transport in the Human Intestinal Epithelial Cells, Caco-2. J. Agric. Food Chem. 2003, 51, 6056-6061
Derde, M., Guérin-Dubiard, C., Lechevalier, V., Cochet, M.F., Jan, S., Baron, F., Gautier, M., Vié, V. and Nau,F., 2014. Dry-heating of lysozyme increases its activity against Escherichia coli membranes. J. Agric. Food Chem. 62 (2014) 1692–1700.
Derde, M., Nau, F., Guérin-Dubiard, C., Lechevalier, V., Paboeuf, G., Jan, S., Baron, F., Gautier, M. and Vié, V., 2015. Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane. Biochimica et Biophysica Acta 1848 (2015) 1065–1073
Diniz, E.M. and Martin, A.M., 1997. Effect of the extract of enzymatic hydrolysis on functional properties of shark protein hydrolysate. Lebensm.Wiss. Technol. 30: 266-272.
Farra, D.C., Domloge, N. and Botto, J.M., 2013. Use of a composition comprising a non-fermented rice peptidic hydrolyzate for stimulating hair growth. BiBTeX, EndNote, RefMan. Patent No.: US 8,541,374 B2
Fitzgerald, R.J. and Murray, B.A., 2006. Bioactive peptides and lactic fermentations. Int J Dairy Technol 2006, 59:118-125.
Fujita, H., Yokoyama, K. and Yoshikawa, M., 2000. Classification and antihypertensive activity of Angiotensin I–converting enzyme inhibitory peptides derived from food proteins. Food Chem. Toxicol. 65:564–569.
Frokjaer, S., 1994. Use of Hydrolysates for Protein Supplementation. Food　Technol. 48: 10-14.
Gill, I., López-Fandiño, R., Jorba, X. and Vulfson, E.N., 1996. Biologically active peptides and enzymatic approaches to their production. Enzyme Microb Technol. 1996 Feb 15;18(3):163-83.
Guéguen, L. and Pointillart, A., 2000.The Bioavailability of Dietary Calcium. J Am Coll Nutr., 19(2), 119S-36S.
Harold, M., Farrell, Jr., Thomas, F., Kumosinski, E.L.M. and Eleanor, M.B., 2002. The Caseins of Milk as Calcium-Binding Proteins. Calcium-Binding Protein Protocols Methods in Molecular Biology™ Volume 172, 2002, pp 97-140
Hartmann, R. and Meiselm H., 2007. Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology 2007, 18:163–169.
Hiidenhovi, J., Aro, H.S. and Kankare, V., 1999. Separation of ovomucin subunits by gel filtration: Enhanced resolution of subunits by using a dual-column system. J. Agric. Food Chem. 47:1004– 1008.
Horiguchi, N., Horiguchi, H. and Suzuki, Y., 2005. Effect of wheat gluten hydrolysate on the immune system in healthy human subjects. Biosci Biotechnol Biochem 2005, 69:2445-2449.
Huang, W., Shen, S.,, Nimalaratne, C., Li, S., Majumder, K. and Wub, J., 2012. Effects of addition of egg ovotransferrin-derived peptides on the oxygen. Food Chemistry 135 (2012) 1600–1607 radical absorbance capacity of different teas
Intarasirisawat, R., Benjakul, S., Visessanguan, W. and Wuc, J., 2012. Antioxidative and functional properties of protein hydrolysate from defatted skipjack (Katsuwonous pelamis) roe. Food Chemistry 135 (2012) 3039–3048
Jiratanan, T. and Liu, R.H., 2004 . Antioxidant activity of processed table beets (Beta vulgaris var. Conditiva) and green beans (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry, 52, 2659–2670.
Jung, W.K., Lee,B.J. and Kim, S.K., 2006. Fish-bone peptide increases calcium solubility and bioavailability in ovariectomised rats. British Journal of Nutrition (2006), 95, 124–128
Jung, W.K., Park, P.J., Byun, H.G., Moon, S.H. and Kim, S.K., 2005. Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous crude proteinase. Food Chem 91, 333-340.
Kim. S. B. and Lim J. W., 2004. Calcium-binding Peptides Derived from Tryptic Hydrolysates of Cheese Whey Protein. Asian-Australasian Journal of Animal Sciences 2004;17(10): 1459-1464.
Kitts, D.D. and Weiler, K., 2003. Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Curr Pharm Des 2003, 9:1309-1323.
Ko, K.Y. and Ahn, D. U., 2008. An economic and simple purification procedure for the large scale production of ovotransferrin from egg white. Poult. Sci. 87:1441–1450.
Ko, S.C., Kim, D. and Jeon, T.J., 2012. Protective effect of a novel antioxidative peptide purified from a marine Chlorella ellipsoidea protein against free radical-induced oxidative stress. Food and Chemical Toxicology 50 (2012) 2294–2302
Kovacs-Nolan, J. K. N., Phillips, M. and Mine, Y., 2005. Advances in the value of eggs and egg components for human health. J. Agric. Food Chem. 53:8421–8431.
Lahl, W. J. and Brum, S. D., 1994. Enzymatic production of protein hydrolysates for food use. Food Technol. 48: 68-71.
Lee, S.H. and Song, K.B., 2009. Isolation of a Calciumbinding Peptide from Enzymatic Hydrolysates of Porcine Blood Plasma Protein. J. Korean Soc. Appl. Biol. Chem. 52(3), 290-294 (2009)
Li-Chan, E.C.Y. and Kim, H-O., 2008. Structure and chemical composition of eggs. In: Mine Y (ed.) Egg Bioscience and Biotechnology, pp. 1–95. Hoboken, NJ: Wiley.
Lo, W.M.Y. and eunice. LI-CHAN, E. C. Y., 2005. Angiotensin I Converting Enzyme Inhibitory Peptides from In Vitro Pepsin-Pancreatin Digestion of Soy Protein. J. Agric. Food Chem. 2005, 53, 3369-3376 3369
Manley, C. H. and Ahmedi, S., 1995. The development of process flavors. Trends Food Sci. Tech. 6: 16-51.
Matsuda, T., Gu, J., Tsuruta, K. and Nakamura, R., 1985. Immunoreactive glycopeptides separated from peptic hydrolysates of chicken egg white ovomucoid. J. Food Sci. 50:592–594.
Megias C, Pedroche J, Yust, M.M., Giron-Calle, J., Alaiz, M., Millan, F. and Vioque, J., 2007. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates. J. Agr. Food Chem. 55: 6509-6514.
Meisel, H., Frister, H. and Schlimme, E., 1989. Biologically active peptides in milk proteins. Zeitschrift fur Ernaehrungswissenschaft . , 28(4), 267–278.
Meisel, H., Goepfert, A. and Gunther, S., 1997. ACE-inhibitory . activities in milk products. Milchwissenschaft, 52(6), 307–311.
Mendis, E., Rajapakse, N. and Kim, S.K., 2005. Antioxidant Properties of a Radical-Scavenging Peptide Purified from Enzymatically Prepared Fish Skin Gelatin Hydrolysate. J. Agric. Food Chem. 2005, 53, 581-587.
Miguel, M. and Aleixandre, A., 2006. Antihypertensive peptides derived from egg proteins. Journal of Nutrition 136: 1457–1460
Miquel, E., Alegrı´a, A., Barbera´, R. and Farre´ R., 2006. Casein phosphopeptides released by simulated gastrointestinal digestion
of infant formulas and their potential role in mineral binding. International Dairy Journal 16 (2006) 992–1000
Mine, Y. and Kovacs-Nolan, J., 2006. New insights in biologically active proteins and peptides derived from hen egg. Worlds Poult Sci J 2006, 62:87-95.
Moon, S. H., Lee, J. H., Lee, Y. J., Chang, K. H., Paik, J. Y., Ahn, D.U. and Paik, H. D., 2013. Screening for cytotoxic activity of ovotransferrin and its enzyme hydrolysates. Poult. Sci. 92:424–434.
Nisbet, A.D., Saundry, R.H., Moir, A.J.G., Fothergill, L.A. and Fothergill, J. E., 1981. The complete amino-acid sequence of hen ovalbumin. Eur. J. Biochem. 115:335–345.
Oe, H., Doi, E. and Hirose, M., 1988. Amino-terminal and carboxylterminal half-molecules of ocotransferrin: preparation by a novel procedure and their interactions. J. Biochem. 103:1066–1072.
Omana, D.A., Wang, J. and Wu, J., 2010. Co-extraction of egg white proteins using ion-exchange chromatography from ovomucin-removed egg white. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878:1771–1776.
Oliveira, F. C., Coimbra, J. S. R., Silva, L.H.M., Rojas, E.E.G. and Silva, M.C.H., 2009. Ovomucoid partitioning in aqueous two-phase system. Biochem. Eng. J. 47:55–60.
Otake, S., Nishiharam, Y., Makimura, M., Hatta, H., Kim, M., Yamamoto, T. and Hirasawa, M., 1991. Protection of rats against dental caries by passive immunization with hen-egg-yolk antibody (IgY). J Dent Res. 1991 Mar;70(3):162-6.
Pellegrini, A., Andreas, J., Hu¨lsmeier, Hunziker, P. and Thomas, U., 2004. Proteolytic fragments of ovalbumin display antimicrobial activity. Biochimica et Biophysica Acta 1672 (2004) 76– 85
Pellegrini, A., Thomas, U., Vonfellenberg, R. and Wild, P., 1992. Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character, J. Appl. Bacteriol. 72 (1992) 180–187.
Pinto, M., Robine-Léon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J. and Zweibaum, A., 1983. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell. 47, 323-330.
Quirós, A., Chichón, R., Recio, I. and López-Fandiño, R., 2007. The use of high hydrostatic pressure to promote the proteolysis and release of bioactive peptides from ovalbumin. Food Chemistry 104 (2007) 1734–1739
Robinson, D S. and Monsey, J.B., 1975. The composition and proposed subunit structure of egg-white beta-ovomucin. The isolation of an unreduced soluble ovomucin. Biochem J. 1975 Apr; 147(1): 55–62.
Ryu, B.M., Himaya, S.W.A., Qian, Z.J., Lee, S.H. and Kim,S.K., 2011. Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli. Peptides 32 (2011) 639–647
Ryu, B.O., Qian Zhong-Ji. and Kim, S.K., 2010. Purification of a peptide from seahorse, that inhibits TPA-induced MMP, iNOS and COX-2 expression through MAPK and NF-B activation, and induces human osteoblastic and chondrocytic differentiation. Chemico-Biological Interactions 184 (2010) 413–422
Schaafsma, G., 1997.Bioavailability of calcium and magnesium. European J. Clin .Nutr.51 :13-16.
Shen, L.H., Luten, J., Robberechtl, H., Bindels, J. and Deelstra, H., 1994. Modification of an in-vitro method for estimating the bioavailability of zinc and calcium from foods. Lebensm Unters Forsch (1994) 199:442-445
Silva, S.V. and Malcata, F.X., 2005. Caseins as source of bioactive peptides. Int Dairy J 2005, 15:1-15.
Simkhada, J.R., Mander, P., Cho S.S. and Yoo, J.C., 2012. A novel fibrinolytic protease from Streptomyces sp. CS684. Process Biochemistry 45 (2010) 88–93
Stadelman, W. J. and Cotterill, O. J., 2001. Egg Science and Technology. 4th ed. Avi Publ. Co., Westport, CT
Tessier, B., Harscoat-Schiavo, C. and Marc I., 2006.Selective separation of peptides contained in a rapeseed (Brassica campestris L.) protein hydrolysate using UF/NF membranes. J. Agr. Food Chem. 54: 3578-3584 (2006)
Ting, B. P. C. P., Pouliot, Y., Gauthier, S. F. and Mine, Y., 2010. Fractionation of egg proteins and peptides for nutraceutical applications. Separation, extraction and concentration processes in the food, beverage and nutraceutical industries 2010 pp. 595-618
Torino, M.I., Limón, R.I., Cristina, M.V., Sari Mäkinen, Pihlanto, A., Concepció, V.V. and Frias, J., 2013. Antioxidant and antihypertensive properties of liquid and solid state fermented lentils. Food Chemistry 136 (2013) 1030–1037
Ng, T.B. 2013. Antifungal and Antibacterial Peptides of Fungal Origin. Handbook of Biologically Active Peptides (Second Edition) Chapter 23.
Vavrusova,M. and Skibsted, L.H., 2014. Calcium nutrition. Bioavailability and fortification. LWT - Food Science and Technology Volume 59, Issue 2, Part 2, December 2014, Pages 1198–1204
Waheed, S., Rahman,S. and Siddique, N., 2014. Calcium supplements as source of trace elements:Adequacy and safety of supplements with vitamin C,vitamin D and phosphate formulations. Applied Radiation and Isotopes 89 (2014)134–140
Zhou, C., Zhang, D., Bai, Y. and Li, S., 2014. Casein phosphopeptide–amorphous calcium phosphate remineralization of primary teeth early enamel lesions. Journal of dentistry 42 ( 2014 ) 21 – 29.
Zhao, L., Cai, X., Huang, S., Wang, S., Huang, S., Hong J. and Rao, P., 2015. Isolation and identification of a whey protein-sourced calciumbinding tripeptide Tyr-Asp-Thr. International Dairy Journal 40 (2015) 16e23
Ying, L., Xiaolan, B., He, L., Jianhua, R., Shuntang, G., 2013. Purification and characterization of caclium-binding soybean protein hydrolysates by Ca2+/Fe3+ immobilized metal affinity chromatography (IMAC). Food Chemistry 141 (2013) 1645–1650