Journal of Food and Nutrition Research
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Journal of Food and Nutrition Research. 2017, 5(10), 763-770
DOI: 10.12691/jfnr-5-10-7
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Purification of Bovine Bone Oligophosphopeptide with High Calcium-binding Activity by Bacillus cereus MBL13 Collagenolytic Protease

Lili Liu1, 2, , Chenliu Yang1, Yang Zhu2, Yuanyuan Meng1 and Xiaoning Dai1

1College of Food and Bioengineering, Henan University of Science and Technology, Luoyang, China

2School of Agriculture and Food Science, University of Queensland, Queensland, Austrilia

Pub. Date: October 10, 2017

Cite this paper:
Lili Liu, Chenliu Yang, Yang Zhu, Yuanyuan Meng and Xiaoning Dai. Purification of Bovine Bone Oligophosphopeptide with High Calcium-binding Activity by Bacillus cereus MBL13 Collagenolytic Protease. Journal of Food and Nutrition Research. 2017; 5(10):763-770. doi: 10.12691/jfnr-5-10-7


A demineralized bovine bone was hydrolyzed by a specific bone-degrading collagenolytic protease extracted from Bacillus cereus MBL13 (isolated from chopped animal bone wastes) to utilize its collagen in nutraceuticals with high calcium bioavailability. Bovine bone peptide (BBP), a novel oligophosphopeptide with a high calcium binding ability (8.25 mmol/g-protein), was isolated from bovine bone hydrolysates by Chelex 100, ultrafiltration, hydroxyapatite chromatography, gel filtration chromatography, and reverse-phase high performance liquid chromatography. The results showed that demineralization treatment can significantly increase hydrolysis (p < 0.05). The amino acid content of BBP showed that the Asp, Ala, Tyr, and Thr contents were remarkable increments compared to the bone hydrolysates. The molecular mass of BBP was found to be around 3.305 kDa through SDS-PAGE and MALDI-TOF mass spectrometry. FT-IR spectra showed characteristic absorption peaks. Moreover, BBP exhibited higher calcium binding activity than that of casein oligophosphopeptide (CPP). Therefore, this study demonstrated that B. cereus MBL13 collagenolytic protease (BCC) could degrade bovine bone collagen, and prepared oligophosphopeptide could be utilized as a nutraceutical with high calcium-binding activity.

bovine bone collagen Bacillus cereus MBL13 collagenolytic protease oligophosphopeptide calcium binding activity

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[1]  Wang, S., Sun, X. and Zhou, D, “Physicochemical and Reactive Oxygen Species Scavenging Properties of Collagen and Collagen Hydrolysates from Farmed Globefish ( Fugu rubripes ) Bone,” Journal of Aquatic Food Product Technology, 26 (5). 527-542. 2017.
[2]  Li, Z.R., Wang, B., Chi, C.F., Zhang, Q.H., Gong, Y.D., Tang, J.J., Luo, H.Y., and Ding, G.F, “Isolation and characterization of acid soluble collagens and pepsin soluble collagens from the skin and bone of Spanish mackerel (Scomberomorous niphonius),” Food Hydrocolloids, 31 (1). 103-113. 2013.
[3]  Cheng, F.Y., Wan, T.C., Liu, Y.T., Chen, C.M., Lin, L.C. and Sakata, R, “Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase,” Animal Science Journal, 80 (1). 91-97. 2009.
[4]  Kumar, B. and Rani, S, “Technical note on the isolation and characterization of collagen from fish waste material,” Journal of Food Science and Technology, 54 (1). 276-78. 2017.
[5]  Pal, G. K. and Suresh, P. V, “Sustainable valorisation of seafood by-products: recovery of collagen and development of collagen-based novel functional food ingredients,” Innovative Food Science & Emerging Technologies, 37, 201-215. 2016.
[6]  Jung, W.K., Park, P.J., Byun, H.G., Moon, S.H. and Kim, S.K, “Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase,” Food Chemistry, 91 (2). 333-340. 2005.
[7]  Jung, W.K., Karawita, R., Heo, S.J., Lee, B.J., Kim, S.K. and Jeon, Y.J, “Recovery of a novel Ca-binding peptide from Alaska Pollack (Theragra chalcogramma ) backbone by pepsinolytic hydrolysis,” Process Biochemistry, 41 (9). 2097-2100. 2006.
[8]  Pal G.K. and Suresh P.V, “Comparative assessment of physico-chemical characteristics and fibril formation capacity of thermostable carp scales collagen,” Materials Science and Engineering: C, 70 (1). 32-40. 2017.
[9]  Lima, C. A., Rodrigues, P. M. B., Porto, T. S., Viana, D. A., Filho, J. L. L., Porto, A. L. F. and Cunha, M. G. C. D, “Production of a collagenase from Candida albicans URM3622,” Biochemical Engineering Journal, 43 (3). 315-320. 2009.
[10]  Huang, G., Ren, L. and Jiang, J, “Purification of a histidine-containing peptide with calcium binding activity from shrimp processing byproducts hydrolysate,” European Food Research & Technology, 232 (2). 281-287. 2011.
[11]  Lee, S.H. and Song, K.B, “Isolation of a calcium-binding peptide from enzymatic hydrolysates of porcine blood plasma protein,” Journal of the Korean Society for Applied Biological Chemistry, 52 (3). 290-294. 2009.
[12]  Wonkyo, J. and Sekwon, K, “Calcium-binding peptide derived from pepsinolytic hydrolysates of hoki (Johnius belengerii) frame,” European Food Research and Technology, 224 (6). 763-767. 2007.
[13]  Sato, R., Noguchi, T. and Naito, H, “Casein Phosphopeptide (CPP) Enhances Calcium Absorption form the Ligated Segment of Rat Small Intestine,” Journal of Nutritional Science and Vitaminology, 32 (1). 67-76. 1986.
[14]  Yuan, Y.V. and Kitts, D.D, “Confirmation of calcium absorption and femoral utilization in spontaneously hypertensive rats fed casein phosphopeptide supplemented diets,” Nutrition Research, 11 (11). 1257-1272. 1991.
[15]  Duarte A.S., Correia A. and Esteves A.C, “Bacterial collagenases- A review,”Critical Reviews in Microbiology, 42(1): 106-126. 2016.
[16]  Pal, G. K. and Pv, S, “Microbial collagenases: challenges and prospects in production and potential applications in food and nutrition,” RSC Advances, 6 (40), 33763-33780. 2016.
[17]  Itoi, Y., Horinaka, M., Tsujimoto, Y., Matsui, H. and Watanabe, K, “Characteristic features in the structure and collagen-binding ability of a thermophilic collagenolytic protease from the thermophile geobacillus collagenovorans mo-1,” Journal of Bacteriology, 188 (18). 6572-9. 2006.
[18]  Kurata, A., Uchimura, K., Kobayashi, T. and Horikoshi, K, “Collagenolytic subtilisin-like protease from the deep-sea bacterium alkalimonas collagenimarina ac40t,” Applied Microbiology & Biotechnology, 86 (2). 589. 2010.
[19]  Bhagwat, P.K., Jhample, S.B., Jalkute, C.B. and Dandge, P.B, “Purification, properties and application of a collagenolytic protease produced by pseudomonas sp. Suk,” Rsc Advances, 6 (69). 65222-65231. 2016.
[20]  Bhagwat, P.K., Jhample, S.B. and Dandge, P.B, “Statistical medium optimization for the production of collagenolytic protease by pseudomonas, sp. suk using response surface methodology,” Microbiology, 84 (4). 520-530. 2015.
[21]  Zhao, G. Y., Zhou, M. Y., Zhao, H. L., Chen, X. L., Xie, B. B., & Zhang, X. Y., He, H.L., Zhou, B.C. and Zhang, Y.Z, “Tenderization effect of cold-adapted collagenolytic protease mcp-01 on beef meat at low temperature and its mechanism,” Food Chemistry, 134 (4). 1738-1744. 2012.
[22]  Watanabe, K, “Collagenolytic proteases from bacteria,” Applied Microbiology & Biotechnology, 63 (5). 520-526. 2004.
[23]  Fricke, B., Drößler, K., Willhardt, I., Schierhorn, A., Menge, S. and Rücknagel, P, “The cell envelope-bound metalloprotease (camelysin) from Bacillus cereus is a possible pathogenic factor,” Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1537 (2). 132-146. 2001.
[24]  Liu, L., Ma, M.H., Cai, Z.X., Yang, X.L. and Wang, W.T, “Purification and properties of a collagenolytic protease produced by Bacillus cereus MBL13 strain,” Food Technology & Biotechnology, 48 (2). 151-160. 2010.
[25]  Nielsen, P.M., Petersen, D. and Dambmann, C, “Improved Method for Determining Food Protein Degree of Hydrolysis,” Journal of Food Science, 66 (5). 642-646. 2010.
[26]  Koga, T., Minamizato, T., Kawai, Y., Miura, K., Takashi, I., Nakatani, Y., Sumita, Y. and Asahina, I, “Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size,” Plos One, 11 (1). e0147235. 2016.
[27]  Schägger, H, “Tricine-SDS-PAGE,” Nature Protocols, 1 (1). 16-22. 2006.
[28]  Huang, H., Li, B., Liu, Z., Wu, H., Mu, X. and Zeng, M, “Purification of a Novel Oligophosphopeptide with High Calcium Binding Activity from Carp Egg Hydrolysate,” Food Science & Technology International Tokyo, 20 (4). 799-807. 2014.
[29]  Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J, “Protein measurement with the Folin phenol reagent,” Journal of Biological Chemistry, 193 (1). 265. 1951.
[30]  Kowalski, Z., Banach, M. and Makara, A, “Technology for manufacturing protein hydrolyzates and dried proteins of meat-bone tissue,” Przemysl Chemiczny, 90 (7). 1346-1352. 2011.
[31]  Ren, X., Ma, L., Chu, J., Wang, Y., Zhuang, Y., Zhang, S., Yang, H. and An, H, “Optimization of Enzymatic Hydrolysis of Channel Catfish Bones for Preparing Antimicrobial Agents,” Journal of Aquatic Food Product Technology, 21 (2). 99-110. 2012.
[32]  Tishinov, K., Christov, P., Neshev, G., Nustorova, M., Paskaleva, D., Vasilevatonkova, E., Gousterova, A. and Nedkov, P, “Investigation of the Possibility for Enzymatic Utlization of Chicken Bones,” Biotechnology & Biotechnological Equipment, 24 (4). 2108-2111. 2010.
[33]  Mecham, D.K. and Olcott, H.S, “Phosvitin, the principal phosphoprotein of egg yolk,” Journal of the American Chemical Society, 71 (11). 3670-3679. 1949.
[34]  Barakat, N.A.M., Khil, M.S., Omran, A.M., Sheikh, F.A. and Kim, H.Y, “Extraction of pure natural hydroxyapatite from the bovine bones bio waste by three different methods,” Journal of Materials Processing Tech, 209 (7). 3408-3415. 2009.
[35]  Jiang, B. and Mine, Y, “Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin,” Journal of Agricultural & Food Chemistry, 48 (4). 990. 2000.
[36]  Hoang, Q.Q., Sicheri, F., Howard, A.J. and Yang, D.S, “Bone recognition mechanism of porcine osteocalcin from crystal structure,” Nature, 425 (6961). 977. 2003.
[37]  Nishimoto, S.K., Waite, J.H., Nishimoto, M. and Kriwacki, R.W, “Structure, activity, and distribution of fish osteocalcin,” Journal of Biological Chemistry, 278 (14). 11843. 2003.
[38]  Beck, S.M., Kai, K. and Arcot, J, “Effect of Low Moisture Extrusion on a Pea Protein Isolate’s Expansion, Solubility, Molecular Weight Distribution and Secondary Structure as Determined by Fourier Transform Infrared Spectroscopy (FTIR),” Journal of Food Engineering, 214.166-174. 2017.
[39]  Krimm, S. and Bandekar, J, “Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins,” Advances in Protein Chemistry, 38 (C). 181-364. 1986.
[40]  Beauchemin, R., Harnois, J., Rouillon, R., Tajmir-Riahi, H.A. and Carpentier, R, “Interaction of polyamines with proteins of photosystem II: Cation binding and photosynthetic oxygen evolution,” Journal of Molecular Structure, 833 (1-3). 169-174. 2007.