臺灣博碩士論文加值系統

明膠是來自動物結締組織中的膠原蛋白水解物，已廣泛用於食品工業改善食品的質地；除此之外，由明膠水解物製備的活性胜肽可直接對人類帶來益處。本研究的目的是以鳳梨酵素 (bromelain)、膠原蛋白酶 (collagenase) 及木瓜酵素 (papain) 水解市售牛骨、豬皮、吳郭魚明膠來探討其抗氧化、血管收縮素轉化酶 (angiotensin converting enzyme，ACE) 抑制及抗健忘潛在之生理活性。從線上資料庫工具 (basic local aligment search tool，BLAST) 分析牛明膠與豬明膠同源性高達 97%，顯示兩者蛋白質序列相似，並自 UniProt KB 資料庫取得牛、豬、吳郭魚之膠原蛋白序列，使用 BIOPEP-UWM 活性胜肽資料庫模擬 bromelain、papain 水解，得知抑制 ACE 之胜肽活性頻率 (AE) 為 0.0919-0.1113，而抗氧化與抗健忘則分別為0.0007-0.0014、 0.0177-0.0428，另外使用腸胃道消化酶分析，抗氧化活性頻率提升至0.0021-0.0027，而抑制 ACE 的關鍵成分多以雙胜肽組成。三種明膠皆以蛋白質為主要成分 (83.31-85.10%)，水解率結果表明 collagenase 為最高 (45.44-53.89%)，且隨著水解率增加，水解物平均分子量越低。牛明膠 collagenase 水解物 (BC) 在體外抗氧化試驗中具有 DPPH 自由基清除活性 22.53％、亞鐵還原力 0.0056 mM Fe2+/mg和超氧自由基清除活性 41.64 %，另金屬離子螯合試驗以 collagenase 酵素水解產物皆較 bromelain 或 papain有顯著抗氧化效果，表示高水解率有高抗氧化表現；然而，papain 水解的豬明膠 (PP) 具有較佳的ACE 抑制作用 (2 mg/mL，60.94%)。接著篩選樣品，根據經濟效益與成本考量，後續選擇 PP 水解1小時 (PP1) 與4小時 (PP4) 的水解物進行比較，從抗氧化與 ACE 抑制能力結果得知兩者並無顯著差異，並使用超過濾膜將 PP1 分為 <1 kD、1-5 kD、5-10 kD，其中 <1 kD 之分離物具抗健忘之活性，可抑制 AChE (acetylcholinesterase) 與 PEP (Prolyl endopeptidase) 達21.24% 和 48.07%。 胺基酸 Gly 和 Pro 可能是主要具有活性的關鍵成分。最後由 D-galactose (DG) 誘導的小鼠衰老 (300 mg/kg/day)，顯示補充 PP1 可改善小鼠認知記憶能力，並測定超氧化物歧化酶活性與總抗氧化力 (trolox equivalent antioxidant capacity，TEAC) 皆有提升，而高劑量 PP1 腦組織切片證實能減緩 DG 誘導腦部氧化壓力造成的神經元破壞。根據上述 PP1 具抗氧化、抗高血壓及抗健忘症之生理活性，且未來有應用於食品保健產業之潛力。

Gelatin is a collagen hydrolysate from animal connective tissues and has been widely used in food industry to improve the texture of variable foods. Furthermore, active peptides derived from gelatin hydrolysates are beneficial to humans. The purpose of this study was to hydrolyze commercial bovine bone, porcine skin, and tilapia gelatins by bromelain, collagenase and papain in search of their antioxidant and ACE-inhibition activities. From the online database (basic local aligment search tool，BLAST) analysis, the homology between bovine and porcine gelatins was up to 97%, which showed that the protein sequences were identical. In addition, these three collagen sequences were obtained from UniProt database, and enzymes (bromelain and papain) were used to simulate the enzyme action by the BIOPEP-UWM. As a result, the frequency (AE) of ACE-inhibition peptides was 0.0919-0.1113, while peptide frequencies were 0.0177-0.0428 and 0.00007-0.0014 for anti-amnesia and antioxidant activities, respectively. In particular, combination of digestive enzymes showed better AE in antioxidant activity, and in silico results indicated that ACE-inhibition peptides consisted of dipeptides mostly. The major components of these gelatins are protein (83.31-85.10%), and the results also showed that the collagenase hydrolysis rate was the highest (45.44-53.89%), with the lower the average molecular weights of hydrolysates as the hydrolysis rate increased otherwise. Bovine gelatin collagenase hydrolysate (BC) had 22.53% DPPH free radical scavenging activity, 0.0056 mM/mg ferrous reduction and the superoxide radical scavenging activity was 41.64% during in vitro antioxidant tests. Besides, the from a metal ion chelation test demonstrated that hydrolysis by collagenase had significantly antioxidant effect compared with the ones hydrolyzed by bromelain or papain. However, porcine gelatin hydrolyzed by papain (PP) showed better ACE-inhibition (60.94%). According to economic benefits and cost considerations, the subsequent hydrolysis of PP for 1 hour (PP1) and 4 hours (PP4) had been conducted. There was no significant difference in antioxidant and ACE-inhibition abilities between PP1 and PP4. Moreover, PP1 was further seprataed into <1 kD, 1-5 kD, 5-10 kD by ultrafiltration membrane, finding that the <1 kD fraction had anti-amnesia activity which can inhibit acetylcholinesterase (AChE) and prolyl endopeptidase (PEP) to 21.24% and 48.07%, respectively. Gly and Pro might play the critical roles in biological activities in PP1. An aging ICR mouse model was established by chronically administering D-galactose (300 mg/kg) for 7 weeks. Results revealed that PP1 can not only inhibit spatial learning and memory impairments but increase superoxide dismutase (SOD) activity and trolox equivalent antioxidant capacity (TEAC) values. Additionally, it proved that DG_HPP1 supplementation attenuated the neuronal degenerations of dentate gyrus area in the hippocampus histology. These results suggested that PP1 have antioxidant, anti-hypertensive and, anti-amnesia physiological activities, being potential application of the nutraceutical food industry in the future.

壹、前言 1
貳、文獻回顧 2
一、膠原蛋白 2
1.1 結構 2
1.2 來源 5
1.3 應用 5
二、蛋白質水解物 7
2.1 酸鹼水解 7
2.2 酵素水解 8
2.3 微生物發酵水解 8
三、生理活性 9
3.1 抗氧化活性 9
3.2 降血壓活性 10
3.2.1 angiotensin II 與腦部腎素 - 血管收縮素–醛固酮系統 (Renin-Angiotensin-Aldosterone System，RAAS) 11
3.3 抗健忘活性 12
3.3.1 脯胺醯內肽酶 12
3.3.2 乙醯膽鹼酯 12
四、食物來源之蛋白質水解物生理活性 13
五、明膠 15
5.1 結構與應用 15
5.2 明膠水解物之生理活性 15
六、 老化與失智症 20
6.1 全球與臺灣人口老化程度 20
6.2 全球與臺灣失智症人口分布及趨勢 21
6.3失智症 22
6.3.1 失智症成因 22
6.3.1退化性失智症 23
6.3.1.1阿茲海默症 (Alzheimer’s Disease，AD) 23
6.3.1.2額顳葉型失智症 (Frontotemporal lobe degeneration，FTLD) 23
6.3.1.3路易氏體失智症 (Dementia with Lewy Bodies，DLB) 23
6.3.2血管性失智症 23
6.3.3其他因素導致之失智症 24
七、D-galactose 誘導老化模型 24
參、實驗架構與方法 26
一、實驗材料 28
1.1 實驗樣品 28
1.2 實驗藥品 28
1.3 蛋白質酵素 28
二、實驗方法 29
2.1 酵素水解 29
2.2 酵素水解物特性分析 29
2.2.1 水解率 (degree of hydrolysis, DH) 29
2.2.2 胜肽含量測定 29
2.2.3 可溶性蛋白質定量 30
2.3 抗氧化活性測定 30
2.3.1 1,1-diphenyl-2-picrylhydrazyl (DPPH) 自由基清除能力 30
2.3.2 金屬螯合能力 30
2.3.3 亞鐵還原氧能力 (ferric reducing antioxidant power， FRAP) 30
2.3.4 超氧自由基清除能力 (superoxide radical scavenging activity，SRSA) 31
2.4 降血壓能力測定 31
2.4.1 ACE (Angiotensin-I converting enzyme) 抑制能力 31
2.5 抗健忘活性測定 32
2.5.1 PEP (prolyl endopeptidase) 抑制能力 32
2.5.2 AChE (acetylcholinesterase) 抑制能力 32
2.6 活性胜肽之鑑定 32
2.6.1 超過濾 (Ultrafiltration) 32
2.6.2 膠體過濾層析 32
2.6.3 胺基酸組成分析 33
2.6.4 生物資訊工具分析 34
2.7 統計分析 34
2.8 動物認知行為能力試驗 (Morris 水迷宮) 35
2.8.1 動物飼養 35
2.8.2 實驗裝置與設計 35
2.8.2 第1天 Morris 水迷宮：可見平台 36
2.8.3 第2–5天Morris水迷宮：參考記憶試驗 (Reference memory task) 36
2.8.4 第6天Morris水迷宮：空間性探測試驗 (Probe test) 37
2.9 小鼠器官採集 37
2.9.1 腦組織採樣與均質液製備 37
2.10 腦組織抗氧化能力分析 37
2.10.1 蛋白質濃度測定 37
2.10.2 硫巴比妥酸反應物質 (TBARS) 分析 37
2.10.3 TEAC (trolox equivalent antioxidant capacity) 總抗氧化力 38
2.10.4 超氧岐化酶 (Superoxide dismutase，SOD) 活性分析 38
2.10.5 GPx (Glutathione peroxidase) 活性 39
2.11 腦組織切片與染色 39
2.12 統計分析 40
肆、結果與討論 42
一、市售明膠之 in silico 分析 42
二、市售明膠之一般成分、水解率、蛋白質分子量分析 43
三、市售明膠之體外抗氧化與 ACE 抑制能力 44
3.1 DPPH 自由基清除能力 44
3.2 亞鐵離子還原力 45
3.3 金屬離子螯合 45
3.4 超氧陰離子清除能力 46
3.5 ACE (Angiotensin-I converting enzyme) 抑制能力 46
四、豬明膠 papain 酵素水解物之生理活性 46
4.1 水解時間對抗氧化及ACE 抑制能力之影響 47
4.2 PP1 分離物之抗氧化、ACE抑制、抗健忘活性 47
五、胺基酸組成分析 48
六、PP1 改善小鼠學習認知行為能力 50
6.1 PP1 對 D-galactose (DG) 誘導小鼠之生理影響 50
6.2 PP1對 DG 誘導小鼠之水迷宮學習記憶改善情形 50
6.3 PP1對 DG 誘導小鼠之腦部組織特徵與抗氧化能力 51
伍、結論 53
陸、未來研究方向 53
柒、參考文獻 54
捌、圖表 67
玖、附錄 94

梁崇正。(2002)。明膠的溶膠-凝膠相變化與微乳液-有機凝膠相變化。國立中央大學，桃園市，臺灣。
黃彥富，湯正明，徐善慧。(2003)。揭開膠原蛋白的神秘面紗。科學發展， 362，pp 44-47。臺北市，臺灣。
黃博斌。(2016)。吳郭魚加工共產物中蛋白質鑑定及其潛在活性胜肽之分析。國立臺灣海洋大學，基隆市，臺灣。
台灣失智症協會 (2016)。五歲分年齡層失智症盛行率。臺北市，臺灣。
台灣失智症協會 (2019)。認識失智症。臺北市，臺灣。
中華民國內政部戶政司。(2019)。台灣老年人口結構。臺北市，臺灣。
Akagündüz, Y., Mosquera, M., Giménez, B., Alemán, A., Montero, P. & Gómez-Guillén, M. C. (2014). Sea bream bones and scales as a source of gelatin and ACE inhibitory peptides. LWT - Food Science and Technology, 55, 579-585.
Akira, T. & Jean‐Jacques, S. (1982). A rapid method for acid hydrolysis of protein with a mixture of trifluoroacetic acid and hydrochloric acid. European Journal of Biochemistry, 124, 585-588.
Alemán, A., Giménez, B., Pérez-Santin, E., Gómez-Guillén, M. C. & Montero, P. (2011). Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chemistry, 125, 334-341.
Ao, J. & Li, B. (2012). Amino acid composition and antioxidant activities of hydrolysates and peptide fractions from porcine collagen. Food Science and Technology International, 18, 425-434.
Aoyagi, T., Wada, T., Nagai, M., Kojima, F., Harada, S., Takeuchi, T., Takahashi, H., Hirokawa, K. & Tsumita, T. (1990). Deficiency of kallikrein-like enzyme activities in cerebral tissue of patients with Alzheimer's disease. Experientia, 46, 94-97.
Augustyn, A., Bauer, P., Duignan, B., Eldridge, A., Gregersen, E., McKenna, A., Petruzzello, M., Rafferty, J. P., Ray, M., Rogers, K., Tikkanen, A., Wallenfeldt, J., Zeidan, A. & Zelazko, A. (2017). Renin-angiotensin system. https://www.britannica.com/science/renin-angiotensin-system Access 07.31 2019.
Aydın, S., Yanar, K., Atukeren, P., Dalo, E., Sitar, M. E., Uslu, E., Caf, N. & Çakatay, U. (2012). Comparison of oxidative stress biomarkers in renal tissues of D-galactose induced, naturally aged and young rats. Biogerontology, 13, 251-260.
Babu, K. M. (2019). 5 - The dyeing of silk. Silk 2 ed., 109-128: Woodhead Publishing.
Baehaki, A., Suhartono, M., Sukarno, S., Syah, D. & Setyahadi, S. (2016). Antioxidant activity of collagen hydrolysates from fish skin with a microbial collagenase. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 7, 1677-1682.
Benzie, I. F. F. & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239, 70-76.
Berillis, P. (2015). Marine collagen: Extraction and applications. Research Trends in Biochemistry, Molecular Biology and Microbiology, 1-13.
Bhagwat, P. K. & Dandge, P. B. (2018). Collagen and collagenolytic proteases: A review. Biocatalysis and Agricultural Biotechnology, 15, 43-55.
Bo-Htay, C., Palee, S., Apaijai, N., Chattipakorn, S. C. & Chattipakorn, N. (2018). Effects of D-galactose-induced ageing on the heart and its potential interventions. Journal of Cellular and Molecular Medicine, 22, 1392-1410.
Borawska, J., Darewicz, M., Vegarud, G. E. & Minkiewicz, P. (2016). Antioxidant properties of carp (Cyprinus carpio L.) protein ex vivo and in vitro hydrolysates. Food Chemistry, 194, 770-779.
Bosch, A. M. (2006). Classical galactosaemia revisited. Journal of Inherited Metabolic Disease, 29, 516-525.
Bosch, L., Alegría, A. & Farré, R. (2006). Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. Journal of Chromatography B, 831, 176-183.
Bougatef, A., Nedjar-Arroume, N., Manni, L., Ravallec, R., Barkia, A., Guillochon, D. & Nasri, M. (2010). Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita) by-products proteins. Food Chemistry, 118, 559-565.
Bromley-Brits, K., Deng, Y. & Song, W. (2011). Morris water maze test for learning and memory deficits in Alzheimer's disease model mice. Journal of Visualized Experiments, 2920-2924.
Burjanadze, T. V. (2000). New analysis of the phylogenetic change of collagen thermostability. Biopolymers, 53, 523-528.
Cebe, T., Yanar, K., Atukeren, P., Ozan, T., Kuruç, A. I., Kunbaz, A., Sitar, M. E., Mengi, M., Aydın, M. S., Eşrefoğlu, M., Aydın, S. & Cakatay, U. (2014). A comprehensive study of myocardial redox homeostasis in naturally and mimetically aged rats. Age, 36, 9728-9728.
Chakrabarti, S., Jahandideh, F. & Wu, J. (2014). Food-derived bioactive peptides on inflammation and oxidative stress. BioMed Research International, 2014, 608979.
Chang, Y.-M., Chang, H.-H., Lin, H.-J., Tsai, C.-C., Tsai, C.-T., Chang, H.-N., Lin, S.-L., PadmaViswanadha, V., Chen, R.-J. & Huang, C.-Y. (2017). Inhibition of cardiac hypertrophy effects in D-galactose-induced senescent hearts by alpinate oxyphyllae fructus treatment. Evidence-Based Complementary and Alternative Medicine, 2017.
Chen, H.-M., Muramoto, K., Yamauchi, F. & Nokihara, K. (1996). Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of Agricultural and Food Chemistry, 44, 2619-2623.
Church, F. C., Swaisgood, H. E., Porter, D. H. & Catignani, G. L. (1983). Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66, 1219-1227.
Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science and Technology, 11, 254-262.
Cushman, D. W. & Ondetti, M. A. (1999). Design of angiotensin converting enzyme inhibitors. Nature Medicine, 5, 1110-1112.
De Maria, S., Ferrari, G. & Maresca, P. (2017). Effect of high hydrostatic pressure on the enzymatic hydrolysis of bovine serum albumin. Journal of the Science of Food and Agriculture, 97, 3151-3158.
Erdmann, K., Cheung, B. W. Y. & Schröder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. Journal of Nutritional Biochemistry, 19, 643-654.
Feng, L., Qiao, Y., Zou, Y., Huang, M., Kang, Z. & Zhou, G. (2014). Effect of Flavourzyme on proteolysis, antioxidant capacity and sensory attributes of Chinese sausage. Meat Science, 98, 34-40.
Fenton, H. J. H. (1894). LXXIII.—Oxidation of tartaric acid in presence of iron. Journal of the Chemical Society, Transactions, 65, 899-910.
Fischer, A. H., Jacobson, K. A., Rose, J. & Zeller, R. (2008). Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harbor Protocols, 3, 4986-4988.
Friess, W. (1998). Collagen – biomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 45, 113-136.
Fujii, N., Satoh, K., Harada, K. & Ishibashi, Y. (1994). Simultaneous stereoinversion and isomerization at specific aspartic acid residues in αA-crystallin from human lens. Journal of Biochemistry, 116, 663-669.
Gómez-Guillén, M., Giménez, B., López-Caballero, M. a. & Montero, M. (2011). Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloids, 25, 1813-1827.
Gelatine Manufacturers of Europe, GME. (2017). Standardised methods for the testing of edible gelatine. 4, Avenue Edmond Van Nieuwenhuyse, 1160 Auderghem, Belgium.
George, A., Malone, J. P. & Veis, A. (1999). The secondary structure of type I collagen N-telopeptide as demonstrated by Fourier transform IR spectroscopy and molecular modeling. Proceedings of the Indian Academy of Sciences - Chemical Sciences, 111, 121.
Gildberg, A. (1993). Enzymic processing of marine raw materials. Process Biochemistry, 28, 1-15.
Gillum, R. F., Yorrick, R. & Obisesan, T. O. (2011). Population surveillance of dementia mortality. International Journal of Environmental Research and Public Health, 8, 1244-1257.
Girgih Abraham, T., Udenigwe Chibuike, C. & Aluko Rotimi, E. (2010). In vitro antioxidant properties of hemp seed (Cannabis sativa L.) protein hydrolysate fractions. Journal of the American Oil Chemists' Society, 88, 381-389.
Girgih, A. T., Nwachukwu, I. D., Hasan, F., Fagbemi, T. N., Gill, T. & Aluko, R. E. (2015). Kinetics of the inhibition of renin and angiotensin I-converting enzyme by cod (Gadus morhua) protein hydrolysates and their antihypertensive effects in spontaneously hypertensive rats. Food and Nutrition Research, 59, 29788.
Gomez-Guillen, M. C., Turnay, J., Fernandez-Diaz, M. D., Ulmo, N., Lizarbe, M. A. & Montero, P. (2002). Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloids, 16, 25-34.
Gonzalez-Perez, O., Gutiérrez-Smith, Y., Guzmán-Muñiz, J. & A Moy-López, N. (2011). Intrauterine stress impairs spatial learning in the progeny of Wistar rats. Revista de Investigación Clínica, 63, 279-286.
Hanani, Z. A. N. (2016). Gelatin. Encyclopedia of Food and Health, 191-195. Oxford: Academic Press.
Harnedy, P. A. & Fitzgerald, R. J. (2013). Ch2-Bioactive proteins and peptides from macroalgae, fish, shellfish and marine processing waste. Marine Proteins and Peptides.
Hartmann, R. & Meisel, H. (2007). Food-derived peptides with biological activity: from research to food applications. Current Opinion in Biotechnology, 18, 163-169.
Hsieh, C. H., Wang, T. Y., Hung, C. C., Hsieh, Y. L. & Hsu, K. C. (2016). Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate. Food and Function, 7, 565-573.
Huang, B.-B., Lin, H.-C. & Chang, Y.-W. (2015). Analysis of proteins and potential bioactive peptides from tilapia (Oreochromis spp.) processing co-products using proteomic techniques coupled with BIOPEP database. Journal of Functional Foods, 19, 629-640.
Hung, M.-Y., Fu, T. Y.-C., Shih, P.-H., Lee, C.-P. & Yen, G.-C. (2006). Du-Zhong (Eucommia ulmoides Oliv.) leaves inhibits CCl4-induced hepatic damage in rats. Food and Chemical Toxicology, 44, 1424-1431.
Iwaniak, A., Minkiewicz, P., Darewicz, M., Sieniawski, K. & Starowicz, P. (2016). BIOPEP database of sensory peptides and amino acids. Food Research International, 85, 155-161.
Jang, A., Jo, C., Kang, K.-S. & Lee, M. (2008). Antimicrobial and human cancer cell cytotoxic effect of synthetic angiotensin-converting enzyme (ACE) inhibitory peptides. Food Chemistry, 107, 327-336.
Je, J.-Y., Park, P.-J., Kwon, J. Y. & Kim, S.-K. (2004). A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. Journal of Agricultural and Food Chemistry, 52, 7842-7845.
Jean-Yves, E., Dominique, L. G., Qin, L. & Robert, G. (1991). Short chain collagens in sponges are encoded by a family of closely related genes. Journal of Biological Chemistry, 266, 21923-21928.
Jia, J.-H., Zhao, Q.-H., Liu, Y., Gui, Y.-Z., Liu, G.-Y., Zhu, D.-Y., Yu, C. & Hong, Z. (2013). Phase I study on the pharmacokinetics and tolerance of ZT-1, a prodrug of huperzine A, for the treatment of Alzheimer's disease. Acta Pharmacologica Sinica, 34, 976-982.
Jin, J.-E., Ahn, C.-B. & Je, J.-Y. (2018). Purification and characterization of antioxidant peptides from enzymatically hydrolyzed ark shell (Scapharca subcrenata). Process Biochemistry, 72, 170-176.
Johnson, K. A., Rogers, G. J., Roe, S. C., Howlett, C. R., Clayton, M. K., Milthorpe, B. K. & Schindhelm, K. (1999). Nitrous acid pretreatment of tendon xenografts cross-linked with glutaraldehyde and sterilized with gamma irradiation. Biomaterials, 20, 1003-1015.
Kadler, K. E., Baldock, C., Bella, J. & Boot-Handford, R. P. (2007). Collagens at a glance. Journal of Cell Science, 120, 1955-1958.
Karel, M., Tannenbaum, S. R., Wallace, D. H. & Maloney, H. (1966). Autoxidation of methyl linoleate in freeze-dried model systems. III. Effects of added amino acids. Journal of Food Science, 31, 892-896.
Kim, S.-K., Byun, H.-G., Park, P.-J. & Shahidi, F. (2001). Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 49, 2992-2997.
Kou, X., Gao, J., Xue, Z., Zhang, Z., Wang, H. & Wang, X. (2013). Purification and identification of antioxidant peptides from chickpea (Cicer arietinum L.) albumin hydrolysates. LWT - Food Science and Technology, 50, 591-598.
Kucharz, E. J. (1992). Structure, heterogeneity, and distribution. The Collagens: Biochemistry and Pathophysiology, 5-29. Berlin, Heidelberg: Springer Berlin Heidelberg.
Labandeira-Garcia, J. L., Rodríguez-Perez, A. I., Garrido-Gil, P., Rodriguez-Pallares, J., Lanciego, J. L. & Guerra, M. J. (2017). Brain renin-angiotensin system and microglial polarization: Implications for aging and neurodegeneration. Frontiers in Aging Neuroscience, 9, 129-129.
Lafarga, T. & Hayes, M. (2014). Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients. Meat Science, 98, 227-239.
Lafarga, T., O'Connor, P. & Hayes, M. (2015). In silico methods to identify meat-derived prolyl endopeptidase inhibitors. Food Chemistry, 175, 337-343.
Lassoued, I., Mora, L., Barkia, A., Aristoy, M. C., Nasri, M. & Toldrá, F. (2015). Bioactive peptides identified in thornback ray skin's gelatin hydrolysates by proteases from Bacillus subtilis and Bacillus amyloliquefaciens. Journal of Proteomics, 128, 8-17.
Li, B., Chen, F., Wang, X., Ji, B. & Wu, Y. (2007). Isolation and identification of antioxidative peptides from porcine collagen hydrolysate by consecutive chromatography and electrospray ionization–mass spectrometry. Food Chemistry, 102, 1135-1143.
Li, J., Liu, Z., Zhao, Y., Zhu, X., Yu, R., Dong, S. & Wu, H. (2018). Novel natural angiotensin converting enzyme (ACE)-Inhibitory peptides derived from sea cucumber-modified hydrolysates by adding exogenous proline and a study of their structure-activity relationship. Marine Drugs, 16, 271-283.
Lin, H.-C., Alashi, A. M., Aluko, R. E., Sun Pan, B. & Chang, Y.-W. (2017). Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates. Food and Nutrition Research, 61, 1391666-1391676.
Liu, Y.-T., Cheng, F.-Y., Takeda, S., Lai, K.-M., Lin, L.-C. & Sakata, R. (2019). Effects of porcine brain hydrolysate on impairment of cognitive learning ability in amyloid β(1–40)-infused rats. Animal Science Journal, 90, 271-279.
Lu, J., Wu, D.-M., Zheng, Y.-L., Hu, B., Zhang, Z.-F., Ye, Q., Liu, C.-m., Shan, Q. & Wang, Y.-J. (2010). Ursolic Acid Attenuates D-Galactose-Induced Inflammatory Response in Mouse Prefrontal Cortex through Inhibiting AGEs/RAGE/NF-κB Pathway Activation. Cerebral Cortex, 20, 2540-2548.
Lunow, D., Kaiser, S., Ruckriemen, J., Pohl, C. & Henle, T. (2015). Tryptophan-containing dipeptides are C-domain selective inhibitors of angiotensin converting enzyme. Food Chemistry, 166, 596-602.
Müller, W. E. G. (2003). The origin of metazoan complexity: Porifera as integrated animals. Integrative and Comparative Biology, 43, 3-10.
Maes, M., Goossens, F., Scharpé, S., Calabrese, J., Desnyder, R. & Meltzer, H. Y. (1995). Alterations in plasma prolyl endopeptidase activity in depression, mania, and schizophrenia: Effects of atidepressants, mood stabilizers, and antipsychotic drugs. Psychiatry Research, 58, 217-225.
Malomo, S. A. & Aluko, R. E. (2016). In vitro acetylcholinesterase-inhibitory properties of enzymatic hemp seed protein hydrolysates. Journal of the American Oil Chemists' Society, 93, 411-420.
Marcuse, R. (1960). Antioxidative effect of amino-acids. Nature, 186, 886-887.
Matmaroh, K., Benjakul, S., Prodpran, T., Encarnacion, A. B. & Kishimura, H. (2011). Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus). Food Chemistry, 129, 1179-1186.
Meinlschmidt, P., Sussmann, D., Schweiggert-Weisz, U. & Eisner, P. (2015). Enzymatic treatment of soy protein isolates: effects on the potential allergenicity, technofunctionality, and sensory properties. Food Science and Nutrition, 4, 11-23.
Mendis, E., Rajapakse, N. & Kim, S.-K. (2005). Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 53, 581-587.
Min, L.-J., Kobayashi, Y., Mogi, M., Tsukuda, K., Yamada, A., Yamauchi, K., Abe, F., Iwanami, J., Xiao, J.-Z. & Horiuchi, M. (2017). Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice. PLOS ONE, 12, 0171515-0171528.
Mogi, M., Iwanami, J. & Horiuchi, M. (2012). Roles of brain angiotensin II in cognitive function and dementia. International Journal Hypertension, 2012, 169649.
Morris, R. G. M., Garrud, P., Rawlins, J. N. P. & O'Keefe, J. (1982). Place navigation impaired in rats with hippocampal lesions. Nature, 297, 681-683.
MRC Statistic. (2016). Gelatin - Global Market Outlook (2016–2022). https://www.marketresearch.com/Stratistics-Market-Research-Consulting-v4058/Gelatin-Global-Outlook-10601257/ Access 05.15 2017.
Mueller, A. S., Bosse, A. C., Most, E., Klomann, S. D., Schneider, S. & Pallauf, J. (2009). Regulation of the insulin antagonistic protein tyrosine phosphatase 1B by dietary Se studied in growing rats. The Journal of Nutritional Biochemistry, 20, 235-247.
Neves, A. C., Harnedy, P. A., O'Keeffe, M. B., Alashi, M. A., Aluko, R. E. & FitzGerald, R. J. (2017). Peptide identification in a salmon gelatin hydrolysate with antihypertensive, dipeptidyl peptidase IV inhibitory and antioxidant activities. Food Research International, 100, 112-120.
Ngo, D.-H., Vo, T.-S., Ryu, B. & Kim, S.-K. (2016). Angiotensin- I- converting enzyme (ACE) inhibitory peptides from Pacific cod skin gelatin using ultrafiltration membranes. Process Biochemistry, 51, 1622-1628.
O'Keeffe, M. B., Norris, R., Alashi, M. A., Aluko, R. E. & FitzGerald, R. J. (2017). Peptide identification in a porcine gelatin prolyl endoproteinase hydrolysate with angiotensin converting enzyme (ACE) inhibitory and hypotensive activity. Journal of Functional Foods, 34, 77-88.
Ohashi, Y., Onuma, R., Naganuma, T., Ogawa, T., Naude, R., Nokihara, K. & Muramoto, K. (2015). Antioxidant properties of tripeptides revealed by a comparison of six different assays. Food Science and Technology Research, 21, 695-704.
Paglia, D. E. & Valentine, W. N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical Medicine, 70, 158-169.
Pal, G. K. & Suresh, P. V. (2016a). Microbial collagenases: challenges and prospects in production and potential applications in food and nutrition. Royal Socience of Chemistry Advances, 6, 33763-33780.
Pal, G. K. & Suresh, P. V. (2016b). Sustainable valorisation of seafood by-products: Recovery of collagen and development of collagen-based novel functional food ingredients. Innovative Food Science and Emerging Technologies, 37, 201-215.
Paxinos, G. & Franklin, K. B. J. (2004). The mouse brain in stereotaxic coordinates. Elsevier Academic Press.
Phillips, M. I. & de Oliveira, E. M. (2008). Brain renin angiotensin in disease. Journal of Molecular Medicine, 86, 715-722.
Pinto, T., Lanctôt, K. L. & Herrmann, N. (2011). Revisiting the cholinergic hypothesis of behavioral and psychological symptoms in dementia of the Alzheimer's type. Ageing Research Reviews, 10, 404-412.
Poli, G., Leonarduzzi, G., Biasi, F. & Chiarpotto, E. (2004). Oxidative stress and cell signalling. Current Medicinal Chemistry, 11, 1163-1182.
Power, A. E., Vazdarjanova, A. & McGaugh, J. L. (2003). Muscarinic cholinergic influences in memory consolidation. Neurobiology of Learning and Memory, 80, 178-193.
Prasasty, V., Radifar, M. & Istyastono, E. (2018). Natural peptides in drug discovery targeting acetylcholinesterase. Molecules, 23.
Prince, M. J., Wimo, A., Guerchet, M. M., Ali, G. C., Wu, Y.-T. & Prina, M. (2015). World Alzheimer Report 2015 - The Global Impact of Dementia. Alzheimer's Disease International.
Provansal, M. M. P., Cuq, J. L. A. & Cheftel, J. C. (1975). Chemical and Nutritional Modifications of Sunflower Proteins Due to Alkaline Processing. Formation of Amino Acid Crosslinks and Isomerization of Lysine Residues. Journal of Agricultural and Food Chemistry, 23, 938-943.
Raghavan, S. & Kristinsson, H. (2009). ACE-inhibitory activity of tilapia protein hydrolysates. Food Chemistry, 117, 582-588.
Rai, A. K., Sanjukta, S. & Jeyaram, K. (2017). Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension. Critical Reviews in Food Science and Nutrition, 57, 2789-2800.
Rajapakse, N., Mendis, E., Jung, W.-K., Je, J.-Y. & Kim, S.-K. (2005). Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Research International, 38, 175-182.
Regenstein, J. M., Chaudry, M. M. & Regenstein, C. E. (2003). The kosher and halal food laws. Comprehensive Reviews in Food Science and Food Safety, 2, 111-127.
Regenstein, J. M. & Zhou, P. (2007). Collagen and gelatin from marine by-products. Maximising the Value of Marine By-Products, 279-303: Woodhead Publishing.
Saiga, A., Iwai, K., Hayakawa, T., Takahata, Y., Kitamura, S., Nishimura, T. & Morimatsu, F. (2008). Angiotensin I-converting enzyme-inhibitory peptides obtained from chicken collagen hydrolysate. Journal of Agricultural and Food Chemistry, 56, 9586-9591.
Saiga, A., Tanabe, S. & Nishimura, T. (2003). Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. Journal of Agricultural and Food Chemistry, 51, 3661-3667.
Samaranayaka, A. G. P. & Li-Chan, E. C. Y. (2008). Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake Merluccius productus. Food Chemistry, 107, 768-776.
Samonina, G., Ashmarin, I. & Lyapina, L. (2002). Glyproline peptide family: Review on bioactivity and possible origins. Pathophysiology, 8, 229-234.
Sanford, A. M. (2018). Lewy body dementia. Clinics in Geriatric Medicine, 34, 603-615.
Selkoe, D. J. (2001). Alzheimer's disease: Genes, proteins, and therapy. Physiological Reviews, 81, 741-766.
Shahidi, F. & Zhong, Y. (2010). Lipid oxidation and improving the oxidative stability. Chemical Society reviews, 39, 4067-4079.
Shimizu, K., Sato, M., Zhang, Y., Kouguchi, T., Takahata, Y., Morimatsu, F. & Shimizu, M. (2010). The bioavailable octapeptide Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro stimulates nitric oxide synthesis in vascular endothelial cells. Journal of Agricultural and Food Chemistry, 58, 6960-6965.
Sink, K. M., Leng, X., Williamson, J., Kritchevsky, S. B., Yaffe, K., Kuller, L., Yasar, S., Atkinson, H., Robbins, M., Psaty, B. & Goff, D. C., Jr. (2009). Angiotensin-converting enzyme inhibitors and cognitive decline in older adults with hypertension: Results from the cardiovascular health study. Archives of Internal Medicine, 169, 1195-1202.
Siswoyo, T. A., Mardiana, E., Lee, K. O. & Hoshokawa, K. (2011). Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds. Journal of Agricultural and Food Chemistry, 59, 5648-5656.
Sobue, G., Ishigaki, S. & Watanabe, H. (2018). Pathogenesis of frontotemporal lobar degeneration: insights from loss of function theory and early involvement of the caudate nucleus. Frontiers in Neuroscience, 12, 473-481.
Song, E., Yeon Kim, S., Chun, T., Byun, H.-J. & Lee, Y. M. (2006). Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials, 27, 2951-2961.
Suetsuna, K., Ukeda, H. & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. Journal of Nutritional Biochemistry, 11, 128-131.
Sugiura, H., Yunoki, S., Kondo, E., Ikoma, T., Tanaka, J. & Yasuda, K. (2009). In vivo biological responses and bioresorption of tilapia scale collagen as a potential biomaterial. Journal of Biomaterials Science, Polymer Edition, 20, 1353-1368.
Suh, H. J., Chung, M. S., Cho, Y. H., Kim, J. W., Kim, D. H., Han, K. W. & Kim, C. J. (2005). Estimated daily intakes of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) antioxidants in Korea. Food Additives and Contaminants, 22, 1176-1188.
Thiansilakul, Y. (2007). Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chemistry, 103, 1385-1394.
Thiansilakul, Y., Benjakul, S. & Shahidi, F. (2007). Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chemistry, 103, 1385-1394.
Toldrá, F., Reig, M., Aristoy, M. C. & Mora, L. (2018). Generation of bioactive peptides during food processing. Food Chemistry, 267, 395-404.
Uluko, H., Zhang, S., Liu, L., Tsakama, M., Lu, J. & Lv, J. (2015). Effects of thermal, microwave, and ultrasound pretreatments on antioxidative capacity of enzymatic milk protein concentrate hydrolysates. Journal of Functional Foods, 18, 1138-1146.
United Nations (2017). World Population Prospects: the 2017 Revision.
van Dijk, M. T. & Fenton, A. A. (2018). On how the dentate gyrus contributes to memory discrimination. Neuron, 98, 832-845.
Vercruysse, L., Van Camp, J. & Smagghe, G. (2005). ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein:  A review. Journal of Agricultural and Food Chemistry, 53, 8106-8115.
Wang, S., Wang, D.-S. & Wang, R. (2008). Neuroprotective activities of enzymatically hydrolyzed peptides from porcine hide gelatin. International Journal of Clinical and Experimental Medicine, 1, 283-293.
Wang, X., Yu, H., Xing, R., Liu, S., Chen, X. & Li, P. (2018). Effect and mechanism of oyster hydrolytic peptides on spatial learning and memory in mice. Royal Society of Chemistry Advances, 8, 6125-6135.
Wani, S. S. & Mashru, R. C. (2014). Sensitive and selective methods for determination of proteolytic activity of formulation containing bromelain and trypsin as proteolytic enzymes. International Journal of Pharmaceutical Sciences and Research, 5, 4838-4845.
Weng, W., Tang, L., Wang, B., Chen, J., Su, W., Osako, K. & Tanaka, M. (2014). Antioxidant properties of fractions isolated from blue shark (Prionace glauca) skin gelatin hydrolysates. Journal of Functional Foods, 11, 342-351.
WHO, World Health Organization. (2019). Dementia. https://www.who.int/news-room/fact-sheets/detail/dementia. Access 07.31.2019
Wood, A., Ogawa, M., Portier, R. J., Schexnayder, M., Shirley, M. & Losso, J. N. (2008). Biochemical properties of Alligator (Alligator mississippiensis) bone collagen. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 151, 246-249.
Woodhead-Galloway, J. (1980). Collagen: the anatomy of a protein The Institute of Biology's Studies in Biology 117, 1-53.
Wright, J. W. & Harding, J. W. (2010). The brain RAS and Alzheimer's disease. Experimental Neurology, 223, 326-333.
Wu, W., Hou, C.-L., Mu, X.-P., Sun, C., Zhu, Y.-C., Wang, M.-J. & Lv, Q.-Z. (2017). H2S donor NaHS changes the production of endogenous H2S and NO in D-galactose-induced accelerated ageing. Oxidative medicine and cellular longevity, 2017, 5707830-5707843.
Xie, Z., Huang, J., Xu, X. & Jin, Z. (2008). Antioxidant Activity of Peptides Isolated from Alfalfa Leaf Protein Hydrolysate. Food Chemistry, 111, 370-376.
Xiong, L., Mao, S., Lu, B., Yang, J., Zhou, F., Hu, Y., Jiang, Y., Shen, C. & Zhao, Y. (2015). Osmanthus fragrans flower extract and acteoside protect against D-galactose-induced aging in an ICR mouse model. Journal of Medicinal Food, 19, 54-61.
Yamamoto, K., Hayashi, M., Murakami, Y., Araki, Y., Otsuka, Y., Kashiwagi, T., Shimamura, T. & Ukeda, H. (2016). Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract. Bioscience, Biotechnology, and Biochemistry, 80, 172-177.
Yamashoji, S., Yoshida, H. & Kajimoto, G. (1979). Effects of amino acids on the photosensitized oxidation of linoleic acid in an aqueous ethanol solution. Agricultural and Biological Chemistry, 43, 1505-1511.
Yanar, K., Aydın, S., Çakatay, U., Mengi, M., Buyukpınarbaşılı, N., Atukeren, P., Sitar, M. E., Sönmez, A. & Uslu, E. (2011). Protein and DNA oxidation in different anatomic regions of rat brain in a mimetic ageing model. Basic & Clinical Pharmacology & Toxicology, 109, 423-433.
Yang, K.-T., Lin, C., Liu, C.-W. & Chen, Y.-C. (2014). Effects of chicken-liver hydrolysates on lipid metabolism in a high-fat diet. Food Chemistry, 160, 148-156.
Yang, X.-R., Zhao, Y.-Q., Qiu, Y.-T., Chi, C.-F. & Wang, B. (2019). Preparation and characterization of gelatin and antioxidant peptides from gelatin hydrolysate of skipjack tuna (Katsuwonus pelamis) bone stimulated by in vitro gastrointestinal digestion. Marine Drugs, 17, 78-95.
Yang, X. R., Qiu, Y. T., Zhao, Y. Q., Chi, C. F. & Wang, B. (2019). Purification and characterization of antioxidant peptides derived from protein hydrolysate of the marine bivalve mollusk Tergillarca granosa. Marine Drugs, 17.
Yoshikawa, M., Fujita, H., Matoba, N., Takenaka, Y., Yamamoto, T., Yamauchi, R., Tsuruki, H. & Takahata, K. (2000). Bioactive peptides derived from food proteins preventing lifestyle-related diseases. Biofactors, 12, 143-146.
Zhang, L., Li, J. & Zhou, K. (2010). Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation. Bioresource Technology, 101, 2084-2089.
Zhang, Y., Olsen, K., Grossi, A. & Otte, J. (2013). Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides. Food Chemistry, 141, 2343-2354.
Zhao, H., Li, J., Zhao, J., Chen, Y., Ren, C. & Chen, Y. (2018). Antioxidant effects of compound walnut oil capsule in mice aging model induced by D-galactose. Food and Nutrition Research, 62, 1371-1380.
Zhen, Y.-Z., Lin, Y.-J., Li, K.-J., Zhang, G.-L., Zhao, Y.-F., Wang, M.-M., Wei, J.-B., Wei, J. & Hu, G. (2016). Effects of rhein lysinate on D-galactose-induced aging mice. Experimental and Therapeutic Medicine, 11, 303-308.
Zheng, L., Yu, H., Wei, H., Xing, Q., Zou, Y., Zhou, Y. & Peng, J. (2018). Antioxidative peptides of hydrolysate prepared from fish skin gelatin using ginger protease activate antioxidant response element-mediated gene transcription in IPEC-J2 cells. Journal of Functional Foods, 51, 104-112.
Zhou, K., Sun, S. & Canning, C. (2012). Production and functional characterisation of antioxidative hydrolysates from corn protein via enzymatic hydrolysis and ultrafiltration. Food Chemistry, 135, 1192-1197.
Zhou, Y., Dong, Y., Xu, Q., He, Y., Tian, S., Zhu, S., Zhu, Y. & Dong, X. (2013). Mussel oligopeptides ameliorate cognition deficit and attenuate brain senescence in D-galactose-induced aging mice. Food and Chemical Toxicology, 59, 412-420.
Zou, Y., Wang, W., Li, Q., Chen, Y., Zheng, D., Zou, Y., Zhang, M., Zhao, T., Mao, G., Feng, W., Wu, X. & Yang, L. (2016). Physicochemical, functional properties and antioxidant activities of porcine cerebral hydrolysate peptides produced by ultrasound processing. Process Biochemistry, 51, 431-443.