近年來,由食物衍生的生物活性肽被開發(fā)出來并用于治療糖尿病。本研究旨在探討糖巨肽作為天然來源DPP-Ⅳ抑制劑的潛力。為了研究源自糖巨肽的DPP-4抑制肽,首先,收集新鮮的小鼠小腸粘膜分泌物和上皮細(xì)胞以分析DPP-4酶的活性,建立DPP-4抑制劑篩選體系,然后,我們使用糖巨肽（CGMP20）作為原料并用胰蛋白酶和木瓜蛋白酶消化。為了更有效地獲得源自糖巨肽來源的DPP-4抑制肽,進(jìn)行單因素實(shí)驗(yàn)以確定四個(gè)因子的中心點(diǎn)。依次測(cè)定DPP-4抑制活性,然后對(duì)二次非線性回歸擬合進(jìn)行響應(yīng)面分析。回歸模型具有良好的相關(guān)性（R²=0.6876）,測(cè)試的準(zhǔn)確度為13.477。根據(jù)該模型預(yù)測(cè)的最佳條件是組合溫度:溫度（40℃）,時(shí)間（1h）,加酶量（4500U/g pro）,蛋白質(zhì)濃度（4%）。通過木瓜蛋白酶酶解并使用半制備液相色譜分離純化水解物。收集到的七個(gè)級(jí)分中的一個(gè)顯示出顯著的DPP-Ⅳ抑制活性。通過HPLC串聯(lián)質(zhì)譜（HPLC-MS/MS）分析這些級(jí)分。最高活性的片段序列是NQDKTEIPT f（130-140）和NQDKTEIPTIN f（140-150）,其IC50值為563.8... 

【文章來源】：中國(guó)農(nóng)業(yè)科學(xué)院北京市

【文章頁數(shù)】：82 頁

【學(xué)位級(jí)別】：博士

【文章目錄】：
摘要
ABSTRACT
Abbreviation
CHAPTER 1 Background and objectives
    1.1 General introduction
    1.2 Milk Proteins
        1.2.1 Overview of bioactive peptides
        1.2.2 Milk-derived bioactive peptides
        1.2.3 Enzymatic hydrolysis by digestive enzymes
        1.2.4 Microbial fermentation
    1.3 Bioactivities from peptides and health significance
        1.3.1 Antidiabetic functionalities of milk protein-derived bioactive peptides
        1.3.2 Effect of milk protein on insulin secretion
        1.3.3 Antidiabetic applications of bioactive peptides
        1.3.4 In vitro antidiabetic effect of peptides
    1.4 Objectives of the study
        1.4.1 The Specific Objectives of this Research are:
CHAPTER 2 Effects of microwave and ultrasound pretreatments on preparation of Glycomacropeptide withtrypsin as a source DPP-IV inhibitory peptide
    2.1 Introduction
    2.2 Materials and methods
        2.2.1 Materials and reagents
        2.2.2 Instruments equipments
        2.2.3 Determination of protein content
        2.2.4 Protease activity assay
        2.2.5 Trichloroacetic acid precipitation -Determination of short peptide content by forinolmethod
        2.2.6 Determination of the degree of hydrolysis of enzymatically digested samples by the o-Phthalaldehyde method (OPA method)
        2.2.7 Microwave and ultrasonic pretreatments
        2.2.8 Enzyme hydrolysis and degree of hydrolysis
        2.2.9 Assay of the DPP-IV-Inhibitory Activity
        2.2.10 Sttistical analysis
    2.3 Results and discussions
        2.3.1 Protein content and enzyme activity
        2.3.2 Effects of Microwaves and Ultrasound on degree of hydrolysis
        2.3.3 Effect on Enzymatic hydrolysis of GMP with different temperature, time, proteinconcentration and enzyme concentration
        2.3.4 Effects of substrate concentration on DPP-V inhibitory activity
        2.3.5 Effects of enzyme concentration, hydrolysis temperature and hydrolysis time on DPP-4inhibitory activity
    2.4 Conclusion
CHAPTER 3 Response surface optimization of dipeptidyal peptidase (DPP-IV) Inhibition ofglycomacropeptides hydrolysates
    3.1 Introduction
    3.2 Materials and methods
        3.2.1 Materials
        3.2.2 Experimental design of glycomacropeptide Concentrate Hydrolysate by CentralComposite (CCD)
        3.2.3 RSM and Generation of the Optimum GMP Hydrolysate.
        3.2.4 Enzyme hydrolysis and degree of hydrolysis
        3.2.5 Determination of DPP-IV Inhibitory Activity
        3.2.6 Statistical Analysis
    3.3 Results and discussion
        3.3.1 Hydrolysates Generated Within the Experimental Design
        3.3.2 The Effects of different Factors affecting on the DPP-IV Inhibitory Activity of TheHydrolysate
        3.3.3 Optimization and validation
    3.4 Conclusion
CHAPTER 4 Dipeptidyl peptidase-IV inhibitory peptides generated from Papain-Treated hydrolysis of aglycomacropeptide (cGMP20) protein
    4.1 Introduction
    4.2 Materials and methods
        4.2.1 Materials
        4.2.2 Preparation of the glycomacropeptide (GMP) hydrolysate
        4.2.3 Separation and Purification of DPP-IV Inhibitory Peptides
        4.2.4 Identification of DPP-IV Inhibitory Peptides by RP-HPLC-ESI-MS/MS
        4.2.5 Assay of the DPP-IV-Inhibitory Activity
        4.2.6 Peptide Synthesis
        4.2.7 Peptide-cutter tool predicts enzymatic cleavage sequence
    4.3 Results and discussion
        4.3.1 DPP-4 inhibitory activity of GMP with different molecular weight ranges
        4.3.2 Identification of peptide sequences
        4.3.3 DPP-IV inhibitory activity of synthetic peptides
        4.3.4 Peptide-cutter tool predicts enzymatic cleavage sequence
    4.4 Conclusion
CHAPTER 5 Effect of Glycomecropeptide (cGMP20) peptides on DPP-IV and GLP-1 in H716 cells
    5.1 Introduction
    5.2 Materials and methods
        5.2.1 Materials and reagents
        5.2.2 Instruments and equipments
        5.2.3 Test design
        5.2.4 cell culture
        5.2.5 Deermination the number of cells
        5.2.6 Cytotoxicity experiment
        5.2.7 Treatment of different concentrations of glycomacropeptide peptides NCI-H716 cells
        5.2.8 Extraction of DPP-4 and GLP-1 in cells
        5.2.9 Determination of intracellular DPP-4 activity
        5.2.10 Determination of GLP-1 secretion
        5.2.11 Statistical analysis
    5.3 Results and discussion
        5.3.1 DPP-4 half inhibition rate of linagliptin and active peptide
        5.3.2 Cell proliferation assay to determine optimal culture concentration
        5.3.3 Cytotoxicity test to determine the optimum culture concentration of the sample to betested
        5.3.4 Effect of sample to be tested on intracellular DPP-IV secretion
        5.3.5 Effect of sample to be tested on intracellular GLP-1 secretion
    5.4 Conculusion
CHAPTER 6 Overall conclusions
    6.1 Innovation points
    6.2 Outlook
References
致謝 (Acknowledgement)
作者簡(jiǎn)歷 (Resume)



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