Title Page
ABSTRACT
국문 초록
PREFACE
Contents
NOMENCLATURE 19
BACKGROUND 23
CHAPTER 1. Preparation of hypoallergenic whey protein hydrolysate by a mixture of Alcalase and Prozyme and evaluation of its digestibility and immunoregulatory properties 33
1.1. Introduction 33
1.2. Materials and Methods 36
1.2.1. Materials 36
1.2.2. Preparation of Whey Protein Hydrolysate 37
1.2.3. Identification of βLG and αLA in Whey Protein Hydrolysate using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 39
1.2.4. Amino-Nitrogen (A-N) Contents and Degree of Hydrolysis (DH) Calculation 40
1.2.5. Establishment of Optimal Mixing Ratio of Alcalase and Prozyme and of Optimal Reaction Conditions using RSM 41
1.2.6. Amino Acid Composition by High-Performance Liquid Chromatography (HPLC) Analysis 46
1.2.7. In vitro Digestibility and Intestinal Permeability Assay 47
1.2.8. Evaluation of Absorption Rate by High-Dose Single Oral Administration 49
1.2.9. Molecular Weight Distribution of Hydrolysates 50
1.2.10. Evaluation of Immunological Responses of Hypoallergenic Hydrolysates 51
1.2.11. Statistical Analysis 52
1.3. Results 53
1.3.1. Selection of Enzyme Combinations for the Preparation of HWPH 53
1.3.2. Mixing Ratio of Alcalase and Prozyme for the Preparation of HWPH 56
1.3.3. Optimization of Enzyme Amount and Hydrolysis Time for the Preparation of HWPH by Alcalase-Prozyme Mixture 58
1.3.4. Amino Acids Composition of HWPH 61
1.3.5. Digestibility and Intestinal Permeability by Caco-2 Cell 63
1.3.6. Evaluation of Absorption Rate of WP and HWPH in Sprague-Dawley (SD) Rats 66
1.3.7. Molecular Weight Distribution of HWPH 68
1.3.8. Effect of HWPH Administration on Cellular Immunity of Mice 70
1.4. Discussion 76
CHAPTER 2. Comparison of nutritional composition and physicochemical characteristics of a standard, partially hydrolyzed, and extensively hydrolyzed formulas 80
2.1. Introduction 80
2.2. Materials and Methods 82
2.2.1. Preparation of Infant Formulas and Macronutrients Contents 82
2.2.2. Amino Acid Composition by High-Performance Liquid Chromatography (HPLC) Analysis 84
2.2.3. Identification of βLG and αLA in Infant Formulas using Gel Electrophoresis 85
2.2.4. In vitro Digestibility Assay 86
2.2.5. Evaluation of Absorption Rate by High-Dose Single Oral Administration 87
2.2.6. Physical Characteristic of Powder Analysis 88
2.2.7. Statistical Analysis 95
2.3. Results 96
2.3.1. Macronutrients Contents of Infant Formulas 96
2.3.2. Amino Acid Composition of Infant Formulas 99
2.3.3. Identification of βLG and αLA in Infant Formulas 103
2.3.4. In vitro Digestibility 105
2.3.5. Evaluation of Absorption Rate of Infant Formulas in ICR Mice 108
2.3.6. Comparison of Powder Characteristic 113
2.4. Discussion 124
CHAPTER 3. A partially hydrolyzed whey formula provides adequate nutrition in high-risk infants for allergy 132
3.1. Introduction 132
3.2. Material and Methods 134
3.2.1. Subjects 134
3.2.2. Materials 135
3.2.3. Study Design and Protocol 137
3.2.4. Ethics 138
3.2.5. Statistical Analysis 139
3.3. Results 140
3.3.1. Baseline Characteristics 140
3.3.2. Formula Intakes and Tolerance 143
3.3.3. Anthropometric Measures 144
3.3.4. Laboratory Parameters and Atopic Dermatitis 149
3.4. Discussion 151
REFERENCES 155
Table 1-1. Characteristics of the various proteases 38
Table 1-2. Combination of experimental runs based on RSM for mixing ratio of enzymes 42
Table 1-3. Combination of experimental runs based on RSM for reaction conditions (enzyme addition amount and hydrolysis time) for mixed enzymes 44
Table 1-4. Composition of amino acids in hypoallergenic whey protein hydrolysates 62
Table 1-5. Changes in cytokines and antibodies in the blood, spleen cells and small intestine after oral administration of WP and HWPH 73
Table 2-1. Specification for Carr's Index (CI) and Hausner Ratio (HR) 90
Table 2-2. Macronutrients contents of infant formulas 98
Table 2-3. Amino acids composition of SF, pHF, and eHF, as well as those of acid hydrolyzed 101
Table 2-4. Glucose and lactose content in the treatment of artificial digestive enzymes 107
Table 2-5. Analysis of protein and A-N content in mice serum over time after oral administration of the sample 110
Table 2-6. Analysis of serum biochemical parameters over time after oral administration of the sample in ICR mice 111
Table 2-7. Loose density (LD), tapped density (TD) and related indices (HR and CI) for SF, pHF, and eHF 116
Table 2-8. DSC temperature of SF, pHF, and eHF 123
Table 2-9. Standard and specification of Infant formulas and Follow-up (on) formulas 127
Table 3-1. Composition of the study formulas 136
Table 3-2. Baseline characteristics according to formula groups 142
Table 3-3. Anthropometric measures at baseline, 12 weeks, and 24 weeks of age 145
Table 3-4. Changes of anthropometric measures from baseline to 24 weeks of age 147
Table 3-5. Laboratory parameters at 24 weeks of age 150
Figure 1-1. Gel electrophoresis of whey protein hydrolysis for the analysis of α-lactalbumin and β-lactoglobulin.... 54
Figure 1-2. Amino-nitrogen (A-N) (A) and degree of hydrolysis (DH) (B) of whey protein hydrolysate by mixing endo- and exo-types protease.... 55
Figure 1-3. A-N content (A, C) and DH (B, D) of whey protein hydrolysate optimized using response surface methodology (RSM).... 57
Figure 1-4. Bands of α-lactalbumin (αLA) and β-lactoglobulin (βLG) as seen in gel electrophoresis according to hydrolysis time of whey protein (WP).... 60
Figure 1-5. Changes in (A) A-N content, (B) digestibility, and (C-F) intestinal permeability according to time in Caco-2 cells exposed to WP and hypoallergenic whey... 64
Figure 1-6. A-N changes in serum of Sprague-Dawley (SD) rats following oral administration of WP and HWPH before and after hydrolysis of WP with mixed enzymes.... 67
Figure 1-7. Molecular weight distribution of WP (A, B, C) and HWPH (D, E, F). Absorbance was measured at 220 nm while eluting with 50 mM ammonium formate buffer (pH 5.5).... 69
Figure 1-8. Changes in immunoglobulin (Ig) E in the blood (A), spleen cells (B), and small intestine (C) in mice after oral administration of WP and HWPH.... 75
Figure 2-1. Band of αLA and βLG of infant formulas in gel electrophoresis. αLA, α-lactalbumin; βLG, β-lactoglobulin; eHF, extensively hydrolyzed formula; M,... 104
Figure 2-2. Total protein and amino-nitrogen contents of in vitro gastrointestinal digested SF, pHF, and eHF.... 106
Figure 2-3. Distribution of particle size of the (A) SF, (B) pHF, and (C) eHF. eHF, extensively hydrolyzed formula; pHF, partially hydrolyzed formula; SF, standard formula. 114
Figure 2-4. Wettability and dispersibility index of SF, pHF, and eHF. eHF, extensively hydrolyzed formula; pHF, partially hydrolyzed formula; SF, standard formula. 118
Figure 2-5. FT-IR graph of (A) SF, (B) pHF, and (C) eHF. eHF, extensively hydrolyzed formula; pHF, partially hydrolyzed formula; SF, standard formula. 120
Figure 2-6. DSC curves of (A) SF, (B) pHF, and (C) eHF. eHF, extensively hydrolyzed formula; pHF, partially hydrolyzed formula; SF, standard formula. 122
Figure 3-1. Flow diagram of the study participants. pHF, partially hydrolyzed formula; SF, standard cow's milk formula. 141