Preparation, Identification and Application of β-Lactoglobulin Hydrolysates with Oral Immune Tolerance
Abstract
:1. Introduction
2. Materials and Methods
2.1. Prediction of T Cell Epitopes of β-LG
2.2. Preparation of β-LG Hydrolysates
2.3. Determination of the Degree of Hydrolysis (DH) and Peptide Content
2.4. Analysis of Amino Acid Sequences and T Cell Epitopes of Hydrolysate Peptide by Mass Spectrometry and T Cell Proliferation Assay
2.5. Oral Tolerance of β-LG Hydrolysate Identified by Animal Experiment
2.5.1. Animal Experiment Scheme
2.5.2. Determination of Specific IgE, IgG1 and IgG2a
2.5.3. Determination of TH1 and Th2 Cytokines
2.5.4. Determination of Histamine
2.5.5. Determination of Chitinase-3-like Protein 1 Content
2.5.6. Determination of Cell Subsets
2.5.7. Preparation and Identification of Whey Powder with Oral Immune Tolerance
2.5.8. Statistical Analysis of Data
3. Results and Analysis
3.1. Prediction of T Cell Epitopes of β-LG
3.2. The DH and Peptide Content of β-LG Hydrolysate
3.3. Analysis of Amino Acid Sequences and T Cell Epitopes of Hydrolysate Peptide by Mass Spectrometry and T Cell Proliferation Assay
3.4. Clinical Symptoms of Mice in Each Group
3.5. Determination of Optical Density (OD) of Specific Antibodies IgE, IgG1 and IgG2a of β-LG
3.6. Determination of Th1-Related Cytokines
3.7. Determination of TH2 Cytokines
3.8. Determination of Histamine Release
3.9. Determination of Chitinase-3-like Protein 1
3.10. Determination Results of Spleen Cell Subsets
3.10.1. Differentiation of TH1 Cell Subsets
3.10.2. Differentiation of TH2 Cell Subsets
3.10.3. Differentiation of Treg Cell Subsets
3.11. Identification of Trypsin Hydrolysate with Oral Immune Tolerance
3.11.1. Observation Results of Intestinal Histopathology
3.11.2. Apoptosis Results of Intestinal Villi Epithelial Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Name of Protease | Determination of Amino Acid Sequences of Peptides by Mass Spectrometry | Partial or Total Repeats with Predicting T Cell Epitopes | Potential T Cell Epitopes and the Sequence Location of β-Lactoglobulin | Stimulation Index by T Cell Proliferation Assay (SI) |
---|---|---|---|---|
Trypsin | ALKALPMH ALPMH IDALNENK IIAEKTKIPAVFK TKIPAVFK TKIPAVFKIDALNENK TPEVDDEALEK TPEVDDEALEKFDK TPEVDDEALEKFDKALK VAGTWYSLAMAASDISLLDAQSAPLR VLVLDTDYK VLVLDTDYKK VYVEELKPTPEGDLEILLQK | LKALPMH | ALKALPMH (AA155~162) | 2.63 ± 0.07 |
ALPMH | ALPMH (AA158~162) | 2.12 ± 0.02 | ||
IDALNENK | IDALNENK (AA100~107) | 2.27 ± 0.03 | ||
KTKIPAVFK | IIAEKTKIPAVFK (AA87~99) | 2.53 ± 0.01 | ||
TKIPAVFK | TKIPAVFK (AA92~99) | 2.41 ± 0.02 | ||
IPAVFKIDALNENK | TKIPAVFKIDALNENK (AA92~107) | 3.12 ± 0.03 | ||
VAGTWYSLAMAAS-DIS | VAGTWYSLAMAASDISLLDAQSAPLR (AA31~56) | 2.64 ± 0.02 | ||
DTDYK | VLVLDTDYK (AA108~116) | 2.12 ± 0.01 | ||
DTDYKK | VLVLDTDYKK (AA108~117) | 2.04 ± 0.02 | ||
VYVEELKPTPE | VYVEELKPTPEGDLEILLQK (AA57~76) | 2.68 ± 0.02 | ||
Protamex | DEALEKFDKALKALPMH DISLLDAQSAPLR DISSLLDAQSAPLRVY KVAGTWYS LDIQKVAGTWYS LLDAQSAPLR LIVTQTMKGLDIQKVAGTWYS LVRTPEVDDEALEKFDKALKALPMH | KALKALPMH | DEALEKFDKALKALPMH (AA146~162) | 2.96 ± 0.03 |
KVAGTWYS | LDIQKVAGTWYS (AA26~37) | 2.31 ± 0.02 | ||
LIVTQTMKGLDIQ | LIVTQTMKGLDIQKVAGTWYS (AA17~37) | 2.59 ± 0.03 | ||
PMH | LVRTPEVDDEALEKFDKALKALPMH (AA138~162) | 2.11 ± 0.02 | ||
Papain | LDAQSAPLR NENKVLVL | NENKV | NENKVLVL (AA104~111) | 2.05 ± 0.02 |
Neutrase | DTDYKKYL KPTPEGDLEILL LDAQSAPLRV LDTDYKKYL VLDTDYKKYL VRTPEVDDEAL | DTDYKKYL | DTDYKKYL (AA112~119) | 2.12 ± 0.01 |
KPTPEGD | KPTPEGDLEILL (AA63~74) | 2.34 ± 0.02 | ||
LDTDYKKYL | LDTDYKKYL (AA111~119) | 2.65 ± 0.02 | ||
VLDTDYKKYL | VLDTDYKKYL (AA110~119) | 2.56 ± 0.01 |
Score | Symptoms | NC Group (n = 10) | PC Group (n = 10) | TH Group (n = 10) | PM Group (n = 10) | PH Group (n = 10) | NPH Group (n = 10) |
---|---|---|---|---|---|---|---|
0 | No symptoms | 10 | 0 | 9 | 8 | 8 | 0 |
1 | Scratching nose and mouth | 0 | 0 | 1 | 2 | 2 | 0 |
2 | Edema around eyes and mouth; decreased activity; increased respiratory rate | 0 | 0 | 0 | 0 | 0 | 2 |
3 | Tachypnea; rashes surround mouth and tail; increased respiratory rate | 0 | 6 | 0 | 0 | 0 | 6 |
4 | No activity after stimulation with trembling and muscle contraction | 0 | 3 | 0 | 0 | 0 | 2 |
5 | Shock and death | 0 | 1 | 0 | 0 | 0 | 0 |
Scoring | 0 | 35.00 ± 0.56 a | 1.00 ± 0.05 b | 2.00 ± 0.08 b | 2.00 ± 0.05 b | 30.00 ± 0.12 a |
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Tian, L.; Zhang, Q.; Cong, Y.; Yan, W. Preparation, Identification and Application of β-Lactoglobulin Hydrolysates with Oral Immune Tolerance. Foods 2023, 12, 307. https://doi.org/10.3390/foods12020307
Tian L, Zhang Q, Cong Y, Yan W. Preparation, Identification and Application of β-Lactoglobulin Hydrolysates with Oral Immune Tolerance. Foods. 2023; 12(2):307. https://doi.org/10.3390/foods12020307
Chicago/Turabian StyleTian, Linghan, Qianqian Zhang, Yanjun Cong, and Wenjie Yan. 2023. "Preparation, Identification and Application of β-Lactoglobulin Hydrolysates with Oral Immune Tolerance" Foods 12, no. 2: 307. https://doi.org/10.3390/foods12020307