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Hydrolysis Character and Immunological Activity of Bovine Milk by Protease from Pseudomonas Fluorescens

Author: SunZuo
Tutor: QiuChengGuang；MuZhiShen
School: Huazhong Agricultural University
Course: Of Food Science
Keywords: The Pseudomonas fluorescens Protease (PFP) Milk protein Enzymatic Characteristics Hydrolysis Immune activity
CLC: S879.1
Type: Master's thesis
Year: 2008
Downloads: 23
Quote: 0
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Abstract


Preliminary exploration Pseudomonas fluorescens protease (Pseudomonas Fluorescens Protease, PFP) the hydrolyzed milk characteristics as well as the immune activity of the hydrolyzate, the first on the PFP hydrolysis characteristics of milk protein pilot study to determine the optimal conditions of hydrolysis , and on this basis, further study of milk PFP hydrolyzate impact on immune function. Of this study include the following three parts: a preliminary study of the 1.PFP enzymatic properties: PFP content, activity, optimum temperature and pH value of the enzyme. By polyacrylamide gel electrophoresis (sodium dodecyl sulfate of polyacrylamide - gelelectrophoresis by SDS-PAGE) determined after the salting-out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography after protease reached electrophoretically pure; by taking brilliant blue method is the determination of the content of the enzyme; optimum temperature and pH value of the enzyme activity and the enzyme was measured by the azocasein method. The results show that: the obtained PFP electrophoretically pure protease; enzyme content 62.37μg/mL; enzyme activity of 17333 U / mL.; The optimum temperature of the enzyme was 40 ° C, the optimum pH value of 7.5. 2.PFP hydrolyzed milk protein characteristics: PFP of the milk six protein hydrolysis characteristics of experimental research to determine the optimum conditions for the formation of immune active peptides. PFP hydrolysis formed in the milk of six protein hydrolyzate by SDS-PAGE and reversed-phase high performance liquid chromatography (Reverse phase-highperformance liquid chromatograph, RP-HPLC) assay, the hydrolysis process parameters are optimized by orthogonal experiment. The results showed that: α_s-CN, β-CN, κ-CN in PFP role be hydrolyzed, BSA, α-la and β-lg three kinds of whey protein were not been hydrolyzed. Determination the PFP casein hydrolyzate formed Immunopeptides RP-HPLC method, the maximum absorption peak at 210 nm detection area of ??optimum hydrolysis process parameters: hydrolysis time of 3 h, the hydrolysis pH 6.5, hydrolysis temperature 40 ° C , protease substrate ratio of 1:200 (v / v). Immune activity 3.PFP hydrolysis of skimmed milk powder product: skim milk powder PFP hydrolyzate on immune function test. Tested mice were fed with normal saline, low-dose (100 mg / Kg.bw) and high dose (250 mg / Kg.bw) PFP hydrolysis skim milk powder hydrolyzate formed immune activity indicators measured after 30 d hydrolyzate impact on immune function were studied. The results show that: the high and low dose fed mice had no significant effect on growth; feeding low doses can strengthen the spleen index (P <0.05), while no significant effect on the thymus index;, high dose feeding enhances monocyte-macrophage phagocytosis fed 24 h low dose and high doses fed to 48 h (P <0.05); significantly improve the delayed type hypersensitivity in mice (P <0.05), and low-dose fed for 48 h and high dose feeding 24 PFP hydrolyzate h Mouse Delayed allergic antibody cell generation capability showed significant enhancement (P <0.01); show skimmed milk powder can improve immune function. Can be drawn through the completion of the study of the three-part test, the PFP is a kind of heat a certain tolerance of the protease, the protease may be the hydrolysis of the casein in the milk, but not hydrolyzed whey protein hydrolyzate containing immune active peptide. PFP formation of immune activity of casein hydrolyzate peptide hydrolysis time of 3 h hydrolysis PH 6.5, hydrolysis temperature 40 ° C, the enzyme and substrate concentration ratio of 1:200 (v / v); the PFP of hydrolysis of skim milk powder can promote small mouse immune organs, stimulate phagocytic function, enhanced delayed hypersensitivity reaction, as well as increase the number of antibodies generated, so you can improve immune function. Reasonable use of the PFP, this study provides a new way to provide a viable technology ideas at the same time as the formation of the immunological activity of the peptide, and rich milk the source Immunopeptides the content.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Comprehensive utilization of livestock and poultry products > By - products and Its Utilization > Comprehensive utilization of dairy products
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