Small molecular milk casein polypeptide and preparing method
A technology of casein and small molecules, which is applied in the field of casein peptides, small molecule casein peptides and their preparation, which can solve the problems of affecting the taste, losing the nutrition and efficacy of the original protein, etc.
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Embodiment 1
[0011] Mix 1L of milk with 50ml of papain, centrifuge at 2000rpm for 5 minutes to obtain a coagulated precipitate; add the coagulated precipitate to water, boil, centrifuge at 2000rpm for 5 minutes, and take the supernatant; add the supernatant to an acidic buffer of pH 4-5 solution, stirred, and centrifuged at 10,000rpm for 30 minutes to obtain a precipitate; the precipitate was dissolved in a pH4.5-5.0 buffer, and the SephadexG25, pH7.2 PBS buffer was used as the mobile phase to separate a molecular weight of 2-3KDa The polypeptide is obtained. The sequence of the polypeptide determined by a solid-phase protein automatic sequencer is VIMYQILPQNFLPILQPAVBVPFLQ, and the purity is 82%.
Embodiment 2
[0013] Mix 1L of milk with 100ml Albizia juniper protease, centrifuge at 3000rpm for 10 minutes to get coagulated precipitate; add the coagulated precipitate to water, boil, centrifuge at 3000rpm for 10 minutes, take the supernatant; add the supernatant to the acidic buffer of pH 4-5 solution, stirred, and centrifuged at 15000rpm for 60 minutes to obtain a precipitate; the precipitate was dissolved in a pH4.5-5.0 buffer, and the SephadexG25, pH7.2 PBS buffer was used as the mobile phase to separate the molecular weight of 2-3KDa The polypeptide is obtained. The sequence of the polypeptide determined by a solid-phase protein automatic sequencer is VIMYQILPQNFLPILQPAVBVPFLQ, and the purity is 78%.
Embodiment 3
[0015] Immunogenicity test: 6 BALB / c white mice, half male and half male, were orally administered with 36 mg / kg of the polypeptide prepared in Example 1, respectively. The drug was administered continuously for 8 weeks, and blood was collected from the orbital venous plexus once a week from 1 to 12 weeks, centrifuged at 12,000 rpm for 2 minutes, the plasma was separated to obtain the supernatant, and stored at -20°C for later use. After dissolving at room temperature, take 0.1 ml of the supernatant to detect the antibody titer in the serum by indirect ELISA, wherein the ELISA detection kit was purchased from Shanghai Sangong. The RA value is obtained by comparing the actual A value (OD450) of each sample with the serum A value of the negative control. RA ≥ 2.1 is positive. Bloodbio-samples are tested after a series of dilutions, and the highest dilution with a positive reaction is the The titer of the sample. The measurement result is displayed. The blood samples collected ...
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