Method for screening oyster bioactive peptides with effect of improving memory in vitro
A technology for in vitro screening and screening methods, applied in compound screening, biochemical equipment and methods, detection of programmed cell death, etc., to achieve the effect of improving memory
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Embodiment 1
[0027] Preparation of oyster active peptide:
[0028] Take 10 parts of fresh oysters, 10g each, homogenate, add deionized water at a liquid-solid ratio of 1-3:1, stir evenly in a water bath at 45°C, and add in according to the mass ratio of oysters to composite protease 1000:8-16 Compound protease (1.0×10 5 U / g, Suleibao Technology Co., Ltd.), adjust the pH to 7-8, keep it warm for 5 minutes, and then enzymatically hydrolyze it at 45-65°C for 3-7h. Passivate the enzyme until the end of the enzymatic hydrolysis reaction, centrifuge, take the supernatant, and freeze-dry to obtain 10 kinds of oyster active peptides. The samples of oyster active peptides are respectively numbered as oyster active peptides 1-10 according to the different enzymatic hydrolysis conditions. The specific enzymatic hydrolysis conditions of each oyster active peptide sample are shown in Table 1.
[0029] Table 1 Enzymatic hydrolysis conditions of oyster active peptide
[0030]
Embodiment 2
[0032] In vitro screening of oyster active peptides that improve memory:
[0033] Take 10 copies of astrocytes in the logarithmic growth phase (numbered astrocytes 1-10) and inoculate them in 24-well plates at a seeding density of 1×10 5 each / mL, inoculum size 0.5mL, cultured for 24h, after the astrocytes grew to 80% of the orifice plate, the original astrocyte culture solution was sucked away, and No. 1-10 oyster active peptides in Example 1 were added successively (0.5mL, 10mg / mL) acted on astrocytes, sucked away the culture medium again after 24h, and added 0.5ml phosphate buffer (0.01M, pH 7.2) to 10 astrocytes respectively, fully Grind astrocytes, centrifuge at 5000r / min for 10min at 4°C, take 0.4mL of the supernatant, and use the AchE test kit (Nanjing Jiancheng Bioengineering Research Institute, article number: A024, refer to the instruction manual for detection methods) to measure the activity of AchE , to screen out the oyster active peptide with the highest inhibito...
Embodiment 3
[0038] In vitro screening of oyster active peptides that improve memory:
[0039] Take 10 copies of astrocytes in the logarithmic growth phase (numbered astrocytes 1-10) and inoculate them in 24-well plates at a seeding density of 1×10 6 each / mL, inoculum size 0.5mL, cultured for 24h, after the astrocytes grew to 90% of the orifice plate, the original astrocyte culture solution was sucked away, and the oyster active peptide No. 1-10 in Example 1 was added successively (0.5mL, 10mg / mL) acted on astrocytes, and after 24h, the culture medium was sucked away again, and 1.0mL phosphate buffer (0.01M, pH 7.4) was added to 10 astrocytes respectively, fully Grind astrocytes, centrifuge at 8000r / min for 15min at 4°C, take 0.5mL of the supernatant, and use the AchE test kit (Nanjing Jiancheng Bioengineering Research Institute, article number: A024, refer to the instruction manual for detection methods) to measure the AchE activity , to screen out the oyster active peptide with the high...
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