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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

Active Publication Date: 2017-05-31
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the in-depth research of modern medicine, it has been found that oysters have a variety of biological activities, such as antibacterial, anti-oxidation, lowering blood pressure, anti-tumor, improving immunity, anti-aging, and cardiovascular protection. Little research has been done on memory

Method used

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  • Method for screening oyster bioactive peptides with effect of improving memory in vitro
  • Method for screening oyster bioactive peptides with effect of improving memory in vitro
  • Method for screening oyster bioactive peptides with effect of improving memory in vitro

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a method for screening oyster bioactive peptides with effect of improving memory in vitro. The method comprises the following screening steps: inoculating astrocytes in a logarithmic phase to a 24-well plate, and culturing for 24 hours; after the astrocytes grow to 80%-90% of the 24-well plate, sucking an astrocyte culture solution, and adding to-be-screened oyster bioactive peptides to act on the astrocytes; sucking the culture solution after 24 hours again, adding phosphate buffer into the astrocytes, fully grinding the astrocytes, centrifuging to obtain supernatant, measuring acetylcholin esterase (AchE) activity with the supernatant, and screening the oyster bioactive peptide with the highest AchE inhibition rate according to the AchE activity. According to the method disclosed by the invention, the astrocytes with the widest distribution in brains of mammals are taken as a mode, and the AchE is directly and accurately extracted to be used for in-vitro activity screening of the oyster bioactive peptides.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for in vitro screening oyster active peptides capable of improving memory. Background technique [0002] With the acceleration of the pace of modern life and the increase of learning and work pressure, sub-health problems such as learning memory and cognitive function decline are becoming more and more serious. Therefore, the research and development of natural active substances that can relieve human mental stress and improve learning and memory ability Significance. [0003] Acetylcholinesterase (AchE) is a key enzyme in biological nerve transmission, mainly present in the neuromuscular junction and cholinergic nervous system, and its main physiological function is to transfer neurotransmitters in the cholinergic synaptic cleft Hydrolyzed into acetic acid and choline, terminating the stimulating effect. The current in vitro screening method for judging active...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12P21/06
CPCC12N2503/00C12P21/06G01N33/5038G01N33/58G01N2500/10
Inventor 王雪芹于华华邢荣娥刘松陈晓琳李荣锋李鹏程
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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