Method for screening crude delta sleep-inducing peptide extract from milk source in virtue of patch clamp technique

A patch clamp technique and sleep-promoting technology, which is applied in the field of separation and purification of sleep-promoting peptides, can solve the problems of lack of high-quality stereo microscopes and water immersion objectives, not widely used, lack of models, etc., to reduce production costs and simple operation , the effect of increasing the sample volume

Active Publication Date: 2016-05-04
GREENCREAM BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At present, the application of patch clamp experiment technology in China is not widespread, and the blind method is the main method. The lack of high-quality stereo microscopes and water immersion objectives is one reason, and the main difficulty is the preparation of brain slices with good activity.
On the other hand, milk-derived products have sleep-modulating activity, but there is a lack of suitable models to evaluate their activity

Method used

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  • Method for screening crude delta sleep-inducing peptide extract from milk source in virtue of patch clamp technique
  • Method for screening crude delta sleep-inducing peptide extract from milk source in virtue of patch clamp technique
  • Method for screening crude delta sleep-inducing peptide extract from milk source in virtue of patch clamp technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Add 100L of pure water to the reaction vessel, heat to 55°C, add 12KG of milk protein, adjust the pH of the solution to 8, start stirring at 300rpm, add 36g of trypsin, and enzymatically hydrolyze for 3 hours to inactivate the enzyme. Add 12g of pepsin, adjust the pH to 5, adjust the temperature to 40°C, and perform enzymatic hydrolysis for 2 hours. After enzymatic hydrolysis, the enzyme was inactivated, the temperature was adjusted to 90°C, and the temperature was kept for 30 minutes. Then adjust the pH of the filtrate to 5, add ethanol to a concentration of 70%, stir well, after 3 hours of precipitation, stand at 4°C for 4 hours, then filter the supernatant, collect the precipitate, dry the precipitate at 55-65°C, and obtain the casein hydrolysis product , high purity, light bitter and salty taste, good color, yield 2.35kg.

[0027] Using ShimadzuPRC-ODS (K) steel column, the above-mentioned isolated and purified product is separated and purified through the preparat...

Embodiment 2

[0030] Take out rats born about 5 weeks old, weighing about 100g-150g, acutely separate their hypothalamus, slice the hypothalamus into 400um slices, and quickly put them into a tube filled with 95% O 2 and 5% artificial cerebrospinal fluid, incubate at 33°C for 30min, take out the incubation tank, and place at room temperature for 20min. A cored hard glass blank with an outer diameter of 1.4mm is used. After polishing, the tip is drawn to a diameter of about 2um and filled with the electrode inner liquid. First observe the structure of the hypothalamus with a 5× objective lens to find the ventrolateral preoptic area, then observe the morphology of a single ventrolateral preoptic area neuron with a 40× water immersion lens and seal it with electrodes. When the tip of the electrode and the cell form After successful sealing of more than 1GΩ, the whole cell mode is formed after the cells are observed to charge and discharge capacitance in the sealing window. First record a blank...

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Abstract

The invention provides a method for screening crude delta sleep-inducing peptide extract from a milk source in virtue of a patch clamp technique. The method comprises the following steps: adding water, trypsin and pepsin in cow's milk for enzymatic hydrolysis and carrying out spray drying after enzyme killing so as to obtain powdery casein hydrolysate; separating the casein hydrolysate through isoelectric precipitation; subjecting a purified product obtained after isoelectric precipitation to preparative liquid chromatography via a reversed-phase steel column so as to separate and purify samples again, dividing the samples into a plurality of sections according to different polarities of the samples and carrying out modeling to evaluate the activity of the crude delta sleep-inducing peptide extract; employing the whole cell patch clamp technique, giving an evoked action potential with a size of 30-60 Pa / 3000-4000 ms, delivering a drug with a dosage of 100 to 150 [mu]mol / L, recording an electrophysiological spectrum and screening out good active sleep-inducing fragments via discharging number and frequency. The method is simple to operate and has good separation effect; and the crude extract can shorten time for going to sleep and adjust sleep rhythm and provides better guarantee for health of patients with insomnia.

Description

technical field [0001] The invention relates to the field of sleep-promoting health care products, in particular to a method and application of separating and purifying sleep-promoting peptides from milk sources. Background technique [0002] As a necessary process of life, sleep is an important part of the body's recovery, integration and memory consolidation, and an indispensable part of health. A World Health Organization study shows that about 27% of people worldwide suffer from insomnia. About 300 million adults suffer from insomnia in my country. At present, insomnia is still a public health problem that has not been paid enough attention to and solved well in the world. [0003] Clinically, chemically synthesized sedative-hypnotics are commonly used to treat insomnia and sleep disorders caused by other diseases. While prolonging the sleep time, these drugs will have different degrees of side effects such as dizziness, fatigue, drowsiness, inattention, and even temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/487G01N1/34
CPCG01N1/34G01N33/48728
Inventor 曹庸陈媛媛苗建银陈云娇刘又豪彭勃孙圣伟
Owner GREENCREAM BIOTECH
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