CN105548531B - The method that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique - Google Patents
The method that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique Download PDFInfo
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- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
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- G—PHYSICS
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Abstract
The present invention provides a kind of method that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique, including：Water and trypsase are added in cow's milk and pepsin is digested, through being spray-dried to obtain powdery casein hydrolysate after enzyme deactivation；Casein hydrolysate is separated by isoelectric precipitation；Using anti-phase steel column, electricity is put into precipitation gained purified by preparing liquid phase, sample is isolated and purified again, according to the difference of sample polarity, sample is divided into some sections, by modeling, runic thing of being slept to the rush carries out activity rating；Using whole-cell patch-clamp recording technique, 30 60PA are given, 3000 4000ms induced action potentials, 100 150 μm of ol/L are administered, record electro physiology collection of illustrative plates, by number and the frequency of discharging, filtered out with preferable dormifacient active fragment.Simple to operate, good separating effect of the invention, gained coarse extraction can not only shorten the time of sleep, can also adjust sleep rhythm, and preferably guarantee is provided for the health of insomniac.
Description
Technical field
The present invention relates to one kind to promote sleep assistant health product field, particularly a kind of side that delta sleep inducing peptide is isolated and purified from newborn source
Method and purposes.
Background technology
Sleep be used as process necessary to life, is that body restores, integrated and the important step of consolidating memory, be it is healthy not
The part that can lack.One of World Health Organization research shows that the whole world there are about 27% people and is stranded by vigilance disorders
Disturb.300,000,000 adults are there are about in China with insomnia disease.Insomnia is still in the world one and not paid attention to fully at present
The public health problem well solved.
Clinically commonly use sleep-disorder caused by chemical synthesis class hypnotic sedative agent treatment insomnia and other diseases.Such medicine
For thing while the length of one's sleep is extended, it is for example dizzy, weak, sleepy, absent minded different degrees of side effect all occur,
Even temporary forgetful and hangover phenomenon, do not play a part of really improving sleep quality.Therefore it provides drug effect is notable, secondary
The medicine for acting on small, safe improvement sleep effect is a vital task of pharmaceutical sanitary field.Wherein, foundation is quick, has
Effect improvement sleep drugs with function method of evaluating drug effect, medicine is screened and evaluated be realize this task premise and
Basis.
In brain there is a series of sleep being made up of nerve nucleus fibers and mediator, awakening regulating system, each other
Between form in existing form the neutral net of contact functional upper restriction again.Preop- tic area and its adjacent substrate forebrain in hypothalamus
Neuron is sleep generation system.The rush dormancy neuron of preop- tic area is predominantly located at hypothalamus abdomen lateral front area (VLPO）To sleep
Generation and maintenance have vital effect.
Since patch clamp technique in 1976 is born, carry out electrophysiological research using patch clamp technique turns into one kind
Important research meanses.Brain slice in vitro refers to the brain tissue thin slice prepared from animal brain area, can deposit in vitro in artificial cerebrospinal fluid
A few hours living to ten a few houres, and can keep in the similar metabolic characteristic and electrophysiological characteristics of body brain tissue.The present invention is by brain
Piece patch clamp technique is combined with infrared videomicroscopy technology, establishes infrared visual slice patch clamp experimental technique, make from
The electrophysiologic study of body brain piece is expanded to the almost all of brain area such as hypothalamus, cerebral cortex, cerebellum, hippocampus, corpus straitum, brain stem
And Spinal Cord Slices.
Contain multiple biological activities polypeptide in cow's milk, the content of active peptides is less in natural cow's milk, can not meet sub- strong
The needs of health people.Cow's milk protein, it is many precursors with potential source biomolecule active material, is acted on, obtained by protease hydrolyzed
To the small peptide with biological function, i.e., newborn source biologically active peptide.These small peptides are typically made up of 2-20 amino acid residue, energy
It is enough that active influence is produced to the many-side such as digestive system, cardiovascular system, immune system of body, there is regulation nerveous system
The physiologically active such as system, antibacterial, anti-oxidant, anti-hypertension, immunological regulation.Milk product is considered to have promotion and slept for a long time
Sleep, alleviate the effect of pressure, a kind of casein hydrolysates that French scientist is had proven in milk product have clearly anti-
Depression, alleviate the bioactivity of pressure, but its specific mechanism and specific material are never clear and definite.
At present, the application of domestic patch-clamp experimental technique is not extensive, and based on blind, lacks the stereoscopic aobvious of high quality
The reason for micro mirror and water immersion objective are one side, and Major Difficulties are to prepare the good brain piece of activity.On the other hand, newborn source product
Activity with regulation sleep, but lack suitable model and its activity is evaluated.
The content of the invention
It is an object of the invention to provide a kind of method that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique,
It utilizes newborn source polypeptide isoelectric point（pH）, solution polarity（Concentration of alcohol）Crude separation, each property section of quantitative analysis are carried out to it
Proportion（Precipitate yield）, and sample is prepared by liquid phase is prepared, casease phosphoeptide accessory substance is isolated and purified.
Gained coarse extraction can not only shorten the time of sleep, can also adjust sleep rhythm, be provided more for the health of insomniac
Good guarantee.
To achieve the above object, embodiment of the present invention is：One kind is screened using patch clamp technique promotees sleep in newborn source
The method of peptide crude extract, comprises the following steps：
（1）Water is added in a reservoir, is warming up to 40 DEG C -60 DEG C, adds cow's milk and protease is digested, enzymatic hydrolysis condition
For：Water and cow's milk volume ratio are 100：10-15, pH value 7.5-8.5, time 2-4h, the protease are trypsase and stomach egg
White enzyme, the addition of trypsase are the 0.2%-0.6% of milk quality, and the addition of pepsin is the 0.06%- of milk quality
0.12%, after the completion of enzymolysis, enzyme deactivation, spray drying, obtain powdery casein hydrolysate；
（2）Casein hydrolysate is separated by isoelectric precipitation, condition pH4.5-6, concentration of alcohol 60%-90%, precipitation
Time is 1-3h；
（3）Using anti-phase steel column, by step（2）Gained sediment to sample separate pure again by preparing liquid phase
Change, according to the difference of sample polarity, sample is divided into some sections, by modeling, runic thing of being slept to the rush carries out activity rating；
（4）The hypothalamus abdomen lateral front area brain piece of acute isolation rat, thickness about 300-500um, then using complete thin
Born of the same parents' patch clamp technique, by depolarising square wave, give 30-60PA, 3000-4000ms induced action potentials, administration 100-150 μ
Mol/L, electro physiology collection of illustrative plates is recorded, by number and the frequency of discharging, filtered out with preferable dormifacient active fragment.
As optimization, step（1）In, water and the cow's milk volume ratio is 100：12.
As optimization, step（1）In, the enzymolysis is carried out in two steps, the first step：PH value of solution 8 is adjusted, opens stirring
300rpm, trypsase is put into, digest 3h, enzyme deactivation, the addition of trypsase is the 0.3% of milk quality；Second step：Regulation
PH is 5, adds pepsin, and temperature is transferred into 40 DEG C, digests 2h, and the addition of stomach cardia is the 0.1% of milk quality.
As optimization, step（1）In, the enzyme-removal temperature is 90 DEG C, 30 minutes time.
As optimization, step（2）In, isoelectric precipitation condition is：PH5, concentration of alcohol 70% are quiet at 4 DEG C after precipitating 3h
Put 4 hours.
The caseic hydrolysate of complicated component is obtained about 20% precipitation by the present invention using simple separating and purifying technology
Product, simple to operate, good separating effect；Again by liquid phase is prepared, further isolated and purified to promoting sleep crude extract, upper prop is simple
It is easy to operate, applied sample amount can be increased, is prepared compared with Multi-example, facilitates the production in later stage, while reduces production cost；The sample of preparation
It is carried out by patch clamp technique to promote sleep activity rating, by the neural elemental study of complicated polyvoltine, is reduced to infrared visible brain
Piece patch-clamp, it is raw to hypothalamus abdomen lateral front area neuron electricity by parameters such as the discharge frequency of neuron, film Changshu, amplitudes
Reason index carries out the correlative study of sleep index, and the evaluation model has universal adaptability, can not only evaluate the electricity of neuron
Physiological property, neuron Related Mechanism can also be studied by ion channel.
Brief description of the drawings
Fig. 1 has higher rush sleeper effect P3 high-efficient liquid phase chromatograms for the present invention；
Fig. 2 is casein hydrolysate high-efficient liquid phase chromatogram of the present invention；
Fig. 3 is Hypothalamus in Rats separating sliced of the present invention；
Fig. 4 is electro physiology of the present invention -- hypothalamus ventral lateral nucleus of thalamus neuron sealing-in success；
Fig. 5 is electro physiology of the present invention -- sleeper effect collection of illustrative plates.
Embodiment
Below in conjunction with actual conditions, the embodiment of the present invention is described in detail.
Embodiment 1：
Adding pure water 100L in reaction vessel, be heated to 55 DEG C, put into 12KG cow's milk proteins, the pH for adjusting solution is 8,
Stirring 300rpm is opened, puts into trypsase 36g, digests 3h, enzyme deactivation.Put into as pepsin 12g, regulation pH is 5, by temperature
40 DEG C are transferred to, digests 2h.After enzymolysis, enzyme deactivation, by temperature adjustment to 90 DEG C, 30 min are incubated.Then filtrate is adjusted into pH5, added
Enter ethanol to concentration 70%, stirring is abundant, and after precipitating 3h, 4 DEG C stand 4 hours, then filter supernatant, collect precipitation, will precipitate
In 55-65 DEG C of drying, casein hydrolysates are obtained, purity is high, has light bitter taste and saline taste, color and luster is good, yield 2.35kg.
Using Shimadzu PRC-ODS (K) steel column, by the above-mentioned thing that isolates and purifies by preparing liquid phase, sample is entered again
Row isolates and purifies, and method is：By the purified casein hydrolysates of crude separation first, processed good system is loaded to after dissolving
On standby steel column, applied sample amount 8ml, flow velocity is：15ml/min, Detection wavelength 214nm.Water (containing 0.1%TFA) and 10% are used successively,
15%, 20%, 30%, 60%, 75% acetonitrile（Containing 0.1%TFA）Elution, every kind of 3-6 times of column volume of solvent washing.According in chromatograms
Appearance time collect each component, removed completely by being concentrated under reduced pressure into acetonitrile under 40 DEG C -60 DEG C, 300 ± 100pa vacuums,
By the spray-dried delta sleep inducing peptide crude extract for producing preparation liquid phase and preparing of residual filtrate.
According to the difference of liquid chromatogram appearance time, sample is divided into six sections, is named as（P1, P2, P3, P4, P5, P6）.
Method is：Casein hydrolysates 0.5kg is taken, is dissolved with ultra-pure water, is made into 1g/ml, each sample introduction 10ml, divides six sections to sleep rush
Dormancy crude extract carries out connecing sample, concentration, is lyophilized into powder and is used to test in next step.Wherein P1 yield 12%, P2 yield 10%, P3 yield
24%, P4 yield 18%, P5 yield 12%, P6 yield 8%.By modeling, this six sections are promoted with sleep runic thing and carries out activity rating.
Embodiment 2：
Raw 5 weeks or so rat is taken out, body weight about 100g-150g, its hypothalamus of acute isolation, is by inferior colliculus brain section
400um, it is put into rapidly full of 95%O2In 5% artificial cerebrospinal fluid, 30min is incubated in 33 DEG C, takes out incubation slot, in room temperature
Lower placement 20min.External diameter is used to have core hard glass blank for 1.4mm, it is diameter about 2um that polishing rear tip, which is drawn, is full of
Liquid in electrode.First with 5 × object lens observe the structure of hypothalamus, abdomen lateral front area is found, then with 40 × immersion Jing Guan
Examine the form of single abdomen lateral front area neuron and carry out sealing-in using electrode, when eletrode tip and iuntercellular form and be more than 1G
After Ω sealing-ins after success, after sealing-in window observes that capacitor charge and discharge occurs in cell, that is, full cell pattern is formed, first records one
Section blank, usual 2-3min, film potential 50mV, is administered after stable.Then by positive control and blank control, analyze rush and sleep
Activity of sleeping is preferably P3.
Claims (4)
- A kind of 1. method that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique, it is characterised in that including following step Suddenly：（1）Water is added in a reservoir, adds cow's milk and protease is digested, the enzymolysis is carried out in two steps, the first step：Heating To 55 DEG C, pH value of solution 8 is adjusted, stirring 300rpm is opened, puts into trypsase, digests 3h, enzyme deactivation, the addition of trypsase is The 0.3% of milk quality；Second step：It is 5 to adjust pH, adds pepsin, and temperature is transferred into 40 DEG C, digests 2h, stomach cardia Addition is the 0.1% of milk quality, after the completion of enzymolysis, enzyme deactivation, spray drying, obtains powdery casein hydrolysate；（2）Casein hydrolysate is separated by isoelectric precipitation, condition pH4.5-6, concentration of alcohol 60%-90%, sedimentation time For 1-3h；（3）Using anti-phase steel column, by step（2）Gained sediment is isolated and purified, root to sample again by preparing liquid phase According to the difference of sample polarity, sample is divided into some sections, by modeling, runic thing of being slept to the rush carries out activity rating；（4）The hypothalamus abdomen lateral front area brain piece of acute isolation rat, thickness 300-500um, then using full cell patch Tongs technology, by depolarising square wave, give 30-60PA, 3000-4000ms induced action potentials, dosing step（3）Isolate and purify 100-150 μm of ol/L of the active fragment gone out, electro physiology collection of illustrative plates is recorded, by number and the frequency of discharging, filtered out with preferable Dormifacient active fragment.
- 2. the method according to claim 1 that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique, its feature It is, step（1）In, water and the cow's milk volume ratio is 100：12.
- 3. the method according to claim 1 that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique, its feature It is, step（1）In, the enzyme-removal temperature is 90 DEG C, 30 minutes time.
- 4. the method according to claim 1 that delta sleep inducing peptide crude extract in newborn source is screened using patch clamp technique, its feature It is, step（2）In, isoelectric precipitation condition is：PH is 5, concentration of alcohol 70%, and after precipitating 3h, 4 hours are stood at 4 DEG C.
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| RU2697248C2 (en) * | 2017-11-27 | 2019-08-13 | Общество с ограниченной ответственностью "А2 Молоко" (ООО "А2 Молоко") | METHOD FOR DETERMINING PRESENCE OF β-CASEIN ALLELES OF A1 AND/OR A2 GROUP IN CATTLE MILK |
| CN111632130B (en) * | 2020-05-09 | 2022-03-18 | 华南农业大学 | Application of milk-derived active peptide CCL-3S in preparation of sleep-promoting product |
| CN112056453B (en) * | 2020-08-31 | 2022-05-24 | 华南理工大学 | Aromatic amino acid-rich sleep improvement zymolyte and preparation method thereof |
| CN113730549B (en) * | 2021-08-31 | 2023-05-23 | 华南农业大学 | Application of milk-derived active peptide CCL-1S in preparation of sleep-promoting product |
| CN113735957B (en) * | 2021-09-18 | 2023-08-25 | 华南农业大学 | Novel sleep-promoting milk-derived active peptide CCL-4S, and preparation method and application thereof |
| CN113754751B (en) * | 2021-09-18 | 2023-08-25 | 华南农业大学 | Novel sleep-promoting milk-derived active peptide CCL-2S, and preparation method and application thereof |
| CN113827698A (en) * | 2021-09-23 | 2021-12-24 | 华南农业大学 | Sleep-promoting compound and application thereof |
| CN117338905B (en) * | 2023-10-23 | 2024-05-03 | 广州绿萃生物科技有限公司 | Hydrolyzed casein peptide with sleep promoting effect, and preparation method and application thereof |
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| US4032637A (en) * | 1972-09-26 | 1977-06-28 | Sandoz Ltd. | Method of promoting sleep |
| US20050080457A1 (en) * | 2003-09-23 | 2005-04-14 | Bradley Greger | Chronically implantable hybrid cannula-electrode system for continuously monitoring electrophysiological signals during infusion of a chemical or pharmaceutical agent |
| WO2013072839A2 (en) * | 2011-11-15 | 2013-05-23 | Koninklijke Philips Electronics N.V. | Dual-mode capacitive measurement |
| CN103571905B (en) * | 2013-10-30 | 2014-12-17 | 广州绿萃生物科技有限公司 | Preparation method of high-purity casein phosphopeptide |
| CN105085651B (en) * | 2015-09-02 | 2018-12-28 | 广州绿萃生物科技有限公司 | A kind of casein phosphopeptide monomer and preparation method thereof |
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Effective date of registration: 20210720 Address after: 510000 room 209, building 2, No. 8, yayingshi Road, high tech Industrial Development Zone, Guangzhou, Guangdong Patentee after: GREENCREAM BIOTECH Co.,Ltd. Address before: 510642 South China Agricultural University, No. 403, Wushan Road, Tianhe District, Guangzhou, Guangdong Patentee before: SOUTH CHINA AGRICULTURAL University |