CN113528603B

CN113528603B - Pig brain proliferation-promoting peptide-in-brain phospholipid co-production method

Info

Publication number: CN113528603B
Application number: CN202110776199.7A
Authority: CN
Inventors: 谌迪; 肖朝耿; 卢文静; 叶沁; 张岑; 张治国
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2024-04-12
Anticipated expiration: 2041-07-09
Also published as: CN113528603A

Abstract

The invention relates to the technical field of food and medicine ingredients, and particularly discloses a pig brain proliferation promoting peptide-in-brain phospholipid co-production method, which comprises the following steps of (a) selecting pig brain and normal saline, mixing the selected pig brain and normal saline in proportion, and homogenizing; (b) Adding trypsin into the homogenized mixture for stirring and enzymolysis; (c) Filtering the substances after enzymolysis to obtain filtrate I and filter residue I. The invention has the characteristics of simple preparation process, mild reaction process and no influence on product quality.

Description

Pig brain proliferation-promoting peptide-in-brain phospholipid co-production method

Technical Field

The invention relates to the technical field of food and medicine ingredients, in particular to a pig brain proliferation promoting peptide-cephalin co-production method.

Background

The pig brain is fresh, tender and delicious, is rich in protein, phospholipid and various minerals, can increase the immunity of organisms, and is a traditional brain-strengthening food.

The pig brain hydrolysate can be used for treating nervous system diseases and preventing age-dependent dementia. Clinically, ziganshina L E, abakumova T and Vernay L find that the pig brain polypeptide can improve symptoms of patients suffering from acute ischemic cerebral apoplexy and delay disease progression. Studies have shown that porcine brain polypeptides have neuroprotective effects on NSC-34 cells, but that too high concentrations of porcine brain polypeptides inhibit proliferation of cells. Shalma H S, zimmermann-Meinzingn S and Johanson C E found that the hydrolysate of pig brain, pig brain polypeptide, can slow down cerebral edema and inhibit neuronal damage; the pig brain hydrolysate can obviously enhance the learning and memory ability of mice. The pig brain polypeptide is also used for vascular type mental retardation with gradual loss of cognitive ability, and the pig brain polypeptide hydrolysis promotes brain metabolism and increases brain activity by improving ATP generation amount of anaerobic metabolism of brain tissues, reducing oxygen free radical generation and other action mechanisms. The pig brain hydrolysate is used for treating brain dysfunction diseases, and the traditional Chinese medicine preparation has been developed in clinic for decades as early as in China in the last century and is used for treating headache, for example, zhennaoning is developed by taking pig brain as a raw material, and the brain active polypeptide prepared by the suckling pig has similar effect with the imported brain active element with high price. Meanwhile, brain active substances have been widely used clinically for over 40 years, and brain active substances are known to be a mixture of 75% free amino acids and 25% short-chain peptides, which are physiologically active ingredients thereof. Therefore, the preparation of the pig brain polypeptide has important pharmaceutical value.

The phospholipid is commonly existing in the protoplasm and the biological membrane of animal and plant cells, has important regulation function on the physiological activity of the biological membrane and the normal metabolism of organisms, and has good emulsifying property and oxidation resistance, so that the phospholipid is widely applied to industries such as food, medicine, cosmetics and the like. Lecithin and cephalin are two important phospholipids, and their chemical names are Phosphatidylcholine (PC) and phosphatidylethanolamine (Phosphatidyl Ethanolamine PE), respectively. Wherein lecithin is a main component constituting human cell membranes, is one of life basic substances for maintaining normal forms and functions of cells, and has important regulation effect on normal metabolism of human bodies. The effects on heart and cerebral vessels are to keep the elasticity and permeability of the vessel wall, soften the vessel, increase the content of high density lipoprotein, reduce or clear the sediment on the inner wall of the vessel, and prevent blood coagulation; the effect on the nervous system is to sharpen the nervous response, improve the memory and prevent amnesia and senile dementia; can effectively prevent and treat liver function diseases and relieve diabetes; can promote the renewal of injured cells and improve the immunity of human body. In the 70 s, lecithin is used as health food in the world, and the total sales amount is inferior to the third place of vitamin C complex and vitamin E complex. The deficiency of lecithin is one of the main causes of modern civilization diseases such as cardiovascular and cerebrovascular diseases. The molecular structure of lecithin has hydrophilic group and hydrophobic group, and the bipolar property makes lecithin show excellent surface activity, belongs to phosphoric acid type amphoteric surfactant with wider application, has the functions of emulsification, thickening, stabilization, dispersion, solubilization, wetting, lubrication and the like, and can be widely applied to industrial and agricultural production such as food, feed, medicine, cosmetics, paint, printing ink, textile, leather, petroleum products, plant protection and the like. The phospholipid in the brain is a good natural surfactant, has unique biological activity and physiological function, and is highly valued by the scientific and technical circles and industry at home and abroad because of no toxicity, no pollution, no stimulation and easy biodegradation.

At present, pig brain is used as a raw material, proliferation-promoting peptide and phospholipid in the brain are prepared separately, the preparation process is complex, and the preparation efficiency is low. For the preparation of proliferation-promoting peptides, brain proteins are mostly hydrolyzed by adopting an acid-base hydrolysis mode, wherein tryptophan is completely destroyed by acid hydrolysis, and serine, tyrosine, threonine and cysteine parts are destroyed; base hydrolysis causes racemization; there are problems in that the reaction process is not easy to control, the reaction is too severe, and acid-base residues can affect the environment. The preparation method of the phospholipid mainly comprises an extraction method, a column chromatography method, a precipitation method, a membrane separation method and the like. The column chromatography can obtain high-purity lecithin with the content of more than 90%, but has very limited treatment capacity, long period, complicated steps and difficult realization of continuous operation, and a plurality of toxic organic solvents are used in the treatment process, so that the popularization and application of the technology in the fields of foods and medicines are limited by the residual solvents. And the purification of PC can be realized by adopting an inorganic salt composite precipitation method. Japanese patent reports that 95% ethanol was mixed with crude egg yolk phospholipids followed by ZnCl ₂ After precipitation and centrifugal separation, znCl is collected ₂ And adding cooled acetone into the phospholipid complex, stirring and filtering under the protection of nitrogen, and evaporating to remove the solvent to obtain the lecithin with the purity of 99.6%. Inorganic salt complexThe co-precipitation method utilizes the selectivity of inorganic salt to phospholipid molecules, and can greatly improve the content of lecithin, but can introduce metal ions in operation to influence the quality of products and cause metal ion pollution.

Therefore, the existing preparation of the proliferation-promoting peptide and the phospholipid in the brain by taking the pig brain as the raw material has the problems of complex preparation process, destroyed amino acid, difficult control of reaction process, environmental influence and influenced product quality.

Disclosure of Invention

The invention provides a pig brain proliferation-promoting peptide-in-brain phospholipid co-production method which is simple in preparation process, mild in reaction process and free from influence on product quality, and aims to solve the technical problems of the preparation of the proliferation-promoting peptide and in-brain phospholipid by taking pig brain as a raw material.

The technical scheme of the invention is as follows: the pig brain proliferation promoting peptide-in-brain phospholipid co-production process includes the following steps,

(a) Selecting pig brain and normal saline, mixing the pig brain and normal saline in proportion, and homogenizing;

(b) Adding trypsin into the homogenized mixture for stirring and enzymolysis;

(c) Filtering the substances after enzymolysis to obtain filtrate I and filter residue I.

According to the invention, pig brain and normal saline are mixed in proportion and then hydrolyzed by trypsin, two initial raw materials for further preparing the pro-proliferative peptide and the phospholipid in the brain are directly obtained after filtration, the preparation process is simple, the cost is low, and the two initial raw materials for further preparing the pro-proliferative peptide and the phospholipid in the brain are directly obtained from the pig brain by enzymolysis by trypsin; the condition of trypsin is mild in the enzymolysis mixed solution, the whole reaction is well controlled, only proper metering reactants and trypsin are needed to be added, other parameters such as the required adding amount, pH, enzymolysis temperature and enzymolysis time of trypsin in the enzymolysis process are needed to be determined, other parameters are not needed to be excessively participated, the reaction process is well controlled, the hydrolyzed amino acid is not damaged, no toxic and harmful substances are produced in the whole process, the environment is protected, the quality of the finally prepared proliferation-promoting peptide and phospholipid finished products in the brain is not influenced, and the added trypsin has good specificity only for pig brain.

Preferably, the method comprises the steps of,

(d) Ultrafiltering the filtrate I in the step (c) sequentially through a cellulose membrane with a molecular weight of 5000Da to 6000Da and a cellulose membrane with a molecular weight of 950Da to 1500 Da;

(e) And (3) vacuum drying and dehydrating the substance subjected to ultrafiltration in the step (d) to obtain an extract I, namely the porcine brain proliferation-promoting peptide.

According to the invention, cellulose membranes with molecular weights of 5000 Da-6000 Da and cellulose membranes with molecular weights of 950 Da-1500 Da are used successively to intercept substances containing pro-proliferative peptides in the filtrate I, and then a physical mode of vacuum drying and dehydration is adopted to obtain a final pig brain pro-proliferative peptide finished product. Wherein the pig brain proliferation promoting peptide is a small molecular weight polypeptide with proliferation effect.

Preferably, the method comprises the steps of,

(f) Freeze-dehydrating the filter residue I in the step (c), taking cold acetone with the temperature of-15 ℃ to-2 ℃, and mixing the cold acetone with the filter residue I after freeze-dehydration;

(g) Standing the mixed substances in the step (f) at a low temperature, and centrifuging after standing is completed to obtain filter residue II;

(h) Evaporating the filter residue II in the step (g) to dryness to obtain brain dry powder;

(i) Taking an alkali ethanol solution, stirring and mixing the alkali ethanol solution and the brain dry powder in the step (h), and filtering to obtain a filtrate II;

(j) And (3) drying the filtrate II in the step (i) in vacuum to obtain a powdery substance, namely the phospholipid extract in the brain.

According to the invention, the filter residue I is subjected to freeze dehydration, so that the subsequent removal of fat in the filter residue by using cold acetone is facilitated, phospholipid components are not dissolved out, the loss of phospholipid is reduced, and meanwhile, the activity of phospholipid molecules in the brain is not influenced by temperature in the dehydration process; cold acetone with the temperature of-15 ℃ to-2 ℃ and low-temperature standing are used for better removing fat in filter residues without dissolving phospholipid components, so that phospholipid loss is reduced, meanwhile, the temperature is controlled so as not to influence the activity of phospholipid molecules in the brain due to the temperature in the degreasing process, and the low-temperature control is also used for preventing the degreasing effect from being influenced due to the volatilization of acetone; the alkali ethanol solution is added to better extract the cephalin in the brain dry powder; the preparation process of the phospholipid in the whole brain is simple, batch treatment can be carried out, the treatment speed is high, continuous operation can be realized, no heavy metal ions are introduced in the treatment process, the alkaline ethanol is completely volatilized in the vacuum drying process, and the quality influence of residues of additives on the final phospholipid extract finished product in the brain is avoided. The evaporating can be performed by vacuum decompression drying, a rotary evaporator can be used, and when the yield is high, large-scale industrial evaporating equipment can be used.

Preferably, the mixing ratio of the pig brain and the normal saline in the step (a) is 1/9-1/2. More preferably, the mixing ratio of the pig brain and the normal saline in the step (a) is 1/7-1/3. More preferably, the mixing ratio of the pig brain and the normal saline in the step (a) is 1/5 to 1/4. The pig brain and the physiological saline with proper proportion can be changed into liquid substances stably, so that the pig brain is fully hydrolyzed by trypsin added later in a better state, and finally the initial raw materials for preparing the proliferation-promoting peptide and the phospholipid in the brain are prepared, and the physiological saline provides a good reaction medium environment for the enzymolysis process of the pig brain.

Preferably, the homogenization time in step (a) is 8 to 20 minutes. More preferably, the homogenization time in step (a) is 10min to 15min. The homogenization time is set to ensure that pig brains are fully mixed in the physiological saline water while ageing is increased, and the pig brains are subjected to enzymolysis by trypsin added later in a better particle state.

Preferably, the stirring time in the step (b) is 20 to 30 minutes. The stirring time is set to ensure that the whole enzymolysis process is more complete and that each pig brain particle can be thoroughly hydrolyzed as far as possible.

Preferably, the trypsin is added in an amount of 5U/mg to 10U/mg. More preferably, the trypsin is added in an amount of 7U/mg to 9U/mg. The adding amount of trypsin is set according to the amount of pig brain, so that the pig brain can be fully hydrolyzed, and the added trypsin is not excessive and becomes an impurity in the subsequent preparation process.

Preferably, the pH value of the enzymolysis process solution in the step (b) is 6-8. More preferably, the pH value of the enzymolysis process solution in the step (b) is 6.5-7.5. The pH value of the solution in the enzymolysis process is kept, so that the activity of trypsin can be fully ensured, and the trypsin can carry out enzymolysis on pig brains in the best state all the time.

Preferably, the enzymolysis temperature in the step (b) is 36-50 ℃. More preferably, the enzymolysis temperature in the step (b) is 40-45 ℃. The temperature is kept, so that the activity of trypsin can be fully ensured, and the trypsin can be used for carrying out enzymolysis on pig brains in the best state all the time.

Preferably, the enzymolysis time in the step (b) is 3 to 8 hours. More preferably, the enzymolysis time in the step (b) is 4 to 6 hours. The enzymolysis time is set by fully considering comprehensive factors such as the enzymolysis pH value, the enzymolysis temperature, the timeliness of the whole reaction process, the reaction rate, the reaction completeness and the like, and finally the enzymolysis time is determined.

Preferably, the method comprises the steps of,

(d1) Ultrafiltering the filtrate I in the step (c) through a cellulose membrane with the molecular weight of 5000 Da-6000 Da; continuously ultrafiltering until the volume of the filtrate I is changed to 1/3.5-1/3 of the original volume to obtain trapped fluid I and passing fluid I; more preferably, the filtrate I in step (c) is ultrafiltered by a cellulose membrane having a molecular weight of 5400Da to 5800 Da;

(d2) Continuing ultrafiltration of the passing liquid I in the step (d 1) by a cellulose membrane with the molecular weight of 950Da to 1500 Da; continuously ultrafiltering until the volume of the passing liquid I is changed to 1/3.5-1/3 of the original volume to obtain trapped liquid II and passing liquid II; more preferably, the passing liquid I in the step (d 1) is continuously ultrafiltered by a cellulose membrane with the molecular weight of 1000Da to 1300 Da;

(e1) And (3) carrying out vacuum drying and dehydration on the obtained solution II to obtain an extract I, namely the porcine brain proliferation-promoting peptide.

The invention firstly uses a cellulose membrane with larger molecular weight to carry out coarse interception on substances containing pro-proliferative peptides in filtrate I in an ultrafiltration mode, so that the residual volume is changed into 1/3.5-1/3 of the initial filtrate I, then uses a cellulose membrane with smaller molecular weight to carry out fine interception on substances containing pro-proliferative peptides in filtrate I in an ultrafiltration mode, and then enables the residual volume to be changed into 1/3.5-1/3 of the volume at the beginning of ultrafiltration, thus the residual mixed solution contains pig brain pro-proliferative peptide molecules with high concentration, and then the water in the residual mixed solution is dehydrated in a vacuum drying and dewatering mode, thus obtaining the pig brain pro-proliferative peptides with high precision.

Preferably, the pressure of the feed water in the ultrafiltration process is 2.81Bar to 3.05Bar, and the reflux pressure in the ultrafiltration process is 0.53Bar to 0.67Bar. The setting of the inlet pressure is to enable the filtrate to obtain good pushing force to pass through the cellulose membrane, and the setting of the reflux pressure is to enable the filtrate to obtain good pulling force to pass through the cellulose membrane, and the pushing-pulling double acting force enables the cellulose membrane to perform good ultrafiltration on the filtrate; the specific pressure value is set, so that the filter liquor can pass through the cellulose membrane well, and meanwhile, the situation that the pig brain proliferation-promoting peptide molecules leak out of the pores of the cellulose membrane due to overlarge pressure is prevented, and the loss of the pig brain proliferation-promoting peptide molecules is avoided.

Preferably, the volume usage of the cold acetone is 2-3 times of the volume of the filter residue I after freeze dehydration. The volume dosage of the cold acetone is set according to the volume of the filter residue I after freeze-dehydration, so that good degreasing of the filter residue I after freeze-dehydration can be ensured, and meanwhile, the cold acetone is prevented from being excessive and entering the subsequent preparation process in the form of impurities.

Preferably, the time for the low-temperature standing is 20 to 30 minutes. More preferably, the low-temperature standing time is 25min to 28min. The setting of low-temperature standing is to fully degrease filter residue I and ensure timeliness of the whole preparation process.

Preferably, the centrifugal speed in the step (g) is 8000rpm to 12000rpm; the centrifugation time is 5-10 min; the operation was repeated 3 times. More preferably, the centrifugal speed in the step (g) is 9500rpm to 11000rpm; the centrifugation time is 7-9 min. The setting of the centrifugal rotation speed is used for ensuring a better centrifugal effect, and the setting of the centrifugal time is used for ensuring timeliness of the whole preparation process while ensuring sufficient centrifugation.

Preferably, the feed liquid ratio of the brain stem powder to the alkali ethanol solution is 1/30-1/5. More preferably, the ratio of the brain dry powder to the alkali ethanol solution is 1/20-1/10. More preferably, the ratio of the brain dry powder to the alkali ethanol solution is 1/18-1/15. The ratio of feed to liquid is set here to ensure that the alkaline ethanol extracts the phospholipid from the brain better.

Preferably, the temperature of the stirring and mixing in the step (i) is 15-52 ℃; the stirring time in the step (i) is 20-60 min. More preferably, the temperature of the stirring and mixing in the step (i) is 20-45 ℃; the stirring time in the step (i) is 30-50 min. More preferably, the temperature of the stirring and mixing in the step (i) is 30-35 ℃; the stirring time in the step (i) is 35-45 min. The stirring and mixing temperature is limited here, so as to create a proper extraction temperature condition for better extracting the phospholipid in the brain from the brain dry powder by using the alkaline ethanol; the setting of the stirring time ensures that the alkaline ethanol fully extracts the phospholipid in the brain from the brain dry powder and simultaneously takes the timeliness of the whole preparation process into consideration.

Preferably, the alkali ethanol solution is prepared by mixing ethanol and ammonia water, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 25-100. More preferably, the volume ratio of the ethanol to the ammonia water in the alkaline ethanol solution is 35-85. More preferably, the volume ratio of the ethanol to the ammonia water in the alkaline ethanol solution is 45-65. The alkaline ethanol solution is prepared by mixing ethanol and ammonia water, heavy metal ions are not introduced in the preparation process of the alkaline ethanol solution, the ammonia water has high volatility, and the ammonia water can be completely volatilized in the subsequent drying process, so that the quality of the final phospholipid extract finished product in the brain is not influenced.

Preferably, the content of phospholipid extract in the brain is detected by HPLC. The accuracy of HPLC detection is high, and the purity of the phospholipid extract in the brain can be accurately obtained.

The invention has the following beneficial effects:

firstly, mixing pig brain and normal saline in proportion, then hydrolyzing by using trypsin, filtering to directly obtain two initial raw materials for further preparing the proliferation-promoting peptide and the phospholipid in the brain, wherein the preparation process is simple, the cost is low, and the two initial raw materials for further preparing the proliferation-promoting peptide and the phospholipid in the brain are directly obtained from the pig brain by using trypsin through enzymolysis; the condition of trypsin is mild in the enzymolysis mixed solution, the whole reaction is well controlled, only proper metering reactants and trypsin are needed to be added, other parameters such as the required adding amount, pH, enzymolysis temperature and enzymolysis time of trypsin in the enzymolysis process are needed to be determined, other parameters are not needed to be excessively participated, the reaction process is well controlled, the hydrolyzed amino acid is not damaged, no toxic and harmful substances are produced in the whole process, the environment is protected, the quality of the finally prepared proliferation-promoting peptide and phospholipid finished products in the brain is not influenced, and the added trypsin has good specificity only for pig brain.

Drawings

FIG. 1 is a graph showing the effect of different concentrations of porcine brain polypeptide on the pro-proliferative activity of NE-4C cells according to the present invention;

FIG. 2 is a graph showing the effect of enzymatic hydrolysis products of different enzymes on the proliferation-promoting activity of NE-4C cells according to the present invention;

FIG. 3 is a graph showing the molecular weight distribution of polypeptides of the pig brain homogenate of the present invention prior to enzymatic hydrolysis;

FIG. 4 is a graph showing the molecular weight distribution of polypeptides obtained by pepsin enzymolysis of a pig brain homogenate according to the present invention;

FIG. 5 is a graph showing the molecular weight distribution of polypeptides obtained by subjecting a pig brain homogenate to trypsin enzymatic hydrolysis in accordance with the present invention;

FIG. 6 is a graph showing the molecular weight distribution of polypeptides obtained by enzymatic hydrolysis of a pig brain homogenate with a complex enzyme according to the invention;

FIG. 7 is a graph showing the effect of trypsin enzyme addition on the activity of porcine brain-proliferation-promoting peptide according to the present invention;

FIG. 8 is a graph showing the effect of trypsin enzymatic hydrolysis time on the activity of porcine brain proliferation-promoting peptide according to the present invention;

FIG. 9 is a graph showing the effect of trypsin enzymatic hydrolysis temperature on the activity of porcine brain-proliferation-promoting peptide according to the present invention;

FIG. 10 is a graph showing the effect of trypsin enzymatic pH on the activity of porcine brain-proliferation-promoting peptide according to the present invention;

FIG. 11 is a bar graph showing the effect of ethanol to ammonia volume ratio on phosphatidylcholine content in the present invention;

FIG. 12 is a bar graph showing the effect of extraction time on phosphatidylcholine content in the present invention;

FIG. 13 is a bar graph showing the effect of extraction temperature on phosphatidylcholine content in the present invention;

FIG. 14 is a bar graph showing the effect of feed liquor ratio of brain powder and alkaline ethanol solution on phosphatidylcholine content in the present invention.

Detailed Description

The invention is further illustrated by the following examples and figures, which are not intended to be limiting.

The pig brain proliferation promoting peptide-in-brain phospholipid co-production process includes the following steps,

(a) Selecting pig brain and normal saline, mixing the pig brain and normal saline in proportion, and homogenizing; the mixing ratio of the pig brain to the normal saline in the step (a) is 1/9-1/2. The homogenization time in the step (a) is 8-20 min.

(b) Adding trypsin into the homogenized mixture for stirring and enzymolysis; the stirring time in the step (b) is 20-30 min. The addition amount of the trypsin is 5U/mg-10U/mg. And (b) the pH value of the enzymolysis process solution in the step (b) is 6-8. The enzymolysis temperature in the step (b) is 36-50 ℃. And (c) the enzymolysis time in the step (b) is 3-8 h.

Wherein the preparation steps of the pig brain proliferation promoting peptide are as follows,

(d) Ultrafiltering the filtrate I in the step (c) sequentially through a cellulose membrane with a molecular weight of 5000Da to 6000Da and a cellulose membrane with a molecular weight of 950Da to 1500 Da; the pressure of the inlet water in the ultrafiltration process is 2.81Bar to 3.05Bar, and the reflux pressure in the ultrafiltration process is 0.53Bar to 0.67Bar.

Wherein the pig brain proliferation-promoting peptide is prepared by the following steps,

(d1) Ultrafiltering the filtrate I in the step (c) through a cellulose membrane with the molecular weight of 5000 Da-6000 Da; continuously ultrafiltering until the volume of the filtrate I is changed to 1/3.5-1/3 of the original volume to obtain trapped fluid I and passing fluid I; the pressure of the inlet water in the ultrafiltration process is 2.81Bar to 3.05Bar, and the reflux pressure in the ultrafiltration process is 0.53Bar to 0.67Bar.

(d2) Continuing ultrafiltration of the passing liquid I in the step (d 1) by a cellulose membrane with the molecular weight of 950Da to 1500 Da; continuously ultrafiltering until the volume of the passing liquid I is changed to 1/3.5-1/3 of the original volume to obtain trapped liquid II and passing liquid II; the pressure of the inlet water in the ultrafiltration process is 2.81Bar to 3.05Bar, and the reflux pressure in the ultrafiltration process is 0.53Bar to 0.67Bar.

Wherein the preparation method of the phospholipid extract in the brain comprises the following steps of,

(f) Freeze-dehydrating the filter residue I in the step (c), taking cold acetone with the temperature of-15 ℃ to-2 ℃, and mixing the cold acetone with the filter residue I after freeze-dehydration; the volume dosage of the cold acetone is 2-3 times of the volume of the filter residue I after freeze dehydration.

(g) Standing the mixed substances in the step (f) at a low temperature, and centrifuging after standing is completed to obtain filter residue II; the low-temperature standing time is 20-30 min. The centrifugal rotating speed in the step (g) is 8000 rpm-12000 rpm; the centrifugation time is 5-10 min; repeating the steps (f) and (g) 3 times.

(i) Taking an alkali ethanol solution, stirring and mixing the alkali ethanol solution and the brain dry powder in the step (h), and filtering to obtain a filtrate II; the ratio of the brain dry powder to the alkali ethanol solution is 1/30-1/25. The temperature of stirring and mixing in the step (i) is 15-20 ℃; the stirring time in the step (i) is 20-60 min. The alkali ethanol solution is prepared by mixing ethanol and ammonia water, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 25-50.

(j) And (3) drying the filtrate II in the step (i) in vacuum to obtain a powdery substance, namely the phospholipid extract in the brain. The content of phospholipid extract in the brain was detected by HPLC.

HPLC detection was performed using LiChrospere 100DIOL chromatographic column (250 mm. Times.4 mm,5 μm); wherein the mobile phase A is n-heptane-isopropanol (65:114), the mobile phase B is n-heptane-isopropanol-water (31:62:12), the isocratic elution is carried out, the flow rate is 1.0mL/min, the column temperature is 40 ℃, and the sample injection amount is 10 mu L. An evaporative light scattering detector was used, the temperature of the evaporator tube was 55deg.C, and the nitrogen flow rate was 2.0L/min. And (3) qualitatively determining according to the retention time of phospholipid standards such as phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and the like, and quantifying by adopting an external standard, wherein the phospholipid extract in the brain can reach as high as 0.762mg/mL through HPLC detection.

The invention uses trypsin to carry out enzymolysis, because the enzymolysis of trypsin has better enzymolysis effect on the pig brain compared with the enzymolysis of other enzymes. In order to demonstrate that trypsin has better enzymatic activity on pig brain than other enzymes, pepsin and lipase are selected as comparison, and the optimal process conditions of trypsin are described.

1. Materials and methods

1.1 materials and reagents

Frozen fresh pig brain (purchased from farmer market). Trypsin (1:250) and pepsin (1:30000), both selected from Shanghai source leaf biotechnology limited; palatase 2000L (lipase, 20000U/g), selected from novelin (china) biotechnology limited; a standard bovine serum albumin selected from the group consisting of the biological engineering (Shanghai) stock company; eagle MEM medium selected from gibco; fetal bovine serum selected from Hyclon company; MTT kit selected from Nanjakyl Biotechnology Co., ltd; poly-L-lysine (cat#P-9155) selected from Sigma Co; glutamax (35050) and Non-essentialAmino Acids, (100X, 11140) is selected from Invitrogen corporation. Other reagents are all of domestic analytical purity.

1.2 major instruments and apparatus

5424R cryocentrifuge is selected from Eppendorf, germany; the Spectra MAX190 full wavelength microplate reader is selected from MD, USA; an ultrafiltration device selected from Millipore corporation, usa; an ultra clean bench selected from HITIK; a carbon dioxide incubator selected from Thermo company; a pH meter selected from Shanghai Sanxingjingsu instruments Co., ltd; ULTRA-TURRAX T1.8basic refiner, selected from IKA company, germany; an electric heating constant temperature water bath pot selected from Shanghai Jing HongJi laboratory Equipment Co., ltd; a pulverizer selected from the group consisting of Deqingbaijie electric company; an oven selected from Shanghai-Heng science instruments Co.

1.3 Experimental methods

1.3.1 screening of enzyme preparation from porcine brain proliferation-promoting polypeptide

Freezing fresh pig brain, naturally thawing, removing blood clot and meninges (pia mater and arachnoid), washing with physiological saline, homogenizing with homogenizer to obtain pig brain homogenate, and sealing in refrigerator for cold storage.

1.3.2NE-4C neural stem cell culture

Eagle MEM, fetal bovine serum, glutamax and Non-essential Amino Acids (100X) were prepared at 88:10:1:1, and preparing a complete culture solution of the mouse neural stem cells under a sterile condition. At least 2h in advance before cell recovery and passage, 15 mug/ml poly-L-lysine was spread in a T25 flask for pretreatment. NE-4C cells were inoculated into a complete medium of mouse neural stem cells under sterile conditions, cultured in a CO2 incubator at 37℃in a 5% CO2 environment, and the complete medium was changed every day. When the cells grow to 70% -80% of the total volume of the culture flask, 0.25% of trypsin is digested, and after pretreatment for 2 hours, the cells are passaged and cultured continuously. Cells required for the assay were taken from the same generation.

1.3.3MTT assay for the cell proliferation-promoting Activity of NE-4C

The specific method for detecting the proliferation promoting activity comprises the following steps: NE-4C cells were seeded into 96-well plates at 2000/well, T25 flasks were pre-treated with 15. Mu.g/mL poly-L-lysine 2h before seeding, and after overnight incubation the medium was removed. The polypeptide content in the enzymolysis liquid is detected by using a Bradford method, the pig brain polypeptide is diluted to 0.5mg/mL by using a complete culture medium, NE-4C cells are cultivated for 48 hours, 50 mu L of MTT working solution is added into each hole, the culture medium is removed after 4 hours, 150 mu L of DMSO is added and mixed uniformly, a shaking table is used for 1min at room temperature, and the light absorption value is detected at 492 nm.

1.3.4 detection of molecular weight distribution of pig brain polypeptide after enzymolysis by chromatography

And (3) characterizing the molecular mass distribution of the pig brain polypeptide by adopting a high-efficiency gel permeation chromatography. Chromatographic conditions: ultrahydrogel TM Linear 2 in series (7.8 mm. Times.300 mm); column temperature: 45 ℃; mobile phase: 0.1mol/L NaNO3; flow rate: 0.6mL/min; sample injection amount: 20. Mu.L. Detection was performed using a RID differential refractive detector. The pig brain polypeptide zymolyte with the concentration of 1.0mg/mL and a series of dextran standard substance solution are prepared by using a mobile phase, and are analyzed after passing through a 0.22 mu m water-based filter membrane. The retention times of the series of standard samples and the corresponding molecular mass logarithmic values are automatically analyzed by GPC software and molecular weight standard curves are generated. The relative molecular mass of each peak in the chromatogram of the sample to be measured is automatically calculated by GPC software based on a standard curve.

1.3.5 Effect of enzyme addition amount, pH, enzymatic hydrolysis temperature and time on proliferation-promoting Activity

Accurately weighing 1.00+/-0.01 g of pig brain homogenate, placing the pig brain homogenate into a 250mL conical flask, adding 200mL of deionized water, magnetically stirring for 30min, respectively carrying out enzymolysis under the optimal conditions of 30mg of enzyme addition, pH7.0, enzymolysis temperature of 42 ℃ and enzymolysis time of 8h, and sequentially researching the influence of enzyme addition, pH, enzymolysis temperature and time on the proliferation promoting activity of the enzymolysis solution.

1.3.6 optimization of extraction process of pig brain proliferation-promoting active peptide

Aiming at the extraction and preparation process of the pig brain proliferation-promoting active peptide, the L9 (34) orthogonal test is adopted to optimize the addition amount (A, mg), pH (B), enzymolysis temperature (C, DEGC) and enzymolysis time (D, h) of trypsin, and the factors and the levels are shown in the table 1.

Table 1: factors and levels

1.3.7 data statistics

The test results were analyzed by SPSS 18.0 software and the differences between groups were compared using One-way ANOVA test to compare the significance of the differences.

2. Results and analysis

2.1 screening of porcine brain-proliferation-promoting peptide preparation enzymes

As shown in FIG. 1, the proliferation-promoting ability of NE-4C cells is improved with the increase of the concentration of the polypeptide in the enzymatic hydrolysate by adopting trypsin for enzymatic hydrolysis of pig brain protein, but when the concentration is higher than 5mg/ml, the proliferation-promoting ability is not continuously enhanced, so that the method is suitable for culturing NE-4C cells when the concentration of pig brain polypeptide is 5 mg/ml.

As can be seen from fig. 2, the proliferation promoting activity of the polypeptide hydrolysate after trypsin enzymatic hydrolysis of pig brain is significantly higher than that of the complete culture medium (P < 0.05) compared with the complete culture medium of the control group, and the enzymatic hydrolysis products of pepsin and lipase have no significant difference (P > 0.05), which is probably related to the relatively high small amount of polypeptide after trypsin enzymatic hydrolysis of pig brain. Many polypeptides that regulate and promote cell proliferation exist in the form of short peptides with relatively small molecular weights, such as silkworm chrysalis protein peptides, osteogenic growth peptides, neuropeptide P substances.

2.2 molecular weight distribution of Polypeptides in pig brain enzymatic hydrolysate

Table 2: molecular weight distribution of polypeptide in pig brain enzymolysis liquid

As can be seen from fig. 3, 4, 5, 6 and table 2, the pig brain homogenate had a low polypeptide content and could not be detected by the instrument before enzymolysis. After pepsin, trypsin and complex enzyme are used for enzymatic hydrolysis of the pig brain protein, wherein the complex enzyme is a mixed enzyme of the pepsin and the trypsin, the molecular weight distribution of the polypeptide is mainly concentrated below 2000Da, and the pepsin and the trypsin are sequentially subjected to enzymatic hydrolysis. In the trypsin enzymatic hydrolysis product, the molecular weight distribution of the polypeptide is mainly concentrated below 500Da, and the polypeptide with the molecular weight has higher bioactivity. After the pig brain is subjected to enzymolysis by trypsin, the polypeptide with the molecular weight of 180 Da-500 Da accounts for 42.42 percent of the total polypeptide content, and the polypeptide with the molecular weight of less than 180Da accounts for 30.61 percent, which is higher than pepsin and complex enzyme enzymolysis products, so that the trypsin is superior to pepsin and complex enzyme in the aspect of preparing small molecular peptides of the pig brain; the abscissa in fig. 3, 4, 5 and 6 represents the retention time in min, and the ordinate represents the peak intensity.

The polypeptide content of the composite enzymolysis liquid is lower than that of other two enzymes, and the polypeptide content is only 0.1 percent and 0.61 percent of the total polypeptide content. The results show that the complex enzyme can increase the proportion of polypeptides with a molecular weight of <2000Da, but the effect of preparing polypeptides with a molecular weight of <180Da is not as good as that of trypsin. Thus, we have found that the pro-proliferative activity of trypsin enzymatic products is related to their higher proportion of short peptides with a molecular weight <180 Da.

2.3 Effect of single factors on pro-proliferative Activity

As shown in FIG. 7, the activity of the porcine brain proliferation-promoting peptide was strongest when the enzyme amounts were 20mg and 30 mg; as can be seen from fig. 8, the proliferation-promoting activity gradually increased with increasing enzymolysis time, but there was no significant difference; from FIG. 9, it is understood that the activity of the prepared porcine brain pro-proliferative peptide is the lowest when the enzymolysis temperature is 30 ℃, and the pro-proliferative activity can be remarkably increased when the enzymolysis temperature is 45 ℃; as can be seen from fig. 10, there was no significant difference in the effect of pH on pro-proliferative activity.

2.4 orthogonal optimization of porcine brain polypeptide preparation

According to the single factor experimental result, the proliferation promoting activity is taken as an index according to the factors and the levels listed in the table 1, and the proliferation promoting activity is calculated by L9 (3 ⁴ ) Orthogonal testExtraction process of pig brain proliferation promoting peptide. The results are shown in Table 3.

Table 3: l9 (3) ⁴ ) Orthogonal test

The influence of four factors on the pig brain proliferation promoting active peptide enzymolysis process is respectively shown in the table 3, and the main factors and the secondary factors affecting the enzymolysis process are respectively shown in the table 3. The preferred level is 25mg of enzyme and the enzymolysis time is 4 hours, the enzymolysis temperature is 45 ℃ and the pH is 7.5. Under optimal conditions, NE-4C has a relative viability of 0.161.

3. Conclusion(s)

The experiment prepares the proliferation-promoting active peptide by enzymatic hydrolysis of pig brain. By taking the proliferation promoting capacity of the enzymolysis liquid as an index, the trypsin is more suitable for preparing the pig brain proliferation promoting peptide than pepsin and complex enzyme. High performance gel permeation chromatography shows that the polypeptide with molecular weight less than 500Da accounts for 73.03% of the total polypeptide content in the trypsin degradation product, especially the polypeptide with molecular weight less than 180Da accounts for 30.67%, which is far higher than 8.75% of pepsin and 5.62% of complex enzyme, and shows that the proliferation promoting activity of the trypsin degradation pig brain product is related to the high proportion of low molecular weight peptide. The orthogonal test is designed according to the enzyme adding amount, the enzymolysis time, the enzymolysis temperature and the pH of trypsin, and the optimal level is 25mg of enzyme adding amount, the enzymolysis time is 4 hours, the enzymolysis temperature is 45 ℃, and the pH is 7.5. Under optimal conditions, NE-4C has a relative viability of 0.161. The pig brain polypeptide prepared by the experiment has a certain proliferation promoting activity, can be used as a functional ingredient to be applied to related foods, and provides a new reference for high-value utilization of pig brain.

Example 1:

(a) Selecting 100g of pig brain and 900ml of physiological saline, mixing the selected pig brain and the physiological saline in proportion, and homogenizing for 15min;

(b) Adding 8U/mg trypsin into the homogenized mixture for stirring and enzymolysis, wherein the stirring time is 25min, the pH value of the solution in the enzymolysis process is 6-8, the enzymolysis temperature is 45 ℃, and the enzymolysis time is 5h;

Comprising the steps of (a) a step of,

(f) Freeze-dehydrating the filter residue I in the step (c), taking cold acetone with the temperature of-15 ℃ to-2 ℃, and mixing the cold acetone with the freeze-dehydrated filter residue I, wherein the volume dosage of the cold acetone is 2.5 times of the volume of the freeze-dehydrated filter residue I;

(g) Standing the mixed substances in the step (f) at a low temperature, centrifuging after standing is completed to obtain filter residue II, wherein the time of standing at the low temperature is 25min, the centrifuging speed is 10000rpm, and the centrifuging time is 8min;

(i) Mixing the alkali ethanol solution and the brain dry powder in the step (h) by stirring, filtering to obtain filtrate II, wherein the temperature of stirring and mixing is 18 ℃ and the stirring time is 40min; the feed liquid ratio of the brain dry powder to the alkali ethanol solution is 1/30, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 100:0.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 4;

table 4: when the volume ratio of ethanol to ammonia water is 100:0, the content of components in the phospholipid extract in the brain

Example 2:

the volume ratio of ethanol to ammonia in the alkaline ethanol solution was 100:0.5, and the other steps and data were the same as in example 1.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 5;

table 5: when the volume ratio of ethanol to ammonia water is 100:0.5, the content of the components in the phospholipid extract in the brain

Example 3:

the volume ratio of ethanol to ammonia in the alkaline ethanol solution was 100:1, and the other steps and data were the same as in example 1.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 6;

table 6: when the volume ratio of ethanol to ammonia water is 100:1, the content of components in the phospholipid extract in the brain

Example 4:

the volume ratio of ethanol to ammonia in the alkaline ethanol solution was 100:2, and the other steps and data were the same as in example 1.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 7;

Table 7: when the volume ratio of ethanol to ammonia water is 100:2, the content of components in the phospholipid extract in the brain

Example 5:

the volume ratio of ethanol to ammonia in the alkaline ethanol solution was 100:3, and the other steps and data were the same as in example 1.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 8;

table 8: when the volume ratio of ethanol to ammonia water is 100:3, the content of components in the phospholipid extract in the brain is

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As can be seen from fig. 11, as the volume ratio of ethanol to ammonia water increases, the content of phosphatidylcholine (lecithin, PC) gradually increases, and when the volume ratio of ethanol to ammonia water in the alkaline ethanol solution is 100/2-100/3, the content of lecithin is significantly higher than other ratios; when the volume ratio of ethanol to ammonia water is 100/3, the prepared lecithin has the highest content.

Example 6:

Comprising the steps of (a) a step of,

(i) Mixing the alkali ethanol solution and the brain dry powder in the step (h) by stirring, filtering to obtain filtrate II, wherein the temperature of stirring and mixing is 18 ℃ and the stirring time is 20min; the feed liquid ratio of the brain dry powder to the alkali ethanol solution is 1/30, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 100:3. Wherein the stirring time is the extraction time.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 9;

Table 9: the component content of the phospholipid extract in the brain when the stirring time in the step (i) is 20min

Example 7:

the stirring time in step (i) was 30min, and the other steps and data were the same as in example 6.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 10;

table 10: the component content of the phospholipid extract in the brain when the stirring time in the step (i) is 30min

Example 8:

the stirring time in step (i) was 40min, and the other steps and data were the same as in example 6.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 11;

table 11: the content of the components in the phospholipid extract in the brain is obtained when the stirring time in the step (i) is 40min

Example 9:

the stirring time in step (i) was 50min, and the other steps and data were the same as in example 6.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 12;

table 12: the content of the component in the phospholipid extract in the brain is obtained when the stirring time in the step (i) is 50min

Example 10:

The stirring time in step (i) was 60min, and the other steps and data were the same as in example 6.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 13;

table 13: the content of the components in the phospholipid extract in the brain is obtained when the stirring time in the step (i) is 60min

As can be seen from fig. 12, the extraction time is in the range of 20min to 60min, and the content of phosphatidylcholine (lecithin, PC) gradually increases as the extraction time increases, and the content of lecithin is the highest when the extraction time is 60min, but the extraction time can be suitably shortened in consideration of the extraction efficiency and the production energy consumption.

Example 11:

Comprising the steps of (a) a step of,

(i) Mixing the alkali ethanol solution and the brain dry powder in the step (h) by stirring, filtering to obtain filtrate II, wherein the temperature of stirring and mixing is 20 ℃ and the stirring time is 60min; the feed liquid ratio of the brain dry powder to the alkali ethanol solution is 1/30, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 100:3. Wherein the stirring time is the extraction time; the temperature of the stirring and mixing is the extraction temperature.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 14;

Table 14: the component content of the phospholipid extract in the brain is obtained when the temperature of stirring and mixing in the step (i) is 20 DEG C

Example 12:

the temperature of the stirred mixture in step (i) was 28℃and the other steps and data were the same as in example 11.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 15;

table 15: the component content of the phospholipid extract in the brain is obtained when the stirring and mixing temperature in the step (i) is 28 DEG C

Example 13:

the temperature of the stirred mixture in step (i) was 36℃and the other steps and data were the same as in example 11.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 16;

table 16: the component content of the phospholipid extract in the brain is obtained when the stirring and mixing temperature in the step (i) is 36 DEG C

Example 14:

the temperature of the stirred mixture in step (i) was 44℃and the other steps and data were the same as in example 11.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 17;

table 17: the component content of the phospholipid extract in the brain when the stirring and mixing temperature in the step (i) is 44 DEG C

Example 15:

the temperature of the stirred mixture in step (i) was 52℃and the other steps and data were the same as in example 11.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 18;

table 18: the component content of the phospholipid extract in the brain is obtained when the stirring and mixing temperature in the step (i) is 52 DEG C

As is clear from fig. 13, the total content of phosphatidylcholine (lecithin, PC) gradually decreased gradually with increasing extraction temperature in the range of 20 to 52 ℃.

Example 16:

Comprising the steps of (a) a step of,

(i) Mixing the alkali ethanol solution and the brain dry powder in the step (h) by stirring, filtering to obtain filtrate II, wherein the temperature of stirring and mixing is 20 ℃ and the stirring time is 60min; the feed liquid ratio of the brain dry powder to the alkali ethanol solution is 1/5, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 100:3. Wherein the stirring time is the extraction time; the temperature of the stirring and mixing is the extraction temperature.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain as shown in table 19;

table 19: when the feed liquid ratio of brain dry powder and alkali ethanol solution is 1/5, the component content of phospholipid extract in brain

Example 17:

the ratio of the brain powder to the alkali ethanol solution was 1/10, and the other steps and data were the same as in example 16.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 20;

table 20: when the feed liquid ratio of brain dry powder and alkali ethanol solution is 1/10, the component content of phospholipid extract in brain

Example 18:

the ratio of the brain powder to the alkali ethanol solution was 1/15, and the other steps and data were the same as in example 16.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 21;

table 21: when the feed liquid ratio of brain dry powder to alkali ethanol solution is 1/15, the component content of phospholipid extract in brain

Example 19:

the ratio of the brain powder to the alkali ethanol solution was 1/25, and the other steps and data were the same as in example 16.

HPLC detection of Phosphatidylcholine (PC), phosphatidylinositol (PI) and Phosphatidylethanolamine (PE) content of phospholipid extract in brain is shown in table 22;

table 22: when the feed liquid ratio of brain dry powder and alkali ethanol solution is 1/25, the component content of phospholipid extract in brain

As is clear from fig. 14, when the ratio of the dry brain powder to the alkali ethanol solution is in the range of 1/10 to 1/25, the total content of phosphatidylcholine (lecithin, PC) tends to gradually increase with the increase of the ratio of the liquid to the solid, and when the ratio of the liquid to the solid is 1/25, the content of lecithin is highest, but in consideration of the excessively high ratio of the liquid to the solid, the concentration cost is increased and the production energy is increased, so that the ratio of the liquid to the solid is preferably controlled to about 1/25.

Claims

1. A pig brain proliferation-promoting peptide-in-brain phospholipid co-production method is characterized by comprising the following steps: comprises the steps of,

(b) Adding trypsin into the homogenized mixture for stirring and enzymolysis;

the stirring time in the step (b) is 20-30 min; the addition amount of the trypsin is 5U/mg-10U/mg; the pH value of the enzymolysis process solution in the step (b) is 6-8; the enzymolysis temperature in the step (b) is 36-50 ℃; the enzymolysis time in the step (b) is 3-8 h;

(c) Filtering the substances subjected to enzymolysis to obtain filtrate I and filter residue I;

(e) Vacuum drying and dehydrating the substances subjected to ultrafiltration in the step (d) to obtain an extract I, namely the porcine brain proliferation-promoting peptide;

(d1) Ultrafiltering the filtrate I in the step (c) through a cellulose membrane with the molecular weight of 5000 Da-6000 Da; continuously ultrafiltering until the volume of the filtrate I is changed to 1/3.5-1/3 of the original volume to obtain trapped fluid I and passing fluid I;

(d2) Continuing ultrafiltration of the passing liquid I in the step (d 1) by a cellulose membrane with the molecular weight of 950Da to 1500 Da; continuously ultrafiltering until the volume of the passing liquid I is changed to 1/3.5-1/3 of the original volume to obtain trapped liquid II and passing liquid II;

(e1) Vacuum drying and dewatering the liquid II to obtain an extract I, namely the porcine brain proliferation-promoting peptide;

the temperature of stirring and mixing in the step (i) is 15-52 ℃; the stirring time in the step (i) is 20-60 min;

the ratio of the brain dry powder to the alkali ethanol solution is 1/25-1/10; the alkali ethanol solution is prepared by mixing ethanol and ammonia water, and the volume ratio of the ethanol to the ammonia water in the alkali ethanol solution is 25-100;

(j) Vacuum drying the filtrate II in the step (i) to obtain powder, namely phospholipid extract in brain;

the pig brain proliferation promoting peptide is pig brain NE-4C cell proliferation promoting peptide.

2. The method for coproducing the porcine brain pro-proliferative peptide-in-brain phospholipid according to claim 1, wherein the method comprises the following steps of: the mixing ratio of the pig brain to the normal saline in the step (a) is 1/9-1/2; the homogenization time in the step (a) is 8-20 min.

3. The method for coproducing the porcine brain pro-proliferative peptide-in-brain phospholipid according to claim 1, wherein the method comprises the following steps of: the pressure of the inlet water in the ultrafiltration process is 2.81Bar to 3.05Bar, and the reflux pressure in the ultrafiltration process is 0.53Bar to 0.67Bar.

4. The method for coproducing the porcine brain pro-proliferative peptide-in-brain phospholipid according to claim 1, wherein the method comprises the following steps of: the volume dosage of the cold acetone is 2-3 times of the volume of the filter residue I after freeze dehydration; the low-temperature standing time is 20-30 min.

5. The method for coproducing the porcine brain pro-proliferative peptide-in-brain phospholipid according to claim 1, wherein the method comprises the following steps of: the centrifugal rotating speed in the step (g) is 8000 rpm-12000 rpm; the centrifugation time is 5-10 min.