CN115669945B - Liposome-coated polypeptide with neurotrophic effect, and preparation method and application thereof - Google Patents
Liposome-coated polypeptide with neurotrophic effect, and preparation method and application thereof Download PDFInfo
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- CN115669945B CN115669945B CN202211307253.4A CN202211307253A CN115669945B CN 115669945 B CN115669945 B CN 115669945B CN 202211307253 A CN202211307253 A CN 202211307253A CN 115669945 B CN115669945 B CN 115669945B
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Abstract
The application relates to a liposome-encapsulated polypeptide with a neurotrophic effect, and a preparation method and application thereof. A liposome encapsulated polypeptide having neurotrophic effects, comprising a core material and a liposome shell; a core material comprising a whey protein polypeptide and 5-hydroxytryptamine; the preparation method of the whey protein polypeptide comprises the following steps: mixing whey protein powder with water, and performing microwave, steam and drying treatment to obtain pretreated whey protein powder; mixing the pretreated whey protein powder with water to obtain whey protein powder water solution, and regulating pH and temperature for enzymolysis; centrifuging the enzymolysis product, collecting supernatant, dialyzing the supernatant, purifying, and freeze-drying to obtain whey protein polypeptide; the molecular weight of the whey protein polypeptide is 2-3kDa. The liposome wrapping polypeptide with the neurotrophic effect has the effects of supplementing brain nutrition, keeping brain healthy and improving memory.
Description
Technical Field
The application relates to the technical field of foods, in particular to a liposome-encapsulated polypeptide with a neurotrophic effect, a preparation method and application thereof.
Background
Memory is the process of the human brain to memorize, hold, reproduce or re-recognize the experienced things, and is the process of people to encode and process a great amount of information obtained in life and study, input and store the information into the brain, and extract the related stored information when necessary, and apply the information to practical activities.
Due to life pressure, poor sleep and long-term malnutrition can lead to near memory decline. Meanwhile, with the increase of age, cerebral cell hypoxia and ischemic injury, cerebral atrophy and cerebral lesion cause far memory decline, so that the memory decline occurs to different degrees for people of all ages in society.
Although proper supplementation of some nutrients may help people improve memory, people's daily diets often fail to meet the body's needs for these nutrients. Therefore, there is a need to provide a product for improving memory.
Disclosure of Invention
In order to improve memory, the application provides a liposome-encapsulated polypeptide with a neurotrophic effect, and a preparation method and application thereof.
The innovation of the application mainly comprises the following steps: firstly, collecting whey protein polypeptides with various molecular weights in a proteolytic mode, and then, screening the mixture of the whey protein polypeptides according to the target molecular weight by adopting a molecular membrane filtration technology to finally obtain the whey protein polypeptides with the target molecular weight. And finally, obtaining the required liposome-coated polypeptide by adopting a high-speed homogenizer and combining a corresponding production process through a large number of experiments. The phase pertinently solves the world problem that the corresponding active ingredients in the whey protein are degraded in the foreseeable gastrointestinal tract digestion process, so that the product efficacy is lost.
According to the liposome-coated polypeptide production process, the technological problem that how to keep the biological activity of the product in the digestion process cannot be solved by the rough mixing of whey protein products in the original industry is solved in a breakthrough manner by separating firstly, then, coating pertinently (can be regarded as a single mixing process among individual raw materials), and finally, the due characteristics of the product are maintained through the ingeniously designed mixing process. The technical disclosure which is completely the same as the technical characteristics of the project is not found in the public literature at home and abroad through the search of a Shanghai science and technology new station of the academy of sciences of China. The conclusion is that the technical project is novel.
In a first aspect, the present application provides a liposome-encapsulated polypeptide with a neurotrophic effect, and a preparation method and an application thereof, and adopts the following technical scheme:
a liposome encapsulated polypeptide having neurotrophic effects, comprising a core material and a liposome shell;
the core material comprises the following components in parts by weight:
30-50 parts of whey protein polypeptide;
20-30 parts of 5-hydroxytryptamine;
the preparation steps of the whey protein polypeptide are as follows:
s1: mixing whey protein powder with water, and performing microwave, steam and drying treatment to obtain pretreated whey protein powder;
s2: mixing the pretreated whey protein powder with water to obtain whey protein powder water solution, regulating the pH and temperature of the whey protein powder water solution, and adding alkaline protease for enzymolysis to obtain an enzymolysis product;
s3: centrifuging the enzymolysis product, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze-drying to obtain whey protein polypeptide;
the molecular weight of the whey protein polypeptide is 2-3kDa.
By adopting the technical scheme, firstly, the whey protein polypeptide and the 5-hydroxytryptamine are compounded, so that on one hand, the stability and the fat solubility of the whey protein polypeptide can be improved, the whey protein polypeptide can be favorably absorbed by a human body, and the efficacy of the whey protein polypeptide is improved. On the other hand, the full activity of the 5-hydroxytryptamine can be maintained, the 5-hydroxytryptamine can permeate through the blood sugar barrier, nutrition is provided for nerve cells, dormant nerve stem cells are activated, regeneration and functional repair of damaged nerve cells are promoted, and the memory is improved.
Secondly, in the preparation of the whey protein polypeptide, the whey protein powder is subjected to microwave and high-temperature steam treatment in advance, so that the whey protein is moderately denatured, the hydrolysis degree of the whey protein is improved, the enzyme exposing sites are facilitated, the enzymolysis of alkaline protease is accelerated, and the efficiency of the enzymolysis of the whey protein polypeptide is improved. Then, the moderately denatured whey protein powder is treated by a specific enzymolysis step and a post-treatment step, so that the activity and the nutritional value of the obtained whey protein polypeptide are improved.
Thirdly, the self-made whey protein polypeptide with the molecular weight of 2-3kDa has higher activity and nutrition value, can enter human cells more easily, promotes the combination of 5-hydroxytryptamine and corresponding receptors, enhances the activity of the receptors, and supplies cell nutrition, thereby supplementing brain nutrition and improving memory.
Therefore, the self-made whey protein polypeptide with the molecular weight of 2-3kDa and 5-hydroxytryptamine are adopted for compounding, and the obtained liposome coated polypeptide with the neurotrophic effect has the effects of supplementing brain nutrition, keeping brain health and improving memory.
Preferably, in the preparation step S1 of the whey protein polypeptide, the power of the microwave treatment is 600w-700w, and the temperature of the steam treatment is 105-115 ℃.
By adopting the technical scheme: after the whey protein powder is subjected to microwave treatment and steam treatment under the conditions, the whey protein powder is not only subjected to enzymolysis, so that the enzymolysis efficiency of the whey protein polypeptide is improved, the obtained whey protein polypeptide can promote cell division, regulate metabolism of cells, improve the number, quality and speed of protein synthesis in the cells to be in a normal state, and further improve the memory.
Preferably, in the step S2 of preparing the whey protein polypeptide, the mass-volume concentration of the aqueous whey protein powder solution is (4-6) g/100ml; the alkaline protease is added in an amount of 3000-4000U/g.
By adopting the technical scheme: the quality concentration of whey protein and the addition amount of alkaline protease in pretreatment are controlled, the activity and the nutritional value of the obtained whey protein polypeptide are higher, the whey protein polypeptide is compounded with 5-hydroxytryptamine, and the obtained liposome with the neurotrophic effect wraps the polypeptide, so that the effect of improving memory is better.
Preferably, in the step S2 of preparing the whey protein polypeptide, the pH value of the whey protein powder aqueous solution is 9-11, the temperature is 55-60 ℃, and the enzymolysis is carried out for 1.5-2 hours.
By adopting the technical scheme: is beneficial to improving the enzymolysis efficiency of whey protein, improving the activity of the obtained enzymolysis product, supplementing brain nutrition and improving memory.
Preferably, the weight ratio of the core material to the liposome is 1 (1.2-1.6).
By adopting the technical scheme: the formed liposome coated polypeptide with the neurotrophic effect has good stability and fat solubility, is favorable for promoting the absorption of the whey protein polypeptide by human bodies, and further improves the effect of improving the memory.
Preferably, the core material further comprises an agaricus blazei polypeptide having a molecular weight of 0.5-1kDa.
By adopting the technical scheme: the agaricus blazei murill polypeptide is an effective component extracted from agaricus blazei murill, and the polypeptide in a plurality of animal and plant proteins shows biological activity after in-vivo or in-vitro release. The agaricus blazei murill polypeptide with the molecular weight of 0.5-1kDa is added, so that the improvement of the in vivo antioxidation capability is stronger, the reduction of cell oxidative damage is facilitated, and the aging is effectively delayed.
In a second aspect, the present application provides a method for preparing a liposome-encapsulated polypeptide with neurotrophic effect, which adopts the following technical scheme:
a method for preparing liposome-encapsulated polypeptide with neurotrophic effect, comprising the following steps:
a1: dissolving lecithin, cholesterol and vitamin E in absolute ethyl alcohol to prepare a lipid film, drying, adding Tween 80 aqueous solution for hydration, shearing, purifying and breaking a film to obtain a liposome;
a2: stirring and mixing whey protein polypeptide, 5-hydroxytryptamine and agaricus blazei murill polypeptide to obtain a core material;
a3: mixing the liposome and the core material, and filtering and drying to obtain the liposome-encapsulated polypeptide with neurotrophic effect.
The unique actions of lecithin in liposomes are: the content of lecithin in the cerebral nerve cells is about 17% -20% of the mass of the cerebral nerve cells. The choline is the basic component of soybean lecithin, and sufficient supply of the lecithin can ensure that sufficient choline and acetyl in human body are synthesized into acetylcholine. The acetylcholine is an information conducting substance in the brain, thereby improving the activation degree of brain cells and improving memory.
Lecithin has effects of emulsifying and decomposing oil, promoting blood circulation, improving serum lipid, scavenging peroxide, reducing cholesterol and neutral fat content in blood, reducing residence time of fat in blood vessel inner wall, promoting atherosclerotic plaque dissipation, and preventing damage of blood vessel inner membrane caused by cholesterol.
By adopting the technical scheme, the liposome has higher embedding rate on the whey protein polypeptide, can protect the whey protein polypeptide, improve the stability of the whey protein polypeptide, delay the release of the whey protein polypeptide, promote the absorption of the whey protein polypeptide and improve the compounding effect of the whey protein polypeptide and 5-hydroxytryptamine. Therefore, the brain nutrition can be effectively supplemented, and the memory can be improved.
In a third aspect, the present application provides an application of a liposome-encapsulated polypeptide with a neurotrophic effect, which adopts the following technical scheme:
use of a liposome-encapsulated polypeptide having a neurotrophic effect in a beverage.
By adopting the technical scheme, the solid beverage has good flavor, and can supplement brain nutrition and improve memory. When the liposome-encapsulated polypeptide with the neurotrophic effect is applied to beverages, the recommended daily dosage of the liposome-encapsulated polypeptide with the neurotrophic effect is less than or equal to 0.2g/kg.
In summary, the present application has the following beneficial effects:
1. the activated whey protein polypeptide with specific molecular weight is preferably adopted in the application, so that the whey protein polypeptide has higher activity and nutritive value, and can promote cell division, regulate cell metabolism and improve the number, quality and speed of intracellular protein synthesis in a normal state; therefore, the whey protein polypeptide with specific molecular weight and the 5-hydroxytryptamine are compounded, so that nutrition can be provided for nerve cells, the nerve cells can be repaired, and the memory can be improved;
2. according to the method, a special channel of a cell membrane is opened through a liposome wrapping technology and directly enters blood, so that the complete activity of whey protein polypeptide and 5-hydroxytryptamine is improved, meanwhile, the whey protein polypeptide and 5-hydroxytryptamine are promoted to permeate through a blood sugar barrier, nutrition is provided for nerve cells, dormant nerve stem cells are activated, regeneration of injured nerve cells and restoration of functions of injured nerve cells are promoted, and the memory is improved.
Drawings
FIG. 1 is a graph of escape latency versus days for 6 groups of rats after molding;
FIG. 2 is a graph of HE staining of hippocampal tissue from 6 groups of rats;
FIG. 3 is a diagram showing the detection of beta-catenin expression in hippocampal immunohistochemistry in 6 groups of rats;
FIG. 4 is a graph showing the expression levels of mRNA of beta-catenin and GSK-3 beta in hippocampal tissues of 6 groups of rats;
FIG. 5 is a graph of bands of expression of beta-catenin, GSK-3 beta protein in hippocampal tissue of each group of rats.
Detailed Description
The present application is described in further detail below with reference to the drawings and examples.
Preparation example
Preparation example 1
A whey protein polypeptide is prepared by the following steps:
s1: mixing 100g of whey protein powder with 100g of water, and then carrying out microwave, water vapor and drying treatment to obtain pretreated whey protein powder;
the power of the microwave treatment is 600w, the time is 0.5min, the temperature of the water vapor treatment is 105 ℃, the time is 5min, the temperature of the drying treatment is 100 ℃, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain whey protein water solution with the mass-volume concentration of 3g/100ml, regulating the pH of the whey protein water solution to 8 and the temperature to 55 ℃, adding alkaline protease 2800u/g, and carrying out enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has the product number of FDG-4001.
S3: inactivating enzyme at 100deg.C for 8min, centrifuging at 10deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and lyophilizing to obtain whey protein polypeptide.
The molecular weight of the whey protein polypeptide obtained by dialysis interception in the preparation example of the present application was detected to be 2kDa.
In the preparation example of the application, the whey protein powder is food grade and purchased from the biological technology company of Shenzhen Jin Fuyuan; 5-hydroxytryptamine, purchased from Beijing Kangjia macro-biosciences limited.
Preparation example 2
A whey protein polypeptide differs from preparation example 1 in that in preparation step S3 of the whey protein polypeptide, the molecular weight of the whey protein polypeptide obtained by dialysis entrapment is 2.5kDa.
Preparation example 3
A whey protein polypeptide differs from preparation example 1 in that in preparation step S3 of the whey protein polypeptide, the molecular weight of the whey protein polypeptide obtained by dialysis entrapment is 3.0kDa.
Preparation example 4
A whey protein polypeptide differs from preparation example 2 in that in preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein aqueous solution is 4g/100ml; the amount of alkaline protease added was 3000U/g.
Preparation example 5
A whey protein polypeptide differs from preparation example 2 in that in preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein aqueous solution is 5g/100ml; the alkaline protease was added at 3500u/g.
Preparation example 6
A whey protein polypeptide differs from preparation example 2 in that in preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein aqueous solution is 6g/100ml; the amount of alkaline protease added was 4000u/g.
Preparation example 7
A whey protein polypeptide differs from preparation example 5 in that in preparation step S2 of the whey protein polypeptide, the pH of the whey protein aqueous solution is 9 and the temperature is 55deg.C, and the enzymolysis is performed for 1.5h.
Preparation example 8
A whey protein polypeptide differs from preparation example 5 in that in preparation step S2 of the whey protein polypeptide, the pH of the whey protein aqueous solution is 10 and the temperature is 58℃and the enzymolysis is carried out for 1.7 hours.
Preparation example 9
A whey protein polypeptide differs from preparation example 5 in that in preparation step S2 of the whey protein polypeptide, the pH of the whey protein aqueous solution is 11, the temperature is 60℃and the enzymolysis is carried out for 2 hours.
Preparation example 10
A whey protein polypeptide differs from preparation example 8 in that in preparation step S1 of the whey protein polypeptide, the power of the microwave treatment is 700w and the temperature of the water vapor treatment is 115 ℃.
PREPARATION EXAMPLE 11
A whey protein polypeptide differs from preparation example 8 in that in preparation step S1 of the whey protein polypeptide, the power of the microwave treatment is 800w and the temperature of the water vapor treatment is 120 ℃.
Preparation example 12
A whey protein polypeptide differs from preparation example 8 in that in preparation step S1 of the whey protein polypeptide, the power of the microwave treatment is 550w and the temperature of the water vapor treatment is 102 ℃.
Examples
Example 1
The liposome-encapsulated polypeptide with neurotrophic effect is prepared by the following steps of:
a1: dissolving lecithin, cholesterol and vitamins in absolute ethanol at 60 ℃ to obtain a mixture;
shearing the mixture at 18000r/min for 5min, homogenizing at 15MPa for 5min, and evaporating at 80deg.C in water bath under reduced pressure to obtain liposome film;
adding Tween 80 water solution with the mass percentage concentration of 0.5% into the liposome film, firstly hydrating for 1h at 50 ℃, then shearing for 5min at 18000r/min, and finally purifying and breaking the film to obtain the liposome.
A2: mixing whey protein polypeptide, 5-hydroxytryptamine and Agaricus blazei polypeptide under stirring to obtain core material.
A3: dissolving the liposome obtained in the step A1 in water to obtain a liposome aqueous solution with the mass percentage concentration of 20 mg/mL; and (3) adding the core material obtained by the step A2 into the liposome aqueous solution, then carrying out ultrasonic stirring at 100w for 1min, and then carrying out filtration membrane filtration and drying treatment to obtain the liposome-encapsulated polypeptide with the neurotrophic effect.
Wherein, the weight ratio of the core material to the liposome is 1:1;
in the core material, the whey protein polypeptide is prepared by using the whey protein polypeptide prepared in preparation example 1.
Examples 2 to 3
A liposome encapsulated polypeptide having a neurotrophic effect, which is different from example 1 in that each component and its corresponding weight are shown in table 1.
TABLE 1 Components and weights (kg) of the components in examples 1-3
Examples 4 to 5
A liposome-encapsulated polypeptide having a neurotrophic effect, which differs from example 2 in that in the preparation step S3 of the whey protein polypeptide, the molecular weight of the whey protein polypeptide obtained by dialysis interception is different; the whey protein polypeptides were prepared as shown in table 2.
Table 2 preparation examples of the use of whey protein polypeptides in examples 4-5
| Examples | Example 2 | Example 4 | Example 5 |
| Preparation example | Preparation example 1 | Preparation example 2 | Preparation example 3 |
Examples 6 to 8
A liposome-encapsulated polypeptide having a neurotrophic effect, which is different from example 4 in that in the preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the aqueous whey protein solution and the addition amount of alkaline protease are different; the whey protein polypeptides were prepared as shown in table 3.
Table 3 shows examples of the preparation of whey protein polypeptides used in examples 6-8.
| Examples | Example 4 | Example 6 | Example 7 | Example 8 |
| Preparation example | Preparation example 2 | Preparation example 4 | Preparation example 5 | Preparation example 6 |
Examples 9 to 11
A liposome-encapsulated polypeptide having a neurotrophic effect, which is different from example 7 in that in the preparation step S2 of the whey protein polypeptide, the pH value, temperature and time of enzymolysis of the aqueous solution of the whey protein polypeptide are different; the whey protein polypeptides were prepared as shown in table 4.
Table 4 examples of the preparation of whey protein polypeptides used in examples 9-11.
| Examples | Example 7 | Example 9 | Example 10 | Example 11 |
| Preparation example | Preparation example 5 | Preparation example 7 | Preparation example 8 | Preparation example 9 |
Examples 12 to 14
A liposome-encapsulated polypeptide having a neurotrophic effect, which is different from example 10 in that in the preparation step S1 of the whey protein polypeptide, the power of the microwave treatment and the temperature of the water vapor treatment are different; the whey protein polypeptide was prepared as shown in Table 5.
Table 5 examples of the preparation of whey protein polypeptides used in examples 12-14.
| Examples | Example 10 | Example 12 | Example 13 | Example 14 |
| Preparation example | Preparation example 8 | Preparation example 10 | PREPARATION EXAMPLE 11 | Preparation example 12 |
Examples 15 to 17
A liposome-encapsulated polypeptide having a neurotrophic effect, which is different from example 10 in that the core material further comprises an agaricus blazei polypeptide; the agaricus blazei polypeptides and their molecular weights are shown in table 6.
Table 6 Agaricus blazei Murill polypeptide (kg) and molecular weight (Da) of examples 15-17
In the above implementation, the agaricus blazei polypeptides used were purchased from Shaanxi Xiazhou biotechnology Co., ltd, and the molecular weights were customized.
Example 18
A liposome encapsulated polypeptide having a neurotrophic effect, which was different from example 15 in that the weight of the liposome was as shown in table 7.
TABLE 7 weight of liposomes (kg) in example 18
Since the weight ratio of the core material to the liposome is 1 (1.2-1.6) in the embodiment of the application, the effect on each property of the liposome-encapsulated polypeptide with the neurotrophic effect obtained by the application is the same, in the embodiment of the application, only the weight ratio of the core material to the liposome is 1:1.2, but the application of other addition weight ratios of the core material to the liposome in the application is not influenced.
Comparative example
Comparative example 1
A liposome encapsulated polypeptide differs from example 2 in that in the preparation step S3 of the whey protein polypeptide, the molecular weight of the whey protein polypeptide obtained by dialysis entrapment is 4kDa.
Comparative example 2
A liposome encapsulated polypeptide differs from example 2 in that in the preparation step S3 of the whey protein polypeptide, the molecular weight of the whey protein polypeptide obtained by dialysis entrapment is 1.5kDa.
Comparative example 3
A liposome-encapsulated polypeptide was different from example 2 in that in the preparation step S1 of whey protein polypeptide, 100g of whey protein powder was mixed with 100g of water and then subjected to only microwave treatment and drying treatment.
The preparation steps of the whey protein polypeptide are as follows:
s1: mixing 100g of whey protein powder with 100g of water, and performing microwave treatment and drying treatment to obtain pretreated whey protein powder;
the power of the microwave treatment is 600w, the time is 0.5min, the temperature of the drying treatment is 100 ℃ and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain whey protein water solution with the mass-volume concentration of 3g/100ml, regulating the pH of the whey protein water solution to 8 and the temperature to 55 ℃, adding alkaline protease 2800u/g, and carrying out enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has the product number of FDG-4001.
S3: inactivating enzyme at 100deg.C for 8min, centrifuging at 10deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and lyophilizing to obtain whey protein polypeptide.
The molecular weight of the whey protein polypeptide obtained by dialysis interception in the preparation example of the present application was detected to be 2kDa.
Comparative example 4
A liposome-encapsulated polypeptide was different from example 2 in that in the preparation step S1 of whey protein polypeptide, 100g of whey protein powder was mixed with 100g of water and then subjected to only steam treatment and drying treatment.
The preparation steps of the whey protein polypeptide are as follows:
s1: mixing 100g of whey protein powder with 100g of water, and then performing steam treatment and drying treatment to obtain pretreated whey protein powder;
the temperature of the water vapor treatment is 105 ℃, the time is 5min, the temperature of the drying treatment is 100 ℃, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain whey protein water solution with the mass-volume concentration of 3g/100ml, regulating the pH of the whey protein water solution to 8 and the temperature to 55 ℃, adding alkaline protease 2800u/g, and carrying out enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has the product number of FDG-4001.
S3: inactivating enzyme at 100deg.C for 8min, centrifuging at 10deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and lyophilizing to obtain whey protein polypeptide.
The molecular weight of the whey protein polypeptide obtained by dialysis interception in the preparation example of the present application was detected to be 2kDa.
Comparative example 5
A liposome-encapsulated polypeptide is different from example 2 in that 100g of whey protein powder is mixed with 100g of water in the preparation step S1 of whey protein polypeptide, and then subjected to steam, microwave and drying treatment.
The preparation steps of the whey protein polypeptide are as follows:
s1: mixing 100g of whey protein powder with 100g of water, and then carrying out steam, microwave and drying treatment to obtain pretreated whey protein powder;
the power of the microwave treatment is 600w, the time is 0.5min, the temperature of the water vapor treatment is 105 ℃, the time is 5min, the temperature of the drying treatment is 100 ℃, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain whey protein water solution with the mass-volume concentration of 3g/100ml, regulating the pH of the whey protein water solution to 8 and the temperature to 55 ℃, adding alkaline protease 2800u/g, and carrying out enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has the product number of FDG-4001.
S3: inactivating enzyme at 100deg.C for 8min, centrifuging at 10deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and lyophilizing to obtain whey protein polypeptide.
The molecular weight of the whey protein polypeptide obtained by dialysis interception in the preparation example of the present application was detected to be 2kDa.
Performance detection
1 materials and methods
1.1 materials
Experimental animals:
72 male SD rats, 12-16 weeks old, with a mass of 250+ -50 g, supplied by Xinjiang university laboratory animal management center, quality qualification code [ SCXK (New) 2019-0002], were routinely housed in SPF-class laboratories, maintained adequate ventilation and lighting conditions, 3-4 animals per cage, and were fed and eaten freely.
Main drugs and reagents:
the experimental sample is the liposome-encapsulated polypeptide with neurotrophic effect obtained in example 2, which is hereinafter referred to as liposome-encapsulated polypeptide;
d-galactose (D-Gal), donepezil hydrochloride was purchased from Shanghai Michelin Biochemical technologies Co., ltd;
beta-catenin (beta-catenin) antibodies were purchased from Abcam, uk;
glycogen synthase kinase 3 beta (GSK-3 beta) antibodies and GAPDH antibodies were all purchased from Beijing Boaosen Marine biotechnology company;
the total RNA extraction kit of the animal tissue, the first chain synthesis kit and the fluorescence quantification kit are purchased from Beijing Tiangen biochemical research Co;
BCA protein content assay kit, SDS-PAGE gel preparation kit, were all purchased from beijing solebao technologies.
The main instrument is as follows:
the Morris water maze video image monitoring analysis control system is purchased from Chengdu Ten technology limited public;
the QutantStudio6 real-time fluorescence PCR instrument, nanddrop000/000C spectrophotometer were all available from Thermo Inc. of America.
1.2 grouping and treatment methods for animals
Model group, donepezil group, liposome-encapsulated polypeptide low dose group, liposome-encapsulated polypeptide medium dose group, and liposome-encapsulated polypeptide high dose group, each group comprising 12. Except for the blank group, D-gal was infused with 150mg/kg for 1 time/day to establish an AD model, and the blank group was infused with an equal amount of physiological saline solution for 60 days. The donepezil group, the liposome-encapsulated polypeptide low dose group, the liposome-encapsulated polypeptide medium dose group and the liposome-encapsulated polypeptide high dose group are modeled and respectively and simultaneously, 0.9 mg/(kg.d) of donepezil and 420, 630 and 840 mg/(kg.d) of liposome-encapsulated polypeptide are respectively and gastrographically administered, and the time is 1 time per day, and 60 days are continuously carried out.
1.3 learning memory ability observations
Morris water maze test was used. And (3) treating each component for 60d, and carrying out Morris water maze experiments which are 6d in total, wherein the Morris water maze experiments are divided into positioning navigation experiments and space exploration experiments.
(1) Positioning navigation experiment: putting each group of rats from the center of a fixed quadrant to the pool wall into water, and recording the time for finding a platform within 120s, namely escape latency; over 120s without finding, guide it to climb up the platform and stay for 10s, training is performed for 5d,2 times/day.
(2) Space exploration experiment: the platform was removed on day 6 for each group of rats and the number of times it entered the effective area (area where the 1.5-fold original platform was located) within 120s was recorded.
1.4 pathological observations of hippocampal tissue
HE staining was used. Treating each group of the rats for 60 days, randomly selecting 4 rats, injecting 1% pentobarbital sodium into 0.45mL/100g of abdominal cavity for anesthesia, placing the rats in an anatomical disc after the rats are in coma, exposing the heart of the rats, and inserting and fixing a perfusion needle in the left ventricle; cutting right auricle, slowly pouring 150mL of sterile physiological saline frozen in advance (4 ℃) until the nasal tip of a rat is pale, and after the color of liver and lung turns from red to white, the clear liquid flows out from the right auricle, and then pouring 200mL of 4% paraformaldehyde; when the four limbs of the rat are convulsion and spasm, the head is broken to take out the brain, and the bilateral hippocampus of the brain is peeled off on an ice table, 4% paraformaldehyde is fixed, and the preparation is carried out for later use.
Sequentially performing gradient ethanol dehydration, tissue transparency, wax dipping and embedding, and cutting the hippocampal tissue into paraffin sections with the thickness of 4 mu m in a coronal manner. After the slices are baked for a plurality of hours, dewaxing, transparence and dehydration treatment are carried out on dewaxing liquid, after aseptic water immersion washing, hematoxylin is dyed for 1min,1% hydrochloric acid ethanol is differentiated, and then the slices are fully washed again; 1% eosin staining for 1min, gradient dewatering, dewaxing, transparent, neutral resin sealing. The individual groups of hippocampal tissue pathology were observed under an optical microscope.
1.5 observation of Positive expression of beta-catenin in hippocampal tissue
Immunohistochemical method was used. Taking coronal slices of each group of hippocampal tissues, soaking the slices in 3% hydrogen peroxide solution for 10min at room temperature after rehydration, washing with distilled water for 3min, and fully washing with Phosphate Buffer (PBS) for 3 times, each time for 5min; placing the mixture into citrate buffer solution, carrying out microwave repair (92-98 ℃) for 12min, cooling to normal temperature at room temperature, and washing with PBS for 3 times; after antigen retrieval, blocking solution was removed by blocking in 5% BSA for 30min, and diluted polyclonal rabbit anti-beta-catenin (1:500) was added dropwise and incubated overnight at 4℃in a refrigerator. Washing 3 times by using PBS in the next day, dripping diluted horseradish peroxidase-labeled secondary antibody on the surface of the sliced tissue, and incubating for 30min at 37 ℃; washing 3 times by PBS, uniformly dripping DAB chromogenic solution on the surface of the tissue for developing for 60s until the tissue is observed to appear brown yellow under a microscope, and ending tap water; hematoxylin counterstain for 1min, hydrochloric acid differentiation, gradient ethanol dehydration and neutral resin sealing. And observing the beta-catenin protein localization and quantity of each group of hippocampal tissues under a microscope.
1.6 detection of GSK-3 beta, beta-catenin mRNA expression in hippocampal tissue
The Real-timePCR method is adopted. The remaining rats of 60d were treated with each fraction, the brain was removed after cardiac perfusion, hippocampal tissue was isolated, frozen in liquid nitrogen and transferred to a-80℃refrigerator for storage. Weighing Hippocampus tissue 20mg, adding lysate and grinding beads, grinding for several times with an automatic grinding instrument, and extracting Hippocampus total mRNA. The purity of mRNA of each sample was measured, and reverse transcription was performed when the absorbance A260/A280 was measured at 260 and 280nm and was 1.9 to 2.0. The primer sequences of GSK-3 beta, beta-catenin and internal reference GAPDH are designed and synthesized by Shanghai Biotechnology Co-Ltd by using a real-time fluorescence quantitative PCR instrument, and the primer sequences are shown in Table 1.
PCR amplification conditions: cycling for 1 time at 95 ℃ for 15 min; cycling for 40 times at 95 ℃, 10s,60 ℃ and 32 s; and drawing a dissolution curve at 65-95 ℃. By 2 －ΔΔCt The relative expression of target gene is calculated by the method.
TABLE 1 primer sequences for GSK-3 beta, beta-catenin and internal reference GAPDH
1.7 detection of GSK-3 beta, beta-catenin protein expression in hippocampal tissue
Westernblotting method was used. Taking out the frozen rat hippocampal tissues in 1.6, shearing into 200-500 mg tissues, adding 250-500 mu LRIPA lysate, protease inhibitor and phosphatase inhibitor, and homogenizing thoroughly in a grinder. Centrifuging at 14000r/min for 10min at 4deg.C, and collecting supernatant. The BCA protein quantification kit measures protein concentration, draws a standard curve, calculates each sample concentration, and normalizes each sample concentration by adding RIPA. And (3) preparing gel by adopting polyacrylamide gel (SDSPAGE), electrophoresis, transferring the protein onto a PVDF film by a wet transfer method, and sealing for 2 hours by using 5% skimmed milk powder. Soaking PVDF membrane in diluted beta-catenin (1:5000) and GSK-3 beta (1:1000) primary antibody, and incubating overnight at 4deg.C; the next day the washed PVDF membrane was immersed in secondary antibody (1:5000) and incubated for 1h at room temperature. After washing the film, soaking the PVDF film in ECL chemiluminescent agent, taking out and exposing and developing by a gel imager. The relative protein expression was calculated by analyzing the band gray values with ImageJ software using GAPDH as an internal reference.
1.8 statistical methods
SPSS22.0 statistical software was used. The measurement data adopts a shape-Wilk normal property test method, is normally distributed and expressed by + -s, the comparison among multiple groups adopts variance analysis, and the comparison among the two groups adopts t test; the non-normal distribution is denoted by M (P25, P75) and the inter-group comparison uses a rank sum test. P <0.05 is statistically significant for the differences.
2 results
2.1 comparison of the number of times rats of each group escape latency and enter the effective region
Referring to fig. 1 and table 2, the escape latency of rats in the model group was significantly prolonged and the number of times of entering the effective area was significantly reduced (P <0.05 in both groups) compared to the blank group. Compared with the model group, the escape latency of rats in the donepezil group and the high-dose group with the liposome coated polypeptide is shortened, the frequency of entering the effective area is obviously increased (P is less than 0.05), and the change of the low-dose group and the medium-dose group with the liposome coated polypeptide is not obvious (P is more than 0.05). The escape latency of rats in donepezil group and in liposome-encapsulated polypeptide high dose group was relatively non-statistically different (P-all > 0.05) from the number of times of entry into the active area.
TABLE 2 comparison of the number of times rats of each group have escaped latency and entered the active area (+/-s)
| Group of | n | Escape latency(s) | Number of times (times) of entering the effective area |
| Blank group | 12 | 26.94±2.49 | 8.50±2.62 |
| Model group | 12 | 39.31±3.70* | 5.88±2.48* |
| DonepezilGroup of | 12 | 28.94±3.34# | 10.88±3.00# |
| Liposome-encapsulated polypeptide low dose group | 12 | 32.75±3.61 | 7.00±2.39Δ |
| Dosage group in liposome-encapsulated polypeptides | 12 | 31.81±2.90 | 7.50±2.88Δ |
| Liposome-coated polypeptide high dose group | 12 | 24.33±1.90#▲ | 8.63±2.56#▲ |
Note that: in comparison with the blank set of the cells, * P<0.05; in comparison with the set of models, # P<0.05; in comparison with the donepezil group, Δ P<0.05; compared with the liposome coated polypeptide low and medium dose groups, ▲ P<0.05。
2.2 comparison of the pathological changes of the hippocampal tissue of rats in groups
Referring to fig. 2, compared with the control group, the model group rat hippocampal pyramidal cells were arranged in disorder, more nerve cells were reduced in volume, nuclei were contracted, and the number of neurons was significantly reduced, and more damage was also occurred to pyramidal cells. Compared with the model group, the low, medium and high dose groups of the donepezil group and the liposome coated polypeptide have obviously increased neuron quantity and complete morphology, wherein most of the sea horse cone cells of the liposome coated polypeptide high dose group and the donepezil group have normal morphology, more compact cell arrangement, clear nucleolus structure and uniform staining.
2.3 comparison of Positive expression of beta-catenin in hippocampal tissue of rats of each group
Referring to FIG. 3, the rat hippocampal tissue β -catenin proteins of each group were mainly localized to the cytoplasm, and positive cells were yellow brown. Compared with a control group, the positive cells of the rat hippocampal tissue beta-catenin protein of the model group have shallow staining, and the positive cells have fewer numbers and more dispersed cells. Compared with the model group, the donepezil group and the liposome wrapping polypeptide are increased in the number of the positive cells of the beta-catenin protein of the rat hippocampal tissue in the low, medium and high dose groups, and the staining is deeper.
2.4 comparison of the expression of GSK-3 beta, beta-catenin mRNA in rat hippocampal tissues of each group
Referring to table 3 and fig. 4, compared to the blank group, the model group rat hippocampal tissue had elevated GSK-3 β mRNA expression and reduced β -catenin mRNA expression (P < 0.05); compared with the model group, the donepezil group and the liposome coated polypeptide of the rat hippocampal tissue GSK-3 beta mRNA expression of the rat hippocampal tissue of the low, medium and high dose groups are reduced, and the beta-catenin mRNA expression is increased (P is less than 0.05). There was no statistical difference (P > 0.05) in the expression of GSK-3 beta, beta-catenin mRNA from both donepezil and liposome-encapsulated polypeptide high dose groups of rat hippocampal tissue.
TABLE 3 comparison of the expression of GSK-3 beta, beta-catenin mRNA in rat hippocampal tissues of each group (+/-s)
| Group of | n | GSK-3βmRNA | β-cateninmRNA |
| Blank group | 4 | 1.01±0.18 | 1.00±0.11 |
| Model group | 4 | 1.81±0.68* | 0.38±0.22* |
| Donepezil group | 4 | 0.79±0.34# | 1.87±0.52# |
| Liposome-encapsulated polypeptide low dose group | 4 | 1.41±0.10 | 1.20±0.52#Δ |
| Dosage group in liposome-encapsulated polypeptides | 4 | 1.16±0.46# | 1.56±0.15# |
| Liposome-coated polypeptide high dose group | 4 | 0.85±0.37# | 1.61±0.16# |
Note that: in comparison with the blank set of the cells, * P<0.05; in comparison with the set of models, # P<0.05; in comparison with the donepezil group, Δ P<0.05; compared with the liposome coated polypeptide low and medium dose groups, ▲ P<0.05。
2.5 comparison of the expression of GSK-3 beta, beta-catenin protein in rat hippocampal tissues of each group
Referring to table 4 and fig. 5, compared to the blank group, the model group rat hippocampal tissue had elevated GSK-3 beta protein expression and reduced beta-catenin protein expression (P < 0.05); compared with the model group, the expression of GSK-3 beta protein of rat hippocampal tissues in the low, medium and high dose groups of donepezil group and liposome-encapsulated polypeptide is reduced, and the expression of beta-catenin protein is increased (P is less than 0.05). There was no statistical difference (P > 0.05) between the expression of GSK-3 beta, beta-catenin protein in rat hippocampal tissue of donepezil group and liposome-encapsulated polypeptide high dose group.
Table 4 comparison of the expression of GSK-3 beta, beta-catenin protein in rat hippocampal tissue of each group (+ -s) s
Note that: in comparison with the blank set of the cells, * P<0.05; in comparison with the set of models, # P<0.05; in comparison with the donepezil group, Δ P<0.05。
based on the detection results, the AD model is built by intraperitoneal injection of D-gal, and the results show that compared with a blank group, the model group has the advantages that the arrangement of the pyramidal cells in the hippocampus region of the rat is disordered, the volume of more nerve cells is reduced, the nuclei are contracted, the number of neurons is obviously reduced, more damage to the pyramidal cells is also caused, the escape latency period is increased, and the frequency of entering an effective area is reduced, so that the AD model is successfully built.
The application result shows that compared with a model group, the escape latency of rats in the donepezil group and the liposome-encapsulated polypeptide group is reduced, the frequency of entering an effective area is increased, and the change of the donepezil group and the liposome-encapsulated polypeptide group in high dosage is more obvious; the liposome wrapping polypeptide is suggested to be helpful for improving the learning and memory capacity of AD rats, and the effect of wrapping the polypeptide by the liposome with high dosage is better and is equivalent to that of donepezil.
The application result shows that the GSK-3 beta mRNA and protein expression of the hippocampal tissue of the model group is higher than that of the blank group, and the beta-catenin mRNA and protein expression is lower than that of the blank group, which indicates that the Wnt/beta-catenin signal pathway in the hippocampus of the AD rat is inhibited; the expression of GSK-3 beta mRNA and protein of rat hippocampal tissues in the low, medium and high dose groups of donepezil and liposome coated polypeptide is lower than that in the model group, the expression of beta-catenin mRNA and protein is higher than that in the model group, and the high dose groups of donepezil and liposome coated polypeptide are not statistically different; the liposome-encapsulated polypeptide can activate Wnt/beta-catenin signal pathway, and the neuroprotection effect of the high-dose liposome-encapsulated polypeptide on AD rats is equivalent to that of donepezil.
In conclusion, the liposome-encapsulated polypeptide has neuroprotective effect on AD rats, and the higher the dosage is, the better the effect is, and the mechanism of the liposome-encapsulated polypeptide is possibly related to activating Wnt/beta-catenin signaling, so that an experimental foundation is laid for early prevention and treatment of AD.
3 study memory Capacity observations of the Liposome-coated Polypeptides having neurotrophic Effect obtained in examples 1 and 3 to 18 and Liposome-coated Polypeptides obtained in comparative examples 1 to 5
1.1 materials laboratory animals: 264 male SD rats were subjected to the same conditions as those for detecting the liposome-encapsulated polypeptide having a neurotrophic effect obtained in example 2.
Main drugs and reagents:
the experimental samples were liposome-encapsulated polypeptides having a neurotrophic effect obtained in examples 1 and 3 to 18 and liposome-encapsulated polypeptides obtained in comparative examples 1 to 5, and the experimental samples were designated as examples experimental samples 1 and 3 to 18 and comparative examples experimental samples 1 to 5 (e.g., liposome-encapsulated polypeptides having a neurotrophic effect obtained in example 1 and designated as example experimental sample 1) according to the numbers of examples and comparative examples;
d-galactose (D-Gal) was purchased from Shanghai Meilin Biochemical technologies Co.
The main instrument is as follows: the conditions for detecting the liposome-encapsulated polypeptide having a neurotrophic effect were the same as those obtained in example 2.
1.2 grouping and treatment methods for animals
Rats were randomly divided into 18 groups of examples, specifically example 1, example 3, example 18, and 5 groups of comparative examples, including comparative example 1, comparative example 5, each group having 12 rats. The abdominal cavities of rats in each of the example group and the comparative example group were injected with 150mg/kg of D-gal1 time/day to establish an AD model, and the injections were continued for 60 days. Rats in the example group and the comparative example group were gavaged with 420 mg/(kg·d) of experimental samples in the corresponding group, 1 time/day, for 60 days (for example, example 1 group was gavaged with example experimental sample 1).
1.3 learning memory ability observations
The Morris water maze test was used under the same conditions as those used in the detection of the liposome-encapsulated polypeptide with neurotrophic effect obtained in example 2.
SPSS22.0 statistical software was used for data in the 1.3 learning memory observations. The measurement data adopts a shape-Wilk normal property test method, is normally distributed and expressed by + -s, the comparison among multiple groups adopts variance analysis, and the comparison among the two groups adopts t test; the non-normal distribution is denoted by M (P25, P75) and the inter-group comparison uses a rank sum test. P <0.05 is statistically significant for the differences.
1.4 study and memory Capacity of the Liposome-coated polypeptide with neurotrophic action obtained in examples 1 and 3-18 and Liposome-coated polypeptide obtained in comparative examples 1-5 the comparison of the escape latency and the number of times of entry into the effective region of each group of rats was compared with the blank group, the escape latency of the model group of rats was significantly prolonged and the number of times of entry into the effective region was significantly reduced (P < 0.05). Compared to the model group, rats in both donepezil and example groups had a shortened escape latency, a significantly increased number of entries into the active area (P <0.05 each), while the comparative group had no significant change (P >0.05 each).
TABLE 5 comparison of the number of times rats of each group have escaped latency and entered the active area (+/-s)
/>
Note that: in comparison with the blank set of the cells, * P<0.05; in comparison with the set of models, # P<0.05; in comparison with the donepezil group, Δ P<0.05。
4 acute toxicity test
1 Material
Experimental animals: 180 male SD mice, 4 weeks old, with a mass of 20+ -2 g, were supplied by laboratory animal management center of Xinjiang medical university, and were routinely housed in SPF class laboratories, maintaining sufficient ventilation and lighting conditions, 3-4 animals were housed per cage, and were fed and eaten freely.
Test sample: the neurotrophic liposomes obtained in examples 1-18 encapsulate polypeptides.
2 animal grouping and processing method
Grouping animals: mice were randomly divided into 18 groups of 10 mice each and scored as groups 1-18.
Acute toxicity experiment: the neurotrophic liposomes obtained in examples 1-18 were coated with the polypeptide, and mice from groups 1-18 were gavaged sequentially at a gavage dose of 25g/kg BW, and then observed continuously for 14 days.
3 results of experiments
Mice did not show significant poisoning performance during the experiment and no death was observed during the observation period. The acute oral toxicity (MTD) of the sample to mice is larger than 25 g/kg.BW, and the acute toxicity is classified according to food safety toxicology evaluation procedure and method (2003 edition), and belongs to non-toxic grade.
The present embodiment is merely illustrative of the present application and not limiting of the present application, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as necessary, but are protected by patent laws within the scope of the claims of the present application.
Claims (1)
1. A liposome-encapsulated polypeptide having a neurotrophic effect, comprising a core material and a liposome shell;
the core material comprises the following components in parts by weight:
30-50 parts of whey protein polypeptide;
20-30 parts of 5-hydroxytryptamine;
20 parts of agaricus blazei murill polypeptide;
the preparation steps of the whey protein polypeptide are as follows:
s1: mixing whey protein powder with water, and performing microwave, steam and drying treatment to obtain pretreated whey protein powder;
s2: mixing the pretreated whey protein powder with water to obtain whey protein powder water solution, regulating the pH and temperature of the whey protein powder water solution, and adding alkaline protease for enzymolysis to obtain an enzymolysis product;
s3: centrifuging the enzymolysis product, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze-drying to obtain whey protein polypeptide;
the molecular weight of the whey protein polypeptide is 2-3kDa;
in the preparation step S1 of the whey protein polypeptide, the power of microwave treatment is 600w-700w, and the temperature of steam treatment is 105-115 ℃;
in the preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein powder aqueous solution is 4-6g/100ml; the addition amount of alkaline protease is 3000-4000U/g;
in the preparation step S2 of the whey protein polypeptide, the pH value of the whey protein powder aqueous solution is 9-11, the temperature is 55-60 ℃, and the enzymolysis is carried out for 1.5-2 hours;
the weight ratio of the core material to the liposome is 1 (1.2-1.6);
the core material further comprises an agaricus blazei polypeptide, wherein the molecular weight of the agaricus blazei polypeptide is 0.5-1kDa;
the preparation method of the liposome-encapsulated polypeptide with the neurotrophic effect comprises the following steps:
a1: dissolving lecithin, cholesterol and vitamin E in absolute ethyl alcohol to prepare a lipid film, drying, adding Tween 80 aqueous solution for hydration, shearing, purifying and breaking a film to obtain a liposome;
a2: stirring and mixing whey protein polypeptide, 5-hydroxytryptamine and agaricus blazei murill polypeptide to obtain a core material;
a3: mixing the liposome and the core material, and filtering and drying to obtain the liposome-encapsulated polypeptide with neurotrophic effect.
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