CN115669945A - 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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- CN115669945A CN115669945A CN202211307253.4A CN202211307253A CN115669945A CN 115669945 A CN115669945 A CN 115669945A CN 202211307253 A CN202211307253 A CN 202211307253A CN 115669945 A CN115669945 A CN 115669945A
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- polypeptide
- whey protein
- liposome
- encapsulated
- neurotrophic
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Abstract
The application relates to the technical field of food, in particular to a liposome-encapsulated polypeptide with a neurotrophic effect, and a preparation method and application thereof. A liposome-encapsulated polypeptide with neurotrophic effect comprises core material and liposome shell; a core material comprising 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 a whey protein powder aqueous solution, adjusting the pH and temperature of the aqueous solution, and performing enzymolysis; centrifuging the enzymolysis product, collecting supernatant, dialyzing the supernatant, purifying, and freeze-drying to obtain whey protein polypeptide; the molecular weight of the lactalbumin polypeptide is 2-3kDa. The liposome-encapsulated polypeptide with the neurotrophic effect has the effects of supplementing brain nutrition, keeping the brain healthy and improving the memory.
Description
Technical Field
The application relates to the technical field of food, in particular to a liposome-encapsulated polypeptide with a neurotrophic effect, and a preparation method and application thereof.
Background
The memory is the process of the human brain to remember, maintain, reproduce or reclassify the experienced things, and is the process of encoding, processing and inputting a large amount of information obtained in life and study, storing the information in the brain, extracting the relevant stored information when necessary and applying the information to practical activities.
Near memory loss can be caused by factors such as living stress, poor sleep and long-term malnutrition. Meanwhile, as the people age, the brain cells suffer from hypoxia and ischemic injury, the cranial nerves are atrophied, and the brain lesion causes the decline of the far memory, so that the memory of people of all ages in the society declines to different degrees.
Although the proper supplement of some nutrient elements can help people to improve memory, the daily diet of people often cannot meet the needs of the body for the nutrient elements. 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 is mainly as follows: firstly, a collection of whey protein polypeptides with various molecular weights is obtained through a proteolysis mode, then, a molecular membrane filtration technology is adopted to screen a mixture of the whey protein polypeptides according to a target molecular weight, and finally, the whey protein polypeptides with the target molecular weight are obtained. And finally, obtaining the required liposome-encapsulated polypeptide by adopting a high-speed homogenizer and combining a corresponding production process through a large number of experiments. This stage is aimed at solving the world problem that the corresponding active ingredients in whey protein are degraded in the foreseeable gastrointestinal digestive process and thus lose the efficacy of the product.
According to the production process of the liposome-coated polypeptide, the technological problem that how to keep the biological activity of the product in the digestion process cannot be solved by extensive mixing of whey protein products in the original industry is solved in a mode of separating firstly, then coating specifically (can be regarded as a single mixing process among individual raw materials), and finally mixing all the raw materials totally, and the due characteristics of the product are maintained ingeniously through a carefully designed mixing process. Through the Shanghai science and technology search of China academy of sciences, the technical disclosure which is completely the same as the technical characteristics of the project is not seen in the published documents at home and abroad. The conclusion is that the technical project is novel.
In a first aspect, the application provides a liposome-encapsulated polypeptide with a neurotrophic effect, a preparation method and an application thereof, and adopts the following technical scheme:
a liposome-encapsulated polypeptide with neurotrophic effect comprises core material and 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 a whey protein powder aqueous solution, adjusting the pH and the temperature of the whey protein powder aqueous 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 lactalbumin 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 fat solubility of the whey protein polypeptide can be increased, the absorption of the whey protein polypeptide by a human body is facilitated, and the efficacy of the whey protein polypeptide is improved. On the other hand, the complete activity of the 5-hydroxytryptamine can be kept, the 5-hydroxytryptamine is favorable for providing nutrition for nerve cells through a blood sugar barrier, the dormant nerve stem cells are activated, the regeneration of damaged nerve cells and the repair of functions of the damaged nerve cells are promoted, and the memory is improved.
Secondly, in the preparation of the lactalbumin polypeptide, the lactalbumin is subjected to microwave and high-temperature steam treatment in advance, so that the lactalbumin is moderately denatured, the hydrolysis degree of the lactalbumin is improved, enzyme exposure sites are facilitated, the enzymolysis of alkaline protease is accelerated, and the efficiency of the enzymolysis of the lactalbumin polypeptide is improved. Then the properly 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 nutritive value, can more easily enter human cells, 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 whey protein polypeptide with the molecular weight of 2-3kDa, which is self-prepared by the application, is compounded with 5-hydroxytryptamine, 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 enzymolysis of the whey protein powder is facilitated, the enzymolysis efficiency of whey protein polypeptide is improved, the obtained whey protein polypeptide can promote cell division, adjust the metabolism of cells, improve the number, quality and speed of protein synthesis in the cells and be in a normal state, and the memory can be further improved.
Preferably, in the preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein powder aqueous solution is (4-6) g/100ml; the addition amount of alkaline protease is 3000-4000U/g.
By adopting the technical scheme: the quality concentration of the whey protein and the adding amount of the alkaline protease in the pretreatment are controlled, the activity and the nutritive value of the obtained whey protein polypeptide are higher, the whey protein polypeptide is compounded with the 5-hydroxytryptamine, and the obtained liposome with the neurotrophic effect wraps the polypeptide, so that the effect of improving the memory is better.
Preferably, in the step S2 of preparing the lactalbumin polypeptide, the pH value of the aqueous solution of the lactalbumin powder is 9-11, the temperature is 55-60 ℃, and the enzymolysis is carried out for 1.5-2h.
By adopting the technical scheme: is beneficial to improving the enzymolysis efficiency of the lactalbumin, 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-encapsulated polypeptide with the neurotrophic effect has good stability and fat solubility, is favorable for promoting the absorption of whey protein polypeptide by a human body, and further improves the effect of improving the memory.
Preferably, the core material further comprises an agaricus blazei murill polypeptide, wherein the molecular weight of the agaricus blazei murill polypeptide is 0.5-1kDa.
By adopting the technical scheme: the Agaricus blazei polypeptide is an effective component extracted from Agaricus blazei, and many of the polypeptides in animal and plant proteins show biological activity after being released in vivo or in vitro. The agaricus blazei murill polypeptide with the molecular weight of 0.5-1kDa is added, so that the antioxidant capacity in vivo is improved more, the oxidative damage of cells is relieved, and the aging is effectively delayed.
In a second aspect, the present application provides a method for preparing liposome-encapsulated polypeptides with neurotrophic effects, which comprises the following steps:
a method for preparing liposome-encapsulated polypeptide with neurotrophic effect comprises the following steps:
a1: dissolving lecithin, cholesterol and vitamin E in absolute ethyl alcohol to prepare a lipid film, drying, adding a Tween 80 aqueous solution for hydration, shearing, purifying, and breaking the membrane to obtain a liposome;
a2: stirring and mixing whey protein polypeptide, 5-hydroxytryptamine and Agaricus blazei Murill polypeptide to obtain core material;
a3: and mixing the liposome and the core material, and filtering and drying to obtain the liposome-coated polypeptide with the neurotrophic effect.
The unique role of lecithin in liposomes is: the content of lecithin in the cranial nerve cells accounts for about 17-20% of the weight of the cranial nerve cells. The choline is the basic component of soybean lecithin, and the sufficient supply of the lecithin can ensure that sufficient choline is synthesized into acetylcholine with acetyl in a human body. Acetylcholine is an information conductor in the brain, thus increasing the degree of activation of brain cells and improving memory.
Lecithin has effects of emulsifying and decomposing oil, promoting blood circulation, improving serum lipid, removing peroxide, reducing cholesterol and neutral fat content in blood, reducing fat retention time on blood vessel inner wall, promoting dissipation of atherosclerotic plaque, and preventing blood vessel intimal injury caused by cholesterol.
By adopting the technical scheme, the embedding rate of the liposome on the whey protein polypeptide is higher, the whey protein polypeptide can be protected, the stability of the whey protein polypeptide is improved, the release of the whey protein polypeptide can be delayed, the absorption of the whey protein polypeptide is promoted, and the compounding effect of the whey protein polypeptide and 5-hydroxytryptamine is improved. 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 liposome-encapsulated polypeptide with neurotrophic effects, which adopts the following technical scheme:
the application of liposome-encapsulated polypeptide with neurotrophic effect in 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 the beverage, 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 application preferably adopts the activated whey protein polypeptide with specific molecular weight, has higher activity and nutritive value, and simultaneously can promote the division of cells, adjust the metabolism of the cells, and improve the quantity, quality and speed of protein synthesis in the cells to be 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 coating technology and directly enters blood, so that the complete activity of the whey protein polypeptide and the 5-hydroxytryptamine is improved, and meanwhile, the whey protein polypeptide and the 5-hydroxytryptamine are promoted to penetrate through a blood sugar barrier, so that nutrition is provided for nerve cells, dormant nerve stem cells are activated, the regeneration of damaged nerve cells and the restoration of functions of the damaged nerve cells are promoted, and the memory is improved.
Drawings
FIG. 1 is a graph of escape latency as a function of days for 6 groups of rats after molding;
FIG. 2 is a graph of HE staining of hippocampal tissue in 6 groups of rats;
FIG. 3 is a graph of 6 groups of rats processed by hippocampal immunohistochemistry and detecting the expression of beta-catenin;
FIG. 4 is a graph showing the mRNA-related expression levels of β -catenin and GSK-3 β in hippocampal tissues of 6 groups of rats;
FIG. 5 is the expression banding pattern of beta-catenin and GSK-3 beta protein in rat hippocampal tissue of each group.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
Preparation examples
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 microwave treatment power is 600w, the time is 0.5min, the temperature of water vapor treatment is 105 deg.C, the time is 5min, and the temperature of drying treatment is 100 deg.C, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain a whey protein aqueous solution with the mass-volume concentration of 3g/100ml, adjusting the pH of the whey protein aqueous solution to 8 at 55 ℃, adding 2800u/g of alkaline protease, and performing enzymolysis for 1.5 hours to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has a product number of FDG-4001.
S3: inactivating enzyme of enzymolysis product at 100 deg.C for 8min, centrifuging at 10 deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze drying to obtain lactalbumin polypeptide.
According to detection, in the preparation example of the application, the molecular weight of the lactalbumin polypeptide obtained by dialysis interception is 2kDa.
In the preparation example, the whey protein powder is food grade and is purchased from Lezhen Jin Fuyuan Biotechnology Limited; 5-hydroxytryptamine, purchased from Kyoto Hongyao Biotechnology, inc., beijing.
Preparation example 2
A whey protein polypeptide, which is different from preparation example 1 in that the molecular weight of the whey protein polypeptide obtained in step S3 of preparing the whey protein polypeptide is 2.5kDa by dialysis interception.
Preparation example 3
A whey protein polypeptide, which is different from preparation example 1 in that the molecular weight of the whey protein polypeptide obtained in step S3 of preparing the whey protein polypeptide is 3.0kDa by dialysis interception.
Preparation example 4
A whey protein polypeptide, which is different from preparation example 2 in that in the 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, which is different from preparation example 2 in that in the preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein aqueous solution is 5g/100ml; the amount of alkaline protease added was 3500u/g.
Preparation example 6
A whey protein polypeptide, which is different from preparation example 2 in that in the 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 is different from the preparation example 5 in that in the preparation step S2 of the whey protein polypeptide, the pH value of a whey protein aqueous solution is 9, the temperature is 55 ℃, and the enzymolysis is carried out for 1.5h.
Preparation example 8
The whey protein polypeptide is different from the preparation example 5 in that in the preparation step S2 of the whey protein polypeptide, the pH value of a whey protein aqueous solution is 10, the temperature is 58 ℃, and the enzymolysis is carried out for 1.7h.
Preparation example 9
A whey protein polypeptide is different from the preparation example 5 in that in the preparation step S2 of the whey protein polypeptide, the pH value of a 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 which is different from preparation example 8 in that in the 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 which is different from preparation example 8 in that in the 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 which is different from preparation example 8 in that in the 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
A liposome-encapsulated polypeptide having neurotrophic activity, the components and their respective weights being as shown in Table 1, and prepared by the steps of:
a1: firstly, dissolving lecithin, cholesterol and vitamins in absolute ethyl alcohol at 60 ℃ to obtain a mixture;
shearing the mixture at 18000r/min for 5min, homogenizing at 15MPa for 5min, and evaporating in 80 deg.C water bath under reduced pressure to obtain liposome film;
adding 0.5% Tween 80 aqueous solution into the liposome film, hydrating at 50 deg.C for 1h, shearing at 18000r/min for 5min, purifying, and breaking membrane to obtain liposome.
A2: stirring and mixing whey protein polypeptide, 5-hydroxytryptamine and Agaricus blazei Murill polypeptide to obtain core material.
A3: dissolving the liposome obtained from A1 in water to obtain liposome aqueous solution with mass percent concentration of 20 mg/mL; and (3) adding the core material obtained in the step A2 into the aqueous solution of the liposome, then carrying out ultrasonic stirring for 1min at 100w, and then carrying out filter membrane filtration and drying treatment to obtain the liposome-coated 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 prepared in preparation example 1 was used.
Examples 2 to 3
A liposome-entrapped polypeptide having neurotrophic activity, which differs from example 1 in that the components and their respective weights are shown in Table 1.
TABLE 1 Components and weights (kg) thereof in examples 1-3
Examples 4 to 5
A liposome-encapsulated polypeptide having neurotrophic activity, which differs from example 2 in that the molecular weight of the whey protein polypeptide obtained by dialysis entrapment in the whey protein polypeptide preparation step S3 is different; whey protein polypeptides were prepared as shown in table 2.
Table 2 preparation examples of whey protein polypeptides used 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 the mass-volume concentration of the whey protein aqueous solution and the amount of alkaline protease added are different in the step S2 of preparing the whey protein polypeptide; the preparation of whey protein polypeptides is shown in table 3.
Table 3 preparation examples 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 the pH value, temperature and enzymatic hydrolysis time of the aqueous solution of whey protein polypeptide are different in the step S2 of preparing whey protein polypeptide; the preparation of whey protein polypeptides was as shown in table 4.
Table 4 preparation examples 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 the power of the microwave treatment and the temperature of the water vapor treatment are different in the step S1 of preparing the whey protein polypeptide; the preparation of whey protein polypeptides was as shown in table 5.
Table 5 preparation examples 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 neurotrophic activity, which is different from example 10 in that the core material further comprises agaricus blazei murill polypeptide; agaricus blazei Murill polypeptide and its molecular weight are shown in Table 6.
TABLE 6 Polypeptides (kg) of Agaricus Blazei Murill and their molecular weights (Da) of examples 15-17
In the above embodiment, the Agaricus blazei Murill polypeptides are purchased from Shanxi Xia Biotech Co., ltd, and have molecular weights customized.
Example 18
A liposome-encapsulated polypeptide having neurotrophic activity, which differs from example 15 in that the weight of the liposome is shown in Table 7.
TABLE 7 weight (kg) of liposomes in example 18
In the present embodiment, the weight ratio of the core material to the liposome is 1 (1.2-1.6), and the influence on the properties of the liposome-encapsulated polypeptide having neurotrophic effects obtained in the present invention is the same, so in the present embodiment, the weight ratio of the core material to the liposome is 1.2.
Comparative example
Comparative example 1
A liposome-encapsulated polypeptide, which differs from example 2 in that, in the step S3 of preparing the whey protein polypeptide, the molecular weight of the whey protein polypeptide retained by dialysis is 4kDa.
Comparative example 2
A liposome-encapsulated polypeptide, which differs from example 2 in that the molecular weight of the whey protein polypeptide obtained in step S3 of preparing the whey protein polypeptide is 1.5kDa as a result of dialysis entrapment.
Comparative example 3
A liposome-encapsulated polypeptide is different from that of example 2 in that, in the step S1 of preparing the whey protein polypeptide, 100g of whey protein powder is mixed with 100g of water, and then only microwave treatment and drying treatment are performed.
The preparation method of the whey protein polypeptide comprises the following steps:
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 microwave treatment power is 600w, the time is 0.5min, the drying temperature is 100 deg.C, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain a whey protein aqueous solution with the mass-volume concentration of 3g/100ml, adjusting the pH of the whey protein aqueous solution to 8 at 55 ℃, adding 2800u/g of alkaline protease, and performing enzymolysis for 1.5 hours to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has a product number of FDG-4001.
S3: inactivating enzyme of enzymolysis product at 100 deg.C for 8min, centrifuging at 10 deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze drying to obtain lactalbumin polypeptide.
According to detection, in the preparation example of the application, the molecular weight of the lactalbumin polypeptide obtained by dialysis interception is 2kDa.
Comparative example 4
A liposome-encapsulated polypeptide is different from that of example 2 in that, in the whey protein polypeptide preparation step S1, 100g of whey protein powder is mixed with 100g of water, and then only steam treatment and drying treatment are performed.
The preparation method of the whey protein polypeptide comprises the following steps:
s1: mixing 100g of whey protein powder with 100g of water, and performing steam treatment and drying treatment to obtain pretreated whey protein powder;
the temperature of the steam treatment is 105 deg.C, and the time is 5min, and the temperature of the drying treatment is 100 deg.C, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain a whey protein aqueous solution with the mass-volume concentration of 3g/100ml, adjusting the pH of the whey protein aqueous solution to 8 at the temperature of 55 ℃, adding 2800u/g of alkaline protease, and performing enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has a product number of FDG-4001.
S3: inactivating enzyme of the enzymolysis product at 100 deg.C for 8min, centrifuging at 10 deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze drying to obtain whey protein polypeptide.
According to detection, in the preparation example of the application, the molecular weight of the lactalbumin polypeptide obtained by dialysis interception is 2kDa.
Comparative example 5
A liposome-encapsulated polypeptide is different from that in example 2 in that, in the preparation step S1 of whey protein polypeptide, 100g of whey protein powder is mixed with 100g of water, and then steam, microwave and drying treatment are performed.
The preparation method of the whey protein polypeptide comprises the following steps:
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 microwave treatment power is 600w, the time is 0.5min, the temperature of water vapor treatment is 105 deg.C, the time is 5min, and the temperature of drying treatment is 100 deg.C, and the time is 8min.
S2: mixing the pretreated whey protein powder with water to obtain a whey protein aqueous solution with the mass-volume concentration of 3g/100ml, adjusting the pH of the whey protein aqueous solution to 8 at the temperature of 55 ℃, adding 2800u/g of alkaline protease, and performing enzymolysis for 1.5h to obtain an enzymolysis product;
the alkaline protease is food-grade alkaline protease for soybean and vegetable protein, and has a product number of FDG-4001.
S3: inactivating enzyme of the enzymolysis product at 100 deg.C for 8min, centrifuging at 10 deg.C and 4000r/min for 10min, filtering, collecting supernatant, dialyzing the supernatant, concentrating, purifying, and freeze drying to obtain whey protein polypeptide.
According to detection, in the preparation example of the application, the molecular weight of the lactalbumin polypeptide obtained by dialysis interception is 2kDa.
Performance detection
1 materials and methods
1.1 materials
Experimental animals:
72 male SD rats, 12-16 weeks old, with a body mass of 250 + -50 g, supplied by the laboratory animal management center of Xinjiang medical university, with quality code [ SCXK (New) 2019-0002], were kept routinely in SPF laboratories, maintaining adequate ventilation and lighting conditions, 3-4 rats per cage, and were fed freely and on a diet.
Main drugs and reagents:
the experimental sample is the liposome-encapsulated polypeptide with the neurotrophic effect obtained in example 2, which is hereinafter referred to as liposome-encapsulated polypeptide;
d-galactose (D-Gal) and donepezil hydrochloride were obtained from Shanghai Michelin Biotech, inc.;
beta-catenin (beta-catenin) antibodies were purchased from Abcam, UK;
the total RNA extraction kit, the first strand synthesis kit and the fluorescence quantitative kit of the animal tissue are purchased from Beijing Tiangen Biochemical research corporation;
the BCA protein content determination kit and the SDS-PAGE gel preparation kit are purchased from Beijing Solaibao science and technology Co.
The main apparatus is as follows:
the Morris water maze video image monitoring analysis control system is purchased from a Gongtai union limited science and technology;
the QutantStaudio 6 real-time fluorescence PCR instrument and the Nanddrop000/000C spectrophotometer are both available from Thermo corporation of America.
1.2 animal grouping and handling method
The kit comprises a model group, a donepezil group, a liposome-encapsulated polypeptide low-dose group, a liposome-encapsulated polypeptide medium-dose group and a liposome-encapsulated polypeptide high-dose group, wherein each group comprises 12 patients. The abdominal cavities of the other groups except the blank group are injected with D-gal150mg/kg for 1 time/day to establish an AD model, and the abdominal cavity of the blank group is injected with the same amount of normal saline and is continuously injected 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 respectively administered by intragastric administration for 0.9 mg/(kg.d) of donepezil, 1 time/day and 60 days continuously while modeling.
1.3 learning and memory Observation
The Morris water maze test was used. The components are treated for 60d, and a Morris water maze experiment is carried out for 6d, and is divided into a positioning navigation experiment and a space exploration experiment.
(1) Positioning navigation experiment: putting each group of rats into water from the center of a fixed quadrant to the wall of the pool, and recording the time for finding the platform within 120s, namely the escape latency; beyond 120s without finding, lead them to climb the platform and stay for 10s, train for 5d,2 times/day.
(2) Space exploration experiment: the platform was removed from each group of rats on day 6 and the number of entries into the available area (1.5 fold of the original platform) was recorded over 120 s.
1.4 Hippocampus histopathological examination
HE staining was used. Treating each component for 60 days, randomly selecting 4 rats, carrying out intraperitoneal injection anesthesia on the rats with 1% sodium pentobarbital of 0.45mL/100g, placing the rats on an anatomical disc after the rats are coma, exposing the heart of the rats, and inserting and fixing a perfusion needle in the left ventricle; cutting right auricle, slowly infusing 150mL of pre-frozen (4 ℃) sterile normal saline until the tip of the nose of a rat becomes pale, the color of the liver and the lung changes from red to white, and infusing 200mL of 4% paraformaldehyde after clear liquid flows out from the right auricle; when the four limbs of the rat twitch and spasm, cutting the head to take the brain, peeling off the hippocampus at the two sides of the brain on an ice bench, fixing with 4% paraformaldehyde, and making into tablets for later use.
After gradient ethanol dehydration, tissue transparency, wax dipping and embedding are carried out in sequence, the hippocampal tissue is cut into paraffin sections with the thickness of 4 mu m in a coronal section. Baking slices for several hours, dewaxing by dewaxing and transparentizing dewaxing solution, dehydrating, washing by sterile water, dyeing by hematoxylin for 1min, differentiating by 1% hydrochloric acid ethanol, and fully washing again; dyeing with 1% eosin for 1min, gradient dehydrating, removing wax, and sealing with neutral gum. Pathological changes of hippocampal tissues of each group were observed under an optical microscope.
1.5 Observation of Hippocampus tissue beta-catenin Positive expression
Immunohistochemistry method is adopted. Taking coronary slice of each group of hippocampal tissue, rehydrating, soaking slice with 3% hydrogen peroxide solution at room temperature for 10min, washing with distilled water for 3min, and washing with Phosphate Buffer Solution (PBS) for 3 times, each time for 5min; placing the mixture in citrate buffer solution, performing microwave repair (92-98 ℃) for 12min, cooling the mixture to the normal temperature at room temperature, and washing the mixture for 3 times by PBS; after antigen retrieval, the blocks were removed by blocking in 5% BSA for 30min, and diluted polyclonal rabbit anti-beta-catenin (1: 500) was added dropwise and incubated overnight in a refrigerator at 4 ℃. Washing with PBS for 3 times the next day, dripping diluted horseradish peroxidase labeled secondary antibody on the surface of the sliced tissue, and incubating in a constant temperature oven at 37 deg.C for 30min; washing with PBS for 3 times, uniformly dripping DAB color development solution on the surface of the tissue for developing for 60s until the tissue is observed to be brownish yellow under a microscope, and stopping running water; counterstaining with hematoxylin for 1min, differentiation with hydrochloric acid, gradient ethanol dehydration, and sealing with neutral gum. And observing the positioning, the quantity and the like of the beta-catenin protein of the hippocampal tissues of each group under a microscope.
1.6 expression detection of GSK-3 beta, beta-catenin mRNA in Hippocampus tissue
The Real-timePCR method was used. Collecting the rest rats treated with each component for 60 days, perfusing heart, taking out brain, separating hippocampal tissue, freezing in liquid nitrogen, and transferring to-80 deg.C refrigerator for storage. 20mg of hippocampus tissue is weighed, lysate and grinding beads are added, and after grinding for a plurality of times by an automatic grinder, total hippocampus mRNA is extracted. The mRNA purity of each sample was measured, and reverse transcription was performed when the absorbance A260/A280 was measured at 260 nm to 280nm and was 1.9 to 2.0. The primers of GSK-3 beta, beta-catenin and internal reference GAPDH were designed and synthesized by Shanghai Bioengineering Co., ltd. And the sequences are shown in Table 1.
PCR amplification conditions: circulating for 1 time at 95 ℃ for 15 min; circulating at 95 deg.C for 10s,60 deg.C for 32s for 40 times; and (4) drawing a dissolution curve at 65-95 ℃. By means of 2 －ΔΔCt The relative expression level of the target gene is calculated by the method.
TABLE 1 primer sequences for GSK-3 beta, beta-catenin and internal reference GAPDH
1.7 Hippocampus tissue GSK-3 beta, beta-catenin protein expression detection
The Westernblotting method was used. The frozen rat hippocampus tissue of each group of 1.6 is taken out, cut into 200-500 mg tissue, added with 250-500 mu LRIPA lysate, protease inhibitor and phosphatase inhibitor, and fully homogenized in a grinding instrument. Centrifuging at 14000r/min for 10min at 4 ℃, and taking supernatant. The BCA protein quantitative kit is used for measuring the protein concentration, drawing a standard curve, calculating the concentration of each sample, and adding RIPA to normalize the concentration of each sample. The protein is transferred to a PVDF membrane by polyacrylamide gel (SDSPAGE) gel preparation, electrophoresis and wet transfer method, and 5% skimmed milk powder is sealed for 2h. Soaking PVDF membrane in diluted primary antibody of beta-catenin (1: 5000) and GSK-3 beta (1: 1000), and incubating overnight in a shaking table at 4 ℃; the next day, the washed PVDF membrane was soaked in a secondary antibody (1: 5000) and incubated at room temperature for 1 hour. And (3) after washing the membrane, soaking the PVDF membrane in an ECL chemiluminescence agent, taking out the PVDF membrane, and exposing and developing the PVDF membrane by using a gel imager. The relative protein expression was calculated by analyzing the band gray values with the aid of the GAPDH reference using the ImageJ software.
1.8 statistical methods
SPSS22.0 statistical software was used. The measured data adopts a Shapiro-Wilk normality test method, the normal distribution is represented by +/-s, the variance analysis is adopted for the comparison of multiple groups, and the t test is adopted for the comparison of two groups; the abnormal distribution is expressed as M (P25, P75), and the comparison among groups adopts the rank sum test. P <0.05 is statistically significant for the differences.
2 results
2.1 comparison of the number of escape latency and entry into the effective area for each group of rats
Referring to fig. 1 and table 2, the escape latency of the model group rats was significantly prolonged and the number of entry into the effective area was significantly reduced (P was < 0.05) compared to the blank group. Compared with the model group, the escape latency of rats in the donepezil group and the liposome-encapsulated polypeptide high-dose group is shortened, the frequency of entering the effective area is obviously increased (P is less than 0.05), and the change of the liposome-encapsulated polypeptide low-dose group and the liposome-encapsulated polypeptide medium-dose group is not obvious (P is more than 0.05). The escape latency and the frequency of entering effective areas of the rats of the donepezil group and the liposome-encapsulated polypeptide high-dose group are not statistically different (P is both > 0.05).
TABLE 2 comparison of the number of escape latencies and entry into the effective area (+ -s) for each group of rats
| Group of | n | Escape latency(s) | Number of times of entering effective area |
| Blank group | 12 | 26.94±2.49 | 8.50±2.62 |
| Model set | 12 | 39.31±3.70* | 5.88±2.48* |
| Donepezil combination | 12 | 28.94±3.34# | 10.88±3.00# |
| Liposome-encapsulated polypeptide low-dose group | 12 | 32.75±3.61 | 7.00±2.39Δ |
| Liposome-encapsulated polypeptide medium dose group | 12 | 31.81±2.90 | 7.50±2.88Δ |
| Liposome-encapsulated polypeptide high dose group | 12 | 24.33±1.90#▲ | 8.63±2.56#▲ |
Note: in comparison with the blank set, the results, * 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 low and medium dose groups of the liposome-encapsulated polypeptide, ▲ P<0.05。
2.2 comparison of pathological changes in Hippocampus tissue in various groups of rats
Referring to fig. 2, in the model group rats, pyramidal cells in the hippocampal region were disorganized, and more nerve cells were reduced in volume, and the cell nuclei were fixed and shrunk, and the number of neurons was significantly reduced, and more damage occurred to pyramidal cells, as compared with the control group. Compared with the model group, the donepezil group and the liposome-encapsulated polypeptide group have the advantages that the number of neurons is obviously increased, the neuron quantity is obviously increased, and the form is complete, wherein most of the forms of the hippocampal pyramidal cells of the liposome-encapsulated polypeptide group and the donepezil group are normal, the cell arrangement is relatively dense, the nucleolus structure is clear, and the coloring is uniform.
2.3 comparison of Positive expression of Hippocampus tissue beta-catenin in various groups of rats
Referring to fig. 3, the β -catenin protein in hippocampal tissues of rats in each group was mainly localized to cytoplasm, and positive cells were yellowish brown. Compared with the control group, the model group rat hippocampal tissue beta-catenin protein positive cells are lightly colored, the number of the positive cells is less, and the cells are scattered. Compared with the model group, the donepezil group and the liposome-encapsulated polypeptide low, medium and high dose groups have the advantages that the number of the beta-catenin protein positive cells of the rat hippocampal tissue is increased, and the staining is deeper.
2.4 comparison of the expression of GSK-3 beta, beta-catenin mRNA in hippocampal tissues of various groups of rats
Referring to Table 3 and FIG. 4, model group rats had elevated GSK-3 β mRNA expression and reduced β -catenin mRNA expression in hippocampal tissue compared to blank group (P all < 0.05); compared with the model group, the rat hippocampal tissue GSK-3 beta mRNA expression of the donepezil group and the rat hippocampal tissue of the liposome-encapsulated polypeptide is reduced, and the beta-catenin mRNA expression is increased (P is less than 0.05). Compared with rat hippocampal tissue GSK-3 beta and beta-catenin mRNA expression, the donepezil group and the liposome-encapsulated polypeptide high-dose group have no statistical difference (P is more than 0.05).
TABLE 3 comparison of GSK-3 beta, beta-catenin mRNA expression in rat hippocampal tissues of each group (+ -s)
| Group of | n | GSK-3βmRNA | β- |
| Blank group | |||
| 4 | 1.01±0.18 | 1.00±0.11 | |
| Model set | 4 | 1.81±0.68* | 0.38±0.22* |
| |
4 | 0.79±0.34# | 1.87±0.52# |
| Liposome-encapsulated polypeptide low- |
4 | 1.41±0.10 | 1.20±0.52#Δ |
| Liposome-encapsulated polypeptide |
4 | 1.16±0.46# | 1.56±0.15# |
| Liposome-encapsulated polypeptide |
4 | 0.85±0.37# | 1.61±0.16# |
Note: in comparison with the blank set, the results, * 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 low and medium dose groups of the liposome-encapsulated polypeptide, ▲ P<0.05。
2.5 comparison of GSK-3 beta, beta-catenin protein expression in Hippocampus tissues of various groups of rats
Referring to Table 4 and FIG. 5, the model group rat hippocampal tissue GSK-3 beta protein expression is increased and beta-catenin protein expression is decreased (P is all < 0.05) compared with the blank group; compared with the model group, the rat hippocampal tissue GSK-3 beta protein expression of the donepezil group and the rat hippocampal tissue of the liposome-encapsulated polypeptide is reduced, and the beta-catenin protein expression is increased (P is less than 0.05). Compared with rat hippocampal tissue GSK-3 beta and beta-catenin protein expression, the donepezil group and the liposome-encapsulated polypeptide high-dose group have no statistical difference (P is more than 0.05).
TABLE 4 comparison of GSK-3 beta, beta-catenin protein expression (+ -s) s in rat hippocampal tissues of each group
Note: in comparison with the blank set, the results, * 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 established by injecting D-gal into the abdominal cavity, the result shows that compared with a blank group, the model group rat hippocampal pyramidal cells are disorderly arranged, the volume of more nerve cells is reduced, the cell nucleus is solidified and shrunk, the number of nerve cells is obviously reduced, the pyramidal cells are also damaged, the escape latency is increased, the number of times of entering an effective area is reduced, and the successful establishment of the AD model is prompted.
The application result shows that compared with the model group, the escape latencies of rats in the donepezil group and the liposome-encapsulated polypeptide low, medium and high dose groups are reduced, the times of entering an effective area are increased, and the change of the donepezil group and the liposome-encapsulated polypeptide high dose group is more obvious; the suggestion that the liposome-encapsulated polypeptide is helpful for improving the learning and memory abilities of AD rats, and the effect of the high-dose liposome-encapsulated polypeptide is better and is equivalent to that of donepezil.
The results of the application show that the GSK-3 beta mRNA and protein expression of rat hippocampal tissues of the model group is higher than that of the blank group, and the beta-catenin mRNA and protein expression are lower than that of the blank group, which indicates that the Wnt/beta-catenin signal channel in AD rat hippocampus is inhibited; the rat hippocampal tissue GSK-3 beta mRNA and protein expression of the donepezil group and the liposome-encapsulated polypeptide low, medium and high dose groups are lower than those of the model group, the beta-catenin mRNA and protein expression are higher than those of the model group, and the donepezil group and the liposome-encapsulated polypeptide high dose group have no statistical difference compared; the results show that the liposome-encapsulated polypeptide can activate Wnt/beta-catenin signaling pathway, and the neuroprotective effect and the Wnt/beta-catenin signaling pathway activation effect of the high-dose liposome-encapsulated polypeptide on AD rats are equivalent to those of donepezil.
In conclusion, the liposome-encapsulated polypeptide has a neuroprotective effect on AD rats, the higher the dosage is, the better the effect is, the mechanism of the liposome-encapsulated polypeptide is probably related to the activation of Wnt/beta-catenin signaling pathway, and an experimental basis is laid for the early prevention and treatment of AD.
3 Observation and examination of learning and memory Capacity of Liposome-Encapsulated Polypeptides having neurotrophic Activity obtained in examples 1 and 3-18 and Liposome-Encapsulated Polypeptides obtained in comparative examples 1-5
1.1 materials experimental animals: the conditions of the male SD rat 264 were the same as those for detecting the liposome-encapsulated polypeptide having neurotrophic effect obtained in example 2.
Main drugs and reagents:
the experimental samples are the liposome-encapsulated polypeptides with the neurotrophic effect obtained in examples 1 and 3-18 and the liposome-encapsulated polypeptides obtained in comparative examples 1-5, and the experimental samples are named as example experimental samples 1 and 3-18 and comparative example experimental samples 1-5 (for example, the liposome-encapsulated polypeptide with the neurotrophic effect obtained in example 1 is named as example experimental sample 1) according to the numbers of the examples and the comparative examples;
d-galactose (D-Gal) was purchased from Shanghai Michelin Biotech, inc.
The main apparatus is as follows: the conditions were the same as those for detecting the liposome-encapsulated polypeptide having neurotrophic activity obtained in example 2.
1.2 animal grouping and handling method
The rats were randomly divided into 18 groups of examples and 5 groups of comparative example, the 18 groups of examples specifically including the example 1 group, the example 3 group to the example 18 group, and the 5 groups of comparative example including the comparative example 1 group to the comparative example 5 group, each group having 12 rats. Rats in each of the example and comparative groups were intraperitoneally injected with D-gal at 150mg/kg for 1 time/day to establish an AD model, and injected continuously for 60 days. The rats of the example group and the comparative group were modeled and simultaneously gazed with 420 mg/(kg. D) for 1 time/day for 60 consecutive days (for example, the rats of the example 1 group were gazed with the experimental sample 1 of the example).
1.3 learning and memory ability Observation
The Morris water maze test is adopted, and the detection conditions are the same as those of the liposome-encapsulated polypeptide with the neurotrophic effect obtained in the example 2.
For the data in the 1.3 learning memory ability observation, SPSS22.0 statistical software is adopted. The measured data adopts a Shapiro-Wilk normality test method, the measured data presents normal distribution and is expressed by +/-s, variance analysis is adopted for multi-group comparison, and t test is adopted for two-group comparison; the abnormal distribution is expressed as M (P25, P75), and the comparison among groups is performed by using a rank sum test. P <0.05 is statistically significant for the differences.
1.4 observation and detection results of learning and memory abilities of the liposome-encapsulated polypeptide with neurotrophic effects obtained in examples 1, 3-18 and the liposome-encapsulated polypeptide obtained in comparative examples 1-5 show that the escape latency and the number of times of entering an effective area of rats in each group are compared with those in a blank group, the escape latency of rats in a model group is obviously prolonged, and the number of times of entering the effective area of rats is obviously reduced (P is less than 0.05). Compared with the model group, the escape latency of rats of the donepezil group and the example group is shortened, and the number of times of entering the effective area is obviously increased (P is less than 0.05), while the comparative example group is not obviously changed (P is more than 0.05).
TABLE 5 comparison of the number of escape latencies and entry times into the available area (. + -. S) for each group of rats
Note: in comparison with the blank set, the results, * 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 body mass of 20 + -2 g, were supplied by the laboratory animal management center of Xinjiang medical university, were housed routinely in SPF-grade laboratories, maintained under adequate ventilation and lighting conditions, housed 3-4 mice per cage, and were free to eat and eat.
Test samples: the liposome-encapsulated polypeptides having neurotrophic effects obtained in examples 1-18.
2 animal grouping and processing method
Animal grouping: mice were randomly divided into 18 groups of 10 mice each and scored as groups 1-18.
Acute toxicity test: the liposome-encapsulated polypeptides having neurotrophic effects obtained in examples 1 to 18 were sequentially gavaged to mice of groups 1 to 18 at a gavage dose of 25 g/kg. BW, and then continuously observed for 14 days.
3 results of the experiment
No obvious toxic signs were observed in mice during the experiment, and no death occurred during the observation period. The acute oral toxicity (MTD) of the sample to mice is more than 25g/kg & BW, and the sample belongs to non-toxic grade according to the acute toxicity grading of food safety toxicology evaluation program and method (2003 edition).
The present embodiment is only for explaining the present application and is not limited to the present application, and those skilled in the art can make modifications without inventive contribution to the present embodiment as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (8)
1. A liposome-encapsulated polypeptide with neurotrophic activity, 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 a whey protein powder aqueous solution, adjusting the pH and the temperature of the whey protein powder aqueous 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 lactalbumin polypeptide is 2-3kDa.
2. The neurotrophic liposome-encapsulated polypeptide of claim 1, which is characterized by: 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 ℃.
3. The liposome-encapsulated polypeptide having a neurotrophic effect according to claim 1, wherein: in the preparation step S2 of the whey protein polypeptide, the mass-volume concentration of the whey protein powder aqueous solution is (4-6) g/100ml; the addition amount of alkaline protease is 3000-4000U/g.
4. The liposome-encapsulated polypeptide having a neurotrophic effect according to claim 1, wherein: in the preparation step S2 of the lactalbumin polypeptide, the pH value of the aqueous solution of lactalbumin powder is 9-11, the temperature is 55-60 ℃, and the enzymolysis is carried out for 1.5-2h.
5. The neurotrophic liposome-encapsulated polypeptide of claim 1, which is characterized by: the weight ratio of the core material to the liposome is 1 (1.2-1.6).
6. The liposome-encapsulated polypeptide having a neurotrophic effect according to claim 5, wherein: the core material further comprises an Agaricus blazei Murill polypeptide, wherein the molecular weight of the Agaricus blazei Murill polypeptide is 0.5-1kDa.
7. A method for preparing liposome-encapsulated polypeptide with neurotrophic effect, which is characterized by comprising the following steps:
a1: dissolving lecithin, cholesterol and vitamin E in absolute ethyl alcohol to prepare a lipid film, drying, adding a Tween 80 aqueous solution for hydration, shearing, purifying, and breaking the membrane to obtain a liposome;
a2: stirring and mixing whey protein polypeptide, 5-hydroxytryptamine and Agaricus blazei Murill polypeptide to obtain core material;
a3: and mixing the liposome and the core material, and filtering and drying to obtain the liposome-coated polypeptide with the neurotrophic effect.
8. Use of a liposome-encapsulated polypeptide having a neurotrophic effect according to any of claims 1 to 6 in a beverage.
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