A composite donkey-hide gelatin small molecule peptide suitable for pre-pregnancy nourishment and a preparation method thereof

Abstract

The present invention relates to a small molecule peptide of donkey-hide gelatin and a preparation method thereof. The present invention uses donkey-hide gelatin as a raw material, performs enzymolysis by a composite enzyme, obtains donkey-hide gelatin small molecule peptide compounds after separation and purification, and then performs light fermentation by Lactobacillus acidophilus ATCC 67707. The prepared donkey-hide gelatin small molecule peptide is easier to absorb, has a digestion and absorption rate far greater than that of donkey-hide gelatin itself, and has an activity far superior to that of the amino acids in donkey-hide gelatin. It also has the effects of enhancing immunity and improving fertility. The method of the present invention of enzymolysis of donkey-hide gelatin with activated carbon is easy to operate, low in cost, short in enzymolysis time, reduced in pollution, high in oil degreasing rate, simple in process flow, and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of donkey-hide gelatin small molecule peptide production, and specifically to a method for preparing donkey-hide gelatin small molecule peptide having multiple functions such as anti-fatigue, anti-oxidation, immunity enhancement, and hematopoietic function promotion. Background Art

[0002] Donkey-hide gelatin is a solid gelatin made by boiling and concentrating the dried or fresh skin of the donkey, an equine animal. It mainly contains protein and peptide components. Donkey-hide gelatin tastes sweet and is neutral in nature. It enters the lung, liver, and kidney meridians. It has the effects of nourishing blood and yin, moistening dryness, and stopping bleeding. It has been revered as a "holy medicine" for blood nourishment by doctors of all dynasties. The protein content of donkey-hide gelatin is 60% to 80%, and it is difficult to digest and absorb due to its large molecular weight. The small molecule donkey-hide gelatin peptide obtained by enzymatic hydrolysis technology can be directly absorbed by the human body in its functional form, with low energy consumption and high absorption rate. In the production of collagen, defatting is part of the pretreatment. Excessive fat content will generate insoluble fatty acid calcium in the subsequent process, and agglomerate, which seriously hinders the subsequent operation. In addition, excessive oil residue will affect the color, transparency and hydrophilic (hydrophobic) properties of the gelatin, so the fat needs to be separated. Donkey-hide gelatin contains gelatin, protein, trace elements and other effective ingredients, and has multiple functions such as anti-fatigue, anti-oxidation, enhancing immunity, and promoting hematopoietic function. Donkey-hide gelatin, which originated in the Qin and Han Dynasties and has a history of more than 2,000 years, is a solid animal glue that is boiled and concentrated from donkey skin. Its main component is protein. Most of them are square blocks or cubes, dark brown, shiny, hard and brittle, with bright cross-section. The fragments are brown and translucent when viewed under light, with a slight smell and a slightly sweet taste. It is used for blood deficiency, chlorosis, dizziness, palpitations, etc. It has the effect of nourishing yin and moistening dryness, and is a good blood tonic. Donkey-hide gelatin is used as the raw material, and after enzymatic hydrolysis by complex enzymes, the small molecule peptide compounds of donkey-hide gelatin obtained after separation and purification are easier to absorb, and their digestion and absorption rate is much greater than that of donkey-hide gelatin itself, and their activity is also much better than the amino acids in donkey-hide gelatin. As a molecular fragment of protein, donkey-hide gelatin small molecule peptides can not only provide the nutrients needed for human growth and development, but also have physiological activities that proteins do not have. Small molecule peptides are gradually favored and respected by contemporary people. At present, donkey-hide gelatin preparations, such as oral liquid, effervescent tablets, and soft capsules, are usually made by directly adding donkey-hide gelatin raw powder.

[0003] The present invention aims to study a method for preparing a pre-pregnancy nourishing donkey-hide gelatin small molecule peptide, which can effectively improve the health problems of pregnant women before pregnancy, and at the same time has multiple functions such as anti-fatigue, anti-oxidation, immunity enhancement, and promotion of hematopoietic function. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a method for preparing donkey-hide gelatin small molecule peptides suitable for pre-pregnancy nourishment; the present invention selects donkey-hide gelatin, which is a solid gelatin made by boiling and concentrating the dry or fresh skin of the donkey of the Equine family. It is known as the three nourishing treasures together with ginseng and deer antlers, and was first recorded in the "Shennong Bencao Jing". Donkey-hide gelatin contains gelatin, protein, trace elements and other effective ingredients, and has multiple effects such as anti-fatigue, anti-oxidation, enhancing immunity, and promoting hematopoietic function. Ginseng is sweet and mild, slightly bitter and not laxative, and has strong medicinal power. It is an important medicine for replenishing qi and strengthening the body. It is good at replenishing vital energy and treating qi deficiency and impotence; it is also good at replenishing the qi of the spleen and lungs and treating various symptoms of spleen and lung qi deficiency; it can also replenish qi and produce body fluid, calm the mind, and improve intelligence, and treat thirst, thirst, restlessness, palpitations and forgetfulness caused by body fluid loss. Yam can strengthen the spleen and stomach, benefit the lungs and relieve cough, moisturize the skin and remove dryness, lower blood sugar and blood lipids, and is very good for the body of pregnant women. Vitamin C can improve immunity and promote the growth of lymphocytes in the human body. It also has a very good antioxidant effect. It can prevent free radicals from harming human health and beautify the skin. It can prevent ultraviolet rays from harming the skin and avoid more dark spots, chloasma, sun spots, etc. on women's skin. It can also relieve the damage to the skin after sunburn, promote the skin to increase metabolism, and discharge the melanin substances that have been formed in the skin. A donkey-hide gelatin composite polypeptide suitable for postpartum recovery and its preparation method are characterized by the following main operating steps:

[0005] After the donkey-hide gelatin is dissolved, it is hydrolyzed with alkaline composite enzymes, wherein the alkaline composite enzymes are alkaline composite enzyme powders mixed with subtilisin, neutral protease and glycosidase.

[0006] After the exploration, Lactobacillus acidophilus culture liquid was prepared and fermented: Lactobacillus acidophilus ATCC 67707 culture liquid was prepared at a concentration of 2.6×10 8 ~3.6×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67707 bacterial liquid; taking 700-800g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, reacting at 35-38°C for 6-12h, and obtaining a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus.

[0007] As a preferred embodiment, the specific steps are as follows:

[0008] S1. Pretreatment of donkey-hide gelatin: Put the raw material of donkey-hide gelatin into a wall-breaking machine for crushing and grinding, and sieve the ground raw material powder through a 200-400 mesh sieve, and take the sieved raw material for use;

[0009] S2. Pretreatment of ginseng, Chinese yam and vitamin C: Take 100g of ginseng, put it in 700-1000g of water, soak it in water for 1h, use a homogenizer with a pore size of 200 mesh to crush and homogenize it. After it is completely crushed, heat the slurry, maintain the temperature when it rises to 70°C, then add 1% enzyme, and stir at a constant temperature for 2-4h for enzymolysis; after the enzymolysis is completed, raise the temperature to 80-120°C and perform high-temperature enzyme inactivation for 15-30 minutes; use a filter with a pore size of 500 mesh to perform coarse filtration, and then pass it through an ultrafiltration membrane with a molecular weight cutoff of 100,000 and an ultrafiltration membrane with a molecular weight cutoff of 10,000 in turn to obtain a ginseng small molecule peptide stock solution for use; peel and chop the Chinese yam, then use a pulper to grind and collect the slurry, dry it in the sun, and obtain Chinese yam powder for use; weigh 0.05g of vitamin C and dissolve it in 100ml for use;

[0010] S3, dissolving and mixing donkey-hide gelatin: weigh 500-800g of the donkey-hide gelatin powder obtained in step S1, add it into a blender, add a certain amount of clean water, stir it thoroughly, and control the stirring speed at 30-40r / min to make the collagen in the donkey-hide gelatin raw material powder fully dissolved in the water;

[0011] S4, filtering and removing impurities from donkey-hide gelatin: pouring out the mixed collagen obtained in step S3, filtering to remove undissolved impurities, collecting the filtered filtrate, standing it, and waiting for the filtrate to cool to room temperature;

[0012] S5, alkaline composite enzymatic hydrolysis: add 5% calcium hydroxide aqueous solution to the mixed solution obtained in step S4, add dropwise while stirring, and observe the pH value of the solution at all times during the stirring process until the pH value of the filtrate is between 7 and 8.5, then add 100-150g of alkaline composite enzyme powder mixed with subtilisin, neutral protease and glycosidase in a ratio of 1:1:1, and after fully mixing, perform enzymatic hydrolysis at a constant temperature and pressure of 50-55°C for 1.5-3h; in an alkaline environment, perform enzymatic hydrolysis on the donkey-hide gelatin raw material that is crushed and mixed into a solution in step S4, so that the components in the donkey-hide gelatin can be fully enzymatically hydrolyzed at a suitable temperature to form donkey-hide gelatin small molecule peptides;

[0013] S6, impurity removal and decolorization: add 50-80g of activated carbon powder to the solution after enzymatic hydrolysis in step S5, and stir continuously for 25-30min at 85-90°C to inactivate the enzymes, and at the same time, remove the odor and grease in the solution by adsorption of activated carbon and decolorize at the same time;

[0014] S7, filtration and concentration: using a filter press method to filter out the activated carbon particles and solid impurities in the solution, adding the filtered solution into a concentrator for concentration to obtain donkey-hide gelatin small molecule peptides;

[0015] S8. Preparation and fermentation of Lactobacillus acidophilus culture liquid: Lactobacillus acidophilus ATCC 67305 (purchased from Weikai Haisi Bioengineering Co., Ltd.) culture liquid was prepared at a concentration of 2.6×10 8 ~3.6×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67305 bacterial liquid; taking 700-800g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, and fermenting it at 35-38°C for 6-12h to obtain a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus;

[0016] S9, shaping: citric acid, stevioside, and fructose syrup are prepared in a ratio of 1:1:1 as a flavoring agent for later use, and the medicine obtained in the steps S8 and S2, Acer truncatum seed oil, and the flavoring agent are mixed and stirred in a ratio of 2:1:1. After stirring evenly, the mixture is cooled to below 35° C. until the mixture forms a paste.

[0017] Preferably, the donkey-hide gelatin used in step S1 is selected high-quality donkey-hide gelatin.

[0018] Preferably, in step S5, the addition amount of the alkaline complex enzyme powder is 0.5%-2% by weight of the donkey-hide gelatin raw material powder, and the alkaline complex enzyme powder is a mixture of subtilisin, neutral protease and glycosidase in a ratio of 1:1:1.

[0019] Preferably, the prepared donkey-hide gelatin small molecule peptide is a low peptide with an average molecular weight below 1000Da.

[0020] Preferably, in step S6, the activated carbon has the highest degreasing rate under the conditions of pH = 7.6, temperature of 26° C., and solid-liquid ratio of 1:40.

[0021] Preferably, the concentration of the Lactobacillus acidophilus ATCC 67305 bacterial solution used in step S8 is 3.6×10 8 cfu / ml.

[0022] Preferably, before step S9, the following flavoring agents need to be prepared: citric acid, stevioside, and fructose syrup, which are prepared in a ratio of 1:1:1 and then added to the mixed solution in S9 to adjust the taste of the donkey-hide gelatin small molecule peptide.

[0023] Preferably, the ratio of the donkey-hide gelatin raw material to the flavoring agent is 1:1.

[0024] The beneficial effects of the present invention are:

[0025] 1. The method of enzymolysis of donkey-hide gelatin with activated carbon of the present invention is convenient to operate, low in cost, short in enzymolysis time, reduced in pollution, high in fat degreasing rate, simple in process flow, and suitable for industrial production. The present invention creatively treats donkey-hide gelatin with composite enzymes first, lays a good foundation for subsequent probiotic fermentation, provides good metabolic protein, moderate energy, and a variety of active substances that stimulate the lactobacillus, and finally obtains donkey-hide gelatin small molecule peptides with high nutritional value and high biological activity.

[0026] 2. The Lactobacillus acidophilus fermentation used in the present invention can also regulate immune function by regulating intestinal function, reduce oxidative stress and inflammation to protect the brain, liver and other organs, help decompose toxins, etc. to help balance hormone levels and enhance fertility.

[0027] 3. The Chinese yam and ginseng used in the present invention have a synergistic effect and can better invigorate qi, benefit the lungs and relieve cough.

[0028] 4. The present invention uses as few auxiliary materials as possible during the production process to reduce the increase in the burden on the body of pregnant women. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0030] Figure 1 It is a comparison chart of blood lipid and blood sugar of Example 1 of the present invention and Comparative Examples 1 and 2.

[0031] Figure 2 It is a comparison chart of degreasing rate between Example 2 of the present invention and Comparative Example 2.

[0032] Figure 3 This is a SEM image of the donkey-hide gelatin small molecule peptide in Example 1 of the present invention.

[0033] Figure 4 is a SEM image of the activated carbon powder in Example 2 of the present invention.

[0034] Figure 5 It is a comparison chart of immunoglobulins in animal experiments in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0036] Example 1

[0037] This embodiment proves that the added yam has a better nourishing effect on the body of pregnant women, can strengthen the spleen and stomach, benefit the lungs and relieve cough, moisturize the skin and remove dryness, lower blood sugar and blood lipids, and is extremely good for the body of pregnant women.

[0038] S1. Pretreatment of donkey-hide gelatin: Put the raw material of donkey-hide gelatin into a wall-breaking machine for crushing and grinding, sieve the ground raw material powder through a 200-mesh sieve, and take the sieved raw material for later use;

[0039] S2. Pretreatment of ginseng, Chinese yam and vitamin C: 100g of ginseng was placed in 850g of water and soaked for 1h. The slurry was crushed and homogenized using a homogenizer with a pore size of 200 mesh. After the slurry was completely crushed, the slurry was heated and maintained at 70°C. Then 1% medium-temperature α-amylase was added and stirred at a constant temperature for 3h for enzymolysis. After the enzymolysis was completed, the temperature was raised to 100°C and the high-temperature enzyme was inactivated for 20 minutes. The ginseng was coarsely filtered using a filter with a pore size of 500 mesh, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 100,000 and an ultrafiltration membrane with a molecular weight cutoff of 10,000 in turn to obtain a ginseng small molecule peptide stock solution for later use. The Chinese yam was peeled and chopped, and then the slurry was collected by grinding with a grinder, and dried to obtain Chinese yam powder for later use. 0.05g of vitamin C was weighed and dissolved in 100ml for later use.

[0040] S3, dissolving and mixing donkey-hide gelatin: weigh 600g of the donkey-hide gelatin powder obtained in step S1, add it into a blender, add a certain amount of clean water, stir it thoroughly, and control the stirring speed at 40r / min to make the collagen in the donkey-hide gelatin raw material powder fully dissolved in the water;

[0041] S4, filtering and removing impurities from donkey-hide gelatin: pouring out the mixed collagen obtained in step S3, filtering to remove undissolved impurities, collecting the filtered filtrate, standing it, and waiting for the filtrate to cool to room temperature;

[0042] S5, alkaline composite enzymatic hydrolysis: add 5% calcium hydroxide aqueous solution to the mixed solution obtained in step S4, add dropwise while stirring, and observe the pH value of the solution at all times during the stirring process until the pH value of the filtrate is between 7 and 8.5, then add 150g of alkaline composite enzyme powder mixed with subtilisin, neutral protease and glycosidase in a weight ratio of 1:1:1, and after fully mixing, perform enzymatic hydrolysis at a constant temperature and pressure of 50°C for 1.5h; in an alkaline environment, perform enzymatic hydrolysis on the donkey-hide gelatin raw material that is crushed and mixed into a solution in step S4, so that the components in the donkey-hide gelatin can be fully enzymatically hydrolyzed at a suitable temperature to form donkey-hide gelatin small molecule peptides;

[0043] S6, impurity removal and decolorization: add 70g of activated carbon powder to the solution after enzymatic hydrolysis in step S5, and stir continuously for 30min at 90°C to inactivate the enzymes, and at the same time, remove the odor and grease in the solution by adsorption of activated carbon and decolorize at the same time;

[0044] S7, filtration and concentration: using a filter press method to filter out the activated carbon particles and solid impurities in the solution, adding the filtered solution into a concentrator for concentration to obtain donkey-hide gelatin small molecule peptides;

[0045] S8. Preparation and fermentation of Lactobacillus acidophilus culture liquid: Lactobacillus acidophilus ATCC 67305 (purchased from Weikai Haisi Bioengineering Co., Ltd.) culture liquid was prepared at a concentration of 2.6×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67305 bacterial liquid; taking 800g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, and fermenting it at 38°C for 12h to obtain a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus;

[0046] S9, shaping: citric acid, stevioside, and fructose syrup are prepared in a ratio of 1:1:1 as a flavoring agent for later use, and the medicine obtained in the steps S8 and S2, Acer truncatum seed oil, and the flavoring agent are mixed and stirred in a ratio of 2:1:1. After stirring evenly, the mixture is cooled to below 35° C. until the mixture forms a paste.

[0047] Comparative Example 1: Except that Chinese yam is not added in step S2, the remaining steps are the same as those in Example 1.

[0048] Comparative Example 2: The ginseng in step S2 is removed, and the remaining steps are the same as those in Example 1.

[0049] Example 2

[0050] This example demonstrates that the activated carbon enzymatic hydrolysis method has a higher degreasing rate, shorter enzymatic hydrolysis time and less pollution.

[0051] S1. Pretreatment of donkey-hide gelatin: Put the raw material of donkey-hide gelatin into a wall-breaking machine for crushing and grinding, sieve the ground raw material powder through a 300-mesh sieve, and take the sieved raw material for later use;

[0052] S2. Pretreatment of ginseng, Chinese yam and vitamin C: 150g of ginseng was placed in 700g of water and soaked for 1h. The slurry was crushed and homogenized using a homogenizer with a pore size of 200 mesh. After the slurry was completely crushed, the slurry was heated and maintained at 70°C. Then 1% medium-temperature α-amylase was added and stirred at a constant temperature for 2h for enzymolysis. After the enzymolysis was completed, the temperature was raised to 80°C and the high-temperature enzyme was inactivated for 15 minutes. The ginseng was coarsely filtered using a filter with a pore size of 500 mesh, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 100,000 and an ultrafiltration membrane with a molecular weight cutoff of 10,000 in turn to obtain a ginseng small molecule peptide stock solution for use. The Chinese yam was peeled and chopped, and then the slurry was collected by a pulping machine, dried, and the Chinese yam powder was obtained for use. 0.05g of vitamin C was weighed and dissolved in 100ml for use.

[0053] S3, dissolving and mixing donkey-hide gelatin: weigh 500g of the donkey-hide gelatin powder obtained in step S1, add it into a blender, add a certain amount of clean water, stir it thoroughly, and control the stirring speed at 30r / min to make the collagen in the donkey-hide gelatin raw material powder fully dissolved in the water;

[0054] S4, filtering and removing impurities from donkey-hide gelatin: pouring out the mixed collagen obtained in step S3, filtering to remove undissolved impurities, collecting the filtered filtrate, standing it, and waiting for the filtrate to cool to room temperature;

[0055] S5, alkaline composite enzymatic hydrolysis: add 5% calcium hydroxide aqueous solution to the mixed solution obtained in step S4, add dropwise while stirring, and observe the pH value of the solution at all times during the stirring process until the pH value of the filtrate is between 7 and 8.5, then add 100g of alkaline composite enzyme powder mixed with subtilisin, neutral protease and glycosidase in a weight ratio of 1:1:1, and after fully mixing, perform enzymatic hydrolysis at a constant temperature and pressure of 55°C for 3h; in an alkaline environment, perform enzymatic hydrolysis on the donkey-hide gelatin raw material that is crushed and mixed into a solution in step S4, so that the components in the donkey-hide gelatin can be fully enzymatically hydrolyzed at a suitable temperature to form donkey-hide gelatin small molecule peptides;

[0056] S6, impurity removal and decolorization: add 50g of activated carbon powder to the solution after enzymatic hydrolysis in step S5, stir continuously for 25min at 85°C to inactivate the enzymes, and at the same time, remove the odor and grease in the solution by adsorption of activated carbon and decolorize at the same time;

[0057] S7, filtration and concentration: using a filter press method to filter out the activated carbon particles and solid impurities in the solution, adding the filtered solution into a concentrator for concentration to obtain donkey-hide gelatin small molecule peptides;

[0058] S8. Preparation and fermentation of Lactobacillus acidophilus culture liquid: Lactobacillus acidophilus ATCC 67305 (purchased from Weikai Haisi Bioengineering Co., Ltd.) culture liquid was prepared at a concentration of 3.6×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67305 bacterial liquid; taking 700g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, and fermenting it at 35°C for 6h to obtain a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus;

[0059] S9, shaping: citric acid, stevioside, and fructose syrup are prepared in a ratio of 1:1:1 as a flavoring agent for later use, and the medicine obtained in the steps S8 and S2, Acer truncatum seed oil, and the flavoring agent are mixed and stirred in a ratio of 2:1:1. After stirring evenly, the mixture is cooled to below 35° C. until the mixture forms a paste.

[0060] Comparative Example 3: The method of activated carbon enzymatic hydrolysis of donkey-hide gelatin was replaced by an ultrasonic combined with a surfactant method, and the rest was the same as Example 2.

[0061] The activated carbon powder in step S6 is removed, and then the grease is removed by ultrasound, and finally the prepared medicine is placed in a Soxhlet extractor; the medicine prepared by removing the grease with the activated carbon powder is placed in a Soxhlet extractor, and finally the remaining amount of grease is compared.

[0062] Table 1 Comparison of degreasing rates between Example 2 and Comparative Example 2

[0063]

[0064] The results show that the degreasing rate of activated carbon powder enzymatic hydrolysis method is higher than that of ultrasonic combined with surfactant method.

[0065] Example 3

[0066] S1. Pretreatment of donkey-hide gelatin: Put the raw material of donkey-hide gelatin into a wall-breaking machine for crushing and grinding, sieve the ground raw material powder through a 200-mesh sieve, and take the sieved raw material for later use;

[0067] S2. Pretreatment of ginseng, Chinese yam and vitamin C: 100g of ginseng was placed in 1000g of water and soaked for 1h. The slurry was crushed and homogenized using a homogenizer with a pore size of 200 mesh. After the slurry was completely crushed, the slurry was heated and maintained at 70°C. Then 1% medium-temperature α-amylase was added and stirred at a constant temperature for 4h for enzymolysis. After the enzymolysis was completed, the temperature was raised to 120°C and the high-temperature enzyme was inactivated for 30 minutes. The ginseng was coarsely filtered using a filter with a pore size of 500 mesh, and then passed through an ultrafiltration membrane with a molecular weight cutoff of 100,000 and an ultrafiltration membrane with a molecular weight cutoff of 10,000 in turn to obtain a ginseng small molecule peptide stock solution for use. The Chinese yam was peeled and chopped, and then the slurry was collected by grinding with a grinder, and dried to obtain Chinese yam powder for use. 0.05g of vitamin C was weighed and dissolved in 100ml for use.

[0068] S3, dissolving and mixing donkey-hide gelatin: weigh 800g of the donkey-hide gelatin powder obtained in step S1, add it into a blender, add a certain amount of clean water, stir it thoroughly, and control the stirring speed at 35r / min to make the collagen in the donkey-hide gelatin raw material powder fully dissolved in the water;

[0069] S4, filtering and removing impurities from donkey-hide gelatin: pouring out the mixed collagen obtained in step S3, filtering to remove undissolved impurities, collecting the filtered filtrate, standing it, and waiting for the filtrate to cool to room temperature;

[0070] S5, alkaline composite enzymatic hydrolysis: add 5% calcium hydroxide aqueous solution to the mixed solution obtained in step S4, add dropwise while stirring, and observe the pH value of the solution at all times during the stirring process until the pH value of the filtrate is between 7 and 8.5, then add 120g of alkaline composite enzyme powder mixed with subtilisin, neutral protease and glycosidase in a ratio of 1:1:1, and after fully mixing, perform enzymatic hydrolysis at a constant temperature and pressure of 45°C for 2h; in an alkaline environment, perform enzymatic hydrolysis on the donkey-hide gelatin raw material that is crushed and mixed into a solution in step S4, so that the components in the donkey-hide gelatin can be fully enzymatically hydrolyzed at a suitable temperature to form donkey-hide gelatin small molecule peptides;

[0071] S6, impurity removal and decolorization: add 80g of activated carbon powder to the solution after enzymatic hydrolysis in step S5, stir continuously for 25min at 85°C to inactivate the enzymes, and at the same time, remove the odor and grease in the solution by adsorption of activated carbon and decolorize at the same time;

[0072] S7, filtration and concentration: using a filter press method to filter out the activated carbon particles and solid impurities in the solution, adding the filtered solution into a concentrator for concentration to obtain donkey-hide gelatin small molecule peptides;

[0073] S8. Preparation and fermentation of Lactobacillus acidophilus culture liquid: Lactobacillus acidophilus ATCC 67707 (purchased from Weikai Haisi Bioengineering Co., Ltd.) culture liquid was prepared at a concentration of 3.0×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67707 bacterial liquid; taking 750g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, and fermenting it at 70°C for 9h to obtain a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus;

[0074] S9, shaping: citric acid, stevioside, and fructose syrup are prepared in a ratio of 1:1:1 as a flavoring agent for later use, and the medicine obtained in the steps S8 and S2, Acer truncatum seed oil, and the flavoring agent are mixed and stirred in a ratio of 2:1:1. After stirring evenly, the mixture is cooled to below 35° C. until the mixture forms a paste.

[0075] Comparative Example 4: In step S8, the fermentation is performed without using the Lactobacillus acidophilus culture ATCC 67305, and the rest is the same as in Example 3.

[0076] Comparative Example 5: The Lactobacillus acidophilus bacterial solution ATCC 67305 in step S8 was replaced with Lactobacillus acidophilus bacterial solution ATCC 67707 (purchased from Weikaihaisi Bioengineering Co., Ltd.).

[0077] Example 4

[0078] Pre-pregnancy conditioning experiment

[0079] Animal grouping, modeling and drug administration

[0080] 60 SPF-grade healthy KM female mice (supplied by Sbefor (Beijing) Biotechnology Co., Ltd., animal production license SCXK (Beijing) 2016-0002), 130 days old, weighing 92±11g, were kept in our company's SPF-grade animal breeding room with relatively constant temperature (25℃), constant humidity (75%), fed with conventional female mouse pellet feed, and had free access to food and water.

[0081] Thirty female mice were taken as experimental research subjects 1, and the female mice were randomly divided into 6 groups, each with 5 mice, including an immunodeficiency model group, a normal group, a positive control folic acid group, an example group (Example 3), and a comparative example group (Comparative Example 4, Comparative Example 5) by a random number table method. The modeling day was the 0th day. From the 3rd day, except for the normal group, the other groups were infused with cyclophosphamide (50 mg / kg) daily to establish a mouse model of immunodeficiency in preparation for pregnancy for a total of 10 days. At the same time as the modeling, according to the human and animal dose conversion method, the example group (Example 3) and the comparative example group (Comparative Example 4 and Comparative Example 5) were fed with 2.0 g / 100 g·d of the composite donkey-hide gelatin small molecule peptide prepared in Example 3, Comparative Example 4, and Comparative Example 5, respectively, and the folic acid group was given folic acid (5 g / kg), and the normal group and the immunodeficiency model group were given an equal amount of physiological saline. The mice were killed 10 days after intragastric administration (12th day of pregnancy), and the spleen and thymus of each group of mice were collected, weighed and recorded for histological morphology observation and spleen lymphocyte proliferation capacity determination. The body immunity and health status of the mice were tested with reference to the existing methods.

[0082] Thirty female mice were selected as experimental research subjects 2. The same model was constructed using the same method as above, and then five healthy male mice were placed in the model. After the experiment was completed, the pregnancy status was checked by dissection. After the animals were euthanized, the uterus was removed and the uterine status was observed.

[0083] Index detection

[0084] According to the ELISA kit instructions, the levels of immunoglobulins IgG, IgA, and IgM in the serum of the experimental research subject one were determined.

[0085] According to observations, all the female mice in Experimental Study 2 were pregnant.

[0086] Table 2 The levels of immunoglobulins IgG, IgA, and IgM in the serum of the experimental subject 1

[0087]

[0088] Table 3 Ovarian weight of experimental subject 1

[0089]

[0090] According to Table 2 and Table 3, the composite donkey-hide gelatin small molecule peptides fermented by Lactobacillus acidophilus liquid ATCC 67305 can enhance immunity and physical fitness. When replaced by Lactobacillus acidophilus liquid ATCC 67707, the enhancement of immunity is not obvious, indicating that the fermentation of Lactobacillus acidophilus liquid ATCC 67305 has specificity for enhancing immunity. According to the results of experimental research object 2, the composite donkey-hide gelatin small molecule peptides prepared by the present invention can improve the fertility of female mice.

[0091] Example 5

[0092] Thirty SPF-grade healthy KM mice (supplied by Beijing Biotechnology Co., Ltd., animal production license SCXK (Beijing) 2016-0002), aged 130 days and weighing 92±11g, were raised in our company's SPF-grade animal breeding room with relatively constant temperature (25°C) and constant humidity (75%), fed with regular feed, and had free access to food and water.

[0093] Thirty mice were taken as research subjects, and the mice were randomly divided into a hyperlipidemia and hyperglycemia model group, a normal group, a positive control biguanide hypoglycemic drug group, an example group (Example 1), and a comparative example group (Comparative Example 1, Comparative Example 2) using a random number table method, with a total of 6 groups, 5 mice in each group. The day of modeling was the 0th day. From the 3rd day, except for the normal group, the other groups were fed with high-fat and high-sugar feed every day to establish a mouse hyperlipidemia and hyperglycemia model for a total of 10 days. At the same time as modeling, according to the human and animal dose conversion method, the example group (Example 1) and the comparative example group (Comparative Example 1 and Comparative Example 2) were respectively fed with 2.0g / 100g·d of the composite donkey-hide gelatin small molecule peptide prepared in Example 1, Comparative Example 1, and Comparative Example 2, the biguanide hypoglycemic drug group was given biguanide hypoglycemic drugs (150mg g / kg), and the normal group and the hyperlipidemia and hyperglycemia model group were given an equal amount of physiological saline. After 10 days of intragastric administration, the mice in each group were fasted for 24 h, blood was collected from the eye sockets, and the mice were placed at 70°C for 30 min and 3000 r / min. -1 After centrifugation for 10 min, serum was separated for index determination.

[0094] Index detection

[0095] The fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) in the serum of mice were measured using an automatic biochemical analyzer.

[0096] Table 4 Serum FBG, TC, TG contents in experimental study subject 2

[0097]

[0098] It can be seen from Table 4 that compared with medicines without yam, medicines with yam can better benefit the lungs and relieve cough, and lower blood sugar and blood lipids. This shows that yam and ginseng have a synergistic effect, which can better replenish qi, benefit the lungs and relieve cough, and have a better effect.

[0099] The above-mentioned embodiments only express the specific implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a composite donkey-hide gelatin small molecule peptide liquid suitable for pre-pregnancy nourishment, characterized in that Here are the steps: The specific steps are as follows: S1. Pretreatment of donkey-hide gelatin: Put the raw material of donkey-hide gelatin into a wall-breaking machine for crushing and grinding, and sieve the ground raw material powder through a 200-400 mesh sieve, and take the sieved raw material for use; S2. Pretreatment of ginseng, yam and vitamin C: take 100g ginseng, put it in 700-1000g water, soak it in water for 1h, use a homogenizer with a pore size of 200 mesh to crush and homogenize it, after it is completely crushed, heat the slurry, maintain the temperature when it rises to 70°C, then add 1% medium-temperature α-amylase, and stir at a constant temperature for 2-4h for enzymolysis; after the enzymolysis is completed, raise the temperature to 80-120°C and perform high-temperature enzyme inactivation for 15-30 minutes; use a filter with a pore size of 500 mesh for coarse filtration, and then pass it through an ultrafiltration membrane with a molecular weight cutoff of 100,000 and an ultrafiltration membrane with a molecular weight cutoff of 10,000 in turn to obtain a ginseng small molecule peptide stock solution for use; peel and chop the yam, then use a pulper to grind and collect the slurry, dry it in the sun, and obtain yam powder for use; weigh 0.05g of vitamin C and dissolve it in 100ml for use; S3, dissolving and mixing donkey-hide gelatin: weigh 500-800g of the donkey-hide gelatin powder sieved in step S1, add it into a blender, add clean water, stir it thoroughly, and control the stirring speed at 30-40r / min to make the collagen in the donkey-hide gelatin raw material powder fully dissolved in the water; S4, filtering and removing impurities from donkey-hide gelatin: pouring out the dissolved collagen obtained in step S3, filtering to remove undissolved impurities, collecting the filtered filtrate, standing it, and waiting for the filtrate to cool to room temperature; S5, alkaline composite enzymatic hydrolysis of donkey-hide gelatin: add 5% calcium hydroxide aqueous solution to the filtrate obtained in step S4, add dropwise while stirring, and observe the pH value of the solution at all times during the stirring process until the pH value of the filtrate is between 7 and 8.5, then add 100-150g of alkaline composite enzyme powder mixed with subtilisin, neutral protease and glycosidase in a weight ratio of 1:1:1, and after fully mixing, perform enzymatic hydrolysis at a constant temperature and pressure of 50-55°C for 1.5-3h; perform enzymatic hydrolysis on the filtrate in step S4 under an alkaline environment, so that the components in the donkey-hide gelatin can be fully enzymatically hydrolyzed at a suitable temperature to form donkey-hide gelatin small molecule peptides; S6, impurity removal and decolorization: add 50-80g of activated carbon powder to the solution after enzymatic hydrolysis in step S5, and stir continuously for 25-30min at 85-90°C to inactivate the enzymes, and at the same time, remove the odor and grease in the solution by adsorption of activated carbon and decolorize at the same time; S7, filtration and concentration: using a filter press method to filter out the activated carbon particles and solid impurities in the solution, adding the filtered solution into a concentrator for concentration to obtain donkey-hide gelatin small molecule peptides; S8. Preparation and fermentation of Lactobacillus acidophilus culture liquid: Preparation of Lactobacillus acidophilus ATCC 67707 culture liquid with a concentration of 2.6×10 8 ~3.6×10 8 cfu / ml of Lactobacillus acidophilus ATCC 67707 bacterial liquid; taking 700-800g of the donkey-hide gelatin small molecule peptide prepared in step S7, adding it to the prepared bacterial liquid, reacting at 35-38°C for 6-12h, and obtaining a Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid fermented by Lactobacillus acidophilus; S9, shaping: citric acid, stevioside, and fructose syrup are prepared in a ratio of 1:1:1 as a seasoning for later use; the Lactobacillus acidophilus-donkey-hide gelatin small molecule peptide liquid in step S8 and the medicine obtained by the ginseng small molecule peptide stock solution, yam powder, and vitamin C in S2, Acer truncatum seed oil and the seasoning are mixed and stirred in a ratio of 2:1:1; after stirring evenly, the mixture is cooled to below 35°C until the mixture forms a paste.

2. The method for preparing a composite donkey-hide gelatin small molecule peptide liquid suitable for pre-pregnancy nourishment according to claim 1, characterized in that: The prepared donkey-hide gelatin small molecule peptide is a low peptide with an average molecular weight below 1000Da.

3. The method for preparing a composite donkey-hide gelatin small molecule peptide liquid suitable for pre-pregnancy nourishment according to claim 2, characterized in that: Before step S9, the following flavoring agents need to be prepared: citric acid, stevioside, and fructose syrup, which are prepared in a ratio of 1:1:1 and then added into S9 to adjust the taste of the donkey-hide gelatin small molecule peptide.

4. The method for preparing a composite donkey-hide gelatin small molecule peptide liquid suitable for pre-pregnancy nourishment according to claim 3, characterized in that: The concentration of the Lactobacillus acidophilus ATCC 67707 bacterial solution used in step S8 is 3.6×10 8 cfu / ml.

5. A composite donkey-hide gelatin small molecule peptide liquid suitable for pre-pregnancy nourishment, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

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