CN116473239B - Protein peptide composition for relieving acute alcoholic liver injury and preparation method thereof - Google Patents
Protein peptide composition for relieving acute alcoholic liver injury and preparation method thereof Download PDFInfo
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- CN116473239B CN116473239B CN202310407425.3A CN202310407425A CN116473239B CN 116473239 B CN116473239 B CN 116473239B CN 202310407425 A CN202310407425 A CN 202310407425A CN 116473239 B CN116473239 B CN 116473239B
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Abstract
The invention provides a protein peptide composition for relieving acute alcoholic liver injury and a preparation method thereof, belonging to the technical field of protein peptides, and comprising the following steps: extracting nostoc sphaeroids kutz protein, mixing with oyster meat and egg white, performing secondary enzymolysis to obtain an enzymolysis product, adding hydrolyzed wheat protein powder, hydrolyzed soybean protein powder, a carbon source, vitamins and inorganic salts into water to prepare a culture medium, inoculating white ginseng fungus and saccharomycetes for fermentation, sterilizing, inoculating lactobacillus plantarum and lactobacillus rhamnosus, fermenting and proliferating, and freeze-drying to prepare a fermentation product, and embedding the fermentation product, together with corn oligopeptide, fructo-oligosaccharide and taurine into a sodium alginate-carboxymethyl cellulose sodium shell layer to prepare the protein peptide composition for relieving acute alcoholic liver injury. The composition contains abundant small peptide substances, has low energy consumption, quick absorption, high absorption rate and difficult saturation of a carrier, has better inhibition effect on the increase of the concentration of ethanol in serum, and has obvious effects of dispelling the effects of alcohol and protecting the liver.
Description
Technical Field
The invention relates to the technical field of protein peptides, in particular to a protein peptide composition for relieving acute alcoholic liver injury and a preparation method thereof.
Background
Alcoholic liver injury (Alcoholic liver disease, ALD) can not only damage the health of drinkers, but can even harm the families causing social problems. The data shows that global excessive alcohol consumption in 2016 resulted in about 300 thousands of deaths. The liver injury caused by one-time overdrinking or long-term consumption of alcohol is classified into chronic and acute liver injury according to the etiology. Alcoholic liver injury is generally thought to be caused by oxidative stress mediated by ethanol and metabolites, immune responses of the body, and liver cell injury.
The early stages of alcoholic liver injury are manifested as fatty liver, mainly due to the accumulation of fat (triglycerides, phospholipids, cholesterol esters, etc.) in hepatocytes. Early studies showed that drinking increases the NAPDH/NAD ratio in hepatocytes, destroying beta-oxidized fatty acids in mitochondria, leading to steatosis. Alcohol intake enhances the lipid supply from the small intestine to the liver, increasing the liver's absorption of fatty acids metabolized from adipose tissue. The long-term drinking can also cause malnutrition absorption, so that the absorption of antioxidants in foods is reduced, oxidation promotion substances in organisms are increased, the oxidation resistance substances are reduced, oxidative stress is promoted, and finally, the hepatic cell injury and even necrosis are caused.
There are many kinds of drugs for treating alcoholic liver injury today. The treatment cycle of the chemical medicine is shorter, but adverse reactions are often accompanied, and the chemical medicine is not suitable for the old and the weak pregnant women to take, and has certain limitation. The side effect of the traditional Chinese medicine is small, but the treatment period is long, besides the verification of part of Chinese patent medicines, other prescription medicines are different from person to person, and the clinical large-scale popularization and application are not facilitated.
With the improvement of living standard and the increase of health consciousness, "homology of medicine and food" has attracted a great deal of attention. Based on the theory, compared with chemical medicines, the protein peptide has the advantages of convenient absorption, wide sources, good safety and the like, can participate in but not interfere with human metabolism, has little adverse reaction, and is suitable for various crowds with renal insufficiency. Because of its high nutritional value and functional activity, it can improve to some extent the malnutrition and complications due to alcoholism in addition to repairing liver injury. When the nutritional status of the patient is improved, the immune function of the patient is also obviously improved. More and more studies begin to utilize natural active substances extracted from foods to intervene in alcoholic liver injury.
Disclosure of Invention
The invention aims to provide a protein peptide composition for relieving acute alcoholic liver injury and a preparation method thereof, which contain abundant small peptide substances, have the advantages of low energy consumption, quick absorption, high absorptivity, difficult saturation of a carrier and the like, have higher contents of alanine, leucine and glutamic acid, have better inhibition effect on the increase of the concentration of ethanol in serum, and have obvious effects of dispelling the effects of alcohol and protecting the liver.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a protein peptide composition for relieving acute alcoholic liver injury, which comprises the steps of extracting nostoc sphaeroids kutz protein, mixing with oyster meat and egg white, carrying out secondary enzymolysis to obtain an enzymolysis product, adding hydrolyzed wheat protein powder, hydrolyzed soybean protein powder, a carbon source, vitamins and inorganic salts into water to prepare a culture medium, inoculating white ginseng fungus and saccharomycetes for fermentation, sterilizing, inoculating lactobacillus plantarum and lactobacillus rhamnosus, fermenting and proliferating, and freeze-drying to prepare a fermentation product, and embedding the fermentation product, corn oligopeptide, fructo-oligosaccharide and taurine into a sodium alginate-carboxymethyl cellulose shell to prepare the protein peptide composition for relieving acute alcoholic liver injury.
As a further improvement of the invention, the method comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, freezing and thawing, recovering to room temperature, adding lysozyme for enzymolysis, inactivating enzyme, filtering, adding ethanol into the filtrate for precipitation, and filtering to obtain nostoc sphaeroids kutz protein liquid;
s2, primary enzymolysis: mincing oyster meat, mixing with egg white and nostoc sphaeroids kutz protein liquid prepared in the step S1, adding primary complex enzyme for enzymolysis, and inactivating enzyme to obtain a primary enzymolysis product;
S3, secondary enzymolysis: adding a secondary complex enzyme into the primary enzymolysis product obtained in the step S2 for enzymolysis, inactivating enzyme, filtering, and freeze-drying to obtain an enzymolysis product;
s4, preparing a culture medium: adding the enzymolysis product obtained in the step S3, hydrolyzed wheat protein powder, hydrolyzed soybean protein powder, a carbon source, vitamins and inorganic salt into water, adjusting the pH value, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating activated white ginseng fungus and saccharomycete strain seed liquid into the culture medium prepared in the step S4, fermenting and culturing, sterilizing and filtering to prepare fermentation liquor;
s6, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution into the fermentation liquor prepared in the step S5, supplementing a carbon source, fermenting, proliferating and culturing, and freeze-drying to obtain a fermentation product;
s7, embedding: dissolving sodium alginate and sodium carboxymethylcellulose in water, adding the fermentation product obtained in the step S6, corn oligopeptide, fructo-oligosaccharide and taurine, stirring and mixing uniformly, adding edible oil, emulsifying, dropwise adding calcium chloride solution, solidifying at normal temperature, and freeze-drying to obtain the protein peptide composition for relieving acute alcoholic liver injury.
As a further improvement of the invention, the solid-to-liquid ratio of nostoc powder to water in the step S1 is 1:5-7g/mL, and the freezing and thawing treatment method is that the nostoc powder is frozen for 2-3h at the temperature of-25 to-20 ℃ and thawed at room temperature; the addition amount of lysozyme is 1-2g/L, the enzymolysis temperature is 35-40 ℃, the time is 2-3h, and ethanol is added until the system ethanol content is 70-80wt% and the precipitation is 5-7h.
As a further improvement of the invention, in the step S2, the mass ratio of oyster meat, egg white, nostoc protein liquid and primary compound enzyme is 5-7:3-5:50-70:0.5-1, the primary compound enzyme is alkaline protease and trypsin, the mass ratio is 5-7:10, and the enzymolysis temperature is 38-42 ℃ and the time is 2-3h.
As a further improvement of the invention, in the step S3, the mass ratio of the primary enzymolysis product to the secondary compound enzyme is 100:1-1.5, the mass ratio of the secondary compound enzyme is 3-5:2, the enzymolysis temperature is 45-55 ℃ and the time is 1-2h.
As a further improvement of the invention, the mass ratio of the enzymolysis product, hydrolyzed wheat protein powder, hydrolyzed soybean protein powder, carbon source, vitamin, inorganic salt and water in the step S4 is 5-10:3-5:2-4:20-30:1-2:2-3:500, wherein the carbon source is at least one of glucose, molasses, maltose, lactose, sucrose, fructose, soluble starch and agar, and the vitamin is at least one of vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin K, vitamin B12, vitamin D1, vitamin E, folic acid and vitamin D3, and the inorganic salt is at least one of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride; the pH value is adjusted to 6.7-7.2.
As a further improvement of the invention, the preparation method of the activated white ginseng fungus and saccharomycete strain seed liquid in the step S5 comprises the steps of respectively inoculating white ginseng fungus and saccharomycete into a Gao' S medium, and carrying out activation culture for 12-16 hours at 37-40 ℃ and 50-70r/min to obtain the strain with the bacterial content of 10 8 -10 9 cfu/mL strain seed solution, wherein the inoculum sizes of the activated white ginseng strain and the activated yeast strain seed solution are respectively 2-3% and 1-2%, the fermentation culture conditions are 37-40 ℃ and 50-70r/min, and the fermentation culture is carried out for 36-48h.
As a further improvement of the invention, the preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution in the step S6 comprises the steps of respectively inoculating lactobacillus plantarum and lactobacillus rhamnosus to Gao' S cultureIn the medium, under the conditions of 37-40 ℃ and 50-70r/min and micro-hypoxia, the medium is subjected to activation culture for 12-16h, and the bacterial content is 10 8 -10 9 cfu/mL strain seed liquid; the inoculum sizes of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solutions are 1.5-2% and 1-2% respectively, the conditions of fermentation proliferation culture are 36-38 ℃,50-70r/min, fermentation culture is carried out for 36-48h under the micro-anoxic condition, the micro-anoxic condition is that the content of carbon dioxide is 3-5v/v%, the content of oxygen is 4-6v/v%, and the balance is nitrogen; the amount of the added carbon source is 20-50g/L, and the carbon source is at least one selected from glucose, molasses, maltose, lactose, sucrose, fructose, soluble starch and agar.
As a further improvement of the invention, in the step S7, the mass ratio of the sodium alginate, the sodium carboxymethylcellulose, the water, the fermentation product, the corn oligopeptide, the fructo-oligosaccharide and the taurine is 5-7:7-10:100:12-15:3-5:2-3:1-2, and the edible oil is at least one selected from peanut oil, corn oil, soybean oil, rapeseed oil, fish oil, sunflower seed oil, sesame oil, linseed oil and olive oil; the emulsifying condition is 12000-15000r/min for emulsifying for 4-7min, and the concentration of the calcium chloride solution is 1-2wt%; the curing time at normal temperature is 30-50min.
The invention further protects a protein peptide composition for relieving acute alcoholic liver injury, which is prepared by the preparation method.
The invention has the following beneficial effects: the damage caused by oxidation is one of main hazards of alcohol, excessive drinking leads to aggregation of alcohol and acetaldehyde in blood and liver, and the liver can form a great amount of active oxygen, active nitrogen and the like in the process of decomposing alcohol for metabolism, so that the oxidation resistance level is reduced, further excessive oxidation stress is caused to generate oxidation products, an oxidation resistance system is damaged, mitochondria is damaged, and liver cell damage and liver dysfunction are caused. Oxidative stress and inflammatory response caused by alcohol metabolism can lead to mitochondrial dependent apoptosis, and accelerate the progress of alcoholic liver injury. The long-term consumption of alcohol in large amounts causes great damage to the liver of the body, resulting in alcoholic liver diseases including alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis and other liver diseases.
Generally, after alcohol is slowly introduced, most of the alcohol is absorbed in the process of passing through stomach, small intestine, large intestine and duodenum, only a very small part (about 2%) is discharged, and most of the absorbed alcohol enters liver for metabolism through blood circulation. Alcohol enters the body through a variety of metabolic pathways, but 90% of alcohol metabolism in the liver is catalyzed by Alcohol Dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH), wherein the ADH pathway is the main metabolic pathway, and Nicotinamide Adenine Dinucleotide (NAD) is present during the action + ) Is coenzyme. During ethanol oxidation, coenzyme I phosphorylates to coenzyme II, cytosolic NADH/NAD + The ratio is obviously increased, so that the tricarboxylic acid circulation is limited, thereby affecting the fatty acid metabolism, causing lipid peroxidation and causing partial fatty acid accumulation. After the organism ingests the protein peptide, the concentration of alanine and leucine in blood is increased, and NAD is increased + Is sufficiently supplemented, the tricarboxylic acid circulation is not limited any more, and the increase of the concentration of ethanol in blood is prevented.
The protein peptide composition for relieving acute alcoholic liver injury achieves efficacy aiming at the absorption and metabolism mechanism of alcohol in vivo by three aspects: 1. reducing the concentration of ethanol in the blood by slowing down the rate of absorption and increasing metabolism in the gastrointestinal tract; 2. accelerating alcohol metabolism in the liver by modulating metabolic enzyme activity; 3. reducing injury by eliminating free radicals generated during alcohol metabolism.
The protein peptide composition for relieving acute alcoholic liver injury has the obvious effects of relieving alcoholism and protecting liver, can improve the activity of Alcohol Dehydrogenase (ADH) in a human body to accelerate the metabolism speed of alcohol, eliminates a large amount of free radicals generated in the alcohol metabolism process to reduce the damage of alcohol to the liver, and contains glutamic acid, alanine and leucine which are beneficial to synthesizing alcohol metabolizing enzyme and relieving drunk symptoms.
The corn oligopeptide added in the invention can well promote the metabolism of alcohol in mice, reduce the activity of glutamic pyruvic transaminase in serum of the mice, has a good protective effect on alcoholic liver injury, activates the activity of alcohol dehydrogenase and accelerates the metabolism of alcohol; and the superoxide dismutase activity is improved, so that the accumulation of harmful free radicals in the body is reduced, lipid peroxidation is inhibited, the content of malondialdehyde is reduced, and the effect of reducing liver injury is achieved. Taurine can obviously reduce the concentration of blood plasma ethanol and improve the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase, has good anti-hangover and anti-drunk effects, and has synergistic effects.
The nostoc sphaeroids kutz powder contains abundant proteins, the amino acid types are abundant, the nostoc sphaeroids kutz protein content exceeds 45 percent, and the nostoc sphaeroids kutz powder contains 17 amino acids in total, wherein the content of glutamic acid, alanine and leucine is relatively abundant. The oyster meat contains more taurine, wherein the components with the effects of dispelling effects of alcohol and preventing drunk comprise oyster glycogen besides taurine, and both the oyster glycogen can effectively prolong the alcohol tolerance time and shorten the drunk time. The taurine can greatly improve the enzyme activities of the alcohol dehydrogenase and the acetaldehyde dehydrogenase, so that the alcohol metabolism in blood is accelerated, the concentration of the blood alcohol is obviously reduced, and the sobering-up effect is achieved. The egg protein is rich in amino acids which are beneficial to promoting ethanol metabolism, and the three are subjected to secondary enzymolysis to prepare the plant and animal mixed protein peptide which has small molecular weight and is easy to absorb, so that the egg protein has better antioxidant capacity, can remove free radicals in human bodies, can obviously improve the promotion of ethanol metabolism, and has obvious protective effect on liver injury.
The hydrolyzed soybean protein can reduce triglyceride content in liver, and promote liver lipid metabolism. Hydrolysis of wheat protein can increase the content of alanine and leucine in blood to make NAD + And stably exist. The two have synergistic effect.
The enzymolysis product is mixed with hydrolyzed wheat protein powder and hydrolyzed soybean protein powder, and the white ginseng fungus and the fermentation product thereof have great effects on reducing hypertension, high cholesterol, protecting liver injury, enhancing organism immunity, inhibiting tumor activity and the like after fermentation of white ginseng fungus and saccharomycetes. The yeast fermentation product has the functions of resisting oxidization and protecting liver, can effectively inhibit hydroxyl free radicals and resisting phospholipid oxidization, and has the auxiliary protection function on liver injury induced by ethanol. The synergistic fermentation product of the two strains has the effects of scavenging free radicals, enhancing immunity, improving blood circulation, maintaining internal environment balance, protecting liver, invigorating stomach, and inhibiting tumor.
Intestinal microbiota plays an important role in the pathogenesis of alcoholic liver injury. Since intestinal bacteria or their metabolites enter the liver through the portal vein, resulting in changes in the intestinal microflora and thus affecting liver function, i.e., via the "gut-liver axis" pathway, chronic alcohol exposure can lead to decreased levels of intestinal butyrate, and the butyrate prodrug Tributyrin (TB) can significantly alleviate ethanol-mediated intestinal barrier dysfunction and liver steatosis and damage.
The probiotics (including lactobacillus plantarum and lactobacillus rhamnosus) are further inoculated into fermentation liquor prepared by fermentation of the white ginseng fungus and the saccharomycetes, the probiotics proliferate rapidly and greatly under the condition of rich nitrogen sources and carbon sources, and a large amount of beneficial products including short-chain fatty acids and the like are produced, so that after entering the intestinal tract, the intestinal tract butyric acid level is obviously improved, the abundance of the beneficial intestinal tract bacteria is improved, the positive regulation is well achieved through the intestinal-liver axis, and the liver injury is obviously protected.
The embedded shell layer comprises sodium alginate and sodium carboxymethyl cellulose, and the sodium alginate and the sodium carboxymethyl cellulose are coordinated and crosslinked with metal ions to form a stable structure, active components are wrapped in the microcapsules to form a slow-release structure, and the slow-release structure is not easy to decompose in the stomach after entering a human body, so that the slow-release structure can be degraded and released after smoothly entering the intestinal tract, a large amount of probiotics, corn oligopeptide, fructo-oligosaccharide, taurine and the like can be directly absorbed by the intestinal tract, and the fructo-oligosaccharide is an effective prebiotic to play a role in effectively improving the intestinal tract probiotics flora.
The protein peptide composition for relieving acute alcoholic liver injury contains abundant small peptide substances, has the advantages of low energy consumption, quick absorption, high absorptivity, difficult saturation of a carrier and the like, has higher content of alanine, leucine and glutamic acid, has better inhibition effect on the increase of the concentration of ethanol in serum, and has obvious effects of dispelling effects of alcohol and protecting liver.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Nostoc powder was purchased from the biological engineering company of Hunan Yandi; lysozyme was purchased from the biological technology limited liability company of Henghua-dao, east of nan Ning; egg white is obtained from egg, and egg and oyster meat are purchased from farmer market; alkaline protease, FDG-2202, 20 ten thousand U/g, trypsin, GDG-2016,4 ten thousand U/g, neutral protease, SDG-2430,5 ten thousand U/g, papain, FDG-2203, 10 ten thousand U/g, purchased from Xia Cheng (Beijing) Biotechnology development Co., ltd; hydrolyzed wheat protein powder with a content of >90% purchased from Shenzhen chenxing biotechnology limited company; hydrolyzed soy protein powder with a content of >93% purchased from Shandong Songmao biotechnology Co., ltd; white ginseng fungus is purchased from Hubei Qianbao edible fungus limited company, and saccharomycete is purchased from Angel Yeast Co., ltd; lactobacillus plantarum, JYLP-326, 100 hundred million cfu/g, lactobacillus rhamnosus, JYLR-005, 100 hundred million cfu/g, purchased from Shandong, california bioengineering Co.
Example 1
The embodiment provides a preparation method of a protein peptide composition for relieving acute alcoholic liver injury, which comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of the nostoc sphaeroids kutz powder to the water is 1:5g/mL, freezing at-25 ℃ for 2 hours, thawing at room temperature, adding lysozyme after returning to room temperature, carrying out enzymolysis for 2 hours at 35 ℃, inactivating the enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 70wt%, precipitating for 5 hours, and filtering to obtain nostoc sphaeroids kutz protein liquid;
s2, primary enzymolysis: mincing 5 parts by weight of oyster meat, mixing with 3 parts by weight of egg white and 50 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.5 part by weight of primary complex enzyme, carrying out enzymolysis for 2h at 38 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 5:10;
s3, secondary enzymolysis: adding 1 part by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S2, carrying out enzymolysis for 1h at 45 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 3:2;
S4, preparing a culture medium: adding 5 parts by weight of the enzymolysis product obtained in the step S3, 3 parts by weight of hydrolyzed wheat protein powder, 2 parts by weight of hydrolyzed soybean protein powder, 10 parts by weight of maltose, 10 parts by weight of glucose, 0.5 part by weight of vitamin D1, 0.5 part by weight of vitamin E, 1 part by weight of sodium chloride, 0.3 part by weight of zinc chloride, 0.3 part by weight of copper chloride and 0.4 part by weight of manganese chloride into 500 parts by weight of water, adjusting the pH value to 6.7, and sterilizing by ultraviolet rays to obtain a culture medium;
s5, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S4, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 2% and 1% respectively, fermenting and culturing for 36 hours at 37 ℃ and 50r/min, sterilizing by ultraviolet rays, and filtering to obtain fermentation liquor;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises inoculating white ginseng fungus and saccharomycete into Gao's medium, respectively, and performing activation culture at 37deg.C and 50r/min for 12 hr to obtain a strain containing 10 8 cfu/mL strain seed liquid;
s6, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the fermentation broth prepared in the step S5, wherein the inoculation amount of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid is 1.5% and 1% respectively, adding glucose, and fermenting and culturing for 36h under the micro-anoxic condition at the temperature of 36 ℃ and at the speed of 50r/min to obtain a fermentation product, wherein the added glucose is 20 g/L;
Preparation method of activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solutionThe method comprises inoculating Lactobacillus plantarum and Lactobacillus rhamnosus respectively into Gao's culture medium, and performing activation culture at 37deg.C and 50r/min under micro-anoxic condition for 12 hr to obtain strain with a bacterial content of 10 8 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 3v/v%, the content of oxygen is 4v/v%, and the balance is nitrogen;
s7, embedding: dissolving 5 parts by weight of sodium alginate and 7 parts by weight of sodium carboxymethylcellulose in 100 parts by weight of water, adding 12 parts by weight of the fermentation product obtained in the step S6, 3 parts by weight of corn oligopeptide, 2 parts by weight of fructo-oligosaccharide and 1 part by weight of taurine, stirring and mixing for 20min, adding 150 parts by weight of peanut oil, emulsifying for 4min at 12000r/min, dropwise adding 5 parts by weight of 1wt% calcium chloride solution, solidifying for 30min at normal temperature, and freeze-drying to obtain the protein peptide composition for relieving acute alcoholic liver injury.
Example 2
The embodiment provides a preparation method of a protein peptide composition for relieving acute alcoholic liver injury, which comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of the nostoc sphaeroids kutz powder to the water is 1:7g/mL, freezing at-20 ℃ for 3 hours, thawing at room temperature, adding lysozyme after returning to room temperature, carrying out enzymolysis for 3 hours at 40 ℃, inactivating the enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 80wt%, precipitating for 7 hours, and filtering to obtain nostoc sphaeroids kutz protein liquid;
S2, primary enzymolysis: mincing 7 parts by weight of oyster meat, mixing with 5 parts by weight of egg white and 70 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 1 part by weight of primary complex enzyme, carrying out enzymolysis for 3h at 42 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 7:10;
s3, secondary enzymolysis: adding 1.5 parts by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S2, carrying out enzymolysis for 2 hours at 55 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 5:2;
s4, preparing a culture medium: adding 10 parts by weight of the enzymolysis product obtained in the step S3, 5 parts by weight of hydrolyzed wheat protein powder, 4 parts by weight of hydrolyzed soybean protein powder, 10 parts by weight of maltose, 10 parts by weight of lactose, 10 parts by weight of sucrose, 1 part by weight of vitamin C, 0.3 part by weight of vitamin B1, 0.3 part by weight of vitamin B2, 0.4 part by weight of vitamin A, 1 part by weight of potassium chloride, 0.5 part by weight of calcium chloride, 0.5 part by weight of magnesium sulfate, 0.5 part by weight of ferric chloride and 0.5 part by weight of zinc sulfate into 500 parts by weight of water, adjusting the pH value to 7.2, and carrying out ultraviolet sterilization to obtain a culture medium;
S5, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S4, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 3% and 2%, respectively, at 40 ℃ and 70r/min, fermenting and culturing for 48 hours, sterilizing by ultraviolet rays, and filtering to obtain fermentation liquor;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises inoculating white ginseng fungus and saccharomycete into Gao's medium respectively, and performing activation culture at 40deg.C and 70r/min for 16 hr to obtain a strain containing 10 9 cfu/mL strain seed liquid;
s6, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the fermentation broth prepared in the step S5, wherein the inoculation amount of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid is 2% and 2% respectively, glucose is added, the added glucose amount is 50g/L,38 ℃ and 70r/min, fermentation culture is carried out for 48 hours under the micro-anoxic condition, and freeze drying is carried out, so that a fermentation product is prepared;
the preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution comprises inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a Gao's culture medium, and performing activation culture at 40deg.C and 70r/min under micro-anoxic condition for 16h to obtain a strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 5v/v%, the content of oxygen is 6v/v%, and the balance is nitrogen;
s7, embedding: dissolving 7 parts by weight of sodium alginate and 10 parts by weight of sodium carboxymethylcellulose in 100 parts by weight of water, adding 15 parts by weight of the fermentation product prepared in the step S6, 5 parts by weight of corn oligopeptide, 3 parts by weight of fructo-oligosaccharide and 2 parts by weight of taurine, stirring and mixing for 20min, adding 150 parts by weight of rapeseed oil, emulsifying for 7min at 15000r/min, dropwise adding 5 parts by weight of 2wt% calcium chloride solution, solidifying for 50min at normal temperature, and freeze-drying to obtain the protein peptide composition for relieving acute alcoholic liver injury.
Example 3
The embodiment provides a preparation method of a protein peptide composition for relieving acute alcoholic liver injury, which comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of nostoc sphaeroids kutz powder to water is 1:6g/mL, freezing at-22 ℃ for 2.5h, thawing at room temperature, adding lysozyme after returning to room temperature, adding 1.5g/L of lysozyme, carrying out enzymolysis at 37 ℃ for 2.5h, inactivating enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 75wt%, precipitating for 6h, and filtering to obtain nostoc sphaeroids kutz protein liquid;
S2, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 6:10;
s3, secondary enzymolysis: adding 1.2 parts by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S2, carrying out enzymolysis for 1.5 hours at 50 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 4:2;
s4, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S3, 4 parts by weight of hydrolyzed wheat protein powder, 3 parts by weight of hydrolyzed soybean protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain a culture medium;
S5, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S4, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 2.5% and 1.5% respectively, the temperature is 38 ℃, the fermentation culture is carried out for 42h at 60r/min, ultraviolet sterilization is carried out, and the fermentation liquid is obtained after filtration;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises the steps of respectively inoculating white ginseng fungus and saccharomycete into a Gao's medium, and performing activation culture at 38 ℃ for 14 hours at 60r/min to obtain the white ginseng fungus and saccharomycete seed liquid with the bacterial content of 10 9 cfu/mL strain seed liquid;
s6, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the fermentation broth prepared in the step S5, wherein the inoculation amount of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid is 1.7% and 1.5% respectively, adding glucose, and fermenting and culturing for 42h under the micro-anoxic condition at the temperature of 37 ℃ and at the speed of 60r/min, and freeze-drying to obtain a fermentation product;
the preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution comprises inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a Gao's culture medium, and performing activation culture for 14h at 38deg.C under 60r/min and micro-anoxic condition to obtain a strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 4v/v%, the content of oxygen is 5v/v%, and the balance is nitrogen;
s7, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S6, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Example 4
The difference compared to example 3 is that the primary complex enzyme is a single alkaline protease.
Example 5
The difference compared to example 3 is that the primary complex enzyme is a single trypsin.
Example 6
The difference compared to example 3 is that the secondary complex enzyme is a single neutral protease.
Example 7
The difference compared to example 3 is that the secondary complex enzyme is a single papain.
Comparative example 1
In comparison with example 3, the difference is that step S1 is not performed.
The method comprises the following steps:
the method comprises the following steps:
s1, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of water, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, performing enzymolysis at 40 ℃ for 2.5h, and inactivating enzyme by ultraviolet rays to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 6:10;
s2, secondary enzymolysis: adding 1.2 parts by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S1, carrying out enzymolysis for 1.5 hours at 50 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 4:2;
s3, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S2, 4 parts by weight of hydrolyzed wheat protein powder, 3 parts by weight of hydrolyzed soybean protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain a culture medium;
S4, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S3, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 2.5% and 1.5% respectively, the temperature is 38 ℃, the fermentation culture is carried out for 42h at 60r/min, ultraviolet sterilization is carried out, and the fermentation liquid is obtained after filtration;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises the steps of respectively inoculating white ginseng fungus and saccharomycete into a Gao's medium, and performing activation culture at 38 ℃ for 14 hours at 60r/min to obtain the white ginseng fungus and saccharomycete seed liquid with the bacterial content of 10 9 cfu/mL strain seed liquid;
s5, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the fermentation broth prepared in the step S4, wherein the inoculation amount of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid is 1.7% and 1.5% respectively, adding glucose, and fermenting and culturing for 42h under the micro-anoxic condition at the temperature of 37 ℃ and at the speed of 60r/min, and freeze-drying to obtain a fermentation product;
the preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution comprises inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a Gao's culture medium, and performing activation culture for 14h at 38deg.C under 60r/min and micro-anoxic condition to obtain a strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 4v/v%, the content of oxygen is 5v/v%, and the balance is nitrogen;
s6, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S5, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 2
The difference from example 3 is that oyster meat was not added in step S2.
The method comprises the following steps:
s2, primary enzymolysis: mixing 10 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product.
Comparative example 3
The difference from example 3 is that no egg white was added in step S2.
The method comprises the following steps:
s2, primary enzymolysis: mincing 10 parts by weight of oyster meat, mixing with 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product.
Comparative example 4
In comparison with example 3, the difference is that the primary complex enzyme hydrolysis is not performed in step S2.
The method comprises the following steps:
s2, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, and stirring for 10min to obtain a mixture.
Comparative example 5
In comparison with example 3, the difference is that step S3 is not performed.
The method comprises the following steps:
the method comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of nostoc sphaeroids kutz powder to water is 1:6g/mL, freezing at-22 ℃ for 2.5h, thawing at room temperature, adding lysozyme after returning to room temperature, adding 1.5g/L of lysozyme, carrying out enzymolysis at 37 ℃ for 2.5h, inactivating enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 75wt%, precipitating for 6h, and filtering to obtain nostoc sphaeroids kutz protein liquid;
s2, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 6:10;
S3, preparing a culture medium: adding 7 parts by weight of the primary enzymolysis product prepared in the step S2, 4 parts by weight of hydrolyzed wheat protein powder, 3 parts by weight of hydrolyzed soybean protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to prepare a culture medium;
s4, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S3, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 2.5% and 1.5% respectively, the temperature is 38 ℃, the fermentation culture is carried out for 42h at 60r/min, ultraviolet sterilization is carried out, and the fermentation liquid is obtained after filtration;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises the steps of respectively inoculating white ginseng fungus and saccharomycete into a Gao's medium, and performing activation culture at 38 ℃ for 14 hours at 60r/min to obtain the white ginseng fungus and saccharomycete seed liquid with the bacterial content of 10 9 cfu/mL strain seed liquid;
s5, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the fermentation broth prepared in the step S4, wherein the inoculation amount of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid is 1.7% and 1.5% respectively, adding glucose, and fermenting and culturing for 42h under the micro-anoxic condition at the temperature of 37 ℃ and at the speed of 60r/min, and freeze-drying to obtain a fermentation product;
The preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution comprises inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a Gao's culture medium, and performing activation culture for 14h at 38deg.C under 60r/min and micro-anoxic condition to obtain a strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 4v/v%, the content of oxygen is 5v/v%, and the balance is nitrogen;
s6, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S5, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 6
In comparison with example 3, the difference is that no hydrolyzed wheat protein powder was added in step S4.
The method comprises the following steps:
s4, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S3, 7 parts by weight of hydrolyzed soy protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain the culture medium.
Comparative example 7
The difference compared to example 3 is that no hydrolyzed soy protein powder was added in step S4.
The method comprises the following steps:
s4, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S3, 7 parts by weight of hydrolyzed wheat protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain the culture medium.
Comparative example 8
The difference compared with example 3 is that the hydrolyzed wheat protein powder and hydrolyzed soy protein powder are not added in step S4.
The method comprises the following steps:
s4, preparing a culture medium: 14 parts by weight of the enzymolysis product obtained in the step S3, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate are added into 500 parts by weight of water, the pH value is adjusted to 7, and ultraviolet sterilization is carried out, so that the culture medium is obtained.
Comparative example 9
The difference from example 3 is that white ginseng fungus is not inoculated in step S5.
The method comprises the following steps:
s5, fermenting: inoculating the activated yeast strain seed liquid into the culture medium prepared in the step S4, wherein the inoculation amount of the activated yeast strain seed liquid is 4%, the temperature is 38 ℃, the fermentation culture is carried out for 42 hours at 60r/min, the ultraviolet sterilization is carried out, and the fermentation liquor is prepared;
the preparation method of the activated yeast strain seed liquid comprises inoculating yeast into Gao's medium, and performing activation culture at 38deg.C and 60r/min for 14 hr to obtain a strain containing 10 9 cfu/mL strain seed solution.
Comparative example 10
The difference compared to example 3 is that no yeast is inoculated in step S5.
The method comprises the following steps:
s5, fermenting: inoculating the activated white ginseng fungus seed liquid into the culture medium prepared in the step S4, wherein the inoculation amount of the activated white ginseng fungus seed liquid is 4%, the temperature is 38 ℃, the fermentation culture is carried out for 42 hours at 60r/min, the ultraviolet sterilization is carried out, and the fermentation liquor is prepared;
the preparation method of the activated white ginseng fungus seed liquid comprises inoculating white ginseng fungus into Gao's medium, activating and culturing at 38deg.C for 14h at 60r/min to obtain the product with a fungus content of 10 9 cfu/mL strain seed solution.
Comparative example 11
In comparison with example 3, the difference is that step S5 is not performed.
The method comprises the following steps:
the method comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of nostoc sphaeroids kutz powder to water is 1:6g/mL, freezing at-22 ℃ for 2.5h, thawing at room temperature, adding lysozyme after returning to room temperature, adding 1.5g/L of lysozyme, carrying out enzymolysis at 37 ℃ for 2.5h, inactivating enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 75wt%, precipitating for 6h, and filtering to obtain nostoc sphaeroids kutz protein liquid;
s2, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 6:10;
s3, secondary enzymolysis: adding 1.2 parts by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S2, carrying out enzymolysis for 1.5 hours at 50 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 4:2;
S4, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S3, 4 parts by weight of hydrolyzed wheat protein powder, 3 parts by weight of hydrolyzed soybean protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain a culture medium;
s5, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid into the culture medium prepared in the step S4, wherein the inoculum sizes of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed liquid are 1.7% and 1.5% respectively, adding glucose, and fermenting and culturing for 42h under the micro-anoxic condition at the temperature of 37 ℃ and at the speed of 60r/min, and freeze-drying to obtain a fermentation product;
the preparation method of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution comprises inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a Gao's culture medium, and performing activation culture for 14h at 38deg.C under 60r/min and micro-anoxic condition to obtain a strain with a bacterial content of 10 9 cfu/mL strain seed liquid;
the micro-anoxic condition is that the content of carbon dioxide is 4v/v%, the content of oxygen is 5v/v%, and the balance is nitrogen;
s6, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S5, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 12
In comparison with example 3, the difference is that step S6 is not performed.
The method comprises the following steps:
the method comprises the following steps:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, wherein the solid-to-liquid ratio of nostoc sphaeroids kutz powder to water is 1:6g/mL, freezing at-22 ℃ for 2.5h, thawing at room temperature, adding lysozyme after returning to room temperature, adding 1.5g/L of lysozyme, carrying out enzymolysis at 37 ℃ for 2.5h, inactivating enzyme by ultraviolet rays, filtering, adding ethanol into the filtrate until the ethanol content of the system is 75wt%, precipitating for 6h, and filtering to obtain nostoc sphaeroids kutz protein liquid;
S2, primary enzymolysis: mincing 6 parts by weight of oyster meat, mixing with 4 parts by weight of egg white and 60 parts by weight of nostoc sphaeroids kutz protein liquid prepared in the step S1, stirring for 10min, adding 0.7 part by weight of primary complex enzyme, carrying out enzymolysis for 2.5 hours at 40 ℃, and carrying out ultraviolet enzyme deactivation to obtain a primary enzymolysis product;
the primary complex enzyme is alkaline protease and trypsin, and the mass ratio is 6:10;
s3, secondary enzymolysis: adding 1.2 parts by weight of secondary complex enzyme into 100 parts by weight of the primary enzymolysis product prepared in the step S2, carrying out enzymolysis for 1.5 hours at 50 ℃, inactivating enzyme by ultraviolet rays, filtering, and freeze-drying to obtain an enzymolysis product;
the secondary complex enzyme is neutral protease and papain, and the mass ratio is 4:2;
s4, preparing a culture medium: adding 7 parts by weight of the enzymolysis product obtained in the step S3, 4 parts by weight of hydrolyzed wheat protein powder, 3 parts by weight of hydrolyzed soybean protein powder, 15 parts by weight of glucose, 10 parts by weight of fructose, 1 part by weight of vitamin C, 0.5 part by weight of vitamin B1, 1.5 parts by weight of sodium chloride, 0.2 part by weight of magnesium sulfate, 0.2 part by weight of ferric chloride, 0.2 part by weight of zinc sulfate, 0.2 part by weight of copper sulfate and 0.2 part by weight of manganese sulfate into 500 parts by weight of water, adjusting the pH value to 7, and sterilizing by ultraviolet rays to obtain a culture medium;
S5, fermenting: inoculating activated white ginseng fungus and saccharomycete seed liquid into the culture medium prepared in the step S4, wherein the inoculum sizes of the activated white ginseng fungus and saccharomycete seed liquid are 2.5% and 1.5% respectively, the temperature is 38 ℃, the fermentation culture is carried out for 42h at 60r/min, ultraviolet sterilization, filtration and freeze drying are carried out, and the fermented product is prepared;
the preparation method of the activated white ginseng fungus and saccharomycete seed liquid comprises the steps of respectively inoculating white ginseng fungus and saccharomycete into a Gao's medium, and performing activation culture at 38 ℃ for 14 hours at 60r/min to obtain the white ginseng fungus and saccharomycete seed liquid with the bacterial content of 10 9 cfu/mL strain seed liquid;
s6, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermented product prepared in the step S5, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 13
In comparison with example 3, the difference is that no corn oligopeptide was added in step S7.
The method is as follows;
s7, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S6, 2.5 parts by weight of fructo-oligosaccharides and 5.5 parts by weight of taurine are added, stirred and mixed for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% of calcium chloride solution is added dropwise, and the mixture is solidified at normal temperature for 40min and freeze-dried, so that the protein peptide composition for relieving acute alcoholic liver injury is prepared.
Comparative example 14
In comparison with example 3, the difference is that taurine is not added in step S7.
The method is as follows;
s7, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 13.5 parts by weight of the fermentation product prepared in the step S6, 2.5 parts by weight of fructo-oligosaccharides and 5.5 parts by weight of corn oligopeptide are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 15
In comparison with example 3, the difference is that no corn oligopeptide and no taurine were added in step S7.
The method is as follows;
s7, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 19 parts by weight of the fermentation product prepared in the step S6 and 2.5 parts by weight of fructo-oligosaccharides are added, stirring and mixing are carried out for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% of calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 16
In comparison with example 3, the difference is that no fermentation product is added in step S7.
The method is as follows;
s7, embedding: 6 parts by weight of sodium alginate and 8.5 parts by weight of sodium carboxymethylcellulose are dissolved in 100 parts by weight of water, 17.5 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine are added, stirred and mixed for 20min, 150 parts by weight of corn oil is added, 13500r/min is emulsified for 5min, 5 parts by weight of 1.5wt% calcium chloride solution is added dropwise, normal temperature curing is carried out for 40min, and freeze drying is carried out, thus obtaining the protein peptide composition for alleviating acute alcoholic liver injury.
Comparative example 17
The difference from example 3 is that embedding is not performed in step S7.
The method comprises the following steps:
s7, stirring and mixing 13.5 parts by weight of the fermentation product obtained in the step S6, 4 parts by weight of corn oligopeptide, 2.5 parts by weight of fructo-oligosaccharide and 1.5 parts by weight of taurine for 20 minutes to obtain the protein peptide composition for relieving acute alcoholic liver injury.
Test example 1 component measurement
The protein peptide compositions for alleviating acute alcoholic liver injury prepared in examples 1 to 7 and comparative examples 1 to 17 were subjected to compositional measurement.
Protein content determination: according to GB 5009.5-2016 "determination of proteins in food safety national Standard food", kjeldahl method is adopted for determination.
Determination of free amino acid content: according to GB 5009.124-2016 (national food safety Standard ) for determination of amino acids in foods, determination of tryptophan is performed by an automatic amino acid analyzer and alkaline hydrolysis.
And (3) measuring the content of the small molecule peptide: according to GB/T22492-2008 and GB/T22729-2008, PBH is dissolved in TCA solution, and then the precipitate is centrifugally separated, and the centrifugal supernatant is collected; the TCA soluble protein content in the centrifugate is measured according to the method of GB 5009.5-2016, and the content of small molecular peptide in the sample can be obtained by subtracting the free amino acid content from the TCA soluble protein content in the centrifugate, namely the content of small molecular peptide/(g/100 g) =TCA soluble protein/(g/100 g) -free amino acid content/(g/100 g). The results are shown in Table 1.
TABLE 1
As shown in the table above, the protein peptide compositions for alleviating acute alcoholic liver injury prepared in examples 1 to 3 of the present invention are rich in proteins, wherein the amino acid content and the small molecule peptide content are high.
Test example 2 sustained and controlled Release test
1g of the acute alcoholic liver injury relieving protein peptide composition prepared in the examples 1-3 and the comparative example 7 is added into 10mL of artificial simulated gastric fluid and 10mL of artificial simulated intestinal fluid to react for 2h and 3h respectively at 37 ℃ and 50r/min, and in addition, the same amount of the acute alcoholic liver injury relieving protein peptide composition is added into 10mL of artificial simulated gastric fluid, placed in a shaking table to react for 2h at 37 ℃ and 50r/min, centrifuged, and 10mL of artificial simulated intestinal fluid is added to continue to react for 3h. Probiotic colony cell counts were performed after the end of the reaction. Survival was calculated according to the following formula:
survival (%) =n t /N 0 ×100%
Wherein N is t For the concentration of probiotics (cfu/g) that survived a certain time of incubation in vitro, N 0 Is the original concentration (cfu/g) of added probiotics.
The release rate was calculated according to the following formula:
release rate (%) = (W) t -W 0 ）/W t ×100%
In which W is t Initial weight (g) for sample; w (W) 0 Weight (g) after a certain time of incubation in vitro.
The results are shown in Table 2.
TABLE 2
As can be seen from the above table, the protein peptide composition for alleviating acute alcoholic liver injury prepared in the embodiments 1-3 of the present invention maintains good integrity in artificial simulated gastric fluid, and releases a large amount of active substances after transferring to artificial simulated intestinal fluid, which indicates that the embedded structure of the protein peptide composition for alleviating acute alcoholic liver injury has the characteristics of pH responsiveness and resistance to simulated gastric fluid, and releases very little amount in the presence of gastric acid, and releases a large amount of lipidic compounds embedded with active substances under the condition of high pH after entering the intestinal tract, thereby having the effects of targeted delivery and controlled release of active components in the intestinal tract.
Test example 3 determination of the Activity of Alcohol Dehydrogenase (ADH)
The fermentation products obtained in step S6 of examples 1 to 7 and comparative examples 1 to 11, or the fermentation product obtained in step S5 of comparative example 12 were formulated as an aqueous solution of 0.5g/mL as a sample solution.
1.5mL of sodium pyrophosphate buffer (pH=8.8) was added to the test tube, and 1.0mL of oxidized coenzyme I (NAD) at 0.027mol/L was added + ) 0.1mL of the sample solution and 0.5mL of the 11.5% ethanol solution were mixed, incubated at 25℃for 7min, 0.1mL of 0.25U/mL ethanol dehydrogenase (ADH) was immediately added, mixed, absorbance at 340nm was measured, and double distilled water was zeroed. After 5min, the change in absorbance at 340nm was observed to determine the amount of reduced coenzyme I (NADH) produced. Blank control was performed with distilled water instead of ovalbumin peptide solution. Calculating the activation rate of alcohol dehydrogenase:
W ADH =(A 1 -A 0 )/A 0 ×100%
W ADH : ADH activation rate,%; a is that 1 : absorbance of the sample at 450 nm; a is that 0 : absorbance of the control solution at 450 nm.
The results are shown in Table 3.
TABLE 3 Table 3
As is clear from the above table, the fermentation products prepared in examples 1 to 3 of the present invention have a higher activation rate for alcohol dehydrogenase.
Test example 3 antioxidant Property test
The fermentation products obtained in step S6 of examples 1 to 7 and comparative examples 1 to 11, or the fermentation product obtained in step S5 of comparative example 12 were formulated as 50mg/mL of an aqueous solution as a sample solution.
1. Measurement of DPPH radical scavenging ability:
ascorbic acid was formulated as a 50mg/mL solution as a positive control. After 100 [ mu ] L of 0.5mmol/L DPPH solution and 100 [ mu ] L of sample solution are uniformly mixed, the mixture is incubated for 30min at 37 ℃ in a dark place. Absorbance at 517nm was measured and recorded asA 1 . Ultrapure water was used as a blank, and the absorbance was measured and recorded asA 0 The method comprises the steps of carrying out a first treatment on the surface of the The absorbance was recorded as the absorbance measured using ultrapure water instead of DPPH solutionA 2 Calculated according to the following formula:
DPPH clearance/% =1- (a) 1 -A 2 )/A 0 ×100%
2. Determination of ABTS cation radical scavenging ability:
a solution of 7mmol/L of ABTS cationic free radical and 4.9mmol/L of potassium persulfate was prepared with 0.1mmol/L of PBS buffer. Mixing the ABTS cation free radical solution with the potassium persulfate solution in equal proportion, and carrying out light-shielding reaction for 14h to obtain the mother solution of the ABTS cation free radical solution. Ascorbic acid was formulated as a 50mg/mL solution as a positive control. Diluting the ABTS cation radical mother solution by 20 times by using 0.1mmol/L PBS buffer solution to obtain the ABTS cation radical working solution. After 20 mu L of sample solution and 200 mu L of ABTS cationic free radical working solution are uniformly mixed, absorbance is measured at 734nm wavelength and is recorded asA 1 . The absorbance was measured using 20. Mu.L of ultrapure water instead of the sample, and was recorded asA 0 . The absorbance value was measured using 0.1mmol/LPBS buffer solution instead of ABTS cationic radical working solution and recorded as A 2 Calculated according to the following formula:
ABTS cation radical clearance/% =1- (a) 1 -A 2 )/A 0 ×100%
The results are shown in Table 4.
TABLE 4 Table 4
As can be seen from the above table, the fermentation products prepared in examples 1-3 of the present invention have good oxidation resistance.
Test example 4 test for alleviating alcoholic liver injury
The Kunming mice were selected, male, and 18-22g of body weight were adaptively bred for 1 week, and then the mice were randomly divided into a normal group, a model group, a positive group (bifendate 150 mg/kg), examples 1-7 and comparative examples 1-17 (0.5 g/kg of the corresponding prepared protein peptide composition for alleviating acute alcoholic liver injury), 6 mice each. Normal group and model group mice are filled with equal volume distilled water, each administration group is dosed according to the corresponding dosage, after 30min, the other groups are filled with 56% white spirit according to the dosage of 11.0mL/kg except the blank group filled with distilled water, and the treatment is carried out once daily for 7 days. The successful modeling was indicated during the experiment if the model mice were observed to have a yellow hair, a listlessness, a loss of appetite, and a significant loss of body weight. No water is forbidden for 24 hours after fasting, eyeballs are taken for blood taking, after the eyeballs are placed for 2 hours, the eyeballs are centrifuged, and upper serum is taken and stored in a low-temperature refrigerator at the temperature of minus 80 ℃.
The ELISA kit is adopted to detect the content of mouse tumor necrosis factor-alpha (TNF-alpha), 200 mu L of serum is taken, and the activity of glutamic pyruvic transaminase (ALT) and aspartic amino transferase (AST) in the serum is detected by a full-automatic biochemical analyzer. The viability of total superoxide dismutase (SOD) and Malondialdehyde (MDA) content of liver tissue were determined according to the method of kit instructions.
The results are shown in Table 5.
TABLE 5
Annotation: * P <0.05 compared to the normal group; # is P <0.05 compared to model group.
From the table above, the protein peptide composition for alleviating acute alcoholic liver injury prepared in the embodiments 1-3 can obviously reduce the content of TNF-alpha, reduce ALT and AST activities, improve SOD activity, reduce MDA content, and have a good effect of alleviating alcoholic liver injury.
Examples 4 and 5 compare with example 3 in which the primary complex enzyme is a single alkaline protease or trypsin. Comparative example 4 compared with example 3, the primary complex enzyme hydrolysis was not performed in step S2. The content of free amino acid and small molecular peptide is reduced, the activation rate of alcohol dehydrogenase is reduced, the oxidation resistance is reduced, and the effect of relieving alcoholic liver injury is reduced. The primary enzymolysis is carried out under the action of alkaline protease and trypsin, so that macromolecular proteins are primarily hydrolyzed into smaller polypeptide chains, the subsequent enzymolysis is promoted, and the addition of the alkaline protease and trypsin has a synergistic effect.
Examples 6 and 7 compare with example 3, the secondary complex enzyme was a single neutral protease or papain. Comparative example 5 compared to example 3, step S3 was not performed. The content of free amino acid and small molecular peptide is reduced, the activation rate of alcohol dehydrogenase is reduced, the oxidation resistance is reduced, and the effect of relieving alcoholic liver injury is reduced. The secondary enzymolysis is carried out under the synergistic effect of neutral protease and papain to prepare the plant and animal mixed protein peptide with small molecular weight and easy absorption, which has better antioxidation capability, can remove free radicals in human bodies, obviously improves the metabolism of ethanol and has obvious protection effect on liver injury, and the two have synergistic effect.
Comparative example 1 compared to example 3, step S1 was not performed. The protein content, the free amino acid content and the small molecular peptide content are reduced, the activation rate of alcohol dehydrogenase is reduced, and the effect of relieving alcoholic liver injury is reduced. The nostoc sphaeroids kutz powder contains abundant proteins, the amino acid types are abundant, the nostoc sphaeroids kutz protein content exceeds 45 percent, the nostoc sphaeroids kutz powder contains 17 amino acids, wherein the contents of glutamic acid, alanine and leucine are relatively abundant, the nostoc sphaeroids kutz cell wall is broken under the effects of freeze thawing treatment and lysozyme, most of the proteins are dissolved, and the extraction efficiency of the nostoc sphaeroids kutz protein is greatly improved.
Comparative example 2 in comparison with example 3, oyster meat was not added in step S2. In comparative example 3, in contrast to example 3, no egg white was added in step S2. The protein content, the free amino acid content and the small molecular peptide content are reduced, the activation rate of alcohol dehydrogenase is reduced, the oxidation resistance is reduced, and the effect of relieving alcoholic liver injury is reduced. The oyster meat contains more taurine, wherein the components with the effects of dispelling effects of alcohol and preventing drunk comprise oyster glycogen besides taurine, and both the oyster glycogen can effectively prolong the alcohol tolerance time and shorten the drunk time. The taurine can greatly improve the enzyme activities of the alcohol dehydrogenase and the acetaldehyde dehydrogenase, so that the alcohol metabolism in blood is accelerated, the concentration of the blood alcohol is obviously reduced, and the sobering-up effect is achieved. The egg proteins are rich in amino acids which are beneficial to promoting ethanol metabolism, and have obvious protective effect on liver injury.
In comparative examples 6 and 7, no hydrolyzed wheat protein powder or hydrolyzed soybean protein powder was added in step S4, as compared with example 3. Comparative example 8 in comparison with example 3, no hydrolyzed wheat protein powder and no hydrolyzed soy protein powder were added in step S4. The protein content, the free amino acid content and the small molecular peptide content are reduced, the activation rate of alcohol dehydrogenase is reduced, the oxidation resistance is reduced, and the effect of relieving alcoholic liver injury is reduced. The hydrolyzed soybean protein can reduce triglyceride content in liver, and promote liver lipid metabolism. Hydrolysis of wheat protein can increase the content of alanine and leucine in blood to make NAD + And stably exist. The two have synergistic effect.
In comparative examples 9 and 10, the white ginseng fungus or yeast was not inoculated in step S5, as compared with example 3. Comparative example 11 was inferior to example 3 in the effect of alleviating alcoholic liver injury without performing step S5. The content of free amino acid and small molecular peptide is reduced, the activation rate of alcohol dehydrogenase is reduced, and the oxidation resistance is obviously reduced. The enzymolysis product is mixed with hydrolyzed wheat protein powder and hydrolyzed soybean protein powder, and the white ginseng fungus and the fermentation product thereof have great effects on reducing hypertension, high cholesterol, protecting liver injury, enhancing organism immunity, inhibiting tumor activity and the like after fermentation of white ginseng fungus and saccharomycetes. The yeast fermentation product has the functions of resisting oxidization and protecting liver, can effectively inhibit hydroxyl free radicals and resisting phospholipid oxidization, and has the auxiliary protection function on liver injury induced by ethanol. The synergistic fermentation product of the two strains has the effects of scavenging free radicals, enhancing immunity, improving blood circulation, maintaining internal environment balance, protecting liver, invigorating stomach, and inhibiting tumor.
Comparative example 12 compared to example 3, step S6 was not performed. The content of free amino acid and small molecular peptide is reduced, the activation rate of alcohol dehydrogenase is reduced, the oxidation resistance is reduced, and the effect of relieving alcoholic liver injury is reduced. The probiotics (including lactobacillus plantarum and lactobacillus rhamnosus) are further inoculated into fermentation liquor prepared by fermentation of the white ginseng fungus and the saccharomycetes, the probiotics proliferate rapidly and greatly under the condition of rich nitrogen sources and carbon sources, and a large amount of beneficial products including short-chain fatty acids and the like are produced, so that after entering the intestinal tract, the intestinal tract butyric acid level is obviously improved, the abundance of the beneficial intestinal tract bacteria is improved, the positive regulation is well achieved through the intestinal-liver axis, and the liver injury is obviously protected.
In comparative examples 13 and 14, no corn oligopeptide or taurine was added in step S7, as compared with example 3. Comparative example 15 in contrast to example 3, no corn oligopeptide and taurine were added in step S7. The content of free amino acid and small molecular peptide is reduced, and the effect of relieving alcoholic liver injury is reduced. The corn oligopeptide added in the invention can well promote the metabolism of alcohol in mice, reduce the activity of glutamic pyruvic transaminase in serum of the mice, has a good protective effect on alcoholic liver injury, activates the activity of alcohol dehydrogenase and accelerates the metabolism of alcohol; and the superoxide dismutase activity is improved, so that the accumulation of harmful free radicals in the body is reduced, lipid peroxidation is inhibited, the content of malondialdehyde is reduced, and the effect of reducing liver injury is achieved. Taurine can obviously reduce the concentration of blood plasma ethanol and improve the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase, has good anti-hangover and anti-drunk effects, and has synergistic effects.
Comparative example 16 compared with example 3, no fermentation product was added in step S7. The protein content, the free amino acid content and the small molecular peptide content are obviously reduced, the TNF-alpha content is obviously improved, the ALT and AST activities are obviously improved, the SOD activity is reduced, and the MDA content is improved.
Compared with the embodiment 3, the comparative example 17 has the advantages that the embedding is not carried out in the step S7, the TNF-alpha content is obviously improved, the ALT and AST activities are obviously improved, the SOD activity is reduced, and the MDA content is improved. The embedded shell layer comprises sodium alginate and sodium carboxymethyl cellulose, and forms a stable structure through coordination and crosslinking with metal ions, active components are wrapped in the microcapsule to form a slow-release structure, and after entering a human body, the active components are not easy to decompose in the stomach, and can be degraded and released after smoothly entering the intestinal tract, so that a large amount of probiotics, corn oligopeptide, fructo-oligosaccharide, taurine and the like can be directly absorbed by the intestinal tract, and the fructo-oligosaccharide is an effective prebiotic to play a role in effectively improving the intestinal tract probiotics flora
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. A method for preparing a protein peptide composition for alleviating acute alcoholic liver injury, comprising the steps of:
s1, extraction of nostoc protein: adding nostoc sphaeroids kutz powder into water, freezing and thawing, recovering to room temperature, adding lysozyme for enzymolysis, inactivating enzyme, filtering, adding ethanol into the filtrate for precipitation, and filtering to obtain nostoc sphaeroids kutz protein liquid; the solid-to-liquid ratio of nostoc powder to water is 1:5-7g/mL, and the freezing and thawing treatment method comprises the steps of freezing for 2-3h at-25 to-20 ℃ and thawing at room temperature; the addition amount of lysozyme is 1-2g/L, the enzymolysis temperature is 35-40 ℃, the time is 2-3h, and ethanol is added until the system ethanol content is 70-80wt%, and the precipitation is 5-7h;
s2, primary enzymolysis: mincing oyster meat, mixing with egg white and nostoc sphaeroids kutz protein liquid prepared in the step S1, adding primary complex enzyme for enzymolysis, and inactivating enzyme to obtain a primary enzymolysis product; the mass ratio of the oyster meat to the egg white to the nostoc sphaeroids kutz protein liquid to the primary complex enzyme is 5-7:3-5:50-70:0.5-1, and the primary complex enzyme is alkaline protease and trypsin, wherein the mass ratio is 5-7:10;
s3, secondary enzymolysis: adding a secondary complex enzyme into the primary enzymolysis product obtained in the step S2 for enzymolysis, inactivating enzyme, filtering, and freeze-drying to obtain an enzymolysis product; the mass ratio of the primary enzymolysis product to the secondary compound enzyme is 100:1-1.5, and the mass ratio of the secondary compound enzyme is 3-5:2;
S4, preparing a culture medium: adding the enzymolysis product obtained in the step S3, hydrolyzed wheat protein powder, hydrolyzed soybean protein powder, a carbon source, vitamins and inorganic salt into water, adjusting the pH value, and sterilizing to obtain a culture medium; the mass ratio of the enzymolysis product to the hydrolyzed wheat protein powder to the hydrolyzed soybean protein powder to the carbon source to the vitamins to the inorganic salt to the water is 5-10:3-5:2-4:20-30:1-2:2-3:500;
s5, fermenting: inoculating activated white ginseng fungus and saccharomycete strain seed liquid into the culture medium prepared in the step S4, fermenting and culturing, sterilizing and filtering to prepare fermentation liquor; the inoculum sizes of the activated white ginseng fungus and the yeast strain seed liquid are respectively 2-3% and 1-2%, the fermentation culture conditions are 37-40 ℃ and 50-70r/min, and the fermentation culture is carried out for 36-48h;
s6, fermenting probiotics: inoculating activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solution into the fermentation liquor prepared in the step S5, supplementing a carbon source, fermenting, proliferating and culturing, and freeze-drying to obtain a fermentation product; the inoculum sizes of the activated lactobacillus plantarum and lactobacillus rhamnosus strain seed solutions are 1.5-2% and 1-2% respectively, the conditions of fermentation proliferation culture are 36-38 ℃,50-70r/min, fermentation culture is carried out for 36-48h under the micro-anoxic condition, the micro-anoxic condition is that the content of carbon dioxide is 3-5v/v%, the content of oxygen is 4-6v/v%, and the balance is nitrogen; the amount of the added carbon source is 20-50g/L;
S7, embedding: dissolving sodium alginate and sodium carboxymethylcellulose in water, adding the fermentation product obtained in the step S6, corn oligopeptide, fructo-oligosaccharide and taurine, stirring and mixing uniformly, adding edible oil, emulsifying, dropwise adding calcium chloride solution, solidifying at normal temperature, and freeze-drying to obtain a protein peptide composition for relieving acute alcoholic liver injury; the mass ratio of the sodium alginate to the sodium carboxymethylcellulose to the water to the fermentation product to the corn oligopeptide to the fructo-oligosaccharide to the taurine is 5-7:7-10:100:12-15:3-5:2-3:1-2.
2. The method according to claim 1, wherein the enzymolysis temperature in step S2 is 38-42 ℃ for 2-3 hours.
3. The preparation method according to claim 1, wherein the enzymolysis temperature in step S3 is 45-55 ℃ for 1-2 hours.
4. The preparation method according to claim 1, wherein the carbon source in the step S4 is at least one selected from glucose, molasses, maltose, lactose, sucrose, fructose, soluble starch and agar, the vitamin is at least one selected from vitamin C, vitamin B1, vitamin B2, vitamin a, vitamin K, vitamin B12, vitamin D1, vitamin E, folic acid and vitamin D3, and the inorganic salt is at least one selected from sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride; the pH value is adjusted to 6.7-7.2.
5. The method according to claim 1, wherein the step S5 comprises inoculating white ginseng fungus and yeast respectively into Gao' S medium, culturing at 37-40deg.C for 50-70r/min under micro-anoxic condition for 12-16 hr to obtain strain containing 10 8 -10 9 cfu/mL strain seed solution.
6. The method according to claim 1, wherein the activated lactobacillus plantarum and lactobacillus rhamnosus seed solution in step S6 is prepared by inoculating lactobacillus plantarum and lactobacillus rhamnosus respectively into a high-temperature culture medium, and performing activation culture at 37-40 ℃ and 50-70r/min for 12-16h to obtain a strain containing 10 8 -10 9 cfu/mL strain seed liquid; the carbon source is at least one selected from glucose, molasses, maltose, lactose, sucrose, fructose, soluble starch and agar.
7. The method according to claim 1, wherein the edible oil in step S7 is at least one selected from peanut oil, corn oil, soybean oil, rapeseed oil, fish oil, sunflower seed oil, sesame oil, linseed oil, and olive oil; the emulsifying condition is 12000-15000r/min for emulsifying for 4-7min, and the concentration of the calcium chloride solution is 1-2wt%; the curing time at normal temperature is 30-50min.
8. A protein peptide composition for alleviating acute alcoholic liver injury prepared by the method of any one of claims 1-7.
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