CN116726150A - Lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase and preparation method and application thereof - Google Patents
Lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase and preparation method and application thereof Download PDFInfo
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- CN116726150A CN116726150A CN202211526719.XA CN202211526719A CN116726150A CN 116726150 A CN116726150 A CN 116726150A CN 202211526719 A CN202211526719 A CN 202211526719A CN 116726150 A CN116726150 A CN 116726150A
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Abstract
The invention provides lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase and a preparation method and application thereof, and belongs to the technical field of probiotics. Extracting the traditional Chinese medicine composition with water, extracting with an organic solvent, mixing the solid with lemon, tomato juice, a carbon source and a nitrogen source, inoculating activated probiotics, and fermenting to obtain a fermentation product; mixing fermentation product, acetaldehyde dehydrogenase, ethanol oxidase, lactobacillus helveticus, enterococcus hainanensis, carbomer and carboxylated chitosan, adding into water, solidifying to obtain inner layer slow-release particles, mixing konjaku glucomannan, chinese yam polysaccharide and corn oligopeptide, obtaining polysaccharide peptide compound, mixing the polysaccharide peptide compound with the inner layer slow-release particles, chinese medicine water extract, chinese medicine organic solvent extract, amino acid and sodium alginate, adding into water, solidifying to obtain lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, and having the effects of dispelling effects of alcohol, relieving hangover, reducing adverse reaction after drinking and protecting liver.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, and a preparation method and application thereof.
Background
The number of happy alcohol users in China is numerous, about 60-75% of adult men often drink alcohol, and the proportion of diseases induced by excessive drinking is increased year by year. Excessive drinking can cause liver injury and corresponding liver diseases, and can cause other diseases, especially physiological dysfunction and death of human body. Alcoholic liver injury caused by excessive drinking has become one of the most important factors for liver diseases, and at the same time, excessive drinking causes hypertension, malignant diseases such as gastric cancer, and metabolic disorders of substances such as proteins. The most common poison causing liver damage is alcohol, and liver damage caused by excessive drinking has become a worldwide medical problem. Therefore, the need for sobering up, dispelling the effects of alcohol and reducing the damages of drinking to human gastrointestinal and liver organs is urgent, and the development of effective anti-alcohol drugs has important significance and broad market prospect.
In vivo degradation of alcohol is mainly responsible for oxidation of ethanol to acetaldehyde by alcohol dehydrogenase in the liver, and the generated acetaldehyde is further converted into harmless acetic acid as a substrate under the catalysis of acetaldehyde dehydrogenase. Acetaldehyde toxicity is higher than ethanol and is the main cause of hangover. The acetaldehyde dehydrogenase is properly supplemented by oral administration, so that the metabolism of alcohol in intestinal tracts can be accelerated, and the metabolism pressure of livers can be reduced, thereby relieving uncomfortable reactions caused by alcohol and slowing down chemical liver injury.
After excessive drinking, the metabolism of the organism can be accelerated, the alcohol decomposition can be accelerated and the damage of the alcohol to the organism can be weakened by taking a large amount of small molecules such as amino acid, vitamin and the like. In addition, the energy can be rapidly supplemented by supplementing carbohydrates such as glucose and lactose, and the ethanol in the intestinal tract can be effectively neutralized by supplementing short-chain fatty acids such as lactic acid and acetic acid, so that the method has a certain effect of relieving uncomfortable symptoms.
The anti-alcohol products in the current market are mainly chemical medicines and traditional Chinese medicine preparations. The chemical for dispelling the effects of alcohol is mainly composed of stimulants, vitamins, amino acids and the like, and can quickly play a role in dispelling the effects of alcohol, but most chemical anti-alcohol mechanisms can only promote alcohol decomposition, so that the chemical has a temporary relieving effect, is difficult to play a role in protecting liver and has a large toxic and side effect. The traditional Chinese medicine preparation for dispelling the effects of alcohol has complex raw material components and complex preparation process, and is difficult to effectively improve various symptoms after being drunk to achieve the effects of dispelling the effects of alcohol rapidly and taking the principal and subordinate symptoms into consideration.
In addition, experiments prove that the occurrence, development and prognosis of enterogenic endotoxemia (intestinal endotoxemia, IETM) and alcoholic liver injury are closely related. The normal intestinal barrier has the barrier function of preventing macromolecular toxic substances such as endotoxin from entering the blood. Ethanol is taken into the intestinal tract, which causes damage to the intestinal mucosa and disturbance of the intestinal flora, and generates a large amount of endotoxin, and if the structure and function of the cell-cell tight junction occludin protein are changed, the intestinal epithelial cell permeability is increased, so that bacterial translocation and endotoxemia occur. Endotoxins cause hepatic cell steatosis, and liver injury can aggravate intestinal microecological disorders and endotoxemia, thereby forming a vicious circle.
At present, the main principle of the anti-alcohol effect through exogenous supplementation of active probiotics is that excessive drinking causes intestinal microecological imbalance to generate a large amount of endotoxin, and the liver is directly or indirectly damaged; the balance of intestinal flora is regulated by supplementing probiotics, which is helpful for reducing the generation and absorption of enterogenic endotoxin, thereby maintaining liver health, but the anti-alcoholic effect of the method is still poor.
Disclosure of Invention
The invention aims to provide lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, and a preparation method and application thereof, which can change the composition of intestinal microorganisms, regulate intestinal flora, improve intestinal immunity, reduce oxidative stress and relieve chemical liver injury, thereby achieving the effects of alleviating hangover, improving hangover, reducing adverse reactions after drinking and protecting liver, and having wide application prospect.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, which comprises the steps of extracting radix puerariae, liquorice, hericium erinaceus and dandelion with water, extracting and filtering with an organic solvent to obtain a traditional Chinese medicine water extract and a traditional Chinese medicine organic solvent extract, mixing solids with lemon, tomato juice, a carbon source and a nitrogen source to prepare a fermentation substrate, inoculating activated lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06, and fermenting to obtain a fermentation product; mixing the fermentation product, acetaldehyde dehydrogenase, ethanol oxidase, lactobacillus helveticus, enterococcus hainanensis and carbomer, adding water, adding an aqueous solution dissolved with carboxylated chitosan, emulsifying, adding a metal ion solution for solidification to obtain inner-layer slow-release particles, mixing konjak glucomannan, chinese yam polysaccharide and corn oligopeptide, performing ultrasonic treatment and ozone synergistic treatment to obtain polysaccharide peptide complex, uniformly mixing the polysaccharide peptide complex with the inner-layer slow-release particles, a Chinese medicinal water extract, a Chinese medicinal organic solvent extract, taurine, alanine and leucine, adding an aqueous solution dissolved with sodium alginate, emulsifying, adding the metal ion solution for solidification, and freeze-drying to obtain the lactobacillus freeze-dried powder containing acetaldehyde dehydrogenase.
As a further improvement of the invention, the method comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: cleaning radix Puerariae, glycyrrhrizae radix, hericium Erinaceus, and herba Taraxaci respectively, drying, pulverizing to obtain Chinese medicinal composition powder, adding into water, heating and boiling for 2-3 times, filtering, mixing filtrates, collecting solid, concentrating the filtrate, and drying to obtain water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, heating and extracting for 2-3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate, and drying to obtain an organic solvent extract;
s3, preparing a fermentation substrate: mixing lemon and tomato, squeezing, removing solids, uniformly mixing the juice with the solids obtained in the step S2, adding a carbon source and a nitrogen source, uniformly mixing, adding sterile water, stirring, mixing, and sterilizing to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into a Gao's medium respectively, and performing activation culture to obtain strain seed liquid;
s5, fermenting: inoculating the strain seed liquid in the step S4 into the fermentation substrate in the step S3, and fermenting and culturing to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing konjak glucomannan, yam polysaccharide and corn oligopeptide, and carrying out ultrasonic treatment and ozone synergistic treatment to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing taurine, alanine and leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing acetaldehyde dehydrogenase and ethanol oxidase to obtain a complex enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing the fermentation product obtained in the step S5, the complex enzyme obtained in the step S8, lactobacillus helveticus, enterococcus hainanensis and carbomer, adding into water, uniformly stirring and mixing, adding an aqueous solution dissolved with carboxylated chitosan, rapidly emulsifying a first SPG film to form emulsion, adding a metal ion solution, and curing at normal temperature to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing the water extract prepared in the step S1, the organic solvent extract prepared in the step S2, the polysaccharide peptide compound prepared in the step S6, the amino acid additive prepared in the step S7 and the inner layer slow release particles prepared in the step S9, adding the mixture into an aqueous solution in which sodium alginate is dissolved, emulsifying a second SPG fast film to form an emulsion, adding a metal ion solution, solidifying, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
As a further improvement of the invention, in the step S1, the mass ratio of the kudzuvine root, the liquorice, the hericium erinaceus and the dandelion is 5-10:1-3:3-5:1-2; the solid-liquid ratio of the traditional Chinese medicine composition powder to water is 1:5-10g/mL; the extraction time is 1-3h; the mixed organic solvent in the step S2 is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 5-7:2-4:1-3; the solid-liquid ratio of the solid to the mixed organic solvent is 1:3-5g/mL; heating to 50-70deg.C for 0.5-1 hr.
As a further improvement of the invention, the mass ratio of the lemon, the tomato, the solid, the carbon source, the nitrogen source and the sterile water in the step S3 is 7-12:5-10:3-5:5-10:3-5:30-50; the carbon source is at least one selected from glucose, maltose, lactose, sucrose, fructose, starch, molasses and agar; the nitrogen source is at least one selected from ammonia water, urea, ammonium salt, nitrate, amino acid and fish meal; the ammonium salt is at least one selected from ammonium chloride, ammonium nitrate and ammonium sulfate; the nitrate is selected from potassium nitrate, sodium nitrate, ferric nitrate and nitric acidAt least one of zinc, aluminum nitrate, magnesium nitrate, manganese nitrate, and copper nitrate; the amino acid is selected from one or more of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine; the condition of the activation culture in the step S4 is 36-39 ℃, and the culture is carried out for 18-24 hours at the rotating speed of 50-70r/min under the humidity of 30-50%; the bacterial seed liquid has a bacterial content of 10 8 -10 9 cfu/mL。
As a further improvement of the invention, the inoculum sizes of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S5 are respectively 3-5%, 1-3% and 0.5-1%, and the conditions of the fermentation culture are 36-40 ℃,30-50% humidity and 50-70r/min rotation speed for 48-72h; in the step S6, the mass ratio of the konjac glucomannan, the yam polysaccharide and the corn oligopeptide is 3-5:1-2:2-4; in the ultrasonic treatment and ozone synergistic treatment, the ultrasonic power is 120-150W, the ozone concentration is 30-70mg/L, the gas flow is 15-25L/h, and the treatment time is 5-7s.
As a further improvement of the invention, the mass ratio of taurine, alanine and leucine in the step S7 is 7-12:1-3:2-4; the mass ratio of the acetaldehyde dehydrogenase to the ethanol oxidase in the step S8 is 10-15:3-5.
As a further improvement of the invention, the mass ratio of the fermentation product, the complex enzyme, the lactobacillus helveticus, the enterococcus haii and the carbomer in the step S9 is 15-20:1-2:0.5-1:2-3:1-3; the content of carboxylated chitosan in the water solution containing carboxylated chitosan is 12-15wt%; the aperture of the first SPG rapid membrane is 300-500nm; the metal ion solution is a solution containing 3-5wt% of at least one of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions; the mass ratio of the water extract, the organic solvent extract, the polysaccharide peptide complex, the amino acid additive and the inner layer slow release particles in the step S10 is 3-5:2-4:0.5-1:0.2-0.5:4-6; the content of sodium alginate in the aqueous solution dissolved with sodium alginate is 7-12wt%; the pore diameter of the second SPG rapid membrane is 10-15 mu m; the metal ion solution is a solution containing 3-5wt% of at least one of calcium ion, magnesium ion, aluminum ion, iron ion and zinc ion.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: cleaning, drying and crushing 5-10 parts by weight of radix puerariae, 1-3 parts by weight of liquorice, 3-5 parts by weight of hericium erinaceus and 1-2 parts by weight of dandelion respectively to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating and boiling for extraction for 1-3 hours, extracting for 2-3 times, filtering, merging filtrate, reserving solids, concentrating filtrate, and drying to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:3-5g/mL, heating to 50-70 ℃ for 0.5-1h, extracting for 2-3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate, and drying to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 5-7:2-4:1-3;
s3, preparing a fermentation substrate: mixing 7-12 parts by weight of lemon and 5-10 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice with 3-5 parts by weight of solids obtained in the step S2, adding 5-10 parts by weight of carbon source and 3-5 parts by weight of nitrogen source, uniformly mixing, adding 30-50 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into Gao's culture medium, respectively, and performing activation culture at 36-39deg.C, 30-50% humidity and 50-70r/min for 18-24 hr to obtain strain seed solution with bacterial content of 10 8 -10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 3-5%, 1-3%, 0.5-1%,36-40 ℃ and 50-70r/min under 30-50% humidity, and fermenting and culturing for 48-72h to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 3-5 parts by weight of konjak glucomannan, 1-2 parts by weight of yam polysaccharide and 2-4 parts by weight of corn oligopeptide, and introducing ozone for treatment under 120-150W ultrasonic waves, wherein the ozone concentration is 30-70mg/L, the gas flow is 15-25L/h, and the treatment time is 5-7s to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 7-12 parts by weight of taurine, 1-3 parts by weight of alanine and 2-4 parts by weight of leucine to obtain an amino acid additive;
S8, preparation of complex enzyme: uniformly mixing 10-15 parts by weight of acetaldehyde dehydrogenase and 3-5 parts by weight of ethanol oxidase to obtain a complex enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 15-20 parts by weight of the fermentation product obtained in the step S5, 1-2 parts by weight of the complex enzyme obtained in the step S8, 0.5-1 part by weight of lactobacillus helveticus, 2-3 parts by weight of enterococcus hainanensis and 1-3 parts by weight of carbomer, adding 50 parts by weight of water, uniformly stirring and mixing, adding 70-80 parts by weight of a carboxylated chitosan aqueous solution containing 12-15wt% and a first SPG fast film with the aperture of 300-500nm, emulsifying to form emulsion, adding 7-12 parts by weight of a solution containing 3-5wt% of at least one of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions, and curing at normal temperature for 20-30min to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 3-5 parts by weight of the water extract prepared in the step S1, 2-4 parts by weight of the organic solvent extract prepared in the step S2, 0.5-1 part by weight of the polysaccharide peptide compound prepared in the step S6, 0.2-0.5 part by weight of the amino acid additive prepared in the step S7 and 4-6 parts by weight of the inner layer slow release particles prepared in the step S9, adding 100 parts by weight of the inner layer slow release particles into a sodium alginate aqueous solution containing 7-12wt% and a second SPG fast film with the aperture of 10-15 mu m to emulsify to form emulsion, adding 7-12 parts by weight of a solution containing at least one of 3-5wt% of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions, curing for 10-15min, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
The invention further protects the lactic acid bacteria freeze-dried powder containing the acetaldehyde dehydrogenase prepared by the preparation method.
The invention further provides application of the lactobacillus freeze-dried powder containing the acetaldehyde dehydrogenase in preparation of products for dispelling the effects of alcohol and protecting the liver.
The invention has the following beneficial effects:
the Chinese medicinal composition comprises kudzuvine root, liquorice, hericium erinaceus and dandelion, wherein water extracts of the kudzuvine root, the liquorice, the hericium erinaceus and the dandelion can improve weight and liver index changes caused by drinking and liver function indexes AST (aspartate aminotransferase Aspartate aminotransferase, AST), ALT (alanine aminotransferase Alanine aminotransferase, ALT) and ALP (alkaline phosphatase alkaline phosphatase, ALP) abnormality; the method has the advantages that the down-regulating effect on the blood lipid indexes TC (total triglyceride Total triglyceride, TG) and TG (total triglyceride Total triglyceride, TG) of the alcohol-induced improvement is achieved; simultaneously, the level of antioxidant indexes GSH (Glutathione, GSH), GST (Glutathione-transferase, GST) and the activity of ADH (alcohol dehydrogenase Alcohol Dehydrogenase, ADH) in the liver are obviously improved. In the mixed organic solvent extract, the ethanol extract can obviously shorten the sobering-up time, reduce the ethanol concentration of blood, and the ethyl acetate extract can reduce the AST and ALT activities in serum; the petroleum ether extract can remarkably improve the activity of SOD (superoxide dismutase Superoxide dismutase, SOD) and GSH in liver, reduce the content of MDA (Malondialdehyde) and liver index, wherein the mixed organic solvent comprises ethanol, ethyl acetate and petroleum ether, and the extract obtained by mixing and extracting the three organic solvents can also simultaneously enhance the activity of ADH and ALDH (aldehyde dehydrogenase Acetaldehyde dehydrogenase, ALDH).
Lemon and tomato can enhance the activity of ADH and ALDH at the same time, and after being mixed with the solid residue of the traditional Chinese medicine composition, the lemon and tomato are added with carbon source and nitrogen source to act as fermentation substrates, a large amount of antioxidant active substances are generated under the action of saccharomycetes and lactobacillus, the level of SOD in the liver is increased, the SOD failure caused by alcohol is protected, the lemon fermentation product can also obviously inhibit the elevation of ALT, AST, liver TG and lipid peroxidation caused by alcohol, and simultaneously can also improve the histopathological change of alcoholic liver injury.
The invention cultures lactobacillus reuteri LR06 and bifidobacterium longum BL5b by mixed fermentation, has the synergistic effect, and the bifidobacterium longum BL5b decomposed substrate provides lactic acid, amino acid and the like, provides needed nutrient substances for the growth and proliferation of lactobacillus reuteri LR06, and provides favorable guarantee for the growth of bifidobacterium longum BL5b in the initial stage of fermentation by formic acid, propionic acid, butyric acid, folic acid and the like produced by lactobacillus reuteri LR06, and the bifidobacterium longum BL5b proliferates in a large amount to produce a large amount of lactic acid and amino acid, promote the growth of streptococcus acidophilus to supplement each other and promote each other, thereby promoting the fermentation of zymophyte, producing a large amount of antioxidant substances and lipid-lowering substances, thereby effectively improving liver oxidative damage caused by alcohol, and lowering serum and liver lipid content by regulating blood lipid. In addition, when liver is damaged, bioactive components such as zinc, B vitamins, and reduced glutathione contained in yeast can be used as synthetic raw materials of organism reaction enzymes, and can be used for effectively removing oxygen ions and free radicals in vivo and protecting liver cells.
The konjak glucomannan, the yam polysaccharide and the corn oligopeptide can reduce the ethanol content entering blood through the adsorption effect on ethanol, can improve the activity of ADH and ALDH in blood, improve the level of SOD and GSH, reduce the MDA content, reduce the acetaldehyde content in blood and brain, the yam polysaccharide can prolong the drunk latency, shorten the drunk maintenance time, improve the activity of ADH and ALDH in the liver of drunk mice, reduce the concentration of ethanol and acetaldehyde in serum, reduce the MDA content in liver tissues, improve the content of SOD and GSH, and the corn oligopeptide can enhance the activity of ADH, clear the activity of free radicals, reduce the damage of alcohol to the liver, shorten the drunk time of cooperative mice and reduce the ethanol content in serum. In the low-power ultrasonic wave and ozone treatment process, the structure of the protein can be changed through the ultrasonic wave effect, so that the combination reaction of the corn oligopeptide substance and the ozone is promoted, and the effect of promoting the ozone modification is achieved; corn oligomerization after short-time oxidation treatment by ozoneThe disulfide bond content of the peptide is increased, so that dimeric peptide is obtained, and the molecular weight of the oligopeptide is primarily increased; because the time is short, other reducing amino acids on the corn oligopeptide are not yet available for oxidation reaction, the property of the protein peptide is not greatly influenced, and meanwhile, the hydrogen bond exposure and the hydrogen bond combination of the polysaccharide are improved, the polysaccharide peptide complex is formed, the antioxidant activity of the polysaccharide peptide complex is improved, the alanine and leucine content in blood can be improved, and the NAD is enabled to be improved + The wine is stable and plays a good role in dispelling the effects of alcohol.
Taurine is produced by regulating Ca in liver cells 2+ Balance, improve the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase in liver, reduce the release of peroxidase, and effectively protect liver cells; the addition of alanine and leucine can increase the content of the two in blood, and make NAD + The wine is stable and plays a good role in dispelling the effects of alcohol.
The acetaldehyde dehydrogenase and the ethanol oxidase are active enzymes directly acting on ethanol degradation, fermentation products, acetaldehyde dehydrogenase, ethanol oxidase, lactobacillus helveticus and enterococcus hainanensis are protected in carboxylated chitosan, after emulsification, carboxylated chitosan is crosslinked to form a wall material of particles under the action of metal ions, the active substances are wrapped in microcapsules to form a slow-release structure, and after the slow-release structure is sent into a human body, probiotics and protease are not easy to be decomposed by the human body in the stomach, so that the probiotics and the protease can be degraded and released after entering the intestinal tract, a large amount of probiotics and protease can directly act on the intestinal tract to effectively regulate intestinal flora, the probiotics can directly act on the degradation of the ethanol by regulating intestinal microbial flora to reduce bacterial translocation and liver inflammation, and the protease can directly enter blood to act on the degradation of the ethanol, so that the damage of the ethanol to the liver is reduced, and the liver is protected.
Finally, embedding each component into sodium alginate to form a microcapsule structure, and freeze-drying, keeping at low temperature, thereby being beneficial to keeping the activity of each component, ensuring that probiotics are not easy to inactivate, reducing the amount of viable bacteria, playing a role in long-term storage, prolonging the service life and improving the effect of active substances.
The lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared by the invention can change the composition of intestinal microorganisms, regulate intestinal flora, improve intestinal immunity, reduce oxidative stress and relieve chemical liver injury, thereby achieving the effects of dispelling the effects of alcohol, relieving hangover, reducing adverse reactions after alcohol, and protecting the liver, and having wide application prospect.
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.
Lactobacillus fermentum LF021, latin name: lactobacillus fermentum the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), with the preservation place being No. 1, no. 3 of North West Lu, korean, beijing, and the preservation number being CGMCC NO 22774, and the preservation date being 2021, 6 months and 24 days. Bifidobacterium longum BL5b, latin name: bifidobacterium longum the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), wherein the preservation place is North Xielu No. 1 and No. 3 in the Korean area of Beijing, the preservation number is CGMCC NO 17056, and the preservation date is 2019, 1 month and 2 days. Lactobacillus reuteri LR06, latin name: lactobacillus reuteri the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center), with the preservation place being No. 1, no. 3, and the preservation number being CGMCC NO 22775, and the preservation date being 2021, 6, 24. Are all provided by Jiangsu New Shenao Biotech Inc.
Konjak glucomannan, the content of which is more than 80%, is purchased from Jinan Jiage biotechnology Co., ltd; rhizoma Dioscoreae polysaccharide, purchased from Shanxi Yuning Biotechnology Co., ltd; corn oligopeptide with a content of more than 99% is purchased from Sichuan green gallop biotechnology company.
Acetaldehyde dehydrogenase, content greater than 99%, purchased from shandong yao homohousing company, ethanol oxidase, viscosity: 150-400 mpa.s available from Jiangsu Phea Biotech Inc.
Example 1
The embodiment provides a preparation method of lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, which specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: cleaning, drying and crushing 5 parts by weight of radix puerariae, 1 part by weight of liquorice, 3 parts by weight of hericium erinaceus and 1 part by weight of dandelion respectively to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 1h, extracting for 2 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3h to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:3g/mL, heating to 50 ℃ for 0.5h, extracting for 2 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
The mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 5:2:1;
s3, preparing a fermentation substrate: mixing and juicing 7 parts by weight of lemon and 5 parts by weight of tomato, removing solids, uniformly mixing juice and 3 parts by weight of the solids obtained in the step S2, adding 5 parts by weight of glucose and 3 parts by weight of urea, uniformly mixing, adding 30 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 36 deg.C and 30% humidity and 50r/min for 18 hr to obtain strain seed solution with bacterial content of 10 8 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 3%, 1%, 0.5%,36 ℃ and 30% humidity, and fermenting and culturing for 48 hours at the rotating speed of 50r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 3 parts by weight of konjak glucomannan, 1 part by weight of yam polysaccharide and 2 parts by weight of corn oligopeptide, and introducing ozone for treatment under 120W ultrasonic waves, wherein the ozone concentration is 30mg/L, the gas flow is 15L/h, and the treatment time is 5s, so as to obtain a polysaccharide peptide compound;
S7, preparation of an amino acid additive: uniformly mixing 7 parts by weight of taurine, 1 part by weight of alanine and 2 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 10 parts by weight of acetaldehyde dehydrogenase and 3 parts by weight of ethanol oxidase to obtain a complex enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 15 parts by weight of the fermentation product prepared in the step S5, 1 part by weight of the complex enzyme prepared in the step S8, 0.5 part by weight of lactobacillus helveticus, 2 parts by weight of enterococcus hainanensis and 1 part by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 70 parts by weight of a carboxylated chitosan aqueous solution containing 12wt% and a first SPG rapid membrane with the pore diameter of 300nm for emulsification to form emulsion, adding 7 parts by weight of a calcium ion solution containing 3wt% for curing at normal temperature for 20min, centrifuging for 15min 3000r/min, and washing solids to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 3 parts by weight of the water extract prepared in the step S1, 2 parts by weight of the organic solvent extract prepared in the step S2, 0.5 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.2 part by weight of the amino acid additive prepared in the step S7 and 4 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 7wt% and a second SPG fast membrane with the aperture of 10 mu m to form emulsion, adding 7 parts by weight of a solution containing at least one of 3wt% of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions, curing at normal temperature for 10min, centrifuging for 15min 3000r/min, washing the solid, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Example 2
The embodiment provides a preparation method of lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, which specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 10 parts by weight of radix puerariae, 3 parts by weight of liquorice, 5 parts by weight of hericium erinaceus and 2 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 3 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:5g/mL, heating to 70 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 7:4:3;
s3, preparing a fermentation substrate: mixing 12 parts by weight of lemon and 10 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 5 parts by weight of solids obtained in the step S2, adding 5 parts by weight of glucose, 3 parts by weight of maltose, 2 parts by weight of lactose, 4 parts by weight of urea and 1 part by weight of ammonium nitrate, uniformly mixing, adding 50 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 39deg.C and 50% humidity and 70r/min for 24 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum sizes of the lactobacillus fermentum LF021, the bifidobacterium longum BL5b and the lactobacillus reuteri LR06 are respectively 5%, 3%, 1%,40 ℃ and 50% humidity, and fermenting and culturing for 72 hours at the rotating speed of 70r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 5 parts by weight of konjak glucomannan, 2 parts by weight of yam polysaccharide and 4 parts by weight of corn oligopeptide, and introducing ozone for treatment under 150W ultrasonic waves, wherein the ozone concentration is 70mg/L, the gas flow is 25L/h, and the treatment time is 7s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 12 parts by weight of taurine, 3 parts by weight of alanine and 4 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 15 parts by weight of acetaldehyde dehydrogenase and 5 parts by weight of ethanol oxidase to obtain a compound enzyme;
S9, preparing inner-layer slow-release particles: uniformly mixing 20 parts by weight of the fermentation product prepared in the step S5, 2 parts by weight of the complex enzyme prepared in the step S8, 1 part by weight of lactobacillus helveticus, 3 parts by weight of enterococcus hainanensis and 3 parts by weight of carbomer, adding into 50 parts by weight of water, stirring and uniformly mixing, adding 80 parts by weight of a carboxylated chitosan aqueous solution containing 15wt% and a first SPG rapid membrane with the aperture of 500nm for emulsification to form emulsion, adding 12 parts by weight of a magnesium ion solution containing 5wt% for solidification at normal temperature for 30min, centrifuging for 15min 3000r/min, and washing solids to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 5 parts by weight of the water extract prepared in the step S1, 4 parts by weight of the organic solvent extract prepared in the step S2, 1 part by weight of the polysaccharide peptide compound prepared in the step S6, 0.5 part by weight of the amino acid additive prepared in the step S7 and 6 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 12wt% and a second SPG rapid membrane with the aperture of 15 mu m to form emulsion, adding 12 parts by weight of a calcium ion solution containing 5wt% to solidify at normal temperature for 15min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Example 3
The embodiment provides a preparation method of lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase, which specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
S9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Example 4
In contrast to example 3, the mixed organic solvent in step S2 was replaced by a single ethanol, and the other conditions were not changed.
Example 5
In contrast to example 3, the mixed organic solvent in step S2 was replaced by a single ethyl acetate, and the other conditions were not changed.
Example 6
In contrast to example 3, the mixed organic solvent in step S2 was replaced by a single petroleum ether, and the other conditions were not changed.
Comparative example 1
In comparison with example 3, the water extraction was not performed in step S1, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting an organic solvent of a traditional Chinese medicine composition: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder into a mixed organic solvent, heating to 60 ℃ to extract for 1h, extracting for 3 times, filtering, combining filtrate, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s2, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S1, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S3, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S4, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S2, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s5, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s6, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s7, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
S8, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S4, 1.5 parts by weight of the complex enzyme prepared in the step S7, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
s9, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 7 parts by weight of the organic solvent extract prepared in the step S1, 0.7 part by weight of the polysaccharide peptide compound prepared in the step S5, 0.35 part by weight of the amino acid additive prepared in the step S6 and 5 parts by weight of the inner layer slow-release particles prepared in the step S8, adding 100 parts by weight of the inner layer slow-release particles into a 10wt% sodium alginate aqueous solution, emulsifying a second SPG fast film with the aperture of 12 mu m to form emulsion, adding 10 parts by weight of a 4wt% calcium ion solution, curing at normal temperature for 12min, centrifuging for 15min 3000r/min, washing the solid, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 2
In contrast to example 3, the organic solvent extraction was not performed in step S2, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S1, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s3, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S4, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S3 to a fermentation substrate in the step S2, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56h at a rotating speed of 60r/min to obtain a fermentation product;
s5, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s6, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s7, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s8, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S4, 1.5 parts by weight of the complex enzyme prepared in the step S7, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S9, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 7 parts by weight of the water extract prepared in the step S1, 0.7 part by weight of the polysaccharide peptide compound prepared in the step S5, 0.35 part by weight of the amino acid additive prepared in the step S6 and 5 parts by weight of the inner layer slow-release particles prepared in the step S8, adding 100 parts by weight of the inner layer slow-release particles into a 10wt% sodium alginate aqueous solution, emulsifying the mixture by a second SPG fast film with the aperture of 12 mu m to form emulsion, adding 10 parts by weight of a 4wt% calcium ion solution, curing at normal temperature for 12min, centrifuging for 15min 3000r/min, washing the solid, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 3
In contrast to example 3, the radix Puerariae was not included in step S1, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: cleaning, drying and crushing 2 parts by weight of liquorice, 11 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion respectively to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
The mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 4
In contrast to example 3, hericium erinaceus was not included in step S1, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 11 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 5
In contrast to example 3, no lemon was added in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: squeezing 17 parts by weight of tomatoes, removing solids, uniformly mixing the juice with 4 parts by weight of the solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 6
In contrast to example 3, no tomato was added in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: squeezing 17 parts by weight of lemon, removing solids, uniformly mixing juice with 4 parts by weight of the solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 7
In contrast to example 3, the solid obtained in step S2 was not added in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate into the juice, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 8
In contrast to example 3, steps S1 and S2 were not performed, the solid obtained in step S2 was not added in step S3, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate into the juice, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S2, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S3, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S2 to a fermentation substrate in the step S1, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56h at a rotating speed of 60r/min to obtain a fermentation product;
s4, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s5, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s6, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
S7, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S3, 1.5 parts by weight of the complex enzyme prepared in the step S6, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
s8, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 0.7 part by weight of the polysaccharide peptide compound prepared in the step S4, 0.35 part by weight of the amino acid additive prepared in the step S5 and 5 parts by weight of the inner layer slow release particles prepared in the step S7, adding 100 parts by weight of the inner layer slow release particles into a 10-wt% sodium alginate aqueous solution, emulsifying a second SPG fast film with the aperture of 12 mu m to form emulsion, adding 10 parts by weight of a 4-wt% calcium ion solution, curing at normal temperature for 12min, centrifuging at 3000r/min for 15min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 9
In comparison with example 3, in step S5, lactobacillus fermentum LF021 was not inoculated, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating Bifidobacterium longum BL5b and Lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing active culture at 37deg.C, 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 into a fermentation substrate in the step S3, wherein the inoculum size of the bifidobacterium longum BL5b and the lactobacillus reuteri LR06 is 2 percent, 0.7 percent and 38 percent respectively, and fermenting and culturing for 56 hours at the rotating speed of 60r/min under the humidity of 40 percent to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
S9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 10
In comparison with example 3, bifidobacterium longum BL5b and lactobacillus reuteri LR06 were not inoculated in step S5, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021 into sterilized Gao's culture medium, activating and culturing at 37deg.C and 40% humidity and 60r/min rotation speed for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating the strain seed liquid of the lactobacillus fermentum LF021 in the step S4 into the fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021 is respectively 4%, and the lactobacillus fermentum is fermented and cultured for 56 hours at the rotating speed of 60r/min under the temperature of 38 ℃ and the humidity of 40%, so as to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 11
In contrast to example 3, the ultrasonic treatment and the ozone co-treatment were not performed in step S6, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide mixture: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide to obtain a polysaccharide peptide mixture;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide mixture prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the pore diameter of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 12
In contrast to example 3, step S6 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s7, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s8, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S7, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S9, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.35 part by weight of the amino acid additive prepared in the step S6 and 5 parts by weight of the inner layer slow-release particles prepared in the step S8, adding 100 parts by weight of the inner layer slow-release particles into a 10wt% sodium alginate aqueous solution, emulsifying the mixture by a second SPG (specific surface active ingredient) fast film with the aperture of 12 mu m to form emulsion, adding 10 parts by weight of a 4wt% calcium ion solution, curing at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing the solid, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 13
In contrast to example 3, no taurine was added in step S7, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 14
In contrast to example 3, step S7 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s8, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S7, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging at 3000r/min for 15min, and washing solids to obtain inner-layer slow-release particles;
S9, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6 and 5 parts by weight of the inner layer slow-release particles prepared in the step S9, adding 100 parts by weight of the inner layer slow-release particles into a 10wt% sodium alginate aqueous solution, emulsifying the mixture by a second SPG fast film with the aperture of 12 mu m to form emulsion, adding 10 parts by weight of a 4wt% calcium ion solution, curing at normal temperature for 12min, centrifuging for 15min 3000r/min, washing the solid, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 15
Compared with example 3, the preparation of the inner layer slow release granule in step S9 is not carried out, the components are simply mixed, and other conditions are not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s9, preparation of a mixture: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 0.7 part by weight of lactobacillus helveticus and 2.5 parts by weight of enterococcus haiides to obtain a mixture;
S10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the mixture prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% of the mixture, quickly emulsifying a second SPG with the pore diameter of 12 mu m to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% of the mixture, curing at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 16
In comparison with example 3, the fermentation treatment in step S5 was not performed, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
S2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
s4, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
S5, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s6, preparing complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
s7, preparing inner-layer slow-release particles: uniformly mixing 1.5 parts by weight of the complex enzyme prepared in the step S6, 0.7 part by weight of lactobacillus helveticus, 2.5 parts by weight of enterococcus hainanensis and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the pore diameter of 400nm for emulsification to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt% for curing at normal temperature for 25min, centrifuging for 15min 3000r/min, and washing the solid to obtain inner-layer slow-release particles;
s8, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S4, 0.35 part by weight of the amino acid additive prepared in the step S5 and 5 parts by weight of the inner layer slow release particles prepared in the step S7, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Comparative example 17
In contrast to example 3, enterococcus faecalis was not added in step S9, and the other conditions were not changed.
The method specifically comprises the following steps:
s1, extracting the traditional Chinese medicine composition with water: respectively cleaning, drying and crushing 7 parts by weight of radix puerariae, 2 parts by weight of liquorice, 4 parts by weight of hericium erinaceus and 1.5 parts by weight of dandelion to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating, boiling and extracting for 2 hours, extracting for 3 times, filtering, combining filtrate, reserving solids, concentrating the filtrate, and drying at 70 ℃ for 3 hours to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:4g/mL, heating to 60 ℃ for extraction for 1h, extracting for 3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate under reduced pressure, and drying at 70 ℃ for 2h to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 6:3:2;
s3, preparing a fermentation substrate: mixing 10 parts by weight of lemon and 7 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice and 4 parts by weight of solids obtained in the step S2, adding 4 parts by weight of glucose, 2 parts by weight of sucrose, 1 part by weight of fructose, 2 parts by weight of urea, 1 part by weight of ammonium chloride and 1 part by weight of potassium nitrate, uniformly mixing, adding 40 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into sterilized Gao's culture medium, respectively, and performing activation culture at 37deg.C and 40% humidity and 60r/min for 21 hr to obtain strain seed solution with bacterial content of 10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 4%, 2%, 0.7%,38 ℃, and under 40% humidity, fermenting and culturing for 56 hours at a rotating speed of 60r/min to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 4 parts by weight of konjak glucomannan, 1.5 parts by weight of yam polysaccharide and 3 parts by weight of corn oligopeptide, introducing ozone for treatment under 135W ultrasonic waves, wherein the ozone concentration is 50mg/L, the gas flow is 20L/h, and the treatment time is 6s, so as to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 10 parts by weight of taurine, 2 parts by weight of alanine and 3 parts by weight of leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing 12 parts by weight of acetaldehyde dehydrogenase and 4 parts by weight of ethanol oxidase to obtain a compound enzyme;
S9, preparing inner-layer slow-release particles: uniformly mixing 17 parts by weight of the fermentation product prepared in the step S5, 1.5 parts by weight of the complex enzyme prepared in the step S8, 3.2 parts by weight of lactobacillus helveticus and 2 parts by weight of carbomer, adding 50 parts by weight of water, stirring and uniformly mixing, adding 75 parts by weight of carboxylated chitosan aqueous solution containing 13.5wt% and a first SPG rapid membrane with the aperture of 400nm, emulsifying to form emulsion, adding 10 parts by weight of aluminum ion solution containing 4wt%, curing at normal temperature for 25min, centrifuging for 15min 3000r/min, and washing solids to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 4 parts by weight of the water extract prepared in the step S1, 3 parts by weight of the organic solvent extract prepared in the step S2, 0.7 part by weight of the polysaccharide peptide complex prepared in the step S6, 0.35 part by weight of the amino acid additive prepared in the step S7 and 5 parts by weight of the inner layer slow release particles prepared in the step S9, adding into 100 parts by weight of a sodium alginate aqueous solution containing 10wt% and a second SPG rapid membrane with the aperture of 12 mu m to emulsify to form an emulsion, adding 10 parts by weight of a calcium ion solution containing 4wt% to solidify at normal temperature for 12min, centrifuging for 15min at 3000r/min, washing solids, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
Test example 1
1g of the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the invention examples 1-3 and comparative example 15 are respectively added into 10mL of artificial simulated gastric fluid and 10mL of artificial simulated intestinal fluid, and react for 2h and 3h at 37 ℃ under the condition of 70r/min, and in addition, the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase with the same amount is firstly added into 10mL of artificial simulated gastric fluid, reacts for 2h at 37 ℃ under the condition of 70r/min, is centrifuged, is added into 10mL of artificial simulated intestinal fluid, and continuously reacts for 3h at 37 ℃ under the condition of 70 r/min. 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) surviving after a certain incubation time, N 0 Is the original concentration (cfu/g) of probiotics.
The results are shown in Table 1.
TABLE 1
As can be seen from the table, in the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the embodiment 1-3 of the invention, probiotics are embedded in the inner layer slow release particles through carboxylated chitosan, in artificial simulated gastric juice, although the outer sodium alginate shell layer collapses, the inner layer slow release particles can keep better integrity, after the inner sodium alginate shell layer and the inner layer slow release particles are transferred into the artificial simulated intestinal juice, the sodium alginate shell layer and the inner layer slow release particles collapse, and the probiotics are exposed, so that the double-layer structure has the characteristics of pH responsiveness and resistance to simulated gastric juice, and has better effect of targeted delivery of probiotics. Comparative example 15 was free of inner layer slow release particles, which showed that the survival rate in artificial simulated gastric fluid and pre-artificial simulated gastric fluid was greatly reduced, and that single sodium alginate shell was not pH responsive.
Test example 2
The freeze-dried powders of lactic acid bacteria containing acetaldehyde dehydrogenase prepared in examples 1 to 3 and comparative example 15 were placed in environments having pH of 5.5, 6, 6.5, 7, and 7.5, respectively, and after storage at room temperature for 6 months, the stability was judged.
The freeze-dried powder of lactic acid bacteria containing acetaldehyde dehydrogenase obtained in examples 1 to 3 and comparative example 15 was stored at 7℃and 17℃and 27℃and 37℃and 47℃for 6 months, respectively, and then the stability was judged.
The stability judging method comprises the following steps: 5g of sample is taken and placed in a centrifuge tube, and after centrifugation is carried out for 30min at the speed of 2500r/min, whether water is separated out and layering phenomenon is observed.
The results are shown in tables 2 and 3.
TABLE 2
TABLE 3 Table 3
As is clear from the above table, the lactic acid bacteria lyophilized powder containing acetaldehyde dehydrogenase prepared in examples 1 to 3 of the present invention has good stability under 6 months storage at pH of 5.5 to 7.5 and at temperature of 7 to 47 ℃. Comparative example 15 is only a single layer embedded and has some stability but is not suitable for extreme pH and higher temperature storage.
Test example 3
SD male rats are selected and randomly divided into a normal group, a model group, a positive group, an example 1-6 group and a comparative example 1-17 group, 8 groups of the SD male rats are subjected to free drinking water and diet, after being adaptively fed for 1 week, 100mg/kg of sea Wang Jinzun is fed to the positive group every day, 100mg/kg of corresponding example or lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the example 1-6 and the comparative example 1-17 group are respectively fed to the stomach, and the normal group and the model group rats are fed with equal amounts of physiological saline and continuously fed for 30 days. After the last administration for 30min, other groups are filled with gastric white spirit (the alcohol degree is 56 degrees) except normal group administration physiological saline, and the gastric white spirit filling dosage is 15mL/kg.
Cutting tails and taking blood after 60 min and 120min after wine filling, putting the blood into a cage after hemostasis, treating the blood to obtain serum, and measuring the mass concentration of ethanol in the blood by using a kit. Then, eyeballs are taken out for blood after 12 hours of wine filling, collected blood is processed to obtain serum, and ALT, AST and TG levels in the serum are detected by using a corresponding kit. After the eyeball is removed and blood collection is completed, the rat is killed by cervical dislocation, the liver is quickly taken out, rinsed by precooled normal saline and sucked by filter paper. Small amounts of liver tissue were cut, homogenized with physiological saline, and centrifuged at 4℃to obtain supernatant. The ADH, ALDH, SOD, MDA, GSH content of liver tissue was determined according to the kit instructions.
The results of ethanol concentration after blood perfusing the rats are shown in Table 4.
TABLE 4 Table 4
Annotation: # is significantly different (P < 0.05) from the normal group; * The differences were significant (P < 0.05) compared to the model group. The following is the same.
As shown in the table above, the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the embodiments 1-3 can obviously reduce the mass concentration of ethanol in blood, and has a certain anti-alcoholic effect.
The results of rat liver ADH and ALDH activity are shown in Table 5.
TABLE 5
As can be seen from the above table, the lactic acid bacteria lyophilized powder containing acetaldehyde dehydrogenase prepared in examples 1 to 3 of the present invention can significantly improve the activity of ADH and ALDH in blood, and accelerate ethanol metabolism by enhancing the activity of ADH and ALDH, thereby reducing the ethanol level in blood.
The results of rat liver injury are shown in Table 6.
As shown in the table above, the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the embodiments 1-3 of the invention can obviously reduce ALT, AST and TG levels in rat serum, and has a certain protective effect on liver.
The results of oxidative stress in rats are shown in Table 7.
TABLE 7
| Group of | SOD activity (U/mg) | MDA content (mmol/mg) | GSH content (μg/mg) |
| Normal group | 435.2±53.2 | 2.10±0.45 | 30.24±2.13 |
| Model group | 262.1±15.4 # | 12.34±1.53 # | 16.78±1.57 # |
| Positive group | 410.7±35.7 * | 3.97±1.24 * | 24.57±1.85 * |
| Example 1 | 415.7±42.1 * | 3.78±1.12 * | 26.31±1.45 * |
| Example 2 | 417.5±39.5 * | 3.80±1.31 * | 26.78±1.10 * |
| Example 3 | 419.2±40.2 * | 3.71±1.04 * | 27.57±1.05 * |
| Example 4 | 395.2±45.2 | 4.52±1.32 | 22.34±1.04 |
| Example 5 | 400.1±52.1 | 5.03±1.19 | 21.15±1.15 |
| Example 6 | 356.2±49.5 | 6.21±1.06 | 20.12±1.19 |
| Comparative example 1 | 302.4±45.7 | 4.14±1.52 | 18.74±1.11 |
| Comparative example 2 | 335.6±47.2 | 7.10±1.32 | 19.45±1.05 |
| Comparative example 3 | 342.1±44.5 | 6.21±1.36 | 19.73±1.09 |
| Comparative example 4 | 338.4±43.6 | 5.79±1.44 | 19.61±1.32 |
| Comparative example 5 | 337.3±39.6 | 7.24±1.12 | 19.56±1.29 |
| Comparative example 6 | 342.5±52.4 | 6.75±1.09 | 19.78±1.35 |
| Comparative example 7 | 351.7±51.6 | 5.47±1.56 | 20.04±1.21 |
| Comparative example 8 | 289.7±51.1 | 8.10±1.39 | 17.68±1.24 |
| Comparative example 9 | 362.1±54.2 | 6.78±1.22 | 19.12±1.39 |
| Comparative example 10 | 354.2±49.6 | 7.14±1.35 | 19.37±1.33 |
| Comparative example 11 | 395.2±44.4 | 5.73±1.52 | 20.78±1.41 |
| Comparative example 12 | 375.4±46.2 | 7.32±1.72 | 18.64±1.22 |
| Comparative example 13 | 395.2±49.6 | 4.22±1.46 | 24.56±1.45 |
| Comparative example 14 | 392.1±42.6 | 4.56±1.21 | 24.12±1.02 |
| Comparative example 15 | 314.5±48.2 | 7.98±1.15 | 17.95±1.11 |
| Comparative example 16 | 345.1±54.2 | 7.42±1.53 | 18.97±1.42 |
| Comparative example 17 | 359.4±43.9 | 7.10±1.28 | 19.24±1.39 |
From the table above, the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase prepared in the embodiments 1-3 of the invention can obviously reduce MDA content in rat serum, increase SOD and GSH levels, and has a certain improvement effect on oxidative stress.
In examples 4, 5 and 6, compared with example 3, the organic solvent mixture in step S2 was replaced by single ethanol, ethyl acetate or petroleum ether, the alcohol content after alcohol in example 4 was significantly increased, the serum AST, ALT viability in example 5 was increased, the liver SOD and GSH viability in example 6 was decreased, the MDA content was increased, and the activity of ADH and ALDH was decreased without the organic solvent extraction in step S2 compared with example 3, the alcohol content after alcohol was significantly increased, the serum AST, ALT viability was increased, the liver SOD and GSH viability was decreased, and the activity of ADH and ALDH was decreased. In the invention, the ethanol extract can obviously shorten the sobering-up time and reduce the concentration of ethanol in blood; the ethyl acetate extract can reduce AST and ALT activity in serum; the petroleum ether extract can remarkably improve the activity of SOD and GSH in liver, reduce the content of MDA and liver index, wherein the mixed organic solvent comprises ethanol, ethyl acetate and petroleum ether, and the extract obtained by mixing and extracting the three organic solvents can also simultaneously enhance the activity of ADH and ALDH.
Comparative example 1 compared with example 3, serum AST and ALT activities were improved, TG content was increased, ADH activity was decreased, and GSH activity was decreased without the step S1 water extraction. In the invention, the water extract can improve the weight and liver index change and liver function indexes AST, ALT and ALP abnormality caused by drinking; the method has the function of down-regulating the blood lipid indexes TC and TG which are improved by alcohol induction; simultaneously, the level of antioxidant indexes GSH and GST is effectively improved, and the activity of ADH in the liver is obviously enhanced.
Compared with the embodiment 3, the comparison examples 3 and 4 do not comprise kudzuvine root or hericium erinaceus in the step S1, other conditions are not changed, GSH and SOD levels are reduced, ADH and ALDH activities are reduced, and the ethanol content in the blood of the rat after the wine filling is obviously increased. In the invention, the kudzuvine root and the hericium erinaceus can obviously improve the antioxidant indexes GSH and SOD levels, obviously enhance the activity of ADH and ALDH in the liver, shorten the sobering-up time and have the synergistic effect.
In comparative examples 5 and 6, no lemon or tomato was added in step S3, the ADH and ALDH activities were decreased, the GSH and SOD levels were decreased, the MDA content was increased, and in comparative example 5, the ALT, AST and liver TG levels were increased, as compared with example 3. Comparative example 7 compared with example 3, the solid obtained in step S2 was not added in step S3, the GSH and SOD levels were reduced, and the ethanol content in the blood of the rat after the wine filling was significantly increased. Comparative example 8 compared with example 3, the solids obtained in step S1 and S2 were not performed, the alcohol content after alcohol was significantly increased, the serum AST and ALT activities were increased, the liver TG level was increased, the liver SOD and GSH activities were decreased, the MDA content was increased, and the ADH and ALDH activities were decreased in step S3 without adding the solids obtained in step S2. According to the invention, the lemon and the tomato can simultaneously enhance the activities of ADH and ALDH, after being mixed with the solid residues of the traditional Chinese medicine composition, a carbon source and a nitrogen source are added to serve as fermentation substrates, a large amount of antioxidant active substances are generated under the action of saccharomycetes and lactobacillus, the level of SOD in the liver is increased, the SOD failure caused by alcohol is protected, the lemon fermentation products can also obviously inhibit the elevation of ALT, AST, liver TG and lipid peroxidation caused by alcohol, and simultaneously, the histopathological change of alcoholic liver injury can also be improved.
Comparative examples 9 and 10 compared with example 3, in step S5, the levels of ALT, AST and TG were increased, the MDA content in the serum of rats was increased, and the SOD and GSH levels were reduced without inoculating lactobacillus fermentum LF021 or bifidobacterium longum BL5b and lactobacillus reuteri LR 06. Comparative example 16, which was not subjected to the fermentation treatment of step S5, had increased ALT, AST and TG levels, increased MDA content in rat serum, decreased SOD and GSH levels, and decreased ADH and ALDH activities, as compared with example 3. Comparative example 17 shows that, compared with example 3, the ALT, AST and TG levels were increased, MDA content in rat serum was increased, SOD and GSH levels were decreased, and ADH and ALDH activities were decreased without adding enterococcus faecalis. The lactobacillus reuteri LR06 and the bifidobacterium longum BL5b are mixed, fermented and cultured, the bifidobacterium longum BL5b has a synergistic effect, a large amount of lactic acid and amino acid are generated, the growth of streptococcus acidophilus is promoted, the lactobacillus reuteri and the bifidobacterium longum are mutually supplemented, the fermentation of zymophyte is promoted, a large amount of antioxidant substances and lipid-lowering substances are generated, so that the liver oxidative damage caused by alcohol is effectively improved, and the blood lipid, serum and liver lipid content are reduced by regulating blood lipid. The bioactive components such as zinc, B vitamins, and reduced glutathione contained in yeast can be used as synthetic raw materials of organism reaction enzymes, and can be used for effectively removing oxygen ions and free radicals in vivo and protecting liver cells. The enterococcus hai can play an obvious role in dispelling the effects of alcohol, and meanwhile, by changing the composition of intestinal microorganisms, the intestinal flora is regulated, the intestinal immunity is improved, the oxidative stress is reduced, and the chemical liver injury is relieved, so that the effects of dispelling the effects of alcohol, improving hangover, reducing adverse reactions after the alcohol and protecting the liver are achieved.
Compared with the example 3, the comparative example 11 has no ultrasonic treatment and ozone synergistic treatment in the step S6, the ethanol content after drinking is obviously improved, the MDA content in the serum of the rat is improved, and the SOD and GSH levels are reduced. In the low-power ultrasonic wave and ozone treatment process, the structure of the protein can be changed through the ultrasonic wave effect, so that the combination reaction of the corn oligopeptide substance and the ozone is promoted, and the effect of promoting the ozone modification is achieved; the disulfide bond content of the corn oligopeptide after being subjected to ozone short-time oxidation treatment is increased, so that dimeric peptide is obtained, and the molecular weight of the oligopeptide is primarily increased; because the time is short, other reducing amino acids on the corn oligopeptide are not yet available for oxidation reaction, the property of the protein peptide is not greatly influenced, meanwhile, the hydrogen bond exposure can be improved, the hydrogen bond combination of the corn oligopeptide and polysaccharide is promoted, a polysaccharide peptide compound is formed, the antioxidant activity of the corn oligopeptide is improved, the content of alanine and leucine in blood can be improved, NAD+ exists stably, and the effect of dispelling the effects of alcohol is achieved.
Comparative example 12 compared with example 3, the ethanol content after alcohol is obviously increased, ALT, AST and TG levels are increased, MDA content in rat serum is increased, SOD and GSH levels are reduced, and ADH and ALDH activities are reduced without performing step S6. The konjak glucomannan, the yam polysaccharide and the corn oligopeptide can reduce the ethanol content entering blood through the adsorption effect on ethanol, can improve the activity of ADH and ALDH in blood, improve the level of SOD and GSH, reduce the MDA content, reduce the acetaldehyde content in blood and brain, the yam polysaccharide can prolong the drunk latency, shorten the drunk maintenance time, improve the activity of ADH and ALDH in the liver of drunk mice, reduce the concentration of ethanol and acetaldehyde in serum, reduce the MDA content in liver tissues, improve the content of SOD and GSH, and the corn oligopeptide can enhance the activity of ADH, clear the activity of free radicals, reduce the damage of alcohol to the liver, shorten the drunk time of cooperative mice and reduce the ethanol content in serum.
Comparative example 13, in which taurine was not added in step S7, showed decreased ADH and ALDH activities and increased ALT, AST and TG levels, as compared with example 3. Comparative example 14, compared with example 3, did not proceed to step S7, and its post-alcohol content was significantly improved. Taurine is produced by regulating Ca in liver cells 2+ Balance, improve the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase in liver, reduce the release of peroxidase, and effectively protect liver cells; the addition of alanine and leucine can increase the content of the two in blood, and make NAD + The wine is stable and plays a good role in dispelling the effects of alcohol.
Comparative example 15 in comparison with example 3, the preparation of the inner layer slow release granule of step S9 was not performed, and the components were simply mixed. The fermentation product, acetaldehyde dehydrogenase, ethanol oxidase, lactobacillus helveticus and enterococcus hainanensis are protected in carboxylated chitosan, after emulsification, carboxylated chitosan is crosslinked to form a wall material of particles under the action of metal ions, a slow-release structure is formed, and after the fermentation product is sent into a human body, probiotics and protease are not easy to decompose in the stomach, so that the probiotics and the protease can be degraded and released after entering the intestinal tract, a large amount of probiotics and protease can directly act on the intestinal tract to effectively regulate the intestinal flora, and the probiotics can reduce bacterial translocation, reduce liver inflammation, the protease can directly enter blood and act on the degradation of ethanol, so that the damage of the ethanol to the liver is reduced, and the effect of protecting the liver is achieved.
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 (10)
1. A preparation method of lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase is characterized in that radix puerariae, liquorice, hericium erinaceus and dandelion are subjected to water extraction and organic solvent extraction and filtration to obtain a traditional Chinese medicine water extract and a traditional Chinese medicine organic solvent extract, solid is mixed with lemon juice, tomato juice, a carbon source and a nitrogen source to prepare a fermentation substrate, and activated lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 are inoculated for fermentation to obtain a fermentation product; mixing the fermentation product, acetaldehyde dehydrogenase, ethanol oxidase, lactobacillus helveticus, enterococcus hainanensis and carbomer, adding water, adding an aqueous solution dissolved with carboxylated chitosan, emulsifying, adding a metal ion solution for solidification to obtain inner-layer slow-release particles, mixing konjak glucomannan, chinese yam polysaccharide and corn oligopeptide, performing ultrasonic treatment and ozone synergistic treatment to obtain polysaccharide peptide complex, uniformly mixing the polysaccharide peptide complex with the inner-layer slow-release particles, a Chinese medicinal water extract, a Chinese medicinal organic solvent extract, taurine, alanine and leucine, adding an aqueous solution dissolved with sodium alginate, emulsifying, adding the metal ion solution for solidification, and freeze-drying to obtain the lactobacillus freeze-dried powder containing acetaldehyde dehydrogenase.
2. The method of manufacturing according to claim 1, comprising the steps of:
s1, extracting the traditional Chinese medicine composition with water: cleaning radix Puerariae, glycyrrhrizae radix, hericium Erinaceus, and herba Taraxaci respectively, drying, pulverizing to obtain Chinese medicinal composition powder, adding into water, heating and boiling for 2-3 times, filtering, mixing filtrates, collecting solid, concentrating the filtrate, and drying to obtain water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, heating and extracting for 2-3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate, and drying to obtain an organic solvent extract;
s3, preparing a fermentation substrate: mixing lemon and tomato, squeezing, removing solids, uniformly mixing the juice with the solids obtained in the step S2, adding a carbon source and a nitrogen source, uniformly mixing, adding sterile water, stirring, mixing, and sterilizing to obtain a fermentation substrate;
s4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into a Gao's medium respectively, and performing activation culture to obtain strain seed liquid;
s5, fermenting: inoculating the strain seed liquid in the step S4 into the fermentation substrate in the step S3, and fermenting and culturing to obtain a fermentation product;
S6, preparing a polysaccharide peptide complex: uniformly mixing konjak glucomannan, yam polysaccharide and corn oligopeptide, and carrying out ultrasonic treatment and ozone synergistic treatment to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing taurine, alanine and leucine to obtain an amino acid additive;
s8, preparation of complex enzyme: uniformly mixing acetaldehyde dehydrogenase and ethanol oxidase to obtain a complex enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing the fermentation product obtained in the step S5, the complex enzyme obtained in the step S8, lactobacillus helveticus, enterococcus hainanensis and carbomer, adding into water, uniformly stirring and mixing, adding an aqueous solution dissolved with carboxylated chitosan, rapidly emulsifying a first SPG film to form emulsion, adding a metal ion solution, and curing at normal temperature to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing the water extract prepared in the step S1, the organic solvent extract prepared in the step S2, the polysaccharide peptide compound prepared in the step S6, the amino acid additive prepared in the step S7 and the inner layer slow release particles prepared in the step S9, adding the mixture into an aqueous solution in which sodium alginate is dissolved, emulsifying a second SPG fast film to form an emulsion, adding a metal ion solution, solidifying, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
3. The preparation method according to claim 2, wherein the mass ratio of the kudzuvine root, the liquorice, the hericium erinaceus and the dandelion in the step S1 is 5-10:1-3:3-5:1-2; the solid-liquid ratio of the traditional Chinese medicine composition powder to water is 1:5-10g/mL; the extraction time is 1-3h; the mixed organic solvent in the step S2 is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 5-7:2-4:1-3; the solid-liquid ratio of the solid to the mixed organic solvent is 1:3-5g/mL; heating to 50-70deg.C for 0.5-1 hr.
4. The preparation method according to claim 2, wherein the mass ratio of lemon, tomato, solids, carbon source, nitrogen source, sterile water in step S3 is 7-12:5-10:3-5:5-10:3-5:30-50; the carbon source is at least one selected from glucose, maltose, lactose, sucrose, fructose, starch, molasses and agar; the nitrogen source is at least one selected from ammonia water, urea, ammonium salt, nitrate, amino acid and fish meal; the ammonium salt is at least one selected from ammonium chloride, ammonium nitrate and ammonium sulfate; the nitrate is at least one selected from potassium nitrate, sodium nitrate, ferric nitrate, zinc nitrate, aluminum nitrate, magnesium nitrate, manganese nitrate and copper nitrate; the amino acid is selected from one or more of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine and phenylalanine; the condition of the activation culture in the step S4 is 36-39 ℃, and the culture is carried out for 18-24 hours at the rotating speed of 50-70r/min under the humidity of 30-50%; the bacterial seed liquid has a bacterial content of 10 8 -10 9 cfu/mL。
5. The preparation method according to claim 2, wherein the inoculum size of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in step S5 is 3-5%, 1-3%, 0.5-1%, respectively, and the conditions of the fermentation culture are 36-40 ℃,30-50% humidity, 50-70r/min rotation speed, and 48-72h; in the step S6, the mass ratio of the konjac glucomannan, the yam polysaccharide and the corn oligopeptide is 3-5:1-2:2-4; in the ultrasonic treatment and ozone synergistic treatment, the ultrasonic power is 120-150W, the ozone concentration is 30-70mg/L, the gas flow is 15-25L/h, and the treatment time is 5-7s.
6. The preparation method according to claim 2, wherein the mass ratio of taurine, alanine and leucine in step S7 is 7-12:1-3:2-4; the mass ratio of the acetaldehyde dehydrogenase to the ethanol oxidase in the step S8 is 10-15:3-5.
7. The preparation method according to claim 2, wherein the mass ratio of the fermentation product, the complex enzyme, the lactobacillus helveticus, the enterococcus haii and the carbomer in the step S9 is 15-20:1-2:0.5-1:2-3:1-3; the content of carboxylated chitosan in the water solution containing carboxylated chitosan is 12-15wt%; the aperture of the first SPG rapid membrane is 300-500nm; the metal ion solution is a solution containing 3-5wt% of at least one of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions; the mass ratio of the water extract, the organic solvent extract, the polysaccharide peptide complex, the amino acid additive and the inner layer slow release particles in the step S10 is 3-5:2-4:0.5-1:0.2-0.5:4-6; the content of sodium alginate in the aqueous solution dissolved with sodium alginate is 7-12wt%; the pore diameter of the second SPG rapid membrane is 10-15 mu m; the metal ion solution is a solution containing 3-5wt% of at least one of calcium ion, magnesium ion, aluminum ion, iron ion and zinc ion.
8. The preparation method according to claim 2, characterized by comprising the following steps:
s1, extracting the traditional Chinese medicine composition with water: cleaning, drying and crushing 5-10 parts by weight of radix puerariae, 1-3 parts by weight of liquorice, 3-5 parts by weight of hericium erinaceus and 1-2 parts by weight of dandelion respectively to obtain traditional Chinese medicine composition powder, adding the traditional Chinese medicine composition powder and water into water, heating and boiling for extraction for 1-3 hours, extracting for 2-3 times, filtering, merging filtrate, reserving solids, concentrating filtrate, and drying to obtain a water extract;
s2, extracting the traditional Chinese medicine composition by using an organic solvent: adding the solid obtained in the step S1 into a mixed organic solvent, wherein the solid-to-liquid ratio of the solid to the mixed organic solvent is 1:3-5g/mL, heating to 50-70 ℃ for 0.5-1h, extracting for 2-3 times, filtering, combining the filtrates, reserving the solid, removing the organic solvent from the filtrate, and drying to obtain an organic solvent extract;
the mixed organic solvent is a mixed solvent of ethanol, ethyl acetate and petroleum ether according to the mass ratio of 5-7:2-4:1-3;
s3, preparing a fermentation substrate: mixing 7-12 parts by weight of lemon and 5-10 parts by weight of tomato, squeezing, removing solids, uniformly mixing juice with 3-5 parts by weight of solids obtained in the step S2, adding 5-10 parts by weight of carbon source and 3-5 parts by weight of nitrogen source, uniformly mixing, adding 30-50 parts by weight of sterile water, stirring and mixing, and sterilizing by ultraviolet rays to obtain a fermentation substrate;
S4, activating strains: inoculating lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 into Gao's culture medium, respectively, and performing activation culture at 36-39deg.C, 30-50% humidity and 50-70r/min for 18-24 hr to obtain strain seed solution with bacterial content of 10 8 -10 9 cfu/mL；
S5, fermenting: inoculating strain seed liquid of lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 in the step S4 to a fermentation substrate in the step S3, wherein the inoculum size of the lactobacillus fermentum LF021, bifidobacterium longum BL5b and lactobacillus reuteri LR06 is respectively 3-5%, 1-3%, 0.5-1%,36-40 ℃ and 50-70r/min under 30-50% humidity, and fermenting and culturing for 48-72h to obtain a fermentation product;
s6, preparing a polysaccharide peptide complex: uniformly mixing 3-5 parts by weight of konjak glucomannan, 1-2 parts by weight of yam polysaccharide and 2-4 parts by weight of corn oligopeptide, and introducing ozone for treatment under 120-150W ultrasonic waves, wherein the ozone concentration is 30-70mg/L, the gas flow is 15-25L/h, and the treatment time is 5-7s to obtain a polysaccharide peptide compound;
s7, preparation of an amino acid additive: uniformly mixing 7-12 parts by weight of taurine, 1-3 parts by weight of alanine and 2-4 parts by weight of leucine to obtain an amino acid additive;
S8, preparation of complex enzyme: uniformly mixing 10-15 parts by weight of acetaldehyde dehydrogenase and 3-5 parts by weight of ethanol oxidase to obtain a complex enzyme;
s9, preparing inner-layer slow-release particles: uniformly mixing 15-20 parts by weight of the fermentation product obtained in the step S5, 1-2 parts by weight of the complex enzyme obtained in the step S8, 0.5-1 part by weight of lactobacillus helveticus, 2-3 parts by weight of enterococcus hainanensis and 1-3 parts by weight of carbomer, adding 50 parts by weight of water, uniformly stirring and mixing, adding 70-80 parts by weight of a carboxylated chitosan aqueous solution containing 12-15wt% and a first SPG fast film with the aperture of 300-500nm, emulsifying to form emulsion, adding 7-12 parts by weight of a solution containing 3-5wt% of at least one of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions, and curing at normal temperature for 20-30min to obtain inner-layer slow-release particles;
s10, preparing lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase: uniformly mixing 3-5 parts by weight of the water extract prepared in the step S1, 2-4 parts by weight of the organic solvent extract prepared in the step S2, 0.5-1 part by weight of the polysaccharide peptide compound prepared in the step S6, 0.2-0.5 part by weight of the amino acid additive prepared in the step S7 and 4-6 parts by weight of the inner layer slow release particles prepared in the step S9, adding 100 parts by weight of the inner layer slow release particles into a sodium alginate aqueous solution containing 7-12wt% and a second SPG fast film with the aperture of 10-15 mu m to emulsify to form emulsion, adding 7-12 parts by weight of a solution containing at least one of 3-5wt% of calcium ions, magnesium ions, aluminum ions, iron ions and zinc ions, curing for 10-15min, and freeze-drying to obtain the lactic acid bacteria freeze-dried powder containing acetaldehyde dehydrogenase.
9. A lactic acid bacterium lyophilized powder containing acetaldehyde dehydrogenase produced by the production method according to any one of claims 1 to 8.
10. Use of the lyophilized lactic acid bacteria powder containing acetaldehyde dehydrogenase according to claim 9 for preparing products for alleviating hangover and protecting liver.
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