CN111406856A - Functional fermented beverage for dispelling effects of alcohol and protecting liver and preparation method thereof - Google Patents
Functional fermented beverage for dispelling effects of alcohol and protecting liver and preparation method thereof Download PDFInfo
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- CN111406856A CN111406856A CN202010165035.6A CN202010165035A CN111406856A CN 111406856 A CN111406856 A CN 111406856A CN 202010165035 A CN202010165035 A CN 202010165035A CN 111406856 A CN111406856 A CN 111406856A
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- fermentation
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- fermented beverage
- liver
- pediococcus acidilactici
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/56—Flavouring or bittering agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
- A23L2/62—Clouding agents; Agents to improve the cloud-stability
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
- A23V2400/413—Acidilactici
Abstract
The invention discloses a functional fermented beverage for relieving alcoholism and protecting liver and a preparation method thereof, belonging to the field of microbial fermentation application. The raw materials of the fermented beverage comprise 5-10g of red dates, 10-15g of kudzu root powder and the balance of water; the probiotics used for fermentation of the functional fermented beverage for dispelling the effects of alcohol and protecting the liver is one of lactobacillus plantarum or pediococcus acidilactici. According to the invention, polysaccharides, vitamins, cellulose, phenolic substances, flavonoids and the like in the red dates and the kudzuvine roots can exert the greatest effect through probiotic fermentation, and the fermented beverage not only preserves the nutritive value of natural plants, but also has the nutrition and health care effects of probiotic fermented products, and has unique taste, obvious effect and good market prospect.
Description
Technical Field
The invention belongs to the field of microbial fermentation application, and particularly relates to a functional fermented beverage for dispelling effects of alcohol and protecting liver and a preparation method thereof.
Background
With the improvement of the rhythm of the living standard, more and more people have an increasing trend of the number of drinking people due to the influence of the aspects of life, pressure, work, communication, remuneration and the like, and in China, the number of drinking people is always increasing in recent years. At present, the drinking rates of men and women in China are 84.1% and 29.3% respectively, wherein 16.1% of men and 2.5% of women drink wine daily. The consumption of Chinese alcoholic beverages increases faster than in any other region of the world, and with the development of economy, the production of alcoholic beverages is steadily increasing, and the alcohol-related injuries are further aggravated. Relevant researches show that the Chinese date is sweet, mild and nontoxic in taste, is a common food and a common medicine, and has the health-care effects of protecting liver, strengthening spleen and stomach and prolonging life when being eaten for a long time or being added into medicinal food; the red dates contain 18 amino acids, and the red dates have a protein promoting effect and have the effects of tonifying spleen and nourishing liver. The triterpenes in the red dates can inhibit the activity of hepatitis viruses and improve the phagocytic capacity of a mononuclear phagocyte system in vivo to a certain extent. Has effects in enhancing immunity and protecting liver. The substance has good therapeutic effect on liver diseases. Pueraria lobata contains pueraria isoflavone and puerarin, and has the effects of improving brain circulation, learning and memory and relieving alcoholism. However, at present, relatively few patents for alleviating hangover and protecting liver are provided for red dates and kudzuvine roots, and the patents mainly include the following patents:
publication No.: 105380051A patent application for sobering up and protecting liver and its preparation method discloses a beverage for sobering up and protecting liver and its preparation method, the composition includes A and B components: the weight parts of the extracts of the substances in the formula A are as follows: 32-44 parts of rhodiola rosea, 24-36 parts of hawthorn, 20-32 parts of kudzu root, 6-16 parts of dark plum and 4-14 parts of liquorice; the weight parts of the extracts of the materials in the formula B are as follows: 28-42 parts of poria cocos, 12-24 parts of wolfberry, 8-20 parts of rhizoma polygonati, 2-12 parts of wrinkled gianthyssop, 1-10 parts of honeysuckle and 1-8 parts of cinnamon have the effects of protecting liver and the like, are beneficial to human health and are used as a beverage for relieving alcoholism and protecting liver.
Publication No.: 108126123A provides a composition, a beverage, a preparation method and an application thereof, which can achieve the effects of relieving alcoholism, protecting liver and inducing diuresis, can promote the discharge of alcohol from urine, resist oxidation, strengthen stomach and protect stomach, and further reduce the harm of alcohol to human body. The paint comprises the following components in parts by weight: the raisin tree is: 1-15 parts; and (3) kudzuvine flower: 1-10 parts; tuckahoe, poria cocos: 1-10 parts; white atractylodes rhizome: 1-10 parts; reed rhizome: 1-10 parts; glossy privet fruit: 1-10 parts; mint: 1-5 parts of reed rhizome; salt algae: 1-5 parts. The invention aims to provide a composition for relieving alcoholism and protecting liver, which comprises various components such as hovenia dulcis thunb, pueraria lobata, poria cocos, bighead atractylodes rhizome, mint, rhizoma phragmitis, glossy privet fruit, dunaliella salina and the like; wherein semen Hoveniae and flos Puerariae Lobatae have effects of relieving hangover and promoting diuresis; the tuckahoe and the atractylodes macrocephala have the effects of tonifying spleen and qi, promoting diuresis and calming heart and are ministerial; the mint has the effects of soothing liver and resolving depression, the reed rhizome has the effects of clearing lung and promoting fluid production, the glossy privet fruit has the effects of tonifying liver and kidney, and the seven medicines are used as adjuvant medicines, mainly have the effects of tonifying spleen and removing dampness, and have the coordination of the functions of the heart, the liver, the spleen, the lung and the kidney. Meanwhile, the dunaliella salina is added to improve immunity and nourish cells.
Publication No.: 110433196A discloses an application of a substance in relieving alcoholism and protecting liver. The solvent extraction method is used for preparing the fat-soluble part of the rubber tree seeds, and the solvent selected for extraction is No. 6 solvent. 100kg of fresh rubber tree seeds are used as raw materials, and 40kg of kernels are prepared after drying and shelling. Then, after the kernels are puffed, the kernels are placed into a sealed leaching container, and 2 times of solvent by weight is added. Stirring and mixing the material and the solvent under the conditions of 55 ℃ (lower than the boiling point of the solvent) and normal pressure to ensure that the material and the solvent are fully contacted. Then standing for precipitation, draining off the solvent, and volatilizing the solvent under the vacuum condition of 120 ℃ (higher than the boiling temperature of the solvent) and 100Pa to leave 18kg of liquid substance, namely the substance of the invention. The invention provides a brand-new substance for relieving alcoholism and protecting liver; the substance is safe and has no side effect, can be used for a long time, can be temporarily remedied before drinking, during drinking or even after drinking, and is used for relieving alcoholism and protecting liver.
The foods for alcoholic liver injury and sobering and protecting liver disclosed by the prior patent technology are health-care foods prepared by taking articles which are not considered to have curative effect on liver injury in traditional Chinese medicines as raw materials, the used raw materials are complex, the preparation method is single, and most of the foods are simple mixed and processed. However, the health food for alleviating hangover and protecting liver prepared by probiotic fermentation has not been reported.
Disclosure of Invention
The invention aims to provide a red date enzyme beverage which is prepared aiming at relieving alcoholism and nourishing liver of people and provides a research idea for production of related products. Not only can retain various active ingredients of the strains, but also the beneficial ingredients in the raw materials have health care effect on organisms. Since the functional beverages and foods on the market have relatively single functions and are mostly simple mixing processes, it is necessary to produce related products using probiotics and combine the effective ingredients thereof.
The red dates have high content of nutrient components and are always considered as 'good tonics'. The red dates are rich in polysaccharides, phenols, cyclic adenosine monophosphate, alkaloids and other bioactive substances. The research shows that polysaccharides, triterpenes and saponins contained in the red dates have the effect of protecting the liver, and the air-swallowed tartaric acid can increase the concentration of serum protein, reduce the content of glutamic-pyruvic transaminase in the serum and reduce the damage of toxic substances to the liver. However, compared with the red dates fermented by probiotics, the unfermented red dates have better effect of reducing the glutamic-pyruvic transaminase in serum. The beverage with the functions of dispelling the effects of alcohol and protecting the liver is produced by fermenting the jujube pulp through lactobacillus plantarum.
The invention aims to obtain the probiotic functional beverage by fermenting red dates and kudzuvine roots by using lactobacillus plantarum (TK9) single bacteria and pediococcus acidilactici (TK530) respectively.
The first purpose of the invention is to provide a functional fermented beverage for relieving alcoholism and protecting liver, which comprises the following raw materials: the mass ratio of the red dates to the kudzu root powder is (5-10): (10-15) mixing, wherein the mass-volume ratio of the total amount of the red dates and the kudzu root powder to water is as follows: 1: (4-8); the probiotics used for fermentation of the functional fermented beverage for dispelling the effects of alcohol and protecting the liver is one of lactobacillus plantarum or pediococcus acidilactici.
Preferably, the lactobacillus plantarum is lactobacillus plantarum (L actinobacillus plantarum) TK9, and the preservation number is CGMCC No. 11891;
preferably, the pediococcus acidilactici is pediococcus acidilactici (L actinobacillus plantarum) TK530 with a preservation number of CGMCC No. 18737;
the second purpose of the invention is to provide a preparation method of the functional fermented beverage for relieving alcoholism and protecting liver, which comprises the following steps:
(1) mixing the red dates, the kudzu root powder and water according to a proportion, and sterilizing to obtain a fermentation medium;
(2) activating the lactobacillus plantarum or pediococcus acidilactici, and inoculating with an inoculum size of 106-108CFU/m L, inoculating in fermentation medium, standing and fermenting at 31-40 deg.C for 12-36 h;
(3) and after the fermentation is finished, adding a stabilizer into the fermentation liquor, and carrying out acid adjustment, flavor adjustment and homogenization to obtain the product.
Preferably, in the step (1), the red dates are washed, dried, pitted and the radix puerariae powder is directly purchased for standby.
Preferably, in the step (1), the sterilization conditions are as follows: 121 ℃ and 20 min.
Preferably, in the step (2), the probiotic activation step is as follows: inoculating 1% of glycerol tubes of probiotics into an activation culture medium for activation, activating at the temperature of 37 ℃ for 24h at the pH of 7.2-7.4 to obtain a first-stage seed solution, transferring the first-stage seed solution into the activation culture medium according to the inoculation amount of 4-10% for secondary activation to obtain a second-stage seed solution after 24h of activation, transferring the second-stage seed solution into the activation culture medium according to the inoculation amount of 1-3% (v/v) for three-time activation, and activating for 24h to obtain a third-stage seed solution.
More preferably, in the step (2), the activation medium is MRS medium, and the sterilization condition is 121 ℃ and 20 min.
Preferably, in the step (2), the fermentation time of the probiotics is 18-26 h.
More preferably, in the step (2), when the probiotic is lactobacillus plantarum TK9, the fermentation time is 18 h.
More preferably, in the step (2), the probiotic is pediococcus acidilactici TK530, and the fermentation time is 24 h;
more preferably, in the step (2), when the probiotic is lactobacillus plantarum TK9, the inoculation amount is 5 × 107CFU/ml。
More preferably, in the step (2), when the probiotic is pediococcus acidilactici TK530, the inoculation amount is 1.64 × 108CFU/ml。
Preferably, in the step (3), the addition amount of the stabilizer accounts for 0.5-1% of the mass percentage of the fermentation broth, and the stabilizer is sodium tripolyphosphate. The addition of the stabilizer prevents the phenomena of floating, flocculation, layering and the like of the jujube pulp.
Preferably, in the step (3), the acid adjustment is performed by using the following raw materials: 0.05 to 0.08 percent of citric acid and 5 to 12 percent of glucose, wherein the percentages are the mass percentage of the raw materials to the fermentation liquor.
Preferably, in the step (3), the raw materials for blending flavor comprise: 0.05-0.1% of edible essence, wherein the percentage is the mass percentage of the raw materials in the fermentation liquor.
Preferably, in the step (3), the homogenizing pressure is 30-40MPa, and the homogenizing time is 5-15 min.
The fermented jujube pulp is homogenized, so that the jujube pulp has more delicate mouthfeel. The red date and kudzu vine root probiotic liquid state fermented beverage is obtained by an emulsification method, stabilizer addition, acid adjustment, flavor adjustment and homogenization.
The lactobacillus plantarum (L actinobacillus plantarum) TK9 has been deposited in the general microbiological center of China Committee for culture Collection of microorganisms at 12.17.2015, and the addresses of the lactobacillus plantarum are 100101 in the letter code of China academy of sciences institute of microbiology No. 3, Sihui Lu 1, Beijing area facing the sun, and the preservation number is CGMCC No.11891, has good gastric acid and bile salt resistance, has 2h survival rate of 88.03% at pH 3 and 0.3% of bile salt of 4h survival rate of 81.06%, has wide antifungal property, does not need to add extra preservatives, and can prolong the preservation time of beverages and foods, and is disclosed in the invention patent publication No. CN108148791A, namely a probiotic preparation for improving water quality in aquaculture and a preparation method thereof.
The pediococcus acidilactici (Pediococcus acidilactici) TK530, which is deposited in the China general microbiological culture Collection center at 25.10.2019, addresses: the microbial code of the institute of microbiology, China academy of sciences, No. 3 Xilu No.1, Beijing, Chaoyang, and the collection number is CGMCC NO. 18737. The bacterium has good acid and bile salt resistance, the survival rate of the bacterium is 98.8 percent after the bacterium is treated for 3 hours in a pH (2) environment, the survival rate of the bacterium is 124.7 percent after the bacterium is treated for 3 hours in a pH (3) environment, and the Pediococcus acidilactici TK530 can normally grow in a pH (4) environment. The result shows that the pediococcus acidilactici TK530 has strong acid resistance and can resist the acid environment in the intestinal tract. The survival rate of the cells after being treated for 3 hours in the environment with 0.15 percent of the bile salt is 122.4 percent, the survival rate of the cells after being treated for 3 hours in the environment with 0.3 percent of the bile salt is 98.8 percent, and the survival rate of the cells after being treated for 3 hours in the environment with 0.45 percent of the bile salt is 87.0 percent. The result shows that the pediococcus acidilactici TK530 has certain bile salt resistance. The bacteria inhibition performance is identified, and the results show that the bacteria inhibition rates of the fermented supernatant to escherichia coli, salmonella and staphylococcus aureus are 32.2%, 37.6% and 13.4% respectively.
Has the advantages that:
in the invention, the red dates have unique flavor and sweet and fragrant taste, the nutritional ingredients of the red dates are full and rich, and the modern science proves that the red dates have a plurality of nutritional and health-care ingredients. Is called 'Baiguowang' and is rich in protein, fat, saccharide, carotene, B vitamins, vitamin C, vitamin P, minerals such as calcium, phosphorus, iron and the like, cyclic adenosine monophosphate and other nutrient components. According to the analysis of Chinese academy of agriculture, Chinese red jujube (dried jujube) sugar 55% -80%, crude protein 2.92%, vitamin C8.7 mg/100g and also contains rich amino acids, 18 kinds, including 8 kinds of amino acids necessary for human body. The red date is a food used as both medicine and food, wherein the red date polysaccharide and flavonoid compound have the effects of reducing blood cholesterol, preventing angiosclerosis, enhancing the immunity of the organism, delaying senility, preventing cancer, inhibiting cancer and the like. The red date also contains cyclic adenosine monophosphate which is an active substance necessary for human energy metabolism and plays a role in physiological regulation of human cells. Has effects in enhancing immunity, protecting liver, improving liver function, treating coronary heart disease and myocardial infarction, and increasing myocardial contraction force. After common lactic acid bacteria are fermented, saccharides in red dates can be converted into lactic acid and the like, so that the acid content in the red date pulp is increased, and after lactobacillus plantarum (TK9) single bacteria and pediococcus acidilactici (TK530) are fermented, the jujube polysaccharide is converted into bacterial polysaccharide, so that the antioxidant effect is increased. The pediococcus acidilactici TK530 has high phenyllactic acid yield, and the yield of phenyllactic acid synthesized by pyruvic acid in red dates is over 9.5ug/g, so that the antibacterial performance and the storage time of the product are obviously improved.
The kudzu root contains daidzin, daidzein, puerarin and the like, and also contains components such as daidzein-4, 7-diglucoside, puerarin-7-xyloside, puerarin, isoflavone glycoside, starch and the like, wherein the kudzu root total isoflavone has the functions of improving brain circulation, covering the function of learning and memory, reducing blood pressure, enhancing immunologic function, resisting tumors, relieving alcoholism and the like. The puerarin can improve the cardiovascular and cerebrovascular circulation quality and reduce the content of blood sugar, has the effects of relieving fever, dispelling the effects of alcohol, resisting oxidation and the like in traditional Chinese medicine, can generate flavor substances by fermenting the red dates with common lactic acid bacteria, and improves the taste of the product. The pueraria lobata fermented by the lactobacillus plantarum TK9 and the pediococcus acidilactici TK530 can produce metabolites such as protease and lipase, and endow the product with more health care functions.
The red dates and the kudzuvine roots are mixed and fermented, the characteristics of various substances are integrated, the red dates contain effective components such as red date polysaccharide, flavonoid, saponin, triterpenes, alkaloids, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and the like, the kudzuvine roots contain substances such as starch, puerarin, soybean flavonoid and arachidic acid, most researches believe that intestinal lactobacillus, bifidobacteria and the like can promote intestinal tract peristalsis and can better absorb beneficial components in raw materials, functional foods fermented by the red dates have an important regulating effect on alcohol dispelling and a liver protecting effect, lactobacillus plantarum 9 and pediococcus acidilactici TK530 have better acid and bile salt resisting capabilities and wide antifungal properties, can also produce metabolic products such as protease and lipase and the like, the functional foods fermented with the red dates have the effects of improving the product antibacterial effect and increasing the beneficial metabolism of the organism and the like, the polysaccharide, the cyclic adenosine monophosphate, the puerarin, the isoflavone, the TK, the lactic acid and the lipase are fermented to further enhance the liver protecting effect of the product, the flavor of common lactobacillus TK, the lactic acid and the lactic acid metabolism of the lactic acid bacteria are improved by fermentation, the production of the.
Drawings
FIG. 1 is an image of pathological section of liver tissue of mouse in experimental example 4, in which (a) is normal group, (b) is model group, (c) is positive group, (d) is product group 1, (e) is product group 2, (f) is product group 3, (g) is product group 4, (h) is product group 5, (i) is product group 6, and (j) is non-fermented group.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to examples and experimental examples, and the specific examples described herein are only for explaining the present invention, but the present invention is not limited to the following specific examples and the scope of protection is not limited thereto.
The% in the examples means, unless otherwise specified, the mass percentage, the percentage of the solution means the number of grams of solute contained in 100m L, and the percentage between the liquids means the volume ratio of the solution at 25 ℃.
The invention adopts Ningxia red jujube, kudzu root and so on as raw materials, and adopts mixed bacteria liquid fermentation of lactobacillus plantarum and pediococcus acidilactici to prepare the product, the following embodiment will explain the invention by taking lactobacillus plantarum TK9 and pediococcus acidilactici TK530 as examples, the selected bacteria should be understood as exemplary and not specific limitation, the method of the invention is applicable to the known strains of the same species with the same function.
The methods of strain activation used in the following examples are, unless otherwise specified:
activating a culture medium: preparing an activation medium as an MRS medium; the sterilization condition is 121 ℃ for 20 min. Inoculating 1% (v/v volume ratio of activated culture medium) into glycerol tubes of Lactobacillus plantarum TK9 and Pediococcus acidilactici TK530 respectively, activating at pH7.2-7.4 and at 37 deg.C; after activation for 24 hours, transferring the strain to an activation culture medium according to the inoculation amount of 4% (v/v) for secondary activation, and obtaining a secondary seed solution after activation for 24 hours; inoculating the strain to an activation culture medium again according to the strain inoculation amount of 1% (v/v) for three times of activation, and obtaining a third-level seed solution after 24 hours of activation.
The present invention will be specifically described below using the above-mentioned strains.
Example 1 preparation of functional fermented beverage for alleviating hangover and protecting liver
(1) Cleaning and pitting fructus Jujubae, soaking, and treating with wall breaking machine;
(2) sterilizing the fructus Jujubae pulp in a sterilizing pot at 80 deg.C for 30 min.
(3) Preparing an activation medium as an MRS medium; sterilizing at 121 deg.C for 20 min;
(4) preparing a fermentation medium: adding 6g of red date and 10g of kudzu root powder into 100ml of water;
(5) activating strains: inoculating 1% of glycerol tubes of single lactobacillus plantarum TK9 in an activation culture medium respectively for activation, wherein the pH is 7.2-7.4, and the activation temperature is 37 ℃; respectively activating for 24h, transferring an activation culture medium according to the inoculum size of 4% (v/v) for secondary activation to obtain a secondary seed solution after 24h of activation, transferring the activation culture medium according to the inoculum size of 1% (v/v) for tertiary activation to obtain a tertiary seed solution after 24h of activation;
(6) fermenting probiotics: inoculating lactobacillus plantarum TK9 according to the inoculation amount of 107CFU/m L was inoculated into the fermentation medium and liquid fermented for 18h at 37 ℃.
(7) After fermentation is finished, 1% of stabilizer sodium polyphosphate is added into the fermentation liquor, 0.06% of citric acid and 10% of glucose are added for acid adjustment, 0.1% of edible essence is added for flavor adjustment, and after mixing, homogenization treatment is carried out, wherein the homogenization pressure is 30MPa, the homogenization time is 15min, and the product is obtained after homogenization. The percentage is the mass percentage of the raw materials in the fermentation liquor.
Example 2 preparation of functional fermented beverage for alleviating hangover and protecting liver
(1) Cleaning and pitting fructus Jujubae, soaking, and treating with wall breaking machine;
(2) sterilizing the fructus Jujubae pulp in a sterilizing pot at 80 deg.C for 30 min.
(3) Preparing an activation medium as an MRS medium; sterilizing at 121 deg.C for 20 min;
(4) preparing a fermentation medium: adding 6g of red date and 10g of kudzu root powder into 100ml of water;
(5) activating strains: inoculating 1% of glycerol tubes of single pediococcus acidilactici TK530 bacteria in an activation culture medium respectively for activation, wherein the pH is 7.2-7.4, and the activation temperature is 37 ℃; respectively activating for 24h, transferring an activation culture medium according to the inoculum size of 4% (v/v) for secondary activation to obtain a secondary seed solution after 24h of activation, transferring the activation culture medium according to the inoculum size of 1% (v/v) for tertiary activation to obtain a tertiary seed solution after 24h of activation;
(6) fermenting probiotics: the pediococcus acidilactici TK530 was inoculated in an amount of 107CFU/m L is inoculated into a fermentation medium, and liquid state fermentation is carried out for 24 hours at the temperature of 37 ℃.
(7) After fermentation is finished, 1% of stabilizer sodium polyphosphate is added into the fermentation liquor, 0.08% of citric acid and 8% of glucose are added for acid adjustment, 0.05% of edible essence is added for flavor adjustment, and after mixing, homogenization treatment is carried out, wherein the homogenization pressure is 35MPa, the homogenization time is 10min, and the product is obtained after homogenization. The percentage is the mass percentage of the raw materials in the fermentation liquor.
Example 3 preparation of functional fermented beverage for alleviating hangover and protecting liver
(1) Cleaning and pitting fructus Jujubae, soaking, and treating with wall breaking machine;
(2) sterilizing the fructus Jujubae pulp in a sterilizing pot at 80 deg.C for 30 min.
(3) Preparing an activation medium as an MRS medium; sterilizing at 121 deg.C for 20 min;
(4) preparing a fermentation medium: the mass ratio of the red dates to the kudzu root powder is 10: 11, mixing, wherein the mass-volume ratio of the total amount of the red dates and the kudzu root powder to water is as follows: 1: 4;
(5) activating strains: inoculating 1% of glycerol tubes of single lactobacillus plantarum TK9 in an activation culture medium respectively for activation, wherein the pH is 7.3, and the activation temperature is 37 ℃; respectively activating for 24h, transferring an activation culture medium according to the inoculum size of 4% (v/v) for secondary activation to obtain a secondary seed solution after 24h of activation, transferring the activation culture medium according to the inoculum size of 1% (v/v) for tertiary activation to obtain a tertiary seed solution after 24h of activation;
(6) fermenting probiotic bacteria, namely inoculating lactobacillus plantarum TK9 according to the inoculation amount of 2 × 107CFU/m L was inoculated into the fermentation medium and liquid fermented for 19h at 37 ℃.
(7) After fermentation is finished, 0.8% of stabilizer sodium polyphosphate is added into the fermentation liquor, 0.07% of citric acid and 8% of glucose are added for adjusting acid, 0.08% of edible essence is added for adjusting flavor, and after mixing, homogenization treatment is carried out, wherein the homogenization pressure is 30MPa, the homogenization time is 15min, and the product is obtained after homogenization. The percentage is the mass percentage of the raw materials in the fermentation liquor.
Example 4 preparation of functional fermented beverage for alleviating hangover and protecting liver
(1) Cleaning and pitting fructus Jujubae, soaking, and treating with wall breaking machine;
(2) sterilizing the fructus Jujubae pulp in a sterilizing pot at 80 deg.C for 30 min.
(3) Preparing an activation medium as an MRS medium; sterilizing at 121 deg.C for 20 min;
(4) preparing a fermentation medium: the mass ratio of the red dates to the kudzu root powder is 8: 10, mixing the red dates and the kudzu root powder, wherein the mass-volume ratio of the total amount of the red dates to the kudzu root powder to the water is as follows: 1: 4.5;
(5) activating strains: inoculating 1% of glycerol tubes of single pediococcus acidilactici TK530 bacteria in an activation culture medium respectively for activation, wherein the pH is 7.2-7.4, and the activation temperature is 37 ℃; respectively activating for 24h, transferring an activation culture medium according to the inoculum size of 4% (v/v) for secondary activation to obtain a secondary seed solution after 24h of activation, transferring the activation culture medium according to the inoculum size of 1% (v/v) for tertiary activation to obtain a tertiary seed solution after 24h of activation;
(6) fermenting probiotic bacteria by inoculating 3 × 10 to Pediococcus acidilactici TK5307CFU/m L was inoculated into the fermentation medium and liquid fermented for 23h at 37 ℃.
(7) After fermentation is finished, 0.8 percent of stabilizer sodium polyphosphate is added into the fermentation liquor, 0.06 percent of citric acid and 10 percent of glucose are added for adjusting acid, 0.06 percent of edible essence is added for adjusting flavor, and after mixing, homogenization treatment is carried out, wherein the homogenization pressure is 40MPa, the homogenization time is 5min, and the product is obtained after homogenization. The percentage is the mass percentage of the raw materials in the fermentation liquor.
Experimental example 1 influence of addition amount of red dates and fermentation parameters on fermentation activity of probiotics
1. Influence of feed-water ratio on probiotic fermentation activity
The embodiment is divided into eight groups, lactobacillus plantarum TK9 and pediococcus acidilactici TK530 are respectively utilized to ferment in fermentation culture media with different material-water ratios, red dates and kudzu root powder are mixed according to the mass ratio of 6:10 in the fermentation culture media, and the total amount of the red dates and the kudzu root powder and water are respectively mixed according to the mass-volume ratios of 1:4, 1:5, 1:6 and 1:7(g/m L). The experimental steps are as follows:
(1) cleaning fructus Jujubae, removing core, and air drying
(2) Preparing an activation medium as an MRS medium; sterilizing at 121 deg.C for 20 min;
(3) preparing a fermentation medium: mixing the red dates and the radix puerariae powder according to the mass ratio of 6:10, and mixing the total amount of the red dates and the radix puerariae powder with water according to the mass-volume ratio.
(4) Activating strains, namely respectively inoculating 1% of glycerol tubes of lactobacillus plantarum TK9 and pediococcus acidilactici TK530 to an activation medium for activation, wherein the pH is 7.2-7.4, and the temperature is controlled to be 37 ℃; after respectively activating for 24 hours, transferring the strain to an activation culture medium again according to the strain inoculation amount of 4% (v/v) for secondary activation, and then activating for 24 hours to obtain a third-level seed solution;
(5) fermenting probiotics: inoculating lactobacillus plantarum TK9 according to the inoculation amount of 107CFU/m L was inoculated to the above different ratios (feed/water ratio: 1:4, 1:5, 1:6, 1:7g/m L) for fermentation cultureIn the culture medium, performing liquid fermentation for 18h at the temperature of 37 ℃; respectively detecting the number of live probiotics in the fermentation liquor;
the pediococcus acidilactici TK530 was inoculated in an amount of 107CFU/m L was inoculated into the fermentation media at different ratios (feed/water ratio: 1:4, 1:5, 1:6, 1:7g/m L) and liquid fermented at 37 ℃ for 24h, and the number of viable probiotic bacteria in the fermentation broth was determined, as shown in Table 1.
Strain number: no.1 is a single bacterium of Lactobacillus plantarum TK9, and No. 2 is a single bacterium of Pediococcus acidilactici TK 530.
TABLE 1 viable count at different feed/water ratios (CFU/m L)
| Different material-water ratios | 1：4 | 1：5 | 1：6 | 1：7 |
| 1 | 3.0×107 | 9.10×107 | 1.3×108 | 1.0×108 |
| 2 | 3.30×108 | 1.67×108 | 2.48×108 | 1.37×108 |
The results are shown in Table 1, wherein the highest viable count of 1.3 × 10 is 1.3 to 2 when the feed-water ratio is 1:6 and the viable count of g/m L is 1 to 68And 2.48 × 108CFU/mL。
2. Influence of fermentation temperature on probiotic fermentation activity
The present example is divided into 8 groups, and lactobacillus plantarum TK9 and pediococcus acidilactici TK530 are respectively utilized to ferment under different fermentation temperature conditions, wherein the fermentation temperatures are respectively as follows: 31. 34, 37 and 40 ℃.
The experimental procedure was as follows:
(1) cleaning fructus Jujubae, removing core, and air drying
(2) Preparing an activation medium: the experimental step (2) of the same experiment example 1, namely the influence of the feed-water ratio on the fermentation activity of the probiotics;
(3) preparing a fermentation culture medium, namely mixing the red dates and the radix puerariae powder according to a mass ratio of 6:10, and mixing the total amount of the red dates and the radix puerariae powder with water according to a mass-volume ratio of 1:6(g/m L).
(4) Activating strains: the experimental procedure (4) of "influence of feed-water ratio on probiotic fermentation activity" as in experimental example 1.
(5) Fermenting probiotics: inoculating lactobacillus plantarum TK9 according to the inoculation amount of 107Respectively inoculating CFU/m L into four groups of same fermentation culture media, and performing liquid fermentation for 18h at different fermentation temperatures, namely 31, 34, 37 and 40 ℃ respectively;
the pediococcus acidilactici TK530 was inoculated in an amount of 107CFU/m L was inoculated into four groups of the same fermentation media, respectively, and liquid fermentation was carried out for 24 hours at different fermentation temperatures, i.e., 31, 34, 37, and 40 ℃ respectively, and the number of viable probiotic bacteria in the fermentation broth was measured, respectively, with the results shown in Table 2.
Strain number: no.1 is a single bacterium of Lactobacillus plantarum TK9, and No. 2 is a single bacterium of Pediococcus acidilactici TK 530.
TABLE 2 viable count at different fermentation temperatures (CFU/m L)
| Different fermentation temperatures | 31℃ | 34℃ | 37℃ | 40℃ |
| Number 1 | 2.3×107 | 4.59×107 | 1.9×108 | 1.26×108 |
| Number 2 | 3.90×107 | 3.95×107 | 4.25×108 | 3.45×108 |
As shown in Table 2, the maximum viable cell count of 1.93 × 10 was measured for inoculated cells No.1 and No. 2 at a fermentation temperature of 37 deg.C8And 4.25 × 108CFU/mL。
3. Influence of inoculum size on probiotic fermentation activity
This example was divided into 8 groups and fermented with Lactobacillus plantarum TK9 and Pediococcus acidilactici TK530, respectively, at different inoculum sizes, 1 × 106、5×106、1×107And 5 × 107CFU/m L medium.
The experimental procedure was as follows:
(1) cleaning fructus Jujubae, removing core, and air drying
(2) Preparing an activation medium: the experimental step (2) of the same experiment example 1, namely the influence of the feed-water ratio on the fermentation activity of the probiotics;
(3) preparing a fermentation culture medium, namely mixing the red dates and the radix puerariae powder according to a mass ratio of 6:10, and mixing the total amount of the red dates and the radix puerariae powder with water according to a mass-volume ratio of 1:6(g/m L).
(4) Activating strains: the experimental procedure (4) of "influence of feed-water ratio on probiotic fermentation activity" as in experimental example 1.
(5) Fermenting probiotic, namely respectively inoculating lactobacillus plantarum TK9 according to different inoculation amounts of 1 × 106、5×106、1×107And 5 × 107CFU/m L is inoculated in five groups of same fermentation culture media, liquid state fermentation is carried out for 18 hours at the temperature of 37 ℃, and the number of live probiotics in the fermentation liquor is respectively detected;
simultaneously, the pediococcus acidilactici TK530 is respectively inoculated according to different inoculation amounts of 1 × 106、5×106、1×107And 5 × 107CFU/m L was inoculated into five groups of the same fermentation media, liquid fermentation was carried out at 37 ℃ for 24 hours, and the number of viable probiotic bacteria in the fermentation broth was measured, respectively, and the results are shown in Table 3.
Strain number: no.1 is a single bacterium of Lactobacillus plantarum TK9, and No. 2 is a single bacterium of Pediococcus acidilactici TK 530.
TABLE 3 viable count at different inoculum sizes (CFU/m L)
| Different inoculation amount | 1×106 | 5×106 | 1×107 | 5×107 |
| Number 1 | 1.03×107 | 1.94×108 | 2.58×108 | 2.62×108 |
| Number 2 | 9.7×107 | 1.18×108 | 1.38×108 | 1.64×108 |
The results are shown in Table 3, where the ratio of feed to water is 1:6, the fermentation temperature is 37 ℃, and the inoculation of No.1 and No. 2 is 5 × 107When the number of viable bacteria was measured, the highest number of viable bacteria was 2.62 × 108And 1.38 × 108CFU/mL。
4. Effect of culture time on probiotic fermentation Activity
This example was divided into 8 groups and fermented at different fermentation times of 12, 18, 24 and 30h using Lactobacillus plantarum TK9 and Pediococcus acidilactici TK530, respectively.
The experimental procedure was as follows:
(1) cleaning fructus Jujubae, removing core, and air drying;
(2) preparing an activation medium: the experimental step (2) of the same experiment example 1, namely the influence of the feed-water ratio on the fermentation activity of the probiotics;
(3) preparing a fermentation culture medium, namely mixing the red dates and the radix puerariae powder according to a mass ratio of 6:10, and mixing the total amount of the red dates and the radix puerariae powder with water according to a mass-volume ratio of 1:6(g/m L).
(4) Activating strains: the experimental procedure (4) of "influence of feed-water ratio on probiotic fermentation activity" as in experimental example 1.
(5) Fermenting probiotic, namely respectively inoculating lactobacillus plantarum TK9 according to the inoculation amount of 5 × 107Inoculating CFU/m L into four groups of same fermentation culture media, and performing liquid state fermentation at 37 deg.C for 12, 18, 24 and 30h respectively;
simultaneously, the lactic acid pediococcus TK530 was inoculated with 5 × 107CFU/m L was inoculated into four groups of the same fermentation media, and liquid fermentation was carried out at 37 ℃ for 12, 18, 24 and 30 hours, respectively, and the number of live probiotic bacteria in the fermentation broth was measured, respectively, and the results are shown in Table 4.
Strain number: no.1 is a single bacterium of Lactobacillus plantarum TK9, and No. 2 is a single bacterium of Pediococcus acidilactici TK 530.
TABLE 4 viable count at different fermentation times (CFU/m L)
| Different fermentation time | 12h | 18h | 24h | 30h |
| Number 1 | 1.39×108 | 2.63×108 | 1.85×108 | 1.45×108 |
| Number 2 | 1.64×108 | 1.80×108 | 2.48×108 | 2.44×108 |
The results are shown in Table 4: the optimal culture time of the liquid fermentation is 18h and 24h after inoculation.
Experimental example 2 liquid fermentation Process for jujube pulp beverage-Change of active ingredients in culture Medium before and after fermentation
In order to verify the influence of different strains on the components of the fermented beverage, lactobacillus plantarum TK9, pediococcus acidilactici TK530, lactobacillus plantarum CGMCC No.16021 and pediococcus acidilactici CGMCC No.16077 are respectively inoculated into a fermentation medium for fermentation, and the change of main effective components in the medium before and after fermentation is measured.
The grouping is as follows: the experiment was divided into five groups, with blank controls: a pre-fermentation medium set; the experimental groups were: lactobacillus plantarum TK9 fermentation group, pediococcus acidilactici TK530 fermentation group; the control group was: lactobacillus plantarum CGMCC No.16021 fermentation group and pediococcus acidilactici CGMCC No.16077 fermentation group.
And in the blank control group, the red dates and the radix puerariae powder are mixed according to the mass ratio of 6:10, and then the total amount of the red dates and the radix puerariae powder are mixed with water according to the mass-volume ratio of 1:6(g/m L).
The method for measuring the main effective components comprises the following steps: measuring flavonoids, radix Puerariae isoflavone and puerarin by high performance liquid chromatography, and measuring organic acid with reference to GB/T5009.157; the polyphenol content is determined by reference GB/T31740.2; the crude polysaccharide is determined by referring to national standard SN/T4260.
The fermentation steps of the strain are as follows:
(1) cleaning and pitting fructus Jujubae, soaking, and treating with wall breaking machine;
(2) sterilizing the fructus Jujubae pulp in a sterilizing pot at 80 deg.C for 30 min.
(3) Preparing an activation medium: 4.925gMRS was added per 100ml of water; sterilizing the activation culture medium at 121 deg.C for 20 min;
(4) preparing a fermentation culture medium, namely mixing the red dates and the radix puerariae powder according to a mass ratio of 6:10, and mixing the total amount of the red dates and the radix puerariae powder with water according to a mass-volume ratio of 1:6(g/m L).
(5) Activating strains: respectively inoculating 1% of glycerol tubes of single bacteria of Lactobacillus plantarum TK9, Pediococcus acidilactici TK530, Lactobacillus plantarum CGMCCNo.16021 and Pediococcus acidilactici CGMCC No.16077 into an activation culture medium for activation, wherein the pH is 7.2-7.4, and the activation temperature is 37 ℃; respectively activating for 24 hours, transferring an activation culture medium according to the inoculum size of 4% (v/v) for secondary activation, obtaining a secondary seed solution after 24 hours of activation, then transferring the activation culture medium according to the inoculum size of 1% (v/v) for tertiary activation, and respectively obtaining a tertiary seed solution of each bacterium after 24 hours of activation;
(6) fermenting probiotic, namely inoculating single lactobacillus plantarum TK9 and lactobacillus plantarum CGMCC No.16021 according to the inoculation amount of 5 × 107The CFU/m L is respectively inoculated in different groups of fermentation culture media and fermented for 18h at 37 ℃, and the obtained fermentation liquid is the products of a lactobacillus plantarum TK9 fermentation group and a lactobacillus plantarum CGMCC No.16021 fermentation group.
Separately inoculating 5 × 10 strains of single bacteria of Pediococcus acidilactici TK530 and Pediococcus acidilactici CGMCC No.160777CFU/m L was inoculated into different groups of fermentation medium, fermented at 37 ℃ for 24h, the obtained fermentation broth was the product of Pediococcus acidilactici TK530 fermentation group and Pediococcus acidilactici CGMCC No.16077 fermentation group, the change of the main effective components in the medium before and after fermentation is detailed in Table 5.
TABLE 5 variation of active ingredients before and after liquid fermentation of Zizyphi fructus
Note: the stages are denoted by numbers: 1-blank control group; 2-lactobacillus plantarum TK9 fermentation group; 3-pediococcus acidilactici TK530 fermentation group; 4-Lactobacillus plantarum CGMCC No.16021 fermentation group; 5-Pediococcus acidilactici CGMCC No.16077 fermentation group
As can be seen from the data in Table 5, through the fermentation of common lactobacillus and the fermentation of lactobacillus plantarum TK9 and pediococcus acidilactici TK530, the polyphenol content and the flavone content generated by the lactobacillus plantarum fermented jujube pulp are higher than those of the common lactobacillus, the polyphenol substances have higher inoxidizability, and the functions of removing free radicals of a human body, reducing blood pressure and the like are achieved. Generally, the protein content in the body of a patient with liver disease is abnormal and is often low, and the amino acid contained in the red dates has the effect of promoting the synthesis of protein and has the effects of tonifying spleen and nourishing liver. The functionality of the jujube pulp product can be further improved.
Experimental example 3: pediococcus acidilactici TK530 physiological characteristics
1. Bacteriostatic rate of supernate of pediococcus acidilactici TK530
The experimental method comprises the following steps:
(1) preparation of supernatant of pediococcus acidilactici TK 530: inoculating 100mml of bacteria with concentration of 10 to 10ml of MRS liquid culture medium7CFU/ml of Pediococcus acidilactici TK530, cultured at 37 ℃ for 24 h. Centrifuging the culture solution at 12000r/min for 10min, and evaporating and concentrating the supernatant to half of the original volume for later use.
(2) Experimental groups: respectively inoculating 10ml of RCM liquid culture medium with 100mml of bacteria concentration of 106CFU/ml of Escherichia coli, Salmonella, Staphylococcus aureus. Then 1ml of pediococcus acidilactici TK530 sterile supernatant was added.
(3) Control group: respectively inoculating 10ml of RCM liquid culture medium with 100mml of bacteria concentration of 106CFU/ml of Escherichia coli, Salmonella, Staphylococcus aureus. Then 1ml of sterile physiological saline was added.
(4) And (3) viable count detection: viable bacteria count was performed after 12h of culture in the experimental group and the control group.
(5) Calculating the bacteriostatic rate:
the results are shown in Table 6:
TABLE 6 bacteriostatic properties of fermentation supernatant of Pediococcus acidilactici TK530
| Bacterial strain | Escherichia coli | Salmonella | Staphylococcus aureus |
| Rate of inhibition of bacteria | 32.2％ | 37.6％ | 13.4％ |
As can be seen from Table 6, the bacteriostatic effect of pediococcus acidilactici TK530 on different pathogenic bacteria is that salmonella is larger than Escherichia coli > Staphylococcus aureus.
It can be seen that the pediococcus acidilactici TK530 of the invention has an especially significant bacteriostatic property for salmonella.
2. Pediococcus acidilactici TK530 antibacterial zone experiment
The experimental method comprises the following steps:
respectively activating Escherichia coli, Salmonella and Staphylococcus aureus with liquid L B culture medium, and gradient diluting the activated bacteria liquid with sterile physiological saline to concentrate to 108CFU/m L, observing the zone of inhibition by double-layer plate method, pouring sterilized water agar into plate, pouring 10ml, pouring 15ml of unset solid L B culture medium after agar is completely solidified, standing for 20min, respectively taking diluted indicator bacterium liquid 150 mu L after L B culture medium is completely solidified, uniformly coating the indicator bacterium liquid on the surface of L B solid culture medium, placing oxford cup, adding 200 mu L bacterium to 10 mu8Placing the suspension of the CFU/ml pediococcus acidilactici TK530 bacteria into a 37 ℃ incubator for cultureAnd (5) 24 h. And measuring the diameter of the inhibition zone by using a vernier caliper.
Wherein the solid L B culture medium comprises tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, and water in balance.
TABLE 7 bacteriostatic properties of fermentation supernatant of Pediococcus acidilactici TK530
| Bacterial strain | Escherichia coli | Salmonella | Staphylococcus aureus |
| Diameter mm of bacteriostatic zone | 28.5 | 34.2 | 24.6 |
In the invention, the pediococcus acidilactici TK530 has remarkable bacteriostatic performance, and the red date fermented beverage obtained by fermenting the pediococcus acidilactici TK530 not only can normally play the functions of dispelling the effects of alcohol and protecting the liver, but also can effectively improve the gastrointestinal environment.
3. Detection of acid and bile salt resistance
Acid-resistant experiment, adjusting pH of MRS liquid culture medium to 2.0, 3.0, 4.0 with HCI with concentration of 0.1 mol/L, sterilizing at 121 deg.C for 20min, cooling, inoculating Lactobacillus paracasei suspension under aseptic condition to maintain initial viable count of culture medium at 1 × 108CFU/m L, culturing at 37 deg.C for 1h, 2h, and 3h, and sampling to count viable count.
Bile salt resistance test: adjusting bile salt of MRS liquid culture medium by using pig bile saltThe content and the addition amount are respectively 0.15 percent, 0.3 percent and 0.45 percent, the lactobacillus paracasei suspension is inoculated under the aseptic condition after the sterilization at the temperature of 121 ℃ for 20min, and the initial viable count in the culture medium is kept at 1 × 108CFU/m L, culturing at 37 deg.C for 1h, 2h, and 3h, and sampling to count viable count.
TABLE 8 acid-resistant detection results of Pediococcus acidilactici TK530
The detection result shows that the survival rate is 98.8% after the treatment for 3h in the environment of pH 2, the survival rate is 124.7% after the treatment for 3h in the environment of pH 3, and the pediococcus acidilactici TK530 can normally grow in the environment of pH 4. The result shows that the pediococcus acidilactici TK530 has strong acid resistance and can resist the acid environment in the intestinal tract. Therefore, the strain can keep better strain activity in an acidic gastrointestinal tract environment, and simultaneously exert better harmful bacterium inhibition performance of the strain.
TABLE 9 Pediococcus acidilactici TK530 bile salt resistance test results
The detection result shows that the survival rate of the cells after being treated for 3 hours in the environment with 0.15 percent of the cholate content is 122.4 percent, the survival rate of the cells after being treated for 3 hours in the environment with 0.3 percent of the cholate content is 98.8 percent, and the survival rate of the cells after being treated for 3 hours in the environment with 0.45 percent of the cholate content is 87.0 percent. The result shows that the pediococcus acidilactici TK530 has certain bile salt resistance. Therefore, the strain can keep better strain activity in intestinal environments containing bile salts, and simultaneously, the strain can better inhibit harmful bacteria.
Experimental example 4: animal experiment research on protective effect of red date fermented liquid beverage on alcoholic liver injury
Dividing the experiment into 10 groups, namely 6 groups including an unfermented jujube pulp group and a product group, a model group, a blank control group and a positive control group, wherein:
the unfermented fructus Jujubae pulp comprises 100m L water, 6g fructus Jujubae, 10g radix Puerariae powder;
product group:
product group 1: lactobacillus plantarum TK9 live bacterial powder;
product group 2: lactobacillus plantarum TK9 fermented jujube pulp, obtained by preparation of example 1, steps (1) - (7);
product group 3: lactobacillus plantarum CGMCC No.16021 viable bacteria powder;
product group 4: lactobacillus plantarum CGMCC No.16021 fermented jujube pulp;
product group 5: pediococcus acidilactici TK530 fermented jujube pulp obtained by the preparation of the steps (1) to (7) of example 2;
product group 6: pediococcus acidilactici CGMCC No.16077 fermented jujube pulp;
wherein product group 1: the preparation method of the live lactobacillus plantarum TK9 comprises the following steps:
(1) preparing an activation medium: same as example 1, step (3);
(2) preparing a fermentation medium: the composition and the method are configured with an activation culture medium;
(3) activating strains: same as example 1, step (5);
(4) fermenting probiotics: performing centrifugation on the fermentation liquor at 8000rpm for 10min to obtain thallus, and freeze-drying to obtain thallus powder in the same step (6) as in example 1;
wherein product group 3: the preparation method of the lactobacillus plantarum CGMCC No.16021 viable bacteria powder is the same as the preparation method of the lactobacillus plantarum TK9 viable bacteria powder, and the only difference is that the strain is the lactobacillus plantarum CGMCC No. 16021.
Wherein product group 4: the preparation steps of the lactobacillus plantarum CGMCC No.16021 fermented jujube pulp are the same as the preparation steps (1) - (7) in the example 1, and the only difference is that the strain is the lactobacillus plantarum CGMCC No. 16021.
Wherein product group 6: the preparation steps of the pediococcus acidilactici CGMCC No.16077 fermented jujube pulp are the same as the preparation steps (1) - (6) in the embodiment 2, and the only difference is that the strain is pediococcus acidilactici CGMCC No. 16077.
A positive control group, namely a product prepared according to the steps (1) - (5) in the embodiment 1 of the Chinese invention patent CN 110521784A, wherein the proportion of the soybean powder, the turmeric powder and the hovenia dulcis thunb powder is 60: 25: 15, the optimal material-water ratio is 1: 1.9, the inoculation ratio of lactobacillus plantarum single bacteria, bifidobacterium animalis single bacteria, lactobacillus paracasei single bacteria, bacillus coagulans single bacteria, pediococcus acidilactici single bacteria and bacillus subtilis single bacteria is 1: 0.1: 10, and the inoculation amount of the bacillus coagulans is 5 × 107cfu/g, the time of each phase of the probiotic fermentation is 24 hours, 30 hours, 20 hours and 14 hours, the product obtained by fermentation is put into a vacuum freeze dryer for freeze-drying for 20 hours, and the probiotic fermented functional food for alleviating hangover and nourishing liver is prepared, the bacterial activity of the product is 1.73 × 1010The bacterial viability of the CFU/g product after being diluted by normal saline is 2.68 × 108CFU/mL。
Model group: the 53-degree white spirit (Beijing Hongxing Erguotou) is sold in the market;
the blank and model groups were given an equal amount of 0.9% saline and the gavage dose was 10ml/kg d (1 time per day).
The experimental steps are as follows:
determination of optimal mouse wine-filling dosage
SPF-level Kunming male mice are randomly divided into 5 groups, 10 mice are fed in each group, the mice are adaptively fed for 5d, the mice are marked and weighed, the mice are fasted for 12h without water supply, the mice are subjected to intragastric gavage according to the wine supply of 6ml/kg, 8ml/kg, 10ml/kg, 12ml/kg and 12.5ml/kg respectively, the drunk state of the mice is observed, and the number of non-drunk mice and the number of dead mice are recorded. The following experiment was performed with the selection of a low alcohol dose for both non-intoxication and mortality.
TABLE 10 Effect of different alcohol doses on mice
The drunkenness rate and the death rate of the mice after being infused with different doses are shown in a table 10. the drunkenness rate of the mice is increased along with the increase of the drunkenness dose, when the drunkenness dose is 10.00m L/kg, the drunkenness rate reaches 100 percent, and the death rate of the mice is gradually increased along with the continuous increase of the drunkenness dose, therefore, the drunkenness rate of the mice can reach the highest drunkenness rate by the drunkenness dose of 10.00m L/kg, and the death rate after being drunk is lower, so the dose is selected as the optimal drunkenness dose.
(2) The invention comprises the following specific experimental steps
1. Experimental grouping and processing method
Healthy male SPF-level Kunming mice are randomly divided into 10 groups, 10 mice in each group are subjected to intragastric administration by an intragastric administration needle (detailed in Table 11) at eight points every morning, the intragastric administration is continuously carried out for 30d, after the corresponding reagents are filled for 4h for the last time, 0.9% physiological saline 0.1m L/10g is filled in a blank control group, 0.1ml/10g of 53-degree white spirit (Beijing Hongxing Erguotou) is filled in other groups, after fasting is carried out for 12h before sacrifice, blood is collected and serum is separated, and livers are weighed and put in a-80 refrigerator for storage.
TABLE 11 model grouping and processing method
(1) Observation of body weight and general morphology.
Calculating the liver index: liver index (liver quality/mouse body mass)
TABLE 12 influence of jujube pulp on mouse body Mass and liver coefficients
| Group of | Initial body mass/g | Final body mass/g | Liver coefficient/(g/100 g) |
| Blank control group | 20.74±1.28 | 37.68±2.29 | 3.68±0.15 |
| Model set | 27.74±4.29 | 39.04±2.91 | 4.12±0.23 |
| Positive control group | 26.05±3.34 | 34.52±3.75 | 3.38±0.15 |
| Product group 1 | 25.47±1.15 | 36.57±2.14 | 3.45±0.04 |
| Product group 2 | 28.04±1.65 | 36.87±2.49 | 3.23±0.12 |
| Product group 3 | 25.38±2.34 | 32.65±1.17 | 3.78±1.22 |
| Product group 4 | 26.09±1.52 | 35.5±2.99 | 3.54±0.25 |
| Product group 5 | 25.27±2.56 | 37.17±2.31 | 3.41±0.24 |
| Product group 6 | 26.28±1.27 | 34.08±1.87 | 3.52±0.32 |
| Unfermented jujube pulp group | 29.16±1.71 | 35.96±3.47 | 4.08±0.23 |
The liver index is the ratio of the liver weight to the body weight of an animal and is a common index in toxicology, and the larger the liver index is, the more serious the condition that the liver of an organism is swollen is. As shown in table 12, the liver index of the model group was higher than that of the normal group, indicating that acute intoxication caused the mice to develop hepatomegaly and liver tissue inflammation. The liver indexes of the positive control group, the unfermented group and the experimental groups 1, 2, 3, 4, 5 and 6 are lower than those of the model, the experimental group 2 has better effect than the experimental groups 1, 3, 4, 5 and 6, wherein the experimental group 2 shows that the fermented jujube pulp and the positive control group can effectively inhibit the inflammation of the liver tissue after drinking.
(2) Influence of anti-alcohol and liver-protecting composition on contents of Triglyceride (TG) and Total Cholesterol (TC) in serum of mouse
TABLE 13 Effect of jujube pulp on mouse triglyceride TG and Total Cholesterol TC levels
Alcoholic fatty liver has a close relationship with blood fat. Excessive alcohol intake can lead to accumulation of liver lipids, and if the liver develops steatosis, the blood lipid concentration will increase. The increased serum TG, TC concentrations in the model group compared to the normal group indicate that alcohol intake may cause cholesterol accumulation and affect lipid metabolism. The positive control group, the unfermented group and the experimental group 1, 2, 3, 4, 5, 6 had decreased serum TG, TC concentrations compared to the model group, indicating that it can improve fat accumulation. The test group 2 and the positive control group showed more remarkable effects than the other test groups 1, 3, 4, 5 and 6, and could reduce the TG and TC concentrations.
(3) Influence of hangover alleviating and liver protecting composition on contents of glutamic-pyruvic transaminase AST and glutamic-oxalacetic transaminase A L T in mice
TABLE 14 Effect on the levels of alanine AST and aspartate aminotransferase A L T in mice
The A L T, AST activity is an important index for reflecting hepatocyte injury, and the activity of hepatocytes is obviously improved when the hepatocytes are severely damaged, and Table 14 shows that the A L T, AST activity of a normal group is improved compared with that of a model group, which indicates that an alcoholic liver injury model is successfully established.
(4) Pathological section observation of liver tissue of mice of each model group
Referring to fig. 1, pathological sections of each group of model mice show that the cells of the normal group of mice are regularly and orderly arranged and have no cell necrosis; the mouse liver cells in the model group are disorderly arranged, the intervals are enlarged and swollen, the liver cells are moderately edematous, and individual liver cells are necrotized in a punctate manner; the positive group has complete liver tissue structure and partial hepatic cell microvesicle steatosis; product 1 cells had slight edema; the cell shape of the product 2 is relatively complete; product 3 cell swelling with edema; the product 4 has fat deformation of cells and fuzzy cell boundary; the product 5 has individual punctate necrosis of cells; product 6 cytoma microvesicle steatosis; the micro-cellular steatosis of the liver cells of the liver tissue part of the unfermented group is caused, and the intercellular gaps are fuzzy; the pathological analysis result shows that the fermented jujube pulp has better effect than the unfermented jujube pulp, and the lactobacillus plantarum TK9 fermented jujube pulp has better effect of preventing alcoholic liver injury than the common lactobacillus fermented jujube pulp.
The beneficial components in the probiotic liquid state fermented beverage mainly comprise puerarin, pueraria isoflavone, polysaccharide, flavonoid and triterpenes, and the red dates comprise 18 amino acids, so that the puerarin, the polysaccharide, the flavonoid and the triterpenes have the effects of promoting protein, invigorating spleen and nourishing liver. The triterpene compounds in fructus Jujubae have liver protecting effect, and puerarin and radix Puerariae isoflavone contained in radix Puerariae have effects of regulating cardiovascular system, relieving hangover and protecting liver. Improve the phagocytic capacity of the mononuclear phagocyte system in vivo to a certain extent. Has good curative effect on enhancing the immunity of the organism, protecting the liver and the like.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the appended claims.
Claims (5)
1. A functional fermented beverage for relieving alcoholism and protecting liver is characterized in that: the functional fermented beverage for relieving alcoholism and protecting liver comprises the following raw materials: the mass ratio of the red dates to the kudzu root powder is (5-10): (10-15) mixing, wherein the mass-volume ratio of the total amount of the red dates and the kudzu root powder to water is as follows: 1: (4-8); the probiotics used for fermentation of the functional fermented beverage for dispelling the effects of alcohol and protecting the liver is one of lactobacillus plantarum or pediococcus acidilactici.
2. The functional fermented beverage for relieving alcoholism and protecting liver according to claim 1, wherein the Lactobacillus plantarum is Lactobacillus plantarum (L actinobacillus plantarum) TK9 with a preservation number of CGMCC No. 11891.
3. The functional fermented beverage for relieving alcoholism and protecting liver according to claim 1, wherein the Pediococcus acidilactici is Pediococcus acidilactici (L actinobacillus plantarum) TK530 with preservation number of CGMCC No. 18737.
4. A method for preparing the functional fermented beverage for relieving alcoholism and protecting liver as claimed in any one of claims 1-3, wherein the fermented beverage is characterized in that: the method comprises the following steps:
(1) mixing the red dates, the kudzu root powder and water according to a proportion, and sterilizing to obtain a fermentation medium;
(2) the probiotic bacteria were activated and inoculated in an amount of 106-108CFU/m L, inoculating into the fermentation medium, standing and fermenting at 31-40 deg.C for 12-36 h;
(3) and after the fermentation is finished, adding a stabilizer into the fermentation liquor, and carrying out acid adjustment, flavor adjustment and homogenization to obtain the product.
5. The method for preparing the functional fermented beverage for alleviating hangover and protecting liver according to claim 4, wherein the fermented beverage comprises: in the step (2), the probiotic activation step is as follows: inoculating 1% of glycerol tubes of the probiotics into an activation culture medium for activation, activating at the temperature of 37 ℃ for 24h at the pH of 7.2-7.4 to obtain a first-stage seed solution, inoculating 4-10% of the first-stage seed solution into the activation culture medium for secondary activation for 24h to obtain a second-stage seed solution, inoculating 1-3% (v/v) of the second-stage seed solution into the activation culture medium for three times of activation, and activating for 24h to obtain a third-stage seed solution; the activation culture medium is MRS culture medium.
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