CN117099850B

CN117099850B - Fermented plant-based yoghurt and preparation method and application thereof

Info

Publication number: CN117099850B
Application number: CN202311380408.1A
Authority: CN
Inventors: 王家明; 赵春然; 郑加栋; 李敬华; 张红梅
Original assignee: Xinyi Tianjin Biotechnology Co ltd
Current assignee: Xinyi Tianjin Biotechnology Co ltd
Priority date: 2023-10-24
Filing date: 2023-10-24
Publication date: 2024-03-12
Anticipated expiration: 2043-10-24
Also published as: CN117099850A

Abstract

The invention belongs to the technical field of microorganisms, and discloses application of lactobacillus plantarum in the aspects of simultaneously dispelling the effects of alcohol and protecting liver and reducing uric acid and a plant-based yoghourt with the functions of dispelling the effects of alcohol and protecting liver and reducing uric acid, wherein the plant-based yoghourt is obtained by fermenting lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 and lactobacillus lactis CICC 6242. The plant-based yoghourt can relieve the effects of alcohol and protect the liver and simultaneously reduce uric acid, has no chemical additive, has the effects of relieving the effects of alcohol and protecting the liver and reducing uric acid, is stable and good, has unique flavor and excellent mouthfeel, has no bitter and astringent mouthfeel, is easy to obtain raw materials, is low in cost and is easy to realize industrialization.

Description

Fermented plant-based yoghurt and preparation method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and in particular relates to fermented plant-based yoghurt capable of dispelling the effects of alcohol, protecting the liver and reducing uric acid, a preparation method and application thereof.

Background

A mechanism for dispelling the effects of alcohol and protecting liver. The essence of the anti-alcohol is that alcohol dehydrogenase (Alcohol Dehydrogenase, ADH) oxidizes ethanol to acetaldehyde, which is then converted from acetaldehyde dehydrogenase (ALDH) to acetic acid, which enters the blood circulation and is metabolized to carbon dioxide, water and energy. Therefore, the anti-alcoholic mechanism is to activate ADH and ALDH production in vivo, increase the activities of ADH and ALDH, accelerate the oxidative decomposition of ethanol in vivo, reduce the concentration of ethanol and acetaldehyde in vivo, shorten the drunk time, relieve the inadaptation of people caused by drunk, and reduce the induction rate of various diseases caused by acetaldehyde poisoning. Excessive alcohol can cause liver damage, and glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) are important indicators for evaluating liver damage, and when liver cell membrane permeability is increased or liver cells are necrotized, ALT and AST levels can be increased.

The existing anti-alcoholic medicines comprise metadoxine, naloxone and the like, and have a relatively large number of adverse reactions although having an anti-alcoholic effect. For example, metadoxine is an activator of acetaldehyde dehydrogenase, which antagonizes the decline of alcohol dehydrogenase activity caused by acute and chronic alcoholism and accelerates the excretion of alcohol and its metabolites acetaldehyde and ketone bodies through urine, but metadoxine may cause peripheral nerve diseases. Naloxone is an antidote for opioid overdose and can also treat alcoholism, but can cause side effects such as mania. The patent publication CN 107455476A discloses a taste-changing sobering-up walnut milk beverage and a processing method thereof, wherein the sobering-up function is realized by promoting the metabolism and decomposition of alcohol of a human body through substances in miracle fruit extract, and the total protein content in the walnut milk protein beverage is up to 0.69mL/100mL, so that the sobering-up effect is good.

The plant polypeptide has liver protecting effect. The study on the gastric mucosa protection and anti-alcohol refreshment and liver protection effects of walnut peptide on acute alcoholism mice (in 2023 of traditional Chinese medicine report) has reported that the walnut peptide can improve the antioxidant capacity of the liver of AAI model mice, lighten gastric mucosa injury and strengthen the memory of mice, has the effects of gastric mucosa protection and anti-alcohol refreshment and liver protection, and the walnut used in the invention is common walnut peptide. SUZUKI Y et al have found that wheat gluten hydrolysate can inhibit hepatic fibrosis in rat liver model induced by carbon tetrachloride, and the hydrolysate has liver protecting and pathological changes preventing effects (Biomedical Research,2011,22 (1): 481-488), and wheat germ is used in the invention.

Hyperuricemia refers to metabolic diseases caused by purine metabolic disturbance and Uric Acid (UA) levels higher than normal. In 2021, the medical guideline of combining hyperuricemia and gout symptoms is provided that the fasting blood uric acid concentration of two times in a normal purine diet state is more than 420 mu mol/L, namely the hyperuricemia is considered. Hyperuricemia occurs mainly due to increased uric acid or decreased uric acid excretion in humans, about 2/3 of the urate excreted from the kidneys, and 1/3 of the urate excreted from the intestinal tract, and the micro-ecosystem constituted by the intestinal flora has been considered as a new target for the treatment of hyperuricemia. At present, the treatment of hyperuricemia mainly depends on drug treatment, such as allopurinol, febuxostat, tribromone, probenecid and the like, but the toxic and side effects of kidneys, gastrointestinal tracts and the like are large, so that the research of intestinal flora has important significance.

Intestinal flora is associated with hyperuricemia. The rising blood uric acid can reversely induce the chronic inflammation of the organism and the intestinal canal and change the environment in the intestinal canal, thereby affecting the distribution of the flora and the change of the quantity and aggravating the hyperuricemia symptom of the organism. Shao et al analyzed 26 stool samples from patients with gout and healthy people by nuclear magnetic resonance hydrogen spectrometry and high throughput sequencing technology, found that the diversity of stool flora of patients with gout was significantly reduced, while the abundance of pathogenic bacterial flora of Bacteroides, rhodomonas and anaerobic rope bacteriaceae was significantly increased. In addition, as early as 1952, buzard et al have first shown by in vitro bacteriostasis experiments that intestinal bacteria have a decomposing effect on uric acid.

In the activity research of lactobacillus plantarum ZXH-1304S for degrading creatinine and uric acid (in 2019 of food industry technology), the content of creatinine and uric acid in a Wistar rat can be remarkably reduced. Chinese patent publication No.: CN115287240a, publication date: the lactobacillus plantarum with hyperuricemia and gout preventing and treating effects and application thereof are disclosed in 11.2022 and 4.A long-term gastric lavage of a hyperuricemia model mouse is mainly adopted, and the lactobacillus plantarum LTJ48 can obviously reduce serum uric acid level, has obvious uric acid reducing effect and has kidney protecting effect on the hyperuricemia model mouse. Chinese patent publication No.: CN115287239a, publication date: the invention discloses lactobacillus plantarum with the capability of degrading nucleoside, purine and uric acid in vitro and application thereof, and discloses lactobacillus plantarum LTJ1 with the capability of degrading nucleoside and purine in vitro and capable of reducing uric acid level at the cellular level, wherein the lactobacillus plantarum LTJ1 has the capability of degrading nucleoside and purine in vitro and can be used for reducing uric acid level at the 2022 and 11-4-month. There is growing evidence that the intestinal flora is not only associated with the occurrence of hyperuricemia but also with the therapeutic effects of hyperuricemia.

The plant-based yoghurt is a dairy product prepared by taking plants as raw materials and fermenting probiotics. The plant-based yoghurt can provide high-quality plant protein for human bodies, and simultaneously, has extremely low cholesterol and trans-fatty acid. Is an excellent office hunger-resistant snack for people with cow milk protein allergy and vegetarian food.

At present, various lactobacillus plantarum is mainly used for in vitro degradation of nucleotide uric acid or measurement of serum creatinine and uric acid level, and verification measurement of uric acid and creatinine level in urine is not available. Lactobacillus plantarum has few reports of functions of dispelling effects of alcohol and protecting liver while reducing uric acid. In the case of daily drinking, the drunk, liver injury and uric acid are often present at the same time, and for this reason, we have carried out the research.

The invention mainly uses mixed strains, namely lactobacillus plantarum OPB15, pediococcus acidilactici and lactobacillus acidophilus, ferments the plant yoghourt, and the fermented plant yoghourt has the functions of dispelling the effects of alcohol and protecting the liver and reducing uric acid.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide fermented plant-based yoghourt capable of dispelling the effects of alcohol, protecting the liver and reducing uric acid, and a preparation method and application thereof.

The technical scheme adopted for solving the technical problems is as follows:

an application of lactobacillus plantarum (Lactobacillus plantarum) in the aspects of simultaneously dispelling the effects of alcohol, protecting liver and reducing uric acid, wherein the lactobacillus plantarum is lactobacillus plantarum OPB15, and the name of the lactobacillus plantarum is: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center (CGMCC) of the China Committee for culture Collection of microorganisms, beijing, chaoyang area, north Chenxi Lu No. 1, 3.

Further, the application is the application in preparing plant-based yoghurt for dispelling the effects of alcohol, protecting liver and reducing uric acid.

Further, the plant-based yogurt has at least one of the following effects:

the thickening of the colonic myolayer reduces the damage of submucosa tissues, the arrangement of intestinal glands is tidy, the tissue morphology is improved, and the intestinal environment is improved;

or has the effects of dispelling effects of alcohol, protecting liver and reducing uric acid;

alternatively, the effect of increasing Alcohol Dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) levels;

alternatively, the effect of reducing the levels of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST);

alternatively, the effect of lowering Uric Acid (UA), creatinine (Cr) and urea nitrogen (BUN) levels.

Further, the plant-based yogurt includes a plant fermented beverage, a metazoan, or an enzyme.

A plant-based yogurt having functions of alleviating hangover, protecting liver, and reducing uric acid at the same time, which is obtained by fermenting lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 (lactobacillus helveticus OPB102 has been described and disclosed in chinese patent publication CN 116790448A), and lactococcus lactis cic 6242 (lactococcus lactis cic 6242 is purchased from the chinese industrial microorganism strain collection management center);

Wherein the name of the lactobacillus plantarum OPB15 is as follows: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center (CGMCC), north Chenxi Lu No. 1, 3, the Korean area of Beijing, and the China general microbiological culture Collection center;

the thallus is characterized in that: gram-positive rod-shaped bacteria, non-spore-forming, non-motile bacteria; colony characteristics: the diameter is about 1.0-1.2 mm, the front surface is round, the center is convex, the edge is neat, the surface is moist and smooth, the surface is opaque and has slightly sour taste; growth characteristics: culturing in MRS culture medium at 37deg.C for 12 hr to end of logarithm; has strong tolerance to simulated gastrointestinal fluid.

A method of preparing a plant-based yoghurt as described above, the method comprising the steps of:

(1) Adding Aronia melanocarpa freeze-dried powder, water-soluble extract and water-insoluble extract, homogenizing the mixture under high pressure for 10 minutes at 3500psi, and then performing instantaneous sterilization at 120-130 ℃ for 1-5 seconds;

(2) Inoculating Lactobacillus plantarum OPB15, lactobacillus helveticus OPB102, and lactococcus lactis CICC 6242, wherein the activity is (1.0-1.8) x 10 respectively ⁸ cfu/mL，(1.0-2.0)×10 ⁸ cfu/mL，(0.9-1.5)×10 ⁷ cfu/mL, the inoculation amount is 3% (calculated by volume), fermentation is carried out after inoculation, the fermentation temperature is 38-42 ℃, and the fermentation time is 24-36 h;

(3) Adding active polypeptide, stirring the mixture uniformly, continuously stirring for 25 minutes, filling, and refrigerating the filled yoghourt for 12 hours at the temperature of 2-6 ℃ to obtain plant-based yoghourt;

wherein, the Aronia melanocarpa freeze-dried powder: water-soluble extract: water insoluble extract: the weight ratio of the active polypeptide is 40-50: 20-30: 10-20: 80 to 100. Preferably, the active polypeptide: aronia melanocarpa freeze-dried powder: water-soluble extract: the mass ratio of the water-insoluble extract is 90:45:25:15.

further, the preparation method of the Aronia melanocarpa freeze-dried powder in the step (1) comprises the following steps:

(1) Selecting fresh or frozen Aronia melanocarpa fruits with soft skin, soft pulp, no mildew and no mechanical damage;

(2) Scalding fresh or frozen Aronia melanocarpa fruits in the step (1) in boiling water for 30-60 s, immediately immersing in cold water for cooling after fishing out, and crushing to obtain Aronia melanocarpa fruit puree;

(3) Adding complex enzyme (one or two of tannase, pectase and protease) into Aronia melanocarpa fruit pulp, inactivating enzyme, and filtering to obtain pulp;

(4) Adding lactobacillus plantarum OPB15 into the pulp for fermentation at 37 ℃ for 36 hours, and filtering to obtain a fermentation product;

(5) Ultrasonically extracting the fermentation product with water for 60-90 min each time to obtain a Aronia melanocarpa fermentation extract;

(6) Filtering the Aronia melanocarpa fermentation extract to obtain filtrate, and vacuum freeze-drying at-80deg.C to obtain Aronia melanocarpa freeze-dried powder.

Further, the preparation method of the water-soluble extract and the water-insoluble extract in the step (1) comprises the following steps:

(1) Pretreatment of raw materials: 40-50 parts of hawthorn, 40-50 parts of celery seed, 30-40 parts of kudzuvine root, 20-30 parts of garlic, 20-30 parts of seaweed, 20-30 parts of American ginseng, 20-30 parts of black matrimony vine, 5-25 parts of golden aster and 5-10 parts of eucommia male pollen, mixing, crushing to obtain traditional Chinese medicine powder, and sieving for later use;

(2) Soaking and decocting: the Chinese medicinal powder is prepared from the following components in percentage by mass: mL is 1: adding water into 6-8, mixing, decocting at 60-80deg.C for 2-4 hr, and filtering to obtain Chinese medicinal decoction and residue;

(3) Aqueous extracts of Chinese medicinal materials: pre-freezing the Chinese medicinal decoction in the step (2) at-20 ℃ and-80 ℃, then putting the pre-frozen Chinese medicinal decoction into a freeze dryer, and freeze-drying the pre-frozen Chinese medicinal decoction at-80 ℃ for 12 hours to obtain water-soluble freeze-dried powder which is a water-soluble extract;

(4) Insoluble extract of Chinese medicinal liquid: drying the residues obtained in the step (2), crushing, sieving to obtain powder, adding 10 times of ethanol, heating and reflux-extracting at 80-85 ℃ for 2h, cooling to room temperature, and vacuum-filtering; heating the filtrate at 55-70deg.C, vacuumizing, rotary evaporating to obtain concentrated solution, pre-freezing at-20deg.C and-80deg.C, lyophilizing at-80deg.C for 12 hr to obtain lyophilized powder of water insoluble substance, which is water insoluble extract.

Further, the preparation method of the active polypeptide in the step (3) comprises the following steps:

taking 80-100 parts by weight of hickory, 30-50 parts by weight of wheat germ, 15-20 parts by weight of black rice and 10-20 parts by weight of fructus cannabis, crushing, adding water, adjusting the pH value to 8.5-9.0, extracting protein by an alkali-dissolution acid precipitation method, freeze-drying the protein, carrying out enzymolysis by protease, centrifuging at 8000-12000rpm for 10-20 minutes, taking supernatant, and freeze-drying to obtain active polypeptide.

The application of the plant-based yoghurt in serving as and/or preparing health-care food for preventing and/or treating the effects of dispelling the effects of alcohol and protecting the liver and reducing uric acid.

The invention has the advantages and positive effects that:

1. the plant-based yoghourt has the effects of dispelling the effects of alcohol, protecting the liver and reducing uric acid. Can change the shape of colon tissue, thicken colon muscle layer, reduce tissue injury under mucous membrane, arrange intestinal gland neatly, improve intestinal environment, and has effects of relieving alcoholic intoxication and protecting liver, and reducing uric acid; can also increase the level of Alcohol Dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) of acute drunk mice model, and reduce the level of glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST); and can also reduce Uric Acid (UA), creatinine (Cr) and urea nitrogen (BUN) levels in serum of hyperuricemia mice.

2. The polysaccharide of the Aronia melanocarpa extract is firstly applied to the plant-based yoghurt, so that the polysaccharide can be used as a prebiotic, promote the propagation of probiotics, improve the intestinal microenvironment, and simultaneously carry out deep fermentation to strengthen the conversion of active ingredients, thereby achieving the effects of dispelling the effects of alcohol, protecting the liver and reducing uric acid. Meanwhile, the hickory, wheat germ, black rice and fructus cannabis are used for preparing the active polypeptide, so that the active polypeptide has a good effect of protecting the liver of an acute drunk mouse. The raw materials are cheap and easy to obtain, and industrialization is easy to realize, so that the problems of liver injury, uric acid increase and the like caused by excessive drinking at present are solved.

3. The invention is prepared by extracting hawthorn, celery seed, kudzuvine root, garlic, seaweed, american ginseng, black matrimony vine, golden silk aster and eucommia male pollen together, separating and extracting water-soluble extract and water-insoluble extract, and better matching according to the weight part ratio. The extraction process can effectively extract bioactive components in the raw materials, and simultaneously the probiotics fermentation can hydrolyze macromolecular substances in the raw materials into small molecules which are easy to absorb and utilize, so that the raw material utilization rate is improved, and the effects of dispelling the effects of alcohol, protecting the liver and reducing uric acid are remarkable.

4. The invention adopts lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 and lactococcus lactis CICC 6242 for mixed fermentation, and the probiotics not only can promote the conversion of active ingredients to small molecules, but also can produce lactic acid, acetic acid and the like, can easily produce esters with ethanol, and reduces the ethanol content of blood; meanwhile, the Chinese herbal medicine can be propagated in a large quantity, promote gastrointestinal peristalsis, increase metabolism and promote further excretion of ethanol.

5. The plant-based yoghourt can relieve the effects of alcohol and protect the liver and simultaneously reduce uric acid, has no chemical additive, has the effects of relieving the effects of alcohol and protecting the liver and reducing uric acid, is stable and good, has unique flavor and excellent mouthfeel, has no bitter and astringent mouthfeel, is easy to obtain raw materials, is low in cost and is easy to realize industrialization.

Drawings

FIG. 1 is a colony morphology of Lactobacillus plantarum OPB15 according to the invention;

FIG. 2 is a microscopic photograph of Lactobacillus plantarum OPB15 according to the invention;

FIG. 3 is a graph of BLAST results versus tree for the Lactobacillus plantarum OPB15 genomic sequence of the invention;

FIG. 4 is a graph showing the histological characteristics of Lactobacillus plantarum OPB15 against the colon of an acute drunk mouse according to the invention; h & E staining (400 x magnification);

FIG. 5 is a graph showing the histological characteristics of Lactobacillus plantarum OPB15 against the colon of an acute drunk mouse according to the invention; AB-PAS dyeing (magnification 400 times)

An application of lactobacillus plantarum (Lactobacillus plantarum) in the aspects of simultaneously dispelling the effects of alcohol, protecting liver and reducing uric acid, wherein the lactobacillus plantarum is lactobacillus plantarum OPB15, and the name of the lactobacillus plantarum is: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.

Detailed Description

The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.

The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.

Preferably, the application is the application in preparing plant-based yoghurt for dispelling the effects of alcohol, protecting liver and reducing uric acid.

Preferably, the plant-based yoghurt has at least one of the following effects:

Preferably, the plant-based yoghurt comprises a plant fermented beverage, a metazoan or an ferment.

A plant-based yoghurt with functions of dispelling effects of alcohol, protecting liver and reducing uric acid, which is obtained by fermenting lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 and lactococcus lactis CICC 6242;

wherein the name of the lactobacillus plantarum OPB15 is as follows: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3 of the Korean area of Beijing, the China general microbiological culture Collection center;

preferably, the preparation method of the Aronia melanocarpa freeze-dried powder in the step (1) comprises the following steps:

Preferably, the preparation method of the water-soluble extract and the water-insoluble extract in the step (1) comprises the following steps:

Preferably, the method for preparing the active polypeptide in the step (3) comprises the following steps:

Specifically, the related preparation and detection are as follows:

example 1: screening of Lactobacillus plantarum OPB15

Isolation and screening of Lactobacillus

(1) 0.1mL of yogurt sample (from the automatic brewing yogurt in the home of the herdsman in Kaisha, sinkiang) was inoculated into skim milk medium, and incubated in a constant temperature incubator at 37℃for 48 hours. A small amount of bacterial liquid is picked by an inoculating loop, streaked and inoculated in an MRS agar culture medium plate.

(2) After 48 hours, different colonies are picked up by an inoculating loop according to the shape, size, color and the like of the colonies, and streaked separation and purification are carried out.

(3) Gram-positive bacteria and catalase-negative bacteria were retained by gram staining and catalase analysis.

(4) Obtaining thallus and colony characteristics of the final strain: gram-positive rod-shaped bacteria, non-sporulating, non-motile bacteria (fig. 1); the diameter is about 1.0-1.2 mm, the front face is round, the center is convex, the edge is neat, the surface is moist and smooth, the surface is opaque and has slightly sour taste (figure 2).

Molecular biological identification of Lactobacillus

(1) Single genome extraction

(A) Culturing the screened lactobacillus overnight;

(B) Using a Soxhaust bacteria genome DNA extraction kit, the product number is 1600, taking 1mL of the bacterial suspension cultured overnight, centrifuging in a 1.5mL centrifuge tube at 12000r/min for 1min, and sucking out the supernatant as much as possible;

(C) Adding 200 mu L of lysozyme with a final concentration of 20mg/mL into the thalli, and treating for 40min at 37 ℃;

(D) Adding solution A (10 mmol/L, pH =8.0 Tris-HCl and 1mmol/L EDTA mixed solution, SDS with concentration of 0.08g/mL and NaCl with concentration of 1.5 mol/L) into the mixed solution obtained in the step (C), blowing by a pipette to fully suspend the thalli, adding 20 mu L rNaseA (10 mg/mL) into the suspension, fully reversing and uniformly mixing, and standing at room temperature for 30min;

(E) Adding 20 mu L of proteinase K into the tube, fully and uniformly mixing, and digesting at 55 ℃ for 60min; inverting the centrifuge tube for several times during digestion until the sample is clear and viscous, and proving that the sample is completely digested;

(F) Adding 200 μl of solution B (mixed solution of phenol: chloroform: isoamyl alcohol=25:24:1) into the inquiry tube, fully inverting and mixing, if white precipitate appears, standing at 75deg.C for 15-30min, and removing the precipitate;

(E) Adding 200 μl of absolute ethanol into the tube, mixing thoroughly, wherein flocculent precipitate may appear without affecting DNA extraction, adding the solution and flocculent precipitate into adsorption column, and standing for 2min;

(F) Centrifuging at 12000rpm for 2min, discarding the waste liquid, and placing the adsorption column into a collecting pipe;

(G) Adding 600 μl of rinse solution (75% ethanol) into the adsorption column, centrifuging at 12000rpm for 1min, discarding the waste liquid, placing the adsorption column into a collecting tube, and repeating for one time;

(H) Centrifuging at 12000rpm for 2min, and placing the adsorption column in a 50 deg.C incubator for several minutes;

(I) Placing the adsorption column into a sterilized 1.5mL centrifuge tube, suspending and dripping 100 μL of the washing liquid (general purpose) preheated by a 65 ℃ water bath into the center of the adsorption film, standing for 5min at room temperature, and centrifuging for 1min at 12000 rpm;

(J) And adding the eluent obtained by centrifugation into an adsorption column, standing for 2min at room temperature, and centrifuging for 2min at 12000rpm to obtain the genome DNA of the lactobacillus.

The 16S rDNA gene sequence of the lactobacillus is as follows:

TTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTC

ATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGAT

CCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTG

AGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCAT

TGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCT

TCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGAT

AATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGA

CAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGC

ATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCC

ACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCC

AGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTT

AGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATAC

TTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCC

ATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAG

TTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAAGCTTTCACATCAGACTTAAA

AAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTAT

TACCGCGGCTGCTGGCACGTAGTAGCCGTGGCTTTCTGGTAATACCGTCATACCTGAAC

AGTTACTCTCAGATATGTTCTTCTTTAACAACNGAGTTTTACGAGCCGAAACCCTTCTC

ACTGCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTT

ACATTTCGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGG

GGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCG

AGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGAT

GGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGC

CACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTC

CACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTC

GTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGG

TATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG

GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTG

ATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGC

AGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA

CACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGG

GTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTG

TTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAG

TACGGCCGCAAGGCTGAACTCAAGGAATTGACGGGGCCCGCACAAGCGGTGGAGCAT

GTGGTTTAATTCGAAGCTACGCGAAGACCTTACCNNTCTGACATACTATGCAAATCTAG

AGAT。

(2)16S r DNAPCR

bacterial 16S r DNA 50. Mu. LPCR reaction System: 24 mu L of Taq enzyme; ddH ₂ O: 18. Mu.L; 27F, 2. Mu.L; 1492R, 2. Mu.L; and (3) a template: 4. Mu.L.

PCR conditions: 94℃for 5min (i.e.step 1); 94℃for 30s (i.e.step 2); 55℃for 30s (i.e.step 3); 30s at 72 ℃ (i.e. step 4); step2 to 430×; and at 72℃for 5min.

Preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;

the obtained PCR product was sent to a professional sequencing company, and the obtained sequencing result was subjected to searching and similarity alignment in Gen Bank using BLAST (see FIG. 3), and identified as a strain of Lactobacillus plantarum, and stored at-80 ℃.

Example 2: lactobacillus plantarum OPB15 has good tolerance to simulated gastrointestinal fluids

In vitro simulated gastric fluid: the pH value of the 0.1mol/L potassium phosphate buffer solution is regulated to 2.5, pepsin (10 g/L) is added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, so that simulated gastric juice can be obtained.

In vitro simulated intestinal juice: the pH value of the 0.1mol/L potassium phosphate buffer solution is regulated to 6.8, pancreatin (10 g/L) and pig bile salt (3 g/L) are added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, thus obtaining the simulated intestinal juice.

Gastric digestion was simulated:

3.0mL of lactobacillus plantarum OPB15 culture solution is taken and centrifuged for 3min, mycelium is collected and placed in a gastric juice simulating environment with pH value of 2.5 of 3.0mL, and gastric juice digestion is simulated under anaerobic condition with the temperature of 37 ℃ and the speed of 90 r/min. Samples were taken at 0, 1.0, 2.0, 3.0 hours, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.

Intestinal digestion was simulated:

3.0mL of lactobacillus plantarum OPB15 culture solution is taken and centrifuged for 3min, mycelium is collected and placed in a simulated intestinal juice environment with the pH value of 6.8 at 3.0mL, and intestinal juice digestion is simulated under anaerobic conditions at 37 ℃ and 120 r/min. Samples were taken at 0, 2.0, 4.0, 6.0, 8.0 hours, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.

The survival rate (%) was calculated as the ratio of the number of viable bacteria at the time of sampling to the number of viable bacteria at the time of 0h in the culture solution, expressed as%. The experimental results are shown in table 1 and table 2, and the results show that lactobacillus plantarum OPB15 has better tolerance to artificial simulated gastrointestinal fluids.

TABLE 1 tolerance of Lactobacillus plantarum OPB15 in artificial simulated gastric fluid

TABLE 2 tolerance of Lactobacillus plantarum OPB15 in artificially simulated intestinal juice

Example 3: lactobacillus plantarum OPB15 has no toxic or side effect on SFP-class Balb/c mice

Suspending Lactobacillus plantarum OPB15 in 100g/L skim milk solution to give 4.0X10 g concentration ⁹ Bacterial suspension of CFU/m L. SPF-class Balb/c mice are bred in an environment with 40 males, the body mass of 18-22 g, the temperature of 20-25 ℃ and the relative humidity (50+/-5)%, and the illumination time of 12h/12 h. The lactobacillus plantarum OPB15 group and the control group are divided. The lactobacillus plantarum OPB15 group was given 0.3mL of this concentrated bacterial suspension once daily for gavage, and the control group was gavaged with the same volume of 100g/L skim milk solution without lactobacillus plantarum OPB15, and the death and weight were recorded for one week.

The results of these tests are shown in Table 3. These results indicate that the feeding concentration is 1.2X10 ⁹ The CFU/lactobacillus plantarum OPB15 has no obvious influence on mice, no obvious change of weight and no death phenomenon. The appearance of the mice has no obvious pathological symptoms.

TABLE 3 weight changes and death in mice

Example 4: preparation method of fermented plant-based yoghurt

(1) Mixing materials: 50 parts of Aronia melanocarpa freeze-dried powder, 30 parts of water-soluble extract and 20 parts of water-insoluble extract are mixed according to the weight proportion, homogenized under high pressure for 10 minutes, the pressure is 3500psi, and then the instant sterilization is carried out for 1 to 5 seconds at the temperature of 120 to 130 ℃;

(2) Fermenting at low temperature: inoculating Lactobacillus plantarum OPB15, lactobacillus helveticus OPB102, and lactococcus lactis CICC6242, the vitality is (1.0-1.8). Times.10 respectively during inoculation ⁸ cfu/mL，(1.0-2.0)×10 ⁸ cfu/mL，(0.9-1.5)×10 ⁷ cfu/mL, the inoculation amount is 3% (calculated by volume), fermentation is carried out after inoculation, the fermentation temperature is 38-42 ℃, and the fermentation time is 36h.

(3) Preparing yoghourt: and adding 100 parts by weight of active polypeptide according to the weight ratio, uniformly stirring the mixture, continuously stirring for 25 minutes, filling, and refrigerating the filled yoghourt for 12 hours at the temperature of 2-6 ℃ to obtain the plant-based yoghourt.

Example 5: preparation method of fermented plant-based yoghurt

(1) Mixing materials: mixing 40 parts by weight of Aronia melanocarpa freeze-dried powder, 20 parts by weight of water-soluble extract and 10 parts by weight of water-insoluble extract according to the weight proportion, homogenizing under high pressure for 10 minutes, wherein the pressure is 3500psi, and then carrying out instantaneous sterilization for 1-5 seconds at 120-130 ℃;

(2) Fermenting at low temperature: inoculating Lactobacillus plantarum OPB15, lactobacillus helveticus OPB102, and lactococcus lactis CICC 6242 with activity of (1.0-1.8) x 10 respectively ⁸ cfu/mL，(1.0-2.0)×10 ⁸ cfu/mL，(0.9-1.5)×10 ⁷ cfu/mL, fermenting after inoculation, wherein the fermentation temperature is 38-42 ℃ and the fermentation time is 24h.

(3) Preparing yoghourt: and adding 80 parts by weight of active polypeptide according to the weight ratio, uniformly stirring the mixture, continuously stirring for 25 minutes, filling, and refrigerating the filled yoghourt for 12 hours at the temperature of 2-6 ℃ to obtain the plant-based yoghourt.

Example 6: preparation method of fermented plant-based yoghurt

(1) Mixing materials: 45 parts by weight of Aronia melanocarpa freeze-dried powder, 25 parts by weight of water-soluble extract and 15 parts by weight of water-insoluble extract are mixed according to the weight proportion, homogenized under high pressure for 10 minutes under 3500psi, and then subjected to instantaneous sterilization at 120-130 ℃ for 1-5 seconds;

(3) Preparing yoghourt: and adding 90 parts by weight of active polypeptide according to the weight ratio, uniformly stirring the mixture, continuously stirring for 25 minutes, filling, and refrigerating the filled yoghourt for 12 hours at the temperature of 2-6 ℃ to obtain the plant-based yoghourt.

Example 7: preparation method of fermented plant-based yoghurt

(2) Fermenting at low temperature: lactobacillus helveticus OPB102 and lactococcus lactis CICC 6242 with activity of (1.0-1.8) x 10 respectively ⁸ cfu/mL，(1.0-2.0)×10 ⁸ cfu/mL，(0.9-1.5)×10 ⁷ cfu/mL, fermenting after inoculation, wherein the fermentation temperature is 38-42 ℃ and the fermentation time is 36h.

Example 8: determination of plant-based yoghurt on acute drunk mice ADH, ALDH, ALT and AST

SPF-class Balb/c mice are bred in an environment with 40 males, the body mass of 18-22 g, the temperature of 20-25 ℃ and the relative humidity (50+/-5)%, and the illumination time of 12h/12 h. The number of mice was randomly divided into 4 groups of 10 mice each, and the normal group, the positive control group (sea Wang Jinzun), the model group (red star Erguotou 56 ° white spirit) and the experimental group were set. The model group, the positive control group and the experimental group were respectively filled with 10.00mL/kg of physiological saline, sea Wang Jinzun and plant-based yoghurt (prepared according to example 5) each day, and the normal group was continuously filled with stomach for 30d without any treatment. After the last administration for 30min, except 10mL/kg normal group gastric lavage physiological saline, each group of mice is administrated with 5mL/kg red star Erguotou 56 degree white wine, after the last gastric lavage for 30min, eyeballs are taken out blood, the centrifugation is carried out for 10min at 3000r/min, serum is taken out, and the concentration of ethanol in the serum is measured by using a blood ethanol kit. Immediately dissecting after blood taking, taking a small amount of liver, taking 0.90% physiological saline as a homogenate medium to prepare 10% liver homogenate, centrifuging to obtain supernatant, and measuring the activity of the mouse liver homogenate ADH and ALDH according to ADH ELISA detection kit and ALDH ELISA detection kit instructions. After 12 hours of no water forbidden, blood is taken from eyeballs, the eyeballs are centrifuged for 10 minutes at 3000r/min, serum is taken, and the activity of ALT and AST in the serum of each group of mice is measured according to the instruction book of the kit.

TABLE 4 influence of each experimental group on blood ethanol concentration of acute drunk micen＝10)

Group of	Blood ethanol concentration/(mg/dL)
		Normal group	0.88±0.16
Model group	501.10±40.22 ^**
		Positive control group	288.21±21.54 ^##
Experimental group	280.01±18.22 ^##

Remarks: in comparison with the normal group, ^** p is less than 0.01; in comparison with the set of models, ^## ＜0.01。

alcohol consumption by mice results in an increase in the concentration of alcohol in the blood. The influence of each experimental group on the ethanol concentration in the blood of the mice is shown in Table 4, and compared with the normal group, the ethanol concentration in the blood of the mice is remarkably improved (p is less than 0.01) in the model group, which indicates that the ethanol content in the blood rises after the mice drink a large amount of wine, and indicates that the establishment of the acute drunk model is successful. The ethanol concentration in the blood of mice was significantly reduced (p < 0.01) in the positive control group (sea Wang Jinzun) and the experimental group (plant-based yoghurt) compared to the model group. Experimental results show that the plant-based yoghurt has the effect of dispelling the effects of alcohol, and can obviously reduce the ethanol content in the blood of mice.

TABLE 5 influence of the groups on acute intoxicated mouse Alcohol Dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST)n＝10)

Group of	ADH(U/mL)	ALDH(U/mL)	ALT(U/L)	AST(U/L)
					Normal group	13.02±3.12	79.33±1.54	15.67±1.21	17.86±2.51
Model group	28.22±1.89 ^**	128.01±2.33 ^**	49.15±2.02 ^**	51.32±4.66 ^**
					Positive control group	85.06±3.33 ^##	230.12±16.41 ^##	13.53±2.54 ^##	34.03±4.16 ^##
Experimental group	57.22±5.01 ^##	188.04±18.18 ^##	20.01±3.09 ^##	28.65±5.45 ^##

ADH and ALDH are key enzymes affecting alcohol metabolism and alcohol content in blood, so that their vitality values can be used for measuring the anti-alcohol effect. As shown in Table 5, after the mice take ethanol, the viability of liver homogenate ADH and ALDH was significantly increased, indicating that a large amount of drinking in a short period resulted in a rapid increase in the viability of liver ADH and ALDH. Compared with a model group, the plant-based yoghurt experimental group can obviously improve ADH activity (p is less than 0.01), and the ADH activity is increased from 28.22U/mL to 57.22U/mL, and the numerical value is similar to that of a positive control group 85.06U/mL, so that the plant-based yoghurt experimental group can obviously accelerate the decomposition speed of ethanol into acetaldehyde and accelerate the catabolism of ethanol in blood. Compared with the model group, the plant-based yoghurt experimental group can obviously improve the activity of ALDH enzyme (p is less than 0.01), and the ALDH enzyme activity is respectively increased to 188.04U/mL from 128.01U/mL, so that the plant-based yoghurt experimental group can improve the oxidation speed of acetaldehyde to acetic acid, and further promote ethanol metabolism. The results show that the plant-based yoghurt has remarkable anti-alcohol effect, and the effect is similar to that of a sea Wang Jinzun positive control group, and can accelerate ethanol metabolism and further reduce the concentration of blood alcohol by increasing the activity of liver ADH and ALDH enzymes.

ALT and AST are enzymes distributed in the cytoplasm and mitochondria of liver cells, which undergo necrotic rupture when mice ingest a large amount of alcohol, and transfer into the blood, resulting in elevated concentrations of ALT and AST in the blood. Therefore, ALT and AST activity values in blood can be used as indexes for judging the damage of the liver cells of the mice. As shown in Table 5, the serum ALT and AST activity of the model group is significantly increased (p < 0.01) compared with that of the normal group, ALT is increased from 15.67U/L to 49.15U/L, and AST is increased from 17.86U/L to 51.32U/L, which indicates that the acute drunk liver injury model is successfully established. ALT and AST activities in the serum of mice in the plant-based yogurt group were reduced to 20.01U/L and 28.65U/L (p < 0.01), respectively, and ALT levels were lower than those in the positive control group, as compared to the model group. The results show that the plant-based yoghurt can effectively reduce ALT and AST levels in blood and well protect liver cells of mice suffering from acute intoxication.

Example 9: effects of each experimental group on hyperuricemia mice Uric Acid (UA), creatinine (Cr) and urea nitrogen (BUN)

SPF-class Balb/c mice are bred in an environment with 40 males, the body mass of 18-20 g, the temperature of 20-25 ℃ and the relative humidity (50+/-5)%, and the illumination time of 12h/12 h. The cells were randomly divided into 4 groups of 10 cells, and the cells were selected from the group consisting of a blank control group, a model group, a positive control group and an experimental group. (1) Blank control group: feeding with normal feed; (2) model group: potassium oxazinate (250 mg/kg, vehicle 5% CMC-Na aqueous solution by mass); (3) positive control group: potassium oxazinate (250 mg/kg, vehicle 5% CMC-Na aqueous solution by mass) +allopurinol (5 mg/kg); (4) experimental group: potassium oxazinate (250 mg/kg, solvent 5% CMC-Na aqueous solution by mass) +vegetable yoghurt (500 mg/kg, prepared in example 5); the other groups were dosed by gavage as described above except for the blank group, which was given an equal volume of vehicle (i.e. 5% cmc-Na in water by mass). All groups were fed continuously for 14d, 1 time per day, with a gavage volume of 0.3mL/100 g. After 14 days, fasting for 12 hours, taking blood from eyeballs, centrifuging for 20 minutes at a rotating speed of 3000r/min, and separating serum; uric Acid (UA), creatinine (Cr) and urea nitrogen (BUN) levels in serum were measured using commercially available kits.

Table 6 effect of each experimental group on hyperuricemia mice UA, cr, and BUN. (n＝10)

Group of	UA(mg/L)	Cr(μmol/L)	BUN(mmol/L)
				Normal group	1.51±0.11	0.35±0.21	6.42±1.33
Model group	7.35±2.06 ^**	0.99±0.13 ^**	18.09±2.51 ^**
				Positive group	1.88±0.42 ^##	0.43±0.21 ^##	14.02±1.17 ^##
Experimental group	1.90±2.51 ^##	0.51±0.17 ^##	12.26±0.55 ^##

Note that: remarks: in comparison with the normal group, ^** p is less than 0.05; in comparison with the set of models, ^## ＜0.05。

as shown in Table 6, the comparison of UA, BUN, cr levels in serum of each group shows that UA levels are significantly higher than those of the negative group (P < 0.05) after the mice of the model group are perfused with the potassium oxyzinate, thus indicating that the model of hyperuricemia mice is successfully modeled. After 14d of continuous administration of the treatment, uric acid levels in serum of the plant-based yogurt experimental group were significantly reduced (P < 0.05) compared to the model group, while serum uric acid levels of mice of the plant-based yogurt group were close to uric acid levels of mice of the positive group. Compared with a model group, the serum creatinine and urea nitrogen levels are obviously reduced (P is less than 0.05), the improvement effect of the plant-based yogurt group on the serum urea nitrogen levels is lower than that of a positive group, the serum creatinine level is reduced from 0.99 mu mol/L to 0.51 mu mol/L, the improvement amplitude is nearly doubled, and the effect is obvious (P is less than 0.05).

Example 10: influence of plant-based yogurt on pathological changes in colon tissue of model mice

For example 9, clean colon tissue samples were cut 2cm with sterile surgical scissors, placed in 10% formalin solution for overnight fixation, and then prepared into paraffin sections of colon tissue. Sections were stained with hematoxylin-eosin (HE staining), alisxin blue-periodate schiff stain (AB-PAS staining). The colon histopathological changes were observed under an optical microscope and stored by photographing (fig. 2).

After HE staining, as shown in FIG. 4, the normal colon tissue morphology is normal, the villi is compact, the arrangement is orderly, the structure is clear, and the intestinal glands of the tissue mucous membrane layers are well-organized. Compared with the model group, the damage of submucosa is increased, intestinal glands of tissue mucous layer are arranged irregularly and disorderly, and villi are loosened and broken. The colon muscle layer of the probiotics group is thickened, the tissue injury of submucosa is reduced, the intestinal glands are orderly arranged and approach to the normal level, and the probiotics group is obviously superior to the model group, so that the experiment group has a certain protection effect on the intestinal tract. The length of the villus of the model group and the depth of the crypt are observed, the villus of the model group is shortest, and the length of the colon villus after being fed by the plant-based yoghurt is recovered, so that the plant-based yoghurt can improve the length of the colon villus to a certain extent.

After AB-PAS staining, a large number of aligned goblet cells were observed in the normal group, while fewer goblet cells were in the model group and the alignment was not ordered, as shown in FIG. 5. The goblet cells of mice fed with the plant-based yoghurt are orderly arranged, colon tissues are in a damaged recovery state, the number of goblet cells is increased, so that the level state of the colon tissues is close to that of a normal level group, and the colonial growth of intestinal flora is facilitated, thereby realizing the effects of promoting metabolism, reducing uric acid, dispelling alcohol and protecting liver.

3. Synergistic effects of the present invention

The freeze-dried powder, the water-soluble extract, the water-insoluble extract, the active polypeptide and the lactobacillus plantarum OPB15 of the invention have a synergistic effect when used or not, and are shown in tables 7 to 9.

(1) The preparation method of comparative examples 1 to 5 in Table 7 is the same as in example 4, except that the Aronia melanocarpa freeze-dried powder, the water-soluble extract, the water-insoluble extract, the weight parts of the active polypeptide and Lactobacillus plantarum OPB15 in comparative example are used or not.

TABLE 7 synergistic effects table of the present invention

As can be seen from Table 7, the use or absence of the freeze-dried powder, the water-soluble extract and the lactobacillus plantarum OPB15 of Aronia melanocarpa and the influence of the active polypeptide on the taste and aroma of the plant-based yoghurt are great; wherein the Aronia melanocarpa and water soluble extract contains plant polysaccharide, and can provide prebiotics for later fermentation and promote rapid proliferation of probiotics. When the parts by weight of the Aronia melanocarpa and the water-soluble extract are increased, the excessive fermentation is promoted, so that the fragrance is released for a plurality of degrees, and the faint scent of plants is weakened instead. Meanwhile, after the freeze-dried powder of the Aronia melanocarpa is more than 50 parts by weight, the astringency is enhanced, and the Aronia melanocarpa is related to certain sour and astringent feeling of the Aronia melanocarpa. The water-soluble extract is excessive, and after the water-soluble extract exceeds 30 parts by weight, the sour and astringent feeling is not increased. Meanwhile, lactobacillus plantarum promotes fragrance release, and fragrance release is weakened when lactobacillus plantarum is not added. In addition, the bitter taste of the yoghurt is greatly influenced by adding excessive active peptide. As is clear from Table 7, in the present invention, when the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the use or absence of Lactobacillus plantarum and the active polypeptide were used together, the influence of the obtained plant-based yogurt on ADH (U/mL) and ALDH (U/mL) was significantly higher than that of the single treatment. In particular, when the parts by weight of the freeze-dried powder and the water-soluble extract of Aronia melanocarpa were increased, as prebiotics, the probiotics were promoted to be rapidly increased, the fermentation was excessive, the anti-hangover effect was weakened, the effects on ADH and ALDH were large, the ADH of comparative examples 1 and 2 were 25.26U/mL and 25.88U/mL, respectively, the ALDH was 80.09U/mL and 85.22U/mL, respectively, and the comparative examples 1 and 2 were closest to the model group level. Meanwhile, the lactobacillus plantarum is not added, the liver protection effect is most weakened, the influence on ALT (U/L) is the greatest in the family with damaged cell membranes, the numerical value is 53.33U/L, and the level is closest to the level of a model group. In addition, the increase of water insoluble substances affects the fermentation level, so that the deep fermentation of probiotics is affected to a certain extent. Therefore, the increase in water insolubles and fermentation without Lactobacillus plantarum had a greater effect on uric acid levels, UA values of 4.59mg/L and 4.30mg/L, respectively, closest to the model group levels, higher than those of comparative example 2, comparative example 2 and comparative example 5. The data show that the effect of example 4 is better than that of comparative examples 1-5, and the effect of the final product is reduced by changing any condition within any required range, so that the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the lactobacillus plantarum OPB15 and the active polypeptide of the invention have a synergistic effect, and the state and the function of the plant-based yoghurt can be obviously improved in a synergistic way. In particular, the Aronia melanocarpa freeze-dried powder: water-soluble extract: water insoluble extract: the weight ratio of the active polypeptide is 40-50: 20-30: 10-20: 80-100, and simultaneously, the lactobacillus plantarum is used, so that the state and the function of the plant-based yoghurt can be improved greatly in a synergistic way.

(2) The preparation method of comparative examples 6 to 10 in Table 8 is the same as in example 5, except that the Aronia melanocarpa freeze-dried powder, the water-soluble extract, the water-insoluble extract, the weight parts of the active polypeptide and Lactobacillus plantarum OPB15 in comparative example are used or not.

TABLE 8 synergistic effects table of the present invention

As can be seen from table 8, whether the freeze-dried powder of Aronia melanocarpa, the water-soluble extract and the lactobacillus plantarum OPB15 are used or not has a great influence on the taste and aroma of the plant-based yoghurt; the Aronia melanocarpa and the water-soluble extract promote the rapid proliferation of probiotics, but when the parts by weight of the Aronia melanocarpa and the water-soluble extract are reduced to below 40 parts, enough prebiotics cannot be provided for the deep fermentation of the probiotics, so that the aroma release is reduced and the fragrance is not strong enough. Meanwhile, lactobacillus plantarum promotes fragrance release, and fragrance release is weakened when lactobacillus plantarum is not added. As can be seen from Table 8, the effects of the obtained plant-based yoghurt on ADH (U/mL) and ALDH (U/mL) are significantly higher than those of single treatment when the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the use or non-use of Lactobacillus plantarum and the active polypeptide are used together in the present invention. When the parts by weight of the freeze-dried powder and the water-soluble extract of Aronia melanocarpa are reduced, the plant polysaccharide prebiotics are reduced, the probiotics are insufficiently fermented, the anti-hangover effect is weakened, the effects on ADH and ALDH are large, the ADH of comparative examples 6 and 7 are respectively 27.33U/mL and 26.91U/mL, the ALDH is respectively 83.11U/mL and 87.09U/mL, and the comparative examples 6 and 7 are closest to the level of the model group. The secondary contributors are the water insoluble extract and active polypeptide, with ADH and ALDH levels better for comparative examples 8 and 10 than for comparative examples 6 and 7. Meanwhile, lactobacillus plantarum is not added, the liver protection effect is the weakest, the influence on ALT (U/L) is the greatest, the numerical value is 55.09U/L, and the model group level is the closest. Meanwhile, the amount of water insoluble substances is reduced, and the effective components for reducing uric acid are also reduced at any time, so that the uric acid level is influenced. Meanwhile, the influence of not using lactobacillus plantarum is also larger. Thus, the UA values of comparative example 8 and comparative example 9 were 5.50mg/L and 4.88mg/L, respectively, closest to the model group level. The data show that the effect of example 5 is better than that of comparative examples 6-10, and the effect of the final product is reduced by changing any condition within any required range, so that the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the lactobacillus plantarum and the active polypeptide of the Aronia melanocarpa have a synergistic effect, and the state and the function of the plant-based yoghurt can be obviously improved in a synergistic manner. In particular, the Aronia melanocarpa freeze-dried powder: water-soluble extract: water insoluble extract: the weight ratio of the active polypeptide is 40-50: 20-30: 10-20: 80-100, and simultaneously, the lactobacillus plantarum is used, so that the state and the function of the plant-based yoghurt can be improved greatly in a synergistic way.

(3) The preparation methods of comparative examples 11 to 15 in Table 9 are the same as in example 6, except that the complex enzyme in comparative example was used or not, the hot water reflux extraction method, the ratio of the mixed powder to water and the solid state fermentation time.

TABLE 9 synergistic effects of the invention

As can be seen from table 9, whether the freeze-dried powder of Aronia melanocarpa, the water-soluble extract and the lactobacillus plantarum OPB15 are used or not has a great influence on the taste and aroma of the plant-based yoghurt; the Aronia melanocarpa and the water-soluble extract promote the rapid proliferation of probiotics, but when the weight parts of the Aronia melanocarpa and the water-soluble extract are reduced to 38, enough prebiotics cannot be provided for the deep fermentation of the probiotics, so that the aroma release is reduced and the fragrance is not strong enough. Meanwhile, it can be seen from Table 9 that the released fragrance in comparative example 14 was weakened when Lactobacillus plantarum was not added. As is clear from Table 9, in the present invention, when the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the use or absence of Lactobacillus plantarum and the active polypeptide were used together, the influence of the obtained plant-based yoghurt on ADH (U/mL) and ALDH (U/mL) was significantly higher than that of the single treatment. The greatest effect on ADH and ALDH levels was achieved by reducing the weight parts of Aronia melanocarpa lyophilized powder, which were 25.41U/mL and 80.52U/mL, respectively, with comparative example 11 closest to the model group level. The secondary influencing factors were water insoluble extracts, ADH and ALDH of comparative example 12 were 27.56U/mL and 88.11U/mL, respectively, close to comparative example 11. The active polypeptide has stronger liver protection effect, and when the addition amount of the active polypeptide is small, the liver protection effect is the weakest, the influence on ALT (U/L) is the largest, the value is 40.31U/L, and the level is closest to the level of a model group. In addition, the UA values of comparative example 8 and comparative example 9 were 6.23mg/L and 5.07mg/L, respectively, closest to the model group level, for uric acid levels, with a greater effect on water insolubles than without the addition of Lactobacillus plantarum. The above data show that the effect of example 6 is better than that of comparative examples 11-15, and the final product effect is reduced by changing any condition within any required range, so that the freeze-dried powder, the water-soluble extract, the water-insoluble extract, the lactobacillus plantarum of the invention have a synergistic effect between the use or non-use of the Aronia melanocarpa and the operation steps of the active polypeptide, and the state and the functional effect of the plant-based yoghurt can be obviously improved by the synergistic effect. In particular, the Aronia melanocarpa freeze-dried powder: water-soluble extract: water insoluble extract: the weight ratio of the active polypeptide is 40-50: 20-30: 10-20: 80-100, and simultaneously, the lactobacillus plantarum is used, so that the state and the function of the plant-based yoghurt can be improved greatly in a synergistic way.

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims

1. A plant-based yoghurt characterized in that: the plant-based yoghurt is obtained by fermenting lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 and lactobacillus lactis CICC 6242;

wherein the name of the lactobacillus plantarum OPB15 is as follows: OPB15, partClass name is: lactobacillus plantarum (L.) KummerLactobacillus plantarum) The preservation number is: CGMCC No. 27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3 of the Korean area of Beijing, the China general microbiological culture Collection center;

the thallus is characterized in that: gram-positive rod-shaped bacteria, non-spore-forming, non-motile bacteria; colony characteristics: the diameter is 1.0-1.2 mm, the front surface is round, the center is convex, the edge is neat, the surface is moist and smooth, the surface is opaque, and the product has sour taste; growth characteristics: culturing in MRS culture medium at 37deg.C for 12 hr to end of logarithm; has tolerance to simulated gastrointestinal fluids;

The method comprises the following steps:

step 1, adding freeze-dried powder, water-soluble extract and water-insoluble extract of Aronia melanocarpa, homogenizing the mixture under high pressure for 10 minutes, wherein the pressure is 3500psi, and then carrying out instantaneous sterilization for 1-5 seconds at 120-130 ℃;

step 2, inoculating lactobacillus plantarum OPB15, lactobacillus helveticus OPB102 and lactococcus lactis CICC 6242, wherein the activity is (1.0-1.8) multiplied by 10 in sequence during inoculation ⁸ cfu/mL，（1.0-2.0）×10 ⁸ cfu/mL，（0.9-1.5）×10 ⁷ cfu/mL, the inoculation amount is 3% (calculated by volume), fermentation is carried out after inoculation, the fermentation temperature is 38-42 ℃, and the fermentation time is 24-36 h;

step 3, adding active polypeptide, stirring the mixture uniformly, continuously stirring for 25 minutes, filling, and refrigerating the filled yoghourt for 12 hours at the temperature of 2-6 ℃ to obtain plant-based yoghourt;

wherein, the weight ratio of the Aronia melanocarpa freeze-dried powder to the water-soluble extract to the water-insoluble extract to the active polypeptide is 40-50: 20-30: 10-20: 80-100;

the preparation method of the Aronia melanocarpa freeze-dried powder in the step 1 comprises the following steps:

(3) Adding complex enzyme into the Aronia melanocarpa fruit primary pulp, inactivating enzyme, and filtering to obtain pulp;

(6) Filtering the Aronia melanocarpa fermented extract to obtain filtrate, and vacuum freeze-drying at-80deg.C to obtain Aronia melanocarpa lyophilized powder;

the preparation method of the water-soluble extract and the water-insoluble extract in the step 1 comprises the following steps:

(4) Insoluble extract of Chinese medicinal liquid: drying the residues obtained in the step (2), crushing, sieving to obtain powder, adding 10 times of ethanol, heating and reflux-extracting at 80-85 ℃ for 2h, cooling to room temperature, and vacuum-filtering; heating the filtrate at 55-70deg.C, vacuumizing, rotary evaporating to obtain concentrated solution, pre-freezing at-20deg.C and-80deg.C, lyophilizing at-80deg.C in a lyophilizer, and lyophilizing for 12 hr to obtain water insoluble lyophilized powder which is water insoluble extract;

the preparation method of the active polypeptide in the step 3 comprises the following steps:

2. Use of the plant-based yoghurt of claim 1 as a health food.