Biological preparation for relieving intestinal discomfort caused by alcohol as well as preparation method and application of biological preparation

Abstract

The invention relates to the technical field of functional microbial preparations, in particular to a biological preparation for relieving intestinal discomfort caused by alcohol and a preparation method and application thereof.The main technical scheme is that the biological preparation is prepared from, by weight, 5-20 parts of bacillus coagulans, 10-30 parts of pediococcus pentosaceus, 5-10 parts of lactobacillus rhamnosus and 0-20 parts of saccharomycetes. 0-5 parts of bifidobacterium longum and 0-5 parts of lactococcus lactis. By constructing an alcohol metabolism-intestinal protection dual-function synergistic system, specific florae such as bacillus coagulans, pediococcus pentosaceus and lactobacillus rhamnosus are scientifically compatible with functional components such as inulin and L-cysteine, alcohol is efficiently metabolized, and a carbon source is generated; the technical problems of low metabolic efficiency, weak intestinal protection and serious gastric acid inactivation of a traditional product are comprehensively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional microbial preparation, and particularly relates to a biological preparation for relieving intestinal discomfort caused by alcohol and a preparation method and application thereof. BACKGROUND

[0002] Appropriate drinking has the effect of exciting nerves, so that the brain inhibition function is weakened, blood vessels are dilated, and blood circulation is strengthened, which can relieve fatigue, excite spirit, increase appetite, and promote digestion and absorption. When the ethanol concentration in the blood reaches 0.05%, the effect of alcohol begins to appear, and excitement and euphoria appear; when the ethanol concentration in the blood reaches 0.1%, people will lose self-control. When it reaches 0.2%, people are in a state of drunkenness; when it reaches 0.4%, people can lose consciousness, coma, and even be in danger of life. Therefore, excessive drinking has many aspects of damage to the body. It can cause multiple neuritis, myocardial lesions, brain lesions, hematopoietic dysfunction, pancreatitis, gastritis and ulcer disease, and can also increase the incidence of hypertension. Long-term heavy drinking also endangers reproductive cells, causes low intelligence of offspring, and the incidence of laryngeal cancer and digestive tract cancer of people who often drink is significantly increased.

[0003] Alcohol is absorbed through the gastrointestinal tract and then metabolized in the liver. Alcohol stays in the stomach for a short time and is less absorbed, mainly through the intestinal tract, and alcohol absorption can cause short-term damage to the human body. At the same time, long-term drinking can also change the structure of intestinal flora, reduce intestinal probiotics and increase pathogenic bacteria. Under healthy human health conditions, the intestinal flora is mainly composed of anaerobic beneficial bacteria, and the oxygen content in the intestine is low; under the action of alcohol, the intestinal flora is in a state of imbalance, the number of pathogenic bacteria in the intestine increases sharply, and the oxygen content in the intestine increases, thereby inhibiting the growth of anaerobic beneficial bacteria and affecting the normal function of intestinal flora. Analysis of intestinal flora of patients with alcoholic liver disease confirms that long-term intake of alcohol by the body can lead to a significant decrease in the number of intestinal probiotics, such as lactobacillus, and an increase in the number of harmful bacteria, such as prevotella, gram-negative bacteria, and gram-positive bacteria. It can be seen that the change in the number of intestinal flora caused by alcohol is mainly manifested in the decrease of intestinal probiotics and the increase of pathogenic bacteria, which leads to damage to the physiological function of the intestine. The disorder of the flora structure will bring a series of health problems.

[0004] However, the existing products for relieving alcohol or protecting the liver can only relieve the short-term discomfort caused by alcohol through enzyme degradation of alcohol or plant extracts homologous to medicine, and cannot solve the long-term effects of drinking, so there is an urgent need for better products to solve these problems. SUMMARY

[0005] The application aims to provide a biological preparation for relieving alcohol-induced intestinal discomfort, a preparation method thereof and application thereof, and a dual-function synergistic system of alcohol metabolism and intestinal protection is constructed, and through synergistic effect of specific flora combination and functional components, the intestinal barrier function is significantly enhanced while alcohol is efficiently decomposed.

[0006] The above technical objective of the application is achieved by the following technical scheme:

[0007] The application provides a biological preparation for relieving alcohol-induced intestinal discomfort, which comprises the following components in parts by weight:

[0008] 5-20 parts of Bacillus coagulans, 10-30 parts of Pediococcus pentosaceus, 5-10 parts of Lactobacillus rhamnosus, 0-20 parts of yeast, 0-5 parts of Bifidobacterium longum and 0-5 parts of Lactococcus lactis.

[0009] Further, the yeast is Saccharomyces boulardii.

[0010] Further, the Pediococcus pentosaceus has ethanol dehydrogenase activity.

[0011] Further, the biological preparation further comprises, in parts by weight: 20-50 parts of inulin, 25-50 parts of L-cysteine and / or 1-5 parts of a pharmaceutically acceptable excipient.

[0012] Further, the biological preparation further comprises, in parts by weight: 0-10 parts of fructooligosaccharide, 10-30 parts of glutathione and / or 0.1-1 part of alpha-tocopherol.

[0013] The application further provides a preparation method of the biological preparation for relieving alcohol-induced intestinal discomfort, comprising the following steps:

[0014] The Bacillus coagulans is aerobically cultured at pH 6.5-7.0, a temperature of 35-37 DEG C and dissolved oxygen DO 30-50% for 16-20 hours;

[0015] The Pediococcus pentosaceus and the Lactococcus lactis are co-cultured at a inoculation ratio of 1:0.2-0.5 under anaerobic conditions at pH 6.2-6.8 and a temperature of 30-32 DEG C for 12-15 hours;

[0016] The Lactobacillus rhamnosus is cultured alone at pH 5.5-6.0 and a temperature of 37 DEG C for 18-24 hours;

[0017] The Bifidobacterium longum is cultured under strict anaerobic conditions at pH 6.8-7.2 and a temperature of 37-39 DEG C for 20-28 hours;

[0018] The yeast is cultured under aeration conditions at pH 4.5-5.5 and a temperature of 25-28 DEG C for 24-36 hours.

[0019] Bacterial cell collection and complex protection:

[0020] Each strain was collected by centrifugation at the end of the logarithmic growth phase and washed with buffer solution;

[0021] Complex freeze-drying:

[0022] After mixing the above wet bacterial cells, add freeze-drying protectant, vacuum freeze-drying: pre-freeze to below -45℃, main drying stage -25℃~20℃, desorption drying stage 25-30℃;

[0023] Bacterial powder standardization:

[0024] The resulting mixed powder is mixed through an 80-100 mesh sieve, and the final product is controlled to have a moisture content of ≤5%, and a total number of viable bacteria of ≥1×10 11 CFU / g.

[0025] Further, the Bacillus coagulans is subjected to heat treatment at 80℃ for 10 minutes before collection to inactivate the vegetative cells and retain the spore form.

[0026] Further, the Lactobacillus rhamnosus is cultured in a separate tank to the middle of the logarithmic phase, and then mixed; the temperature difference is controlled to be ≤5℃ during mixing, and 0.1-0.5% trehalose is added as a compatibility agent.

[0027] The application also provides a biological agent for relieving alcohol-induced intestinal discomfort, and the biological agent is prepared into probiotic tablet candies, probiotic pills or probiotic oil droplets.

[0028] Further, the tablet candies are used for preventive use before drinking, the probiotic oil droplets are used for emergency treatment after drinking, and the probiotic pills are used for intestinal repair of long-term alcohol consumers.

[0029] In summary, the application has the following beneficial effects:

[0030] The present application realizes three breakthrough effects by constructing an "alcohol metabolism-intestinal protection" dual-function synergistic system: (1) alcohol metabolism bacteria (Pediococcus pentosaceus + Lactococcus lactis) efficiently metabolize alcohol and produce carbon sources to provide proliferation nutrients for protective bacteria (Bifidobacterium longum, etc.); (2) acid-resistant bacteria (spore-forming Bacillus coagulans) increase the survival rate of live bacteria in the stomach acid stage, ensuring intestinal targeted release; (3) L-cysteine and bacterial metabolites synergistically repair the intestinal barrier, and the tablet candy (before drinking) can reduce the blood alcohol peak, the oil drop liquid (after drinking) can increase the acetaldehyde discharge rate, and the health pill (long-term) can restore the intestinal flora diversity to a healthy level, thereby comprehensively solving the three technical problems of low metabolic efficiency, weak intestinal protection, and severe inactivation in the stomach of traditional products. DETAILED DESCRIPTION

[0031] To further illustrate the technical means and effects adopted by the present application to achieve the predetermined object of the application, the specific embodiments, features and effects of a biological agent for relieving alcohol-induced intestinal discomfort, a preparation method and application thereof according to the present application are described in detail as follows.

[0032] The biological agent for relieving alcohol-induced intestinal discomfort provided in the present embodiment comprises the following components by weight:

[0033] Bacillus coagulans 5-20 parts, Pediococcus pentosaceus 10-30 parts, Lactobacillus rhamnosus 5-10 parts, yeast 0-20 parts, Bifidobacterium longum 0-5 parts, and Lactococcus lactis 0-5 parts.

[0034] It can be understood that the biological agent constructs an "alcohol metabolism-intestinal protection" dual-function synergistic system, and the core principle thereof is that Pediococcus pentosaceus (containing ethanol dehydrogenase) and Lactococcus lactis form an "alcohol metabolism engine" to efficiently metabolize ethanol into acetic acid, and at the same time provide carbon sources for Bacillus coagulans and Bifidobacterium longum, etc. to promote the secretion of antibacterial peptides and mucin. The system forms a metabolism-protection closed loop (alcohol metabolism bacteria metabolites directly nourish intestinal protection bacteria); and pH self-adaptation (acid-resistant Bacillus coagulans spores ensure high survival rate in the stomach, and Bifidobacterium longum realizes intestinal targeted release); and synergistic effect (L-cysteine and short-chain fatty acids metabolized by bacteria repair tight junctions together, and improve the intestinal leakage improvement rate.

[0035] In some preferred embodiments, the yeast is Saccharomyces boulardii.

[0036] Saccharomyces boulardii has a unique ethanol metabolic pathway, and the pyruvate decarboxylase it secretes can accelerate the conversion of acetaldehyde to acetic acid, forming a complementary metabolic network with the ethanol dehydrogenase of Pediococcus pentosaceus, which can improve the acetaldehyde clearance rate and maintain high activity in the stomach acid environment, providing early protection for other bacterial flora.

[0037] The secreted protease can degrade bacterial toxins (such as Clostridium difficile toxin B) to stimulate the production of intestinal secretory IgA, enhance mucosal immune defense, and reduce alcohol-induced intestinal inflammatory factors (TNF-α reduction) by regulating the NF-κB pathway.

[0038] The production of short-chain fatty acids (butyric acid, etc.) promotes the proliferation of protective flora (such as Bifidobacterium) and inhibits the colonization of pathogenic bacteria (such as Escherichia coli) in the intestine, maintains the stability of intestinal pH (6.5-7.0), optimizes the survival environment of other probiotics, and plays a rapid alcohol-eliminating role in tablet candies (oral mucosa rapidly absorbs metabolites), synergizes with α-tocopherol in oil drop liquid dosage forms, and continuously releases active substances in enteric-coated pills.

[0039] In some preferred embodiments, the Pediococcus pentosaceus has ethanol dehydrogenase activity.

[0040] It can be understood that by ADH (ethanol dehydrogenase) to oxidize ethanol to acetaldehyde (Km value as low as 2.3 mM, high affinity), while expressing ALDH (aldehyde dehydrogenase) to convert acetaldehyde to harmless acetic acid, forming a complete metabolic pathway of "ethanol-acetaldehyde-acetic acid" with Bacillus coagulans, utilizing the NAD+ / NADH balance system (oxidizing 1 molecule of ethanol to regenerate 1 molecule of NAD+), the acetic acid produced by metabolism is used as a carbon source by other probiotics. Combined with L-cysteine, the speed of intestinal barrier function repair is accelerated by 2.2 times.

[0041] In some preferred embodiments, the biological preparation further comprises, in terms of parts by weight: inulin 20-50 parts, L-cysteine 25-50 parts, and / or pharmaceutically acceptable excipients 1-5 parts.

[0042] It can be understood that the scientific combination of inulin, L-cysteine and pharmaceutical adjuvants in the biological preparation forms a triple synergistic protection mechanism: inulin as a high-efficiency probiotic selectively promotes the proliferation of protective flora such as Bifidobacterium, and the short-chain fatty acids (SCFAs) produced by its fermentation not only reduce the intestinal pH to 5.2 to inhibit pathogenic bacteria, but also improve the acetaldehyde degradation efficiency of Pediococcus pentosaceus; L-cysteine plays a role through two pathways, on the one hand, as a glutathione precursor to improve the antioxidant capacity of the liver, directly neutralize acetaldehyde toxins, on the other hand, stimulate intestinal epithelial cell proliferation (increase Ki-67 positive rate) and inhibit cell apoptosis (reduce caspase-3 activity); the pharmaceutical adjuvant system ensures the targeted release of active ingredients through trehalose freeze-drying protection and precise control of the disintegration time (15±2 minutes). The three components form a three-dimensional protection network of "metabolic support-oxidative defense-mechanical repair", and the combination can improve intestinal permeability, reduce serum endotoxin, and shorten the relief time of discomfort after drinking, showing synergistic effect compared with traditional preparations.

[0043] In some preferred embodiments, the biological preparation further comprises, calculated by weight fraction: fructooligosaccharides 0-10 parts, glutathione 10-30 parts and / or alpha-tocopherol 0.1-1 part.

[0044] It can be understood that the combination of fructooligosaccharides, glutathione and alpha-tocopherol in the biological preparation forms a multi-level intestinal protection network: fructooligosaccharides and inulin synergistically construct a gradient probiotic system, precisely promoting the proliferation of probiotics (increasing the number of Lactococcus lactis), while maintaining the balance of intestinal osmotic pressure; glutathione directly neutralizes acetaldehyde toxins and forms a circulating antioxidant system with L-cysteine (increasing GSH regeneration efficiency), effectively repairing mitochondrial function; alpha-tocopherol blocks lipid peroxidation of cell membranes (reducing MDA), enhances bacterial stability, and provides long-term protection. The synergistic effect of the three components significantly improves the antioxidant capacity and barrier function of the intestine.

[0045] The preparation method of the biological preparation for relieving alcohol-induced intestinal discomfort provided by the specific embodiment includes the following steps:

[0046] S1, Bacillus coagulans is aerobically cultured at pH 6.5-7.0, temperature 35-37℃, dissolved oxygen DO 30-50% for 16-20 hours;

[0047] S2, Pediococcus pentosaceus and Lactococcus lactis are co-cultured at a inoculation ratio of 1:0.2-0.5 under anaerobic conditions at pH 6.2-6.8, 30-32℃ for 12-15 hours;

[0048] S3, Lactobacillus rhamnosus is cultured alone at pH 5.5-6.0, 37℃ for 18-24 hours;

[0049] S4, culturing Bifidobacterium under strict anaerobic conditions at pH 6.8-7.2, 37-39℃ for 20-28 hours;

[0050] S5, culturing yeast under aeration conditions at pH 4.5-5.5, 25-28℃ for 24-36 hours;

[0051] S6, cell collection and composite protection: centrifugal collection of each strain at the end of logarithmic growth and washing with buffer;

[0052] S7, composite freeze-drying: adding freeze-drying protectant after mixing the above wet cells, vacuum freeze-drying: pre-freezing to below -45℃, main drying stage -25℃-20℃, desorption drying stage 25-30℃;

[0053] S8, standardization of bacterial powder: mixing the obtained mixed powder through 80-100 mesh screen, controlling the final product moisture ≤5%, total viable bacteria ≥1×10 11 CFU / g.

[0054] It can be understood that the preparation method of the biological preparation realizes the optimized preparation of high-efficiency alcohol-eliminating and intestine-protecting bacteria by combining the precise cultivation in stages with the composite freeze-drying process. The differentiated culture conditions are designed according to the characteristics of each strain: Bacillus coagulans forms acid-resistant spores in an oxygen-rich environment (DO 30-50%), Pediococcus pentosaceus and Lactococcus lactis are co-cultured at a specific ratio (1:0.2-0.5) to significantly improve the ethanol dehydrogenase activity to 158 U / mg, and strict anaerobic conditions ensure high survival rate of Bifidobacterium. Gradient freeze-drying technology (pre-freezing -45℃→ main drying -25-20℃→ desorption drying 25-30℃) is used with trehalose-degreased milk composite protectant to improve the freeze-drying survival rate of the bacterial flora. The total viable bacteria of the final product are ≥1×10 11 CFU / g, 80-100 mesh standardization screening ensures the uniformity of the preparation, has excellent intestinal release rate and alcohol metabolism efficiency, and can effectively repair the intestinal barrier, solving the key technical problems of mutual inhibition of traditional probiotic preparations, inactivation of gastric acid and attenuation of efficacy.

[0055] In some preferred embodiments, the Bacillus coagulans is subjected to heat treatment at 80℃ for 10 minutes before collection to inactivate vegetative cells and retain spore form.

[0056] It can be understood that the Bacillus coagulans is subjected to a heat treatment process of 80℃ for 10 minutes, by selectively inactivating vegetative cells (inactivation rate 99.9%) and retaining the highly heat-resistant spore form, the heat stability advantage of the specific pyridinedicarboxylic acid calcium complex (15% of dry weight) of the spore is fully exerted. The treatment makes the spore gastric acid survival rate jump from 30% to 98%, and the intestinal targeting release (germination rate 95% at pH≥6.8), while bringing triple promotion: 40% increase in purity to avoid immunogenicity; 60% increase in efficiency of bacterial population coaggregation due to exposure of surface adhesion sites; 10 times increase in stability (viable bacteria decrease rate <5%) shown in 45℃ acceleration test. The process not only guarantees the long-acting shelf life of 36 months of the spores, but also realizes the advantage of 30-minute rapid effect, so that the alcohol metabolism enzyme activity retention rate is >90%, perfectly solving the core problems of traditional probiotics, such as gastric acid inactivation and low colonization efficiency.

[0057] In some preferred embodiments, the Lactobacillus rhamnosus and the yeast are cultured in separate tanks to the mid-log phase, and then mixed; the temperature difference is controlled to be ≤5℃, and 0.1-0.5% trehalose is added as a compatibility agent.

[0058] It can be understood that the technical solution adopts the process of culturing the Lactobacillus rhamnosus and the yeast in separate tanks to the mid-log phase (OD600=0.6-0.8) and then mixing, by strictly controlling the temperature difference to be ≤5℃ and adding 0.1-0.5% trehalose as a compatibility agent, three breakthrough effects are realized: first, the phased culture effectively avoids the inhibition of yeast acid production (pH≤4.5) on lactobacilli, and the trehalose forms a protective hydration layer to reduce the exchange of toxic metabolites of the bacteria, so that the survival rate of mixing is improved; second, the precise selection of the mid-log phase mixing ensures that the glutathione precursor material secreted by the yeast reaches the peak value, significantly enhancing the antioxidant capacity of lactobacilli, and the bacteriocin produced by lactobacilli is also in the best antibacterial state (the diameter of the inhibition zone increases by 5mm); finally, the addition of trehalose not only reduces the risk of osmotic shock, but also promotes the formation of coaggregation biofilm, so that the intestinal colonization efficiency is improved. The process makes the viable bacteria yield of the final product reach more than 5×10^10 CFU / mL, the acetaldehyde clearance efficiency is increased to 92%, the intestinal mucus secretion is increased by nearly 1 times (28mg / g), the product shelf life is extended by 50% (the viable bacteria retention rate is >80% for 24 months), the alcohol effect time is shortened to 25 minutes, and the problem of strain antagonism in co-culture of probiotics is perfectly solved.

[0059] The specific embodiment also provides an application of a biological agent for relieving alcohol-induced intestinal discomfort, and the biological agent is made into a probiotic pressed tablet candy, a probiotic pill or a probiotic oil droplet.

[0060] In some preferred embodiments, the compressed tablet candy is for preventive use before drinking, the probiotic oil droplet liquid is for emergency treatment after drinking, and the probiotic pill is for intestinal repair of long-term alcohol consumers.

[0061] The application will be further described in connection with specific embodiments.

[0062] Example 1

[0063] Antialcoholic compressed tablet candy

[0064] Component formula (parts by weight): Bacillus coagulans 12 parts, Pediococcus pentosaceus 18 parts, Lactobacillus rhamnosus 8 parts, Saccharomyces boulardii 8 parts, Bifidobacterium 3 parts, Lactococcus lactis 2 parts, inulin 35 parts, fructooligosaccharide 6 parts, L-cysteine 40 parts, microcrystalline cellulose 3 parts, magnesium stearate 1 part, and hydroxypropyl methylcellulose (HPMC) coating agent in an appropriate amount (coating weight gain 3%).

[0065] Preparation method:

[0066] 1) Strain culture:

[0067] Bacillus coagulans (aerobic culture, spore formation): culture medium LB liquid medium (pH 6.8), culture conditions: temperature 37℃±1℃, dissolved oxygen (DO) 40%±5%, time 18h (to spore formation rate ≥90%) post-treatment: 80℃ heat treatment for 10min, inactivate vegetative cells, retain acid-resistant spores.

[0068] Pediococcus pentosaceus & Lactococcus lactis (co-culture, anaerobic), culture medium: MRS liquid medium (pH 6.5), inoculation ratio: Pediococcus pentosaceus: Lactococcus lactis = 1:0.3, culture conditions: temperature: 31℃±1℃, anaerobic environment (N2 replacement, O2<0.1%), time: 14h (to OD600=1.2-1.5).

[0069] Lactobacillus rhamnosus (single culture, microaerophilic), culture medium: MRS liquid medium (pH 5.8), culture conditions: temperature 37℃±1℃, microaerophilic (5% CO2), time 20h (to OD600=1.0).

[0070] Bifidobacterium longum (strict anaerobic culture), culture medium: modified MRS (add 0.05% cysteine-HCl, pH 7.0), culture conditions: temperature 38℃, strict anaerobic (anaerobic workstation, O2<0.01%), time: 24h (to OD600=0.8).

[0071] B. subtilis (aerobic culture), medium: LB liquid medium (pH 6.8), condition: 37℃ aerobic culture for 18h (DO 40%), post-treatment: heat treatment at 80℃ for 10min to reserve spores.

[0072] (2) Cell collection and complex protection

[0073] Centrifugal collection: centrifuge each bacterial solution at 4℃, 8000rpm for 10min, discard the supernatant, and wash twice with 0.85% sterile normal saline.

[0074] Complex protection: mix the wet cells, add freeze-drying protective agent (trehalose: skim milk = 1:2, total weight 5%), freeze-dry, mix the obtained mixed powder through an 80-100 mesh sieve, control the final product moisture ≤5%, and the total number of viable bacteria ≥1×10 11 CFU / g.

[0075] (3) Tabletting

[0076] Mix the above freeze-dried bacterial powder, inulin, fructooligosaccharide, L-cysteine and microcrystalline cellulose in a V-type mixer for 30min.

[0077] Tabletting: pressure 10kN, tablet weight 1.0g / tablet, coating: enteric coating (Eudragit L30D-55, weight gain 3%).

[0078] Example 2

[0079] This Example 2 provides a probiotic oil droplet liquid

[0080] Component formula (parts by weight): B. coagulans 15 parts, P. pentosaceus 25 parts, B. subtilis 8 parts, L. rhamnosus 8 parts, B. longum 3 parts, L. lactis 3 parts, sunflower seed oil 40 parts, perilla seed oil 20 parts, α-tocopherol (α-VE) 0.5 parts, enteric coating material (Eudragit L30D-55) 3 parts.

[0081] Preparation method:

[0082] (1) Strain culture

[0083] B. coagulans culture: medium: LB liquid medium (pH 6.8), condition: 37℃ aerobic culture for 18h (DO 40%), post-treatment: heat treatment at 80℃ for 10min to reserve spores;

[0084] P. pentosaceus and L. lactis co-culture: medium: MRS liquid medium (pH 6.5), condition: 31℃ anaerobic culture for 14h (N2 environment);

[0085] Culture of other strains:

[0086] Lactobacillus rhamnosus: 37°C microaerobic culture for 20h (pH 5.8), Bifidobacterium longum: 38°C strict anaerobic culture for 24h, Saccharomyces boulardii: 27°C aerobic culture for 30h.

[0087] (2) Cell treatment

[0088] Centrifugation (8000rpm, 10min, 4°C), washed twice with physiological saline, mixed with lyophilization protectant (trehalose: skim milk = 1:2), freeze-dried (-45°C pre-freezing, gradient drying).

[0089] (3) Oil phase preparation

[0090] Sunflower seed oil and perilla seed oil were mixed (60°C stirring to dissolve α-tocopherol), and freeze-dried bacterial powder (passed through a 100-mesh sieve) was added and homogenized.

[0091] (4) Capsule preparation

[0092] Soft capsules were prepared using a rotary die method (0.5g / capsule), enteric-coated (Eudragit L30D-55, 3% weight gain), and dried (30°C, 40% relative humidity, 24h).

[0093] Example 3

[0094] Example 3 provides a probiotic pill

[0095] Component formulation (parts by weight): Bacillus coagulans 15 parts (acid-resistant spore preparation), Pediococcus pentosaceus 25 parts (ADH activity ≥ 150 U / mg), Saccharomyces boulardii 10 parts, Lactobacillus rhamnosus 8 parts, Bifidobacterium longum 3 parts, Lactococcus lactis 3 parts, inulin 40 parts, fructooligosaccharide (FOS) 5 parts, vitamin C 3 parts, L-cysteine 35 parts, glutathione 20 parts, microcrystalline cellulose 3 parts, and silicon dioxide 1 part.

[0096] Preparation method:

[0097] (1) Strain culture stage

[0098] Bacillus coagulans: culture medium: LB liquid medium (pH 6.8), culture conditions: 37°C, DO 40%, 18h, post-treatment: 80°C heat treatment for 10min to retain spores.

[0099] Pediococcus pentosaceus and Lactococcus lactis co-culture: culture medium: MRS liquid medium (pH 6.5), inoculation ratio 1:0.3, culture conditions: 31°C anaerobic culture for 14h.

[0100] Other strains culture: Lactobacillus rhamnosus: MRS medium (pH 5.8), 37℃ microaerobic culture for 20h; Bifidobacterium longum: modified MRS medium (+0.05% cysteine), 38℃ strict anaerobic culture for 24h; Saccharomyces boulardii: YPD medium (pH 5.0), 27℃ aerobic culture for 30h.

[0101] (2) Bacterial cell treatment, centrifugal collection (8000rpm, 10min, 4℃), washing with physiological saline for 2 times, adding freeze-drying protective agent (trehalose: skim milk = 1:2, total weight 5%), freeze-drying: pre-freezing at -45℃ for 4h, main drying at -25℃→20℃ (24h), resolving drying at 25-30℃ (8h).

[0102] (3) Powder preparation

[0103] Inulin, fructooligosaccharide are sieved through 100 mesh, vitamin C, L-cysteine, glutathione are crushed to 80 mesh; three-stage mixing: first stage: mixing of freeze-dried probiotic powder (V-type mixer, 15min), second stage: adding active ingredients (vitamin C, L-cysteine, glutathione) and mixing for 20min, third stage: adding prebiotics and excipients and mixing for 30min.

[0104] Sub-packing: aluminum foil bag is filled with nitrogen and packaged (5g / bag), moisture control ≤5%.

[0105] Example 4

[0106] The present example 4 provides a biological agent

[0107] Component formula (weight parts): Bacillus coagulans 15 parts, Pediococcus pentosaceus 25 parts, Lactobacillus rhamnosus 8 parts, Saccharomyces boulardii 10 parts, Bifidobacterium longum 3 parts, Lactococcus lactis 3 parts.

[0108] Preparation method:

[0109] (1) Strain culture process

[0110] Bacillus coagulans: culture medium LB liquid medium (pH 6.8), culture parameters: 37℃, dissolved oxygen 40%, 18h; post-treatment: 80℃ heat treatment for 10min to reserve spores.

[0111] Pediococcus pentosaceus and Lactococcus lactis: co-culture ratio 1:0.3, culture medium: MRS liquid medium (pH 6.5), culture conditions: 31℃ anaerobic culture for 14h.

[0112] Lactobacillus rhamnosus: culture medium: MRS (pH 5.8), culture parameters: 37℃ microaerobic culture for 20h.

[0113] Bifidobacterium longum: Special medium: Modified MRS (add 0.05% Cysteine-HCl), culture condition: 38℃, strict anaerobic culture for 24h.

[0114] Saccharomyces boulardii: Medium: YPD (pH 5.0), culture parameters: 27℃, aeration amount 0.5vvm, 30h.

[0115] (2) Bacterial cell treatment: centrifugal collection: 8000rpm x 10min (4℃), washing procedure: 0.85% physiological saline washing for 2 times, adding 5% freeze-drying protective agent (trehalose: skim milk = 1:2).

[0116] (3) Freeze-drying process: pre-freezing stage: -45℃ for 4h, main drying: -25℃→20℃ (24h, vacuum degree 0.05mbar), analytical drying: 25-30℃ (8h, vacuum degree 0.01mbar).

[0117] (4) Product preparation: sieving treatment: 80-100 mesh, moisture control: ≤5% (Karl Fischer method), viable bacteria standard: ≥1×10 11 CFU / g.

[0118] Comparative Example 1

[0119] Comparative Example 1 provides a hangover-type tablet candy

[0120] The formula is as follows: Bacillus coagulans 12 parts, Pediococcus pentosaceus 18 parts, Lactobacillus rhamnosus 8 parts, (canceling Saccharomyces boulardii, Bifidobacterium longum, Lactococcus lactis), and the remaining components are the same as in Example 1.

[0121] The preparation method is exactly the same as the process parameters of Example 1, only the culture steps of the three strains are reduced.

[0122] Comparative Example 2

[0123] Comparative Example 2 provides a magneto-rheological fluid and a preparation method thereof

[0124] The formula is as follows: strain group is the same as Example 1, canceling inulin and fructooligosaccharide, L-cysteine is changed to 20 parts, and the excipients are the same as in Example 1.

[0125] The preparation method is the same as Example 1.

[0126] Performance test

[0127] The following performance tests are performed on Examples 1-4 and Comparative Examples 1 and 2.

[0128] I. Test method

[0129] Viable bacteria count

[0130] Method: GB 4789.35-2016 Food microbiology test Lactic acid bacteria test, condition: gastric acid simulation solution (pH 2.0, containing 0.3% pepsin, 37℃ for 2h), intestinal fluid simulation solution (pH 6.8, containing 0.1% trypsin, 37℃ for 6h).

[0131] Alcohol metabolism capacity

[0132] In vitro model: initial concentration of ethanol 100mg / dL, anaerobic culture at 37℃ for 3h, HPLC detection of acetaldehyde and acetic acid content (chromatographic column: Agilent ZORBAX SB-C18).

[0133] Intestinal barrier function

[0134] Animal experiment:

[0135] SD rat alcoholic intestinal injury model (gavage 50% ethanol, 5g / kg), immunohistochemical detection of ZO-1 and Occludin protein expression, ELISA detection of serum LPS level.

[0136] Stability test

[0137] Accelerated test: 40℃ / 75%RH, 0 / 1 / 3 / 6 months sampling.

[0138] Viable bacteria retention rate: plate count method.

[0139] II. Comparison of test results

[0140] 1. Viable bacteria performance

[0141]

[0142] 2. Alcoholism efficacy

[0143]

[0144] 3. Intestinal protection effect

[0145]

[0146]

[0147] 4. Dosage form characteristics

[0148] Test parameters Example 1 (tablets) Example 2 (oil drop) Example 3 (pills) Disintegration / release time (min) 12 ± 2 (gastric juice) 45 ± 5 (enteric) 15 ± 3 (water) Peroxide value (meq / kg) - 2.1±0.3 - Water activity (Aw) 0.25±0.02 0.18±0.01 0.22±0.02

[0149] From the above results, (1) the acetaldehyde removal rate of the complete flora combination (Example 1) was 41-73% higher than that of Comparative Example 1 (lacking 3 species) (p<0.01), and the absence of S. boulardii led to a 46% decrease in mucus secretion; (2) the intestinal ZO-1 expression of the inulin / L-cysteine-containing group (Example 1) was 35-42% higher than that of Comparative Example 2, and the oil droplets liquid lacking alpha-tocopherol (Comparative Example 2) had a 300% excess peroxide value; (4) heat treatment of spores increased the survival rate in gastric acid by 27% (compared to the untreated group); and the gradient freeze-drying process increased the stability of live bacteria by 1.9 times.

[0150] Note: All data are mean ± SD, n = 6, p < 0.05 (t-test) compared with the corresponding example group

[0151] The above description is only the preferred embodiments of the present application, and does not limit the present application in any form. Although the present application has been shown and described as above, it is not intended to limit the present application, and any person skilled in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application still belong to the scope of the technical solution of the present application.

Claims

1. A biological agent for alleviating alcohol-induced intestinal discomfort, characterized in that, The following components are included by weight parts: Bacillus coagulans 5-20 parts, Pediococcus pentosaceus 10-30 parts, Lactobacillus rhamnosus 5-10 parts, yeast 0-20 parts, Bifidobacterium longum 0-5 parts and Lactococcus lactis 0-5 parts.

2. The biological agent for alleviating alcohol-induced intestinal discomfort according to claim 1, wherein The yeast is Saccharomyces boulardii.

3. The biological agent for alleviating alcohol-induced intestinal discomfort according to claim 1, wherein The Pediococcus pentosaceus has ethanol dehydrogenase activity.

4. The biological agent for alleviating alcohol-induced intestinal discomfort according to claim 1, wherein The biological preparation further includes, calculated by weight fraction: inulin 20-50 parts, L-cysteine 25-50 parts and / or pharmaceutically acceptable adjuvant 1-5 parts.

5. The biological agent for alleviating alcohol-induced intestinal discomfort according to claim 1, wherein The biological preparation further includes, calculated by weight fraction: fructooligosaccharides 0-10 parts, glutathione 10-30 parts and / or alpha-tocopherol 0.1-1 part.

6. A method for preparing a biological agent for relieving alcohol-induced intestinal discomfort according to claim 1, characterized in that, The following steps are included: Bacillus coagulans is aerobically cultured at pH 6.5-7.0, temperature 35-37℃, dissolved oxygen DO 30-50% for 16-20 hours; Pediococcus pentosaceus and Lactococcus lactis are co-cultured at a inoculation ratio of 1:0.2-0.5 under anaerobic conditions at pH 6.2-6.8, 30-32℃ for 12-15 hours; Lactobacillus rhamnosus is cultured alone at pH 5.5-6.0, 37℃ for 18-24 hours; Bifidobacterium longum is cultured under strict anaerobic conditions at pH 6.8-7.2, 37-39℃ for 20-28 hours; Yeast is cultured at pH 4.5-5.5, 25-28℃ under aeration conditions for 24-36 hours; Bacterial cell collection and composite protection: Each strain is collected by centrifugation at the end of the logarithmic growth phase and washed with buffer; Composite freeze-drying: After mixing the above wet bacterial cells, add freeze-drying protectant and vacuum freeze-dry: pre-freeze to below -45℃, main drying stage -25℃~20℃, desorption drying stage 25-30℃; Bacterial powder standardization: The resulting mixed powder is mixed through a 80-100 mesh screen, controlling the final product moisture < 5%, total viable count > 1 x 10 11 CFU / g.

7. A method for preparing a biological agent for relieving alcohol-induced intestinal discomfort according to claim 6, characterized in that, The Bacillus coagulans is heat-treated at 80℃ for 10 minutes before collection to inactivate vegetative cells and retain spore morphology.

8. The method of claim 6, wherein the biological agent is prepared by the steps of: a) obtaining a culture of Lactobacillus fermentum; b) mixing the culture with a carrier; and c) lyophilizing the mixture. The Lactobacillus rhamnosus and yeast are mixed after separate tank cultivation to the middle logarithmic phase; the temperature difference is controlled to be ≤5℃ during mixing, and 0.1-0.5% trehalose is added as a compatibility agent.

9. Use of a biological agent for alleviating alcohol-induced intestinal discomfort according to any one of claims 1 to 5, characterized in that, The biological preparation is made into probiotic tablet candies, probiotic pills or probiotic oil droplets.

10. Use according to claim 9, characterized in that, The tablet candies are used for preventive use before drinking, the probiotic oil droplets are used for emergency treatment after drinking, and the probiotic pills are used for intestinal repair of long-term alcohol consumers.

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