Lactobacillus plantarum and fermented products that enhance intestinal immunity

Abstract

The present invention discloses a plant lactobacillus PC037 and a fermented product for improving intestinal immunity. The plant lactobacillus PC037 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 13, 2021, with a deposit number of CGMCC No. 24096. The plant lactobacillus PC037 can increase the repair speed of the intestinal mucosa and promote the enhancement of intestinal immunity. The plant lactobacillus fermented product for improving intestinal immunity provided by the present invention can regulate the frequency of flatulence, promote the enhancement of intestinal immunity, and reduce the number of diarrhea caused by pathogen infection during travel.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, in particular to a Lactobacillus plantarum capable of improving intestinal immunity and a fermented product. Background Art

[0002] The intestinal mucosa is the largest mucosal immune organ in animals, accounting for 70% of infections. An intact and healthy intestinal mucosa is crucial for strong intestinal immunity, playing a greater role in combating pathogen invasion and metastasis. Unlike the systemic immune system, it primarily involves gut-associated lymphoid tissue and secretory IgA. The intestinal mucosal immune system can not only respond to stimulation to produce local mucosal immune responses but also induce systemic immune responses.

[0003] The intestinal microecology is a unified entity where the intestinal flora and the intestinal mucosa coexist and interact, maintaining a dynamic equilibrium. The intestinal microecological barrier, composed of beneficial bacteria, opportunistic pathogens, and harmful bacteria, can total up to 100 trillion bacteria and plays a vital role in maintaining human health and immunity. The biological barrier it forms can defend against infection and prevent or inhibit the invasion of pathogens. Normal intestinal flora continuously stimulate the mucosa, activating mucosal immunity and maintaining normal mucosal immunity. They also act as an immune barrier against pathogens through both clearance and rejection.

[0004] Research has found that the intestinal microbiota can regulate the balance between effector and regulatory T cells, thereby promoting immune homeostasis. Certain species of Clostridium and Bacteroides fragilis have been found to stimulate the differentiation of colonic Treg cells, which in turn induce IL-10 secretion, thereby alleviating pathological inflammation and maintaining intestinal health. This suggests that the intestinal microbiota and the intestinal mucosal barrier are interdependent and mutually influential, acting as the guardians of the body's internal immune environment.

[0005] When the human body's intestinal immunity is low, it is more susceptible to infection by pathogens, intestinal mucosal damage is aggravated, and intestinal flora is unbalanced, causing intestinal spasms, excessive intestinal fluid secretion, intestinal rumbling, harmful bacteria breeding, repeated intestinal inflammation and other adverse reactions, which manifest as one or more symptoms such as abdominal pain, abdominal distension, diarrhea, constipation, indigestion, bloody stools, etc., seriously affecting people's lives and endangering human health.

[0006] Lactobacillus plantarum is a common lactic acid bacterium found in fermented cheese, fermented vegetables, fermented meat products, silage, and the intestines. It has a strong tolerance to gastric acid and bile salts and a strong ability to kill pathogens. It has multiple beneficial effects, including preventing cancer and cardiovascular and cerebrovascular diseases, inhibiting harmful bacteria, regulating immunity and the gastrointestinal tract, and reducing cholesterol and nitrite levels. Studies have shown that Lactobacillus plantarum produces several metabolites, such as EPS, SCFAs, and CLA, which have significant effects on alleviating intestinal diseases. EPS, a heterogeneous structure secreted by the strain, helps antagonize pathogens, inhibit colonic inflammatory cell infiltration, alleviate tissue lipid peroxidation, and enhance human immunity. In cases of intestinal inflammation, EPS can activate macrophages, promote T cell proliferation, and inhibit TNF-α expression, thereby inhibiting the progression of intestinal inflammation. Due to individual strain differences, the beneficial effects of these bacteria vary. Even for the same strain, changes in the fermentation substrate can affect the beneficial effects. Summary of the Invention

[0007] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a Lactobacillus plantarum that improves intestinal immunity, which can increase the repair speed of intestinal mucosa and promote the enhancement of intestinal immunity.

[0008] The second purpose of the present invention is to provide a plant lactobacillus fermented product that can improve intestinal immunity, regulate flatulence frequency, promote intestinal immunity enhancement, and reduce the incidence of diarrhea caused by pathogen infection during travel.

[0009] The third object of the present invention is to provide a method for preparing a Lactobacillus plantarum fermentation product for improving intestinal immunity.

[0010] One of the purposes of the present invention is achieved by the following technical solution:

[0011] A plant lactobacillus for improving intestinal immunity, wherein the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) PC037, the plant lactobacillus PC037 was deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2021, with the deposit number CGMCC No. 24096.

[0012] As an optional solution of the present invention, the Lactobacillus plantarum PC037 exerts the effect of improving intestinal immunity through at least one of the following items (I) to (IV):

[0013] (I) Improve the repair speed of intestinal mucosa;

[0014] (II) Reduce intestinal inflammation;

[0015] (III) Adjust the exhaust frequency;

[0016] (IV) Reduce the frequency of diarrhea.

[0017] As an optional solution of the present invention, the Lactobacillus plantarum PC037 improves the repair speed of the intestinal mucosa by one or more of the following: reducing the number of days required for the DAI score to be less than 3%, reducing MPO activity, reducing TGF-β content, and increasing EGF content.

[0018] The second object of the present invention is achieved by adopting the following technical solution:

[0019] The present invention provides a product for improving intestinal immunity. The active ingredient of the product comprises live bacteria and / or inactivated bacteria of Lactobacillus plantarum PC037. The Lactobacillus plantarum PC037 is the Lactobacillus plantarum described in one of the purposes of the present invention.

[0020] As an optional solution of the present invention, the products include health products and medicines.

[0021] As an optional solution of the present invention, the product includes a solid product, a liquid product, and a semi-solid product.

[0022] As an optional solution of the present invention, the products include probiotic freeze-dried powder and probiotic fermentation products.

[0023] The present invention also provides a fermented product for improving intestinal immunity, which is made by processing a probiotic fermentation product. The probiotic fermentation product is formed by fermenting a composition with Lactobacillus plantarum PC037. The composition includes the following components in parts by weight: 30 to 40 parts of oats, 10 to 20 parts of brown rice, 5 to 17 parts of Oyster mushrooms, 12 to 25 parts of Morels, 10 to 20 parts of Dictyophora, 6 to 15 parts of glucose, 5 to 15 parts of walnuts, and 800 to 1000 parts of purified water.

[0024] Oats are rich in dietary fiber, with the bran containing up to 30% of the fiber, which has a honeycomb-like appearance. Small-molecule glucans, primarily β-glucans, are the most viscous. Found primarily in the endosperm and the aleurone cell walls of the endosperm, β-glucans are the highest soluble fiber in oats, reaching a maximum concentration of 6%. β-glucans can enhance intestinal immunity and have certain anti-cancer effects. They can also regulate intestinal probiotics, maintain intestinal moisture and stool volume, and promote the proliferation of beneficial bacteria, thereby forming a protective film. This reduces the retention time of excrement and prevents the reabsorption of metabolic toxins, helping to prevent intestinal diseases such as colorectal cancer.

[0025] Brown rice, the product of hulled rice, primarily consists of the bran layer, germ, and endosperm. The aleurone layer and germ are rich in many bioactive ingredients, such as vitamin E, linoleic acid, rice bran protein, rice germ protein, GABA, glutamic acid, inositol hexaphosphate, and γ-oryzanol. These ingredients have been shown to enhance immunity, remove oxygen free radicals, lower blood lipids, prevent cardiovascular and cerebrovascular diseases, and slow aging. Furthermore, the dietary fiber in the bran layer helps promote gastrointestinal motility, excreting food residue and decomposed toxins. This helps treat digestive tract disorders such as stomach problems, constipation, and hemorrhoids, as well as prevent cancer. According to the Compendium of Materia Medica, brown rice has the miraculous effect of "harmony of the five internal organs and improving complexion."

[0026] Tricholoma is a rare edible and medicinal mushroom in my country. Its mild nature and sweet flavor are known to benefit the intestines and stomach, dispelling heat and relieving exterior symptoms. It is suitable for treating indigestion, abdominal distension, stomach pain, and diarrhea. Pharmacological studies have shown that Tricholoma is rich in selenium, polysaccharides, lectins, sterols, and other chemical components. The selenium content is as high as 2.565 μg / g, which can enhance immunity, fight aging, inhibit cancer cell proliferation, and protect the heart from ischemia / hypoxia.

[0027] Morels are a rare and precious fungus used as both a medicine and food worldwide. They are highly nutritious and medicinally valuable. Rich in polysaccharides, enzymes, amino acids, pyrone antibiotics, and fatty acids, they possess significant anti-tumor and anti-viral properties, as well as immune-boosting properties. According to the Compendium of Materia Medica, morels are described as "neutral, sweet, and non-toxic," and have the benefits of "benefiting the stomach and intestines, aiding digestion, resolving phlegm and regulating qi, tonifying the kidneys and promoting qi, and nourishing the brain and mind."

[0028] Dictamnus, also known as bamboo fungus and bamboo ginseng, contains polysaccharides and a variety of trace elements. Dictamnus polysaccharide is a heteropolysaccharide, a highly active macromolecular substance, mainly distributed in the cell wall of the spore body of Dictamnus. The polysaccharide components are galactose, glucose, mannose and xylose, which can enhance the phagocytic function of neutrophils and promote the formation of antibodies in the spleen, thereby realizing immune regulation function. In addition, Dictamnus also contains a large amount of rare element germanium, which can enhance the ability of macrophages to kill foreign viruses, regulate and enhance the body's own immune function and disease prevention and anti-cancer ability.

[0029] Walnuts are rich in various active ingredients, such as unsaturated fatty acids, high-quality protein, polyphenol compounds, vitamins and minerals. They have strong antioxidant properties and can improve the body's immunity by regulating IL-10 and IL-17A. In addition, the protein content in walnuts is 15-18%, mainly composed of globulin, gluten, albumin and alcohol-soluble protein. They are rich in 18 kinds of amino acids and have a high amino acid score and a balanced proportion. They can be used as a source of high-quality protein for the human body.

[0030] The fermented product for improving intestinal immunity provided by the present invention has a composition whose raw material selection and formula compatibility have been verified through multiple experiments, and an optimal formula is finally obtained. The composition is rich in nutrition and is very novel and creative. The composition is then processed and manufactured using an innovative process. In particular, Lactobacillus plantarum PC037 is selected for deep fermentation. This not only biotransforms nutrients such as protein, dietary fiber, glucan, selenium, germanium, and polysaccharides in the composition, but also produces a variety of prebiotic factors such as more easily absorbed amino acids, short-chain fatty acids, mucopolysaccharides, and bacteriocins. The resulting fermented product effectively reduces the incidence of diarrhea caused by pathogenic bacteria infection among travelers during travel, thereby improving the human intestinal immunity through internal regulation.

[0031] As an optional solution of the present invention, the composition includes the following components in parts by weight: 30 parts of oats, 20 parts of brown rice, 17 parts of button mushrooms, 12 parts of morels, 10 parts of bamboo fungus, 6 parts of glucose, 5 parts of walnuts, and 800 parts of purified water.

[0032] As an optional solution of the present invention, the probiotic fermentation product is produced by fermenting a composition with Lactobacillus plantarum PC037, and the fermentation includes a first fermentation stage and a second fermentation stage.

[0033] The third object of the present invention is achieved by adopting the following technical solution:

[0034] A method for preparing a fermented product for improving intestinal immunity as described in the second purpose, comprising the following steps:

[0035] 1) Select dry, mold-free, and insect-free oats, brown rice, morels, bamboo fungus, and walnuts. Wash and drain all of these ingredients, and weigh the formulated amounts of oats and brown rice. Soak the morels and bamboo fungus, then wash and squeeze out the water, and weigh the formulated amounts of morels and bamboo fungus. Shell the walnuts and take the kernels according to the formulated amount. Select fresh button mushrooms, wash and drain, and weigh the formulated amount of glucose.

[0036] 2) Soak the brown rice in clean water for 12–24 hours, then pour it into a colander. Cover the colander with sterilized, soaked, and wrung-out gauze. Rinse with 30–40°C warm water and spray with 30–40°C warm water 1–5 times daily. Allow the rice to germinate for 20–30 hours. Collect the germinated brown rice when the sprouts reach 0.5–0.9 cm in length.

[0037] 3) Broken oats, germinated brown rice, and a portion of glucose were treated, and purified water was added during the process to prepare a broken oats slurry. The breaking parameters were 22,000-28,000 rpm for 1-3 minutes. The pH was adjusted to 6.2-6.6, and the temperature was maintained at 115-135°C for 15-20 minutes. The mixture was then cooled to 36-38°C to prepare Solution 1.

[0038] 4) After activating Lactobacillus plantarum PC037 for 2 to 5 generations, collect the bacterial suspension and centrifuge it at 1 to 4°C and 5000 to 8000 rpm for 15 to 20 minutes. Collect the precipitate and dilute it with sterile saline until the viable count is 1 × 10 9 ~1×10 10 CFU / mL, resuspend to obtain bacterial suspension;

[0039] 5) Add the bacterial suspension to Solution 1 at a ratio of 3-6%, stir evenly, and ferment at a constant pH of 36-38°C for 12-24 hours. Filter through a 60-80 mesh filter and collect the filtrate as Solution 2.

[0040] 6) Solution No. 2 was pre-frozen at -15 to -25°C for 18 to 36 hours, then warmed to 1 to 5°C and placed in an ice bath for ultrasonic disruption at 700 to 800 MHz, with an on-time of 1 to 5 seconds, a 1 to 5 second pause, and a duration of 20 to 40 minutes to obtain a fermentation lysate.

[0041] 7) Crack the mushrooms, morels, bamboo fungus, walnuts, and the remaining glucose, adding the remaining purified water to make a cracked slurry at a speed of 22,000-28,000 rpm for 1-5 minutes, maintaining at 115-135°C for 15-20 minutes, then cooling to 36-38°C. Add the fermentation lysate, stir evenly, and adjust the pH to 6.2-6.6 to prepare Solution 3.

[0042] 8) Add the bacterial suspension to solution No. 3 at a ratio of 5-10%, stir evenly, and ferment at a constant pH of 36-38°C for 8-12 hours to prepare the fermentation broth.

[0043] 9) Place the fermentation liquid on a plate and freeze it at -15 to -25°C for 12 to 24 hours. Then take it out and freeze-dry it in a freeze dryer for 12 to 24 hours. Grind it into 60 to 80 mesh with a grinder to obtain the fermentation product.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The Lactobacillus plantarum PC037 provided by the present invention can increase the repair speed of intestinal mucosa and promote the enhancement of intestinal immunity.

[0046] (2) The fermented product for improving intestinal immunity provided by the present invention can regulate the frequency of flatulence, promote the enhancement of intestinal immunity, and reduce the incidence of diarrhea caused by pathogenic bacteria infection during travel.

[0047] (3) The fermented product of the present invention adopts an innovative two-stage fermentation method. In the first stage of fermentation, Lactobacillus plantarum PC037 fully proliferates, transforming various nutrients such as amino acids, proteins, unsaturated fatty acids, dietary fiber, vitamins, etc. in oats and brown rice, and produces probiotic factors such as uronic acid polymers, short-chain fatty acids, and bacteriocins. After filtering to remove crude fiber, it is lysed to release active enzymes in the cells. In the second stage of fermentation, it proliferates to the most vigorous stage, transforming rare elements, polysaccharides, amino acids, and other nutrients in Agaricus edodes, Morchella esculenta, Dictyophora indicus, and walnuts, while also undergoing secondary biotransformation of the fermentation lysate. The resulting fermented product is rich in active probiotics and postbiotics, has a stronger affinity for the human intestine, can effectively improve intestinal immunity, build a strong gastrointestinal mucosa, reduce the risk of intestinal infection by pathogens, and protect intestinal health.

[0048] Biomaterial preservation information: Lactobacillus plantarum PC037, preservation number is CGMCC No.24096, classification name: Lactobacillus plantarum Lactobacillus plantarum , was deposited on December 13, 2021 in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101). The abbreviation of the depository is CGMCC. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the raw materials, equipment, etc. used in the following embodiments can be purchased through commercial channels.

[0050] The embodiment of the present invention provides a Lactobacillus plantarum PC037 capable of improving intestinal immunity, which is obtained through animal experiment screening.

[0051] System 1: Screening of the best bacteria to improve intestinal immunity

[0052] 1. Experimental strains

[0053] Lactobacillus plantarum CICC 25282 (Deposit number: CICC 25282), Lactobacillus plantarum CICC 24936 (Deposit number: CICC 24936), Lactobacillus plantarum GDMCC 1.4956 (Deposit number: GDMCC 1.4956), Lactobacillus plantarum CICC 25283 (Deposit number: CICC 25283), Lactobacillus plantarum PC037.

[0054] 2. Sample preparation

[0055] After Lactobacillus plantarum was activated for 3 generations, the bacterial suspension was collected and centrifuged at 4°C and 6000 r / min for 20 min. The precipitate was collected and the viable bacterial count was adjusted to 1×10 with sterile saline. 9 CFU / mL, and resuspend to obtain bacterial suspension.

[0056] 3. Experimental Animals

[0057] C57BL / 6 female mice, 8 weeks old, weighing 19 g ± 1 g, were randomly divided into 7 groups, 10 mice each, as shown in Table 1.

[0058] 4. The SPF animal room was maintained at a temperature of 24°C with a 12-hour light and dark cycle. Animals were provided with ample food and water during both animal husbandry and experimental procedures. After acclimation to the mice for 5 days, the initial weight of each group was recorded. All mice in all groups, except Group A, were allowed to drink freely a 3% DDS solution for 5 days. The 3% DDS solution was replaced every 2 days, and after the fifth day, the DDS solution was removed and replaced with normal drinking water. Starting on the sixth day, all mice were gavage-administered with samples listed in Table 1 at a dose of 10 mL / kg for 10 consecutive days.

[0059] Table 1 Mouse groups and gavage samples

[0060]

[0061] 5. The mice were weighed and DAI scores were taken every day. Fecal occult blood was detected using a fecal occult blood kit. The scoring criteria are shown in Table 2. The number of days required for the DAI score to be <3% was recorded. The results are shown in Table 3.

[0062] Table 2 DAI scoring criteria

[0063]

[0064]

[0065] Where: W: Percentage of body weight loss. A: Initial body weight of the mouse. B: Body weight of the mouse on the day of scoring.

[0066] DAI score = weight loss score + blood in stool score + stool characteristics score

[0067] 6. Six days after oral administration, 5 mice were randomly selected from each group. The selected mice were fasted for 12 hours and anesthetized with 7% chloral hydrate, and the colon tissues were obtained.

[0068] (1) Weigh 50 mg of colon tissue and prepare a 10% homogenate with 0.5% HTBA solution. Centrifuge at 14,000 r / min for 15 min at 4°C. Take 80 μL of the supernatant and add 120 μL of 0.0005% phenyldianisidine dihydrochloride containing 0.1% H2O2. Measure the A460 absorbance every 30 s with a microplate reader. Calculate the MPO activity based on the average change rate of the absorbance within 2 min. Calculate the average value. The results are shown in Table 4.

[0069] (2) Weigh 0.5 g of colon tissue and prepare a 10% homogenate with phosphate buffer solution (pH 7.4). Centrifuge at 5000 rpm for 10 min at 4°C, and collect the supernatant. Determine the TGF-β and EGF content according to the ELISA kit instructions. Calculate the average value. Set up two replicate wells for each sample. The results are shown in Table 4.

[0070] 7. Results and Analysis

[0071] (1) Scoring of mice

[0072] Table 3 The number of days required for mice to reach DAI score < 3%

[0073]

[0074] DDS-induced colonic injury in mice is a well-established experimental enteritis model, and the induced colonic injury model is stable. Results showed that compared with group A, the number of days required for mice in group B to reach a DAI score of <3% was significantly increased, indicating that the intestinal mucosal injury mouse model was successfully established. Compared with group B, the number of days required for mice in groups C through G to reach a DAI score of <3% was reduced, indicating that Lactobacillus plantarum has a repairing effect on damaged intestinal mucosa. Different strains have different repair rates, with mice in group G requiring the fewest days to reach a DAI score of <3%, indicating the fastest recovery. This suggests that Lactobacillus plantarum PC037 can accelerate the repair of intestinal mucosa, contributing to the establishment of a complete and healthy intestinal mucosa and enhancing intestinal immunity.

[0075] (2) Changes in colon indicators in mice

[0076] Table 4 Changes in colonic indices of mice

[0077]

[0078] MPO is a marker of neutrophil activation. Its level and activity represent the function and status of neutrophils. In inflammatory responses, the release and activation of MPO participate in the natural immune response to fight against the invasion of pathogens. TGF-β maintains the integrity of the intestinal mucosa by regulating the renewal and damage repair of the intestinal mucosal epithelium. EGF is produced by colonic goblet cells, which can induce the differentiation and proliferation of colon cells and is the main regulatory factor that promotes intestinal mucosal repair. The results showed that compared with group A, the MPO activity and TGF-β content of mice in group B were significantly increased, and the EGF content was significantly decreased, indicating that the intestinal mucosa of the mice was damaged, leading to aggravated inflammation, the mucosa was not effectively restored, and the intestinal immunity was low. Compared with group B, the MPO activity and TGF-β content of mice in groups C to G decreased, while the EGF content increased, indicating that Lactobacillus plantarum is beneficial for reducing intestinal inflammation and repairing intestinal mucosa. However, the effectiveness of different strains varies. The MPO activity and TGF-β content of mice in group G were the lowest, and the EGF content was the highest, indicating that the intestinal mucosa of mice in group G recovered the best and had the strongest immune barrier. In other words, Lactobacillus plantarum PC037 is most effective in repairing intestinal mucosa, can effectively improve intestinal immunity, and regulate the intestinal immune system to maintain homeostasis.

[0079] Based on the experimental results in Tables 3 and 4, Lactobacillus plantarum PC037 was selected as the best strain for improving intestinal immunity.

[0080] System 2: Screening of the best formula for Lactobacillus plantarum fermentation products

[0081] 1. Sample preparation

[0082] Table 5 Weight parts of raw materials in the formulations of different embodiments

[0083]

[0084] A method for preparing a fermented product for improving intestinal immunity is prepared according to the following method:

[0085] 1. Select dry, mold-free, and insect-free oats, brown rice, morels, bamboo fungus, and walnuts. Wash and drain the oats and brown rice, and weigh the oats and brown rice according to the weight listed in Table 5. Soak the morels and bamboo fungus, then wash and squeeze out the water. Weigh the morels and bamboo fungus according to the weight listed in Table 5. Shell the walnuts, remove the kernels, and weigh several portions according to the weight listed in Table 5. Select fresh shiitake mushrooms, wash and drain, and weigh several portions according to the weight listed in Table 5. Select qualified glucose and weigh several portions according to the weight listed in Table 5.

[0086] 2. Soak the brown rice in clean water for 24 hours, then pour it into a colander, cover the colander with gauze that has been sterilized at high temperature and wrung out, rinse with 30℃ warm water, spray with 30℃ warm water three times a day, germinate after about 28 hours, and collect the germinated brown rice when the sprouts reach 0.7 cm in length.

[0087] 3. Broken the oats, germinated brown rice, and 1 / 2 part by weight of glucose, and add 500 parts of purified water to prepare a broken slurry. The broken parameter is 24000r / min, and the duration is 3min. Adjust the pH to 6.5, maintain at 120℃ for 20min, and then cool to 37℃ to obtain Solution No. 1.

[0088] 4. After Lactobacillus plantarum PC037 was activated for 3 generations, the bacterial solution was collected and centrifuged at 4°C and 6000 rpm for 20 min. The precipitate was collected and diluted with sterile saline until the viable count was 1×10 9 CFU / mL, and resuspend to obtain bacterial suspension.

[0089] 5. Add bacterial suspension to solution No. 1 at a ratio of 6%, stir evenly, ferment at a constant pH of 37°C for 24 hours, filter with an 80-mesh filter, and collect the filtrate as solution No. 2.

[0090] 6. Solution No. 2 was pre-frozen at -20°C for 24 h, then heated to 4°C and placed in an ice bath for ultrasonic disruption. The ultrasonic disruption conditions were 750 MHz, 3 s on, 5 s off, and 30 min to obtain the fermentation lysate.

[0091] 7. Broken the wall of Agaricus tiliaceus, Morchella oleracea, Dictyophora japonica, walnut and 1 / 2 part by weight of glucose, add 300 parts of purified water to prepare broken wall slurry, the broken wall parameter is 24000r / min, continuous 3min, keep at 120℃ for 20min, then cool to 37℃, add fermentation lysate, stir evenly, adjust pH to 6.5, and use it as solution No. 3.

[0092] 8. Add bacterial suspension to solution No. 3 at a ratio of 6%, stir evenly, and ferment at a constant pH of 37°C for 10 hours to prepare the fermentation liquid.

[0093] 9. Put the fermentation liquid onto a plate and freeze it at -20℃ for 12 hours. Then take it out and freeze dry it in a freeze dryer for 24 hours. Use a grinder to grind it into 60 mesh to obtain the Lactobacillus plantarum fermentation product.

[0094] 2. Crowd Testing

[0095] 1. Recruit 110 male adults with low intestinal immunity as subjects, aged 20 to 55 years.

[0096] Inclusion criteria: ≥25 flatulences per day, currently on a 21-day trip with diarrhea, and not eating gas-producing foods such as potatoes, white radishes, and other tubers within 7 days before the trial.

[0097] Exclusion criteria: patients with acute and critical digestive system diseases such as gastrointestinal obstruction and intestinal perforation; patients with abnormal kidney and liver function.

[0098] 2. The subjects were randomly divided into 11 groups, each with 10 participants, as shown in Table 6. Group A consumed 150 mL of purified water, while the remaining groups each consumed one Lactobacillus plantarum fermented product, as shown in Table 6. Each Lactobacillus plantarum fermented product was consumed by mixing 1 gram of the product with 150 mL of 37°C purified water. All participants consumed the product once daily, half an hour after lunch, for 15 days.

[0099] Table 6 Grouping of subjects

[0100]

[0101] 3. On the 1st and 16th days, the number of bowel movements and diarrhea episodes (within 3 days) of the subjects were recorded and the average values ​​were calculated. The results are shown in Table 7.

[0102] 4. Results and Analysis

[0103] Table 7 Test results of subjects

[0104]

[0105] When intestinal immunity is low, harmful bacteria can breed in the intestines, leading to increased intestinal gas production, which manifests as an abnormal increase in the amount of flatus expelled by the human body. In addition, during travel, it is also easy to be infected by pathogens, leading to the occurrence of diarrhea. The results show that, whether compared with Group A or compared with Day 1, the number of flatus expulsions and the number of diarrhea episodes during travel for the subjects in Groups B to K on Day 16 were all reduced, indicating that fermented products can enhance intestinal immunity and reduce the number of flatus expulsions and the number of diarrhea episodes during travel. Since the formulations of the fermented products in different groups are different, the effects they can play are also different. The number of flatus expulsions and the number of diarrhea episodes during travel for the subjects in Group J on Day 16 were the lowest, indicating that the fermented product consumed by the subjects in Group J was the most effective, namely the plantarum fermented product of Example 9. Therefore, the formulation of Example 9 was selected as the optimal formulation of the plantarum fermented product. The resulting fermented product can regulate the intestinal microecological balance, reduce the number of flatus expulsions and the number of diarrhea episodes caused by pathogen infection during travel, and is conducive to enhancing intestinal immunity.

[0106] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A fermented product for improving intestinal immunity, characterized in that: The fermented product is prepared by fermenting the following raw materials in parts by weight with Lactobacillus plantarum PC037: 30-40 parts of oats, 10-20 parts of brown rice, 5-17 parts of button mushrooms, 12-25 parts of morels, 10-20 parts of bamboo fungus, 6-15 parts of glucose, 5-15 parts of walnuts, and 800-1000 parts of purified water; the brown rice is used after germination; The plant lactobacillus PC037 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 13, 2021, with the deposit number CGMCC No. 24096.

2. The fermented product for improving intestinal immunity according to claim 1, characterized in that The raw materials are: 30 parts of oats, 20 parts of brown rice, 17 parts of button mushrooms, 12 parts of morels, 10 parts of bamboo fungus, 6 parts of glucose, 5 parts of walnuts and 800 parts of purified water.

3. The fermented product for improving intestinal immunity according to claim 1, characterized in that The fermentation includes a first stage fermentation and a second stage fermentation.

4. A method for preparing a fermented product for improving intestinal immunity according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) Select dry, mold-free, and insect-free oats, brown rice, morels, bamboo fungus, and walnuts. Wash and drain all of these ingredients, and weigh the formulated amounts of oats and brown rice. Soak the morels and bamboo fungus, then wash and squeeze out the water, and weigh the formulated amounts of morels and bamboo fungus. Shell the walnuts and take the kernels according to the formulated amount. Select fresh button mushrooms, wash and drain, and weigh the formulated amount of glucose. 2) Soak the brown rice in clean water for 12–24 hours, then pour it into a colander. Cover the colander with sterilized, soaked, and wrung-out gauze. Rinse with 30–40°C warm water and spray with 30–40°C warm water 1–5 times daily. Allow the rice to germinate for 20–30 hours. Collect the germinated brown rice when the sprouts reach 0.5–0.9 cm in length. 3) Broken oats, germinated brown rice, and a portion of glucose were treated, and purified water was added during the process to prepare a broken oats slurry. The breaking parameters were 22,000-28,000 rpm for 1-3 minutes. The pH was adjusted to 6.2-6.6, and the temperature was maintained at 115-135°C for 15-20 minutes. The mixture was then cooled to 36-38°C to prepare Solution 1. 4) After activating Lactobacillus plantarum PC037 for 2 to 5 generations, collect the bacterial suspension and centrifuge it at 1 to 4°C and 5000 to 8000 rpm for 15 to 20 minutes. Collect the precipitate and dilute it with sterile saline until the viable count is 1 × 10 9 ~1×10 10 CFU / mL, resuspend to obtain bacterial suspension; 5) Add the bacterial suspension to Solution 1 at a ratio of 3-6%, stir evenly, and ferment at a constant pH of 36-38°C for 12-24 hours. Filter through a 60-80 mesh filter and collect the filtrate as Solution 2. 6) Solution No. 2 was pre-frozen at -15 to -25°C for 18 to 36 hours, then warmed to 1 to 5°C and placed in an ice bath for ultrasonic disruption at 700 to 800 MHz, with an on-time of 1 to 5 seconds, a 1 to 5 second pause, and a duration of 20 to 40 minutes to obtain a fermentation lysate. 7) Crack the mushrooms, morels, bamboo fungus, walnuts, and the remaining glucose, adding the remaining purified water to make a cracked slurry at a speed of 22,000-28,000 rpm for 1-5 minutes, maintaining at 115-135°C for 15-20 minutes, then cooling to 36-38°C. Add the fermentation lysate, stir evenly, and adjust the pH to 6.2-6.6 to prepare Solution 3. 8) Add the bacterial suspension to solution No. 3 at a ratio of 5-10%, stir evenly, and ferment at a constant pH of 36-38°C for 8-12 hours to prepare the fermentation broth. 9) Place the fermentation liquid on a plate and freeze it at -15 to -25°C for 12 to 24 hours. Then take it out and freeze-dry it in a freeze dryer for 12 to 24 hours. Grind it into 60 to 80 mesh with a grinder to obtain the fermentation product.

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