Phytobacterium plantarum for improving intestinal immunity and fermented product

Abstract

The invention discloses a plant lactobacillus PC037 capable of improving intestinal immunity and a fermented product. The plant lactobacillus PC037 is preserved in the China General Microbiological Culture Collection Center on December 13, 2021, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 24096. The plant lactobacillus PC037 can improve the repair speed of the intestinal mucosa and promote the enhancement of the immunity of the intestinal tract. The plant lactobacillus fermented product capable of improving the immunity of the intestinal tract, provided by the invention, can be used for adjusting the exhaust frequency, promoting the enhancement of the immunity of the intestinal tract and reducing the occurrence frequency of diarrhea caused by pathogenic bacteria infection of travelers in a travel process.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and particularly relates to a Lactiplantibacillus plantarum and a fermented product for improving intestinal immunity. Background Art

[0002] The intestinal mucosa is the largest mucosal immune organ in the animal body. 70% of the infections in the body occur on the mucosa. A complete and sound intestinal mucosa is an important guarantee for strong intestinal immunity and can play a greater role in combating pathogen invasion and metastasis. Different from the systemic immune system, it mainly involves gut-associated lymphoid tissue and secretory IgA. The intestinal mucosal immune system can not only be stimulated to produce local mucosal immune responses, but also induce systemic immune responses.

[0003] The intestinal microecosystem is a unified body in which the intestinal flora coexists and interacts with the intestinal mucosa and is in a dynamic balance. The intestinal microecological barrier composed of beneficial bacteria, opportunistic pathogens, and harmful bacteria in the intestinal flora has a total bacterial count of up to 100 trillion, which plays an important role in maintaining human health and immunity. The formed biological barrier can defend against infections and prevent or inhibit the invasion of pathogenic bacteria. The normal intestinal flora continuously stimulates the mucosa, activates mucosal immunity, and maintains the normal mucosal immune level in the intestine. At the same time, it shows the effects of immune clearance and immune rejection on pathogenic bacteria.

[0004] Research has found that the intestinal flora can regulate the balance of effector / regulatory T cells, thereby promoting immune homeostasis. Some species of Clostridium and Bacteroides fragilis have been found to stimulate the differentiation of colonic Treg cells and then induce the secretion of IL-10, thereby reducing pathological inflammation and maintaining intestinal health. Thus, it can be seen that the intestinal flora and the intestinal mucosal barrier are interdependent and influence each other, and they are the "guardians" of immune stability in the body's internal environment.

[0005] When the intestinal immunity of the human body is low, it is more likely to be infected by pathogens, the intestinal mucosa damage is aggravated, the intestinal flora is imbalanced, causing various adverse reactions such as intestinal spasm, excessive intestinal fluid secretion, borborygmus, growth of harmful bacteria, and repeated intestinal inflammation, manifested as one or more symptoms such as abdominal pain, abdominal distension, diarrhea, constipation, dyspepsia, and bloody stools, seriously affecting people's lives and endangering human health.

[0006] Lactiplantibacillus plantarum is a common lactic acid bacterium in fermented cheese, fermented vegetables, fermented meat products, silage, the intestine, etc. It has strong tolerance to gastric acid and bile salts and strong bactericidal ability against pathogenic bacteria, and has various probiotic effects such as preventing cancer and cardiovascular and cerebrovascular diseases, inhibiting harmful bacteria, regulating immunity and the gastrointestinal tract, and reducing cholesterol and nitrite content. Research shows that Lactiplantibacillus plantarum can metabolize several metabolites, such as EPS, SCFA, and CLA, etc., which have a significant effect on alleviating intestinal diseases. EPS is a heterogeneous structure secreted by the strain, which helps to antagonize pathogenic bacteria, inhibit infiltration of inflammatory cells in the colon, relieve tissue lipid peroxidation, and enhance human immunity. In the state of intestinal inflammation, EPS can activate macrophages, promote the proliferation of T cells, and inhibit the expression of TNF-α, thereby inhibiting the development of intestinal inflammation. Due to the individual differences of the strains, the performance of their probiotic effects is uneven. Even for the same strain, when the fermentation substrate changes, it will also affect the exertion of its probiotic effect. 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 Lactiplantibacillus plantarum for improving intestinal immunity, which can improve the repair speed of intestinal mucosa and promote the enhancement of intestinal immunity.

[0008] Another objective of the present invention is to provide a fermented product of Lactiplantibacillus plantarum for improving intestinal immunity, which can regulate the exhaust frequency, promote the enhancement of intestinal immunity, and reduce the occurrence frequency of diarrhea caused by pathogenic bacteria infection during travel for travelers.

[0009] Another objective of the present invention is to provide a preparation method of a fermented product of Lactiplantibacillus plantarum for improving intestinal immunity.

[0010] One of the objectives of the present invention is achieved by adopting the following technical solutions: A Lactiplantibacillus plantarum for improving intestinal immunity, the Lactiplantibacillus plantarum is Lactiplantibacillus plantarum ( Lactobacillus plantarum ) PC037, and the Lactiplantibacillus plantarum PC037 was deposited with the China General Microbiological Culture Collection Center on December 13, 2021, and the deposit number is CGMCC No. 24096.

[0011] As an optional scheme of the present invention, the Lactiplantibacillus plantarum PC037 exerts the effect of improving intestinal immunity from at least one of the following items (Ⅰ) to (Ⅳ): (Ⅰ)Improve the repair speed of repairing intestinal mucosa; (Ⅱ)Reduce intestinal inflammation; (Ⅲ)Regulate the exhaust frequency; (Ⅳ)Reduce the number of diarrhea.

[0012] As an alternative embodiment of the present invention, the Lactiplantibacillus plantarum PC037 plays a role in enhancing the repair rate of intestinal mucosa through one or more of the following: reducing the number of days required for the DIA score to be less than 3%, reducing the MPO activity, reducing the TGF-β content, and increasing the EGF content.

[0013] The second object of the present invention is achieved by the following technical solution: The present invention provides a product for enhancing intestinal immunity, and the active ingredient of the product includes live bacteria and / or inactivated bacteria of Lactiplantibacillus plantarum PC037; the Lactiplantibacillus plantarum PC037 is the Lactiplantibacillus plantarum described in the first object of the present invention.

[0014] As an alternative embodiment of the present invention, the product includes food, health products, and drugs.

[0015] As an alternative embodiment of the present invention, the product includes solid products, liquid products, and semi-solid products.

[0016] As an alternative embodiment of the present invention, the product includes probiotic freeze-dried powder and probiotic fermentation products.

[0017] The present invention also provides a fermented product for enhancing intestinal immunity, which is processed from a probiotic fermentate. The probiotic fermentate is fermented from a composition by Lactiplantibacillus plantarum PC037. The composition includes the following components in parts by weight: 30-40 parts of oats, 10-20 parts of brown rice, 5-17 parts of Tricholoma gambosum, 12-25 parts of Morchella esculenta, 10-20 parts of Dictyophora indusiata, 6-15 parts of glucose, 5-15 parts of walnuts, and 800-1000 parts of purified water.

[0018] Oats are rich in dietary fiber, with a content as high as 30% in the bran, and the appearance is honeycomb-like. Among them, the small-molecule glucan has the greatest viscosity, mainly β-glucan. β-glucan is mainly distributed in the endosperm and the cell wall of the aleurone layer of the endosperm of oats. It is the most abundant soluble fiber in oats, with a maximum content of 6%. It has the effects of enhancing intestinal immunity and certain anti-cancer effects, and can also regulate intestinal probiotics, maintain and increase intestinal moisture and fecal volume, promote the proliferation of beneficial bacteria, thereby forming a protective film, reducing the retention time of excreta, and avoiding the reabsorption of metabolic toxins, which is beneficial to the prevention of intestinal diseases such as colorectal cancer.

[0019] Brown rice is the product of paddy after hulling, mainly composed of bran layer, germ and endosperm. Its aleurone layer and embryo are rich in many bioactive components such as VE, linoleic acid, rice bran protein, rice germ protein, GABA, glutathione, inositol hexaphosphate, γ-oryzanol, etc., which have the effects of improving human immune function, removing oxygen free radicals in the body, reducing blood lipid, preventing cardiovascular and cerebrovascular diseases, delaying aging, etc. In addition, the dietary fiber in its bran layer helps to promote gastrointestinal peristalsis, making food residues and decomposed toxins excreted faster, and has a certain effect on digestive tract diseases such as stomach diseases, constipation, hemorrhoids and cancer prevention. According to the "Compendium of Materia Medica", brown rice has the wonderful use of "harmonizing the five internal organs and improving complexion".

[0020] Tricholoma gambosum is a rare edible and medicinal mushroom in China. Its medicinal property is mild and its taste is sweet. It can promote the intestine and benefit the stomach, and has the effect of dissipating heat and relieving the exterior. It is suitable for symptoms such as indigestion, abdominal distension, stomachache, diarrhea, etc. Pharmacological studies show that Tricholoma gambosum contains a variety of chemical components such as rich selenium elements, polysaccharide compounds, lectins, sterol compounds, etc. The selenium element is as high as 2.565 μg / g, which can improve human immunity, resist aging, inhibit the proliferation of cancer cells, and has a protective effect on ischemic / hypoxic heart.

[0021] Morchella esculenta is a world-renowned rare and precious medicinal and edible fungus, rich in nutrition and extremely high medicinal value. It is rich in components such as polysaccharides, enzymes, amino acids, pyranone antibiotics, fatty acids, etc., and has significant effects in anti-tumor, anti-viral and enhancing immune activity. According to the "Compendium of Materia Medica" records, Morchella esculenta "has a mild nature and a sweet taste without toxicity", and has the effects of "benefiting the intestines and stomach, promoting digestion, resolving phlegm and regulating qi, tonifying the kidney and receiving qi, nourishing the brain and refreshing the mind".

[0022] Dictyophora indusiata, also known as Dictyophora phalloidea and Dictyophora rubrovolvata, contains polysaccharides and various trace elements. Dictyophora indusiata polysaccharide is a heteropolysaccharide, a macromolecule with high activity, mainly distributed in the cell wall of the sporophyte of Dictyophora indusiata. Among them, the polysaccharide component is galactose, glucose, mannose and xylose, which can enhance the phagocytic function of neutrophils and promote the spleen to form antibodies, realizing the immune regulation function. In addition, Dictyophora indusiata also contains a large amount of rare element germanium, and germanium element 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.

[0023] Walnuts are rich in a variety of active components, such as unsaturated fatty acids, high-quality proteins, polyphenolic compounds, vitamins and minerals, etc., with strong antioxidant properties. It 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, glutelin, albumin and prolamin, rich in 18 kinds of amino acids and with a high amino acid score and balanced proportion, which can be used as a source of high-quality protein for the human body.

[0024] The fermented product for enhancing intestinal immunity provided by the present invention has been verified through multiple experiments in terms of the raw material selection and formula compatibility of the composition, and finally the optimal formula is obtained. It is rich in nutrition, highly novel and creative, and is processed by innovative technology. In particular, Lactiplantibacillus plantarum PC037 is selected for deep fermentation, which can not only biotransform the nutritional components such as protein, dietary fiber, glucan, selenium, germanium, and polysaccharide in the composition, but also produce a variety of probiotic factors such as more easily absorbable amino acids, short-chain fatty acids, mucopolysaccharides, and bacteriocins. The obtained fermented product can effectively reduce the occurrence frequency of diarrhea caused by pathogen infection during travel of travelers, and improve the intestinal immunity of the human body in an internal regulation manner.

[0025] As an alternative embodiment of the present invention, the composition comprises the following components by weight: 30 parts of oats, 20 parts of brown rice, 17 parts of Tricholoma gambosum, 12 parts of Morchella esculenta, 10 parts of Dictyophora indusiata, 6 parts of glucose, 5 parts of walnuts, and 800 parts of purified water.

[0026] As an alternative embodiment of the present invention, the probiotic fermentate is fermented from the composition by Lactiplantibacillus plantarum PC037, and the fermentation includes a first-stage fermentation and a second-stage fermentation.

[0027] The third object of the present invention is achieved by the following technical solution: A preparation method of a fermented product for enhancing intestinal immunity as described in the second object, which comprises the following steps: 1) Select oats, brown rice, Morchella esculenta, Dictyophora indusiata, and walnuts that are dry, free of mildew and insect damage. Wash the above raw materials and drain the water. Weigh the formulated amount of oats and brown rice; after soaking Morchella esculenta and Dictyophora indusiata, wash them, squeeze out the water, and weigh the formulated amount of Morchella esculenta and Dictyophora indusiata; remove the shell of walnuts and take the kernels according to the formulated amount; select fresh Tricholoma gambosum, wash it, drain the water, and weigh it according to the formulated amount; weigh the formulated amount of glucose; 2) Soak the brown rice in clear water for 12 - 24 h, then pour it into a draining basin, cover the draining basin with a gauze that has been disinfected at high temperature and soaked and wrung dry, rinse it with warm water at 30 - 40 °C, spray it with warm water at 30 - 40 °C 1 - 5 times a day, and germinate it for 20 - 30 h. When the bud length reaches 0.5 - 0.9 cm, collect the germinated brown rice; 3) Subject oats, germinated brown rice, and part of the glucose to cell wall breaking treatment, and add part of the purified water to make a cell wall breaking slurry during the process. The cell wall breaking parameters are 22000 - 28000 r / min for 1 - 3 min, adjust the pH to 6.2 - 6.6, maintain at 115 - 135 °C for 15 - 20 min, and then cool to 36 - 38 °C to obtain Solution 1; 4) After activating Lactiplantibacillus plantarum PC037 for 2 to 5 generations, collect the bacterial liquid, centrifuge it at 1 to 4 °C and 5000 to 8000 r / min for 15 to 20 min, collect the precipitate, and dilute it with sterile normal saline to a viable cell count of 1×10 9 ~1×10 10 CFU / mL, resuspend to obtain a bacterial suspension; 5) Add the bacterial suspension to Solution 1 at an addition ratio of 3 to 6%, stir evenly, ferment at a constant pH of 36 to 38 °C for 12 to 24 h, then filter through a 60 to 80 mesh sieve, and collect the filtrate as Solution 2; 6) After pre-freezing Solution 2 at -15 to -25 °C for 18 to 36 h, warm it up to 1 to 5 °C, place it in an ice bath for ultrasonic disruption. The ultrasonic disruption conditions are 700 to 800 MHz, ultrasonic for 1 to 5 s, stop for 1 to 5 s, and continue for 20 to 40 min to obtain a fermentation lysate; 7) Subject Tricholoma gambosum, Morchella esculenta, Dictyophora indusiata, walnuts, and the remaining part of glucose to cell wall breaking treatment, and add the remaining part of pure water during the process to make a cell wall breaking slurry. The cell wall breaking parameters are 22000 to 28000 r / min and continue for 1 to 5 min, maintain at 115 to 135 °C for 15 to 20 min, then cool to 36 to 38 °C, add the fermentation lysate, stir evenly, and adjust the pH to 6.2 to 6.6 as Solution 3; 8) Add the bacterial suspension to Solution 3 at an addition ratio of 5 to 10%, stir evenly, and ferment at a constant pH of 36 to 38 °C for 8 to 12 h as the fermentation broth; 9) Spread the fermentation broth on a plate and place it in a freezer at -15 to -25 °C for 12 to 24 h, then take it out and place it in a freeze dryer for freeze drying for 12 to 24 h, and crush it with a pulverizer to 60 to 80 mesh to obtain the fermented product.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Lactiplantibacillus plantarum PC037 provided by the present invention can improve the repair speed of intestinal mucosa and promote the enhancement of intestinal immunity.

[0029] (2) The fermented product for enhancing intestinal immunity provided by the present invention can regulate the exhaust frequency, promote the enhancement of intestinal immunity, and reduce the occurrence frequency of diarrhea caused by pathogen infection during travel for travelers.

[0030] (3) The innovative fermented product of the present invention adopts a two-stage fermentation method. Lactiplantibacillus plantarum PC037 multiplies sufficiently in the first-stage fermentation, transforming various nutrients such as amino acids, proteins, unsaturated fatty acids, dietary fiber, and vitamins in oats and brown rice, and producing probiotic factors such as uronic acid polymers, short-chain fatty acids, and bacteriocins. After filtering out crude fiber, cell lysis treatment is carried out to release intracellular active enzymes. In the second-stage fermentation, it multiplies to the most vigorous stage, transforming rare elements, polysaccharides, amino acids and other nutrients in Tricholoma gambosum, Morchella esculenta, Dictyophora indusiata, and walnuts, and at the same time, carrying out secondary biological transformation on the fermentation cell lysis products. The obtained 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 pathogenic bacteria, and protect intestinal health.

[0031] Biological material preservation information: Lactiplantibacillus plantarum PC037, preservation number is CGMCC No. 24096, taxonomic name: Lactiplantibacillus plantarum Lactobacillus plantarum , which was preserved in the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) on December 13, 2021, and the abbreviation of the preservation unit is CGMCC. Specific implementation manners

[0032] Next, in combination with specific implementation manners, the present invention will be further described. It should be noted that, on the premise of no conflict, the following-described embodiments or technical features can be arbitrarily combined to form new embodiments. The raw materials, equipment, etc. used in the following embodiments can be obtained through commercial channels except for special limitations.

[0033] The embodiment of the present invention provides a Lactiplantibacillus plantarum PC037 for improving intestinal immunity, which is obtained by animal experiment screening.

[0034] System 1: Screening of the best strains for improving intestinal immunity 1. Experimental strains Lactiplantibacillus plantarum CICC 25282 (preservation number: CICC 25282), Lactiplantibacillus plantarum CICC 24936 (preservation number: CICC 24936), Lactiplantibacillus plantarum GDMCC 1.4956 (preservation number: GDMCC 1.4956), Lactiplantibacillus plantarum CICC 25283 (preservation number: CICC 25283), Lactiplantibacillus plantarum PC037.

[0035] 2. Sample preparation After the Lactobacillus plantarum was activated for three generations, the bacterial liquid was collected and centrifuged at 4 °C and 6000 r / min for 20 min. The precipitate was collected and the viable count was adjusted to 1×10 9 CFU / mL with sterile physiological saline, resuspended to obtain a bacterial suspension.

[0036] 3. Experimental animals Female C57BL / 6 mice, 8 weeks old, weighing 19 g ± 1 g. The mice were randomly divided into 7 groups with 10 mice in each group. The grouping is shown in Table 1. 4. The temperature in the SPF-class animal room was set at 24 °C, and a lighting system of 12 hours of light and 12 hours of darkness was adopted. During the animal feeding and experiment process, the food and water supply were sufficient. After the mice were adaptively fed for 5 days, the original weights of the mice in each group were recorded. Except for the mice in group A, the mice in the other groups were allowed to freely drink 3% DDS solution for 5 days, and the 3% DDS solution was changed every 2 days. On the 5th day, the DDS solution was removed and replaced with normal drinking water. Starting from the 6th day, all the mice were gavaged with the samples. The types of samples are shown in Table 1, and the gavage dose was 10 mL / kg for 10 consecutive days.

[0037] Table 1 Grouping of mice and gavage sample conditions Group Type of intragastric administration sample Group A Normal saline Group B Normal saline Group C Bacterial suspension of Lactobacillus plantarum CICC 25282 Group D Bacterial suspension of Lactobacillus plantarum CICC 24936 Group E Bacterial suspension of Lactobacillus plantarum GDMCC 1.4956 Group F Bacterial suspension of Lactobacillus plantarum CICC 25283 Group G Bacterial suspension of Lactobacillus plantarum PC037 5. The mice were weighed and DAI scored every day. The fecal occult blood condition was detected using a fecal occult blood kit. The scoring criteria are shown in Table 2, and the number of days required when the DIA score < 3% was recorded. The results are shown in Table 3.

[0038] Table 2 DIA scoring criteria Score (points) Weight loss score Hematochezia score Fecal character score 1 1%～5% Mild occult blood Slightly soft feces 2 5%～10% Severe occult blood Soft feces 3 10%～15% A small amount of blood can be seen in the feces Unformed feces 4 >15% Severe bloody stool Watery stool

[0039] Among them, in the formula, W: percentage of weight loss. A: original weight of the mouse. B: weight of the mouse on the day of scoring.

[0040] DIA score = weight loss score + blood in stool score + fecal consistency score 6. After the 6th day of gavage, 5 mice were randomly selected from each group. The selected mice were fasted for 12 h, anesthetized with 7% chloral hydrate, and the colon tissue was taken.

[0041] (1) Weigh 50 mg of colon tissue, make it into a 10% homogenate with 0.5% HTBA solution, centrifuge at 14000 r / min at 4 °C for 15 min, take 80 μL of the supernatant, add 120 μL of 0.0005% benzidine dihydrochloride containing 0.1% H2O2, and measure the absorbance value of A460 every 30 s with an enzyme-labeled instrument. Calculate the MPO activity according to the average change rate of the absorbance value within 2 min, and find the average value. The results are shown in Table 4.

[0042] (2)Weigh 0.5 g of colon tissue, make it into a 10% homogenate with phosphate buffer solution at pH 7.4, centrifuge at 5000 r / min for 10 min at 4 °C, and take the supernatant. Determine the contents of TGF-β and EGF according to the operation instructions of the ELISA kit, and calculate the average value. Set 2 replicate wells for each sample. The results are shown in Table 4.

[0043] 7. Results and Analysis (1)Scoring of mice Table 3 Days required for the DIA score of mice to be < 3% Group Number of days (days) Group A 0 Group B 9.125±0.64 Group C 7.250±0.46 Group D 8.375±0.52 Group E 7.125±0.64 Group F 7.750±0.87 Group G 5.375±0.52 The colon injury induced by DDS in mice is a recognized experimental colitis model, and the induced colon injury has a stable model. The results show that compared with group A, the number of days required for the DIA score of mice in group B to be < 3% increased significantly, indicating that the mouse model of intestinal mucosal injury was successfully established. Compared with group B, the number of days required for the DIA score of mice in groups C - G to be < 3% decreased, indicating that Lactiplantibacillus plantarum has a repair effect on the damaged intestinal mucosa, and different strains have different repair speeds for the intestinal mucosa. The number of days required for the DIA score of mice in group G to be < 3% was the lowest, indicating that the intestinal mucosa recovered the fastest, that is, Lactiplantibacillus plantarum PC037 can improve the repair speed of the intestinal mucosa, is conducive to building a complete and healthy intestinal mucosa, and improves intestinal immunity.

[0044] (2)Changes in colon indexes of mice Table 4 Changes in colon indexes of mice

[0045] MPO is a marker of neutrophil activation, and its level and activity represent the function and status of neutrophils. In the inflammatory response, the release and activation of MPO are involved in the innate immune response to combat the invasion of pathogens. TGF-β maintains the integrity of the intestinal mucosa by regulating the renewal and damage repair of intestinal mucosal epithelium. EGF is produced by colonic goblet cells, can induce the differentiation and proliferation of colon cells, and is the main regulatory factor promoting intestinal mucosal repair. The results showed that compared with group A, the MPO activity and TGF-β content in group B mice increased significantly, while the EGF content decreased significantly, indicating that the intestinal mucosal injury in mice led to exacerbated inflammation, the mucosa was not effectively restored, and the intestinal immunity was low. Compared with group B, the MPO activity and TGF-β content in group C - G mice decreased, while the EGF content increased, indicating that Lactiplantibacillus plantarum is beneficial to reducing intestinal inflammation and repairing intestinal mucosa, but the effective degree of different strains is different. The MPO activity and TGF-β content in group G mice were the lowest, and the EGF content was the highest, indicating that the intestinal mucosa of group G mice recovered the best and the immune barrier was the strongest, that is, Lactiplantibacillus plantarum PC037 had the best effect on repairing intestinal mucosa, could effectively improve intestinal immunity, and regulate the intestinal immune system to maintain homeostasis. Based on the experimental results in Table 3 and Table 4, Lactiplantibacillus plantarum PC037 was selected as the best strain to improve intestinal immunity.

[0046] System Two: Screening of the Optimal Formulation of Fermented Products of Lactiplantibacillus plantarum I. Sample Preparation Table 5 Raw Material Weight Parts of Formulations in Different Examples

[0047] A preparation method of a fermented product for improving intestinal immunity is prepared according to the following method: 1. Select oats, brown rice, Morchella esculenta, Dictyophora indusiata, and walnuts that are dry, free of mildew, and insect damage. Wash the raw materials such as oats and brown rice, drain the water, and weigh oats and brown rice according to the weight parts in Table 5. After soaking and washing the raw materials such as Morchella esculenta and Dictyophora indusiata, squeeze out the water, and weigh Morchella esculenta and Dictyophora indusiata according to the weight parts in Table 5. Shell the walnuts, take the kernels, and weigh several portions according to the weight parts in Table 5. Select fresh Tricholoma gambosum, wash it, drain the water, and weigh several portions according to the weight parts in Table 5. Select qualified glucose and weigh several portions according to the weight parts in Table 5.

[0048] 2. Soak brown rice in clean water for 24 h, then pour it into a colander, cover the colander with a gauze that has been disinfected at high temperature and soaked and wrung dry, rinse it with warm water at 30 °C, spray it with warm water at 30 °C three times a day, and germinate it for about 28 h. When the bud length reaches 0.7 cm, collect the germinated brown rice.

[0049] 3. Broke the oats, germinated brown rice and 1 / 2 weight part of glucose, adding 500 parts of pure water to prepare the broken slurry. The breaking parameters were 24000r / min for 3min, and the pH was adjusted to 6.5. The temperature was kept at 120℃ for 20min, and then cooled to 37℃ as Solution No. 1.

[0050] 4. After Lactobacillus plantarum PC037 was activated for 3 generations, the bacterial solution was collected and centrifuged at 4°C and 6000 r / min for 20 min. The precipitate was collected and diluted with sterile saline until the number of viable bacteria was 1×10 9 CFU / mL, resuspend to obtain bacterial suspension.

[0051] 5. Add bacterial suspension to solution No. 1 at a ratio of 6%, stir evenly, ferment at 37°C constant pH for 24 hours, filter with an 80-mesh filter, and collect the filtrate as solution No. 2.

[0052] 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 on to obtain the fermentation lysate.

[0053] 7. Break the wall of Oyster mushroom, Morchella, Dictyophora, walnut and 1 / 2 weight part of glucose, add 300 parts of pure water to prepare the broken wall slurry, the breaking 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, as solution No. 3.

[0054] 8. Add bacterial suspension to solution No. 3 at a ratio of 6%, stir evenly, and ferment at 37°C constant pH for 10 hours as the fermentation liquid.

[0055] 9. Put the fermentation liquid onto a plate and spread it flat, freeze it at -20℃ for 12 hours, take it out, put it in a freeze dryer for freeze drying for 24 hours, and grind it into 60 mesh with a grinder to obtain the Lactobacillus plantarum fermentation product.

[0056] 2. Crowd Testing 1. Recruit 110 male adults with low intestinal immunity as subjects, aged 20 to 55 years old.

[0057] Inclusion criteria: Passing gas more than 25 times a day, traveling abroad for 21 days with diarrhea symptoms, and not eating foods that are prone to gas production, such as potatoes, white radishes, and other tubers, within 7 days before the test.

[0058] Exclusion criteria: patients with acute and critical digestive system diseases such as gastrointestinal obstruction and intestinal perforation; patients with abnormal kidney and liver function.

[0059] 2. Randomly divide the subjects into 11 groups, with 10 people in each group. The grouping method is shown in Table 6. The members of Group A consume 150 mL of pure water, and each of the other groups consumes 1 kind of Lactiplantibacillus plantarum fermentation product corresponding to the fermentation product as shown in Table 6. The consumption method of the Lactiplantibacillus plantarum fermentation product is to take 1 g of the Lactiplantibacillus plantarum fermentation product and mix it evenly with 150 mL of pure water at 37 °C for drinking. The consumption time for all subjects is half an hour after lunch every day, once a day, for 15 consecutive days.

[0060] Table 6 Grouping of Subjects Group Type of corresponding sample consumed Group A Purified water Group B Fermented product of Lactobacillus plantarum in Example 1 Group C Fermented product of Lactobacillus plantarum in Example 2 Group D Fermented product of Lactobacillus plantarum in Example 3 Group E Fermented product of Lactobacillus plantarum in Example 4 Group F Fermented product of Lactobacillus plantarum in Example 5 Group G Fermented product of Lactobacillus plantarum in Example 6 Group H Fermented product of Lactobacillus plantarum in Example 7 Group I Fermented product of Lactobacillus plantarum in Example 8 Group J Fermented product of Lactobacillus plantarum in Example 9 Group K Fermented product of Lactobacillus plantarum in Example 10 3. On the 1st day and the 16th day, record the number of defecations and the number of diarrhea (within 3 days) of the subjects on the same day, and calculate the average value. The results are shown in Table 7.

[0061] 4. Results and Analysis Table 7 Test Results of Subjects

[0062] When the intestinal immunity is low, harmful bacteria will grow in the intestine, leading to an increase in intestinal gas production, manifested as an abnormal increase in the amount of human flatulence. In addition, during travel, it is also easy to be infected by pathogenic bacteria, resulting in diarrhea. The results show that whether compared with Group A or with the 1st day, the number of flatulence and the number of diarrhea during travel of the subjects in Groups B - K on the 16th day are both reduced, indicating that the fermentation product can enhance intestinal immunity and reduce the number of flatulence and the number of diarrhea during travel. Since the formulations of the fermentation products in different groups are different, the effects achieved are also different. The number of defecations and the number of diarrhea during travel of the subjects in Group J on the 16th day are the lowest, indicating that the fermentation product consumed by the subjects in Group J has the best effect, that is, the Lactiplantibacillus plantarum fermentation product of Example 9. Therefore, the formulation of Example 9 is selected as the best formulation of the Lactiplantibacillus plantarum fermentation product. The obtained fermentation product can regulate the balance of the intestinal microecology, reduce the number of flatulence and the number of diarrhea caused by pathogenic bacteria infection during travel, and is beneficial to enhancing intestinal immunity.

[0063] The above embodiments are only the 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 substitutions made by those skilled in the art based on the present invention belong to the scope of protection required by the present invention.

Claims

1. A Lactiplantibacillus plantarum for enhancing intestinal immunity, characterized in that, The Lactiplantibacillus plantarum is Lactiplantibacillus plantarum ( Lactobacillus plantarum ) PC037, and the Lactiplantibacillus plantarum PC037 was deposited at the General Microbiological Center of the China General Microbiological Culture Collection Center on December 13, 2021, with the deposit number of CGMCC No. 24096.

2. The Lactiplantibacillus plantarum for enhancing intestinal immunity according to claim 1, wherein The plant lactobacillus PC037 plays a role in improving intestinal immunity through at least one of the following items (I) to (IV): (I) Improve the repair rate of intestinal mucosa; (II) Reduce intestinal inflammation; (III) Adjust the exhaust frequency; (IV) Reduce the frequency of diarrhea.

3. The Lactiplantibacillus plantarum for enhancing intestinal immunity according to claim 2, wherein The Lactobacillus plantarum PC037 increases the repair speed of the intestinal mucosa by one or more of the following: reducing the number of days required for the DIA score to be less than 3%, reducing MPO activity, reducing TGF-β content, and increasing EGF content.

4. A product for enhancing intestinal immunity, characterized in that, The active ingredients of the product include live bacteria and / or inactivated bacteria of Lactobacillus plantarum PC037; the Lactobacillus plantarum PC037 is the Lactobacillus plantarum described in claim 1.

5. The product according to claim 4, wherein, The products include solid products, liquid products and semi-solid products.

6. The product according to claim 4, wherein The products include probiotic freeze-dried powder and probiotic fermented products.

7. A fermented product for enhancing intestinal immunity, characterized in that, The invention is made by processing probiotic fermentation products, wherein the probiotic fermentation products are fermented by Lactobacillus plantarum PC037 on a composition, and the composition comprises the following components in parts by weight: 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.

8. The fermented product for enhancing intestinal immunity according to claim 7, wherein, The composition comprises 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.

9. The fermented product for enhancing intestinal immunity according to claim 7, wherein, The probiotic fermented product is obtained by fermenting a composition by Lactobacillus plantarum PC037, and the fermentation includes a first fermentation stage and a second fermentation stage.

10. A method for preparing a fermented product for enhancing intestinal immunity according to any one of claims 7-9, characterized in that, The following steps are involved: 1) Select dry, mildew-free, and insect-free oats, brown rice, morels, bamboo fungus, and walnuts, wash the above raw materials, drain the water, and weigh the oats and brown rice in the formula amount; soak the morels and bamboo fungus, wash them, squeeze the water, and weigh the morels and bamboo fungus in the formula amount; shell the walnuts and take the kernels in the formula amount; select fresh button mushrooms, wash them, drain the water, and weigh them in the formula amount; weigh the glucose in the formula amount; 2) Soak the brown rice in clean water for 12 to 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 to 40°C warm water, spray with 30 to 40°C warm water 1 to 5 times a day, germinate for 20 to 30 hours, and collect the germinated brown rice when the sprouts reach 0.5 to 0.9 cm in length; 3) Broken oats, germinated brown rice and part of glucose, adding part of purified water to make broken slurry, the breaking parameters are 22000-28000r / min, lasting 1-3min, adjusting pH to 6.2-6.6, maintaining at 115-135℃ for 15-20min, and then cooling to 36-38℃, as solution No. 1; 4) After the Lactiplantibacillus plantarum PC037 is activated for 2 to 5 generations, collect the bacterial liquid, centrifuge it at 1 to 4 °C and 5000 to 8000 r / min for 15 to 20 min, collect the precipitate, and dilute it with sterile normal saline to a viable bacteria count of 1×10 9 ~1×10 10 CFU / mL, resuspend to obtain a bacterial suspension; 5) Add bacterial suspension to solution No. 1 at a ratio of 3-6%, stir evenly, ferment at 36-38℃ constant pH for 12-24h, filter with a 60-80 mesh filter, collect the filtrate as solution No. 2; 6) After the solution No. 2 is pre-frozen at -15 to -25 °C for 18 to 36 h, it is heated to 1 to 5 °C and placed in an ice bath for ultrasonic disruption. The ultrasonic disruption conditions are 700 to 800 MHz, ultrasonic for 1 to 5 s, stop for 1 to 5 s, and continue for 20 to 40 min to obtain a fermentation lysate product; 7) The Tricholoma gambosum, Morchella esculenta, Dictyophora indusiata, walnut, and the remaining part of glucose are subjected to cell wall breaking treatment. During this process, the remaining part of pure water is added to make a cell wall breaking slurry. The cell wall breaking parameters are 22000 to 28000 r / min, continue for 1 to 5 min, maintain at 115 to 135 °C for 15 to 20 min, then cool to 36 to 38 °C, add the fermentation lysate product, stir evenly, and adjust the pH to 6.2 to 6.6 to obtain the solution No. 3; 8) Add the bacterial suspension to the solution No. 3, and the addition ratio is 5 to 10%. Stir evenly and ferment at a constant pH of 36 to 38 °C for 8 to 12 h to obtain a fermentation broth; 9) Spread the fermentation broth on a plate and freeze it at -15 to -25 °C for 12 to 24 h, then take it out and place it in a freeze dryer for freeze drying for 12 to 24 h, and crush it to 60 to 80 meshes with a pulverizer to obtain the fermentation product.

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