CN114540247B - Lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus fermentum CCFM1226 capable of improving intestinal tract IgA level and relieving enteritis and application thereof, belonging to the technical field of microorganisms. The lactobacillus fermentum CCFM1226 can improve the fecal IgA level of a normal mouse, the lactobacillus fermentum CCFM1226 can improve the IgA level of the normal mouse by 1231.5 percent, compared with the lactobacillus fermentum of the same species and different strains, the lactobacillus fermentum CCFM1226 can obviously improve the intestinal secretion IgA level and can stimulate the intestinal tract to produce IgA in a non-inflammation dependent mode; and the strain has the capability of relieving colitis induced by DSS. The lactobacillus fermentum CCFM1226 can be used for preparing functional microbial agents, foods and medicines capable of improving the intestinal secretion IgA level of a host and relieving enteritis and capable of being taken into gastrointestinal tracts, and has wide application prospect.

Description

A strain of Lactobacillus fermentum CCFM1226 that can increase intestinal IgA levels and relieve enteritis and its applications

Technical field

The present invention relates to a strain of Lactobacillus fermentum CCFM1226 that can increase intestinal IgA levels and alleviate enteritis and its application, and belongs to the field of microbial technology.

Background technique

Immunoglobulin A (IgA) is the most abundant immunoglobulin produced in the mammalian intestine; it is a component of serum immunoglobulin, and most IgA passes through the mucosa of the intestine, respiratory tract, biliary tract, and reproductive tract. secreted. In the human body, the structure of IgA mainly exists in monomeric and bimeric forms. According to the distribution of IgA in the body, it can be divided into serotype and secretory type. Serotype IgA is monomeric and has a weak immune effect. Secretory IgA comes in two forms and three forms. It is the main component of the body's mucosal defense system and is widely distributed in milk, saliva, and mucosal secretions of the gastrointestinal tract, respiratory tract, and genitourinary tract. It can inhibit the attachment of microorganisms to the respiratory epithelium, slow down the reproduction of viruses, and has an important immune barrier function. It has antibody activity against certain viruses, bacteria and general antigens, and is the first line of defense to prevent pathogens from invading the body. IgA in the intestine is generally considered to be secretory IgA.

Most lymphoid tissues and immune cells in the intestinal mucosal immune system have certain connections with IgA. At the same time, there is abundant evidence that intestinal bacteria play an important role in mucosal immunity. On the one hand, the mucosal immune system needs to mount an effective defense against pathogens that invade the mucosa, and on the other hand, it needs to develop appropriate tolerance against microorganisms that are symbiotic with the body on the mucosa. IgA has the function of resisting pathogens and controlling mucosal inflammatory reactions, and its production is closely related to mucosal bacteria. The adhesion and wrapping functions of IgA can avoid direct contact between harmful antigens in the intestinal lumen and the host intestinal epithelium, and play an important role in maintaining the integrity of the intestinal barrier. At the same time, IgA also regulates the composition and balance of intestinal flora. IgA neutralizes microbial toxins and pathogens with high affinity, and the low-affinity binding system protects the intestines from commensal bacteria.

In recent years, with in-depth research on the relationship between intestinal flora and human health, a large number of studies have confirmed that probiotics improve human health by regulating immunity. Probiotics are safe and have no side effects. A large number of clinical and animal experimental studies have shown that probiotics can relieve intestinal infection and inflammation by regulating the content of IgA.

Contents of the invention

The first object of the present invention is to provide a strain of Lactobacillus fermentum CCFM1226, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on January 27, 2022, with a deposit number of GDMCC No: 62242.

In one embodiment of the present invention, the Lactobacillus fermentum comes from the feces of infants. The extracted whole genome is sent to a professional sequencing company, and the whole genome of the bacteria is sequenced using a second-generation sequencer. The obtained sequence results are used BLAST searched and compared similarity in GeneBank, and the sequencing results identified it as Lactobacillus fermentum, named Lactobacillus fermentum CCFM1226.

In one embodiment of the invention, Lactobacillus fermentum CCFM1226 has the following characteristics:

(1) Bacterial characteristics: Gram stain positive, non-spore forming, non-motile bacteria.

(2) Colony characteristics: milky white, shiny, raised, opaque, smooth and neat.

(3) Growth characteristics: Under constant temperature conditions of 37°C, culture in MRS medium for about 12 hours to reach the end of logarithmic phase.

(4) It has strong tolerance to simulated gastrointestinal fluid.

The second object of the present invention is to provide a microbial preparation containing the above-mentioned Lactobacillus fermentum CCFM1226.

In one embodiment of the present invention, the number of Lactobacillus fermentum CCFM1226 in the microbial preparation is ≥1×10 ⁶ CFU/mL or ≥1×10 ⁶ CFU/g.

In one embodiment of the present invention, the number of Lactobacillus fermentum CCFM1226 in the microbial preparation is ≥1×10 ⁹ CFU/mL or ≥1×10 ⁹ CFU/g.

In one embodiment of the present invention, the microbial preparations include but are not limited to functional foods, health products or drugs.

In one embodiment of the present invention, the microbial preparation contains one of a live strain of Lactobacillus fermentum CCFM1226, a dry strain of Lactobacillus fermentum CCFM1226, a metabolite of Lactobacillus fermentum CCFM1226 strain, and an inactivated strain of Lactobacillus fermentum CCFM1226 or more.

In one embodiment of the invention, the drug has at least one of the following effects:

(a) Increase the IgA content in feces;

(b) Significantly relieve enteritis.

In one embodiment of the invention, the drug acts on mammals, including but not limited to humans.

In one embodiment of the invention, the medicament further contains a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of fillers, wetting agents, disintegrants, binders or lubricants.

In one embodiment of the present invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one of ethanol or glycerol. or more; the disintegrant is one or more of sodium carboxymethyl starch, croscarmellose sodium, crospovidone or low-substituted hydroxypropyl cellulose; the binder It is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talc or silicon dioxide, or Various.

The third object of the present invention is to provide a product containing the above-mentioned Lactobacillus fermentum CCFM1226 or the above-mentioned microbial preparation.

In one embodiment of the invention, the product is food, medicine or health care product.

In one embodiment of the present invention, the food is solid food, liquid food, semi-solid food or fermented food.

In one embodiment of the present invention, the fermented food includes dairy products, soy products, and fruit and vegetable products.

In one embodiment of the invention, the dairy products include milk, sour cream, and cheese.

In one embodiment of the present invention, the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

In one embodiment of the present invention, the medicine contains pharmaceutically acceptable carriers or excipients.

In one embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of fillers, wetting agents, disintegrants, binders or lubricants.

In one embodiment of the present invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one of ethanol or glycerol. or more; the disintegrant is one or more of sodium carboxymethyl starch, croscarmellose sodium, crospovidone or low-substituted hydroxypropyl cellulose; the binder It is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talc or silicon dioxide, or Various.

The fourth object of the present invention is to provide the application of Lactobacillus fermentum CCFM1226 in the preparation of products that improve intestinal secretion of IgA and resist inflammation.

In one embodiment of the invention, the medicament is used to increase the IgA content in feces.

In one embodiment of the invention, the medicament is used to relieve enteritis.

In one embodiment of the present invention, the product is a functional bacterial agent, medicine or health product.

In one embodiment of the present invention, the medicine contains pharmaceutically acceptable carriers or excipients.

In one embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of fillers, wetting agents, disintegrants, binders or lubricants.

In one embodiment of the present invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the wetting agent is one of ethanol or glycerol. or more; the disintegrant is one or more of sodium carboxymethyl starch, croscarmellose sodium, crospovidone or low-substituted hydroxypropyl cellulose; the binder It is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talc or silicon dioxide, or Various.

The present invention also claims the use of Lactobacillus fermentum CCFM1226 in the preparation of fermented food.

In one embodiment of the present invention, the application includes but is not limited to using the Lactobacillus fermentum CCFM1226 as a fermentation microorganism to use food raw materials for fermentation.

In one embodiment of the present invention, the fermented food includes dairy products, soy products, and fruit and vegetable products.

In one embodiment of the invention, the dairy products include milk, sour cream, and cheese.

In one embodiment of the present invention, the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

beneficial effects

The invention provides Lactobacillus fermentum CCFM1226. The Lactobacillus fermentum CCFM1226 can be used to prepare functional microbial agents, foods and medicines that increase the content of IgA in feces. It has broad application prospects, and is specifically reflected in:

(1) The effect of Lactobacillus fermentum CCFM1226 on the fecal IgA level of mice. Compared with blank mice, intragastric administration of Lactobacillus fermentum CCFM1226 can increase the IgA level of normal mice by 1231.5%. Compared with different strains of Lactobacillus fermentum of the same species, Significantly increases the level of intestinal IgA secretion.

(2) Compared with the model group, the Lactobacillus fermentum CCFM1226 group showed a certain improvement in the degree of crypt damage, swelling of goblet cells, and reduced diffusion. The depth of inflammatory infiltration and the degree of inflammatory cell infiltration were significantly lighter than those in other Lactobacillus groups. Therefore, Lactobacillus fermentum group CCFM1226 has a certain ability to alleviate DSS-induced colitis.

Preservation of biological materials

Lactobacillus fermentum CCFM1226, classified and named: Lactobacillus fermentum, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on January 27, 2022. The deposit number is GDMCC No: 62242, and the deposit address is Courtyard 59, No. 100 Xianlie Middle Road, Guangzhou City Building 5th floor.

Description of the drawings

Figure 1: Effect of oral administration of Lactobacillus on fecal IgA of normal mice; among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (compared with the normal mouse blank group Compare).

Figure 2: The effect of improving enteritis after oral administration of Lactobacillus.

Figure 3: Animal grouping and experimental methods for enteritis experiment.

Figure 4: Effects of lactobacillus in each group on colon inflammation levels of IL-6 in mice after intragastric administration.

Figure 5: Effects of lactobacilli of each group on colon inflammation levels of IL-17 in mice after intragastric administration.

Figure 6: Effects of lactobacilli in each group on colon inflammation levels of IL-1β in mice after intragastric administration.

Detailed ways

Lactobacillus fermentum JCM1173 involved in the following examples was obtained from Japan Collection of Microorganisms (JCM).

Lactobacillus fermentum SL241 involved in the following examples is disclosed in "Yan Zhao et.al., Phylogenetic and comparative genomic analysis of Lactobacillus fermentumStrains and the Key Genes Related to their Intestinal Anti-inflammatory Effects. Engineering, 2021, DOI: https: //doi.org/10.1016/j.eng.2020.09.016"; the Lactobacillus rhamnosus PAL1 involved was disclosed in "Tian Peijun. Research on Bifidobacterium breve CCFM1025 with the function of alleviating depression [D]. Jiangnan University" in the paper.

Healthy female C57BL/6J mice involved in the following examples were purchased from Viton Lever.

The culture media involved in the following examples are as follows:

MRS liquid medium: 10g peptone, 10g beef extract, 5g yeast powder, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate heptahydrate, sulfuric acid tetrahydrate Manganese 0.25g, distilled water 1000mL.

Preparation method: Add the above ingredients into distilled water, heat to dissolve completely, adjust the pH to 6.2~6.4, pack into Erlenmeyer flasks, sterilize at 121°C for 15 minutes.

MRS solid medium: Add 15-20g of agar to the MRS liquid medium.

The preparation and staining methods of the colon paraffin sections involved in the following examples are as follows:

The experimental steps for making colon paraffin sections are:

(1) Take a 1cm section of the distal colon 1cm away from the anus and fix it with 4% paraformaldehyde for 72 hours; (2) Rinse the fixed colon tissue with running water for 8 hours and then perform dehydration treatment, and then pass the sample through 70%, 80%, Dehydrate 90% ethanol solutions of all levels for 30 minutes each, then put 95% and 100% ethanol solutions twice, 20 minutes each time; (3) Put the colon sample into a 1:1 xylene and alcohol mixture for 15 minutes, and then put it in Add xylene I and xylene II for 15 minutes each; (4) Transfer the colon tissue to a mixture of half and half xylene and paraffin for 15 minutes, then add paraffin I and paraffin II for 1 hour each, and keep the temperature at 60°C; (5 ) Use a Lycra paraffin embedding machine to embed the colon in the re-melted wax block; (6) Use a tissue slicer to slice the embedded tissue into 5 μm thick slices; (7) Paste the slices and dry them, and place them at 62 1h in oven.

The HE dyeing process is as follows:

(1) Paraffin sections were dewaxed in xylene I and xylene II for 5 minutes each, then placed in 100%, 95%, 90%, 80%, and 70% alcohol solutions for 3 to 5 minutes each, and then placed in distilled water for 3 minutes; (2) Stain with hematoxylin for 20 seconds; (3) Wash away unbound hematoxylin with distilled water; (4) Stain with eosin for 2 seconds, enter 95% ethanol I, II, and 70% ethanol in sequence, and take them out quickly. Then put it into 80% ethanol for 50 to 55 seconds, and into absolute ethanol for 2 minutes; (5) Place the slices in a 1:1 mixture of xylene and alcohol for 1 minute, and then put them into xylene I and xylene II for 2 to 3 minutes each; (6) Seal the slide with neutral gum.

The HE-stained sections were scanned and photographed using a Pannoramic MIDI digital slide scanner.

Example 1: Screening of Lactobacillus fermentum CCFM1226

1. Isolation and screening of Lactobacilli

(1) Take 1g of fresh feces from infants and enrich the sample in MRS medium containing sorbitol for 12 hours;

(2) Perform gradient dilution of the enriched sample and spread it on an MRS solid plate added with 0.02% olfactory cresyl violet, and culture it for 24 to 48 hours;

(3) Select single colonies with obvious color change circles and consistent with the basic morphology of lactic acid bacteria for plate streak purification, and screen and isolate lactic acid bacteria;

(4) Cultivate the above single colony in liquid MRS culture medium for 24 hours and then perform Gram staining, and select Gram-positive bacteria for subsequent tests.

2. Preliminary identification of Lactobacilli: Calcium Dissolution Circle Measurement

(1) Cultivate the lactic acid bacteria screened in step 1 in liquid sorbitol MRS culture solution for 24 hours, and then take 1 mL of the culture and centrifuge it at 8000×g for 2 minutes;

(2) Wash twice with 0.05M KH ₂ PO ₄ solution;

(3) Resuspend the obtained bacterial slurry, streak it on the solid medium of Sorbitol MRS-0.75% CaCO ₃ , and culture it for 24 hours;

(4) Select colonies with obvious calcium-dissolving rings, convex round shape, fine white color, and no mycelium. After Gram staining, the bacteria are rod-shaped and are initially determined to be Lactobacilli.

3. Molecular biological identification of Lactobacilli

1. Single bacterial genome extraction

(1) Cultivate the Lactobacillus screened in step 1 overnight; take 1 mL of the bacterial suspension cultured overnight in a 1.5 mL centrifuge tube, centrifuge at 10000 r/min for 2 minutes, discard the supernatant to obtain the bacteria; blow with 1 mL of sterile water After washing the bacterial cells, centrifuge at 10000r/min for 2 minutes and discard the supernatant to obtain the bacterial cells; add 200 μL SDS lysis solution and bathe in 80°C water bath for 30 min; add 200 μL of phenol-chloroform solution to the bacterial cell lysis solution, where the composition of the phenol-chloroform solution is The volume ratio is Tris saturated phenol: chloroform: isoamyl alcohol = 25:24:1. After inverting and mixing, centrifuge at 12000 rpm for 5 to 10 minutes, and take 200 μL of the supernatant; add 400 μL of ice ethanol or ice isopropanol to 200 μL of the supernatant. , let stand at -20℃ for 1 hour, centrifuge at 12000rpm for 5~10min, discard the supernatant; add 500μL 70% (volume percentage) ice ethanol to resuspend the pellet, centrifuge at 12000rpm for 1~3min, discard the supernatant; dry in an oven at 60℃, or let it air naturally Dry;

(2) Use 50 μL ddH ₂ O to redissolve the pellet in preparation for PCR.

2. 16S rDNA PCR

Bacterial 16S rDNA 50 μL PCR reaction system: 10× Taq buffer, 5 μL; dNTP, 5 μL; primer 27F, 0.5 μL; primer 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template, 0.5 μL; ddH ₂ O, 38 μL.

PCR conditions: 95℃ 5min; 95℃ 10s; 55℃ 30s; 72℃ 30s; step2-4 30×; 72℃ 5min; 12℃ 2min.

Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 minutes, and then perform gel imaging;

Send the obtained PCR product to a professional sequencing company. The sequence of CCFM1226-27F_C02.ab1 is shown in SEQ ID NO.1, and the sequence of CCFM1226-1492R_B02.ab1 is shown in SEQ ID NO.2. The obtained sequencing results are compared with those in GenBank using BLAST. Search and similarity comparison were performed, and the strain identified as Lactobacillus fermentum was stored at -80°C.

3. Whole genome sequencing

The extracted whole genome was sent to a professional sequencing company, and the whole genome of the bacteria was sequenced using a second-generation sequencer. The obtained sequence results were searched and compared in GenBank using BLAST. The sequencing results were identified as Lactobacillus fermentum and named It is Lactobacillus fermentum CCFM1226, stored at -80°C for later use.

Example 2: Lactobacillus fermentum CCFM1226 can increase the IgA content in feces

Specific steps are as follows:

(1) Take Lactobacillus rhamnosus PAL1, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 and Lactobacillus fermentum JCM1173 out of the refrigerator at -80°C, and streak them into MRS solid medium at 37°C. Cultivate for 48 hours, pick a single colony in the MRS liquid medium, and culture it at 37°C for 24 hours to obtain seed liquid respectively. The prepared seed liquid is inoculated into the new MRS liquid at a volume of 2% (v/v). In the culture medium, culture for 24 hours at 37°C, and then culture again in the same way for one generation to obtain Lactobacillus rhamnosus PAL1 fermentation broth, Lactobacillus fermentum SL241 fermentation broth, Lactobacillus fermentum CCFM1226 fermentation broth, and Lactobacillus fermentum JCM1173 fermentation broth. liquid;

Then, the above-mentioned Lactobacillus fermentation broth was centrifuged for 5 minutes at 6000 r/min and 4°C, and then resuspended in 0.01 MPBS to prepare bacterial suspensions for animal experiments.

(2) Take 30 healthy female C57BL/6J mice weighing 12 to 14 g. After adaptive culture for 1 week, they are randomly divided into 5 groups, namely Lactobacillus fermentum CCFM1226 group, Lactobacillus fermentum SL241 group, and rhamnosus milk. Bacillus PAL1, blank group, and Lactobacillus fermentum JCM1173 group.

The experimental grouping and processing methods are shown in Table 1:

Table 1 Grouping of experimental animals

On the 7th day of the experiment, fresh feces from mice were collected and frozen at -80°C.

At the 21st point of the experiment, the mice were fasted and water-free for 12 hours. Isoflurane anesthesia was used. The isoflurane was volatilized through oxygen flow. Generally, the concentration of 2 to 3% was used to induce anesthesia and 1.5-2% was used to maintain it. Generally, the concentration of mice was The anesthesia time is 2-3 minutes. After anesthesia, cervical dislocation is used to make the patient die.

IgA was measured according to the kit method.

After intragastric administration of lactobacilli in each group, the effects on the fecal IgA levels of mice in the first week are shown in Figure 1. Compared with blank mice (the fecal IgA content of mice is: 5.97±1.89μg/mg protein), Lactobacillus gastric fermentum CCFM1226 (mouse feces IgA content: 79.49±34.45μg/mg protein) can increase the IgA level of normal mice by 1231.5%.

After oral administration of Lactobacillus fermentum SL241, the IgA content of mice's feces was: 15.88±10.49μg/mg protein. After oral administration of Lactobacillus rhamnosus PAL1, the IgA level of mice was: 7.33±2.22μg/mg protein. After intragastric administration of Lactobacillus fermentum JCM1173, the IgA content of mouse feces was: 19.33±6.35 μg/mg protein. It can be seen that the IgA content of mouse feces after intragastric administration of Lactobacillus fermentum SL241, and the IgA content of mouse feces after intragastric administration of Lactobacillus rhamnosus PAL1 IgA and IgA in the feces of mice after oral administration of Lactobacillus fermentum JCM1173 did not change significantly compared with the blank group; administration of Lactobacillus fermentum CCFM1226 significantly increased the level of intestinal secretion of IgA compared with different strains of Lactobacillus fermentum.

Example 3: Lactobacillus fermentum CCFM1226 improves enteritis

Specific steps are as follows:

(1) Take Lactobacillus rhamnosus PAL1, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 and Lactobacillus fermentum JCM1173 out of the refrigerator at -80°C, and streak them into MRS solid medium at 37°C. Cultivate for 48 hours, pick a single colony in the MRS liquid medium, and culture it at 37°C for 24 hours to obtain seed liquid respectively. The prepared seed liquid is inoculated into the new MRS liquid at a volume of 2% (v/v). In the culture medium, culture for 24 hours at 37°C, and then culture again in the same way for one generation to obtain Lactobacillus rhamnosus PAL1 fermentation broth, Lactobacillus fermentum SL241 fermentation broth, Lactobacillus fermentum CCFM1226 fermentation broth, and Lactobacillus fermentum JCM1173 fermentation broth. liquid;

Then, the Lactobacillus fermentation broth was centrifuged at 6000 r/min and 4°C for 5 min, and then resuspended in 0.01M PBS to prepare bacterial suspensions for animal experiments.

(2) The experimental animals were kept in a barrier environment of (23±2)℃, relative humidity (50±10)%, 12h light, and 12h alternating night and day.

Thirty-six mice, six in each group, were randomly divided into 6 groups, namely Lactobacillus fermentum CCFM1226 group, Lactobacillus fermentum SL241, Lactobacillus rhamnosus PAL1, Lactobacillus fermentum JCM1173 group, blank group and model group ( As shown in Figure 3).

The animals were adapted to feeding for one week. During the adaptation period, all groups were given normal drinking water and feed.

Starting from the second week, the blank group: 0.2 mL of 0.01M PBS solution was administered daily for 14 days.

Model group: 0.2 mL of 0.01 M PBS solution was administered daily for 7 days. On days 8 to 14, 0.2 mL of 0.01 M PBS solution was administered daily, and 2.5% (w/v) DSS solution was added to the drinking water.

Lactobacillus group (Lactobacillus rhamnosus PAL1 group / Lactobacillus fermentum SL241 group / Lactobacillus fermentum CCFM1226 group / Lactobacillus fermentum JCM1173 group): Each animal is given a daily gavage containing 1×10 ⁹ CFU/mL of Lactobacillus bacterial suspension. 0.2 mL for 7 days. From days 8 to 14, each animal was given 0.2 mL of 1×10 ⁹ CFU/mL Lactobacillus suspension by intragastric administration every day, and 2.5% (w/v) DSS was added to the drinking water.

After 2 weeks of the experiment, the mice were sacrificed, the colon tissue was taken, and paraffin sections of the colon were made and stained for observation. The results are shown in Figure 2.

The results showed that compared with the model group, the Lactobacillus fermentum CCFM1226 group had a certain improvement in crypt damage, goblet cell swelling, and reduced diffusion. The depth of inflammatory infiltration and the degree of inflammatory cell infiltration were significantly lighter than those of Lactobacillus rhamnosus. PAL1 group, Lactobacillus fermentum SL241 group and Lactobacillus fermentum JCM1173 group. Lactobacillus fermentum group CCFM1226 has a certain ability to alleviate DSS-induced colitis.

Example 4: Application of Lactobacillus fermentum CCFM1226

Specific steps are as follows:

Lactobacillus fermentum CCFM1226 can be used to prepare tablets. The specific preparation process of tablets is as follows:

Pick a single colony of Lactobacillus fermentum CCFM1226 obtained in Example 1 and insert it into the MRS liquid medium, and culture it at 37°C for 24 hours to obtain an activation solution; insert the activation solution into the MRS liquid at an inoculum volume of 1% (v/v) In the culture medium, incubate at 37°C for 24 hours to obtain the first-level seed liquid; insert the first-level seed liquid into the MRS liquid medium at an inoculation amount of 1% (v/v), and culture at 37°C for 24 hours to obtain the second-level seed. liquid; insert the secondary seed liquid into the MRS liquid medium at an inoculum volume of 1% (v/v), and culture it at 37°C for 24 hours to obtain the bacterial liquid; centrifuge the bacterial liquid at 6000g for 15 minutes to collect the precipitate; adjust the precipitate with pH After washing twice with 7.4 PBS buffer, centrifuge again at 6000g for 10 minutes to obtain the cells; resuspend the Lactobacillus fermentum CCFM1226 bacteria with a protective agent solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose. When the cell concentration is 1×10 ¹⁰ CFU/mL, the Lactobacillus fermentum CCFM1226 bacterial liquid is obtained; the Lactobacillus fermentum CCFM1226 bacterial liquid is freeze-dried to obtain Lactobacillus fermentum CCFM1226; the freeze-dried bacterial powder accounts for 10% of the total share, and is added in sequence Total weight: 2% stearic acid as lubricant, 3% CMC Na, 15.5% galactooligosaccharide, 7.8% xylooligosaccharide, 7.8% inulin, lactitol, erythritol, xylitol, and add Other excipients such as starch are compressed to obtain tablets.

Taking 1g of the above tablets and administrating them to normal mice every day for one week can promote intestinal secretion of IgA, increase the IgA content in feces, and regulate immune homeostasis.

Example 5: Application of Lactobacillus fermentum CCFMCCFM1226

Specific steps are as follows:

Lactobacillus fermentum CCFMCCFM1226 can be used to prepare bacterial powder. The specific preparation process of bacterial powder is as follows:

Pick a single colony of Lactobacillus fermentum CCFM1226 obtained in Example 4 and insert it into the MRS liquid medium, and culture it at 37°C for 24 hours to obtain an activation solution; insert the activation solution into the MRS liquid at an inoculum volume of 1% (v/v) In the culture medium, incubate at 37°C for 24 hours to obtain the first-level seed liquid; insert the first-level seed liquid into the MRS liquid medium at an inoculation amount of 1% (v/v), and culture at 37°C for 24 hours to obtain the second-level seed. liquid; insert the secondary seed liquid into the MRS liquid medium at an inoculum volume of 1% (v/v), and culture it at 37°C for 24 hours to obtain the bacterial liquid; centrifuge the bacterial liquid at 6000g for 15 minutes to collect the precipitate; adjust the precipitate with pH After washing twice with 7.4 PBS buffer, centrifuge again at 6000g for 10 minutes to obtain the cells; resuspend the Lactobacillus fermentum CCFMCCFM1226 bacteria with a protective agent solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose. When the cell concentration reaches 1×10 ¹⁰ CFU/mL, the Lactobacillus fermentum CCFM1226 bacterial liquid is obtained; the Lactobacillus fermentum CCFM1226 bacterial liquid is freeze-dried to obtain bacterial powder.

The above-mentioned bacterial powder containing 1×10 ⁹ CFU of total viable bacteria was administered to normal mice every day for one week, which can promote intestinal secretion of IgA, increase the content of IgA in feces, and regulate immune homeostasis.

Example 6: Utilizing the present invention to produce fermented food containing Lactobacillus fermentum CCFM1226

Fresh vegetables are selected, washed and juiced, followed by high-temperature instant sterilization. After high-temperature heat sterilization at a temperature of 140°C for 2 seconds, the temperature is immediately cooled to 37°C, and then the Lactobacillus fermentum CCFM1226 bacterial culture starter prepared in the present invention is added. The concentration is made to reach 10 ⁸ CFU/mL or more, and the mixture is refrigerated and stored at a temperature of 4° C., thereby obtaining a fruit and vegetable beverage containing live Lactobacillus fermentum CCFM1226 bacteria of the present invention.

The present invention can be used to ferment and prepare other fermented foods using Lactobacillus fermentum CCFM1226. The fermented foods include solid foods, liquid foods, and semi-solid foods. The fermented food includes dairy products, soy products, and fruit and vegetable products. The dairy products include milk, sour cream, and cheese; the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

The fermented food can promote intestinal secretion of IgA, increase the IgA content in feces, regulate immune homeostasis, and can also alleviate enteritis.

Example 7: Lactobacillus fermentum CCFM1226 stimulates IgA production in a non-inflammatory manner

The specific implementation is the same as that of Example 2. The difference is that colon tissue is taken to detect interleukin-6 (IL-6), interleukin-17 (IL-17), and interleukin-1β (IL-1β) respectively. The tissue processing method is according to R&D enzyme The immunoassay kit method requires processing.

The effects of lactobacilli in each group on the colon inflammation level of mice are shown in Figure 4-6.

(1) In terms of IL-6 levels, compared with blank mice (the IL-6 content in the mouse colon is: 308.4±146.8pg/mg protein), Lactobacillus fermentum CCFM1226 (the IL-6 content in the mouse colon is: 308.4±146.8pg/mg protein) :391.8±180.7pg/mg protein) slightly increased, but there was no significant difference; it can be seen that Lactobacillus fermentum CCFM1226 of the present invention does not cause significant changes in inflammatory factors, but can significantly increase IgA in mice. content.

After oral administration of Lactobacillus fermentum SL241, the content of IL-6 in the colon of mice was 417.7±200.5pg/mg protein; after oral administration of Lactobacillus rhamnosus PAL1, the content of IL-6 in the colon of mice was 291.3±161.8pg/mg. mg protein; after oral administration of Lactobacillus fermentum JCM1173, the content of IL-6 in the colon of mice was 253.0±50.27pg/mg protein.

(2) In terms of IL-17 levels, compared with blank mice (the IL-17 content in the mouse colon is: 415.2±182.5pg/mg protein), Lactobacillus fermentum CCFM1226 (the IL-17 content in the mouse colon is: 415.2±182.5pg/mg protein) :593.5±241.5pg/mg protein) Although it slightly increased the IL-17 content, there was no significant difference, but it could significantly increase the IgA content in mice.

After oral administration of Lactobacillus fermentum SL241, the content of IL-17 in the colon of mice was 496.8±93.92pg/mg protein; after oral administration of Lactobacillus rhamnosus PAL1, the content of IL-17 in the colon of mice was 428.9±95.37pg/mg. mg protein; after oral administration of Lactobacillus fermentum JCM1173, the content of IL-17 in the colon of mice was 345.7±77.44pg/mg protein.

In terms of IL-1β level, compared with blank mice (mouse colon IL-1β content: 186.7±41.44pg/mg), Lactobacillus fermentum CCFM1226 (mouse colon IL-1β content: 187.7±42.35 pg/mg protein), there was no significant difference; it can be seen that Lactobacillus fermentum CCFM1226 of the present invention does not cause obvious changes in inflammatory factors, but can significantly increase the IgA content in mice.

After being fed with Lactobacillus fermentum SL241, the content of IL-1β in the colon of mice was 179.2±7.75pg/mg protein; after being fed with Lactobacillus rhamnosus PAL1, the content of IL-1β in the colon of mice was 182.2±6.15pg/mg protein. ; After instillation of Lactobacillus fermentum JCM1173, the IL-1β content in mouse colon was 183.3±35.12pg/mg protein.

In summary, Lactobacillus fermentum CCFM1225 promotes the secretion of IgA in the host intestine without stimulating inflammation in the colon.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A strain of Lactobacillus fermentum CCFM1226 that can increase intestinal IgA levels and relieve enteritis and its application

<130> BAA220007A

<160> 2

<170> PatentIn version 3.3

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Claims (9)

1. Lactobacillus fermentumLactobacillus fermentum) CCFM1226, deposited with the Cantonese microorganism strain collection at 1.27 of 2022 under the accession number GDMCC No:62242.

2.a microbial preparation comprising the lactobacillus fermentum CCFM1226 of claim 1.

3. The microbial preparation according to claim 2, wherein the microbial preparation contains one or more of a viable strain of lactobacillus fermentum CCFM1226, a dried strain of lactobacillus fermentum CCFM1226 and an inactivated strain of lactobacillus fermentum CCFM1226.

4. A product comprising the lactobacillus fermentum CCFM1226 of claim 1 or comprising the microbial preparation of claim 2 or 3; the product is food, health product or medicine.

5. The product of claim 4, wherein the food product is: including solid food, liquid food, semi-solid food or fermented food, the said fermented food includes any one of dairy products, bean products, fruit and vegetable products, the said dairy products include any one of milk, sour cream, cheese; the fruit and vegetable product comprises any one of cucumber, carrot, beet, celery and cabbage product.

6. Use of lactobacillus fermentum CCFM1226 according to claim 1 for the preparation of a product for increasing intestinal secretory IgA, wherein said product is a health product or a functional bacterial agent.

7. The use according to claim 6 for the preparation of a product for increasing the content of IgA in faeces.

8. Use of lactobacillus fermentum CCFM1226 according to claim 1 for the preparation of a fermented food product.

9. Use of lactobacillus fermentum CCFM1226 according to claim 1 for the manufacture of a medicament for alleviating colitis.