CN114540247A - Lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof - Google Patents
Lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof Download PDFInfo
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- CN114540247A CN114540247A CN202210266770.5A CN202210266770A CN114540247A CN 114540247 A CN114540247 A CN 114540247A CN 202210266770 A CN202210266770 A CN 202210266770A CN 114540247 A CN114540247 A CN 114540247A
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Abstract
The invention discloses a lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof, and belongs to the technical field of microorganisms. The lactobacillus fermentum CCFM1226 can improve the fecal IgA level of a normal mouse, the lactobacillus gasketed CCFM1226 can improve the IgA level of the normal mouse by 1231.5%, compared with the lactobacillus fermentum of different strains, the intestinal secretory IgA level is obviously improved, and the intestinal IgA production can be stimulated in a non-inflammation-dependent manner; and the strain has remission capability on DSS-induced colitis. The lactobacillus fermentum CCFM1226 disclosed by the invention can be used for preparing functional microbial agents, foods and medicines capable of being ingested into the gastrointestinal tract and capable of improving the secretory IgA level of the intestinal tract of a host and relieving enteritis, and has wide application prospects.
Description
Technical Field
The invention relates to lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof, and belongs to the technical field of microorganisms.
Background
Immunoglobulin a (IgA) is the most abundant Immunoglobulin produced in the mammalian intestinal tract; it is a component of serum immunoglobulins, most IgA is secreted through the mucosa of the intestinal, respiratory, biliary and reproductive tracts. In humans, the structure of IgA exists mainly in monomeric and dimeric forms. According to the distribution of IgA in the body, it can be divided into serotypes and secretory types. The serotype IgA is a monomer and has weak immune effect. Secretory IgA has diploids and trisomes, is the main component of mucosal defense system of organism, and is widely distributed in milk, saliva, and mucous membrane secretion of gastrointestinal tract, respiratory tract and genitourinary tract. It can inhibit the attachment of microbe to respiratory tract epithelium, slow down virus propagation, has important immunity barrier effect, has antibody activity to some viruses, bacteria and common antigen, and is the first defense line to prevent pathogen from invading body. IgA in the intestinal tract is generally considered secretory IgA.
Most lymphoid tissues and immune cells in the intestinal mucosal immune system are associated with IgA. Meanwhile, there is a lot of evidence that intestinal bacteria play an important role in mucosal immunity. The mucosal immune system needs on the one hand to elicit an effective defense against pathogens that invade the mucosa and on the other hand to develop an appropriate tolerance against microorganisms that are symbiotic with the body on the mucosa. IgA has the effects of combating pathogens and controlling mucosal inflammatory responses, the production of which is of great importance in relation to mucosal bacteria. The IgA adhesion and encapsulation can avoid the direct contact between harmful antigen in intestinal cavity and host intestinal epithelium, and has important effect on maintaining the integrity of intestinal barrier, and the IgA also regulates the composition and balance of intestinal flora. IgA neutralizes microbial toxins and pathogens with high affinity, and low affinity binding systems protect the intestinal tract from the invasion of commensal bacteria.
In recent years, with the intensive research on the relationship between the intestinal flora and the human health, a great deal of research proves that the probiotics can improve the human health by regulating immunity. The probiotics have the characteristics of safety, no side effect and the like, and a large number of clinical and animal experimental researches show that the probiotics can relieve intestinal infection and inflammation by regulating and controlling the content of IgA.
Disclosure of Invention
The first purpose of the invention is to provide a Lactobacillus fermentum (CCFM 1226 which has been deposited in the Guangdong province collection of microorganisms at 27 th month 1/2022 with the deposit number GDMCC No: 62242.
in one embodiment of the present invention, the Lactobacillus fermentum is derived from feces of infants, the extracted whole genome is sent to a professional sequencing company, the whole genome of the bacteria is sequenced by using a second generation sequencer, the obtained sequence result is searched and compared with similarity in GeneBank by using BLAST, and the sequence result is identified as Lactobacillus fermentum (Lactobacillus fermentum) CCFM 1226.
In one embodiment of the invention, Lactobacillus fermentum (CCFM 1226 has the following characteristics:
(1) the characteristics of the thallus are as follows: gram-positive, non-sporulating, non-motile bacteria.
(2) Colony characteristics: is milky white, glossy, convex, opaque, smooth and regular.
(3) Growth characteristics: the medium was incubated in MRS medium for about 12h to the end of log at 37 ℃ under constant temperature conditions.
(4) Has strong tolerance to simulated gastrointestinal fluid.
It is a second object of the present invention to provide a microbial preparation comprising the above lactobacillus fermentum CCFM 1226.
In one embodiment of the invention, the number of Lactobacillus fermentum CCFM1226 in the microbial preparation is more than or equal to 1 x 106CFU/mL or more than or equal to 1X 106CFU/g。
In one embodiment of the invention, the number of Lactobacillus fermentum CCFM1226 in the microbial preparation is more than or equal to 1 x 109CFU/mL or more than or equal to 1X 109CFU/g。
In one embodiment of the present invention, the microbial preparation includes, but is not limited to, functional foods, health products or medicines.
In one embodiment of the invention, the microbial preparation comprises one or more of a viable strain of lactobacillus fermentum CCFM1226, a dry strain of lactobacillus fermentum CCFM1226, a metabolite of the lactobacillus fermentum CCFM1226 strain, and an inactivated lactobacillus fermentum CCFM1226 strain.
In one embodiment of the present invention, the medicament has at least one of the following effects:
(a) improving the IgA content in the excrement;
(b) can obviously relieve enteritis.
In one embodiment of the invention, the drug acts on a mammal, including but not limited to a human.
In one embodiment of the present invention, the medicament further comprises 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 a filler, a wetting agent, a disintegrant, a binder, or a lubricant.
In one embodiment of the invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.
The third purpose of the invention is to provide a product, wherein the product contains the lactobacillus fermentum CCFM1226 or the microbial preparation.
In one embodiment of the invention, the product is a food, pharmaceutical or nutraceutical product.
In one embodiment of the invention, the food product is a solid food product, a liquid food product, a semi-solid food product or a fermented food product.
In one embodiment of the invention, the fermented food product comprises dairy products, soy products, fruit and vegetable products.
In one embodiment of the invention, the dairy product comprises milk, sour cream, cheese.
In one embodiment of the invention, the fruit and vegetable products comprise cucumber, carrot, beet, celery, cabbage products.
In one embodiment of the invention, the pharmaceutical product comprises a pharmaceutically acceptable carrier or excipient.
In one embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.
In one embodiment of the invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.
The fourth purpose of the invention is to provide the application of the lactobacillus fermentum CCFM1226 in preparing products for improving intestinal tract secretion IgA and resisting inflammation.
In one embodiment of the invention, the medicament is for increasing IgA content in stool.
In one embodiment of the invention, the medicament is for alleviating enteritis.
In one embodiment of the invention, the product is a functional microbial inoculum, a drug or a health product.
In one embodiment of the invention, the pharmaceutical product comprises a pharmaceutically acceptable carrier or excipient.
In one embodiment of the present invention, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.
In one embodiment of the invention, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium fumarate stearate, talcum powder or silicon dioxide.
The invention also claims the application of the lactobacillus fermentum CCFM1226 in the preparation of fermented food.
In one embodiment of the present invention, said use includes, but is not limited to, fermentation with a food material using said lactobacillus fermentum CCFM1226 as a fermenting microorganism.
In one embodiment of the invention, the fermented food product comprises dairy products, soy products, fruit and vegetable products.
In one embodiment of the invention, the dairy product comprises milk, sour cream, cheese.
In one embodiment of the invention, the fruit and vegetable products comprise cucumber, carrot, beet, celery, cabbage products.
Advantageous effects
The invention provides a lactobacillus fermentum CCFM1226, wherein the lactobacillus fermentum CCFM1226 can be used for preparing functional microbial agents, foods and medicines for improving IgA content in excrement, has wide application prospects, and is specifically embodied in that:
(1) influence of lactobacillus fermentum CCFM1226 on mouse fecal IgA levels compared to the control mice, lactobacillus gasseri CCFM1226 was able to increase IgA levels of normal mice by 1231.5%, significantly increasing IgA levels secreted from intestinal tract compared to lactobacillus fermentum of the same strain.
(2) Compared with the model group, the lactobacillus fermentum CCFM1226 group has the advantages that the crypt damage degree is improved to a certain extent, the goblet cell swelling and the dispersion degree are reduced to a certain extent, the inflammatory infiltration depth and the inflammatory cell infiltration degree are obviously lower than those of other lactobacillus groups, and therefore the lactobacillus fermentum CCFM1226 group has a certain relieving capacity on DSS-induced colitis.
Biological material preservation
Lactobacillus fermentum CCFM1226, classified and named: lactobacillus fermentum has been deposited at 27.1.2022 in Guangdong province culture Collection center with the deposit number GDMCC No: 62242, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Drawings
FIG. 1: the effect of lactobacillus gasseri on fecal IgA in normal mice; wherein P <0.05, P <0.01, P <0.001, P <0.0001 (compared to the normal mouse blank group).
FIG. 2: improving enteritis after lactobacillus gasseri administration.
FIG. 3: animal grouping of enteritis experiment and experiment method.
FIG. 4: effect of lactobacillus in each group on level of IL-6 of colonic inflammation in mice after intragastric administration.
FIG. 5: effect of lactobacillus in each group on level of colonic inflammation IL-17 in mice after intragastric administration.
FIG. 6: effect of lactobacillus in each group on level of colonic inflammation IL-1 β in mice after gavage.
Detailed Description
Lactobacillus fermentum JCM1173, referred to in the following examples, was obtained from the Japanese Collection of Microorganisms (JCM).
Lactobacillus fermentum SL241, referred to in the following examples, is disclosed in "Yan Zhao et al, polymeric and synthetic genetic analysis of Lactobacillus fermentum Strains and the Key Genes Related to the invention endogenous Anti-inflammatory effects engineering,2021, DOI https:// DOI. org/10.1016/j. eng.2020.09.016"; the lactobacillus rhamnosus PAL1 was referred to as published in the "replenisher. study of bifidobacterium breve CCFM1025 with antidepressant action [ D ]. university of south of the river" paper.
Healthy female C57BL/6J mice, referred to in the examples below, were purchased from Witongliwa.
The media involved in the following examples are as follows:
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 2g of dipotassium phosphate, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 801 mL of tween, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate tetrahydrate and 1000mL of distilled water.
The preparation method comprises the following steps: adding the components into distilled water, heating to completely dissolve, adjusting pH to 6.2-6.4, subpackaging in triangular flasks, sterilizing at 121 ℃ for 15 min.
MRS solid medium: 15-20 g of agar is added on the basis of the MRS liquid culture medium.
The method of making and staining the paraffin sections of the colon referred to in the following examples is as follows:
the experimental steps for preparing the colon paraffin section are as follows:
(1) fixing a section of distal colon 1cm away from anus 1cm for 72h with 4% paraformaldehyde; (2) washing the fixed colon tissue with running water for 8h, dehydrating, sequentially dehydrating the sample with 70%, 80%, and 90% ethanol solution for 30min, respectively, and placing at 95% and 100% for 2 times, each for 20 min; (3) putting colon samples into mixed solution of xylene and alcohol at a ratio of 1: 1 for 15min, and then putting xylene I and xylene II for 15min respectively; (4) transferring the colon tissue to mixed liquid of xylene and paraffin for 15min, adding paraffin I and paraffin II, and waxing for 1h respectively, and keeping the temperature at 60 ℃; (5) embedding the colon in a re-melted wax block by using a leica paraffin embedding machine; (6) slicing the embedded tissue by a tissue slicer to a thickness of 5 μm; (7) drying after sticking, and placing in an oven at 62 ℃ for 1 h.
HE staining procedure was as follows:
(1) dewaxing the paraffin sections by dimethylbenzene I and dimethylbenzene II for 5min respectively, then putting the paraffin sections into 100%, 95%, 90%, 80% and 70% alcohol solutions for 3-5 min respectively, and then putting the paraffin sections into distilled water for 3 min; (2) staining with hematoxylin for 20 s; (3) washing away unbound hematoxylin with distilled water; (4) eosin is dyed for 2s, the eosin is sequentially added into 95% ethanol I, II and 70% ethanol, and is quickly taken out, and then the eosin is added into 80% ethanol for 50-55 s and is added into absolute ethanol for 2 min; (5) putting the slices into a mixed solution of xylene and alcohol with the ratio of 1: 1 for 1min, and then putting the slices into xylene I and xylene II for 2-3min respectively; (6) encapsulating with neutral gum.
Scan photographs of HE stained sections were taken using a Pannoramic MIDI digital section scanner.
Example 1: screening of Lactobacillus fermentum CCFM1226
1. Isolation and screening of Lactobacillus
(1) 1g of fresh excrement of the infant is taken, and a sample is enriched for 12 hours in a culture medium containing sorbitol MRS;
(2) performing gradient dilution on the enriched sample, coating the enriched sample on an MRS solid plate added with 0.02% of olcresol purple, and culturing for 24-48 h;
(3) selecting single bacterial colony with obvious color changing ring and according with the basic shape of the lactobacillus to perform plate streaking purification, and screening and separating the lactobacillus;
(4) and culturing the single colony in a liquid MRS culture solution for 24h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.
2. Preliminary identification of lactobacillus: caldolytic ring assay
(l) Culturing the lactobacillus screened in the step 1 in a liquid sorbitol MRS culture solution for 24 hours, and then centrifuging 1mL of culture 8000 Xg for 2 min;
(2) with 0.05M KH2PO4Washing the solution twice;
(3) resuspending the resulting bacterial sludge and streaking on sorbitol MRS-0.75% CaCO3Culturing for 24 hours on the solid culture medium;
(4) selecting bacterial colonies which are obvious in calcium-dissolving ring, round in convex surface, fine, dense, white and sterile mycelia, and preliminarily determining lactobacillus by observing the bacteria in a rod shape through a microscope after gram staining.
3. Molecular biological identification of lactobacillus
Extraction of genome of single bacterium
(1) Culturing the lactobacillus screened in the step 1 overnight; taking l mL of the overnight-cultured bacterial suspension to be placed in a 1.5mL centrifuge tube, centrifuging for 2min at 10000r/min, and removing the supernatant to obtain thalli; purging the thalli with l mL of sterile water, centrifuging for 2min at 10000r/min, and removing the supernatant to obtain the thalli; adding 200 μ L SDS lysate, and water bathing at 80 deg.C for 30 min; adding 200 mu L of phenol-chloroform solution into the thallus lysate, wherein the composition and volume ratio of the phenol-chloroform solution are Tris saturated phenol, chloroform and isoamylol 25:24:1, reversing and mixing uniformly, centrifuging at 12000rpm for 5-10 min, and taking 200 mu L of supernatant; adding 400 mu L of glacial ethanol or glacial isopropanol into 200 mu L of supernatant, standing for 1h at-20 ℃, centrifuging at 12000rpm for 5-10 min, and removing the supernatant; adding 500 mu L of 70% (volume percentage) of glacial ethanol for heavy suspension and precipitation, centrifuging at 12000rpm for 1-3 min, and removing the supernatant; oven drying at 60 deg.C, or naturally air drying;
(2) with 50. mu.L ddH2The pellet was re-dissolved with O for PCR.
Two, 16S rDNA PCR
PCR conditions were as follows: 5min at 95 ℃; 10s at 95 ℃; 30s at 55 ℃; 30s at 72 ℃; step 2-430X; 5min at 72 ℃; 2min at 12 ℃.
Preparing 1% agarose gel, mixing the PCR product with 10000 × loading buffer, loading 2 μ L, running at 120V for 30min, and performing gel imaging;
the obtained PCR product was sent to professional sequencing company, wherein 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, and the obtained sequencing result was subjected to search and similarity comparison in GenBank using BLAST, and the strain identified as Lactobacillus fermentum was stored at-80 ℃.
Third, whole genome sequencing
And (3) sending the extracted whole genome to a professional sequencer, sequencing the whole genome of the strain by using a second-generation sequencer, searching and comparing similarity of obtained sequence results in GenBank by using BLAST, and identifying the sequencing result as Lactobacillus fermentum, wherein the Lactobacillus fermentum CCFM1226 is preserved at-80 ℃ for later use.
Example 2: lactobacillus fermentum CCFM1226 capable of increasing IgA content in feces
The method comprises the following specific steps:
(1) respectively taking out strains of Lactobacillus rhamnosus PAL1, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 and Lactobacillus fermentum JCM1173 in a refrigerator at-80 ℃, streaking the strains in an MRS solid culture medium, culturing for 48h at 37 ℃, selecting a single colony in an MRS liquid culture medium, culturing for 24h at 37 ℃ to respectively obtain seed solutions, inoculating the prepared seed solutions into a new MRS liquid culture medium by the volume amount of 2% (v/v), culturing for 24h at 37 ℃, and culturing for one generation again according to the same manner to respectively obtain Lactobacillus rhamnosus PAL1 fermentation broth, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 fermentation broth and Lactobacillus fermentum JCM1173 fermentation broth;
and then respectively centrifuging the lactobacillus fermentation liquor at 6000r/min and 4 ℃ for 5min, and then carrying out heavy suspension by using 0.01M PBS to respectively prepare bacterial suspensions for animal experiments.
(2) 30 healthy female C57BL/6J mice weighing 12-14 g were taken, adaptively cultured for 1 week, and then randomly divided into 5 groups, i.e., a Lactobacillus fermentum CCFM1226 group, a Lactobacillus fermentum SL241 group, a Lactobacillus rhamnosus PAL1 group, a blank group, and a Lactobacillus fermentum JCM1173 group.
The experimental grouping and treatment methods are shown in table 1:
TABLE 1 Experimental animal groups
On day 7 of the experiment, fresh mouse feces were collected and frozen at-80 ℃.
In the experiment 21, a mouse is fasted for 12 hours without water prohibition, isoflurane anesthetic is used, isoflurane is volatilized through oxygen airflow, the concentration of the isoflurane is generally 2-3% for inducing anesthesia, the isoflurane is maintained to be used for 1.5-2%, the anesthesia time of the general mouse is 2-3min, and the mouse dies by means of cervical dislocation after the anesthesia.
IgA was measured according to the kit method.
The effect of Lactobacillus fermentum on fecal IgA levels in the first week of mice after intragastric administration for each group is shown in FIG. 1, and Lactobacillus fermentum CCFM1226 (fecal IgA content of mice: 79.49. + -. 34.45. mu.g/mg protein) can increase IgA levels in normal mice by 1231.5% compared to naive mice (fecal IgA content of mice: 5.97. + -. 1.89. mu.g/mg protein).
After the lactobacillus fermentum SL241 is perfused, the content of the mouse fecal IgA is as follows: 15.88. + -. 10.49. mu.g/mg protein, IgA levels in mice after gavage with Lactobacillus rhamnosus PAL1 were: 7.33. + -. 2.22. mu.g/mg protein, after administration of L.fermentum JCM1173, the content of IgA in the mouse feces is: 19.33 +/-6.35 mu g/mg protein, and the mouse excrement IgA after the lactobacillus gasseri SL241, the mouse excrement IgA after the lactobacillus rhamnosus PAL1 and the mouse excrement IgA after the lactobacillus gasseri JCM1173 are not obviously changed compared with the blank group; compared with lactobacillus fermentum of the same strain, lactobacillus gasseri CCFM1226 can obviously improve the intestinal secretory IgA level.
Example 3: lactobacillus fermentum CCFM1226 for improving enteritis
The method comprises the following specific steps:
(1) respectively taking out strains of Lactobacillus rhamnosus PAL1, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 and Lactobacillus fermentum JCM1173 in a refrigerator at-80 ℃, streaking the strains in an MRS solid culture medium, culturing for 48h at 37 ℃, selecting a single colony in an MRS liquid culture medium, culturing for 24h at 37 ℃ to respectively obtain seed solutions, inoculating the prepared seed solutions into a new MRS liquid culture medium by the volume amount of 2% (v/v), culturing for 24h at 37 ℃, and culturing for one generation again according to the same manner to respectively obtain Lactobacillus rhamnosus PAL1 fermentation broth, Lactobacillus fermentum SL241, Lactobacillus fermentum CCFM1226 fermentation broth and Lactobacillus fermentum JCM1173 fermentation broth;
and then, respectively centrifuging the lactobacillus fermentation liquor at 6000r/min and 4 ℃ for 5min, and then carrying out heavy suspension by using 0.01M PBS to respectively prepare bacterial suspensions for animal experiments.
(2) The experimental animals are raised in a barrier environment with the temperature of 23 +/-2 ℃, the relative humidity of 50 +/-10 percent, 12h of illumination and 12h of alternate night.
36 mice, six per group, were randomly divided into 6 groups, lactobacillus fermentum CCFM1226, lactobacillus fermentum SL241, lactobacillus rhamnosus PAL1, lactobacillus fermentum JCM1173, blank and model groups (as shown in figure 3).
The adaptive breeding is carried out for one week, and all groups are given normal drinking water and feed in the adaptive period.
Starting from week two, blank group: gavage 0.01M PBS solution 0.2mL daily, water ad libitum, for 14 days.
Model group: gavage 0.01M PBS solution 0.2mL daily for 7 days, gavage 0.01M PBS solution 0.2mL daily for 8-14 days, and adding 2.5% (w/v) DSS solution into drinking water.
Lactobacillus group (lactobacillus rhamnosus PAL1 group/lactobacillus fermentum SL241 group/lactobacillus fermentum CCFM1226 group/lactobacillus fermentum JCM1173 group): each gavage should contain 1 × 10 of the above ingredients every day90.2mL of CFU/mL lactobacillus suspension, lasting for 7 days, 8-14 days, containing 1 × 10 per gavage per day9CFU/mL Lactobacillus suspension 0.2mL, and drinking water added with 2.5% (w/v) DSS.
After 2 weeks of experiment, the mice were sacrificed and colon tissues were taken, and paraffin sections were made and observed after staining, and the results are shown in fig. 2.
The results show that the lactobacillus fermentum CCFM1226 group has improved crypt damage, goblet cell swelling, reduced diffusion, and significantly lower inflammatory infiltration depth and inflammatory cell infiltration than the lactobacillus rhamnosus PAL1, lactobacillus fermentum SL241, and lactobacillus fermentum JCM1173 groups. The lactobacillus fermentum group CCFM1226 has a certain ability to alleviate DSS-induced colitis.
Example 4: application of lactobacillus fermentum CCFM1226
The method comprises the following specific steps:
the lactobacillus fermentum CCFM1226 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:
selecting a single colony of the lactobacillus fermentum CCFM1226 obtained in the example 1, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 24h to obtain an activation solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a first-level seed solution; inoculating the primary seed solution into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a secondary seed solution; inoculating the secondary seed liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a bacterial liquid; centrifuging 6000g of the bacterial liquid for 15min, and collecting precipitates; washing the precipitate with PBS buffer solution with pH of 7.4 twice, and centrifuging again for 10min at 6000g to obtain thallus; the lactobacillus fermentum CCFM1226 cells were resuspended to a cell concentration of 1X 10 using a protectant solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose10CFU/mL to obtain a lactobacillus fermentum CCFM1226 bacterial liquid; freeze-drying the lactobacillus fermentum CCFM1226 bacterial liquid to obtain lactobacillus fermentum CCFM 1226; the freeze-dried mushroom powder accounts for 10 percent of the total content, then stearic acid accounting for 2 percent of the total weight is sequentially added as a lubricant, CMC Na accounting for 3 percent, galacto-oligosaccharide accounting for 15.5 percent, xylo-oligosaccharide accounting for 7.8 percent, inulin accounting for 7.8 percent, lactitol, erythritol and xylitol, and other auxiliary materials such as starch and the like are added for tabletting to obtain the tablet.
The tablet 1g is taken and administered to normal mice per day for one week continuously, so that intestinal tract secretion IgA can be promoted, the IgA content in feces can be increased, and immune homeostasis can be regulated and controlled.
Example 5: application of lactobacillus fermentum CCFMCCFM1226
The method comprises the following specific steps:
the lactobacillus fermentum CCFMCCFM1226 can be used for preparing bacterial powder, and the bacterial powder is prepared by the following specific preparation process:
selecting a single colony of the lactobacillus fermentum CCFM1226 obtained in the example 4, inoculating the single colony into an MRS liquid culture medium, and culturing at 37 ℃ for 24h to obtain an activation solution; inoculating the activated solution into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a first-level seed solution; inoculating the primary seed liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a secondary seed liquid; inoculating the secondary seed liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing at 37 ℃ for 24h to obtain a bacterial liquid; centrifuging 6000g of the bacterial liquid for 15min, and collecting precipitates; washing the precipitate with PBS buffer solution with pH of 7.4 twice, and centrifuging again for 10min at 6000g to obtain thallus; the lactobacillus fermentum CCFMCCFM1226 cells were resuspended to a cell concentration of 1X 10 using a protectant solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose10CFU/mL to obtain a lactobacillus fermentum CCFM1226 bacterial liquid; and (3) freeze-drying the lactobacillus fermentum CCFM1226 bacterial liquid to obtain bacterial powder.
Taking the total number of viable bacteria of 1 × 109The bacterial powder of CFU is administered to normal mice by intragastric administration every day for one week, and can promote intestinal tract secretion of IgA, increase IgA content in feces, and regulate immune homeostasis.
Example 6: fermented food containing the bacterium lactobacillus fermentum CCFM1226 produced by the invention
Selecting fresh vegetables, cleaning, juicing, carrying out high-temperature instant sterilization, carrying out high-temperature heat sterilization at 140 ℃ for 2 seconds, immediately cooling to 37 ℃, and inoculating the lactobacillus fermentum CCFM1226 microbial inoculum starter prepared by the invention to enable the concentration of the lactobacillus fermentum to reach 108And (3) storing the mixture at a temperature of more than 4 ℃ by refrigeration at a CFU/mL ratio, thus obtaining the fruit and vegetable beverage containing the lactobacillus fermentum CCFM1226 viable bacteria.
The invention can be used for preparing other fermented foods by fermenting lactobacillus fermentum CCFM1226, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
The fermented food can promote intestinal tract to secrete IgA, improve IgA content in feces, regulate and control immune homeostasis, and relieve enteritis.
Example 7: lactobacillus fermentum CCFM1226 stimulates the production of IgA in a non-inflammatory manner
The specific implementation manner is the same as that in example 2, except that colon tissues are taken and interleukin-6 (IL-6), interleukin-17 (IL-17) and interleukin-1 beta (IL-1 beta) are respectively detected, and the tissue treatment method is carried out according to the requirements of an R & D enzyme linked immunosorbent assay kit method.
The effect of the level of colonic inflammation in mice after intragastric administration of lactobacillus in each group is shown in fig. 4-6.
(1) At the IL-6 level, the content of the IL-6 in the colon of the lactobacillus fermentum CCFM1226 (the content of the IL-6 in the colon of the mouse is 391.8 +/-180.7 pg/mg protein) is slightly increased compared with that of a blank mouse (the content of the IL-6 in the colon of the mouse is 308.4 +/-146.8 pg/mg protein), but the difference is not significant; therefore, the lactobacillus fermentum CCFM1226 provided by the invention does not cause obvious change of inflammatory factors, and can significantly improve the IgA content in mice.
After the L.fermentum SL241 is perfused, the content of IL-6 in the colon of the mouse is 417.7 +/-200.5 pg/mg protein; after the lactobacillus rhamnosus PAL1 is irrigated, the content of IL-6 in the colon of the mouse is 291.3 +/-161.8 pg/mg protein; after the administration of Lactobacillus fermentum JCM1173, the IL-6 content in the colon of the mouse is 253.0 +/-50.27 pg/mg protein.
(2) At the IL-17 level, compared with a blank mouse (the IL-17 content of the colon of the mouse is 415.2 +/-182.5 pg/mg protein), the content of IL-17 in the lactobacillus fermentum CCFM1226 (the IL-17 content of the colon of the mouse is 593.5 +/-241.5 pg/mg protein) is improved in a small range, but the IgA content in the mouse can be obviously improved without significant difference.
After the L.fermentum SL241 is perfused, the content of IL-17 in the colon of the mouse is 496.8 +/-93.92 pg/mg protein; after the lactobacillus rhamnosus PAL1 is perfused, the content of IL-17 in the colon of the mouse is 428.9 +/-95.37 pg/mg protein; after the administration of Lactobacillus fermentum JCM1173, the content of IL-17 in the colon of the mouse is 345.7 +/-77.44 pg/mg protein.
At the IL-1 beta level, compared with a blank mouse (the IL-1 beta content of the colon of the mouse is 186.7 +/-41.44 pg/mg), the lactobacillus fermentum CCFM1226 (the IL-1 beta content of the colon of the mouse is 187.7 +/-42.35 pg/mg protein) has no significant difference; therefore, the lactobacillus fermentum CCFM1226 provided by the invention does not cause obvious change of inflammatory factors, and can significantly improve the IgA content in mice.
After lactobacillus fermentum SL241 is poured, the content of IL-1 beta in the colon of the mouse is 179.2 +/-7.75 pg/mg protein; after lactobacillus rhamnosus PAL1 is irritated, the content of IL-1 beta in the colon of the mouse is 182.2 +/-6.15 pg/mg protein; after lactobacillus fermentum JCM1173, the IL-1 beta content in the colon of the mouse is 183.3 +/-35.12 pg/mg protein.
In conclusion, lactobacillus fermentum CCFM1225 promotes IgA secretion from the host gut in a manner that does not stimulate inflammation in the colon.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> lactobacillus fermentum CCFM1226 capable of improving intestinal IgA level and relieving enteritis and application thereof
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Claims (10)
1. Lactobacillus fermentum (CCFM 1226, which was deposited at 27.1.2022 in the Guangdong province culture Collection with the deposit number GDMCC No: 62242.
2.a microbial preparation comprising the Lactobacillus fermentum CCFM1226 according to claim 1.
3. The microbial preparation of claim 2, wherein the microbial preparation comprises one or more of a viable strain of lactobacillus fermentum CCFM1226, a dry strain of lactobacillus fermentum CCFM1226, a metabolite of lactobacillus fermentum CCFM1226, and an inactivated strain of lactobacillus fermentum CCFM 1226.
4. A product comprising lactobacillus fermentum CCFM1226 according to claim 1, or comprising a microbial preparation according to claim 2 or 3.
5. The product of claim 4, wherein the product is a food, pharmaceutical or nutraceutical product.
6. The product according to claim 4 or 5, characterized in that the food product is: comprises solid food, liquid food, semi-solid food or fermented food, the fermented food comprises any one of dairy products, bean products and fruit and vegetable products, and the dairy products comprise any one of milk, sour cream and cheese; the fruit and vegetable product comprises any one of cucumber, carrot, beet, celery and cabbage product.
7. Use of lactobacillus fermentum CCFM1226 according to claim 1 for the manufacture of an anti-inflammatory product for increasing intestinal IgA secretion.
8. Use according to claim 7 for the preparation of a product having at least one of the functions (a) to (b):
(a) improving the IgA content in the excrement;
(b) can obviously relieve enteritis.
9. The use according to claim 7 or 8, wherein the product is a pharmaceutical, nutraceutical or functional bacterial agent.
10. Use of lactobacillus fermentum CCFM1226 according to claim 1 for the preparation of a fermented food product.
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