CN114540247B - 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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- CN114540247B CN114540247B CN202210266770.5A CN202210266770A CN114540247B CN 114540247 B CN114540247 B CN 114540247B CN 202210266770 A CN202210266770 A CN 202210266770A CN 114540247 B CN114540247 B CN 114540247B
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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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
Technical Field
The invention relates to a lactobacillus fermentum CCFM1226 capable of improving intestinal tract IgA level and relieving enteritis and application thereof, belonging to the technical field of microorganisms.
Background
Immunoglobulin a (IgA) is the most produced Immunoglobulin in the intestinal tract of mammals; it is a component of serum immunoglobulins, and most of IgA is secreted by the mucosa of the intestinal tract, respiratory tract, biliary tract and genital tract. In humans, the structure of IgA exists mainly in the form of monomers and dimers. According to the distribution of IgA in the body, it can be divided into serotypes and secretes. Serotype IgA is monomeric and has weaker immune effects. Secretory IgA has two and three bodies, is the main component of the body mucosa defense system, and is widely distributed in milk, saliva, gastrointestinal tract, respiratory tract and genitourinary tract mucosa secretion. It can inhibit the adhesion of microbe to respiratory tract epithelium, slow down the propagation of virus, has important immune barrier function, has antibody activity to some viruses, bacteria and general antigens, and is the first defense line for preventing pathogen from invading organism. IgA in the gut is widely known as secretory IgA.
Most lymphoid tissues and immune cells in the intestinal mucosal immune system are in a certain relationship with IgA. Meanwhile, a great deal of evidence shows that intestinal bacteria play an important role in mucosal immunity. The mucosal immune system needs to elicit an effective defense against pathogens of mucosal invasion on the one hand and to develop proper tolerance against microorganisms on the mucosa that are symbiotic with the organism on the other hand. IgA has the effect of combating pathogens and controlling mucosal inflammatory reactions, the production of which has an important relationship with mucosal bacteria. The adhesion and encapsulation of IgA prevents direct contact of the host intestinal epithelium with the harmful antigens in the intestinal lumen, and is important in maintaining the integrity of the intestinal barrier, while IgA also regulates the composition and balance of the intestinal flora. IgA neutralizes microbial toxins and pathogens with a high affinity, and a low affinity binding system protects the gut from commensal bacteria.
In recent years, with the intensive research on the relationship between intestinal flora and human health, a great deal of research has confirmed that probiotics improve human health by modulating immunity. The probiotics have the characteristics of safety, no side effect and the like, and a great 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 object of the present invention is to provide a strain of lactobacillus fermentum (Lactobacillus fermentum) CCFM1226 deposited at the collection of microorganism strains in the cantonese province at 1/27 of 2022 under the accession number GDMCC No:62242.
in one embodiment of the present invention, the lactobacillus fermentum is derived from the stool of infants, the extracted whole genome is sent to a professional sequencing company, the whole genome of the bacteria is sequenced by a second generation sequencer, the obtained sequence result is searched in GeneBank by using BLAST and is subjected to similarity comparison, and the sequencing result is identified as lactobacillus fermentum and is named lactobacillus fermentum (Lactobacillus fermentum) CCFM1226.
In one embodiment of the invention, lactobacillus fermentum (Lactobacillus fermentum) CCFM1226 has the following characteristics:
(1) Characteristics of the cells: gram-positive, no sporulation, non-motile bacteria.
(2) Colony characteristics: it is milky white, glossy, convex, opaque, smooth and tidy.
(3) Growth characteristics: the culture was carried out in MRS medium at a constant temperature of 37℃for about 12 hours to the end of the log phase.
(4) Has strong tolerance to simulated gastrointestinal fluid.
A second object of the present invention is to provide a microbial preparation comprising the above Lactobacillus fermentum CCFM1226.
In one embodiment of the present invention, the amount of Lactobacillus fermentum CCFM1226 in the microbial preparation is not less than 1X 10 6 CFU/mL or ≡1X10. 6 CFU/g。
In one embodiment of the present invention, the amount of Lactobacillus fermentum CCFM1226 in the microbial preparation is not less than 1X 10 9 CFU/mL or ≡1X10. 9 CFU/g。
In one embodiment of the invention, the microbial agent includes, but is not limited to, a functional food, a nutraceutical, or a pharmaceutical.
In one embodiment of the invention, the microbial preparation contains one or more of a viable 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.
In one embodiment of the invention, the medicament has at least one of the following effects:
(a) Increasing the content of IgA in the feces;
(b) Obviously relieving 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 invention, the medicament further comprises a pharmaceutically acceptable carrier.
In one embodiment of the invention, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, wetting agent, disintegrant, binder, or 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 disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked 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 stearate fumarate, talcum powder or silicon dioxide.
It is a third object of the present invention to provide a product comprising the above Lactobacillus fermentum CCFM1226 or the above microbial preparation.
In one embodiment of the invention, the product is a food, pharmaceutical or health 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 comprises dairy products, bean 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 product comprises cucumber, carrot, beet, celery and cabbage products.
In one embodiment of the invention, the pharmaceutical product contains a pharmaceutically acceptable carrier or adjuvant.
In one embodiment of the invention, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, wetting agent, disintegrant, binder, or 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 disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked 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 stearate fumarate, talcum powder or silicon dioxide.
The fourth object of the invention is to provide the application of the lactobacillus fermentum CCFM1226 in preparing products for improving intestinal secretion IgA and resisting inflammation.
In one embodiment of the invention, the medicament is for increasing the IgA content of feces.
In one embodiment of the invention, the medicament is for alleviating enteritis.
In one embodiment of the invention, the product is a functional microbial agent, a pharmaceutical product or a health care product.
In one embodiment of the invention, the pharmaceutical product contains a pharmaceutically acceptable carrier or adjuvant.
In one embodiment of the invention, the pharmaceutically acceptable carrier includes, but is not limited to: one or more of a filler, wetting agent, disintegrant, binder, or 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 disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked 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 stearate fumarate, talcum powder or silicon dioxide.
The invention also claims the application of the lactobacillus fermentum CCFM1226 in preparing fermented food.
In one embodiment of the invention, the use includes, but is not limited to, fermentation using the lactobacillus fermentum CCFM1226 as a fermenting microorganism, using a food material.
In one embodiment of the invention, the fermented food comprises dairy products, bean 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 product comprises cucumber, carrot, beet, celery and cabbage products.
Advantageous effects
The invention provides a lactobacillus fermentum CCFM1226, wherein the lactobacillus fermentum CCFM1226 can be used for preparing functional microbial inoculum, food and medicine for improving the content of IgA in feces, and has wide application prospect, and the specific expression is as follows:
(1) The effect of lactobacillus fermentum CCFM1226 on the IgA level of the feces of mice is compared with that of blank mice, and the gastric lavage lactobacillus fermentum CCFM1226 can improve the IgA level of normal mice by 1231.5 percent and obviously improve the secretion IgA level of intestinal tracts compared with the same different strains of lactobacillus fermentum.
(2) Compared with a model group, the lactobacillus fermentum CCFM1226 has the advantages that the damage degree of the crypt is improved to a certain extent, the goblet cells are swollen, the diffusion degree is reduced, and the inflammatory infiltration depth and the inflammatory infiltration degree are obviously lighter than those of other lactobacillus groups, so that the lactobacillus fermentum CCFM1226 has certain alleviation capability on the colitis induced by DSS.
Preservation of biological materials
Lactobacillus fermentum CCFM1226, classified under the name: lactobacillus fermentum, which was deposited at the Cantonese microorganism strain collection at 1.27 of 2022 under the accession number GDMCC No:62242, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
Drawings
Fig. 1: influence of Lactobacillus curvatus on fecal IgA of normal mice; wherein P <0.05, P <0.01, P <0.001, P <0.0001 (compared to normal mouse blank).
Fig. 2: the effect of improving enteritis after the lactobacillus gastrosis is filled.
Fig. 3: animal grouping and experimental method for enteritis experiment.
Fig. 4: effects of each group of Lactobacillus on the IL-6 level of colon inflammation in mice after gastric lavage.
Fig. 5: effects of each group of Lactobacillus on the IL-17 level of colon inflammation in mice after gastric lavage.
Fig. 6: effects of each group of Lactobacillus on the IL-1. Beta. Level of colon inflammation in mice after gastric lavage.
Detailed Description
The Lactobacillus fermentum JCM1173 referred to in the examples described below was obtained from the Japanese collection of microorganisms (Japan Collection of Microorganisms, JCM).
Lactobacillus fermentum SL241 as referred to in the examples below is disclosed in "Yan Zhao et al Phylogenetic and comparative genomic analysis of Lactobacillus fermentum Strains and the Key Genes Related to their Intestinal Anti-insolator Effects.Engineering,2021,DOI:https:// doi.org/10.1016/j.eng.2020.09.016"; the related lactobacillus rhamnosus PAL1 is disclosed in "Tian Peijun. The study of bifidobacterium breve CCFM1025 with depression relieving function [ D ]. University of south of the river" paper.
Healthy female C57BL/6J mice referred to in the examples below were purchased from Vetolihua.
The following examples relate to the following media:
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast powder, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 1mL of Tween 80, 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 above components into distilled water, heating to dissolve completely, adjusting pH to 6.2-6.4, packaging into triangular flask, sterilizing at 121deg.C for 15min.
MRS solid medium: 15-20 g of agar is added on the basis of MRS liquid culture medium.
The following examples relate to the following methods for the preparation and staining of paraffin sections of colon:
the experimental steps for preparing the colon paraffin section are as follows:
(1) Taking a section of distal colon 1cm away from the anus 1cm, and fixing with 4% paraformaldehyde for 72h; (2) Washing the fixed colon tissue with running water for 8h, dehydrating, sequentially dehydrating the sample with 70%, 80% and 90% ethanol solutions for 30min, and adding 95% and 100% ethanol solution for 2 times each for 20min; (3) Placing a colon sample into a mixed solution of xylene and alcohol in a ratio of 1:1 for 15min, and then placing the colon sample into each of xylene I and xylene II for 15min; (4) Transferring colon tissue to mixed solution of xylene and paraffin for 15min, adding paraffin I and paraffin II, and allowing paraffin permeation for 1 hr, and maintaining at 60deg.C; (5) Embedding the colon in the remelted wax block by a lycra paraffin embedding machine; (6) Slicing the embedded tissue with a tissue slicer to a thickness of 5 μm; and (7) sticking the sheet, airing, and placing in an oven at 62 ℃ for 1h.
The HE staining process is as follows:
(1) Dewaxing paraffin sections by using xylene I and xylene II for 5min respectively, then putting the paraffin sections into alcohol solutions of 100%, 95%, 90%, 80% and 70% levels for 3-5 min respectively, and then putting the paraffin sections into distilled water for 3min; (2) staining with hematoxylin for 20s; (3) washing the unbound hematoxylin with distilled water; (4) Eosin dyeing for 2s, sequentially adding into 95% ethanol I, II and 70% ethanol, quickly taking out, adding into 80% ethanol for 50-55 s, and adding into absolute ethanol for 2min; (5) Slicing, putting the slices into a mixed solution of dimethylbenzene and alcohol in a ratio of 1:1 for 1min, and then putting the slices into dimethylbenzene I and dimethylbenzene II for 2-3min respectively; (6) sealing the sheet by neutral resin.
HE stained sections were scanned using a pannarac MIDI digital section scanner.
Example 1: screening of Lactobacillus fermentum CCFM1226
1. Isolation and screening of Lactobacillus
(1) Taking 1g of fresh feces of an infant, and enriching a sample in a sorbitol-containing MRS medium for 12 hours;
(2) The enriched sample is coated on an MRS solid flat plate added with 0.02 percent of olfactory cresol purple after gradient dilution, and is cultured for 24 to 48 hours;
(3) Selecting single bacterial colony which has obvious color-changing ring and accords with the basic form of the lactobacillus, carrying out flat plate streak purification, and screening and separating the lactobacillus;
(4) The single colony is cultured in liquid MRS culture solution for 24 hours, then gram staining is carried out, and gram positive bacteria are selected for subsequent tests.
2. Preliminary identification of lactobacillus: method for measuring calcium dissolving ring
(l) Culturing the lactobacillus screened in the step 1 in liquid sorbitol MRS culture solution for 24 hours, and centrifuging the L mL culture at 8000 Xg for 2min;
(2) With 0.05M KH 2 PO 4 Washing the solution twice;
(3) Resuspension of the resulting bacterial sludge, streaking at sorbitol MRS-0.75% CaCO 3 Culturing for 24 hours on a solid culture medium;
(4) And selecting bacterial colonies which are obvious in calcium dissolving ring, have a round convex surface, are fine and white and have no mycelium, and observing the bacterial colonies into a rod shape by a microscope after gram staining, namely primarily judging the bacterial colonies as lactobacillus.
3. Molecular biological identification of lactobacillus
1. Single genome extraction
(1) Culturing the lactobacillus screened in the step 1 overnight; taking bacterial suspension L cultured overnight, centrifuging in a 1.5mL centrifuge tube at 10000r/min for 2min, and discarding the supernatant to obtain thalli; washing the thalli with l mL of sterile water, centrifuging for 2min at 10000r/min, and discarding the supernatant to obtain thalli; 200 mu L of SDS lysate is added, and water bath is carried out for 30min at 80 ℃; adding 200 mu L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises the components and the volume ratio of Tris saturated phenol and chloroform and isoamyl alcohol=25:24:1, mixing the mixture reversely, centrifuging the mixture 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 at-20 ℃ for 1h, centrifuging at 12000rpm for 5-10 min, and discarding the supernatant; adding 500 mu L70% (volume percent) of ice-ethanol to re-suspend the sediment, centrifuging at 12000rpm for 1-3 min, and discarding the supernatant; oven drying at 60deg.C, or naturally air drying;
(2) 50 μL ddH is used 2 O was redissolved and precipitated for PCR.
2. 16S rDNA PCR
Bacterial 16S rDNA 50. Mu. LPCR reaction System: 10×Taq buffer, 5. Mu.L; dNTP, 5. Mu.L; primer 27F, 0.5. Mu.L; primer 1492R, 0.5. Mu.L; taq enzyme, 0.5. Mu.L; template, 0.5 μl; ddH 2 O，38μL。
PCR conditions: 95 ℃ for 5min;95 ℃ for 10s; 30s at 55 ℃; 30s at 72 ℃; step 2-4X; 72 ℃ for 5min; and 2min at 12 ℃.
Preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;
and (3) sending the obtained PCR product to a professional sequencing company, wherein the sequence of CCFM1226-27F_C02.ab1 is shown as SEQ ID NO.1, the sequence of CCFM 1226-14992 R_B02.ab1 is shown as SEQ ID NO.2, and carrying out search and similarity comparison on the obtained sequencing result and a strain identified as lactobacillus fermentum in GenBank by using BLAST, and storing at the temperature of-80 ℃.
3. Whole genome sequencing
The extracted whole genome is sent to a professional sequencing company, a second generation sequencer is used for sequencing the whole genome of the bacterium, the obtained sequence result is searched in GenBank by using BLAST and is subjected to similarity comparison, the sequencing result is identified as lactobacillus fermentum, and the lactobacillus fermentum is named lactobacillus fermentum (Lactobacillus fermentum) CCFM1226 and is preserved at the temperature of minus 80 ℃ for later use.
Example 2: lactobacillus fermentum CCFM1226 can increase IgA content in feces
The method comprises the following specific steps:
(1) Respectively taking out lactobacillus rhamnosus PAL1, lactobacillus fermentum SL241, lactobacillus fermentum CCFM1226 and lactobacillus fermentum JCM1173 strains at the temperature of minus 80 ℃, marking the strains in an MRS solid culture medium, culturing the strains for 48 hours at the temperature of 37 ℃, picking single bacterial colonies in the MRS liquid culture medium, culturing the strains for 24 hours at the temperature of 37 ℃ to respectively obtain seed solutions, inoculating the prepared seed solutions into a new MRS liquid culture medium in a volume amount of 2% (v/v), culturing the seed solutions for 24 hours at the temperature of 37 ℃, and culturing the seed solutions again for one generation in the same way to respectively obtain lactobacillus rhamnosus PAL1 fermentation broth, lactobacillus fermentum SL241 fermentation broth, lactobacillus fermentum CCFM1226 fermentation broth and lactobacillus fermentum JCM1173 fermentation broth;
and then respectively centrifuging the lactobacillus fermentation liquid at 6000r/min and 4 ℃ for 5min, and then re-suspending the lactobacillus fermentation liquid by using 0.01M PBS to prepare bacterial suspensions respectively for animal experiments.
(2) 30 healthy female C57BL/6J mice with the weight of 12-14 g are taken, and after being adaptively cultured for 1 week, the mice are randomly divided into 5 groups, namely, 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 processing methods are shown in table 1:
TABLE 1 grouping of experimental animals
On day 7 of the experiment, fresh feces from mice were collected and frozen at-80 ℃.
In experiment 21, mice are fasted without water inhibition for 12 hours, isoflurane anesthetic is used, isoflurane is volatilized through oxygen airflow, concentration of 2-3% is generally used for induced anesthesia, 1.5-2% is maintained, the anesthesia time of the mice is generally 2-3min, and the mice die by cervical dislocation after anesthesia.
IgA was measured according to the kit method.
After the stomach is filled with each group of lactobacillus, the effect on the IgA level of the first week of the mice is shown in FIG. 1, and compared with a blank mouse (the content of the IgA in the feces of the mice is 5.97+/-1.89 mug/mg protein), the CCFM1226 of the lactobacillus fermentum (the content of the IgA in the feces of the mice is 79.49+/-34.45 mug/mg protein) can improve the IgA level of the normal mice by 1231.5 percent.
After the lactobacillus fermentum SL241 was fed, the content of mouse fecal IgA was: 15.88+ -10.49 μg/mg protein, igA levels in mice after L.rhamnosus PAL1 were: 7.33+/-2.22 mug/mg protein, and after the lactobacillus curvatus JCM1173 is filled, the content of the IgA in the feces of the mice is as follows: 19.33+/-6.35 mug/mg protein, and the result shows that the mouse fecal IgA after the lactobacillus curvatus SL241 and the mouse fecal IgA after the lactobacillus curvatus PAL1 are subjected to the stomach-filling and the mouse fecal IgA after the lactobacillus curvatus JCM1173 are not obviously changed compared with the blank group; the gastric lavage lactobacillus fermentum CCFM1226 significantly increases the intestinal tract secretory IgA levels compared to the same different strains of lactobacillus fermentum.
Example 3: lactobacillus fermentum CCFM1226 for improving enteritis
The method comprises the following specific steps:
(1) Respectively taking out lactobacillus rhamnosus PAL1, lactobacillus fermentum SL241, lactobacillus fermentum CCFM1226 and lactobacillus fermentum JCM1173 strains at the temperature of minus 80 ℃, marking the strains in an MRS solid culture medium, culturing the strains for 48 hours at the temperature of 37 ℃, picking single bacterial colonies in the MRS liquid culture medium, culturing the strains for 24 hours at the temperature of 37 ℃ to respectively obtain seed solutions, inoculating the prepared seed solutions into a new MRS liquid culture medium in a volume amount of 2% (v/v), culturing the seed solutions for 24 hours at the temperature of 37 ℃, and culturing the seed solutions again for one generation in the same way to respectively obtain lactobacillus rhamnosus PAL1 fermentation broth, lactobacillus fermentum SL241 fermentation broth, lactobacillus fermentum CCFM1226 fermentation broth and lactobacillus fermentum JCM1173 fermentation broth;
and then, respectively centrifuging lactobacillus fermentation liquid at 6000r/min and 4 ℃ for 5min, and then, re-suspending the lactobacillus fermentation liquid by using 0.01M PBS to prepare bacterial suspensions for animal experiments.
(2) The experimental animals are kept in a barrier environment with alternating temperatures of (23+/-2)%, relative humidity of (50+/-10)%, 12 hours of illumination and 12 hours of night.
36 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 FIG. 3).
And (3) feeding for one week, and all groups in the adaptation period are given normal drinking water and feed.
Starting from the second week, the blank group: the stomach was irrigated 0.01M PBS solution 0.2mL daily, with free water for 14 days.
Model group: 0.2mL of 0.01M PBS solution is filled into the stomach every day for 7 days, 8 to 14 days, 0.2mL of 0.01M PBS solution is filled into the stomach every day, and 2.5% (w/v) DSS solution is added into drinking water.
Lactobacillus group (lactobacillus rhamnosus PAL1 group/lactobacillus fermentum SL241 group/lactobacillus fermentum CCFM1226 group/lactobacillus fermentum JCM1173 group): each stomach is filled with 1×10 medicines each day 9 CFU/mL lactobacillus bacterial suspension 0.2mL for 7 days, 8-14 days, each stomach being 1X 10 per day 9 CFU/mL Lactobacillus suspension 0.2mL, 2.5% (w/v) DSS was added to the drinking water.
After 2 weeks of the experiment, the mice were sacrificed, colon tissues were taken, paraffin sections of the colon were prepared and stained for observation, and the results are shown in fig. 2.
The results show that compared with the model group, the lactobacillus fermentum CCFM1226 group has a certain improvement of crypt injury degree, cup-shaped cell swelling and dispersion degree, and the inflammatory infiltration depth and inflammatory cell infiltration degree are obviously lighter than those of lactobacillus rhamnosus PAL1 group, lactobacillus fermentum SL241 group and lactobacillus fermentum JCM1173 group. The lactobacillus fermentum group CCFM1226 has certain alleviation capability on DSS-induced colitis.
Example 4: application of lactobacillus fermentum CCFM1226
The method comprises the following specific steps:
lactobacillus fermentum CCFM1226 may be used to prepare tablets, the specific preparation process of which is as follows:
picking single colony of lactobacillus fermentum CCFM1226 obtained in example 1, inoculating into MRS liquid culture medium, and culturing at 37deg.C for 24 hr to obtain activated strainA liquid; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain first-stage seed solution; inoculating the first-level seed liquid into MRS liquid culture medium according to an inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain a second-level seed liquid; inoculating the secondary seed solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain bacterial solution; centrifuging 6000g of bacterial liquid for 15min, and collecting precipitate; washing the precipitate with PBS buffer solution with pH of 7.4 twice, and centrifuging 6000g for 10min again to obtain thallus; lactobacillus fermentum CCFM1226 strain was resuspended to a cell concentration of 1X 10 with a protectant solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose 10 CFU/mL, obtaining lactobacillus fermentum CCFM1226 bacterial liquid; freeze-drying the lactobacillus fermentum CCFM1226 bacterial liquid to obtain lactobacillus fermentum CCFM1226; the freeze-dried bacterial powder accounts for 10% of the total weight, 2% of stearic acid is sequentially added as a lubricant, 3% of CMC Na,15.5% of galacto-oligosaccharide, 7.8% of xylo-oligosaccharide, 7.8% of inulin, lactitol, erythritol and xylitol, and other auxiliary materials such as starch are added for tabletting to obtain tablets.
1g of the tablet is taken for daily gastric lavage of a normal mouse, and the tablet can promote intestinal secretion of IgA, improve the content of IgA in feces and regulate and control immune homeostasis for one week.
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 specific preparation process of the bacterial powder is as follows:
picking single colony of lactobacillus fermentum CCFM1226 obtained in the example 4, inoculating into MRS liquid culture medium, and culturing at 37 ℃ for 24 hours to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain first-stage seed solution; inoculating the first-level seed liquid into MRS liquid culture medium according to an inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain a second-level seed liquid; inoculating the secondary seed solution into MRS liquid culture medium according to the inoculum size of 1% (v/v), and culturing at 37 ℃ for 24 hours to obtain bacterial solution; centrifuging 6000g of bacterial liquid for 15min, and collecting precipitate; the pellet was washed twice with PBS buffer at pH 7.4Then, 6000g is centrifuged again for 10min to obtain thalli; lactobacillus fermentum CCFMCCFM1226 strain was resuspended to a cell concentration of 1X 10 with a protectant solution containing 130g/L skim milk, 20g/L trehalose and 20g/L sucrose 10 CFU/mL, obtaining lactobacillus fermentum CCFM1226 bacterial liquid; and freeze-drying the lactobacillus fermentum CCFM1226 bacterial liquid to obtain bacterial powder.
Taking 1×10 total viable bacteria 9 The bacterial powder of the CFU is used for lavaging normal mice daily for one week, so that the secretion of IgA in intestinal tracts can be promoted, the content of IgA in feces can be improved, and the immune homeostasis can be regulated.
Example 6: the invention is used for preparing the fermented food containing the lactobacillus fermentum CCFM1226
Cleaning fresh vegetable, squeezing juice, high-temp. instantaneous sterilizing, high-temp. sterilizing at 140 deg.C for 2 seconds, immediately cooling to 37 deg.C, then inoculating lactobacillus fermentum CCFM1226 microbial inoculum starter prepared by said invention to make its concentration be 10 8 And (3) refrigerating and preserving the mixture at the temperature of 4 ℃ above CFU/mL, so as to obtain the fruit and vegetable beverage containing the lactobacillus fermentum CCFM1226 viable bacteria.
The invention can be used for preparing other fermented foods by using lactobacillus fermentum CCFM1226 for fermentation production, 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 secretion of IgA, increase IgA content in feces, regulate immune homeostasis, and relieve enteritis.
Example 7: lactobacillus fermentum CCFM1226 stimulates IgA production in a non-inflammatory manner
The specific embodiment is the same as example 2, except that colon tissues are taken, interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-1β (IL-1β) are detected respectively, and the tissue treatment method is carried out according to the R & D ELISA kit method.
The effect of colon inflammation levels in mice after lavage with each group of lactobacillus is shown in figures 4-6.
(1) At the IL-6 level, lactobacillus fermentum CCFM1226 (content of mouse colon IL-6: 391.8.+ -. 180.7pg/mg protein) was slightly elevated but not significantly different compared to the blank mice (content of mouse colon IL-6: 308.4.+ -. 146.8pg/mg protein); therefore, the lactobacillus fermentum CCFM1226 does not cause obvious change of inflammatory factors, but can obviously improve the IgA content in mice.
After the lactobacillus fermentum SL241 is filled, the content of the colon IL-6 of the mice is 417.7+/-200.5 pg/mg protein; after the lactobacillus rhamnosus PAL1 is irrigated, the content of the colon IL-6 of the mice is 291.3 +/-161.8 pg/mg protein; after the Lactobacillus curvatus JCM1173, the content of IL-6 in the colon of the mice is 253.0+ -50.27 pg/mg protein.
(2) At the IL-17 level, lactobacillus fermentum CCFM1226 (mouse colon IL-17 content: 593.5.+ -. 241.5pg/mg protein) significantly increased IgA content in mice, although slightly increased, without significant differences, compared to the blank mice (mouse colon IL-17 content: 415.2.+ -. 182.5pg/mg protein).
After the lactobacillus fermentum SL241 is filled, the content of the colon IL-17 of the mice is 496.8 +/-93.92 pg/mg protein; after the lactobacillus rhamnosus PAL1 is irrigated, the content of the colon IL-17 of the mice is 428.9 +/-95.37 pg/mg protein; after the Lactobacillus curvatus JCM1173, the content of the colon IL-17 in the mice is 345.7 +/-77.44 pg/mg protein.
At the IL-1β level, there was no significant difference in Lactobacillus fermentum CCFM1226 (content of mouse colon IL-1β: 187.7.+ -. 42.35pg/mg protein) compared to the blank mice (content of mouse colon IL-1β: 186.7.+ -. 41.44pg/mg protein); therefore, the lactobacillus fermentum CCFM1226 does not cause obvious change of inflammatory factors, but can obviously improve the IgA content in mice.
After the lactobacillus fermentum SL241 is filled, the content of the colon IL-1 beta of the mice is 179.2+/-7.75 pg/mg protein; after lactobacillus rhamnosus PAL1 is filled, the content of the colon IL-1 beta of the mice is 182.2+/-6.15 pg/mg protein; after the lactobacillus fermentum JCM1173 is filled, the content of the colon IL-1 beta of the mice is 183.3+/-35.12 pg/mg protein.
In conclusion, lactobacillus fermentum CCFM1225 promotes intestinal secretory IgA in the host in a manner that does not stimulate inflammation of the colon.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 Jiangnan
<120> Lactobacillus fermentum CCFM1226 capable of increasing intestinal IgA level and relieving enteritis and application thereof
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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.
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