CN112708577A - Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof - Google Patents
Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof Download PDFInfo
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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Abstract
The lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation functions and the application thereof, and the strain has better acid resistance, cholate resistance, hydrophobicity and self-polymerization capability, and the fermented milk has better inhibition capability on staphylococcus aureus, escherichia coli and salmonella. In vitro cell experiments show that the strain has good adhesion capability and immunoregulation capability, and can be used for production of foods such as fermented milk, lactobacillus beverages and the like, functional foods and medicines.
Description
Technical Field
The invention relates to the technical field of biology, in particular to lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof.
Background
Probiotics are defined as: viable microorganisms which, when ingested in sufficient quantities, can have a beneficial effect on human health. Most of them have been generally proven to have health functions such as improving the intestinal environment of consumers, performing immune regulation, etc. The first step in the beneficial effect of the strain on the host is adhesion, and high adhesion prevents the strain from being eliminated by the peristaltic action of the digestive tract.
The adhesion of the strain is beneficial to maintaining the normal flora structure of the intestinal tract and maintaining the integrity of the form and the function of the intestinal mucosa. Many immune cells, including lymphocytes, dendritic cells and macrophages, are present in the gastrointestinal tract, and thus a high adhesion capacity represents more contact time. In addition, the highly adhesive probiotic lactic acid bacteria are gathered in large quantities in the intestinal tract, and can generate enough concentration of bacteriostatic components, such as acidic substances, biological enzymes, antimicrobial peptides and the like, so that the activity of pathogenic bacteria is inhibited.
Probiotics need to maintain a certain viability during digestion in order to be able to adhere to and immunoregulatory in the intestine, so their tolerance to gastrointestinal conditions including low pH of gastric acid and bile salts is always a consideration when selecting probiotics. In vitro studies of strains, the ability to tolerate artificial gastric juices and bile salts is often a priority.
The adhesion property of the lactobacillus is measured in vitro by a direct method and an indirect method, wherein the direct method is to evaluate the adhesion capability of the lactobacillus by an in vitro adhesion test of the lactobacillus and an intestinal cell line Caco-2, and the indirect method is to indirectly reflect the adhesion capability of the lactobacillus by measuring the physicochemical property of the cell surface of the lactobacillus. Surface Hydrophobicity (CSH) is one of the important physicochemical properties of the Surface of lactic acid bacteria, and the microbial adhesion hydrocarbon method (bat), measurement of the self-polymerization ability of bacteria, and measurement of the Surface charge of bacteria are common methods for reflecting Surface Hydrophobicity.
The strain is derived from traditional fermented products of Yunan Dali, such as dairy fan, pickle, cheese, pickled Chinese cabbage, chili sauce and the like, the capabilities of the strain, including acid resistance, cholate resistance, surface hydrophobicity, self-polymerization capability and Caco-2 cell adhesion capability, are researched, and the immunoregulation capability of the screened high-adhesion lactic acid bacteria is researched.
Disclosure of Invention
The invention aims to provide lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof.
The first purpose of the invention is to provide Lactobacillus fermentum DALI02(Lactobacillus fermentum) which is preserved in the China general microbiological culture Collection center at 7-5.2018, wherein the address is No. 3 of Xilu 1 of Beijing university and the institute of microbiology of China academy of sciences, the Korean district, and the preservation number is CGMCC 16064. Obtained by the following steps:
(1) separating the lactic acid bacteria from the traditional fermented product by the culture medium;
(2) through the artificial gastric juice tolerance and bile salt resistance tests of the isolated strain, a strain with good tolerance is obtained;
(3) after fermented milk curds are prepared by using different strains, the bacteriostatic ability is measured;
(4) testing the self-polymerization capability and hydrophobicity of the strain and the adhesion capability of Caco-2 cells to obtain a strain with good adhesion capability, namely DALI 02;
(5) the immunomodulatory effect of DALI02 on RAW264.7 was studied.
The invention also provides application of the lactobacillus fermentum DALI02 and application of the lactobacillus fermentum DALI02 in production of foods (including functional foods) and medicines, and particularly application of the lactobacillus fermentum DALI02 in preparation of fermented milk with immunoregulation effect.
The lactobacillus fermentum DALI02 has good acid resistance, cholate resistance, hydrophobicity, self-polymerization ability and bacteriostatic ability, and the fermented milk has good inhibitory ability to staphylococcus aureus, escherichia coli and salmonella. In vitro cell experiments show that the lactobacillus fermentum DALI02 has high intestinal adhesion capacity and immunoregulation function, and can be used for producing fermented milk, lactobacillus beverage and other foods, functional foods and medicines.
Drawings
FIG. 1 is a gram-stained microscopic image of Lactobacillus fermentum DALI 02.
Detailed Description
In this example, lactic acid bacteria were isolated from collected samples by a conventional spread plate separation method, and strains having acid and bile salt resistance were selected by simulating the environment of the gastrointestinal tract of a human body, and strains having high intestinal adhesion were selected by hydrophobicity, self-aggregation, and adhesion to Caco-2 cells. The immunomodulatory potential of the strain was determined by determining the effect of DALI02 on the amount of TNF-alpha and IL-6 secreted in RAW 264.7.
Related culture medium formula
(1) 12% (mass concentration) skim milk medium: dissolving 120g of skimmed milk powder with double distilled water, diluting to 1000mL, sterilizing at 115 ℃ for 20min, and cooling;
(2) 12% (mass concentration) of whole milk: dissolving 120g of whole milk powder with double distilled water, fixing the volume to 1000mL, sterilizing at 105 ℃ for 5min under high pressure, and cooling;
(3) MRS culture medium: weighing 10g of beef extract, 5g of yeast extract, 10g of peptone, 20g of glucose, 5g of anhydrous sodium acetate, Tween-801 mL and K2HPO4 2g，MgSO4·7H2O 0.58g， MnSO4·4H2O0.25g and 1000mL of distilled water, adjusting the pH value to 6.4, sterilizing at 121 ℃ for 15min, and cooling for later use;
(4) preparing artificial gastric juice: accurately measuring 20mL of hydrochloric acid with the molar concentration of 1mol/L, and adding distilled water to adjust the pH values to respectively reach 3.0. Then, NaCl is added respectively to make the mass fraction of the mixture reach 0.3%, then 1g pepsin is added per 100mL, after the mixture is fully dissolved, a microporous filter membrane with the pore diameter of 0.20 mu m is used for filtering and sterilizing in an aseptic operation platform, and the mixture is transferred into a sterilized bottle for low-temperature preservation for later use.
(5) Preparing simulated bile salt: adding ox bile salt into liquid culture medium to make its mass concentration be 0.30 g/100mL, sterilizing with high pressure steam at 121 deg.C for 15min, and cooling to below 37 deg.C for use.
The method comprises the following specific steps:
1. sample collection
Storing traditional fermented product of Yunan Dali, such as milk fan, sauerkraut, cheese, sauerkraut, chili sauce, etc. in sterilized sampling bottle, and storing at 4 deg.C.
2. Isolation of lactic acid bacteria
0.1g of sample is weighed and cultured in MRS medium at 42 ℃ for 24h for enrichment. The cultures were plated on MRS plates and then incubated at 42 ℃ for 48 h. After culturing, colonies appearing on the agar medium were collected and further purified, eliminating non-lactic acid bacteria strains. The isolated strains were cultured in a broth culture containing 12% (W/W) skim milk, lyophilized, stored at-18 ℃, and then used for various tests for detecting bacterial characteristics.
3. Comparison of acid resistance and bile salt resistance of the strains
Inoculating the strains of the two activated generations into artificial gastric juice with different pH values of 3.0 according to the inoculation amount of 3 percent (mass percentage). Mixing, culturing at 37 deg.C, sampling at 0h and 3h, serial diluting with sterile water at a weight ratio of 1:10, culturing with MRS solid culture medium, and counting viable bacteria.
The tolerance of artificial gastric juice is 3h of viable count/0 h of viable count
Inoculating the strains of the two activated generations into the treated culture medium according to the inoculation amount of 3 percent (mass percentage), culturing for 3 hours at 37 ℃, respectively measuring the viable count of the culture medium with different concentrations, and calculating the tolerance of the strains to bile salts.
The tolerance of bile salt is 3h of viable count/0 h of viable count
And (3) carrying out physiological and biochemical tests, curd performance and acid production characteristic researches on the 87 preliminarily screened bacteria, screening and obtaining 20 bacteria which have good curd characteristics, medium acid production rate and weak post-acidification, and identifying the 20 bacteria as lactic acid bacteria. 20 strains of lactic acid bacteria were inoculated into artificial gastric juice and medium containing bile salt, respectively, and the tolerance results are shown in Table 1.
TABLE 1 acid and bile salt resistance of lactic acid bacteria
As can be seen from Table 1, the survival rates of different lactic acid bacteria in artificial gastric juice are different, and after 3 hours, the survival rate is from 17.98% to 92.81%, wherein the survival rate of 6 strains (90-2-2, A, vacuole 8, c, 17, DALI02) is more than 80%, and the acid-resistant survival rate of the strain DALI02 is 81.14%; and for the bile salt resistance of the strain, the survival rate after 3 hours is 0.01-23.67%, wherein the survival rate of 5 strains (Y-5, m113, h, 7, DALI02) is more than 10%, and the survival rate of the strain DALI02 is 21.12%. Therefore, the strain has better acid resistance and cholate resistance.
4. Bacteriostatic activity of lactobacillus fermented milk on three harmful bacteria
The antibacterial activity of lactic acid bacteria is determined by adopting a punching method, 0.2mL of each pathogenic bacteria fermentation liquid is respectively absorbed, an aseptic smearing rod is used for uniformly coating on an LB solid culture medium flat plate, the flat plate is placed in an ultra-clean workbench for about 30min, 2 holes with the diameter of 9mm are uniformly punched on each flat plate after the bacterial liquid on the surface is fixed on the surface of the flat plate (based on no visible water drop on the flat plate), 0.2mL of a sample to be detected is absorbed and added in the holes, standing culture is carried out for 48h at 37 ℃, 3 parallel averaging values are carried out, the size of an antibacterial ring is determined, and a picture is taken. And (3) judging standard: no bacteria inhibition: no obvious bacteriostatic zone exists; low-degree bacteriostasis: the diameter of the bacteriostatic zone is 1.10-1.50 cm; moderate bacteriostasis: the diameter of the bacteriostatic zone is 1.60-2.00 cm; high bacteriostasis: the diameter of the inhibition zone is more than 2.00 cm.
Lactic acid bacteria are important dominant bacteria in the digestive tract of the body, not only are the digestion and absorption of food facilitated, but also the pathogenic bacteria can be effectively inhibited, so that diseases can be prevented. The bacteriostatic activity of the 20 screened lactic acid bacteria on staphylococcus aureus, escherichia coli and salmonella is measured, and the measurement result is shown in table 2.
TABLE 2 inhibition of three pathogenic bacteria by lactic acid bacteria
Note: the zone diameter includes the pore size (0.9 cm).
The screened strain fermented milk has certain inhibition capacity on harmful bacteria, wherein the inhibition zones of k, 8, DALI02 and B22 on staphylococcus aureus, salmonella and escherichia coli exceed 2.00cm, and the strain DALI02 is highly antibacterial on staphylococcus aureus, escherichia coli and salmonella respectively by 2.30cm, 2.54 cm and 2.39 cm.
5. Comparison of the hydrophobicity and self-polymerization Capacity of lactic acid bacteria
The surface hydrophobicity of the strain is reflected by the affinity of the lactobacillus to hydrocarbon, and the cell surface hydrophobicity of the lactobacillus is measured by adopting a microorganism adhesion hydrocarbon method. Taking 4mL of bacterial suspension, adding 400 mu L of dimethylbenzene, mixing for 60s by vortex, and standing for 15min for layering; taking water phase, using PBS buffer as blank control, measuring OD600And (6) value and recording. Each group was run in parallel with 10 tubes, and OD values were recorded separately for 3 independent experiments. Calculated by the following formula:
surface hydrophobicity (as-is OD)600Aqueous OD600) OD as received600
3mL of the bacterial suspension was vortexedAfter mixing for 10s, the absorbance A at 600nm was determined0Standing the bacterial suspension at room temperature for 2h, and measuring supernatant AtAnd then repeating the experimental determination.
Self-polymerization ═ a0-At)/A0
TABLE 3 self-polymerization and hydrophobicity Capacity of the strains
As is clear from Table 3, the polymerization ability of different lactic acid bacteria was different (3.79% to 41.98%), indicating that proteins or glycoproteins on the cell surface of lactic acid bacteria showed differences. The total number of lactic acid bacteria with the self-polymerization percentage of more than 20 percent is 15, and the total number of lactic acid bacteria with the self-polymerization percentage of more than 25 percent is 13; the hydrophobicity of different lactic acid bacteria is different (0.88% -39.79%), and 5 strains with hydrophobicity more than 10% exist. The self-polymerization capability and the hydrophobic capability of the strain DALI02 are 26.49 percent and 22.48 percent, and the strain has good hydrophobic capability and self-polymerization capability.
6. Adhesion of lactic acid bacteria to Caco-2
A1 mmol stock solution was prepared from a solid powder of cell proliferation tracer fluorescent probe (cFDA-SE) in sterile dimethyl sulfoxide (DMSO). The specific configuration method comprises the following steps: 5mgcFDA-SE was weighed out and dissolved in 8.969 mLDMSO solvent. The membrane was then filtered to remove bacteria (0.22 μm) and stored at-20 ℃ until use. Re-suspending the thallus in PBS solution to prepare thallus concentration of 108CFU/mL, every 1mL lactobacillus solution adding 20 u LcFDA-SE stock solution, shading under the condition of standing and labeling for 15min, 8000r/min centrifugation for 10min to collect the thallus, and using sterile PBS buffer solution to wash three times. To remove excess fluorescent dye. The cells were resuspended in sterile PBS buffer. cFDA-SE labeling was analyzed by flow cytometry (excitation wavelength 488 nm).
Cultured Caco-2 cells were digested and diluted to 5X 10 with a high sugar minimal essential medium solution5Concentration per mL. 1mL of cell suspension was added to a 12-well cell culture plate and incubated at 37 ℃ and 5% CO2Cultured in a monolayer. The strain was washed once with PBS, resuspended in 1mL sterile PBS, and added to the cells. At 37 ℃ and 5% CO2After 2h incubation in the incubator of (1), non-adhering strains were removed by washing 3 times with cold PBS. The cell suspension in the wells was collected and its fluorescence intensity was measured. The adhesion capacity was evaluated as follows:
percent adhesion ═ cell suspension fluorescence intensity/raw fluorescence intensity
TABLE 4 adhesion rate of lactic acid bacteria to Caco-2 cells
| Strain name | Adhesion Rate% | Strain name | Adhesion Rate% |
| S7 | 18.50±0.67c | h | 21.40±0.24a |
| Y-5 | 4.69±0.11m | 7 | 16.56±0.30e |
| 90-2-2 | 12.60±0.22h | Bubble 9 | 6.29±0.07l |
| m113 | 4.23±0.23mn | d | 14.79±0.10g |
| A | 11.49±0.16i | h5 | 17.25±0.17d |
| m | 2.27±0.07o | c | 11.89±0.17i |
| k | 3.91±0.08n | 17 | 6.58±0.11l |
| x3 | 20.67±0.26b | DALI02 | 21.10±0.72ab |
| N | 8.47±0.07j | g7 | 15.99±0.31f |
| Bubble 8 | 7.07±0.12k | B22 | 12.44±0.15h |
Note:abin comparison with the same column, different letters indicate differences (p < 0.05).
The adhesion effect of the lactobacillus to Caco-2 represents that the adhesion condition of the strain to cells is shown in Table 4, the difference of the adhesion capability of different lactobacillus is large, the surface structure and the surface components of the lactobacillus are an important factor influencing the adhesion performance of the lactobacillus, and many researches show that the adhesion factors (such as surface layer protein, peptidoglycan, lipoteichoic acid and other components) existing on the surface of the lactobacillus have large influence on the adhesion performance, the adhesion rate of the strain is 2.27-21.40%, and more than 15% of 7 strains (S7, x3, h, 7, h5, DALI02 and g7) are provided, wherein the adhesion rate of the strain DALI02 is 21.10%, and the strain has the high adhesion capability potential of intestinal tracts.
7. Immunomodulatory effects of Lactobacillus fermentum DALI02 on RAW264.7
At 2X 10 in 96-well plates6Cells were cultured at a density of 100. mu.L/well and incubated for 24 h. A sample diluted in Dulbecco's modified Eagle's medium (100. mu.L) and the same volume of lipopolysaccharide (LPS, 2 ug/mL) were added simultaneously. After 24h incubation, 300. mu.L of the culture broth was collected after centrifugation (4 ℃, 8000rpm, 10min) and stored at-20 ℃ for later use. The effect of the strains on IL-6 and TNF-. alpha.was evaluated using an ELISA kit (Solarbio, China).
TABLE 5 immunomodulatory effects of DALI02
| TNF-α(pg/mL) | IL-6(pg/mL) | |
| Blank group | 265.75±12.11b | 4.20±0.25b |
| Model set | 1650.47±33.98a | 8.66±0.21a |
| DALI02 intervention group | 281.86±47.51b | 4.28±0.93b |
Note: different letters indicate differences (p < 0.05).
After LPS 24h modeling, the secretion of TNF-alpha and IL-6 in the model group is obviously increased, which indicates that the inflammation model modeling is successful, and after DALI02 intervenes for 24h, the proinflammatory factor is obviously reduced, and has no significant difference (p is less than 0.05) with the blank group, so that the strain DALI02 has good immune regulation effect.
8. Identification of lactic acid bacteria
(1) Physiological and biochemical identification
Selecting a single colony from the purified solid culture medium of the strain of the invention, inoculating the single colony into a sugar fermentation micro-identification tube, and sealing the tube with sterile paraffin oil. Placing into a constant temperature incubator at 37 ℃ for culture. And (3) observing the color change condition of the liquid in each tube after 24h and 48h respectively, if the color change is positive, and if the color change is negative, preliminarily determining the species of the isolated strains by contrasting Bergey's Manual of bacteria identification. The strain is preliminarily identified to be lactobacillus fermentum.
(2) API authentication
The strains of the invention were grown on MRS agar plates for 48h at 37 ℃ under anaerobic conditions, and the resulting cultures were then suspended in a suspension medium, the turbidity of which was identical to that of McFarland 2. Then, the cultured cells were inoculated into an API50CHL reagent strip, the upper layer of the reagent strip for measuring sugar fermentation was covered with a layer of sterile paraffin, and anaerobic culture was carried out at 37 ℃ to examine the enzyme activity and the fermentation ability for various sugars.
Enzyme activity and sugar fermentation capacity were analyzed using an API recognition software program. The results showed that the enzyme activity and sugar fermentation ability of this strain were similar to those of Lactobacillus fermentum (Table 6), with a coincidence rate of 99.3%.
TABLE 6 API50CHL System identification results
(3)16S rDNA sequence identification
Taking the prepared lactobacillus genome DNA as a template for PCR amplification, and adopting a PCR reaction system as follows: 10 XBuffer 5.0 μ L, Mg2+3.0. mu.L, 2.5mmol/L dNTP4.0. mu.L, 10.0pmol of each of the primers Lab1 and Lab2 1.5. mu. L, Taq, 1.5U of the enzyme, 5.0. mu.L of the template DNA, and ddH2And O is supplemented to 50.0 mu L. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 64 deg.C for 45s, extension at 72 deg.C for 1min, and circulating for 35 times; extension at 72 ℃ for 8 min.
The PCR product was sent to Biotechnology engineering (Shanghai) Co., Ltd for sequencing, and the sequencing results were submitted to BLAST for on-line comparison to obtain results, indicating that the strain of the present invention was 100% Lactobacillus fermentum.
9. Strain preservation
The strain provided by the invention is lactobacillus fermentum DALI02 which is preserved in China general microbiological culture Collection center of the culture Collection management Committee of microorganisms 7-5.7.2018, and the preservation number is CGMCC 16064 at the address of No. 3 of Xilu No. 1 of Beijing City Kogyo-Yang district and the microbiological research institute of Chinese academy of sciences.
Example 1: preparation of fermented milk Using Lactobacillus fermentum DALI02
Inoculating activated 2 generation lactobacillus DALI02 culture at 3% (mass percent) into skimmed milk (12% mass concentration) with heat treatment at 95 deg.C/5 min, culturing at 37 deg.C for 40h, and cooling to 4 deg.C to prepare starter with viable count of about 108cfu/mL。
Heating whole milk (mass concentration 12%) to about 50 deg.C, adding 6% sucrose, dissolving completely, preheating recovered whole milk to about 60 deg.C, and homogenizing under 20 MPa. And then, carrying out heat treatment on the recovered whole milk at the temperature of 95 ℃/5min, cooling to 38 ℃, inoculating lactobacillus DALI02 starter according to the inoculation amount of 5%, fermenting at 37 ℃ until curd, cooling, and storing at 4 ℃ to obtain the fermented milk with high adhesive force and immunoregulation capability.
Example 2: fermented milk beverage prepared from Lactobacillus fermentum DALI02
42kg of skim milk powder is dissolved by water at 50 ℃ to prepare 350kg of recovered skim milk, after full dissolution, the recovered skim milk is subjected to heat treatment at 95 ℃/5min, cooled to 37 ℃, inoculated with 5% (mass percent) of lactobacillus DALI02 starter, fermented at 37 ℃ for about 24h, the terminal acidity is controlled to be about 160 DEG T, 650kg of sugar sterilized at 110 ℃/5s and stabilizer mixed solution (the mixed solution comprises 12% of sucrose, 0.1% of monoglyceride, 0.1% of sucrose ester and 0.4% of pectin, which are mass concentrations) is added, the mixture is preheated to 60 ℃, homogenized under the pressure of 20MPa, then subjected to heat treatment at 75 ℃/15s, cooled to about 15 ℃, and subjected to aseptic filling, so that 1000kg of bacillus DALI02 fermented milk is obtained.
Example 3: probiotic lactic acid bacteria freeze-dried powder prepared by using lactobacillus fermentum DALI02
Mixing skimmed milk powder, fructo-oligosaccharide, yeast powder, peptone and purified water at 12.0%, 2.0%, 0.8%, 0.7% and 84.5% by weight, preheating to 60 deg.C, and homogenizing under 20 MPa. Then the mixture is subjected to heat treatment at 95 ℃/30min and cooled to 37 ℃. The DALI02 starter prepared in example 1 was inoculated at an inoculum size of 5%, cultured at 37 ℃ for 10h, centrifuged, and freeze-dried under vacuum until the water content was less than 3%, thus obtaining freeze-dried powder of Lactobacillus fermentum DALI 02.
Claims (4)
1. Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function has a preservation number of CGMCC 16064.
2. The method for preparing lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulatory function as claimed in claim 1, comprising the steps of:
(1) separating the lactic acid bacteria from the traditional fermented product by the culture medium;
(2) through the artificial gastric juice tolerance and bile salt resistance tests of the isolated strain, a strain with good tolerance is obtained;
(3) after fermented milk curds are prepared by using different strains, the bacteriostatic ability is measured;
(4) the self-polymerization capability, the hydrophobicity and the adhesion capability of Caco-2 cells of the strain are tested to obtain a strain with good adhesion capability, namely DALI 02;
(5) the immunomodulatory effect of DALI02 on RAW264.7 was studied.
3. The application of lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function is characterized in that the lactobacillus fermentum DALI02 is applied to the production of food and medicines.
4. The use of lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulatory function according to claim 3, wherein lactobacillus fermentum DALI02 is used for preparing fermented milk with immunoregulatory effect.
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