Application of lactobacillus helveticus AFY07 in preparation of intestinal protection drugs, food or health care products

Abstract

The invention provides application of lactobacillus helveticus AFY07 in preparation of intestinal protection drugs, food or health care products, and relates to the technical field of microorganisms. The lactobacillus helveticus AFY07 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30983. Research finds that the lactobacillus helveticus AFY07 can enhance the intestinal function, so that the intestinal structure returns to normal, the intestinal structure is protected, and the intestinal permeability is reduced.

Description

Technical Field

[0001] The invention provides application of Lactobacillus helveticus AFY07 in preparing intestinal protective medicines, foods or health products, belonging to the technical field of microorganisms. Background Art

[0002] Food allergies can cause more serious intestinal damage. Studies have found that when an allergic reaction occurs, the body will experience changes such as increased eosinophils, intestinal barrier damage, mucosal structural disorder, villus disorder, and increased intestinal permeability. At the same time, the expression of tight junction proteins in intestinal epithelial cells of food allergy patients is reduced, and the gaps are significantly widened, resulting in increased intestinal mucosal permeability. The mechanism is closely related to the proliferation and activation of mast cells. Therefore, an effective intestinal protection method is the key to overcoming food allergies. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a use of Lactobacillus helveticus AFY07 in the preparation of intestinal protective drugs, foods or health products.

[0004] The Lactobacillus helveticus AFY07 described in the present invention is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 30983, a microbial classification name of Lactobacillus helveticus, and a deposit date of June 11, 2024.

[0005] The method for obtaining Lactobacillus helveticus AFY07 of the present invention is as follows:

[0006] 1. Take 1mL of naturally fermented yogurt sample, dilute it 10 times with sterile saline to 10-6, then take 100μL of 10-4, 10-5, 10-63 gradient bacterial solution to plate, culture at 37℃ for 24-48h, observe and record the colony morphology. Pick colonies of different morphologies on the plate for streaking separation, and after culturing at 37℃ for 48h, pick single colonies of different morphologies on the plate for streaking separation again, and repeat this 2 to 3 times until pure single colonies with consistent morphology are obtained.

[0007] 2. Inoculate the purified suspected target strain into MRS broth, culture at 37℃ for 18-24h, and then use a bacterial genomic DNA extraction kit to extract DNA. Number the extracted DNA and store it in a -20℃ freezer for later use.

[0008] 3. The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3'), 1 μL of downstream primer 1495R (5'-CTA CGG CTACCTTGT TAC GA-3'), 12.5 μL of 2×TaqplusBuffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile dd H2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃5min; 94℃30s, 55℃30s, 72℃1min, a total of 29 cycles, and finally 72℃ extension for 5min.

[0009] Then 5 μL of the amplified product was taken for agarose gel electrophoresis detection, the agarose concentration was 1.5%, the electrophoresis conditions were 110V, 45min. The successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequenced sequence was compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0010] After oral administration of Lactobacillus helveticus AFY07, the intestinal mucosa and villus structure of the jejunal tissue of food allergic mice were relatively intact, the infiltration of inflammatory cells was significantly reduced, and the overall morphology was close to normal, indicating that Lactobacillus helveticus AFY07 may have the effect of protecting the integrity of intestinal tissue morphology. The expression levels of Occludin, Tjpl and JAM1 in the jejunum of allergic mice after oral administration of Lactobacillus helveticus AFY07 increased, indicating that Lactobacillus helveticus may enhance the barrier function of the intestine by promoting the expression of tight junction protein genes in the jejunum. The research of the present invention shows that Lactobacillus helveticus AFY07 can enhance the intestinal function, restore the intestinal structure to normal, protect the intestinal structure, and reduce intestinal permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the expression of immunoglobulins in mouse serum;

[0012] Figure 2 is the cytokine content in mouse serum;

[0013] Figure 3 H&E staining of mouse jejunum for pathological observation.

[0014] Figure 4 mRNA expression of tight junction proteins in mouse jejunum

[0015] Figure 5 The mRNA expression of intestinal mucosal immune factors in the mouse jejunum. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] 1 Materials and reagents

[0018] Table 1 Experimental materials and reagents

[0019]

[0020]

[0021] 2. Instruments and Equipment

[0022] Table 2 Instruments and equipment

[0023]

[0024] 3 Experimental methods

[0025] 3.1 Animal Model

[0026] Thirty-two four-week-old BALB / c mice were randomly divided into four groups: high-dose AFY07 group, low-dose AFY07 group, normal group, and allergy group, with 8 mice in each group. The normal group used AIN-95G standard feed throughout the experimental period and had free access to water. In addition to normal feeding of AIN-95G, free access to water, and oral gavage of corresponding lactic acid bacteria, the allergy group, high-dose AFY07 group, and low-dose AFY07 group were sensitized by intraperitoneal injection of lactoglobulin on days 7, 14, and 21. Each mouse was injected with 0.2 ml of sensitization solution (20 mg lactoglobulin, 10 ml saline, and 10 ml Freund's adjuvant). On day 28, lactoglobulin stimulator was gavaged twice, with 1 mg / g lactoglobulin stimulator solution per mouse, and normal saline was gavaged as a control. The allergic symptoms of mice were evaluated, blood was collected from the eyeballs after anesthesia, and the mice were killed by dislocation. The thymus and spleen of the mice were weighed and recorded, and about 5 cm of jejunum was cut and fixed in 4% paraformaldehyde.

[0027] 3.3 Determination of mouse body weight, thymus and spleen weight

[0028] Body weight measurement: Place the beaker securely on the electronic scale, adjust to zero and confirm that the pointer is at 0 again, gently place the mouse in the groove, and quickly read the measurement results when the BALB / c mouse is in a static state and record the measurement data with an accuracy of 0.01. On the 28th day, dissect the mouse, weigh and record the weight of the thymus and spleen, and calculate the corresponding organ index.

[0029] 3.5 Determination of mouse immunoglobulin and cytokine content

[0030] On the 28th day, blood was collected from the retroorbital plexus and then centrifuged at 4000 rpm for 20 min at 4°C; the serum was then stored at -80°C until use. The levels of immunoglobulins IgE and IgM, cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, TNF-α, TGF-γ, and IFN-β in the serum were measured by ELISA according to the instructions.

[0031] 3.6 H&E staining of mouse jejunal tissue sections

[0032] The jejunal tissue was fixed, dehydrated, embedded and stained, and finally observed under a microscope and photographed.

[0033] 3.7 Determination of mRNA expression in mouse jejunum tissue

[0034] Total RNA was extracted from jejunal tissue and reverse transcribed into cDNA. SYBR, DDW, upper and lower primers (Table 4) and template cDNA were added in sequence. The Ct value was obtained by 40 cycles of 95℃15s, 55℃30s, and 72℃35s. -ΔΔCt The expression level of target gene mRNA was calculated by this method.

[0035] Table 3 qPCR primer sequences

[0036]

[0037] 3.8 Data Processing

[0038] SPSS 23.0 software was used to perform statistical analysis on the data of each group. The Duncan method was used to analyze the mean and variance of each group, and different letters ad indicate significant differences in the mean values ​​(p<0.05).

[0039] 4 Results and analysis

[0040] 4.1 Effects of food allergy on body weight and organ parameters of mice

[0041] Table 4 Weight loss rate and organ indexes of mice (unit: %)

[0042]

[0043] In Table 4, the weight loss rate of mice in the normal group was the lowest, and the weight loss rate of mice in the allergic group was the highest. After intervention with the AFY07 high-dose group and the AFY07 low-dose group, the weight loss rate of mice decreased, but was still higher than that of the normal group. The thymus index and spleen index were ranked from large to small as follows: the allergic group, the AFY07 low-dose group, the AFY07 high-dose group, and the normal group (p < 0.005).

[0044] 4.2 Effect of Lactobacillus helveticus AFY07 on immunoglobulin expression in mouse serum

[0045] The increase in serum specific IgE levels is an important evaluation indicator for immediate hypersensitivity reactions. Figure 1 The expression of immunoglobulins IgE and IgM were ranked from high to low in the order of allergic group, AFY07 low-dose group, AFY07 high-dose group and normal group, among which IgE in AFY07 high-dose group was reduced by about 17.21% compared with that in the allergic group (p<0.05).

[0046] 4.3 Effect of Lactobacillus helveticus AFY07 on cytokine levels in mouse serum

[0047] Figure 2 The results are as follows: The levels of cytokines in the serum of mice. The levels of IL-4, IL-5, IL-10, IL-13, IL-17A, and TNF-α in the serum of mice in the allergic group were significantly increased. After oral administration of Lactobacillus helveticus AFY07, the levels decreased significantly. The high-dose AFY07 group had a more significant effect on IL-13, which decreased by about 43.77% (p < 0.05). In addition, the levels of IFN-γ and TGF-β in the serum of mice in the high-dose AFY07 group were significantly higher than those in the allergic group, approaching those in the normal group. The trends of the low-dose AFY07 group and the high-dose AFY07 group were consistent, but there were still significant differences (p < 0.05).

[0048] 4.4 Observation of jejunal histopathology in food allergic mice

[0049] like Figure 3 As shown in the figure, the integrity of the jejunal intestinal mucosa and the villus structure of the BLG-induced mice were damaged, and significant edema appeared in the submucosal layer, accompanied by the infiltration of immune cells. In the low-dose AFY07 group, the swelling of the villi was alleviated to a certain extent, and the infiltration of inflammatory cells was also reduced. In the high-dose AFY07 treatment group, the symptoms of jejunal tissue damage were further alleviated, and the villus structure was close to normal, but a small amount of inflammatory cells continued to infiltrate.

[0050] 4.5 Effect of Lactobacillus helveticus AFY07 on tight junction proteins in the jejunum of food allergic mice

[0051] like Figure 4As shown in the figure, the mRNA expression levels of Occludin, TJP1 and JAM1 in the jejunum tissue of mice in the allergic group were significantly lower than those in the normal group (p<0.05), indicating that the intestinal barrier function of mice with food allergy was damaged to a certain extent. Compared with the allergic group, the expression levels of Occludin, TJP1 and JAM1 in the jejunum of mice in the high-dose group of Lactobacillus helveticus AFY07 were increased, and the difference between the two groups was statistically significant (p<0.05). The low-dose group of AFY07 showed the same trend as the high-dose group of AFY07 (p<0.05).

[0052] 4.6 Effects of Lactobacillus helveticus AFY07 on intestinal mucosal immune responses in the jejunum of food-allergic mice

[0053] exist Figure 5 In the study, the expression of IL-5, IL-6, IL-10, IL-25 and TSLP in the jejunum tissue of food allergic mice after AFY07 intervention was significantly reduced, while the expression of Foxp3 was significantly increased, by about 1.8 times. The effect of the high-dose AFY07 group was more significant than that of the low-dose AFY07 group, and the difference was statistically significant (p<0.05).

[0054] The present invention shows that Lactobacillus helveticus AFY07 can reduce the content of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) and Th17 (IL-17A) in β-lactoglobulin-sensitized mice, and increase the content of Th1 cytokine Treg cells (TGF-β) and Th1 cytokine (IFN-γ).

[0055] The tight junction protein expression of intestinal epithelial cells in patients with food allergies is reduced, and the gaps are significantly widened, resulting in increased intestinal mucosal permeability, and its mechanism is closely related to the proliferation and activation of mast cells. The experiment showed that the mRNA expression levels of Occludin, Tjpl and JAM1 in the allergic group were significantly lower than those in the normal group, indicating that the intestinal barrier function of food allergic mice was damaged to a certain extent.

[0056] In summary, Lactobacillus helveticus AFY07 is an important intestinal bacterium that can effectively inhibit the secretion of Th2 cytokines in allergic mice and regulate the Th1 / Th2 immune balance. In addition, it can also enhance intestinal function, restore the intestinal structure to normal, protect the intestinal structure, and reduce intestinal permeability.

[0057] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. An application of Lactobacillus helveticus AFY07 in the preparation of intestinal protective drugs, foods or health products, It is characterized in that The Lactobacillus helveticus AFY07 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.30983.

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