Patent Application
20240390436
The present invention relates to the technical field of microorganisms, in particular to a Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) inhibiting Helicobacter Pylori and an application thereof.
Helicobacter Pylori (Hp) is a helical, flagellated and micro-aerobic Gram-negative bacterium. Helicobacter pylori infection is one of the most common chronic bacterial diseases of the human beings. Helicobacter pylori is also a first-class human carcinogen recognized by the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC). When Helicobacter pylori infects the stomach and is colonized, a lot of urease may be secreted to convert urea in gastric mucus into ammonia to neutralize gastric acid, so as to avoid being affected by the gastric acid, and then Helicobacter Pylori may proliferate greatly in the stomach and duodenum to destroy the gastric mucosal barrier, induce the cell apoptosis of the gastric mucosa, and lead to tissue damage and ulcer formation. Therefore, the Helicobacter Pylori infection is related to gastrointestinal malignant and non-malignant diseases, such as gastritis, chronic dyspepsia, mucosa-associated lymphoid tissue lymphoma, gastric adenocarcinoma, etc. The most common cause of gastric cancer is Helicobacter Pylori infection, so inhibiting the Helicobacter Pylori infection is a most effective method for preventing the gastric cancer.
Three or four different medicine or antibiotics may be usually used for treating the Helicobacter Pylori infection so as to achieve sufficient eradication rate. In recent years, due to the increasing drug resistance of more and more Helicobacter Pylori strains to the antibiotics, the effectiveness of numerous treatment solutions also decreases; and moreover, some antibiotics may cause more side effects and adverse effects. Therefore, relevant researchers in the art are committed to exploring an effective treatment method with no side effect, such as using probiotics combined with antibiotics to treat or eradicate the Helicobacter Pylori infection, wherein the probiotics can reduce the drug resistance of the antibiotics, and has an action of balancing intestinal flora and improving intestinal microecology.
Lactic acid bacteria (LAB) belong to a probiotic that is generally recognized as safe (GRAS) and is widely used in medicine, food and other industries. The lactic acid bacteria can ferment carbohydrates into lactic acid, which can be used for manufacturing yogurt, cheese, Chinese sauerkraut, beer, wine, pickles, pickled food and other fermented food. The common lactic acid bacteria include Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, Pediococcus, Leuconostoc, etc. The lactic acid bacteria can have numerous beneficial effects human hosts, including anti-allergic effects, antibacterial action, regulation of intestinal microbial flora, stimulation of immune system and prevention of colon cancer, and there are some researches on the application of the lactic acid bacteria strains in anti-Helicobacter Pylori.
In order to better meet the clinical practice requirements, it is necessary to develop diversified and effective probiotics to inhibit and treat diseases related to the Helicobacter Pylori infection.
The present invention provides a Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) inhibiting Helicobacter Pylori and an application thereof.
The present invention provides a Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18). The strain is preserved in China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: Institute of Microbiology, China Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, 100101), and classified and named Bifidobacterium animalis subsp. lactis with a preservation number CGMCC No. 25896.
The Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) was isolated from feces of healthy infants. The strain was identified by bacterial morphology, physiology and 16S rDNA (SEQ ID NO. 1) sequencing, and an identification result was Bifidobacterium animalis subsp. lactis, named Bifidobacterium animalis subsp lactis ATM-209 (GOLDGUT-BB18). The strain has good gastric acid tolerance, has an obvious effect for inhibiting the growth of Helicobacter Pylori, can significantly reduce the load of Helicobacter Pylori in the stomach and relieve the inflammation caused by pathogenic infection in animal experiments, and provides strain resources for developing food (such as dietary supplements, and health products), medicine and the like inhibiting the Helicobacter Pylori.
The Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) has the following microbiological traits:
Positive in gram stain, straight rod or curved short rod under a light microscope, in single or paired arrangement, V-shaped or curved shape; and colonies formed on an MRS solid culture medium are smooth, convex, entire-edged, milky white, glistening and soft in tissues.
The Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) can grow well under an artificial gastric acid condition, and can inhibit the growth of Helicobacter Pylori in an in-vitro culture experiment with a diameter of a bacteriostatic ring reaching 18.0 mm, and can inhibit the in-viivo colonization of Helicobacter Pylori and relieve the inflammation caused by the pathogenic infection in animal experiments.
The Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) can be cultured by the following method: the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) is inoculated to a culture medium and subjected to anaerobic culture at 35-37° C., wherein the culture time may be 12-24 h. The culture medium can adopt the conventional culture medium in the art capable of making the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) grow, and preferably adopt an MRS meat soup culture medium (casease digesta 10.0 g/L, beef extract powder 10.0 g/L, yeast extract powder 4.0 g/L, triammonium citrate 2.0 g/L, sodium acetate 5.0 g/L, magnesium sulfate heptahydrate 0.2 g/L, manganese sulfate tetrahydrate 0.05 g/L, glucose 20.0 g/L, dipotassium phosphate 2.0 g/L, and Tween 80 1.0 g/L, pH=5.7±0.2), and the solid culture medium is added with 1.5% agar.
Through safety assessment, the acute oral toxicity test is carried out according to relevant provisions of GB 15193.3-2014, and results show that behaviors and metal status of mice are good, and the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) belongs to an actual non-toxic level; according to relevant provisions of Food Strains Safety Evaluation Procedures (Draft for Comment) of Food Safety National Standard, after sequencing the whole genome of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18), sequences of the whole genome are compared with sequences stored in latest versions of CARD, ResFinder, Argannot and NDARO, and in combination with an antibiotics drug resistance test experiment, the results prove that the strain does not contain a transferable antibiotics resistant gene; and the sequences of the whole genome are compared with latest sequences in VFDB, PAI, DB, MvirDB and CGE databases, and the results show that the strain does not contain any toxic factor.
The present invention provides a microbial preparation, and the microbial preparation includes Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18).
Preferably, in the above microbial preparation, the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) presents in a form of live bacteria.
The above microbial preparation may be a solid preparation (such as bacterial powder) or liquid preparation (such as emulsion preparations).
The present invention provides a method for preparing the above microbial preparation. The method includes a step of culturing the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18).
Preferably, the culture is anaerobic culture under a condition of 35-37° C.
Preferably, the culture is anaerobic culture in liquid culture medium to obtain a bacterial solution.
The bacterial solution may be directly made into the liquid preparation or may be made into the liquid preparation by concentration and/or by adding other ingredients, or may be dried to prepare solid preparations, or bacteria in the bacterial solution may be isolated for preparing the solid preparations.
Based on the function of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18), the present invention provides following applications of the strain:
The present invention provides an application of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation in preparing a product inhibiting Helicobacter Pylori.
The present invention provides an application of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation in inhibiting Helicobacter Pylori.
Inhibiting the Helicobacter Pylori includes in-vitro inhibition of the growth of Helicobacter Pylori, or in vivo inhibition of colonization, infection, proliferation and growth of Helicobacter Pylori.
The present invention provides an application of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation in preparing a product for preventing, relieving or treating Helicobacter Pylori infection or diseases caused by the Helicobacter Pylori infection.
The diseases caused by the Helicobacter Pylori infection are preferably gastrointestinal diseases, including gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and the like.
The present invention provides an application of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation in preparing a product for preventing, relieving or treating inflammation caused by Helicobacter Pylori infection.
The inflammation caused by the Helicobacter Pylori infection includes inflammation of gastric mucosa tissues, duodenal inflammation, etc.
The above products include food, feed, or medicine.
The present invention provides an application of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation in preparing food, feed or medicine.
In the present invention, the food includes ordinary food, dietary supplements and health products.
The present invention provides a food. The food includes Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation.
Besides the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation, the food may also include raw materials allowed in the food field. The food may be a dietary supplement, a health product or fermented food.
The present invention provides medicine. The medicine includes Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation.
Besides the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) or the microbial preparation, the medicine may also include ingredients in allowed the medicine field (such as filler, formant, lubricant, wetting agent, diluting agent, etc.) The preparation types of the medicine can be solid preparations (such as powder, granules, capsules, tablets, etc.) or liquid preparations (such as oral liquid, etc.).
The present invention at least has the following beneficial effects: the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) provided by the present invention grows well in an environment with gastric acid, has good acid tolerance, and can well adapt to the gastric environment; and the strain has an obvious effect for inhibiting the growth of Helicobacter Pylori, can inhibit the in-vivo colonization of Helicobacter Pylori and reduce the load of Helicobacter Pylori in the animal experiments, and can relieve the inflammation caused by the Helicobacter Pylori infection, thereby playing a role in regulating the microbial flora of digestive tracts.
The Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) can be used for preparing the products for preventing, relieving or treating the Helicobacter Pylori infection or the diseases caused by the Helicobacter Pylori infection, and has broad application prospects in clinical prevention, treatment and inhibition of diseases related to Helicobacter pylori infection and gastrointestinal health food.
To more clearly describe the technical solutions in the present invention or in the prior art, the drawings required to be used in the description of the embodiments or in the prior art are simply presented below. Apparently, the following drawings show some embodiments of the present invention, so for those ordinary skilled in the art, other drawings can also be obtained according to these drawings without contributing creative labor.
To make the purposes, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention are described clearly and completely in conjunction with accompanying drawings in the present invention. Apparently, the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments obtained by a person of the ordinary skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
The strain of Helicobacter Pylori used in the following embodiments is Helicobacter Pylori NCTC11639, which is donated by Institute of Digestive Diseases in the First Affiliated Hospital of Nanchang University.
1. Isolation of Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18)
The strain of the Bifidobacterium animalis subsp. lactis used in the present invention was isolated from feces of healthy infants.
An MRS culture medium was used for isolating samples and screening the strain, and the MRS culture medium included the following components: casease digesta 10.0 g/L, beef extract powder 10.0 g/L, yeast extract powder 4.0 g/L, triammonium citrate 2.0 g/L, sodium acetate 5.0 g/L, magnesium sulfate heptahydrate 0.2 g/L, manganese sulfate tetrahydrate 0.05 g/L, glucose 20.0 g/L, dipotassium phosphate 2.0 g/L, and Tween 80 1.0 g/L, the pH was 5.7±0.2, and 1.5% agar was added to form the MRS solid culture medium.
1 g of feces sample was placed into 10 mL of MRS liquid culture medium prepared in step 1.2, and uniformly mixed for culture at 36° C., 1 mL of enriched liquid was absorbed in a super clean bench to perform 10-times gradient dilution, 100 μL of bacterial solution at three dilution gradients, i.e. 10−5, 10−6 and 10−7 were selected and coated on a culture dish containing sterile MRS solid culture medium and stood and cultured for 24-48 h at 36° C. under an aerobic condition, until there were obvious single bacterial colonies formed, a plate grown with 70-120 single colonies was selected from the culture medium, typical colonies were selected and subjected to scribing purification multiple times on the MRS solid plate culture medium, and the culture with the same colony morphology on the whole plate was subjected to strain identification.
2. Identification of Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18)
After the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) was cultured for 24 h in the MRS solid culture medium, the colonies were smooth, convex, complete in edges, milky white, glistening and soft in texture, as shown in
Colony smears of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18): positive in gram staining, straight-rod or curved short rod under a light microscope, single or paired arrangement, V-shaped or curved shape, as shown in
2.3 16S rDNA Identification
Identification unit: Tsingke Biotech Co., Ltd.
Identification sequence: The sequence of 16S rDNA is as shown by SEQ ID No. 1.
Identification result: according to a sequence contrast result and physiological and biochemical results of the strain, the strain was determined to be Bifidobacterium animalis subsp. lactis.
The overall pH condition in a gastric environment of the human body is strongly acidic, so that the acid tolerance of the strain is an important index for evaluating whether the strain can survive and colonize in the gastric acid environment. Artificial gastric fluid was used for simulating the gastric environment; in a gastric acid digestion group, 100 mL of artificial gastric fluid was added into a 96-pore plate containing 100 μL of bacterial solution of Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18), and the mixture was mixed uniformly by blowing, and subjected to anaerobic co-incubation for 2 h at 37° C.; and in a control group, 100 μL of PBS was added into the 96-pore plate containing 100 μL of bacterial solution of Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18), and the mixture was uniformly mixed by blowing, and subjected to anaerobic co-incubation for 2 h at 37° C., and after the incubation, the pore plate was taken out.
An incubation sample in the gastric acid digestion group was diluted with PBS to 10E-5; and the incubation sample in the control group was diluted with PBS to 10E-6. The diluted sample was coated on an MRS agar plate and subjected to anaerobic culture for 16 h at 37° C., then colony counting was carried out, a survival rate of the bacteria was calculated with a calculation formula as follows: survival rate/%=(variable count in the gastric acid digestion group logCFU/mL/variable count in the control group logCFU/mL)*100%.
After being digested for 2 h in gastric acid, the survival rate of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) was 121% (as shown in
Helicobacter Pylori NCTC11639 (abbreviated as H. pylori) was resuscitated by a blood plate culture medium, then inoculated to a BD liquid culture medium, and cultured for 48 h under a microaerophilic condition at 37° C.; and the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) was activated by the MRS solid culture medium and inoculated to the MRS liquid culture medium to be cultured for 24 h at 37° C.
The microaerophilic condition refers to an environment with an oxygen concentration of 5% (percentage by volume), and a carbon dioxide concentration of 10% (percentage by volume).
Bacteriostasis test was carried out by a punching method, an overnight cultured bacterial solution of Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) was centrifuged at 6000 rpm for 10 min, and then supernatant was collected and sterilized by a 0.22 μm filter head to obtain strain fermentation supernatant. The Helicobacter Pylori was coated on an MH solid culture medium to 10E9 CFU, the plate was punched by a puncher after being dried, the strain fermentation supernatant was added into pores, and PBS was used as a negative control. The blood plate was cultured for 48 h in a microaerophilic incubator at 37° C., and a diameter of a bacteriostatic ring was measured. Compared with the PBS control group, the fermentation supernatant of the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) significantly increased the diameter of the bacteriostatic ring (as shown in
Grouping: 36 Mongolian male gerbils at the age of 6-8 weeks were adaptively fed for one week and then randomly divided into 4 groups, i.e. a blank group (6 gerbils), Hp infection group (intragastric injection with H. pylori, 12 gerbils), an antibiotic drug group (first intragastric injection with H. pylori, and then intragastric injection with bismuth-containing quadruple, 6 gerbils), and a probiotic group (first intragastric injection with H. pylori, and then intragastric injection with Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18), 12 gerbils).
Modeling: after the gerbils for modeling were subjected to fasting and refraining from water for 24 h, intragastric injection with 108 CFU of bacterial solution of Helicobacter Pylori was carried out by using a mouse gavage needle, and the intragastric injection lasted for 10 days.
Intervention: after confirming the success of modeling, the gerbil model was fed with antibiotic drugs and probiotics. The gerbils in the antibiotic drug group were treated with bismuth quadruple; the gerbils in the probiotic group were subjected to intragastric injection with 108 CFU of probiotics by using the mouse gavage needle, lasting for 21 days. The probiotics selected the bacterial solution with vigorous growth at logarithmic phase of growth. The gerbils in the Hp infection group and the gerbils in the blank group were also subjected to intragastric injection with normal saline by using a mouse gavage needle, lasting for 21 days.
After 21-day intervention, the mice were killed to take gastric tissues which were fixed by 10% formalin, and then the fixed gastric tissues were sectioned by paraffin for warthin-Starry silver nitrate staining. Mucins on surfaces of spirochetes may be specifically bond with silver to be in black. In the silver nitrate staining diagram of gastric tissues in the Hp infection group, black short rod-like particles and hyphae can be seen, the stained degree is most serious, and the stained area is the largest. The antibiotic treatment can change the infection situation, and the Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18) can slightly improve the infection situation (as shown in
Gastric tissue sections of four groups of experimental gerbils after gastric injection in embodiment 4 were collected for hematoxylin-eosin staining. Results are shown in
Hp may generate a large number of urease in the stomach, so that the Hp infection may be judged by detecting the urease. The final color of urease yellow test paper is regarded as negative, and regarded as positive if the final color is rose or light red. The gastric mucosa tissues of four groups of experimental gerbils after gastric injection in embodiment 4 were taken with a toothpick, and were placed on the center of the urease yellow test paper, then a self-adhesive sticker was laminated to a plastic film, so that the self-adhesive sticker was tightly combined with the plastic film, and 1-3 min later, the color change of the test paper was observed, as shown in
The gastric mucosa tissues of four groups of experimental gerbils after intragastric injection in embodiment 4 were collected, added with RIPA lysate and fully homogenized, and then proteins were extracted from the tissues. The total protein content was determined by a BCA kit. The contents of proinflammatory cytokine-6 (IL-6) and proinflammatory cytokine-8 (IL-8) were determined by an enzyme-linked immunosorbent assay kit. It can be seen from
In conclusion, the present invention obtains Bifidobacterium animalis subsp. lactis through isolation and screening, which is named as Bifidobacterium animalis subsp. lactis ATM-209 (GOLDGUT-BB18). The experiments for in-vitro acid tolerance and growth inhibition of Helicobacter Pylori of the Bifidobacterium animalis subsp. lactis prove that the Bifidobacterium animalis subsp. lactis has strong acid tolerance to the gastric environment, and has an effect for inhibiting the growth of Helicobacter Pylori. It is further discovered that the Bifidobacterium animalis subsp. lactis can effectively reduce the in vivo growth of Helicobacter Pylori and relieve the gastric tissue inflammation caused by the Helicobacter Pylori infection through the Mongolian gerbil models. Therefore, the strain is suitable for the digestive tract environment, has excellent capacity for resisting the Helicobacter Pylori, and has broad application prospect in the aspect of resisting the gastrointestinal pathogen Helicobacter Pylori.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention rather than limiting the present invention. Although the present invention is described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments may be modified or some technical features may be equivalently substituted. However, these modifications or substitutions do not make the essence of the corresponding technical solutions depart from the spirit and scope of the technical solutions of various embodiments of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310578935.7 | May 2023 | CN | national |
