Bifidobacterium bifidum and application thereof

Abstract

The invention provides bifidobacterium bifidum and application thereof, and relates to the technical field of microorganisms. The invention provides Bifidobacterium bifidum NKU FB3-13, which is preserved in the Guangdong Microbial Culture Collection Center, the preservation number of which is GDMCC No: 64747, and the preservation number of which is GDMCC No: 64747. The invention also provides a preparation method of the Bifidobacterium bifidum NKU FB3-13. The strain has good acid resistance and can well adapt to the stomach environment; the strain can effectively reduce the colonization number of helicobacter pylori on gastric epithelium, can greatly reduce the colonization number of helicobacter pylori in animal experiments, relieves inflammation caused by helicobacter pylori infection, and reduces stomach intestinal metaplasia. The preparation method has a wide application prospect in the fields of clinical prevention, treatment and alleviation of diseases related to helicobacter pylori infection, gastrointestinal health-care food and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Bifidobacterium bifidum and an application thereof. Background Art

[0002] Helicobacter pylori (Hp) is a spirally curved, flagellated, microaerophilic Gram-negative bacterium. Helicobacter pylori infection is one of the most common chronic bacterial diseases in humans. Helicobacter pylori is also a Class I human carcinogen recognized by the World Health Organization (WHO) and the International Agency for Research on Cancer. When Helicobacter pylori infects and colonizes the stomach, it secretes a large amount of urease to convert urea in gastric mucus into ammonia to neutralize gastric acid, thereby stimulating gastric tissue to secrete more gastrin to promote gastric acid secretion, thereby destroying the inherent barrier of the gastric mucosa, inducing apoptosis of gastric mucosal cells, leading to tissue damage and inflammation, and even promoting intestinal metaplasia of gastric tissue, thereby inducing gastric atrophy and even gastric cancer. Previous studies have shown that the most common cause of gastric cancer is Helicobacter pylori infection, so inhibiting Helicobacter pylori infection is the most effective way to prevent gastric cancer.

[0003] Treatment of Helicobacter pylori infection usually uses 4-drug combination and antibiotics to kill Helicobacter pylori, but more and more Helicobacter pylori strains are becoming more resistant to antibiotics, making many treatment options ineffective and greatly increasing the probability of recurrence. On the other hand, due to the extensive use of antibiotics, the patient's gastrointestinal flora is greatly damaged, causing gastrointestinal discomfort, loss of appetite, constipation or diarrhea and other adverse reactions, which greatly reduces the patient's quality of life. Therefore, relevant researchers in this field are committed to exploring effective treatment methods without side effects, such as using probiotics to intervene or prevent Helicobacter pylori infection. Because some probiotics can produce antimicrobial peptides, or preferentially occupy the colonization sites in the host's stomach, or preferentially bind to external strains such as Helicobacter pylori, so that Helicobacter pylori cannot colonize or adhere to the host's gastric epithelium, thereby preventing or reducing the number of Helicobacter pylori in the body, while regulating the gastrointestinal flora and protecting the host's health.

[0004] At present, there are many products on the market that use probiotics to intervene or prevent Helicobacter pylori infection. Most of them are lactic acid bacteria, and fewer use bifidobacteria with better probiotic effects, and almost no use of Bifidobacterium bifidum for intervention. Bifidobacterium bifidum is a bifidobacterium with extremely high probiotic value and generally has excellent adhesion and colonization capabilities. Therefore, the field needs to develop new strains of Bifidobacterium bifidum that have antagonistic effects on Helicobacter pylori. Summary of the invention

[0005] The purpose of the present invention is to provide a Bifidobacterium bifidum with excellent acid and bile salt resistance, which can maintain a high number of live bacteria in the human gastrointestinal tract to play a probiotic role, reduce the colonization of Helicobacter pylori in the stomach, has high safety and is not prone to side effects.

[0006] Another object of the present invention is to provide a fermentation broth of Bifidobacterium bifidum, a method for preparing the fermentation broth, a preparation, and the use of the Bifidobacterium bifidum, the fermentation broth or the preparation in the preparation of a product for reducing the colonization of Helicobacter pylori in the stomach, preventing intestinal metaplasia of the host's gastric tissue, reducing gastric atrophy and gastric cancer, alleviating inflammation in the host's gastrointestinal tract, systemic, and blood, and alleviating abnormal gastric acid secretion.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a bifidobacterium bifidum, characterized in that the bifidobacterium bifidum is bifidobacterium bifidum NKU FB3-13, and the preservation number is GDMCC No: 64747.

[0009] The present invention also provides a fermentation liquid comprising the above-mentioned Bifidobacterium bifidum.

[0010] The present invention also provides a method for preparing the fermentation broth, which comprises inoculating the Bifidobacterium bifidum into a culture medium for culturing to obtain the fermentation broth.

[0011] The present invention also provides a preparation comprising the above-mentioned Bifidobacterium bifidum or the above-mentioned fermentation broth.

[0012] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in preparing a product for inhibiting Helicobacter pylori.

[0013] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in preparing products for preventing and treating gastrointestinal diseases.

[0014] Preferably, the gastrointestinal diseases include intestinal metaplasia of gastric tissue, gastritis and gastric cancer.

[0015] Preferably, the gastrointestinal disease comprises gastric atrophy.

[0016] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in preparing a product for alleviating abnormal gastric acid secretion.

[0017] The present invention also provides a food, which comprises the Bifidobacterium bifidum or the fermentation liquid.

[0018] In the present invention, the concentration of live Bifidobacterium bifidum in the product can be selected from 1×10 8 ~1×10 10CFU / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention screens out the Bifidobacterium bifidum NKU FB3-13 strain from the feces of healthy infants. The strain has better acid and bile salt resistance and can maintain a high number of live bacteria in the human gastrointestinal tract to play a probiotic role. At the same time, the Bifidobacterium bifidum NKU FB3-13 does not have hemolysis, amino acid decarboxylase and nitrate reductase, and has high safety and has the potential to be taken by humans as food or medicine.

[0021] The present invention verifies through multi-aspect index determination that the strain can effectively reduce the colonization of Helicobacter pylori in the host, while reducing the host's stomach, blood and systemic inflammation, relieving symptoms of abnormal gastric acid secretion, protecting gastric function, reducing the degree of gastric intestinal metaplasia, and maintaining the host's gastrointestinal health. At the same time, the strain has a strong adhesion and colonization ability, so as to exert a long-term probiotic effect in the human gastrointestinal tract.

[0022] Biodeposit Information:

[0023] The Bifidobacterium bifidum NKU FB3-13 of the present invention has a strain name of Bifidobacterium bifidum NKUFB3-13, is classified and named Bifidobacterium bifidum, and is preserved in Guangdong Microbiological Culture Collection Center (GDMCC). The preservation time is: June 14, 2024. The preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is: GDMCCNo:64747. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a colony photo of Bifidobacterium bifidum NKU FB3-13 on BS solid medium.

[0025] Figure 2 This is a Warthin-Starry silver nitrate-stained section of mouse stomach tissue. The left, middle and right are the blank group, NKUFB3-13 group and model group respectively. The arrow indicates Helicobacter pylori.

[0026] Figure 3 The figure shows the number of Helicobacter pylori in the gastric mucosa of mice. The statistical analysis method was one-way analysis of variance, **** indicates that the p value is less than 0.0001.

[0027] Figure 4 The relative expression of IL6, an inflammatory factor in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, **** indicates that the p value is less than 0.0001.

[0028] Figure 5 Relative expression of IL-10, an inflammatory factor in mouse gastric tissue. The statistical analysis method was one-way ANOVA, ** indicates a p value less than 0.01, **** indicates a p value less than 0.0001.

[0029] Figure 6 The relative expression of TNFa, an inflammatory factor in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, * indicates a p value less than 0.05, **** indicates a p value less than 0.0001.

[0030] Figure 7 The concentration of inflammatory factor IL-8 in mouse serum was plotted. The statistical analysis method was one-way ANOVA, ** indicates p value is less than 0.01, **** indicates p value is less than 0.0001.

[0031] Figure 8 The concentration of IgG in mouse serum was plotted. The statistical analysis method was one-way ANOVA, * indicates p value is less than 0.05, **** indicates p value is less than 0.0001.

[0032] Fig. 9 The concentration of gastrin in mouse gastric tissue was plotted. The statistical analysis method was one-way ANOVA, * indicates p value is less than 0.05, *** indicates p value is less than 0.001.

[0033] Fig.10 This is a section of mouse gastric tissue stained with Schiff-Alsen blue. The left, middle and right are the blank group, NKU FB3-13 group and model group respectively. The purple-red part is acidic mucus.

[0034] Fig.11 The concentration of mucin Muc2 in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, ** indicates a p value less than 0.01, **** indicates a p value less than 0.0001.

[0035] Fig.12 The concentration of tight junction protein Occludin in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, **** indicates that the p value is less than 0.0001.

[0036] Fig.13 The concentration of tight junction protein Tjp2 in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, * indicates p value less than 0.05, **** indicates p value less than 0.0001.

[0037] Fig.14 The concentration of tight junction protein Tjp3 in mouse gastric tissue. The statistical analysis method was one-way analysis of variance, **** indicates that the p value is less than 0.0001.

[0038] Fig.15 This is a Muc5ac immunohistochemical section of mouse gastric tissue. The left, middle and right are the blank group, NKU FB3-13 group and model group respectively. The yellow part is Muc5ac protein. DETAILED DESCRIPTION

[0039] The invention provides a bifidobacterium bifidum, wherein the bifidobacterium bifidum is bifidobacterium bifidum NKU FB3-13, and the preservation number is GDMCC No:64747.

[0040] The Bifidobacterium bifidum NKU FB3-13 strain of the present invention is isolated from healthy infant feces and deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2024. After growing in BS solid medium for 48 hours, the colonies are small, off-white, smooth and slightly convex, with neat or irregular edges, slightly shiny and soft texture. Gram staining of the bacteria is positive under an optical microscope.

[0041] The strain of the present invention is tested for acid resistance and bile salt resistance, and the results show that the strain has better acid and bile salt resistance, and can maintain a higher number of viable bacteria in the human gastrointestinal tract to play a probiotic role. The strain of the present invention is tested for hemolysis, and the colony on the blood plate has no hemolysis circle, that is, it is non-hemolytic. The strain of the present invention is tested for amino acid decarboxylase, and the results show that the color of the two amino acid decarboxylase culture tubes of Bifidobacterium bifidum NKU FB3-13 remains unchanged, which is yellow, showing a negative reaction, that is, the strain does not have ornithine and lysine decarboxylase. The strain of the present invention is tested for nitrate reductase, and the experimental results show that Bifidobacterium bifidum NKU FB3-13 has no color change in nitrate culture medium, that is, the strain does not have nitrate reductase. The above results all illustrate that the Bifidobacterium bifidum NKU FB3-13 of the present invention has better acid and bile salt resistance, and can maintain a higher number of viable bacteria in the human gastrointestinal tract to play a probiotic role. Meanwhile, Bifidobacterium bifidum NK U FB3-13 does not have hemolytic activity, amino acid decarboxylase and nitrate reductase, and has high safety, and has the potential to be taken by humans as food or medicine.

[0042] The present invention also provides a fermentation broth, which is the fermentation broth of the above-mentioned Bifidobacterium bifidum NKU FB3-13 strain. The method for preparing the fermentation broth comprises inoculating the Bifidobacterium bifidum NKU FB3-13 in a culture medium and culturing it to obtain a fermentation broth. As an implementable embodiment, the culture medium may optionally include a BS liquid culture medium; the culture method may optionally be to inoculate the Bifidobacterium bifidum NKU FB3-13 in a BS liquid culture medium, and perform anaerobically culturing in an anaerobic incubator at 35 to 40°C for 18 to 36 hours to obtain the fermentation broth; the culture temperature is preferably 37°C.

[0043] The present invention also provides a preparation comprising the above-mentioned Bifidobacterium bifidum NKU FB3-13 or the above-mentioned fermentation broth. The preparation type of the present invention is not particularly limited, and various dosage forms can be prepared by selecting appropriate excipients according to actual needs. As an implementable embodiment, the preparation can optionally include a lyophilized powder preparation of the strain that exerts a probiotic function or other preparations containing the strain.

[0044] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in preparing a product for inhibiting Helicobacter pylori.

[0045] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in the preparation of a product for preventing and treating gastrointestinal diseases. Preferably, the gastrointestinal diseases include gastric intestinal metaplasia, gastritis and gastric cancer. Preferably, the gastrointestinal diseases include gastric atrophy.

[0046] The present invention also provides the use of the Bifidobacterium bifidum, the fermentation liquid or the preparation in preparing a product for alleviating abnormal gastric acid secretion.

[0047] The present invention also provides a food, which comprises the Bifidobacterium bifidum or the fermentation liquid.

[0048] The Bifidobacterium bifidum NKU FB3-13 strain of the present invention can reduce the colonization of Helicobacter pylori in the body, thereby protecting the host's gastric health; effectively prevent intestinal metaplasia of the host's gastric tissue, and reduce the probability of gastric atrophy and gastric cancer. It can also effectively reduce the number of Helicobacter pylori colonization in mice, while reducing gastric and systemic inflammation, protecting gastric function, relieving abnormal gastric acid secretion, and maintaining the host's gastrointestinal health.

[0049] The present invention does not specifically limit the product type. As an implementation method, the product includes food or medicine. In the product, the Bifidobacterium bifidum NKU FB3-13 or fermentation broth or preparation can be the only active ingredient with the function of maintaining the health of the host's gastrointestinal tract, and can also contain other active ingredients. The product of the present invention can be used alone or in combination with other products with antioxidant functions. The present invention does not specifically limit the concentration of live bacteria of Bifidobacterium bifidum NKU FB3-13 in the product for reducing the colonization of Helicobacter pylori in the body. It can be conventionally selected according to actual needs. Preferably, the concentration of live bacteria of Bifidobacterium bifidum in the product is 1×10 8 ~1×10 10 CFU / mL.

[0050] In order to further illustrate the present invention, the Bifidobacterium bifidum NKU FB3-13 and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The test materials and their sources used in the present invention include the following:

[0052] Culture medium and indicator solution used in the examples:

[0053] BS liquid culture medium: peptone 10g / L, liver extract powder 5g / L, beef extract powder 3g / L, yeast extract powder 5g / L, trypticase peptone 8g / L, soluble starch 0.5g / L, sodium chloride 1g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, glucose 10g / L, ferrous sulfate heptahydrate 0.01g / L, manganese sulfate 0.005g / L, L-cysteine ​​0.5g / L.

[0054] BS solid culture medium: peptone 10g / L, liver extract powder 5g / L, beef extract powder 3g / L, yeast extract powder 5g / L, tryptic peptone 8g / L, soluble starch 0.5g / L, sodium chloride 1g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, glucose 10g / L, ferrous sulfate heptahydrate 0.01g / L, manganese sulfate 0.005g / L, L-cysteine ​​0.5g / L, agar 20g / L.

[0055] Fermentation medium: peptone 10g / L, liver extract powder 5g / L, beef extract powder 3g / L, yeast extract powder 5g / L, tryptic peptone 8g / L, soluble starch 0.5g / L, sodium chloride 1g / L, dipotassium hydrogen phosphate 1g / L, potassium dihydrogen phosphate 1g / L, glucose 20g / L, diammonium hydrogen citrate 2g / L, magnesium sulfate heptahydrate 0.58g / L, ferrous sulfate heptahydrate 0.01g / L, manganese sulfate 0.005g / L, L-cysteine ​​0.5g / L, Tween-80 1mL / L.

[0056] Bacteria protective agent: 140g / L skim milk powder, 100g / L trehalose, 5g / L sodium glutamate, 15g / L glycerol.

[0057] Ornithine decarboxylase culture medium: peptone 5g / L, beef extract 5g / L, glucose 0.5g / L, vitamin B6 0.005g / L, agar 6g / L, 0.2% bromocresol purple 5mL / L, 0.2% cresol red 2.5mL / L, L-ornithine 10g / L.

[0058] Lysine decarboxylase medium: peptone 5g / L, beef extract 5g / L, glucose 0.5g / L, vitamin B6 0.005g / L, agar 6g / L, 0.2% bromocresol purple 5mL / L, 0.2% cresol red 2.5mL / L, L-lysine 10g / L.

[0059] Columbia blood agar medium: 10g / L casein pancreatic digest, 3g / L heart pancreatic digest, 1g / L corn starch, 5g / L meat gastric digest, 5g / L yeast extract powder, 5g / L sodium chloride, 15g / L agar. After sterilization, cool to 45-50℃, then add 10mL / L defibrinated sheep blood and mix well.

[0060] Nitrate medium: peptone 10g / L, yeast extract powder 3g / L, potassium nitrate 2g / L.

[0061] Urease indicator solution: sodium chloride 9g / L, urea 1.2g / L, phenol red 14mg / L.

[0062] The bacterial materials used in the examples are:

[0063] Staphylococcus aureus ATCC 25923 was purchased from Mingzhou Biotechnology, catalog number B81854 (A).

[0064] Bifidobacterium animalis BB12 was purchased from Beina Biotechnology, product number BNCC192320.

[0065] The laboratory-preserved Helicobacter pylori SS1 was purchased from Chengdu Myric Biotechnology Co., Ltd., with the catalog number MRC-Hp-SS1.

[0066] Other biological materials used in the examples:

[0067] GES-1 human gastric epithelial cells are GES-1 cells, purchased from Beina Biotechnology, catalog number BNCC337969.

[0068] Female Balb / c mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Nankai University.

[0069] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0070] Example 1

[0071] 1. Strain screening

[0072] The sample comes from the feces of healthy infants about 40 days old. About 1g of fecal sample is placed in a 50mL centrifuge tube, which is pre-filled with 9mL of anaerobic phosphate buffer solution. After fully vortexing and mixing, the fecal bacteria buffer is gradiently diluted and fully coated on BS solid culture medium, and then placed in an anaerobic incubator for 48h. After the colony grows, the suspected bifidobacterium colony is picked and examined microscopically by Gram staining to observe the morphological characteristics of the bacteria, and Gram-positive bacteria are selected as the target strain. After streaking and purification in BS solid culture medium, a single colony is picked and streaked twice, and the strain that still has the colony morphology of bifidobacterium is used as the target strain, named NKU FB3-13. The target colony of this strain is picked to amplify the bacteria in BS liquid culture medium.

[0073] 2. Morphological observation and microscopic identification

[0074] The target strain NKU FB3-13 was grown in BS solid medium for 48 hours. The colonies were small, off-white, smooth and slightly convex, with neat or irregular edges, slightly shiny and soft texture. Figure 1 shown.

[0075] The colony was smeared and stained with Gram stain and then observed under a microscope. The Gram stain was positive for the bacteria, which were straight rods or short curved rods, some with slightly forked ends, arranged singly or in pairs, and often clustered.

[0076] 3. Molecular Biology Identification

[0077] The genomic DNA of NKU FB3-13 was extracted using a bacterial genome kit, and then the total DNA was used as a template and 16SrRNA universal primers 27F and 1492R were used for PCR amplification. The sequence of primer 27F is: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1), and the sequence of primer 1492R is: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO.2). The amplified product was identified by 1.5% agarose gel electrophoresis and sent to Qingke Biotechnology Co., Ltd. for gene sequence determination. After comparison with the NCBI official website, the NKU FB3-13 strain was determined to be Bifidobacterium bifidum, and the specific sequence is as follows:

[0078]

[0079] The Bifidobacterium bifidum NKU FB3-13 was deposited in the Guangdong Provincial Microbiological Culture Collection Center, abbreviated as GDMCC, on June 14, 2024. The culture collection number is GDMCC No: 64747, and the collection address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0080] 4. Strain activation and preservation

[0081] A single colony of the isolated NKU FB3-13 was inoculated into 10 mL of anaerobic substituted BS medium and placed in an anaerobic workstation at 37°C for 24 hours as the first generation. 0.2 mL of the first generation bacterial solution was inoculated into 10 mL of BS medium for anaerobic culture for 24 hours as the second generation. 0.2 mL of the second generation bacterial solution was inoculated into 10 mL of BS medium for anaerobic culture for 24 hours as the third generation, thereby completing activation.

[0082] After centrifuging the third generation activated bacteria, remove the lower layer of bacterial sludge, mix the bacterial sludge and bacterial protective agent in a ratio of 1:1, pre-freeze at -80°C overnight, and then freeze-dry for storage.

[0083] Example 2

[0084] 1. Acid resistance test: Take the strain of Bifidobacterium bifidum NKU FB3-13 and culture it to the third generation stable period, and inoculate it into BS liquid culture medium with pH values ​​equal to 3, 3.5 and 4 at an inoculum amount of 2% (v / v), and set the BS culture medium with pH value of 6.5 as the control, and place them in an anaerobic incubator at 37°C for anaerobic culture; dilute and apply the bacterial suspension of 0h and 2h respectively, and place the plate after coating in an anaerobic incubator at 37°C for anaerobic culture until colonies grow, and finally count the colonies on the plate. The results are shown in Table 1.

[0085] Table 1 The number of viable bacteria of Bifidobacterium bifidum NKU FB3-13 at different pH values ​​(lg CFU / mL)

[0086] time pH3 pH 3.5 pH4 Control group 0h 8.7 8.9 8.8 9.0 2h 5.1 6.8 8.3 9.1

[0087] 2. Bile salt tolerance test: Take the strain of Bifidobacterium bifidum NKU FB3-13 and culture it to the third generation stable period, and inoculate it into BS liquid culture medium with pig bile salt (purchased from Shanghai McLean Biochemical Technology Co., Ltd., item number B822609) at a 2% (v / v) inoculation amount, respectively, with a concentration of 0.05%, 0.1% and 0.2%, and a BS liquid culture medium without bile salt added as a control, and place them in a 37°C anaerobic incubator for anaerobic culture; take the bacterial suspension of 0h and 3h for dilution and coating, and place the coated plate in a 37°C anaerobic incubator for anaerobic culture until colonies grow, and finally count the colonies on the plate. The results are shown in Table 2.

[0088] Table 2 The number of viable bacteria of Bifidobacterium bifidum NKU FB3-13 at different bile salt concentrations (lg CFU / mL)

[0089] time 0.05％ 0.1％ 0.2％ Control group 0h 8.8 8.9 8.7 8.9 3h 8.8 6.3 3.7 9.1

[0090] 3. Hemolytic test: Take the Bifidobacterium bifidum NKU FB3-13 strain and culture it to the third generation stable period, then use Staphylococcus aureus ATCC 25923 as a positive control, streak it on a Columbia blood agar plate, and place it in a dark and anaerobic culture at 37°C for 48 hours, and observe and record whether there is a hemolytic circle. If a transparent blood lysis area appears around the colony, it is β hemolysis, and a green area (α hemolysis) or no hemolytic area (γ hemolysis) around the colony is considered to indicate that the strain is non-hemolytic. The experimental results show that the colony of Bifidobacterium bifidum NKU FB3-13 on the blood plate does not produce a hemolytic circle, that is, it is non-hemolytic.

[0091] 4. Amino acid decarboxylase test: Take the Bifidobacterium bifidum NKU FB3-13 strain and culture it to the third generation stable period, inoculate it with the pre-prepared liquid ornithine and lysine decarboxylase culture medium at a 2% (v / v) inoculation amount, add an appropriate amount of sterile paraffin oil to each inoculation tube, and then place it at 37°C for anaerobic culture for 48 hours to observe and record the change in liquid color. If the culture medium turns purple or purple, it shows a positive reaction, then the strain has amino acid decarboxylase; if the color of the culture medium does not change, it is yellow, showing a negative reaction, then the strain does not have amino acid decarboxylase. The experimental results show that the color of the two amino acid decarboxylase culture tubes of Bifidobacterium bifidum NKU FB3-13 does not change, it is yellow, showing a negative reaction, that is, the strain does not have ornithine and lysine decarboxylase.

[0092] 5. Nitrate reductase test: Take the strain of Bifidobacterium bifidum NKU FB3-13 and culture it to the third generation stable period. Take the blank nitrate medium as the negative control, inoculate the pre-prepared nitrate medium at a 2% (v / v) inoculation amount, add an appropriate amount of sterile paraffin oil to each inoculation tube, and then place it at 37°C for anaerobic culture for 48h; then add 10 drops of 5% potassium iodide solution and 10 drops of 5% starch solution in turn, vortex mix and observe and record the color changes in each culture tube. The experimental results show that Bifidobacterium bifidum NKU FB3-13 has no color change in nitrate medium, that is, the strain does not have nitrate reductase.

[0093] The above examples show that Bifidobacterium bifidum NKU FB3-13 has good acid and bile resistance, and can maintain a high number of viable bacteria in the human gastrointestinal tract to play a probiotic role. At the same time, Bifidobacterium bifidum NKU FB3-13 does not have hemolytic activity, amino acid decarboxylase and nitrate reductase, and has high safety, and has the potential to be taken by humans as food or medicine.

[0094] Example 3

[0095] 1. Hydrophobicity determination:

[0096] The strains of Bifidobacterium bifidum NKU FB3-13 and Bifidobacterium animalis BB12 were cultured to the third generation stable phase, and the cells were collected by centrifugation at 4500 rpm for 5 minutes, washed with PBS, and resuspended to adjust the strain concentration to 10 8 CFU / mL. Then, each resuspended bacterial solution was mixed with xylene in a 1:1 (volume ratio), vortexed evenly, and allowed to stand at room temperature. After full stratification, the lower aqueous phase was taken to measure the OD 600 Value. Animal Bifidobacterium BB12 was used as a control. The hydrophobicity calculation formula is as follows:

[0097] Hydrophobicity (%) = (M0-M) / M0×100%

[0098] Where: M0 is the initial OD 600 Value; M is the OD in the lower aqueous phase after stratification 600 value.

[0099] After full stratification, the test calculation results are as follows: the hydrophobicity of Bifidobacterium bifidum NKU FB3-13 is 97.2±2.4%, and the hydrophobicity of Bifidobacterium animalis BB12 is 94.4±1.9%.

[0100] 2. Determination of self-aggregation ability: Take the strains of Bifidobacterium bifidum NKU FB3-13 and Bifidobacterium animalis BB12 and culture them to the third generation stable period, collect the cells by centrifugation at 4500 rpm for 5 minutes, wash them with PBS, resuspend them, and adjust the strain concentration to 10 8 CFU / mL, followed by anaerobically incubation at 37°C, and taking the upper layer of the bacterial suspension at 0h, 3h, 6h, 12h and 24h after the start of the culture and measuring the OD 600 Value. Animal Bifidobacterium BB12 was used as a control. The self-aggregation rate was calculated as follows:

[0101] Self-aggregation rate (%) = (M t -M0) / M0×100%

[0102] Where: M0 is the initial OD of the bacteria at 0h 600 Value; M tis the OD of the bacterial suspension in the upper layer at time t 600 The results are shown in Table 3.

[0103] Table 3 Self-aggregation rate of different bifidobacteria

[0104] time Bifidobacterium bifidum NKUFB3-13 Bifidobacterium animalis BB12 0h 0.0％ 0.0％ 3h 28.1±3.1％ 26.4±2.7％ 6h 49.6±4.2％ 46.2±3.8％ 12h 61.4±5.2％ 62.7±4.3％ 24h 79.3±3.6％ 78.6±2.1％

[0105] The above examples show that Bifidobacterium bifidum NKU FB3-13 has excellent hydrophobicity and self-aggregation properties, which indicates that the strain is likely to have strong adhesion and colonization capabilities so as to exert a probiotic effect in the human gastrointestinal tract for a long time.

[0106] Example 4

[0107] Determination of the coaggregation ability of Bifidobacterium bifidum NKU FB3-13 and Helicobacter pylori SS1: Helicobacter pylori SS1 preserved in the laboratory was spread on Columbia blood agar plates, cultured in a microaerobic environment at 37°C for 48 hours, and then the bacterial sludge was scraped off with a spatula, washed with PBS, resuspended, and the strain concentration was adjusted to OD 1. Bifidobacterium bifidum NKU FB3-13 and Bifidobacterium animalis BB12 were cultured to the third generation stable period, centrifuged at 8000 rpm for 5 minutes, washed with PBS, resuspended, and the strain concentration was adjusted to OD 1. Take 20 mL each of Helicobacter pylori SS1 bacterial suspension and Bifidobacterium bifidum NKU FB3-13 bacterial suspension or take 20 mL each of Helicobacter pylori SS1 bacterial suspension and Bifidobacterium animalis BB12 bacterial suspension in a 50 mL centrifuge tube, vortex for 10 seconds to mix, and then incubate at 37°C. At 0h, 3h, 6h, 12h, and 24h of incubation, carefully aspirate 2 mL of the upper liquid, and measure the absorbance of the mixed bacterial suspension of Bifidobacterium bifidum NKU FB3-13, Bifidobacterium animalis BB12 and Helicobacter pylori SS1 at 600nm. The formula for calculating the co-aggregation rate is as follows:

[0108] Coagulation rate (%) = [(A B +A H ) / 2-A t ] / [(A B +A H ) / 2]×100%

[0109] Where: A B is the OD of Bifidobacterium t 600 Value; A H is the OD of Helicobacter pylori at t 600 Value; A t is the OD of the mixed bacterial suspension at time t 600 The results are shown in Table 4.

[0110] Table 4 Coaggregation rate of different Bifidobacteria and Helicobacter pylori SS1

[0111]

[0112]

[0113] Determination of the ability of Bifidobacterium bifidum NKU FB3-13 to inhibit the adhesion of Helicobacter pylori SS1 to GES-1 human gastric epithelial cells: After the GES-1 cells were digested and resuspended, the concentration of GES-1 cells was adjusted to 50,000 / mL, and 0.1 mL of the above-adjusted GES-1 cell resuspension was added to each well of a 96-well plate, and the cells were cultured at 37°C and 5% CO2 for 12 h. After the GES-1 cells adhered to the wall, the GES-1 cells were washed 3 times with PBS buffer, and then 0.1 mL of DMEM medium and 10% resuspended GES-1 cells were added. 7 10 μL of Helicobacter pylori SS1 and Bifidobacterium bifidum NKU FB3-13 resuspension at a concentration of 10 CFU / mL each 7 10 μL of each CFU / mL resuspension of Helicobacter pylori SS1 and animal Bifidobacterium BB12 was used as different treatment groups, and a model group with Helicobacter pylori SS1 alone and a blank group with only GES-1 cells were set up. After culturing in an incubator at 37°C and 5% CO2 for 3 hours, the cells were washed 5 times with PBS buffer to clean the unadsorbed Helicobacter pylori. Finally, 200 μL of urease reagent was added to each group of cells, and the cells were cultured in an incubator at 37°C and 5% CO2 for 2 hours to obtain the culture solution. The absorbance of the culture solution of different groups at a wavelength of 550nm was measured by an enzyme marker. The adhesion rate calculation formula is as follows:

[0114] Adhesion rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of model group - OD value of blank group) × 100%

[0115] When the adhesion rate of the model group was defined as 100%, the adhesion rate of the Bifidobacterium animalis BB12 group was 66.3±9.1%, and the adhesion rate of the Bifidobacterium bifidum NKU FB3-13 group was 40.1±7.9%.

[0116] The above examples show that Bifidobacterium bifidum NKU FB3-13 has extremely strong co-aggregation properties with Helicobacter pylori SS1, and extremely strong ability to reduce the adhesion of Helicobacter pylori SS1 to human gastric epithelial cells, which means that taking Bifidobacterium bifidum NKU FB3-13 is likely to reduce the colonization of Helicobacter pylori in the body, thereby protecting the host's gastric health.

[0117] Example 5

[0118] Animal grouping and modeling: 18 female Balb / c mice, 3 weeks old, were randomly divided into 3 groups after one week of adaptation, including 6 blank groups, 6 Helicobacter pylori SS1 model groups, and 6 Bifidobacterium bifidum NKU FB3-13 groups. During the modeling period, the mice were fasted for 2 hours but not water, and then the NKU FB3-13 group was gavaged for 10 9 CFU Bifidobacterium bifidum NKU FB3-13, the blank group and the model group were gavaged with an equal volume of saline; after an interval of 4 hours, the NKU FB3-13 group and the model group were gavaged with 10 9 CFU Helicobacter pylori SS1, the blank group was gavaged with an equal volume of saline; continued fasting for 2 hours and then resumed normal diet. The model was established for 5 weeks, and then gavage of Bifidobacterium and Helicobacter pylori was stopped for 2 days to wait for the mice to empty the stomach of the uncolonized strains. Finally, the mice were killed, and the stomach tissue and serum were taken to measure the relevant indicators.

[0119] Warthin-Starry silver nitrate staining: mucin on the surface of Helicobacter pylori specifically binds to silver and appears black. Based on this principle, mouse gastric tissue sections were stained. The results are as follows: Figure 2 A large number of black short rod-shaped particles and hyphae can be observed in the slices of the model group, which is the most seriously infected and has the largest area; the NKU FB3-13 group has a small amount of black lumpy hyphae, and the infected area is small and not serious; the blank group has no black short rod-shaped particles or hyphae and no infection.

[0120] Gastric tissue urease assay: Take fresh and uncontaminated gastric tissue, rinse the gastric contents with PBS, then cut about 50 mg of gastric tissue and place it in 1 mL of urease indicator solution, incubate at 37°C for 2 hours, and measure its OD value with a spectrophotometer at OD550 nm. The data of the model group is taken as 100% of the urease activity, and the urease activity of other groups is calculated. The results showed that the urease activity of the NKU FB3-13 group was 33.6±6.2%, and the urease activity of the blank group was 2.8±1.9%. This shows that oral administration of Bifidobacterium bifidum NKU FB3-13 can effectively reduce the urease activity in the host stomach and maintain gastric health.

[0121] Assessment of gastric colonization of Helicobacter pylori: Colonies were scraped from the Helicobacter pylori SS1 plate and diluted in PBS to OD 600 The value is 1, and the number of live bacteria in the bacterial suspension is counted using a hemocytometer to calculate the OD value of the bacterial suspension. 600 The coefficient of the value and the number of viable bacteria was calculated. Then the Helicobacter pylori suspension was diluted in sequence to prepare the OD 600The dilution bacterial suspension was 0.5, 0.25, 0.125, and 0.0625, and 0.1 g of blank group mouse feces was added thereto respectively. Total DNA was extracted using a bacterial DNA extraction kit, and then the qPCR absolute quantitative method and Helicobacter pylori specific primers HpG1-F and HpG1-R were used. The sequence of the specific primer HpG1-F was: 5'-CAAGCCGCTCAAAAAGAGGC-3' (SEQ ID NO.4); the sequence of the specific primer HpG1-R was: 5'-AAGCGCTAAAAGGCTCGTCT-3' (SEQ ID NO.5). The Helicobacter pylori suspension with feces was quantitatively added, and after the Ct value was determined, the standard curve of Helicobacter pylori was drawn with the number of Helicobacter pylori as the horizontal axis and the Ct value as the vertical axis. The total bacterial DNA in the gastric mucosa of mice was extracted using a bacterial DNA extraction kit, and the total bacterial DNA in the gastric mucosa of mice was amplified using primers HpG1-F and HpG1-R. The measured Ct value was substituted into the standard curve of Helicobacter pylori to calculate the number of Helicobacter pylori in the gastric mucosa. The results are shown in Figure 3 The number of bacteria in the NKU FB3-13 group was about 1543±422 CFU, and the number of bacteria in the model group was about 10122±3108 CFU, that is, the number of bacteria in the NKU FB3-13 group was only 15.2% of that in the model group. This shows that oral administration of Bifidobacterium bifidum NKUFB3-13 can effectively reduce the number of Helicobacter pylori in the host's gastric mucosa and maintain gastric health.

[0122] Evaluation of gastric tissue inflammation: mRNA was extracted from mouse gastric tissue, reverse transcribed into cDNA, and then the expression levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor (TNF-α) in mouse gastric tissue were determined by qPCR. Figure 4-6 The expression levels of the three typical inflammatory factors in the NKU-FB3-13 group were significantly lower than those in the model group. In addition, the mouse serum was taken and the concentrations of interleukin-8 (IL-8) and Helicobacter pylori-specific IgG in the mouse serum were determined using an ELISA kit. The results are as follows Figure 7-8 As shown in the figure, the concentrations of IL-8 and IgG in the serum of mice in the NKU-FB3-13 group were significantly lower than those in the model group. This indicates that oral administration of Bifidobacterium bifidum NKU FB3-13 can effectively downregulate the level of inflammation in the host's gastric tissue and blood, and maintain the host's gastric and systemic health.

[0123] Gastric function assessment: Mouse gastric tissue homogenate was obtained, and then the gastrin content in the mouse gastric tissue was determined using an ELISA kit. Gastric tissue sections were also taken and stained with Schiff-Alsen Blue (purchased from Beijing Solebow Technology Co., Ltd., catalog number G1285). Figure 9-10As shown, the gastrin content in the NKU-FB3-13 group was significantly lower than that in the model group. The content of purple-red acidic mucus in the slices of the model group was also significantly less than that in the NKU-FB3-13 group and the blank group, indicating that the model group had abnormal gastric acid secretion, while the NKU-FB3-13 group alleviated this phenomenon.

[0124] Evaluation of gastric intestinal metaplasia: Mouse gastric tissue homogenate was obtained, and then the content of mucin Muc2 in mouse gastric tissue was determined using an ELISA kit. Mouse gastric tissue was also obtained to extract mRNA, which was reverse transcribed into cDNA and then the expression levels of Occludin, Tjp2, and Tjp2 in mouse gastric tissue were determined using the qPCR method. Mouse gastric tissue sections were also obtained, and immunohistochemical sections were prepared using mouse Muc5ac-specific antibodies. The results are shown in Figure 2. Figure 11-15 As shown in the figure, the content or expression of intestinal tissue marker mucin and intestinal tissue marker tight junction protein in the NKU-FB3-13 group was significantly lower than that in the model group. This indicates that oral administration of Bifidobacterium bifidum NKUFB3-13 can effectively prevent intestinal metaplasia of host gastric tissue and reduce the probability of gastric atrophy and gastric cancer.

[0125] The above examples show that Bifidobacterium bifidum NKU FB3-13 can effectively reduce the number of Helicobacter pylori colonization in mice, while alleviating gastric and systemic inflammation, protecting gastric function, reducing the degree of gastric intestinal metaplasia, and maintaining the health of the host's gastrointestinal tract.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Bifidobacterium bifidum, characterized in that The Bifidobacterium bifidum is Bifidobacterium bifidum NKU FB3-13, and its deposit number is GDMCC No:64747.

2. A fermentation broth, characterized in that: The method comprises the Bifidobacterium bifidum according to claim 1.

3. The method for preparing the fermentation liquid according to claim 2, characterized in that: The method comprises inoculating the bifidobacterium bifidum into a culture medium for culturing to obtain a fermentation liquid.

4. A preparation, characterized in that The method comprises the Bifidobacterium bifidum according to claim 1 or the fermentation broth according to claim 2.

5. Use of the Bifidobacterium bifidum according to claim 1, the fermentation liquid according to claim 2 or the preparation according to claim 4 in the preparation of a product for inhibiting Helicobacter pylori.

6. Use of the Bifidobacterium bifidum according to claim 1, the fermentation liquid according to claim 2 or the preparation according to claim 4 in the preparation of products for preventing and treating gastrointestinal diseases.

7. The use according to claim 6, characterized in that: The gastrointestinal diseases include intestinal metaplasia of gastric tissue, gastritis and gastric cancer.

8. The use according to claim 6, characterized in that: The gastrointestinal disorders include gastric atrophy.

9. Use of the Bifidobacterium bifidum according to claim 1, the fermentation liquid according to claim 2, or the preparation according to claim 4 in the preparation of a product for alleviating abnormal gastric acid secretion.

10. A food, characterized in that: The food comprises the Bifidobacterium bifidum according to claim 1 or the fermentation liquid according to claim 2.

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