CN114214230B - Lactobacillus North with helicobacter pylori copolymerization capability and application thereof - Google Patents
Lactobacillus North with helicobacter pylori copolymerization capability and application thereof Download PDFInfo
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- CN114214230B CN114214230B CN202111517474.XA CN202111517474A CN114214230B CN 114214230 B CN114214230 B CN 114214230B CN 202111517474 A CN202111517474 A CN 202111517474A CN 114214230 B CN114214230 B CN 114214230B
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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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- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/60—Drinks from legumes, e.g. lupine drinks
- A23L11/65—Soy drinks
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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Abstract
The invention discloses a Lactobacillus northbound strain with helicobacter pylori copolymerization capability and application thereof, belonging to the technical field of microorganisms and medicines. The invention provides a lactobacillus northbound CCFM1207, wherein the lactobacillus northbound CCFM1207 can specifically form a copolymer with helicobacter pylori, and the specific expression is as follows: the 120min copolymerization rate of the Lactobacillus northleonia CCFM1207 and helicobacter pylori can reach 58.40 percent, and the Lactobacillus northleonia CCFM1207 has great application prospect in the aspect of eliminating helicobacter pylori (without aiming at diagnosis and treatment of diseases) and preparing helicobacter pylori adhesion remover.
Description
Technical Field
The invention relates to a Lactobacillus North with helicobacter pylori copolymerization capability and application thereof, belonging to the technical field of microorganisms and medicines.
Background
Helicobacter pylori (Helicobacter pylori, h. Pyri) was found in 1982 by the australian scholars bary marshall and Luo Bin wal and proved to infect the stomach and cause gastritis, gastric ulcers and duodenal ulcers. H. pyri is spiral or S-shaped and is a microaerophilic gram negative bacterium. H.pyri is mostly rod-shaped or spiral when in the growth phase and spherical when aged or dead. At present, all people around the world are infected with H.pyri and have high infection rate, the detection rate in the population in developing countries is 70-90%, and the detection rate in developed countries is 25-50%. The infection rate of H.pyri is closely related to the health condition, and children are generally easy to infect and can survive in human bodies for a long time, and the incidence rate of H.pyri rises with the increase of age and is related to the social and economic conditions, the race and other factors.
The H.pyri has adhesiveness, can be fixed in the stomach for a long time, and is not expelled from the body together with food due to peristalsis of the stomach. This is due to the fact that the surface protein of H.pyri contains a variety of adhesion molecules and is able to adhere firmly to gastric epithelial cells. Helicobacter pylori infection is a long-term and chronic process, and the human body is generally difficult to spontaneously clear after infection, and helicobacter pylori automatically disappears in the human body unless eradication treatment is carried out or serious intestinal metaplasia occurs in gastric mucosa of the human body.
Currently, the Maastricht-5 consensus and the fifth national helicobacter pylori infection treatment consensus in 2016 in China both recommend bismuth tetrad (PPI+bismuth+2 antibacterial agents) as the primary empirical treatment eradication of Hp. In addition, certain chemical compositions and traditional Chinese medicine components have corresponding beneficial effects on the treatment of helicobacter pylori infection.
For example, a chemical pharmaceutical composition for oral treatment of adult helicobacter pylori infection is disclosed in the chinese patent application publication No. CN111184867a, which comprises fosfomycin or a pharmaceutically acceptable salt thereof, fosfomycin trometamol and an aminoglycoside at least in an amount commonly used for adults. However, since the composition is composed of chemical drugs, the composition has corresponding unavoidable side effects on human health.
For another example, an allicin preparation for treating helicobacter pylori is disclosed in chinese patent application publication No. CN112641891a, and the allicin preparation comprises allicin, coptis chinensis, clove leaf, peppermint leaf, dandelion, scutellaria baicalensis, malt, liquorice, poria cocos, probiotic powder, codonopsis pilosula and yam, and has high sensitivity to helicobacter pylori, and can effectively inhibit the propagation of helicobacter pylori and maintain the balance of gastrointestinal flora. However, the preparation has more components, complex manufacturing principle and higher cost.
Autoaggregation and synergistic co-aggregation are common biological properties of lactobacillus and are also important pathways for the development of probiotic action. Copolymerization of lactobacillus with host protozoa or pathogenic bacteria is an important strategy to achieve pathogenic bacteria control or reduction. For example, a strain of Lactobacillus brevis (Lactobacillus brevis) BBE-Y52 isolated from the oral cavity is disclosed in Chinese patent publication No. CN102690768B, which is capable of producing extracellular polysaccharides and promoting the copolymerization and aggregation of lactic acid bacteria with other microorganisms in the oral environment to form a biofilm, thereby resisting the colonization of pathogenic bacteria. For example, the application potential of enterococcus faecium 132 and lactobacillus paracasei 201 with cholesterol reducing function to the prevention and control of 6 food-borne pathogenic bacteria is disclosed in Chinese patent application publication No. CN112662791A, wherein the copolymerization of the lactobacillus paracasei 201 and pathogenic bacteria is stronger, and the copolymerization inhibition rate of the lactobacillus paracasei 201 and pathogenic bacteria to escherichia coli, salmonella typhimurium, staphylococcus aureus, listeria monocytogenes and bacillus cereus is 20-40%.
Lactobacillus reuteri DSM17648 (Pyloplass) TM ) Is the most widely used probiotics with helicobacter pylori co-aggregation at present, and clinical experiments show that the bacterial powder in an inactivated state can still reduce the helicobacter pylori load in a patient. Subsequently, patent document CN103648511A discloses a method for improving the formation of a copolymer of DSM 17688 and helicobacter pylori to give a more excellent copolymerization effect.
Thus, we can recognize helicobacter pylori by using lactobacillus, and attach it to the surface to form a copolymerized bacterial body. The co-polymerized bacteria are finally excreted, which promotes the reduction of the proliferation of helicobacter pylori in the stomach. Therefore, the composition can remarkably inhibit the activity of helicobacter pylori, has gastric acid regulating capability, helps to kill Hp, has effective gastric field planting capability and excellent gastric acid and bile salt resisting property, and has high gastrointestinal pass rate. The copolymerization reaction can obviously improve the phenomena of stomachache, gastric acid, gastrectasia and the like caused by chronic gastritis and gastrointestinal ulcer, and is a key link for recovering the chronic gastritis and the gastrointestinal ulcer.
Thus, it has been an important point and difficulty in research to obtain a bacterium which can efficiently form a copolymer with helicobacter pylori.
Disclosure of Invention
The invention provides a lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207, wherein the lactobacillus northbound CCFM1207 is preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:62013, the preservation date is 2021, 10 and 25.
The Lactobacillus northlesii CCFM1207 is derived from fresh fecal samples of healthy people, the strain is subjected to sequencing analysis, the 16SrDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in GeneBank, and the homology with Lactobacillus is as follows: 98.95%; the results showed that the strain was Lactobacillus North, designated Lactobacillus North (Lactobacillus kitasatonis) CCFM1207. The colony of the lactobacillus northbound CCFM1207 on the MRS solid culture medium is white and micro-transparent, round or irregular, convex and rough in surface.
The invention provides a product, which contains the lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207.
In one embodiment of the invention, the product is a food, pharmaceutical or health product.
In one embodiment of the invention, the content of Lactobacillus northbound in the product is at least 5×10 9 CFU/mL or 5X 10 9 CFU/g。
In one embodiment of the invention, the food is a health food; or the food is dairy product, bean product or fruit and vegetable product produced by using starter containing Lactobacillus North (Lactobacillus kitasatonis) CCFM 1207; or the food is a beverage or snack containing lactobacillus northbound (Lactobacillus kitasatonis) CCFM 1207.
In one embodiment of the invention, the preparation method of the starter comprises inoculating the Lactobacillus northcus (Lactobacillus kitasatonis) CCFM1207 into a culture medium according to the inoculum size accounting for 2-4% (v/v) of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing thalli with normal saline for 3 times, and then re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension by vacuum freezing to obtain the starter.
In one embodiment of the invention, the mass ratio of the lyoprotectant to the thallus is 2:1.
In one embodiment of the invention, the lyoprotectant comprises 130g/L skimmed milk powder.
In one embodiment of the invention, the medium comprises 87.7% water by weight of the total medium, 10% skim milk by weight of the total medium, 0.5% glucose by weight of the total medium, 1.5% tryptone by weight of the total medium, and 0.3% yeast extract by weight of the total medium.
In one embodiment of the invention, the pH of the medium is 6.8.
In one embodiment of the invention, the pharmaceutical product contains lactobacillus northbound CCFM1207, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and/or additives.
In one embodiment of the invention, the excipient comprises a binder, filler, disintegrant, and/or lubricant.
In one embodiment of the invention, the additive comprises a solubilizer, a co-solvent and/or a preservative.
In one embodiment of the invention, the medicament is in the form of powder, granule, capsule, tablet, pill or oral liquid.
The invention also provides a microbial preparation containing the lactobacillus northbound (Lactobacillus kitasatonis) CCFM 1207.
In one embodiment of the present invention, the content of Lactobacillus northbound in the microbial preparation is at least 5×10 9 CFU/mL or 5X 10 9 CFU/g。
In one embodiment of the invention, the microbial agent is in a solid, liquid or powder form.
The invention also provides an application of the Lactobacillus northlewensis (Lactobacillus kitasatonis) CCFM1207 in preparing products for preventing and/or treating helicobacter pylori infection, wherein the application is not aimed at diagnosing and treating diseases.
In one embodiment of the invention, the product is a food, pharmaceutical or health product.
In one embodiment of the invention, theThe content of Lactobacillus North is at least 5×10 9 CFU/mL or 5X 10 9 CFU/g。
In one embodiment of the invention, the food is a health food; or the food is dairy product, bean product or fruit and vegetable product produced by using starter containing Lactobacillus North (Lactobacillus kitasatonis) CCFM 1207; or the food is a beverage or snack containing lactobacillus northbound (Lactobacillus kitasatonis) CCFM 1207.
In one embodiment of the invention, the preparation method of the starter comprises inoculating the Lactobacillus northcus (Lactobacillus kitasatonis) CCFM1207 into a culture medium according to the inoculum size accounting for 2-4% (v/v) of the total mass of the culture medium, and culturing for 18h at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing thalli with normal saline for 3 times, and then re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension by vacuum freezing to obtain the starter.
In one embodiment of the invention, the mass ratio of the lyoprotectant to the thallus is 2:1.
In one embodiment of the invention, the lyoprotectant comprises 130g/L skimmed milk powder.
In one embodiment of the invention, the medium comprises 87.7% water by weight of the total medium, 10% skim milk by weight of the total medium, 0.5% glucose by weight of the total medium, 1.5% tryptone by weight of the total medium, and 0.3% yeast extract by weight of the total medium.
In one embodiment of the invention, the pH of the medium is 6.8.
In one embodiment of the invention, the pharmaceutical product contains lactobacillus northbound CCFM1207, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise excipients and/or additives.
In one embodiment of the invention, the excipient comprises a binder, filler, disintegrant, and/or lubricant.
In one embodiment of the invention, the additive comprises a solubilizer, a co-solvent and/or a preservative.
In one embodiment of the invention, the medicament is in the form of powder, granule, capsule, tablet, pill or oral liquid.
The invention also provides application of the Lactobacillus northleonia (Lactobacillus kitasatonis) CCFM1207 in the aspect of copolymerizing helicobacter pylori without aiming at diagnosis and treatment of diseases.
The invention also provides an inhibitor for reducing helicobacter pylori load, which comprises the Lactobacillus northlewkii (Lactobacillus kitasatonis) CCFM1207.
Advantageous effects
1. The invention provides a Lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207, wherein the Lactobacillus northbound CCFM1207 can specifically bind to the existing helicobacter pylori in the stomach and prevent the helicobacter pylori from growing continuously to form a pathogenic bacteria biomembrane which colonizes the stomach. The concrete steps are as follows:
the Lactobacillus North CCFM1207 has the capability of copolymerizing and collecting pathogenic bacteria helicobacter pylori, and the copolymerization rate can reach 58.40% in 120 minutes, so that macroscopic flocculent copolymer is formed.
Therefore, the Lactobacillus northleonia (Lactobacillus kitasatonis) CCFM1207 has great application prospect in reducing the helicobacter pylori load (not aiming at diagnosis and treatment of diseases) of helicobacter pylori positive patients and preparing helicobacter pylori specific binding antagonists.
2. The copolymerization collection test is carried out by simulating human gastric juice environment in vitro, and the Lactobacillus northleyi (Lactobacillus kitasatonis) CCFM1207 has stronger acid resistance, can effectively gather helicobacter pylori, achieves higher copolymerization rate in a short time and forms flocculent interpolymer.
Meanwhile, the stability of the co-aggregation is tested by different incubation rotating speed tests, and the tests prove that the lactobacillus northbound CCFM1207 can play a better role in copolymerizing the pylorus at the incubation rotating speed of 0-250r/min, which indicates that the co-aggregation can overcome the shearing force formed by people in physiological processes such as feeding, digestion and the like.
Therefore, the lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207 has great application prospect in preparing products (such as foods or medicines) for preventing and/or treating helicobacter pylori infection.
3. The lactobacillus northbound (Lactobacillus kitasatonis) is separated from lactobacillus acidophilus in 2003, and the lactobacillus acidophilus is incorporated into a list of strains for food issued by sanitation at present, so that the product with the lactobacillus northbound CCFM1207 as an active ingredient does not cause drug resistance of helicobacter pylori, and meanwhile, adverse reaction of patients is not caused in the treatment process.
Preservation of biological materials
Lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207, taxonomic designation Lactobacillus kitasatonis, was deposited at the Cantonese microorganism strain collection at 10 and 25 of 2021 under the accession number GDMCC No:62013, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
Drawings
Fig. 1: growth curve of lactobacillus northbound.
Fig. 2: screening of Lactobacillus co-aggregated with helicobacter pylori.
Fig. 3: influence of ultrasound on the copolymerization of Lactobacillus and helicobacter pylori.
Fig. 4: influence of incubation speed on the copolymerization of Lactobacillus and helicobacter pylori.
Fig. 5: macroscopic view of Lactobacillus North and helicobacter pylori interpolymer.
Fig. 6: scanning electron microscope pictures of the lactobacillus northbound and helicobacter pylori interpolymers; wherein, figures (a) and (b) represent Lactobacillus northsonii CCFM1207 and helicobacter pylori aggregates, respectively, at different shooting times (5000X, 12000X) of the microscopic electron microscope.
Detailed Description
The helicobacter pylori referred in the following examples is helicobacter pylori SS1 from the NTCC national collection of typical cultures; other lactobacillus species involved in the following examples are all derived from the university of south of the Yangtze river biotechnology research center; the NaCl referred to in the examples below was purchased from the national drug group; the Columbia medium referred to in the examples below was purchased from OXOID, UK; the sterile defibrinated sheep blood referred to in the examples below was purchased from Hangzhou New Sharp company; BHI broth as referred to in the examples below was purchased from Qingdao sea blog company.
Lactobacillus rhamnosus SLT6 referred to in the examples below originates from the university of jiang biotechnology research center.
The following examples refer to the respective Lactobacillus:
lactobacillus reuteri 17648 is disclosed in patent WO2007073709 A1;
lactobacillus casei GL1-GL13, lactobacillus reuteri LYS1, LYS3, LYS4, LYS5, lactobacillus rhamnosus SLT3, SLT4, SLT5, SLT6, lactobacillus acidophilus S1, S2, S3, S6, S8 and Lactobacillus fermentum FJ1-FJ12 are all derived from the university of Jiangnan Biotechnology research center.
The media and reagents involved in the following examples were as follows:
MRS solid Medium (g/L): 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 80 1mL/L, agar 20g/L, cysteine amino acid salt 0.5g/L.
MRS liquid Medium (g/L): 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 80 1mL/L, cysteine amino acid salt 0.5g/L.
Columbia blood plates: 39g of solid powder of Columba culture medium is dissolved in 1L of water, sterilized for 15min at 121 ℃, cooled to 55-60 ℃, added with 7.5% (v/v) of sterile defibrinated sheep blood, and poured into a plate after being uniformly mixed.
BHI liquid Medium (g/L): 10.0g/L tryptone, 17.5g/L beef heart infusion powder, 5.0g/L sodium chloride, 2.0g/L glucose, disodium hydrogen phosphate (12H) 2 O) 2.5g/L, pH value 7.4.+ -. 0.2.
Artificial gastric juice (g/L): pepsin (pig pepsin) 3g/L, sodium chloride 5g/L, and adjusting pH to 4.0+ -0.2.
The detection method involved in the following examples is as follows:
the method for detecting the number of living bacteria comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus detection is adopted.
The method for detecting and calculating the copolymerization rate comprises the following steps:
OD values before/after the reaction (0 min/T min) of lactobacillus and mixed bacterial liquid are respectively measured, and the copolymerization rate is calculated according to the following formula: copolymerization ratio (%) = [ (OD) 600 breast pole +OD 600 pylorus )-2×OD 600 mixing t min ]/(OD 600 breast pole +OD 600 pylorus ) X 100. (specific examples of the copolymerization ratio measurement method are described in Chen X, tian F, liu X, et al In vitro screening of lactobacilli with antagonistic activity against Helicobacter pylori from traditionally fermented foods [ J ]].Journal of Dairy Science,2010,93(12):5627-5634)。
The preparation method of helicobacter pylori cells in the following examples is as follows:
helicobacter pylori was streaked onto Columbia platelets in a three-gas incubator at 37℃C (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid; inoculating the seed solution into BHI culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid; centrifuging 8000g of helicobacter pylori bacterial liquid for 10min to obtain helicobacter pylori bacterial;
wherein, columbia blood plate: 39g of solid powder of Columba culture medium is dissolved in 1L of water, sterilized for 15min at 121 ℃, cooled to 55-60 ℃, added with 7.5% (v/v) of sterile defibrinated sheep blood, and poured into a plate after being uniformly mixed.
The preparation method of lactobacillus rhamnosus thallus involved in the following examples is as follows:
streaking lactobacillus rhamnosus on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial liquid for 10min by 8000g to obtain lactobacillus rhamnosus bacterial cells.
The preparation method of Lactobacillus North cell in the following examples is as follows:
streaking the lactobacillus northbound on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial liquid for 10min by 8000g to obtain the lactobacillus North bacterial cells.
Example 1: screening and identification of Lactobacillus North
1. Screening
Fresh feces from healthy people are taken as samples, the samples are pretreated and stored in a refrigerator at the temperature of minus 80 ℃ in 30% glycerol, after being taken out and thawed, the uniformly mixed samples are absorbed with 0.5mL of samples and added into 4.5mL of 0.9% physiological saline for gradient dilution, proper gradient diluent is selected to be coated on MRS solid culture medium, the culture is carried out for 48 hours at the temperature of 37 ℃, typical bacterial colonies are picked up and streaked on an MRS flat plate for purification, single bacterial colonies are picked up and transferred to liquid MRS liquid culture medium for enrichment, and 30% glycerol is stored, thus obtaining bacterial strain CCFM1207.
2. Authentication
Extracting genome of the strain CCFM1207, amplifying and sequencing 16S rDNA of the strain CCFM1207 (completed by Shanghai Meiji biological medicine technology Co., ltd.), sequencing and analyzing, wherein the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, comparing the sequence in GenBank, and has homology with lactobacillus genus: 98.95%; the results showed that the strain was Lactobacillus North, designated Lactobacillus North (Lactobacillus kitasatonis) CCFM1207.
Example 2: culture of Lactobacillus North CCFM1207 thallus
The lactobacillus northbound (Lactobacillus kitasatonis) CCFM1207 prepared in example 1 is inoculated into MRS solid culture medium, and after culturing for 48 hours at 37 ℃, bacterial colonies are observed and are observed under a microscope, and the bacterial colonies are found to be white and micro-transparent, round or slightly irregular, convex and rough in surface.
Inoculating Lactobacillus northlesii (Lactobacillus kitasatonis) CCFM1207 into MRS liquid culture medium, culturing at 10-50deg.C for 48 hr, and measuring OD of the culture solution by enzyme-labeling instrument at intervals 600 The Lactobacillus northbound CCFM1207 was found to grow optimally at 30-37℃and cultured for 18-24 hours to reach the stationary phase of growth, and the results are shown in FIG. 1.
Example 3: action of the pathogenic bacteria helicobacter pylori in the co-aggregation of Lactobacillus North
The method comprises the following specific steps:
(1) Culturing helicobacter pylori:
helicobacter pylori was streaked onto Columbia platelets in a three-gas incubator at 37℃C (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid;
inoculating the seed solution into BHI liquid culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid;
centrifuging 8000g of helicobacter pylori bacterial solution for 10min to obtain helicobacter pylori bacterial body, and regulating bacterial solution concentration to OD with artificial gastric juice (pH=4) 600 =0.5, a helicobacter pylori suspension was obtained.
(2) Culture of Lactobacillus North CCFM1207 and Lactobacillus rhamnosus SLT6
Respectively streaking Lactobacillus North CCFM1207 and Lactobacillus rhamnosus SLT6 on MRS solid culture medium, and culturing at 37deg.C for 48 hr to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution;
inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial solution at 8000g for 10min to obtain Lactobacillus North and Lactobacillus rhamnosus thallus, respectively, and adjusting with PBS buffer solution (pH=7.42) to obtain bacterial solutions with OD concentration 600 Lactobacillus northbound CCFM1207 bacterial suspension and lactobacillus rhamnosus SLT6 bacterial suspension=0.5.
(3) And (3) fully oscillating and mixing the helicobacter pylori suspension (2 mL) prepared in the step (1) with 2mL of Lactobacillus northsonii CCFM1207 suspension and 2mL of Lactobacillus rhamnosus SLT6 suspension (equal volume ratio) respectively to prepare a mixed solution of Lactobacillus northsonii and helicobacter pylori, and the mixed solution of Lactobacillus rhamnosus SLT6 and helicobacter pylori.
(4) And (3) respectively standing and incubating the mixed solution prepared in the step (3) for 0min,4min,10min,30min,60min,90min,120min,150min and 180min at the room temperature of 26 ℃.
OD values before/after the reaction (0 min/T min) of the lactobacillus and the mixed bacterial liquid were measured, respectively, and copolymerization rates were calculated. The results are shown in table 1 and fig. 2:
table 1: copolymerization ratio of Lactobacillus and helicobacter pylori
Further investigation of the time factor of this copolymerization occurrence revealed that rhamnose SLT6 did not undergo strong copolymerization against H.pylori for 0-180 min, whereas the copolymerization rate of Lactobacillus northleyi CCFM1207 with H.pylori was up to 40% or more in 10min, and increased with time. It was thus concluded that Lactobacillus northbound CCFM1207 has the ability to adhere and accumulate helicobacter pylori in a short period of time.
(5) Co-aggregation experiment of external artificial simulated gastric juice environment of different strains
Streaking the lactobacillus to be tested on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial solution at 8000g for 10min to obtain lactobacillus thallus, and adjusting bacterial solution concentration to OD with PBS (pH=7.42) 600 ＝0.5。
The 39 strains of lactobacillus are respectively prepared into bacterial suspension according to the method.
And (3) mixing the helicobacter pylori suspension prepared in the step (1) with 2mL of each of 39 strains of lactobacillus suspension, and sufficiently shaking for 10s to uniformly mix. And standing and incubating for 120min at the room temperature of 26 ℃. OD values after the reaction (T min) of the mixed bacteria liquid of Lactobacillus and helicobacter pylori were measured, respectively, and copolymerization rates were calculated, and the results are shown in Table 2 and FIG. 2.
Table 2: copolymerization ratio of different Lactobacillus and helicobacter pylori after the mixed bacterial liquid is reacted
According to FIG. 2, lactobacillus northgicus CCFM1207 with better effect is obtained through screening experiments of in vitro artificial simulated gastric juice environment co-aggregation of cheese, racemosa, acidophilic lactobacillus fermentum and helicobacter pylori, and the copolymerization rate can reach more than 42%.
Example 4: effect of ultrasound on copolymerization of Lactobacillus North and helicobacter pylori
The method comprises the following specific steps:
(1) Culturing helicobacter pylori:
helicobacter pylori was streaked onto Columbia platelets in a three-gas incubator at 37℃C (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid;
inoculating the seed solution into BHI culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid; centrifuging 8000g of helicobacter pylori bacterial solution for 10min to obtain helicobacter pylori bacterial body, and regulating bacterial solution concentration to OD with artificial gastric juice (pH=4) 600 For use, =0.5, helicobacter pylori suspension was obtained.
(2) Culture of Lactobacillus North CCFM1207 and Lactobacillus rhamnosus SLT6
Respectively streaking Lactobacillus North CCFM1207 and Lactobacillus rhamnosus SLT6 on MRS solid culture medium, and culturing at 37deg.C for 48 hr to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution;
inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging the bacterial solution at 8000g for 10min to obtain Lactobacillus North and Lactobacillus rhamnosus thallus, respectively, and adjusting with PBS buffer solution (pH=7.42) to obtain bacterial solutions with OD concentration 600 Lactobacillus northbound CCFM1207 bacterial suspension and lactobacillus rhamnosus SLT6 bacterial suspension=0.5.
(3) Ultrasonic group: performing ultrasonic treatment on the lactobacillus northbound CCFM1207 bacterial suspension and the lactobacillus rhamnosus SLT6 bacterial suspension prepared in the step (2) for 12min at an ultrasonic frequency of more than 2000Hz, respectively taking 2mL of bacterial suspension after the ultrasonic treatment is finished, respectively mixing the bacterial suspension with the helicobacter pylori bacterial suspension (2 mL) prepared in the step (1) in an equal volume ratio, sufficiently vibrating, and standing and incubating for 120min at the room temperature of 26 ℃; respectively preparing mixed liquor 1 of lactobacillus northbound and helicobacter pylori, and mixed liquor 2 of lactobacillus rhamnosus SLT6 and helicobacter pylori;
Ultrasound-free group: respectively mixing 2mL of the Lactobacillus northleonia CCFM1207 bacterial suspension and 2mL of the Lactobacillus rhamnosus SLT6 bacterial suspension prepared in the step (2) with the helicobacter pylori bacterial suspension (2 mL) prepared in the step (1) in an equal volume ratio, sufficiently shaking, standing and incubating for 120min at the room temperature of 26 ℃; respectively preparing mixed liquor 3 of lactobacillus northbound and helicobacter pylori, and mixed liquor 4 of lactobacillus rhamnosus SLT6 and helicobacter pylori;
OD values before/after the reaction (0 min/T min) of lactobacillus and mixed bacterial liquid are respectively measured, and the copolymerization rate is calculated according to the following formula: copolymerization ratio (%) = [ (OD) 600 breast pole +OD 600 pylorus )-2×OD 600 mixing t min ]/(OD 600 breast pole +OD 600 pylorus ) X 100. The results are shown in Table 3 and FIG. 3.
Table 3: effect of ultrasound on the copolymerization of Lactobacillus and helicobacter pylori
The results show that the co-polymerization of the sonicated Lactobacillus is impaired, but the changes are insignificant (p > 0.05) and do not completely disappear (or appear), so that the co-polymerization of Lactobacillus with helicobacter pylori is an ultrasound insensitive interaction with no significant correlation to the integrity of the Lactobacillus cells.
Example 5: influence of incubation rotation speed on copolymerization of Lactobacillus North and helicobacter pylori
The method comprises the following specific steps:
(1) Culturing helicobacter pylori:
helicobacter pylori was found on a columbia plate (columbia plate: 39g of solid powder of Columba culture medium is dissolved in 1L of water, sterilized for 15min at 121 ℃, cooled to 55-60 ℃, added with 7.5% (v/v) of aseptic defibrinated sheep blood, evenly mixed and poured into a plate) and streaked, and then the mixture is subjected to 37 ℃ sterilizationIn a three-gas incubator (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid;
inoculating the seed solution into BHI culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid; centrifuging 8000g of helicobacter pylori bacterial solution for 10min to obtain helicobacter pylori bacterial body, and regulating bacterial solution concentration to OD with artificial gastric juice (pH=4) 600 =0.5, a helicobacter pylori suspension was obtained.
(2) Culture of Lactobacillus North CCFM1207 and Lactobacillus rhamnosus SLT6
Respectively streaking lactobacillus northbound and lactobacillus rhamnosus on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; and centrifuging 8000g of bacterial liquid for 10min to obtain lactobacillus northbound and lactobacillus rhamnosus thalli, and regulating the concentration of the bacterial solution to be OD600 < = 0.5 by using PBS (pH=7.42), so as to prepare bacterial suspensions respectively.
(3) And (3) respectively taking the helicobacter pylori bacterial suspension prepared in the step (1) and 2mL of each of the helicobacter pylori bacterial suspension prepared in the step (2), and sufficiently vibrating and mixing.
The incubation is carried out at room temperature of 26 ℃ for 120min under the conditions of standing still for 0r/min, 150r/min and 250r/min respectively. OD values before/after the reaction (0 min/T min) of Lactobacillus and the mixed bacterial solution were measured, and copolymerization rates were calculated, and the results are shown in Table 4 and FIG. 4.
Table 4: effect of incubation Rate on the Comerisation
(Note: helicobacter pylori solution OD before mixing) 600 ＝0.543)
Different incubation rotational speed tests test the influence of different shear forces on the stability of coagglomeration, and FIG. 4 shows that the copolymerization rate of Lactobacillus northbound CCFM1207 on helicobacter pylori is always kept above 40%, the copolymerization rate is highest under the condition of 150r/min rotational speed, but has no significant difference (p > 0.05) with standing, and the copolymerization rate is lowest under the condition of 250r/min rotational speed. The copolymerization capability of lactobacillus and pylorus does not show sensitivity to the rotating speed of 0-250 r/min, and the peristaltic motion of stomach can form shearing force and rotating speed with a certain size in the physiological processes of feeding, digestion and the like, and the experiment proves that the co-agglutination of the lactobacillus northleum CCFM1207 and the pylorus helicobacter has stability, and is suitable for the development of oral probiotics and the like.
Example 6: effect of temperature on copolymerization of Lactobacillus North and helicobacter pylori
The method comprises the following specific steps:
(1) Culturing helicobacter pylori:
helicobacter pylori was streaked onto Columbia platelets in a three-gas incubator at 37℃C (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid;
inoculating the seed solution into BHI liquid culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid;
centrifuging 8000g of helicobacter pylori bacterial solution for 10min to obtain helicobacter pylori bacterial body, and regulating bacterial solution concentration to OD with artificial gastric juice (pH=4) 600 =0.5, a helicobacter pylori suspension was obtained.
(2) Culture of Lactobacillus North CCFM1207
Streaking the Lactobacillus northbound CCFM1207 on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution;
inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial solution for 10min to obtain Lactobacillus North bacterial cells, and regulating with PBS buffer solution (pH=7.42) to obtain bacterial solutions with OD concentration 600 Lactobacillus northbound CCFM1207 bacterial suspension=0.5.
(3) And (3) respectively taking the helicobacter pylori bacterial suspension prepared in the step (1) and 2mL of each of the helicobacter pylori bacterial suspension prepared in the step (2), and sufficiently vibrating and mixing. Incubating at 4deg.C, 37deg.C and 80deg.C for 120min, respectively. (the strain was pasteurized at 80 to 85℃and treated for 10 to 15 minutes to inactivate), the OD values before/after the reaction (0 min/T min) of the Lactobacillus and the mixed liquor were measured, respectively, and the copolymerization ratio was calculated, and the results are shown in Table 5.
Table 5: influence of temperature on the copolymerization
As can be seen from the above table, the effect of temperature on the stability of the coagglutination is not remarkable, and the lactobacillus northleonia CCFM1207 is completely inactivated at 80 ℃ for 120min, but the copolymerization rate of the lactobacillus northleonia CCFM1207 on helicobacter pylori is not reduced, so that the experiment proves that the coagglutination of the lactobacillus northleonia CCFM1207 and the helicobacter pylori is independent of the activity of the lactobacillus and is completely the physical effect among strains, and the discovery helps us to greatly reduce the production cost when developing probiotics.
Example 7: electron microscope image of co-aggregation of lactobacillus northbound and helicobacter pylori
The method comprises the following specific steps:
(1) Culturing helicobacter pylori:
Helicobacter pylori was streaked onto Columbia platelets in a three-gas incubator at 37℃C (85% N) 2 、10％CO 2 、5％O 2 ) Culturing for 3 days to obtain single colony; single colonies were picked and inoculated into BHI medium containing 5% (v/v) fetal bovine serum in a three-air incubator (85% N) at 37 ℃ 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain seed liquid;
inoculating the seed solution into BHI liquid culture medium at an inoculum size of 2% (v/v), and culturing in a three-gas incubator (85% N) at 37deg.C 2 、10％CO 2 、5％O 2 ) Culturing for 4 days to obtain helicobacter pylori bacterial liquid;
the helicobacter pylori bacterial solution is centrifuged for 10min at 8000g to obtain helicobacter pylori bacterial body, and the concentration of the bacterial solution is regulated to be OD600 = 0.5 by using artificial gastric juice (pH = 4), so as to obtain helicobacter pylori bacterial suspension.
(2) Culture of Lactobacillus North CCFM1207
Streaking the Lactobacillus northbound CCFM1207 on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution;
inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; the bacterial solution is centrifuged for 10min by 8000g to prepare lactobacillus northbound bacterial cells, and the lactobacillus northbound bacterial cells are regulated by PBS buffer solution (pH=7.42) to prepare lactobacillus northbound CCFM1207 bacterial suspension with bacterial solution concentration of OD 600=0.5.
(3) And (3) respectively taking the helicobacter pylori bacterial suspension prepared in the step (1) and 2mL of each of the helicobacter pylori bacterial suspension prepared in the step (2), and sufficiently vibrating and mixing. After standing at 26℃for 120min, the mixture of Lactobacillus northbound CCFM1207 and helicobacter pylori was stratified as shown in FIG. 5. Centrifuging the mixed solution to obtain an aggregate, placing the aggregate in 2.5% glutaraldehyde solution for fixation at 4 ℃ overnight, collecting the fixed aggregate, dehydrating the aggregate by a gradient of 70% -100% ethanol, and freeze-drying for 48 hours to obtain an aggregate sample to be photographed. After palladium sputtering and metal spraying treatment, shooting is carried out by using a cold field emission scanning electron microscope, and fig. 6 is obtained.
FIG. 5 shows macroscopic manifestations of Lactobacillus plantarum CCFM1207 incubated with H.pylori for 120min, in which the co-polymer formed therein was precipitated in the mixture and the absorbance of the upper solution was decreased, whereas the co-polymerized Lactobacillus rhamnosus SLT6 did not appear. FIGS. 6 (a) and (b) respectively show the aggregation of Lactobacillus northleonia CCFM1207 and helicobacter pylori under different shooting times (5000X, 12000X) of a microscopic electron microscope, wherein the Lactobacillus has a more regular rod-like form, the helicobacter pylori has an irregular curved shape, and a scanning electron microscope shows that the two bacteria aggregate and accumulate with each other to form a combined copolymer block.
Example 8: application of lactobacillus northbound
The lactobacillus northbound CCFM1207 can be used for preparing bacterial powder, and the specific preparation process of the bacterial powder is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Example 9: application of lactobacillus northbound
The Lactobacillus North CCFM1207 can be used for preparing capsule products, and the specific preparation process of the capsule products is as follows
The Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; picking single coloniesInoculating in MRS liquid culture medium, culturing at 37deg.C for 18 hr for activation, and activating for two generations to obtain activating solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; the bacterial suspension is added into sodium alginate solution with the concentration of 30g/L to the concentration of 2 multiplied by 10 9 After CFU/mL, stirring fully to uniformly disperse cells of lactobacillus northbound CCFM1207 in the sodium alginate solution to obtain a mixed solution; extruding the mixed solution into a calcium chloride solution with the concentration of 20g/L to form colloidal particles; after the formed colloidal particles are stationary and solidified for 30min, filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; filling the powder into a medicinal capsule to obtain a capsule product;
the preparation method of the culture medium comprises the following steps: the medium was obtained by dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the medium, and then adjusting the pH to 6.8.
Example 10: application of lactobacillus northbound
The lactobacillus northbound CCFM1207 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Weighing 25.7 parts by weight of lactobacillus northbound CCFM1207 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of carboxymethyl starch sodium, 0.8 part by weight of talcum powder, 1.0 part by weight of sucrose and 1.0 part by weight of water to obtain raw materials; mixing the raw materials to obtain wet particles; tabletting the wet granules by a tablet press of a pharmaceutical machinery factory in the middle south, and drying by a small-sized drug dryer of Yikang traditional Chinese medicine machinery Co., ltd.
Example 11: application of lactobacillus northbound
The lactobacillus northbound CCFM1207 can be used for preparing fermented milk, and the specific preparation process of the fermented milk is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Mixing lactobacillus northbound CCFM1207 bacterial powder with commercial dry powder starter lactobacillus bulgaricus and commercial dry powder starter streptococcus thermophilus according to the mass ratio of 1:1:1 to obtain starter; adding sugar into fresh milk until the concentration is 50g/L to obtain a mixed solution; homogenizing the mixed solution at 65deg.C and 20MPa, and sterilizing at 95deg.C for 5min to obtain fermentation raw material; cooling the fermentation raw material to 35 ℃, inoculating a starter into the fermentation raw material in an inoculum size of 0.03% (v/v), and fermenting at 35 ℃ for 16 hours to obtain fermented milk; and (3) placing the fermented milk at 42 ℃ for 4 hours for curd, and then refrigerating at 4 ℃ for 24 hours for after-ripening to obtain a fermented milk finished product.
Example 12: application of lactobacillus northbound
The Lactobacillus North CCFM1207 can be used for preparing soymilk, and the specific preparation process of the soymilk is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Soaking soybean at 80deg.C for 2 hr, removing soybean hull to obtain peeled soybean; removing the soaking water from peeled soybean, adding boiling water, and pulping to obtain soybean milk; maintaining the temperature of the soybean milk at a temperature higher than 80 ℃ for 12min to obtain cooked soybean milk; filtering cooked soybean milk with 150 mesh sieve, and centrifuging to obtain coarse soybean milk; heating the crude soymilk to 140-150 ℃, then rapidly introducing the heated crude soymilk into a vacuum cooling chamber for vacuumizing, so that the peculiar smell substances in the crude soymilk are rapidly discharged along with water vapor to obtain cooked soymilk; cooling cooked soybean milk to 37deg.C Adding Lactobacillus North CCFM1207 powder into cooked soybean milk until the concentration is not lower than 1×10 6 CFU/mL to give soymilk (soymilk is stored cold at 4deg.C).
Example 13: application of lactobacillus northbound
The lactobacillus northbound CCFM1207 can be used for preparing fruit and vegetable beverages, and the specific preparation process of the fruit and vegetable beverages is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
The components of the protective agent comprise: 130g/L skimmed milk powder.
Cleaning fresh fruits and vegetables, and squeezing to obtain fruit and vegetable juice; sterilizing the fruit and vegetable juice at 140 ℃ for 2 seconds to obtain sterilized fruit and vegetable juice; cooling the sterilized fruit and vegetable juice to about 37deg.C, adding Lactobacillus northleonia CCFM1207 powder to the sterilized fruit and vegetable juice to a concentration of not less than 1×10 6 CFU/mL to obtain fruit and vegetable beverage (fruit and vegetable beverage is required to be refrigerated and stored at 4 ℃).
Example 14: application of lactobacillus northbound
The lactobacillus northbound CCFM1207 can be used for preparing milk drinks, and the specific preparation process of the milk drinks is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; picking single colony inoculationCulturing in MRS liquid culture medium at 37deg.C for 18 hr for activation, and continuously activating for two generations to obtain activating solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
The preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Sterilizing the skimmed milk at 95deg.C for 20min, and cooling to 4deg.C to obtain raw materials; adding Lactobacillus northwest CCFM1207 powder to the raw materials until the concentration is not lower than 1×10 6 CFU/mL, the milk beverage (the milk beverage is required to be refrigerated and stored at 4 ℃).
Example 15: application of lactobacillus northbound
Lactobacillus northbound CCFM1207 can be used for preparing chocolate, and the specific preparation process of the chocolate is as follows:
the Lactobacillus North CCFM1207 is streaked on MRS solid culture medium, and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into a culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain lactobacillus northbound CCFM1207 bacterial powder;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 130g/L skimmed milk powder.
Mixing cocoa mass and white granulated sugar in a mass ratio of 1:1-1:3, heating, and uniformly stirring to obtain chocolate melt; firstly, emulsifying agent (liquid lecithin, soybean lecithin, sorbitan monolaurate) and lactobacillus northbound CCFM1207 bacterial powder by using emulsifying agent: bacterial powder = 80-90: and (3) uniformly mixing the materials according to the mass ratio of 10-20, then carrying out fine grinding, acid removal, water removal, crystallization and temperature adjustment, and finally, selecting a proper model for casting molding to obtain the chocolate (the chocolate is required to be refrigerated and stored at the temperature of 4 ℃).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> Lactobacillus North having capability of copolymerizing helicobacter pylori and application thereof
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Claims (10)
1. Lactobacillus NorthLactobacillus kitasatonis) The method is characterized in that the lactobacillus northcus is preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCC No:62013, the preservation date is 2021, 10 and 25.
2. A product comprising lactobacillus northbound as claimed in claim 1.
3. The product of claim 2, wherein the product is a food or pharmaceutical product.
4. A product according to claim 2 or 3, wherein the lactobacillus northbound is present in the product in an amount of at least 5 x 10 9 CFU/mL or 5X 10 9 CFU/g。
5. A product according to claim 3, wherein the food product is a dairy product, a soy product or a fruit and vegetable product produced using a starter culture comprising lactobacillus northjensis according to claim 1; or the food is a beverage or snack containing lactobacillus northbound according to claim 1.
6. A microbial preparation comprising lactobacillus northbound according to claim 1.
7. The microbial preparation according to claim 6, wherein the content of Lactobacillus northsonii in the microbial preparation is at least 5X 10 9 CFU/mL or 5X 10 9 CFU/g。
8. Use of lactobacillus northbound as claimed in claim 1 in the manufacture of a product for the prophylaxis and/or treatment of helicobacter pylori infection.
9. The use according to claim 8, wherein the product is a food or pharmaceutical product.
10. The use according to claim 9, wherein the content of lactobacillus northbound in the product is at least 5 x 10 9 CFU/mL or 5X 10 9 CFU/g。
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| CN104388344A (en) * | 2014-11-05 | 2015-03-04 | 中国农业科学院哈尔滨兽医研究所 | New strain of lactobacillus kitasatonis and application thereof |
| CN111471626A (en) * | 2020-05-29 | 2020-07-31 | 江南大学 | Lactobacillus helveticus capable of inhibiting helicobacter pylori and application thereof |
| CN111548970A (en) * | 2020-05-29 | 2020-08-18 | 江南大学 | Lactobacillus crispatus capable of preventing and/or treating helicobacter pylori infection |
| CN111607538A (en) * | 2020-05-29 | 2020-09-01 | 江南大学 | Lactobacillus rhamnosus and application thereof in inhibiting helicobacter pylori |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388344A (en) * | 2014-11-05 | 2015-03-04 | 中国农业科学院哈尔滨兽医研究所 | New strain of lactobacillus kitasatonis and application thereof |
| CN111471626A (en) * | 2020-05-29 | 2020-07-31 | 江南大学 | Lactobacillus helveticus capable of inhibiting helicobacter pylori and application thereof |
| CN111548970A (en) * | 2020-05-29 | 2020-08-18 | 江南大学 | Lactobacillus crispatus capable of preventing and/or treating helicobacter pylori infection |
| CN111607538A (en) * | 2020-05-29 | 2020-09-01 | 江南大学 | Lactobacillus rhamnosus and application thereof in inhibiting helicobacter pylori |
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| 张旺 ; 刘琳琳 ; 王云峰 ; 王宏磊 ; 祁小乐 ; 高玉龙 ; 王永强 ; 高宏雷 ; 李凯 ; 刘长军 ; 张艳萍 ; 卢彤岩 ; 何高明 ; 王笑梅 ; 崔红玉 ; .北里乳杆菌97-1产细菌素97-1的抑菌活性及生物特性的研究.中国预防兽医学报.2015,(12),第42-45页. * |
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