CN116763829A - Lactobacillus plantarum LZ010 metazoan composition, preparation method thereof and application thereof in inhibiting helicobacter pylori - Google Patents
Lactobacillus plantarum LZ010 metazoan composition, preparation method thereof and application thereof in inhibiting helicobacter pylori Download PDFInfo
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- CN116763829A CN116763829A CN202311057492.3A CN202311057492A CN116763829A CN 116763829 A CN116763829 A CN 116763829A CN 202311057492 A CN202311057492 A CN 202311057492A CN 116763829 A CN116763829 A CN 116763829A
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- lactobacillus plantarum
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a lactobacillus plantarum LZ010 metazoan composition, a preparation method thereof and application thereof in inhibiting helicobacter pylori, belonging to the technical field of microecological bactericides. Comprises the following components in parts by weight: 30-40 parts of metazoan, 10-25 parts of xylitol, 20-30 parts of fructo-oligosaccharide and 0.006-0.008 part of calcium carbonate; the metagen consists of thallus and metabolite of lactobacillus plantarum LZ 010; the lactobacillus plantarum (Lactobacillus plantarum) LZ010 has a preservation number of CGMCC No.24258 in the China general microbiological culture Collection center, and a preservation date of 2022, 01 and 06 days, and a preservation institution address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. The Lactobacillus plantarum LZ010 metazoan composition has obvious inhibiting effect on the growth of helicobacter pylori and has the capability of resisting helicobacter pylori.
Description
Technical Field
The invention belongs to the technical field of microecological bactericides, and in particular relates to a lactobacillus plantarum LZ010 metazoan composition, a preparation method thereof and application of the composition in inhibiting helicobacter pylori.
Background
Helicobacter pylori (Hp) was first discovered in 1983 by the australian pathologist Warren and the gastroenterologist Marshall. Through years of verification and research, the stomach is found to have helicobacter pylori, and erosive gastritis, peptic ulcer, gastrointestinal hemorrhage and even gastric cancer have close relation with the helicobacter pylori. Helicobacter pylori infection of the stomach, which is usually transmitted by saliva, most commonly by closely contacted people, by mouth water, especially without eating separately, by removing dishes from a dish, and by saliva. Helicobacter pylori is usually parasitic on the gastric mucosal surface of the human body, almost all the infected persons are accompanied with chronic gastritis of different degrees, most patients may have no symptoms, and some symptoms may be manifested by dyspepsia related symptoms such as epigastric discomfort, abdominal distension, hiccups and premature satiety, some patients may have halitosis, and helicobacter pylori infection mainly causes chronic gastritis, peptic ulcer, even gastric cancer and other diseases.
Helicobacter pylori infection is now mainly treated with anti-helicobacter pylori drugs. Although helicobacter pylori is sensitive to many antimicrobial drugs in vitro, it is not as desirable to administer it in vivo. Drug resistance problems arise due to the wide application of antibacterial regimens for the treatment of helicobacter pylori infection.
The concept of metaverses (postbiotics) was first proposed by spanish scientists Katerina Tsilingiri in 2013. In 2018, international food journal "Trends in Food Science & Technology" indicated that metaverses would lead the global health industry to enter the next-stage industrial innovation. The metazoan is the metabolite component of the probiotics after the probiotics are processed and is generally called as including thalli and metabolites. Japanese studies have demonstrated that metazoan have an enhanced immunity over the original viable bacteria, retain a high degree of physiological activity even through high temperature action or treatment with gastrointestinal digestive fluids, and have various health benefits to the human body.
Therefore, how to effectively utilize metaplasia to inhibit helicobacter pylori infection in daily life is a great problem nowadays.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a lactobacillus plantarum LZ010 metazoan composition, a preparation method thereof and application of inhibiting helicobacter pylori, so as to solve the technical problems. The Lactobacillus plantarum LZ010 metagen provided by the invention can effectively inhibit the growth of helicobacter pylori, reduce the number of helicobacter pylori, maintain the balance of bacterial groups in gastrointestinal tracts, reduce the risk of stomach illness, and has wide market prospect in the aspect of preparing related products for inhibiting helicobacter pylori.
The technical scheme of the invention is as follows:
in a first aspect, the invention provides a lactobacillus plantarum LZ010 metazoan composition comprising the following components in parts by weight: 30-40 parts of metazoan, 10-25 parts of xylitol, 20-30 parts of fructo-oligosaccharide and 0.006-0.008 part of calcium carbonate. The metagen consists of thallus and metabolite of lactobacillus plantarum LZ 010; the lactobacillus plantarum (Lactobacillus plantarum) LZ010 has a preservation number of CGMCC No.24258 in the China general microbiological culture Collection center, and a preservation date of 2022, 01 and 06 days, and a preservation institution address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Preferably, the composition comprises the following components in parts by weight: 35 parts of metazoan, 20 parts of xylitol, 25 parts of fructo-oligosaccharides and 0.007 part of calcium carbonate.
Preferably, the preparation method of the metagen comprises the following steps:
(1) Bacterial screening and preservation
A proper amount of lactobacillus plantarum LZ010 is selected, diluted and coated on a TPY solid flat-plate culture medium, cultured for 24-48 hours at the constant temperature of 37 ℃, typical single bacterial colony is selected and streaked on the solid flat-plate culture medium, then the single bacterial colony is extracted for liquid expansion culture, and 10 percent of glycerol is frozen for preservation.
(2) Seed culture
Inoculating the preserved Lactobacillus plantarum LZ010 into sterilized TPY liquid culture medium, and standing and culturing at 37deg.C for 12-24 hr.
(3) Fermentation culture
Inoculating the cultured seeds into a degreasing emulsion fermentation medium, culturing at 37 ℃ for 12-36h, and controlling the pH value to be 6.0-7.0 (ammonia water adjustment) in the culturing process to obtain high-density lactobacillus plantarum LZ010 fermentation liquor.
(4) Cell treatment
After fermentation, heating the fermentation liquor to 70 ℃, maintaining for 15min, cooling to 30 ℃, crushing by using a high-pressure homogenizer, circulating until the thalli are completely crushed, and concentrating by using a reverse osmosis membrane until the volume of the feed liquid is one third, thereby obtaining lactobacillus plantarum LZ010 metaplastic concentrate; and (5) spray-drying the concentrated solution to obtain lactobacillus plantarum LZ010 metazoan.
Preferably, the skim milk liquid fermentation medium in (3) is: 100g/L of skim milk powder, 10g/L of yeast peptone, 5g/L of yeast powder, 1.5g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of diammonium hydrogen citrate, 0.001g/L of nicotinamide, 0.002g/L of VB, 0.001g/L of VB and pH=7.0+/-0.1,0.22 mu m liquid filter filtration and sterilization.
In a second aspect, the invention provides a preparation method of a post-production meta-composition containing lactobacillus plantarum LZ010, which comprises the following steps:
(1) Mixed auxiliary materials
Xylitol, fructo-oligosaccharide and calcium carbonate are put into water to be fully dissolved; then adding lactobacillus plantarum LZ010 metaplasia and stirring uniformly.
(2) Drying
Sterilizing the feed liquid by a pasteurization method, and controlling the moisture of the powder by regulating and controlling the air inlet temperature; adjusting the frequency of the atomizer, controlling the granularity of the powder, and obtaining the Lactobacillus plantarum LZ010 metazoan composition through spray drying.
Preferably, the lactobacillus plantarum LZ010 metacomposition particle size (D90) < 100 mesh.
Preferably, the lactobacillus plantarum LZ010 metacomposition has a moisture of <5%.
In a third aspect, the invention provides the use of a post-metacomposition comprising Lactobacillus plantarum LZ010 for the preparation of a helicobacter pylori inhibiting product.
The beneficial effects of the invention are as follows:
(1) During the culture process of the lactobacillus plantarum LZ010, the lactobacillus plantarum is in a stable growth environment by regulating and controlling the fermentation pH value, the activity of the strain keeps high activity and consistency, the metabolites are more abundant, and the lactobacillus plantarum fermentation liquid with high quality can be produced.
(2) The Lactobacillus plantarum metazoan can still keep higher physiological activity through high-temperature action or treatment in artificial gastrointestinal digestive juice, thereby indicating that the Lactobacillus plantarum LZ010 metazoan composition can be used in daily life of people.
(3) The helicobacter pylori is inoculated into a culture medium containing the Lactobacillus plantarum LZ010 metazoan composition for culture, and has obvious inhibition effect on the growth of the helicobacter pylori, which indicates that the Lactobacillus plantarum LZ010 metazoan has the capability of resisting the helicobacter pylori.
(4) The excellent characteristics of the lactobacillus plantarum LZ010 metazoan lead the lactobacillus plantarum LZ010 metazoan to have wide market prospect in various forms of beneficial products such as probiotic beverages, dairy products, probiotic candies and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a graph showing growth of Lactobacillus plantarum LZ010 MRS plate medium in example 1 of the present invention.
FIG. 2 is a gram stain of Lactobacillus plantarum LZ010 in example 1 of the present invention.
FIG. 3 is a diagram showing the bacteriostasis of Lactobacillus plantarum LZ010 metaplastic composition against helicobacter pylori according to example 6 of the present invention.
In the figure, 1-the bacteriostasis effect of the Lactobacillus plantarum LZ010 metaplastic composition prepared in the example 3 is shown; 2-antibacterial effect graph of Lactobacillus plantarum LZ010 metaplastic composition prepared in example 4; 3-antibacterial effect graph of Lactobacillus plantarum LZ010 metaplastic composition prepared in example 5.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
Separation, purification and identification of lactobacillus plantarum LZ010
1. Sample collection
The sample of the invention is collected in the acid mother water used for pickle fermentation in the city of Leshan, sichuan province.
Taking 1g of pickle acid mother water in Sichuan Leshan city, carrying out gradient dilution, adopting an MRS solid culture medium to carry out anaerobic culture for 72 hours at 37 ℃, selecting single bacterial colony to carry out anaerobic culture for 48 hours at 37 ℃ in a liquid MRS culture medium, carrying out gram staining and contact enzyme test on the separated and purified bacterial strain, preliminarily determining the bacterial strain with positive gram staining and negative contact enzyme test as probiotics, adding sterilized glycerol to 30%, subpackaging to a preservation tube, and preserving in a refrigerator at-80 ℃.
2. Identification of strains
(1) Observation of colony/thallus morphology
The selected probiotic strain was inoculated onto MRS solid medium, and subjected to stationary culture at 37℃for 72 hours, and morphological characteristics of colonies were observed, and the results are shown in FIG. 1.
Colony morphology: white, clean edge, wet surface and opaque.
(2) Morphological characteristics
Plate strains were taken and stained strictly according to the gram staining method, and the strain morphology was observed under an optical microscope with an oil microscope:
gram staining: rod-shaped, single or paired, gram positive, results are shown in figure 2.
(3) Physiological characteristics
The physiological and biochemical tests of Lactobacillus plantarum strains were carried out according to the general bacterial System identification handbook and the Berger System bacteriology handbook, the physiological characteristics of which are shown in tables 1.1 and 1.2 below.
TABLE 1.1 physiological Properties of Lactobacillus plantarum Strain
| Culture temperature | 37℃ | Oxygen demand | Anaerobic system |
| Contact enzyme | - | Oxidase enzyme | - |
TABLE 1.2 Biochemical Properties of Lactobacillus plantarum Strain
| Glycerol | - | Inositol (inositol) | - | Inulin | - |
| Erythritol | - | Mannitol (mannitol) | - | Melezitose | - |
| D-arabinose | - | Sorbitol | - | Raffinose | + |
| L-arabinose | + | alpha-methyl-D-mannosides | - | Starch | - |
| D-ribose | + | alpha-methyl-D-glucoside | - | Glycogen | - |
| D-xylose | - | N-acetyl-glucosamine | - | Xylitol | - |
| L-xylose | - | Amygdalin (amygdalin) | + | Gentiobiose | + |
| Ardong alcohol | - | Arbutin | - | D-melezitose | - |
| beta-methyl-D-xylosides | - | Radix Schefflerae Arboricolae | + | D-lyxose | - |
| D-galactose | - | Salicin | + | D-tagatose | - |
| D-glucose | + | Cellobiose | - | D-fucose | - |
| D-fructose | - | Maltose | + | L-fucose | - |
| D-mannose | - | Lactose and lactose | + | D-arabitol | - |
| L-sorbose | - | Melibiose | + | L-arabitol | - |
| L-rhamnose | - | Sucrose | + | Gluconate salt | - |
| Dulcitol | - | Trehalose | - | 2-keto-gluconate | - |
(4) Molecular biological identification
The probiotic strain is crossed with China center for sequencing and identification, and a 16S rRNA gene sequence is recorded in patent 202211522892.2, namely lactobacillus plantarum LZ010 for reducing blood pressure and blood fat and application thereof.
According to the comprehensive analysis of experimental data such as colony morphology, morphological characteristics, physiological and biochemical characteristics, 16S rRNA gene sequence, tuf gene sequence and the like of a strain, referring to the "Berger system bacteriology handbook", the strain is identified as lactobacillus plantarum (Lactobacillus plantarum) LZ010, the preservation number of the strain in the China general microbiological culture Collection center is CGMCC No.24258, the preservation date is 2022, 01 and 06 days, and the preservation agency address is shown as follows: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Example 2
Preparation of Lactobacillus plantarum LZ010 metazoan
1. Strain activation
Under the aseptic environment, a small amount of glycerol tube is taken to store strains, streaked on a sterilized solid MRS culture medium, and the flat plate is placed in a constant temperature incubator at 37 ℃ for culturing for 24 hours.
2. Seed liquid preparation
Under the aseptic environment, single colony with larger diameter, smooth surface and regular edge on the flat plate culture medium is picked up by an inoculating loop, and then is evenly mixed with the sterilized MRS liquid culture medium, and is subjected to static culture for 18 hours at 37 ℃.
3. Fermentation culture
Inoculating the cultured seeds into a high-temperature sterilized skim milk liquid fermentation medium according to the inoculation amount of 2%, wherein the skim milk liquid fermentation medium is as follows: skim milk powder 100g/L, yeast peptone 10g/L, yeast powder 5g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate heptahydrate 1.5g/L, diammonium hydrogen citrate 1.5g/L, nicotinamide 0.001g/L, VB 1.002 g/L, VB 7.001 g/L, and pH=7.0.+ -. 0.1.
Culture conditions: tank pressure 0.01mpa,37 ℃, stirring rotation speed 50 rpm, pH=6.5+ -0.1 (ammonia water automatic adjustment), fermenting for 24h, and obtaining lactobacillus plantarum LZ010 fermentation liquor with high density.
4. Cell treatment
After fermentation, heating the fermentation liquor to 70 ℃, maintaining for 15min, cooling to 30 ℃, crushing by using a high-pressure homogenizer, circulating until thalli are completely crushed, and concentrating by using a reverse osmosis membrane to one third of the volume of the feed liquid so as to increase the post-biotics concentration of lactobacillus plantarum LZ010 and obtain post-biotics concentrated solution of lactobacillus plantarum; and (5) spray-drying the concentrated solution to obtain lactobacillus plantarum LZ010 metazoan.
Example 3
Preparation of Lactobacillus plantarum LZ010 metazoan composition
(1) Mixed auxiliary materials
Adding 20 parts of xylitol, 25 parts of fructo-oligosaccharide and 0.007 part of calcium carbonate into water, and fully dissolving; then, 35 parts of Lactobacillus plantarum LZ010 metaplasia prepared in example 2 was added and stirred uniformly.
(2) Drying
The feed liquid is subjected to filtration sterilization by a pasteurization method, and the water content of the powder is controlled to be 4.5 percent by regulating and controlling the air inlet temperature; the frequency of the atomizer was adjusted to control the particle size of the powder = 90 mesh (D90), and the lactobacillus plantarum LZ010 metaplastic composition was obtained by spray drying.
(3) Packaging arrangement
And (5) subpackaging the Lactobacillus plantarum LZ010 metaplastic composition according to weight by a full-automatic subpackaging machine.
Example 4
Preparation of Lactobacillus plantarum LZ010 metazoan composition
(1) Mixed auxiliary materials
10 parts of xylitol, 20 parts of fructo-oligosaccharide and 0.006 part of calcium carbonate are put into water for full dissolution; then 30 parts of lactobacillus plantarum LZ010 metaplasia prepared in example 2 is added and stirred uniformly.
(2) Drying
The feed liquid is subjected to filtration sterilization by a pasteurization method, and the water content of the powder is controlled to be 4.7% by regulating and controlling the air inlet temperature; the frequency of the atomizer was adjusted to control the particle size of the powder = 80 mesh (D90), and the lactobacillus plantarum LZ010 metazoan composition was obtained by spray drying.
(3) Packaging arrangement
And (5) subpackaging the Lactobacillus plantarum LZ010 metaplastic composition according to weight by a full-automatic subpackaging machine.
Example 5
Preparation of Lactobacillus plantarum LZ010 metazoan composition
(1) Mixed auxiliary materials
25 parts of xylitol, 30 parts of fructo-oligosaccharide and 0.008 part of calcium carbonate are put into water for full dissolution; then 40 parts of lactobacillus plantarum LZ010 metaplasia prepared in example 2 is added and stirred uniformly.
(2) Drying
The feed liquid is subjected to filtration sterilization by a pasteurization method, and the water content of the powder is controlled to be 4.4 percent by regulating and controlling the air inlet temperature; the frequency of the atomizer was adjusted to control the particle size of the powder = 90 mesh (D90), and the lactobacillus plantarum LZ010 metaplastic composition was obtained by spray drying.
(3) Packaging arrangement
And (5) subpackaging the Lactobacillus plantarum LZ010 metaplastic composition according to weight by a full-automatic subpackaging machine.
Example 6
Determination of the tolerance and helicobacter pylori inhibiting Capacity of Lactobacillus plantarum LZ010 metaplastic composition in the gastrointestinal tract
The artificially prepared gastrointestinal fluid is used for treating the Lactobacillus plantarum LZ010 metaplastic composition, and simultaneously, the oxford cup method is used for measuring the inhibiting effect of the Lactobacillus plantarum LZ010 metaplastic composition on helicobacter pylori.
Preparing simulated artificial gastric juice: taking a proper amount of PBS solution, adding protease to the concentration of 50U/mL, adjusting the pH value to 2.0 by using dilute HCl with the concentration of 1mol/L, and filtering and sterilizing by using a microporous filter membrane with the thickness of 0.22 mu m to prepare the simulated artificial gastric fluid.
Preparing simulated artificial intestinal juice: an appropriate amount of PBS solution was taken and pH was adjusted to 8.0 with 0.4% (w/w) NaOH. Adding 1g trypsin into 100ml above liquid, mixing, filtering with 0.22 μm microporous membrane, and sterilizing to obtain simulated artificial intestinal juice.
Preparation of helicobacter pylori suspension: culturing helicobacter pylori glycerol tube strain under aseptic condition on brain heart infusion agar (10% defibrinated sheep blood) plate streak, and culturing at 37deg.C under standing micro-oxygen condition for 72 hr. Washing the thallus with PBS buffer solution at 8000rpm for 2min, re-suspending with PBS buffer solution, and diluting to thallus concentration of 10 7 CFU/mL, namely helicobacter pyloriA bacillus suspension.
Preparing a double-layer plate: uniformly spreading sterilized brain heart infusion agar (10% defibrinated sheep blood) at 40-50deg.C into a culture dish, and cooling on a horizontal table top to obtain bottom layer. And (3) adding 0.1 times of helicobacter pylori suspension into brain heart infusion agar (10% defibrinated sheep blood is added) at the temperature of 40-50 ℃, uniformly mixing, pouring into a plate, uniformly coating the plate on a bottom layer, placing the plate on a horizontal table surface to cool the plate, taking the plate as a fungus layer, and uniformly placing 4 oxford cups at a medium distance in blood leveling for later use.
And (3) taking a proper amount of the lactobacillus plantarum LZ010 metaplastic composition prepared in the examples 3-5, respectively adding the metaplastic composition into artificial gastric juice and artificial intestinal juice, and respectively digesting for 2 hours and 4 hours by constant-temperature shaking at 37 ℃.
Bacteriostasis test: 0.2ml of digestive juice is dripped into each oxford cup, and the mixture is cultured for 24 hours under the condition of standing and micro-oxygen at 37 ℃ to measure the diameter of a bacteriostasis ring. A plate without Lactobacillus plantarum LZ010 metaplastic was used as a blank. The specific results are shown in table 1 below:
TABLE 1 antibacterial ability of Lactobacillus plantarum LZ010 metazoan compositions
| Group of | Antibacterial circle/mm |
| Blank space | 0 |
| Example 3 | 17.5 |
| Example 4 | 15.2 |
| Example 5 | 16.8 |
From the table above, the Lactobacillus plantarum LZ010 metazoan composition is digested in the artificial gastric juice with pH=2.0 for 2 hours, and in the artificial intestinal juice with pH=8.0 for 4 hours, and still has an inhibiting effect on helicobacter pylori, which fully demonstrates that the Lactobacillus plantarum LZ010 metazoan composition has a very strong inhibiting effect on helicobacter pylori.
Example 7
Effect of LZ010 prebiotic composition after Lactobacillus plantarum on helicobacter pylori adhesion
2g of the Lactobacillus plantarum LZ010 metazoan composition prepared in example 3 was taken and dissolved in 10mL of sterile water for use.
Culture of human gastric mucosal epithelial cells: the frozen tube containing the cell suspension of the human gastric mucosal epithelial cells is rapidly placed into a water bath with the temperature of 37 ℃ for shaking and thawing, and is transferred into a centrifuge tube which is prepared in advance and contains DMEM culture medium and 10% fetal bovine serum culture medium for uniform mixing. Centrifugation was performed at 1000rmp for 4 minutes, the supernatant was discarded, and the supernatant was added to DMEM medium+10% fetal bovine serum medium and then homogenized by blowing. All cell suspensions were then transferred to 96-well plates and placed at 37℃in 5% CO 2 And (3) standing and culturing in a saturated humidity cell culture box. After the cells were attached, the culture supernatant was discarded, and the cells were rinsed 3 times with sterile PBS buffer for use.
Preparation of helicobacter pylori infection of human gastric mucosal epithelial cells: helicobacter pylori suspension (concentration stomach 10) 7 CFU/mL), adding DMEM medium and 10% fetal bovine serum medium, standing in a cell incubator with 5% CO2 and saturated humidity at 37 ℃ for 2 hours, and rinsing the cells with sterile PBS buffer solution for 3 times to obtain the human gastric mucosal epithelial cells infected with helicobacter pylori. The number of bacteria adhered to 100 cells under 20 random fields (adhesion index) was calculated on an oil microscope (x 100).
LZ010 prebiotic composition after addition of Lactobacillus plantarum to helicobacter pylori infected human gastric mucosal epithelial cellsThe solution and DMEM medium plus fetal bovine serum medium were incubated at 37deg.C with 5% CO 2 The cells were allowed to stand in a saturated humidity cell incubator for 2 hours, rinsed with sterile PBS buffer for 3 times for methanol fixation, gram stained, and the number of bacteria adhered to 100 cells (adhesion index) under 20 random fields was calculated on an oil microscope (X100). The specific results are shown in Table 2.
TABLE 2 adhesion Rate of helicobacter pylori to human gastric mucosal epithelial cells
| Group of | Adhesion rate/% |
| Control group | 100±0.1 |
| Experimental group | 46.73±8.5 |
As is clear from the results shown in Table 2, the adhesion rate of helicobacter pylori to human gastric mucosal epithelial cells was greatly reduced from 100% to 46.73% after the treatment of helicobacter pylori-infected human gastric mucosal epithelial cells with Lactobacillus plantarum LZ010 metaplastic composition. From the results, the Lactobacillus plantarum LZ010 metaplastic composition obviously reduces the adhesion of the infected helicobacter pylori to human gastric mucosa epithelial cells and reduces the colonization of the helicobacter pylori epithelial cells, thereby effectively inhibiting the harm of the helicobacter pylori to human gastrointestinal tracts.
Example 8
Determination of the ability of Lactobacillus plantarum LZ010 metazoan compositions to inhibit helicobacter pylori in vivo
Inhibition of H.pylori was determined by feeding a healthy mouse with a Lactobacillus plantarum LZ010 metacomposition (product prepared in example 3).
Mouse modeling: male healthy mice of about 30g body weight are selected at 6-8 weeks of age. The stomach was irrigated with 1mg/mL amoxicillin, 0.5mg/mL clarithromycin mixed antibiotic solution, 1 mL/piece, 2 times/day for a total of 3 days. After 7 days 1mL of fresh helicobacter pylori suspension (1X 10) 7 CUF/mL). After 2 days, the Hp kit is used for detecting the infection condition of the mice, thereby determining that the modeling is successful.
Mice that were modeled successfully were grouped into air-self control and experimental groups. The mice of the experimental group were fed with Lactobacillus plantarum LZ010 metazoan composition, and the blank group was fed normally for 1 month continuously. After the end of feeding, the stomach tissue of the mice was homogenized, subjected to gradient dilution, and subjected to plate culture by brain heart infusion agar (10% defibrinated sheep blood, 5g/mL vancomycin, 1. Mu.g/mL polymyxin, 3. Mu.g/mL amphotericin B, 100g/mL TMP) for 72 hours to detect the number of viable bacteria of Hp, and the specific results are shown in Table 3.
TABLE 3 helicobacter pylori viable count test results
| Group of | HP viable count (CUF/mL) |
| Blank control group | 4.8×10 5 |
| Experimental group | 2.7×10 3 |
As can be seen from Table 3, mice were found to have Hp infection, the control group was infected more severely, and the mice in the experimental group had a smaller number of Hp infection, indicating that the long-term administration of Lactobacillus plantarum LZ010 metaplastic composition had a significant effect of inhibiting Hp.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. The lactobacillus plantarum LZ010 metazoan composition is characterized by comprising the following components in parts by weight: 30-40 parts of metazoan, 10-25 parts of xylitol, 20-30 parts of fructo-oligosaccharide and 0.006-0.008 part of calcium carbonate; the metagen consists of thallus and metabolite of lactobacillus plantarum LZ 010; the lactobacillus plantarum (Lactobacillus plantarum) LZ010 has a preservation number of CGMCC No.24258 in the China general microbiological culture Collection center, and a preservation date of 2022, 01 and 06 days, and a preservation institution address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
2. Lactobacillus plantarum LZ010 metaplastic composition according to claim 1, characterized in that it comprises the following components in parts by weight: 35 parts of metazoan, 20 parts of xylitol, 25 parts of fructo-oligosaccharides and 0.007 part of calcium carbonate.
3. Lactobacillus plantarum LZ010 metazoan composition according to claim 1, characterized in that the metazoan preparation method is as follows:
(1) Bacterial screening and preservation
Selecting a proper amount of lactobacillus plantarum LZ010, diluting and coating the lactobacillus plantarum LZ010 on a TPY solid flat-plate culture medium, culturing the lactobacillus plantarum on the solid flat-plate culture medium at the constant temperature of 37 ℃ for 24-48 hours, selecting a typical single colony to streak on the solid flat-plate culture medium, extracting the single colony to perform liquid expansion culture, and freezing and preserving 10% of glycerol;
(2) Seed culture
Inoculating the preserved lactobacillus plantarum LZ010 into a sterilized TPY liquid culture medium, and standing and culturing for 12-24 hours at 37 ℃;
(3) Fermentation culture
Inoculating the cultured seeds into a defatted emulsion fermentation medium, culturing at 37 ℃ for 12-36h, and controlling the pH value to be 6.0-7.0 in the culturing process to obtain a high-density lactobacillus plantarum fermentation liquid;
(4) Cell treatment
After fermentation, heating the fermentation liquor to 70 ℃, maintaining for 15min, cooling to 30 ℃, crushing by using a high-pressure homogenizer, circulating until thalli are completely crushed, and concentrating by using a reverse osmosis membrane until the volume of the feed liquid is one third, thus obtaining lactobacillus plantarum metaplasium concentrated solution; and (5) spray-drying the concentrated solution to obtain lactobacillus plantarum LZ010 metazoan.
4. The lactobacillus plantarum LZ010 metacomposition according to claim 3, wherein the skim milk liquid fermentation medium in (3) is: 100g/L of skim milk powder, 10g/L of yeast peptone, 5g/L of yeast powder, 1.5g/L of monopotassium phosphate, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of diammonium hydrogen citrate, 0.001g/L of nicotinamide, 0.002g/L of VB, 0.001g/L of VB and pH=7.0+/-0.1,0.22 mu m liquid filter filtration and sterilization.
5. A method for preparing the lactobacillus plantarum LZ010 metacomposition according to claim 1, characterized by the following specific steps:
(1) Mixed auxiliary materials
Xylitol, fructo-oligosaccharide and calcium carbonate are put into water to be fully dissolved; then adding lactobacillus plantarum LZ010 metaplasia and uniformly stirring;
(2) Drying
Sterilizing the feed liquid by a pasteurization method, and controlling the moisture of the powder by regulating and controlling the air inlet temperature; adjusting the frequency of the atomizer, controlling the granularity of the powder, and obtaining the Lactobacillus plantarum LZ010 metazoan composition through spray drying.
6. The method of claim 5, wherein the lactobacillus plantarum LZ010 metaplastic composition particle size D90 < 100 mesh.
7. The method of claim 5, wherein the lactobacillus plantarum LZ010 metaplastic composition has a moisture of <5%.
8. Use of a lactobacillus plantarum LZ010 metazoan composition according to claim 1 for the preparation of a helicobacter pylori inhibiting product.
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| CN117736939A (en) * | 2024-02-18 | 2024-03-22 | 广州同康生物科技有限公司 | Lactobacillus acidophilus BN10 against helicobacter pylori and metazoan |
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| CN112940983A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus plantarum preparation capable of increasing anti-helicobacter pylori effect and preparation method thereof |
| CN113980878A (en) * | 2021-12-29 | 2022-01-28 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum for resisting helicobacter pylori infection and application thereof |
| CN116286458A (en) * | 2022-11-30 | 2023-06-23 | 天津小薇生物科技有限公司 | Lactobacillus plantarum LZ010 capable of reducing blood pressure and blood fat and application thereof |
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| KR20030077895A (en) * | 2002-03-27 | 2003-10-04 | (주) 피엘바이오 | Lactobacillus plantarum isolated from kimchi with inhibiting activities on helicobacter pylori |
| CN112940983A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus plantarum preparation capable of increasing anti-helicobacter pylori effect and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117736939A (en) * | 2024-02-18 | 2024-03-22 | 广州同康生物科技有限公司 | Lactobacillus acidophilus BN10 against helicobacter pylori and metazoan |
| CN117736939B (en) * | 2024-02-18 | 2024-04-23 | 广州同康生物科技有限公司 | Lactobacillus acidophilus BN10 against helicobacter pylori and metazoan |
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