CN117736939B - Lactobacillus acidophilus BN10 against helicobacter pylori and metazoan - Google Patents
Lactobacillus acidophilus BN10 against helicobacter pylori and metazoan Download PDFInfo
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- CN117736939B CN117736939B CN202410179212.4A CN202410179212A CN117736939B CN 117736939 B CN117736939 B CN 117736939B CN 202410179212 A CN202410179212 A CN 202410179212A CN 117736939 B CN117736939 B CN 117736939B
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Abstract
The invention discloses a Lactobacillus acidophilus and metaplasia for resisting helicobacter pylori, wherein the Lactobacillus acidophilus is Lactobacillus acidophilus (Lactobacillus acidophilus) BN10, and is preserved in China general microbiological culture collection center (CGMCC) No.29107 at the date of 11/22 of 2023. The lactobacillus acidophilus BN10 provided by the invention can produce abundant lactic acid and bacteriocin after fermentation, can reduce stomach pH, inhibit growth and reproduction of helicobacter pylori, and is beneficial to regulating stomach microenvironment to normal level. In addition, the lysate is rich in active ingredients such as various enzymes, amino acids, vitamins, polypeptides, minerals and the like, and can promote proliferation of probiotics in the stomach and promote the forward balance of the microecology in the stomach.
Description
Technical Field
The invention relates to the technical field of probiotics, in particular to Lactobacillus acidophilus BN10 and metazoan for resisting helicobacter pylori.
Background
Helicobacter pylori is prone to induce a series of symptoms such as peptic ulcer, gastritis, gastric cancer, micro-ecological imbalance in stomach, halitosis, etc., and has been listed as a class I carcinogen by the world health organization. Along with the development of the catering industry, the infection rate of helicobacter pylori climbs year by year, and the daily life of people is seriously influenced. In addition, helicobacter pylori has the characteristics of easy escape, strong adhesion, self-contained gastric acid neutralization layer, strong replication capacity, strong toxin secretion capacity and the like, is positioned below a gastric mucus layer, is fixedly planted on the surface of gastric mucosa epithelial cells, and has relatively high difficulty in completely removing after human infection.
The lactobacillus metazoan is used as an emerging microecological preparation, is an inanimate microorganism and/or component preparation thereof beneficial to the health of a host, and comprises inactivated thallus cells, thallus components and thallus metabolites, wherein the thallus components comprise lipoteichoic acid, peptidoglycan, cell surface proteins and the like, and the thallus metabolites comprise organic acids, bacteriocins, extracellular polysaccharide, short-chain fatty acids, proteins/polypeptides and the like, and have the advantages of safety, stability, rich nutrition, easy absorption, quick effect and the like.
At present, the method for removing helicobacter pylori on the market mainly uses antibiotics, and the antibiotics are required to be frequently taken when the treatment is carried out by using an antibiotic method because the recurrence rate of helicobacter pylori is high. In addition, the antibiotics have strong side effects, damage liver and gastrointestinal microecology after long-term consumption, and simultaneously, the helicobacter pylori variation is easy to generate drug resistance, so that the helicobacter pylori can not be completely eradicated, and the eradication difficulty is further increased. Therefore, there is a need for a product that is remarkable in effect, natural, free of side effects, and capable of acting directly on helicobacter pylori to eliminate it without disrupting the balance of the intragastric flora.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide helicobacter pylori resistant lactobacillus acidophilus BN10 which can produce rich lactic acid and bacteriocin after fermentation, can reduce stomach pH, inhibit growth and reproduction of helicobacter pylori, and is beneficial to regulating stomach microenvironment to normal level.
The second object of the invention is to provide a probiotic metazoan which is prepared by fermenting, lysing and pectin-adding the composition by lactobacillus acidophilus BN 10.
One of the purposes of the invention is realized by adopting the following technical scheme:
A strain of helicobacter pylori resistant lactobacillus acidophilus is lactobacillus acidophilus (Lactobacillus acidophilus) BN10, wherein the lactobacillus acidophilus (Lactobacillus acidophilus) BN10 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 29107 at the date of 2023, 11 and 22.
The lactobacillus acidophilus BN10 provided by the invention can produce abundant lactic acid and bacteriocin after fermentation, can reduce stomach pH, inhibit growth and reproduction of helicobacter pylori, and is beneficial to regulating stomach microenvironment to normal level. In addition, the lysate is rich in active ingredients such as various enzymes, amino acids, vitamins, polypeptides, minerals and the like, and can promote proliferation of probiotics in the stomach and promote the forward balance of the microecology in the stomach.
As a preferred embodiment of the present invention, the Lactobacillus acidophilus BN10 is effective against helicobacter pylori by at least one of the following items (I) to (III):
(I) producing abundant lactic acid and/or bacteriocin, lowering stomach pH, inhibiting growth and reproduction of helicobacter pylori;
(II) directly acting on helicobacter pylori to cause the helicobacter pylori to rupture and die;
(III) improving the richness of beneficial bacteria in the stomach, regulating the steady state of the bacteria in the stomach and promoting the microecological forward balance of the stomach.
As a preferred embodiment of the present invention, the active ingredient of the product comprises live and/or inactivated cells of Lactobacillus acidophilus BN 10.
As a preferred embodiment of the invention, the product comprises a probiotic freeze-dried powder, a probiotic lysate and a probiotic ferment.
As a preferred aspect of the present invention, the products include foods, medicines and health products.
The second purpose of the invention is realized by adopting the following technical scheme:
A prebiotic of a probiotic, obtained by fermenting a composition by lactobacillus acidophilus BN10 to obtain a fermented product, and subsequently lysing the fermented product, the composition comprising the following components in weight percent: 10-35 parts of rice, 10-35 parts of cabbage, 10-35 parts of white radish and 10-35 parts of kelp.
The prebiotics provided by the invention can directly act on helicobacter pylori through gastric mucosa, so that irregular shrinkage occurs, bacterial films are broken, and helicobacter pylori is finally eliminated; can improve smoothness of gastric mucosa surface and reduce helicobacter pylori adhesion.
The raw materials in the composition belong to plant raw materials and comprise grains and vegetables.
The rice contains rich nutrients such as protein, fat, starch, vitamins, minerals and the like, wherein the starch can be decomposed to produce glucose after saccharification, and can be utilized by microorganisms to produce rich lactic acid, and the lactic acid is also called 2-hydroxy propionic acid, so that the growth and propagation of helicobacter pylori can be inhibited.
Cabbage is rich in calcium, phosphorus, iron, vitamin C, vitamin U, sulforaphane, molybdenum, etc., and has effects in killing helicobacter pylori, inhibiting cancer cell growth, accelerating ulcer healing, improving stomach immunity, and preventing gastric ulcer occurrence and development.
The white radish is rich in mustard oil, so that gastrointestinal peristalsis can be promoted, helicobacter pylori adhesion can be reduced, helicobacter pylori is accelerated to be discharged out of the body, and in addition, the white radish also contains lignin, so that the phagocytic capacity of a human body on cancer cells can be improved, and the disease resistance of the human body is improved.
Kelp is rich in fucoidin, is a water-soluble polysaccharide substance rich in L-fucose and organic sulfate radical, can effectively reduce the field planting amount of helicobacter pylori in the stomach, relieve the inflammatory reaction of gastric mucosa, regulate the balance of gastric flora, increase the abundance of beneficial bacteria in the gastric mucosa and reduce the abundance of harmful bacteria.
As a preferred embodiment of the present invention, the metafurther comprises 0.1 to 0.7 parts of pectin.
The prebiotics provided by the invention can react with acid liquor to form a colloid film after entering the stomach by adding pectin components, and the colloid film covers the surface of helicobacter pylori thallus to prevent helicobacter pylori from escaping, so that the helicobacter pylori is directly hit by anti-humor active components, the anti-humor effect of the prebiotics is enhanced, a protective layer can be formed on the surface of gastric mucosa, the smoothness of the surface of gastric mucosa is improved, and the adhesion of the helicobacter pylori is reduced.
As a preferred embodiment of the present invention, the metazoan is obtained by fermenting a composition with lactobacillus acidophilus BN10 to obtain a fermented product, lysing the fermented product, and then adding pectin; the composition comprises the following components in percentage by weight: 35 parts of rice, 35 parts of cabbage, 20 parts of white radish and 10 parts of kelp; the weight part of pectin is 0.5 part.
The third purpose of the invention is realized by adopting the following technical scheme:
a method for preparing a probiotic metazoan, comprising the following steps:
Gelatinization: pasting rice with a formula amount to obtain pasting liquid A;
Saccharification: saccharifying the gelatinized liquid to obtain saccharified liquid B;
breaking the wall: breaking the wall of cabbage and white radish according to the formula amount to obtain a base material C; adding water into the kelp with the formula amount, and breaking the wall to obtain a base material D;
enzymolysis: carrying out enzymolysis and centrifugation on the base material C, and taking supernatant to obtain a base material E;
extracting: centrifuging the base material D, and taking supernatant to obtain a base material F;
curing: curing the saccharification liquid B to obtain a base material G;
PEF cooperates with medium temperature: uniformly mixing the base material E, the base material F and the base material G, preheating, performing high-voltage pulse electric field enzyme deactivation and sterilization, and cooling to obtain a base material H;
Activating strains: activating lactobacillus acidophilus BN10 to obtain fermentation seed liquid I;
Fermentation: inoculating the fermentation seed liquid I into the base material H for fermentation to obtain fermentation liquid J;
Lysis: repeatedly freezing and thawing the fermentation liquor J at ultralow temperature by liquid nitrogen, and performing ultrasonic oscillation to obtain lysate K;
Preparing pectin: adding pectin with a formula amount into water, heating, stirring and dissolving, adding into the lysate K while the pectin is hot, and stirring uniformly to obtain a base material L;
And (3) sterilization: preheating the base material L, and performing high-voltage pulse electric field enzyme deactivation and sterilization to obtain the Lactobacillus acidophilus BN10 metaplasia.
As a preferred scheme of the invention, the preparation method of the probiotic metazoan comprises the following steps of:
Gelatinization: placing the rice with the dosage into a saccharification pot, adding 2-5 times of boiling water, continuously stirring, and introducing steam to gelatinize for 20-35 min at 100-130 ℃ to obtain saccharification liquid A;
Saccharification: standing and saccharifying the gelatinized liquid A for 12-20 hours at 45-60 ℃ under the action of 0.3-0.8% saccharifying enzyme, and immediately and sequentially filtering with a 100-200 mesh screen and a 300-400 mesh screen when the saccharification is finished, thereby obtaining saccharified liquid B;
Breaking the wall: breaking the wall of the prepared cabbage and white radish for 1-4 min under the condition that the rotating speed is 20000-28000 r/min to obtain a base material C; adding 2-10 times of distilled water into the kelp with the dosage, and breaking the wall for 1-5 min under the condition that the rotating speed is 20000-28000 r/min to obtain a base material D;
enzymolysis: adding 0.1-0.6% myrosinase into the base material C, carrying out enzymolysis and ultrasonic treatment at 20-35 ℃, keeping the ultrasonic power at 150-250W for 50-85 min, placing in a centrifuge, centrifuging for 10-18 min at 2000-4000 r/min, and taking the supernatant to obtain a base material E;
extracting: placing the base material D in a pot, stirring for 35-55 min under the conditions of 450-700 r/min and 70-90 ℃, placing in a centrifuge, centrifuging for 10-20 min under the conditions of 2500-2500 r/min, and taking the supernatant to obtain the base material F;
Curing: homogenizing the saccharification liquid B, introducing steam, maintaining at 90-100 ℃ for 15-25 min, and cooling to 40-50 ℃ to obtain a base material G;
PEF cooperates with medium temperature: uniformly mixing the base material E, the base material F and the base material G, preheating for 3-8 min at 40-50 ℃, performing high-voltage pulse electric field enzyme deactivation and sterilization at the electric field strength of 25-40 kv/cm and the flow rate of 10-20 mL/min and the water bath temperature of 40-50 ℃, and cooling to 35-40 ℃ to obtain a base material H;
Preparing a liquid culture medium: 0.8-1.2% of casein peptone, 0.8-1.2% of beef extract, 0.3-0.8% of yeast extract, 1.5-2.5% of glucose, 0.3-0.8% of sodium acetate, 0.15-0.25% of citric acid diamine, 0.08-0.13% of tween 80, 0.15-0.25% of dipotassium hydrogen phosphate, 0.01-0.03% of magnesium sulfate heptahydrate, 0.003-0.008% of manganese sulfate heptahydrate, pH 5-6 and sterilization at 120-121 ℃ for 18-22 min;
Activating strains: inoculating lactobacillus acidophilus BN10 strain preserved in a freeze-drying tube into 3-8 mL of liquid culture medium, placing the liquid culture medium in a constant temperature incubator for static culture at 35-38 ℃ for 20-24 hours to obtain first-generation activated bacterial liquid; inoculating the first-generation activated bacterial liquid into 4-6 mL of liquid culture medium with the inoculation amount of 8-12%, activating for 2 times, and culturing at 35-38 ℃ for 20-24 hours to obtain a third-generation activated bacterial liquid; centrifuging the third-generation activated bacterial liquid for 8-12 min under the condition of 7500-8500 r/min, taking the precipitate to obtain bacterial cells, washing the bacterial cells with normal saline for 1-4 times, and finally re-suspending the bacterial cells with equal volume of normal saline to obtain fermentation seed liquid I;
Fermentation: inoculating the fermentation seed liquid I into the base material H according to the addition amount of 3-8% for anaerobic fermentation, controlling the fermentation temperature to be 35-40 ℃, stirring the fermentation seed liquid at the rotation speed of 250-350 r/min, keeping the pH to be 5-6, and keeping the fermentation period to be 65-75H to obtain a fermentation liquid J;
lysis: repeatedly freezing and thawing the fermentation liquor J for 2-4 times at ultralow temperature by liquid nitrogen, and then carrying out ultrasonic oscillation in an ice water bath for 2-5 times, wherein the ultrasonic power is 350-450W, and each time is 5-10 min to obtain a lysate K;
Preparing pectin: adding 8-12 times of distilled water into the pectin according to the formula, boiling by steam, stirring while boiling until the pectin is dissolved, adding the pectin into the lysate K while hot, and stirring while adding to obtain a base material L;
And (3) sterilization: preheating the base material L for 3-8 min at 40-48 ℃, and then carrying out high-voltage pulse electric field enzyme deactivation and sterilization at the electric field strength of 25-40 kV/cm and the flow rate of 10-20 mL/min and the water bath temperature of 40-55 ℃ to obtain the lactobacillus acidophilus BN10 metaplasia.
According to the preparation method of the prebiotics provided by the invention, the PEF is adopted to act on the phospholipid bilayer of the microbial cell membrane in cooperation with the medium temperature technology, so that the cell membrane becomes thinner to cause perforation, and finally the death of the microorganism is caused. The high-voltage pulse electric field (plused ELECTRIC FIELDS, PEF) technology is a sterilization and enzyme-deactivation technology which applies a double-electrode to apply microsecond (or nanosecond) level pulse to a treatment material, can not only cause irreversible damage of microbial cell membranes and further cause loss of functions and even death of the microbial cells, and realizes a rapid and efficient sterilization and enzyme-deactivation effect, but also has the advantages of short action time, low temperature (generally not more than 50 ℃), less energy consumption, small change of physicochemical properties of food and good reservation of nutrition and flavor of the food. The effect of temperature on microorganisms is based on the change of permeability of cell membranes, and when the temperature is lower than 30 ℃, the phospholipid bilayer structure of the cell membranes is compact and ordered and has a gel structure; as the temperature increases, the structure becomes less ordered, even losing its elastic properties, and converts to a liquid crystal state. Therefore, PEF and mild heat are combined, so that the advantages of the PEF and the mild heat are better exerted, and the sterilization and enzyme deactivation effects are improved.
Meanwhile, the lysis technology provided by the invention is a repeated freeze thawing combined ultrasonic wall breaking technology, and the method comprises the steps of loosening cell walls of bacteria, increasing permeability, and increasing protein and nucleic acid overflows in cytoplasm and cell breakage through a repeated freeze thawing method; the cell disruption is achieved by ultrasonic treatment, namely by ultrasonic high-intensity sonic energy input, the method is related to ultrasonic cavitation, a negative pressure region is locally formed in metaplasia cells containing bacterial cells (lactobacillus acidophilus BN 10) by the input of high-intensity sonic energy, a large number of cavitation bubbles and tiny bubbles are formed in the negative pressure region, the bubbles in the negative pressure region have extremely poor stability, the cells are broken soon after being generated, larger bursting pressure is generated at the moment of breaking a large number of bubbles, so that severe shock waves are generated, the cell wall structure is impacted under the action of the shock waves to break the content, and large protein, nucleic acid molecules, phospholipid, glycolipid, lipoteichoic acid, polysaccharide and other molecules are broken into smaller molecules, so that the nutritional ingredients of the bacterial cells are fully released.
Compared with the prior art, the invention has the beneficial effects that:
(1) The lactobacillus acidophilus BN10 provided by the invention can produce abundant lactic acid and bacteriocin after fermentation, can reduce stomach pH, inhibit growth and reproduction of helicobacter pylori, and is beneficial to regulating stomach microenvironment to normal level. In addition, the lysate is rich in active ingredients such as various enzymes, amino acids, vitamins, polypeptides, minerals and the like, and can promote proliferation of probiotics in the stomach and promote the forward balance of the microecology in the stomach.
(2) The prebiotics provided by the invention are prepared by processing the compositions of rice, cabbage, white radish and kelp by lactobacillus acidophilus BN10 through links of submerged fermentation, lysis, blending and the like, and the obtained prebiotics are rich in active ingredients such as rich lactic acid, anti-claustrin, various enzymes, amino acids, vitamins, polypeptides, minerals and the like, can directly act on helicobacter pylori to cause the bacterial rupture and death of the helicobacter pylori, can also improve the richness of beneficial bacterial groups in the stomach, regulate the microecology in the stomach to normal level, and reduce the breeding of pathogenic bacteria.
Biological material preservation information: the lactobacillus acidophilus is lactobacillus acidophilus BN10, the preservation number is CGMCC No.29107, and the classification name is: lactobacillus acidophilus Lactobacillus acidophilus was deposited at China general microbiological culture Collection center (address: north Star Xiyu No. 1, 3 of the Korean area of Beijing, national academy of sciences of China, and postal code: 100101) at 11 and 22 days of 2023, and the preservation unit is abbreviated as CGMCC.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below. The raw materials, equipment and the like used in the examples described below are commercially available except for special restrictions.
According to the embodiment of the invention, the strain which has the strongest lactic acid production capacity and can produce anti-claustrine bacteriocin and lysate to promote the growth of the stomach probiotics is screened out through a cell experiment.
System one: screening of bacterium with strongest lactic acid producing ability
1. Experimental strains: bifidobacterium bifidum, lactobacillus helveticus, lactobacillus casei, lactobacillus mucilaginosus, lactobacillus salivarius, streptococcus salivarius subspecies thermophilus.
2. Preparation of a culture medium:
(1) MRS modified liquid medium (seed liquid): glucose 5 g/L, tryptone 10 g/L, beef extract 10 g/L, yeast extract 5 g/L, sodium acetate 5 g/L, ammonium citrate 2 g/L, K 2HPO4 2 g/L, tween 80 1g/L, mgSO 4·7H2O 0.2 g/L,MnSO4·H2 O0.05 g/L, pH 5.5, and sterilization at 121deg.C for 20min;
(2) Glucose fermentation medium: 100g of glucose, 10g of peptone, 5g of yeast extract, 1mL of tween-80, 2g of dipotassium hydrogen phosphate, 2g of monopotassium phosphate, 0.2g of magnesium sulfate, 2g of tri-ammonium citrate, 1g of sodium citrate, 0.01g of ferrous sulfate, 0.25g of manganese sulfate, 0.01g of sodium chloride, 1000mL of pure water, pH of 5.5 and sterilization at 121 ℃ for 20min.
3. The experimental method comprises the following steps:
(1) Activating strains: taking out the strain preservation tube, thawing at 30deg.C, sucking 100 μl of bacterial liquid with a pipetting gun, inoculating into 10 mL MRS modified liquid culture medium, standing at 37deg.C for culturing 12 h, activating for 2 times continuously, centrifuging to obtain bacterial cells, washing with physiological saline for 3 times, and resuspending with equal volume of physiological saline as seed liquid;
(2) Fermentation: inoculating the seed solution into glucose fermentation medium with inoculum size of 3%, shake culturing at 37deg.C and 120r/min for 24 hr, collecting fermentation broth, and measuring lactic acid content in fermentation broth by high performance liquid chromatography to obtain the results shown in Table 1.
The method for detecting the lactic acid content in the fermentation broth comprises the following steps:
(1) Sample treatment: taking 2mL of sample into a centrifuge tube, centrifuging for 8min under 10000r/min, taking 1mL of supernatant, diluting in the centrifuge tube with 10 times of dilution, ultrasonically degassing for 8min under 400W of power, filtering with a filter membrane with 0.22 mu m to obtain sample detection stock solution, and filling the stock solution into a 2mL sample injection bottle for later use;
(2) And (3) preparing a lactic acid standard curve: and respectively weighing 1g lactic acid chromatographic grade standard solution, and using ultrapure water to fix the volume to 100 mL, namely the standard stock solution of the lactic acid with the concentration of 10 g/L. 10 mL lactic acid standard stock solution was drawn into a 50mL volumetric flask and the volume was fixed to 50mL with ultrapure water. The standard stock solutions of 0.1 mL, 0.25 mL, 0.5 mL, 1.00 mL, 2.5 mL, 5 mL and 10 mL acid are respectively sucked, the volume is fixed to 10 mL by ultrapure water, namely the standard working solution of the acid is prepared at present, and all samples are filtered by a 0.22 mu m filter membrane before sample injection.
(3) Chromatographic conditions: the chromatographic column is Bio-Rad HPX-87H ion-exclusion column, and the mobile phase is 5 mmol/L H 2SO4; column temperature 40 ℃; an ultraviolet detector (210 nm) and a differential refractive detector; the flow rate was 0.6 mL/min. The sample injection amount was 20. Mu.L.
(4) And drawing a standard curve according to the peak area and the corresponding standard substance concentration, and calculating the lactic acid content.
4. Results and analysis
TABLE 1 fermentation of lactic acid produced by different lactic acid bacteria strains
Lactic acid is used as an antibacterial agent, has the effects of 4 times of malic acid, citric acid, propionic acid and acetic acid, can destroy the steady state of helicobacter pylori, inhibit the growth and reproduction of helicobacter pylori, and can reduce gastric mucosal injury and mediate the protective effect of gastric mucosal by inhibiting local gastritis reaction.
As can be seen from Table 1, the lactic acid content produced after fermentation of the strains of the different genera is different, the difference is more remarkable, the lactic acid production of the strains of the Lactobacillus mucilaginosus and Lactobacillus in combination is lower, both are smaller than 10g/L, and both are higher, both are larger than 15g/L. Among them, lactobacillus helveticus of lactobacillus has the highest lactic acid production amount, and thus, lactobacillus was selected for the next study.
And a second system: bacterial species screening capable of producing anti-claustrophobic bacteriocins
1. Experimental strains:
(1) Lactic acid bacteria: lactobacillus crispatus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus helveticus, lactobacillus acidophilus, lactobacillus johnsonii;
(2) Standard strain of helicobacter pylori.
2. Preparation of a culture medium:
(1) MRS modified liquid medium (seed liquid): glucose 5 g/L, tryptone 10 g/L, beef extract 10 g/L, yeast extract 5 g/L, sodium acetate 5 g/L, ammonium citrate 2 g/L, K 2HPO4 2 g/L, tween 80 1g/L, mgSO 4·7H2O 0.2 g/L,MnSO4·H2 O0.05 g/L, pH 5.5, and sterilization at 121deg.C for 20min.
(2) Preparation of Columbia medium: weighing 13.3g of Columbia culture medium, dissolving in 270mL of distilled water, shaking, mixing, sterilizing at 121deg.C for 20min, cooling to 50deg.C, adding calf serum with total volume of 10%, shaking, mixing, pouring into a plate, and preserving at 4deg.C.
(3) Preparing brain heart leaching liquid: weighing brain-heart leaching solution powder 3.7 g-100 mL, mixing with distilled water, sterilizing at 121deg.C under high pressure 20 min, cooling to 50deg.C, adding calf serum 10% of total volume, mixing with light shaking, and preserving at 4deg.C.
3. The experimental method comprises the following steps:
(1) Activating strains: taking out the strain preservation tube, thawing at 30deg.C, sucking 100 μl of bacterial liquid by a pipette, inoculating to 10 mL MRS modified liquid culture medium, standing at 37deg.C for culturing 12 h, activating for 2 times, centrifuging to obtain bacterial cells, washing with physiological saline for 3 times, and resuspending with equal volume of physiological saline as seed liquid.
(2) Preparing a fermentation supernatant concentrated solution: inoculating the seed solution into MRS culture medium with inoculum size of 3%, shake culturing at 37deg.C and 120r/min for 24 hr, centrifuging at 7000r/min and 4deg.C for 10min, collecting supernatant, filtering the supernatant with (0.22 μm) filter membrane to obtain cell-free fermentation supernatant, vacuum freeze drying at-70deg.C and 60Pa for 12 hr to obtain solid powder, dissolving the solid powder with physiological saline, and preparing into fermentation supernatant concentrate with concentration of 0.25 g/mL.
(3) Removing the organic acid: and (3) regulating the fermentation supernatant concentrated solution to neutral pH=6.5 to obtain strain test solution.
(4) Helicobacter pylori bacterial culture: taking out the strain preservation tube of helicobacter pylori, thawing to 4deg.C, centrifuging at 7000r/min and 4deg.C for 10min, collecting precipitate, coating on Columbia culture medium, and culturing in three-gas incubator (85% nitrogen, 5% oxygen, 10% carbon dioxide) at 37deg.C for 3d to obtain helicobacter pylori colony.
(5) Helicobacter pylori suspension preparation: and (3) taking helicobacter pylori bacterial colonies, adding the helicobacter pylori bacterial colonies into brain heart infusion culture medium containing 10% calf serum, blowing the helicobacter pylori bacterial colonies uniformly by using a 1mL liquid transfer device, and placing the helicobacter pylori bacterial colonies in a three-gas incubator for shake culture at 37 ℃ for 3d to obtain helicobacter pylori suspension.
(6) Helicobacter pylori plate preparation: the bacterial suspension of helicobacter pylori is diluted to 1.0X10 6 CFU/mL, 100 mu L is added on Columbia culture medium of 10% calf serum, the mixture is closely smeared by a sterile inoculation loop, and then the mixture is placed in a three-gas incubator for culturing for 4 hours at 37 ℃ to obtain helicobacter pylori flat plate.
(7) Bacteriostasis test: 2 mu L of the fermentation supernatant concentrate was dropped onto a helicobacter pylori plate, carefully translated, re-placed in a three-gas incubator, cultured for 3d at 37℃and the diameter of the inhibition zone was measured and recorded, and the results are shown in Table 2.
4. Results and analysis
TABLE 2 diameter of zone of inhibition of helicobacter pylori by bacteriocins produced by fermentation of different strains
Animals, plants and bacteria can produce antibacterial peptides (antimicrobial peptides, AMPs), wherein the antibacterial peptides produced by bacteria (especially lactobacillus) or archaea are called bacteriocins, are synthesized by ribosomes and have remarkable antibacterial activity at a certain concentration, and have the effects of treating stomach diseases and inhibiting the growth of helicobacter pylori.
As can be seen from Table 2, bacteriocins produced by different strains after fermentation have different degrees of difference in the diameter of the bacteriostasis circle of helicobacter pylori, wherein the bacteriostasis circle of the strain test solution of Lactobacillus helveticus is the smallest, and then the bacteriostasis effect of the strain test solution of Lactobacillus delbrueckii subspecies bulgaricus is equivalent to that of the strain test solution of Lactobacillus johnsonii and Lactobacillus crispatus, and the diameter of the bacteriostasis circle of the strain test solution of Lactobacillus acidophilus is the largest, namely the bacteriocin produced by the fermentation of Lactobacillus acidophilus has the strongest inhibition effect on helicobacter pylori, so that Lactobacillus acidophilus strains are selected for the next study.
And (3) a system III: strain selection of lysate to promote growth of the stomach probiotic population
1. Experimental strains: lactobacillus acidophilus CICC 20248 (accession number: CICC 20248), lactobacillus acidophilus BN10, lactobacillus acidophilus CICC 6075 (accession number: CICC 6075).
Wherein Lactobacillus acidophilus CICC 20248 (accession number: CICC 20248) and Lactobacillus acidophilus CICC 6075 (accession number: CICC 6075) are purchased from China center for type culture Collection of microorganisms.
2. Experimental animals: SPF-class SD male rats were selected for 50 animals, 5 weeks old, and had a body weight of 230g.
3. The experimental method comprises the following steps:
(1) Activating strains: taking out the strain preservation tube, thawing at 30deg.C, sucking 100 μl of bacterial liquid by a pipette, inoculating to 10 mL MRS modified liquid culture medium, standing at 37deg.C for culturing 12h, activating for 2 times, centrifuging to obtain bacterial cells, washing with physiological saline for 3 times, and resuspending with equal volume of physiological saline to obtain bacterial suspension.
(2) Lysate preparation: repeatedly freezing and thawing the bacterial suspension for 3 times at ultralow temperature by liquid nitrogen, and ultrasonically oscillating (3 min, 400W) for 3 times in an ice-water bath at 1 ℃ for 8min each time to obtain lysate, namely lysate.
(3) Gastritis rats with disturbed gastric flora were modeled:
After the SD male rats were adaptively bred for one week, the rats were randomly divided into five groups of 10 animals each, namely, a blank group, a model group, a group, B group and C group, by a random number table method according to body weight.
Normal feed feeding and free drinking water for blank group.
The model group, the A group, the B group and the C group are modified by adopting five factors mainly including MNNG, and then the comprehensive modeling is carried out:
Factor one: MNNG:150 mug/mL MNNG solution is free to drink, the operation process needs to avoid light, the solution is replaced every 48 hours, and other drinking water is not needed.
Factor II: ranitidine hydrochloride: SPF-grade rat feed containing 3% ranitidine was prepared to eat freely, during which no other food was taken.
Factor three: hunger and satiety disorder: i.e. 3 days for each monday, three and five satietys, 4 days for each monday, four, six and one day, and the stomach is infused on the fasted days without water inhibition.
Factor four: on a fasted day, a 15% NaCl solution (heated to 50 ℃) of 55-65 ℃ is filled with 2mL/100g on an empty stomach twice a week.
Factor five: the fasted day, 30% ethanol 1 mL/100g is filled on an empty stomach twice a week.
After the rats are adaptively fed for one week, the model group freely drinks MNNG and 3% ranitidine Ding Siliao to start modeling, and the model group starts modeling for the first week, feeds on a single day, and fasts on two days without water inhibition, and the fasted days alternately irrigate the stomach with NaCl and ethanol for 10 weeks.
(4) Experimental administration
The blank group and the model group do not perform drug intervention, the A group, the B group and the C group all start to perform drug intervention at the 10 th week, the drug intervention period is 8 weeks, and the MNNG and the special feed feeding are stopped, and the normal water and the normal feed are recovered to be drunk.
Blank group gastric lavage physiological saline 1ml/100g;
Model group lavage physiological saline 1ml/100g;
1ml/100g of lysate of group A Lactobacillus acidophilus CICC 20248;
1ml/100g of lysate of group B Lactobacillus acidophilus BN 10;
Group C Lactobacillus curvatus CICC 6075 (accession number: CICC 6075) lysate 1ml/100g.
(5) Sample collection: after the last administration of rats of each group is finished, the rats are fasted for 24 hours, water is not forbidden, 10% chloral hydrate (0.3 ml/kg) is used for intraperitoneal injection for anesthesia the next day, abdominal opening is carried out under the xiphoid process, stomach tissues and other tissues are separated by using a pair of toothless forceps, the stomach tissues are clamped by two hemostatic forceps to be the cardiac and lower pylorus of the upper end respectively, the stomach tissues are integrally removed into a culture dish, then the culture dish is cut along the large curve of the stomach, 5ml of 0.9% ice physiological saline is used for slowly flushing the stomach contents, after the filter paper is used for drying the water, the stomach tissues are cut into tissue strips with the length of about 1cm multiplied by 3mm, and the tissue strips are put into an EP tube and stored in a refrigerator at the temperature of minus 80 ℃ for standby.
4. 16S amplicon sequencing
(1) Extracting genomic DNA, namely extracting the genomic DNA of a sample by adopting an SDS method, taking a proper amount of the sample into a centrifuge tube, and diluting the sample to 1 ng/mu l by using sterile water.
(2) PCR amplification, template: diluted genomic dna→primer: specific primers with Barcode are used according to the selection of the sequencing region; the primer of the 16S V3-V4 region is 515F-806R & gtenzyme and buffer solution: phusion of NEW ENGLAND Biolabs Inc. was used-High-FIDELITY PCR MASTER Mix with GCBuffer → reaction program: the 98℃pre-denaturation step 1 min to 30 cycles involved (98℃denaturation step 10s,50℃annealing step 30s,72℃stable extension step 30s,72℃extension step 5 min). Fwdarw.PCR products were detected by electrophoresis using agarose gel at 2% concentration.
PCR reaction system: DNA polymerase Phusion Master Mix (2×) 15 [ mu ] L, primer (2 [ mu ] M) 3 [ mu ] L (6 [ mu ] M), template DNA (1 ng/[ mu ] L) 10 [ mu ] L (5-10 ng), and H2O 2 [ mu ] L;
(3) Mixing and purifying PCR products: and (3) carrying out equal concentration sample mixing according to the concentration of the PCR product, firstly, fully and uniformly mixing, then, using agarose gel with the concentration of 1 xTAE of 2% to electrophorese and purify the PCR product, selecting a sequence with the main band size of 400-450bp, and tapping and recovering a target band. The product purification kit used was a thermo scientific company GeneJET gel recovery kit.
(4) Sequencing: the target bands were sequenced on-machine using NovaSeq 6000,6000 and analyzed according to the sequencing results, the results are shown in table 3.
5. Results and analysis
TABLE 3 comparison of gastric mucosal flora Alpha diversity in rats of each group
The greater the Shannon index, the higher the diversity of the microbial community and the more uniform the species distribution.
The smaller the simpson index, the higher the community diversity and the more uniform the species distribution.
The Chao1 index and the ACE index are based on the number of OTU in the community sample estimated by different statistical methods.
The PD_w hole_tree value represents the relatedness of species within the community, and the higher the index, the closer the relatedness.
Observed _features index is the number of OTUs visually observed.
As can be seen from table 3, the Shannon index, the Chao1 index, the ACE index, and the Observed _patterns index of rats in the model group significantly decreased, and the simpson index and the pd_white_tree value significantly increased, compared with the blank group, indicating that the intragastric flora imbalance was caused, the intragastric microecological balance was destroyed, the flora abundance was significantly decreased, and the harmful bacteria were accumulated. Under the intervention of different lactobacillus acidophilus lysate, the intragastric flora of the gastritis rat is improved to different degrees, wherein the intervention effect of the B group is best, namely the lysate of the lactobacillus acidophilus BN10 of the gastritis rat obviously improves the diversity of gastric mucosa flora of a model of the gastritis rat, so that the more uniform the species distribution is, the steady state of the intragastric flora is regulated.
And a fourth system: formula screening of helicobacter pylori resistant Lactobacillus acidophilus BN10 metagen
1. Sample preparation
TABLE 4 Lactobacillus acidophilus BN10 post-production tuple formulation screening
Note that: the formulation of comparative example 1 was replaced with 100 parts distilled water, comparing that the preparation process of comparative example 1 did not include steps 1-8.
The probiotic metazoans of examples 1-6 and comparative examples 1-4 were prepared as follows:
1. Raw material selection: selecting rice (millet), cabbage, white radish (red radish) and kelp which have complete appearance, no insect damage, fresh and no decay, and cleaning for standby;
2. Gelatinization: weighing rice (millet) according to the weight parts of table 4, placing the rice (millet) into a saccharification pot, adding 4 times of boiling water, continuously stirring, and gelatinizing for 30min at 120 ℃ by introducing steam to obtain saccharification liquid A;
3. saccharification: standing and saccharifying the gelatinized liquid A for 16h at the temperature of 51 ℃ under the action of 0.5% saccharifying enzyme, and immediately and sequentially filtering with a 100-mesh screen and a 300-mesh screen when the saccharification is finished, thereby obtaining saccharified liquid B;
4. Breaking the wall: weighing cabbage, white radish (red radish) and kelp according to the weight parts of table 4, and breaking the wall of the cabbage and the white radish (red radish) for 2min under the condition that the rotating speed is 24000r/min to obtain a base material C; adding 5 times of distilled water into the kelp, and breaking the wall for 2min at 24000r/min to obtain a base material D;
5. Enzymolysis: adding 0.3% myrosinase into the base material C, performing enzymolysis at 30deg.C, performing ultrasound (with ultrasound power of 200W), holding for 65min, centrifuging in a centrifuge at 3000r/min for 15min, and collecting supernatant to obtain base material E;
6. extracting: placing the base material D in a pot, stirring for 45min at 600r/min and 80 ℃, placing in a centrifuge, centrifuging for 15min at 3000r/min, and taking supernatant to obtain base material F;
7. curing: mixing saccharification liquid B, introducing steam, maintaining at 95deg.C for 20min, and cooling to 45deg.C to obtain base material G;
8. PEF cooperates with medium temperature: uniformly mixing the base material E, the base material F and the base material G, preheating for 5min at 45 ℃, performing high-voltage pulse electric field enzyme deactivation and sterilization under the conditions of the electric field intensity of 35 kV/cm, the flow speed of 15 mL/min and the water bath temperature of 45 ℃, and cooling to 37 ℃ to obtain the base material H;
9. Preparing a liquid culture medium: 1.0% of casein peptone, 1.0% of beef extract, 0.5% of yeast extract, 2.0% of glucose, 0.5% of sodium acetate, 0.2% of citric acid diamine, 0.1% of tween 80, 0.2% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate heptahydrate, 0.005% of manganese sulfate heptahydrate, pH 5.5 and sterilization at 121 ℃ of 20 min;
10. activating strains: inoculating lactobacillus acidophilus BN10 strain preserved in a freeze-drying tube into a liquid culture medium of 5mL, placing the culture medium in a constant-temperature incubator for static culture at 37 ℃ for 20-24 hours to obtain first-generation activated bacterial liquid; the first generation activated bacterial liquid is inoculated into a liquid culture medium of 5mL for 2 times with 10 percent of inoculum size, the culture temperature is 37 ℃ and the culture time is 20-24 hours, so as to obtain a third generation activated bacterial liquid; centrifuging the third generation activated bacteria liquid for 10min at 8000r/min, collecting precipitate to obtain bacteria, washing the bacteria with physiological saline for 3 times, and finally re-suspending with equal volume of physiological saline of 5mL to obtain fermentation seed liquid I;
11. Fermentation: inoculating the fermentation seed liquid I into the base material H according to the addition amount of 5% for anaerobic fermentation, controlling the fermentation temperature at 37 ℃, stirring at 300r/min, keeping the pH at 5.5, and fermenting for 72H to obtain a fermentation liquid J;
12. Lysis: repeatedly freezing and thawing fermentation liquor J for 3 times at ultralow temperature by liquid nitrogen, and performing ultrasonic oscillation (3 min, 400W) for 3 times in an ice-water bath at 1 ℃ for 8min each time to obtain lysate K;
13. Preparing pectin: weighing pectin according to the weight parts of table 4, adding 10 times of distilled water, introducing steam for boiling, stirring while boiling until dissolving, adding into the lysate K while hot, and stirring while adding to obtain a base material L;
14. And (3) sterilization: preheating the base material L at 45deg.C for 5min, and performing high-voltage pulse electric field enzyme deactivation and sterilization at electric field strength of 35 kV/cm, flow rate of 15 mL/min and water bath temperature of 45deg.C to obtain Lactobacillus acidophilus BN10 metaplasia.
2. Crowd testing
1. Group of subjects: 30 H.pylori infected persons, age 22-50 years, 19 men and 11 women.
Inclusion criteria for the population tested: (1) Under the fasting state, adopting 13C expiration test, and determining that the DOB value is more than or equal to 4 and the Hp is positive; (2) self or family voluntarily signs the informed consent.
Exclusion criteria (1) combine severe barycenter, liver, kidney, etc. organ organic disease; (2) complex ulcers; (3) allergic to the sample of the study.
2. Test grouping: the group of subjects was randomly divided into 10 groups of 3 persons, each group being designated as group A, group B, group C, group D, group E, group F, group G, group H, group I, group J, respectively.
3. The test method comprises the following steps: the tested group took Lactobacillus acidophilus BN10 metaplasia daily 2 times daily, 50mL each time, 1h after meal, for 27 consecutive days.
Wherein group A people eat the Lactobacillus acidophilus BN10 metaplasia of example 1.
The group B population eat the Lactobacillus acidophilus BN10 metaplasia of example 2;
the group C population eat the Lactobacillus acidophilus BN10 metaplasia of example 3;
the group D population consumed the Lactobacillus acidophilus BN10 metaplasia of example 4;
the group E population consumed the Lactobacillus acidophilus BN10 post-production of example 5;
group F people eat the post-metaplasia of Lactobacillus acidophilus BN10 of example 6;
the group G crowd eats the lactobacillus acidophilus BN10 metaplasia of the comparative example 1;
the H group of people eat the post-metaplasia of the lactobacillus acidophilus BN10 of the comparative example 2;
the group I crowd eats the post-metaplasia of the lactobacillus acidophilus BN10 of the comparative example 3;
the group J population consumed the Lactobacillus acidophilus BN10 metaplasia of comparative example 4.
Positive conditions were tested at 8 am every day, and in the fasting state, 13C breath test was used to record the time to negative, and the results are shown in table 5.
4. Results and analysis
TABLE 5 time to negative (unit: day) for the population tested
As can be seen from Table 5, after taking Lactobacillus acidophilus BN10 metaplasia of different examples, the time of negative transfer exhibited by the human group tested was different to different extent, indicating that different formulations affected the fermentation effect of the strain and the effect of the product in human body after eating. The group G people eat the product of the comparative example 1as a pure thallus after-lysis product, which shows that under the condition of no composition fermentation, only the pure thallus after-lysis product is eaten, and after 24 days, the phenomenon of negative turning can still occur, thus showing that the Lactobacillus acidophilus BN10 has a certain improvement effect on the stomach microecology of helicobacter pylori infected people. The group C people eat the product of the example 3, and after 6 continuous days, the product shows the phenomenon of turning negative, and the effect is remarkable, so the formula of the example 3 is selected as the optimal formula of the Lactobacillus acidophilus BN10 metaplasia. Comparative example 2 the composition lacks pectin compared to example 3; comparative example 3 rice was replaced with millet compared to example 3; comparative example 4 compared to example 3, the replacement of white radish with red radish, and comparative examples 2-4 compared to example 3, all increased the days to change the negative.
The lactobacillus acidophilus BN10 metazoan provided by the embodiment of the invention is prepared by processing lactobacillus acidophilus BN10 through links of core formula (rice, cabbage, white radish, kelp) deep fermentation, lysis, blending and the like, and the obtained metazoan is rich in active ingredients such as rich lactic acid, anti-humor bacteriocin, multiple enzymes, amino acids, vitamins, polypeptides, minerals and the like, and can directly act on helicobacter pylori to cause the thallus to rupture and die, and can also improve the richness of beneficial bacteria groups in the stomach, regulate the microecology in the stomach to normal level and reduce pathogenic bacteria breeding. In addition, the pectin component is added in particular, so that the metaplasia can react with acid liquor to form a colloid film after entering the stomach, and the colloid film is covered on the surface of helicobacter pylori thallus to prevent helicobacter pylori from escaping, so that the helicobacter pylori is directly hit by the anti-humor active component, the anti-humor effect of the metaplasia is enhanced, a protective layer can be formed on the surface of the gastric mucosa, the smoothness of the surface of the gastric mucosa is improved, and the adhesion of the helicobacter pylori is reduced.
System five, rupture of bacterial film test
1. Experimental strains: standard strain of helicobacter pylori
2. Preparation of a culture medium:
(1) Preparation of Columbia medium: weighing 13.3g of Columbia culture medium, dissolving in 270mL of distilled water, shaking, mixing, sterilizing at 121deg.C for 20min, cooling to 50deg.C, adding calf serum with total volume of 10%, shaking, mixing, pouring into a plate, and preserving at 4deg.C;
(2) Preparing brain heart leaching liquid: weighing brain-heart leaching solution powder 3.7 g-100 mL, mixing with distilled water, sterilizing at 121deg.C under high pressure 20 min, cooling to 50deg.C, adding calf serum 10% of total volume, mixing with light shaking, and preserving at 4deg.C.
3. Helicobacter pylori bacterial culture: taking out the strain preservation tube of helicobacter pylori, thawing to 4deg.C, centrifuging at 7000r/min and 4deg.C for 10min, collecting precipitate, coating on Columbia culture medium, and culturing in three-gas incubator (85% nitrogen, 5% oxygen, 10% carbon dioxide) at 37deg.C for 3d to obtain helicobacter pylori colony.
4. Helicobacter pylori suspension preparation: and (3) taking helicobacter pylori bacterial colonies, adding the helicobacter pylori bacterial colonies into a brain heart infusion culture medium containing 10% calf serum, blowing the helicobacter pylori bacterial colonies uniformly by using a 1mL liquid transfer device, and diluting the helicobacter pylori bacterial colonies to 0.5 McO after shaking culture for 3d at 37 ℃ in a three-gas incubator to obtain Hp bacterial suspension.
5. Experimental method
The Hp bacterial suspension was combined with the metazoan of example 3 at 1:1, mixing the materials in proportion to prepare a mixed solution serving as an experimental group. The bacterial suspension without added metazoan is used as a control group. Add to 24 well plates, 2mL per well, repeat 3 times per group. The 24-well plates were incubated at 37℃in a microaerophilic environment and the K + concentration in the bacterial suspensions of each group was measured by full-automatic biochemical analyzer before (T 0), after 1 (T 1）、2（T2）、3（T3）、4（T4）、5（T5）、6（T6) and 7h (T 7) of the intervention, and the results are shown in Table 6.
6. Results and analysis
TABLE 6 concentration of K + (mmol/L) in each group of bacterial suspensions at different time points
As can be seen from table 6, compared with T 0, the concentrations of K + in both the T 1～T7 experimental group and the control group were significantly increased, the concentration of K + in the T 1～T4 experimental group was significantly higher than that in the control group, the concentration of K + in the experimental group was rapidly increased within 2 hours, the peak was reached for 3 hours, then the peak was stabilized, and the concentration of K + in the control group was gradually increased after 1 hour until the peak was stabilized after 5 hours. The metazoan of example 3 was suggested to disrupt the cell membrane of Hp, significantly reduce the number of Hp cells, and resulted in massive leakage of K +, with progressive bacterial death and stabilization of k+ concentration over time.
The prebiotics provided by the embodiment of the invention can directly act on helicobacter pylori through gastric mucosa, so that irregular shrinkage occurs, bacterial films are broken, and helicobacter pylori is finally eliminated; can improve smoothness of gastric mucosa surface and reduce helicobacter pylori adhesion.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (6)
1. A probiotic metazoan, characterized in that the metazoan is obtained by fermenting a composition with lactobacillus acidophilus (Lactobacillus acidophilus) BN10 to obtain a fermentation product, and subsequently lysing the fermentation product, the composition comprising the following components in weight percent: 10-35 parts of rice, 10-35 parts of cabbage, 10-35 parts of white radish and 10-35 parts of kelp; the Lactobacillus acidophilus (Lactobacillus acidophilus) BN10 is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 22 of 2023, and the preservation number is CGMCC No.29107.
2. The probiotic metagen according to claim 1, wherein said metagen further comprises 0.1-0.7 parts of pectin.
3. The probiotic metazoan according to claim 1, wherein the metazoan is obtained by fermenting a composition with lactobacillus acidophilus BN10 to obtain a fermented product, lysing the fermented product, followed by adding pectin; the composition comprises the following components in percentage by weight: 35 parts of rice, 35 parts of cabbage, 20 parts of white radish and 10 parts of kelp; the weight part of pectin is 0.5 part.
4. The prebiotic of claim 1, wherein the active ingredient of the prebiotic comprises lactobacillus acidophilus (Lactobacillus acidophilus) BN10, the lactobacillus acidophilus (Lactobacillus acidophilus) BN10 exerting an anti-helicobacter pylori effect by at least one of the following items (i) to (iii):
(I) producing abundant lactic acid and/or bacteriocin, lowering stomach pH, inhibiting growth and reproduction of helicobacter pylori;
(II) directly acting on helicobacter pylori to cause the helicobacter pylori to rupture and die;
(III) improving the richness of beneficial bacteria in the stomach, regulating the steady state of the bacteria in the stomach and promoting the microecological forward balance of the stomach.
5. The preparation method of the probiotic metazoan is characterized by comprising the following steps of:
Gelatinization: pasting rice with a formula amount to obtain pasting liquid A;
saccharification: saccharifying the gelatinized liquid A to obtain saccharified liquid B;
breaking the wall: breaking the wall of cabbage and white radish according to the formula amount to obtain a base material C; adding water into the kelp with the formula amount, and breaking the wall to obtain a base material D;
enzymolysis: carrying out enzymolysis and centrifugation on the base material C, and taking supernatant to obtain a base material E;
extracting: centrifuging the base material D, and taking supernatant to obtain a base material F;
curing: curing the saccharification liquid B to obtain a base material G;
PEF cooperates with medium temperature: uniformly mixing the base material E, the base material F and the base material G, preheating, performing high-voltage pulse electric field enzyme deactivation and sterilization, and cooling to obtain a base material H;
activating strains: activating lactobacillus acidophilus (Lactobacillus acidophilus) BN10 to obtain fermentation seed liquid I;
Fermentation: inoculating the fermentation seed liquid I into the base material H for fermentation to obtain fermentation liquid J;
Lysis: repeatedly freezing and thawing the fermentation liquor J at ultralow temperature by liquid nitrogen, and performing ultrasonic oscillation to obtain lysate K;
Preparing pectin: adding pectin with a formula amount into water, heating, stirring and dissolving, adding into the lysate K while the pectin is hot, and stirring uniformly to obtain a base material L;
And (3) sterilization: preheating the base material L, and performing high-voltage pulse electric field enzyme deactivation and sterilization to obtain lactobacillus acidophilus BN10 metaplasia;
The Lactobacillus acidophilus (Lactobacillus acidophilus) BN10 is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 22 of 2023, and the preservation number is CGMCC No.29107.
6. The method for preparing the prebiotic of claim 5, comprising the steps of:
Gelatinization: placing rice with the formula amount into a saccharification pot, adding 2-5 times of boiling water, continuously stirring, and introducing steam to gelatinize for 20-35 min at 100-130 ℃ to obtain gelatinized liquid A;
Saccharification: standing and saccharifying the gelatinized liquid A for 12-20 hours at 45-60 ℃ under the action of 0.3-0.8% saccharifying enzyme, and immediately and sequentially filtering with a 100-200 mesh screen and a 300-400 mesh screen when the saccharification is finished, thereby obtaining saccharified liquid B;
Breaking the wall: breaking the wall of cabbage and white radish in the formula amount for 1-4 min under the condition that the rotating speed is 20000-28000 r/min to obtain a base material C; adding 2-10 times of distilled water into the kelp in the formula amount, and breaking the wall for 1-5 min under the condition that the rotating speed is 20000-28000 r/min to obtain a base material D;
enzymolysis: adding 0.1-0.6% myrosinase into the base material C, carrying out enzymolysis and ultrasonic treatment at 20-35 ℃, keeping the ultrasonic power at 150-250W for 50-85 min, placing in a centrifuge, centrifuging for 10-18 min at 2000-4000 r/min, and taking the supernatant to obtain a base material E;
extracting: placing the base material D in a pot, stirring for 35-55 min under the conditions of 450-700 r/min and 70-90 ℃, placing in a centrifuge, centrifuging for 10-20 min under the conditions of 2500-2500 r/min, and taking the supernatant to obtain the base material F;
Curing: homogenizing the saccharification liquid B, introducing steam, maintaining at 90-100 ℃ for 15-25 min, and cooling to 40-50 ℃ to obtain a base material G;
PEF cooperates with medium temperature: uniformly mixing the base material E, the base material F and the base material G, preheating for 3-8 min at 40-50 ℃, performing high-voltage pulse electric field enzyme deactivation and sterilization at the electric field strength of 25-40 kv/cm and the flow rate of 10-20 mL/min and the water bath temperature of 40-50 ℃, and cooling to 35-40 ℃ to obtain a base material H;
Preparing a liquid culture medium: 0.8-1.2% of casein peptone, 0.8-1.2% of beef extract, 0.3-0.8% of yeast extract, 1.5-2.5% of glucose, 0.3-0.8% of sodium acetate, 0.15-0.25% of citric acid diamine, 0.08-0.13% of tween 80, 0.15-0.25% of dipotassium hydrogen phosphate, 0.01-0.03% of magnesium sulfate heptahydrate, 0.003-0.008% of manganese sulfate heptahydrate, pH 5-6 and sterilization at 120-121 ℃ for 18-22 min;
Activating strains: inoculating lactobacillus acidophilus (Lactobacillus acidophilus) BN10 strain preserved by a freeze-drying tube into 3-8 mL of liquid culture medium, placing the liquid culture medium in a constant temperature incubator for static culture at 35-38 ℃ for 20-24 hours to obtain first-generation activated bacterial liquid; inoculating the first-generation activated bacterial liquid into 4-6 mL of liquid culture medium with the inoculation amount of 8-12%, activating for 2 times, and culturing at 35-38 ℃ for 20-24 hours to obtain a third-generation activated bacterial liquid; centrifuging the third-generation activated bacterial liquid for 8-12 min under the condition of 7500-8500 r/min, taking the precipitate to obtain bacterial cells, washing the bacterial cells with normal saline for 1-4 times, and finally re-suspending the bacterial cells with equal volume of normal saline to obtain fermentation seed liquid I;
Fermentation: inoculating the fermentation seed liquid I into the base material H according to the addition amount of 3-8% for anaerobic fermentation, controlling the fermentation temperature to be 35-40 ℃, stirring the fermentation seed liquid at the rotation speed of 250-350 r/min, keeping the pH to be 5-6, and keeping the fermentation period to be 65-75H to obtain a fermentation liquid J;
lysis: repeatedly freezing and thawing the fermentation liquor J for 2-4 times at ultralow temperature by liquid nitrogen, and then carrying out ultrasonic oscillation in an ice water bath for 2-5 times, wherein the ultrasonic power is 350-450W, and each time is 5-10 min to obtain a lysate K;
Preparing pectin: adding 8-12 times of distilled water into the pectin according to the formula, boiling by steam, stirring while boiling until the pectin is dissolved, adding the pectin into the lysate K while hot, and stirring while adding to obtain a base material L;
And (3) sterilization: preheating the base material L for 3-8 min at 40-48 ℃, and then carrying out high-voltage pulse electric field enzyme deactivation and sterilization at the electric field strength of 25-40 kV/cm and the flow rate of 10-20 mL/min and the water bath temperature of 40-55 ℃ to obtain the lactobacillus acidophilus BN10 metaplasia.
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| CN101228262B (en) * | 2005-07-21 | 2011-04-20 | 株式会社益力多本社 | Novel bacterium belonging to the genus bifidobacterium and utilization of the same |
| IT201800009755A1 (en) * | 2018-10-24 | 2020-04-24 | Berardino Vaira | New probiotics useful for eradicating helicobacter pylori infection |
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