CN114908020B - Lactobacillus plantarum for resisting helicobacter pylori infection and application of lactobacillus plantarum in edible herbal enzyme product - Google Patents
Lactobacillus plantarum for resisting helicobacter pylori infection and application of lactobacillus plantarum in edible herbal enzyme product Download PDFInfo
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- CN114908020B CN114908020B CN202210639283.9A CN202210639283A CN114908020B CN 114908020 B CN114908020 B CN 114908020B CN 202210639283 A CN202210639283 A CN 202210639283A CN 114908020 B CN114908020 B CN 114908020B
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Abstract
The invention relates to the technical field of microorganisms, in particular to Lactobacillus plantarum (RH JS 001) for resisting helicobacter pylori infection and application thereof in eating herbal ferment products. The Lactobacillus plantarum (Lactobacillus plantarum) RH JS001 is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 Xilu No.1 Beijing province of the Korean district, and the preservation number is: CGMCC No.23703. The Lactobacillus plantarum RH JS001 has good acid resistance, can effectively inhibit the growth of helicobacter pylori, and is proved by experiments to have the capability of resisting the infection of the helicobacter pylori and can also obviously reduce the inflammatory reaction of the stomach of a mouse infected by the helicobacter pylori.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum (A) for resisting helicobacter pylori infectionLactobacillus plantarum) RH JS001 and application thereof in edible herbal enzyme products.
Background
Helicobacter Pylori (HP), a gram-negative, spirochete microaerophilic bacterium, colonizes the gastric mucosal surface and between mucosal layers, was originally discovered and suggested by australian scholars Marshall and Warren in 1982 to be associated with the development of peptic ulcers and chronic gastritis. A great deal of modern medical research indicates that helicobacter pylori is the main pathogenic cause of acute and chronic gastritis and gastric and duodenal ulcers, and may be associated with the onset of gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) malignant lymphoma. The World Health Organization (WHO) has listed it as a class I carcinogen.
As for the treatment of helicobacter pylori infection, the scientists in China have been continuously exploring, and the most popular scheme at present is a triple therapy of taking a Proton Pump Inhibitor (PPI) and two antibiotics (two selected from clarithromycin, amoxicillin, tetracycline, metronidazole and the like) simultaneously. However, in the actual treatment process, as the drug resistance of helicobacter pylori increases, the eradication rate of triple therapy is continuously decreased, and the proton pump inhibitor causes dyspepsia, and a large amount of the antibacterial agent also disrupts the balance of the normal flora of the human body.
In the current situation, the search for a new therapy for helicobacter pylori is not always ready. Therefore, probiotic therapy is in force. Probiotics, which are generally defined as living microorganisms, can provide health benefits by improving the host's intestinal microbial balance when ingested in certain amounts. These health benefits include reduction of lactose intolerance, enhancement of the immune system, prevention and treatment of cancer, reduction of cholesterol levels, reduction of gastrointestinal tract disorders including antibiotic-associated diarrhea, increased eradication of the gastric pathogen helicobacter pylori, and the like. However, chinese patents CN111118106A and CN113980878A do not combine the application effect of in vivo strains, chinese patents CN111548970A and CN111607538A do not screen lactobacillus from the ability of inhibiting helicobacter pylori, and the above patents only limit the application of probiotics in products to powder such as inhibitor and capsule.
Nowadays, people pay more attention to traditional Chinese medicine health preservation, edible herbal enzyme which is fermented by probiotics and takes medicinal and edible raw materials as fermentation substrates is undoubtedly a new track. A large number of researches show that the hericium erinaceus, the liquorice, the scutellaria baicalensis, the mulberry leaves, the ginger, the poria cocos, the lucid ganoderma, the hawthorn, the red dates, the sealwort, the Chinese yam, the coix seeds, the medlar, the dendrobium officinale and other medicinal and edible raw materials have the effect of inhibiting the growth of helicobacter pylori. Considering that the function of the probiotics for resisting helicobacter pylori infection has strain dependence, strains which are edible and have the ability of inhibiting the growth of the helicobacter pylori are continuously screened and applied to the edible herbal ferment products, and the probiotics and the edible herbal ferment product have practical significance and broad prospect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the lactobacillus plantarum (A), (B), (C) and (C) for resisting the helicobacter pylori infectionLactobacillus plantarum) RH JS001 and application thereof in edible herbal enzyme products.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the present invention provides a Lactobacillus plantarum strain comprising the 16rRNA sequence shown in SEQ ID No.1Lactobacillus plantarum）。
The inventor of the invention surprisingly screens out a strain of lactobacillus plantarum (F.) (from farmer self-made sour radish in Sichuan provinceLactobacillus plantarum) RH JS001, the 16S rDNA sequence of the strain is shown in SEQ ID NO.1 after sequencing analysis, the sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Lactobacillus plantarum.
Based on the above findings, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) RH JS001, lactobacillus plantarum (Lactobacillus plantarum) The RHJS001 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo sunward, and the preservation number is as follows: CGMCC No.23703, the preservation time is 2021 year, 11 months and 1 day.
Lactobacillus plantarum of the present invention: (Lactobacillus plantarum) The RH JS001 has good acid resistance, can effectively inhibit the growth of helicobacter pylori, and is proved by animal experiments to have the capability of resisting the infection of the helicobacter pylori and can also obviously reduce the inflammatory reaction of the stomach of a mouse infected by the helicobacter pylori.
Lactobacillus plantarum: (A)Lactobacillus plantarum) The sizes of the inhibition zones of the bacterial suspension and the supernatant of the RH JS001 to the helicobacter pylori can respectively reach 18.50 +/-1.70 mm and 16.90 +/-2.10 mm.
The present invention provides the above Lactobacillus plantarum (A), (B)Lactobacillus plantarum) Inoculating the fermentation liquor formed by the raw material liquid.
Preferably, lactobacillus plantarum (A)Lactobacillus plantarum) The RH JS001 is inoculated into the raw material liquid according to the inoculation amount of 2-6%.
The third object of the present invention is to provide the above Lactobacillus plantarum (Lactobacillus plantarum) Or the application of the fermentation liquor in the preparation of the helicobacter pylori infection resisting preparation.
In a fourth aspect, the present invention provides the above Lactobacillus plantarumLactobacillus plantarum) Or the application of the fermentation liquor in preparing the edible herbal enzyme product.
As a preferred embodiment of the use according to the invention, the Lactobacillus plantarum (A)Lactobacillus plantarum) Or the application of the fermentation liquor thereof in inhibiting helicobacter pylori in the fermentation process.
The edible herbal enzyme is prepared by fermenting the following components by Lactobacillus plantarum (Lactobacillus plantarum); the components comprise dendrobium officinale or lucid ganoderma.
In some embodiments of the present invention, the substrate is, lactobacillus plantarum (A)Lactobacillus plantarum) And (2) inoculating RH JS001 into the raw material liquid to obtain fermentation liquid, wherein the fermentation conditions are as follows: fermenting at 30-40 deg.C for 48-72 hr, and standing for culture.
The invention provides a preparation method of lactobacillus plantarum: (A)Lactobacillus plantarum) The edible herbal ferment obtained by fermentation can inhibit the growth of helicobacter pylori.
In certain embodiments, the inhibition zone of the dendrobium officinale ferment on helicobacter pylori can reach 12.40 +/-0.90 mm.
In some embodiments, the inhibition zone of the ganoderma lucidum ferment to the helicobacter pylori can reach 16.10 +/-1.40 mm.
In a sixth aspect, the present invention provides a composite herbal enzyme, which comprises the following components prepared from the lactobacillus plantarum (b), c, dLactobacillus plantarum) Fermenting to obtain the finished product;
the components comprise at least three of herba Dendrobii, ganoderma, hericium Erinaceus, glycyrrhrizae radix, fructus crataegi, rhizoma Polygonati, rhizoma Dioscoreae, coicis semen and fructus Lycii.
As a preferred embodiment of the composite herbal ferment disclosed by the invention, the composite herbal ferment comprises the following components in parts by weight:
1-8 parts of dendrobium officinale, 1-8 parts of lucid ganoderma, 5-25 parts of hericium erinaceus, 5-20 parts of liquorice, 2-16 parts of hawthorn, 2-16 parts of rhizoma polygonati, 2-16 parts of Chinese yam, 1-3 parts of semen coicis and 1-8 parts of medlar.
The present invention provides a herbal ferment product, which includes a composite herbal ferment beverage a and/or a composite herbal ferment beverage B.
As a preferred embodiment of the herbal ferment product of the present invention, the composite herbal ferment beverage a comprises the following components in parts by weight:
3-15 parts of compound herbal enzyme, 0.01-0.2 part of hericium erinaceus powder, 0.1-2 parts of red date powder, 0.01-0.2 part of medlar juice powder and 0.1-2 parts of fructo-oligosaccharide.
As a preferred embodiment of the herbal ferment product of the present invention, the composite herbal ferment beverage B comprises the following components in parts by weight:
3-15 parts of compound herbal enzyme, 0.1-2 parts of lemon powder, 0.001-0.01 part of mint powder, 0.01-0.2 part of persimmon extract and 0.1-2 parts of fructo-oligosaccharide.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides lactobacillus plantarum (A)Lactobacillus plantarum) The RH JS001 has good acid resistance, can inhibit the growth of helicobacter pylori, and has great application prospect in the aspect of inhibiting the growth of the helicobacter pylori.
Drawings
FIG. 1 shows Lactobacillus plantarum (C)Lactobacillusplantarum) 16rDNA sequencing plot of RH JS 001;
FIG. 2 shows Lactobacillus plantarum (Lactobacillusplantarum) Phylogenetic tree of RH JS 001.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
Example 1 Lactobacillus plantarum for inhibiting the growth of helicobacter pylori ((R))Lactobacillusplantarum) Screening and identification of RH JS001
1. Screening of lactic acid bacteria strains:
the strain is separated from self-made pickled radishes made by Sichuan farmers. Weighing 1 g of sample under aseptic condition, preparing the sample into suspension by using 0.85% physiological saline, diluting the suspension to a proper gradient, coating the diluent on an MRS solid culture medium, carrying out anaerobic culture at 37 ℃ for 48h, selecting a plurality of single colonies with the characteristics of lactic acid bacteria, and repeatedly carrying out purification culture; the picked single colony is inoculated in an MRS liquid culture medium, cultured for 24 h at 37 ℃, and continuously activated for three times by 2 percent of inoculum concentration to ensure the activity of the strain for subsequent experiments.
The MRS liquid culture medium comprises the following components: 10.0g of peptone, 10.0g of beef powder, 5.0g of yeast powder and glucose (C) 6 H 12 O 6 •H 2 O) 20.0 g, magnesium sulfate (MgSO) 4 •7H 2 O) 0.1 g, sodium acetate (CH) 3 COONa•3H 2 O) 5.0g, ammonium citrate (C) 6 H 17 N 3 O 7 ) 2.0 g of dipotassium hydrogen phosphate (K) 2 HPO 4 •7H 2 O) 2.0 g, manganese sulfate (MnSO) 4 •4H 2 O) 0.05 g and Tween 801.0 g.
2. Screening of lactic acid bacteria inhibiting growth of helicobacter pylori:
(1) Will be purchased from ATCCThe helicobacter pylori with the strain number of ATCCA43504 is taken out at-80 ℃, is coated in a Columbia blood agar culture medium containing 5 percent sheep blood (v/v), is cultured for 48 hours at 37 ℃ in a microaerobic environment, then a single colony is picked, and is repeatedly subjected to purification culture; inoculating the single colony to a helicobacter pylori liquid culture medium, culturing at 37 ℃ for 48h, centrifuging at 4 ℃ for 10min at 2600g, and filtering the supernatant with a 0.22-micron sterile filter membrane to obtain a supernatant; suspending thallus with helicobacter pylori liquid culture medium to obtain suspension, and adjusting concentration of the suspension to 10 8 CFU/mL。
The helicobacter pylori liquid culture medium comprises the following components: 10.0g of peptone, 10.0g of bovine brain extract powder, 9.0 g of bovine heart extract powder, 5.0g of sodium chloride (NaCl), and glucose (C) 6 H 12 O6•H 2 2.0 g of O), disodium hydrogen phosphate (Na) 2 HPO 4 •12H 2 O）2.5 g。
(2) Centrifuging the activated strain culture solution at 4 deg.C for 10min at 2600g, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain supernatant; resuspending the thallus with 0.85% (w/v) physiological saline to obtain a bacterial suspension, and adjusting the concentration of the bacterial suspension to 10 8 CFU/mL。
(3) Bacteriostatic zone experiment:
100 mu L of helicobacter pylori suspension is coated on a Columbia blood agar culture medium containing 5% sheep blood, four Oxford cups are placed on the coated plate, 100 mu L of the prepared bacterial suspension of the strain and the supernatant of the strain are respectively injected into the aperture of the Oxford cup, MRS liquid culture medium is used as a negative control, and 0.025% metronidazole solution is used as a positive control. Culturing at 37 deg.C under micro-aerobic condition for 48h, measuring the diameter of the zone with vernier caliper, and repeating the experiment for 3 times.
3. And (3) identification of strains:
extracting genome DNA of a strain to be identified by adopting a genetic genome DNA kit, sending the extracted genome to a genetic biological engineering (Shanghai) limited company for PCR amplification and sequencing, carrying out BLAST homology analysis and comparison on the obtained sequencing result in an NCBI database, identifying the strain, constructing a phylogenetic tree, and combiningAs shown in fig. 1. As shown in FIG. 1, the strain is Lactobacillus plantarum (A)Lactobacillus plantarum) Named as Lactobacillus plantarum RH JS001, is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is: CGMCC No.23703, is available in No. 3 Hospital No.1 Xilu, north Cheng, the area facing Yang, beijing, and has a preservation time of 11 months and 1 day in 2021 year.
Example 2 Lactobacillus plantarum (Lactobacillusplantarum) Acid resistance evaluation of RH JS001
(1) Preparing artificial gastric juice:
the artificial gastric juice contains 0.85% (w/v) NaCl and 0.30% (w/v) pepsin, the pH values of the artificial gastric juice are respectively adjusted to 2.0, 2.5 and 3.0 by using 0.1M HCl, and the artificial gastric juice is filtered and sterilized by a 0.22 mu M filter membrane for later use.
(2) And (3) testing gastric acid resistance:
mixing Lactobacillus plantarum (A)Lactobacillus plantarum) Inoculating 2% of RH JS001 into MRS liquid culture medium, culturing at 37 ℃ for 24 h, centrifuging at 2600g at 4 ℃ for 10min, discarding supernatant, collecting bacterial sludge, washing bacterial sludge twice with 0.85% (w/v) physiological saline, suspending in 0.85% (w/v) physiological saline, and adjusting the concentration of bacterial suspension to 10 8 CFU/mL. Mixing 1.0 mL of the bacterial suspension with 9.0 mL of sterile artificial gastric juice with pH of 2.0, 2.5 and 3.0, respectively, culturing at 37 deg.C and 50 rpm for 2h, measuring the number of viable bacteria at one time by plate counting method, and calculating the survival rate.
The test results are shown in table 1.
TABLE 1 Lactobacillus plantarum: (Lactobacillusplantarum) Acid resistance results of RH JS001
As can be seen from Table 1, the Lactobacillus plantarum (A) according to the inventionLactobacillusplantarum) The RH JS001 has good gastric acid resistance, the survival rate can reach 82.25 plus or minus 0.53 percent after being incubated in artificial gastric juice with the pH value of 2.0 for 2 hours; the survival rate can reach 92.44 +/-0.42 percent after the artificial gastric juice with the pH value of 2.5 is incubated for 2 hours; at a pH of 3.0The survival rate can reach 95.43 plus or minus 0.35 percent after incubation in the artificial gastric juice for 2 hours.
Example 3 Lactobacillus plantarum: (Lactobacillusplantarum) Verification that RH JS001 inhibits growth of helicobacter pylori
(1) Taking out helicobacter pylori purchased from ATCC and having a strain number of ATCCA43504 from-80 ℃, coating the helicobacter pylori in a Columbia blood agar culture medium containing 5% sheep blood (v/v), culturing the helicobacter pylori in a microaerobic environment at 37 ℃ for 48 hours, picking out a single colony, and repeatedly performing purification culture; inoculating the single colony in a helicobacter pylori liquid culture medium, culturing at 37 ℃ for 48h, centrifuging at 4 ℃ at 2600g for 10min, and filtering the supernatant with a 0.22-micron sterile filter membrane to obtain a supernatant; suspending thallus with helicobacter pylori liquid culture medium to obtain bacterial suspension, and adjusting the concentration of the bacterial suspension to 10 8 CFU/mL。
(2) Activated lactobacillus plantarum (A)Lactobacillusplantarum) Centrifuging the RH JS001 culture solution for 10min at 2600g at 4 ℃, and taking the supernatant to pass through a 0.22-micron sterile filter membrane to obtain a supernatant; resuspending the cells with 0.85% (w/v) physiological saline to obtain a cell suspension, and adjusting the concentration of the cell suspension to 10 8 CFU/mL。
(3) Bacteriostatic zone experiment:
spreading 100 μ L of helicobacter pylori suspension on Columbia blood agar medium containing 5% sheep blood, placing four Oxford cups on the spread plate, and collecting 100 μ L of the above-prepared extractLactobacillusplantarum) Bacterial suspension of RH JS001, lactobacillus plantarum (L.) (Lactobacillus plantarum) The supernatant of RHJS001 is injected into the aperture of an Oxford cup, MRS liquid culture medium is used as a negative control, and 0.025% metronidazole solution is used as a positive control. Culturing at 37 deg.C under micro-oxygen environment for 48h, measuring the diameter of the zone with vernier caliper, and repeating the experiment for 3 times.
The test results are shown in table 2.
TABLE 2 Lactobacillus plantarum: (Lactobacillusplantarum) Bacteriostatic results of RH JS001
| Diameter of bacteriostatic circle (mm) | |
| Lactobacillus plantarum suspension | 18.50±1.70 |
| Lactobacillus plantarum supernatant | 16.90±2.10 |
| Positive control group | 12.30±1.20 |
| Negative control group | 0 |
As can be seen from Table 2, the negative control, MRS medium, did not inhibit the growth of helicobacter pylori, lactobacillus plantarum: (Lactobacillus plantarum) The RH JS001 bacterial suspension and the supernatant can obviously inhibit the growth of helicobacter pylori. Lactobacillus plantarum (described in Chinese patent CN 113980878A) for resisting helicobacter pylori infectionLactobacillus plantarum) LP05, when culturing for 48h, the diameter of the inhibition zone of the bacterial suspension is 15.8 +/-2.2 mm, the diameter of the inhibition zone of the supernatant is more than 14.5 +/-2 mm, and the lactobacillus plantarum (A), (B) and (C) provided by the inventionLactobacillus plantarum) The RH JS001 has better capability of inhibiting the growth of helicobacter pylori.
Example 4 animal test
(1) Grouping of mice and establishment of helicobacter pylori infection model
38 healthy male Kunming mice, 4-6 weeks old, were selected. All mice were first intragastrically mixed with antibiotic solution (azithromycin 10 mg/mL),10mg/mL ampicillin and 1.2mg/mL gentamicin) for 1 week, and the dosage is 0.1 mL per day. Mice were divided into blank, model and intervention groups, 12 each, and 14 model groups. Except for 12 mice prepared to be left as a blank group and gavaged with 1mL of 0.85% (m/v) physiological saline, 26 mice were gavaged with helicobacter pylori (10) 8 CFU/mL) fresh suspension 0.5 mL, every other day 1 times, total 8 times. At the same time, the model group perfuses 0.5 mL of 0.85% (m/v) physiological saline, and the intervention group perfuses 0.5 mL of lactobacillus plantarum (M/V) prepared in example 1Lactobacillus plantarum) RHJS001 bacterial suspension (8.2X 10) 8 CFU/day). After 1 week of the last gastric lavage, 2 mice in the model group are killed randomly, the gastric mucosa is taken and judged by adopting a gastric helicobacter pylori diagnostic kit, and if the gastric mucosa is positive, the infection model is established successfully.
(2) Treatment of mice
Killing all mice 1 week after last gastric lavage, cutting the stomach tissues of the mice, taking the stomach mucosa for histopathological special silver staining to evaluate the distribution condition of helicobacter pylori, and observing the infection classification of the stomach mucosa by HE staining microscopy; serum is taken from orbital venous plexus of mice, and the content of IL-1 beta and TNF-alpha is detected by an ELISA method.
The test results are shown in tables 3 and 4.
TABLE 3 comparison of the gastric tissue of mice for helicobacter pylori infection (unit: only)
| Grouping | Blank group | Model set | Intervention group |
| HP（-） | 11 | 0* | 5# |
| HP（+） | 1 | 1* | 4# |
| HP（++） | 0 | 4* | 2# |
| HP（+++） | 0 | 7* | 1# |
Note: in comparison with the blank set, the results, * P<0.01; in comparison with the set of models, # P<0.05; -, +, +++, ++++ respectively represent negative/no inflammation, mild positive/mild inflammation, moderate positive/moderate inflammation and severe positive/severe inflammation of the degree of inflammation of H.pylori.
As can be seen from Table 3, the number of animals infected with helicobacter pylori in gastric tissue of the model group mice was significantly increased compared to the blank group; compared with the model group, the number and the degree of helicobacter pylori infection of the mice in the intervention group are obviously reduced, and the difference has statistical significance, which indicates that the lactobacillus plantarum (A), (B), (C and C)Lactobacillus plantarum) RH JS001 has the ability to alleviate helicobacter pylori infection.
TABLE 4 comparison of IL-1. Beta. And TNF-. Alpha.levels in the sera of various groups of mice
| Grouping | Blank group | Model set | Intervention group |
| IL-1β | 103.83±10.47 | 220.81±13.20** | 140.28±10.39*# |
| TNF-α | 82.38±4.92 | 267.38±2.81** | 119.39±2.49# |
Note: in comparison to the blank set, the data is, * P<0.05， ** p<0.01; in comparison to the set of models, # P<0.05。
as can be seen from Table 4, in the case of intragastric Lactobacillus plantarum (A)Lactobacillusplantarum) After the RH JS001 bacterial suspension, the expression of inflammatory factors IL-1 beta and TNF-alpha in the stomach of the mouse is reduced, which indicates that the lactobacillus plantarum (b: (b))Lactobacillus plantarum) The RH JS001 can reduce the inflammatory reaction of the stomach of the mouse.
Example 5 Lactobacillus plantarum (Lactobacillusplantarum) Application of RH JS001 in dendrobium officinale enzyme
(1) Pretreatment of raw materials:
selecting fresh and mildew-free dendrobium officinale, cleaning with flowing water, peeling, and cutting into small pieces for later use;
(2) Preparing a dendrobium officinale stock solution:
juicing the dendrobium officinale and water according to the proportion of 1-20, and adding 3-5% of white granulated sugar according to the mass ratio to obtain a dendrobium officinale stock solution for later use;
(3) Enzymolysis:
adding 1-3% of cellulase (enzyme activity 20000U/g) and 1-2% of pectinase (enzyme activity 10000U/g) in the mass ratio into the stock solution of the dendrobium officinale prepared in the step (2), and performing enzymolysis at 45-55 ℃ for 30-60 min to obtain an enzymolysis solution of the dendrobium officinale for later use;
(4) Pasteurization:
pasteurizing the dendrobium officinale enzymolysis liquid obtained in the step (3) at 80-90 ℃ for 5-15 min, and then cooling to room temperature for later use;
(5) And (3) fermenting lactic acid bacteria:
the Lactobacillus plantarum prepared in example 1 (A) was used in a sterile environmentLactobacillus plantarum) RHJS001 bacterial suspension (8.2X 10) 8 CFU/mL) is inoculated into the sterile dendrobium officinale enzymolysis liquid with the inoculation amount of 2-6%, and the dendrobium officinale fermentation primary liquid is obtained after the constant temperature culture at 37 ℃ for 48-72 h;
(6) Centrifugal filtration:
centrifuging the primary fermentation solution of Dendrobium officinale at 4 ℃ at 2600g for 10min, and filtering the supernatant with 300-mesh filter cloth to obtain a fermentation stock solution of Dendrobium officinale;
(7) Seasoning and canning:
adding white granulated sugar with the mass ratio of 1-5% into the dendrobium officinale fermentation stock solution prepared in the step (6) for seasoning, and filling into a sterilized container in an aseptic manner to obtain the dendrobium officinale enzyme.
Example 6 Lactobacillus plantarum (Lactobacillusplantarum) Application of RH JS001 in lucid ganoderma enzyme
(1) Pretreatment of raw materials:
selecting fresh and mildew-free Ganoderma, cleaning with flowing water, peeling, and cutting into small pieces;
(2) Preparing a ganoderma lucidum stock solution:
juicing Ganoderma and water at a ratio of 1;
(3) Enzymolysis:
adding 1-3% of cellulase (enzyme activity 20000U/g) and 1-2% of pectinase (enzyme activity 10000U/g) in the mass ratio into the ganoderma lucidum stock solution prepared in the step (2), and performing enzymolysis at 45-55 ℃ for 30-60 min to obtain ganoderma lucidum enzymatic hydrolysate for later use;
(4) Pasteurization:
pasteurizing the ganoderma lucidum enzymatic hydrolysate obtained in the step (3) at 80-90 ℃ for 5-15 min, and then cooling to room temperature for later use;
(5) And (3) fermenting lactic acid bacteria:
the Lactobacillus plantarum prepared in example 1 (A), (B), (C)Lactobacillus plantarum) RHJS001 bacterial suspension (8.2X 10) 8 CFU/mL) is inoculated into sterile ganoderma lucidum enzymatic hydrolysate with the inoculum size of 2-6%, and is cultured at the constant temperature of 37 ℃ for 48-72 h to obtain ganoderma lucidum fermentation primary liquid;
(6) Centrifugal filtration:
centrifuging the primary Ganoderma fermentation solution at 4 deg.C at 2600g for 10min, and filtering the supernatant with 300 mesh filter cloth to obtain Ganoderma fermentation stock solution;
(7) Seasoning and canning:
adding white granulated sugar with the mass ratio of 1-5% into the ganoderma lucidum fermentation stock solution prepared in the step (6) for seasoning, and filling the mixture into a sterilized container in an aseptic manner to obtain the ganoderma lucidum ferment.
Example 7 Lactobacillus plantarum: (Lactobacillusplantarum) Application of RH JS001 in composite herbal enzyme
(1) Pretreatment of raw materials:
selecting fresh and moldy and unbreakable Dendrobium officinale, lucid ganoderma, hericium erinaceus, liquorice, hawthorn, rhizoma polygonati, chinese yam, coix seed and medlar, cleaning with flowing water, peeling, and cutting into small blocks for later use;
(2) Preparation of a compound herbal stock solution formula I:
juicing the traditional Chinese medicine composition in the step (1) and water according to a ratio of 1;
preparing a compound herbal stock solution formula II:
juicing the traditional Chinese medicine composition in the step (1) and water according to a ratio of 1;
preparing a third formula of the composite herbal stock solution:
juicing the traditional Chinese medicine composition in the step (1) and water according to a ratio of 1;
(3) Enzymolysis:
adding 1-3% of cellulase (enzyme activity 20000U/g) and 1-2% of pectinase (enzyme activity 10000U/g) in the mass ratio into the composite herbal stock solution prepared in the step (2), and performing enzymolysis at 45-55 ℃ for 30-60 min to obtain a composite herbal enzymolysis solution for later use;
(4) And (3) pasteurization:
pasteurizing the composite herbal enzymolysis liquid obtained in the step (3) at 80-90 ℃ for 5-15 min, and then cooling to room temperature for later use;
(5) Fermenting lactic acid bacteria:
the Lactobacillus plantarum prepared in example 1 (A) was used in a sterile environmentLactobacillus plantarum) Inoculating the RHJS001 bacterial suspension (8.2 x 108 CFU/mL) into the sterile composite herbal enzymatic hydrolysate with the inoculation amount of 2-6%, and culturing at the constant temperature of 37 ℃ for 48-72 h to obtain a composite herbal fermentation primary solution;
(6) Centrifugal filtration:
centrifuging the composite herbal fermentation primary liquid at 4 ℃ at 2600g for 10min, and filtering the supernatant with 300-mesh filter cloth to obtain a composite herbal fermentation primary liquid;
(7) Seasoning and canning:
and (5) adding 1-5% by mass of white granulated sugar into the composite herbal fermentation stock solution prepared in the step (6) for seasoning, and filling the mixture into a sterilized container in an aseptic manner to obtain the composite herbal enzyme with three formulas.
Example 8A Compound herbal ferment beverage A
(1) Preparing a composite herbal ferment beverage A:
a composite herbal enzyme beverage A, which mainly comprises 3 parts of composite herbal enzyme prepared in the embodiment 7, 0.05 part of hericium erinaceus powder, 0.2 part of red date powder, 0.1 part of medlar juice powder and 0.3 part of fructo-oligosaccharide.
(2) And (3) sterilization and filling:
and (2) subpackaging the composite herbal ferment beverage prepared in the step (1), carrying out pasteurization at 80-90 ℃ for 5-15 min, and then cooling to room temperature to obtain a finished product of the composite herbal ferment beverage.
Example 9A Compound herbal ferment beverage B
(1) Preparing a composite herbal ferment beverage B:
a composite herbal ferment beverage B, which mainly comprises 3 parts by weight of composite herbal ferment prepared in embodiment 7, 0.2 part by weight of lemon powder, 0.01 part by weight of mint powder, 0.02 part by weight of persimmon extract and 0.4 part by weight of fructo-oligosaccharide.
(2) And (3) sterilization and filling:
and (2) subpackaging the composite herbal ferment beverage prepared in the step (1), carrying out pasteurization at 80-90 ℃ for 5-15 min, and then cooling to room temperature to obtain a finished product of the composite herbal ferment beverage.
Example 10 verification of inhibition of growth of helicobacter pylori by herbal ferment and its product
(1) Taking out helicobacter pylori purchased from ATCC and having a strain number of ATCCA43504 from-80 ℃, coating the helicobacter pylori in a Columbia blood agar culture medium containing 5% sheep blood (v/v), culturing the helicobacter pylori in a microaerobic environment at 37 ℃ for 48 hours, picking out a single colony, and repeatedly performing purification culture; inoculating the picked single colony to the faintCulturing in a liquid culture medium of Bacillus bifidus at 37 deg.C for 48h, centrifuging at 4 deg.C under 2600g for 10min, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain supernatant; suspending thallus with helicobacter pylori liquid culture medium to obtain suspension, and adjusting concentration of the suspension to 10 8 CFU/mL。
(2) Bacteriostatic zone experiment:
100 mu L of helicobacter pylori bacterial suspension is coated on a Columbia blood agar culture medium containing 5% sheep blood, an Oxford cup is placed on the coated flat plate, 100 mu L of dendrobium officinale enzyme, lucid ganoderma enzyme, composite herbal enzyme beverage A and composite herbal enzyme beverage B are respectively taken and injected into the aperture of the Oxford cup, MRS liquid culture medium is used as a negative control, and 0.025% metronidazole solution is used as a positive control. Culturing at 37 deg.C under micro-aerobic condition for 48h, measuring the diameter of the zone with vernier caliper, and repeating the experiment for 3 times.
The test results are shown in table 5.
TABLE 5 bacteriostatic results of herbal enzymes and products thereof
| Group of | Diameter of bacteriostatic circle (mm) |
| Dendrobium officinale enzyme | 12.40±0.90 |
| Lucid ganoderma enzyme | 14.20±1.70 |
| Compound herbal enzyme formula I | 16.10±1.40 |
| Compound herbal enzyme formula II | 16.89±1.10 |
| Compound herbal enzyme formula III | 15.98±1.20 |
| Composite herbal enzyme beverage A | 18.47±2.10 |
| Composite herbal enzyme beverage B | 18.19±1.30 |
| Positive control | 10.40±1.30 |
| Negative control | 0 |
As can be seen from Table 5, the strain produced by Lactobacillus plantarum: (Lactobacillusplantarum) The herbal ferment fermented by RH JS001 has an inhibiting effect on the growth of helicobacter pylori, and when the lactobacillus plantarum RH JS001 is cultured for 48 hours, the diameters of the inhibition zones of the dendrobium officinale ferment, the lucid ganoderma ferment, the compound herbal ferment formula I, the compound herbal ferment formula II, the compound herbal ferment formula III, the compound herbal ferment beverage A and the compound herbal ferment beverage B which are fermented by the lactobacillus plantarum RH JS001 can respectively reach 12.40 +/-0.90 mm, 14.20 +/-1.70 mm, 16.10 +/-1.40 mm, 16.89 +/-1.10, 15.98 +/-1.20, 18.47 +/-2.10 mm and 18.19 +/-1.30 mm.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Keren and universe (Shanghai) Daihu research institute Co., ltd
<120> Lactobacillus plantarum for resisting helicobacter pylori infection and application thereof in edible herbal enzyme products
<130> 2022-05-19
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1029
<212> DNA
<213> Artificial Synthesis
<400> 1
ccaaccttca actgtcactt aggcggctgg ttcctaaaag gttaccccac cgactttggg 60
tgttacaaac tctcatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg 120
cggcatgctg atccgcgatt actagcgatt ccgacttcat gtaggcgagt tgcagcctac 180
aatccgaact gagaatggct ttaagagatt agcttactct cgcgagttcg caactcgttg 240
taccatccat tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca 300
tccccacctt cctccggttt gtcaccggca gtctcaccag agtgcccaac ttaatgctgg 360
caactgataa taagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc 420
tgacgacaac catgcaccac ctgtatccat gtccccgaag ggaacgtcta atctcttaga 480
tttgcatagt atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg 540
ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcagccttgc ggccgtactc 600
cccaggcgga atgcttaatg cgttagctgc agcactgaag ggcggaaacc ctccaacact 660
tagcattcat cgtttacggt atggactacc agggtatcta atcctgtttg ctacccatac 720
tttcgagcct cagcgtcagt tacagaccag acagccgcct tcgccactgg tgttcttcca 780
tatatctacg catttcaccg ctacacatgg agttccactg tcctcttctg cactcaagtt 840
tcccagtttc cgatgcactt cttcggttga gccgaaaggc tttcacatca gacttaaaaa 900
accgcctgcg ctcgctttac gcccaataaa tccggacaac gcttgccacc tacgtattac 960
cgcggctgct ggcacgtagt tagccgtggc tttctggtta aataccgtca ataacctgaa 1020
acagttact 1029
Claims (10)
1. A Lactobacillus plantarum (A)Lactobacillus plantarum) Characterized in that the Lactobacillus plantarum (A), (B)Lactobacillus plantarum) The 16r RNA sequence of (1) is shown as SEQ ID No. 1;
said Lactobacillus plantarum: (Lactobacillus plantarum) Is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and the preservation numbers are: CGMCC No.23703, with preservation time of 2021 year, 11 months and 1 day.
2. The Lactobacillus plantarum (L) according to claim 1Lactobacillus plantarum) Inoculating the fermentation liquid formed by the raw material liquid.
3. The Lactobacillus plantarum (F) (L) of claim 1Lactobacillus plantarum) Or the use of the fermentation broth according to claim 2 for the preparation of a preparation against helicobacter pylori infection.
4. The Lactobacillus plantarum (L) according to claim 1Lactobacillus plantarum) Or the use of the fermentation broth of claim 2 in the preparation of an edible herbal enzyme product.
5. An edible herbal enzyme, which is characterized by comprising the following components fermented by the lactobacillus plantarum (lactobacillus plantarum) of claim 1;
the components comprise dendrobium officinale or lucid ganoderma.
6. A composite herbal enzyme is characterized in that,the composite herbal ferment comprises the following components which are prepared by the lactobacillus plantarum (lactobacillus) as claimed in claim 1Lactobacillus plantarum) Fermenting to obtain the finished product;
the components comprise herba Dendrobii, ganoderma, hericium Erinaceus, glycyrrhrizae radix, fructus crataegi, rhizoma Polygonati, rhizoma Dioscoreae, coicis semen and fructus Lycii.
7. The complex herbal ferment of claim 6, wherein the complex herbal ferment comprises the following components in parts by weight:
1-8 parts of dendrobium officinale, 1-8 parts of lucid ganoderma, 5-25 parts of hericium erinaceus, 5-20 parts of liquorice, 2-16 parts of hawthorn, 2-16 parts of rhizoma polygonati, 2-16 parts of Chinese yam, 1-3 parts of semen coicis and 1-8 parts of medlar.
8. An edible herbal enzyme product comprising the composite herbal enzyme beverage of claim 6 or 7.
9. The edible herbal ferment product of claim 8, wherein the composite herbal ferment beverage comprises the following components in parts by weight:
3-15 parts of compound herbal ferment as claimed in claim 6 or 7, 0.01-0.2 part of hericium erinaceus powder, 0.1-2 parts of red date powder, 0.01-0.2 part of medlar juice powder and 0.1-2 parts of fructo-oligosaccharide.
10. The edible herbal ferment product of claim 8, wherein the composite herbal ferment beverage comprises the following components in parts by weight:
3-15 parts of the composite herbal ferment as claimed in claim 6 or 7, 0.1-2 parts of lemon powder, 0.001-0.01 part of mint powder, 0.01-0.2 part of persimmon extract and 0.1-2 parts of fructo-oligosaccharide.
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