CN116790448B - Lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea and application thereof - Google Patents
Lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea and application thereof Download PDFInfo
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- CN116790448B CN116790448B CN202311051207.7A CN202311051207A CN116790448B CN 116790448 B CN116790448 B CN 116790448B CN 202311051207 A CN202311051207 A CN 202311051207A CN 116790448 B CN116790448 B CN 116790448B
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Abstract
The invention belongs to the technical field of biology, and discloses lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea and application thereof, wherein the name is: OPB102, class name: lactobacillus helveticus with deposit No.: CGMCC No.26806, preservation unit: china general microbiological culture Collection center (China Committee for culture Collection). The OPB102 can endure the gastrointestinal environment, and can be used for preparing functional microbial agents, foods and medicines for relieving constipation or diarrhea; at the same time, it can restore damaged colon tissue, improve villus density and increase goblet cell number.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea and application thereof.
Background
Constipation is a common intestinal disease, mainly manifested by difficult defecation, dry and hard feces and less than 3 times per week, and the incidence rate of constipation tends to rise year by year. Short-term constipation affects normal work and life of people, long-term constipation can even cause kidney diseases and cardiovascular diseases, and therefore people should pay attention to prevention and treatment of constipation. Constipation is affected by a variety of factors including changes in life and eating habits, changes in mental state, etc. Research shows that intestinal flora in intestinal tracts of constipation patients is disturbed, bifidobacteria and lactobacillus are reduced, and pathogenic bacteria and conditional pathogenic bacteria are increased. This suggests that we can regulate intestinal flora by supplementing probiotics in vitro, so as to restore normal intestinal movement and achieve the purpose of relieving constipation.
Diarrhea is also a common bowel disorder, as opposed to constipation. As environmental pollution is increasing, the use of chemical food additives, frequent international trips, and antibiotic treatment and chemotherapy, results in 5% of patients suffering from diarrhea each year. Of these, 25% of patients are faced with antibiotic-induced diarrhea problems due to long-term antibiotic treatment, and treatment of antibiotic-associated diarrhea with probiotics has demonstrated significant effects of probiotics on the regulation of intestinal microbiota balance. Another study showed that the use of mixed cultures of Lactobacillus acidophilus, bifidobacterium and Saccharomyces boulardii had a good effect on traveler diarrhea prevention. Likewise, the use of probiotic lactic acid bacteria and bifidobacteria may reduce the time of acute diarrhea in children by 30% to 40%.
Probiotics and intestinal tract regulating function. Probiotics are essential elements for human health, and can synthesize various vitamins, participate in digestion of food, promote intestinal peristalsis, inhibit growth of pathogenic bacteria, decompose harmful and toxic substances, etc. The probiotics can directly or indirectly regulate intestinal flora composition of human body, improve mucosa barrier integrity, and increase intestinal probiotic amount in symbiotic or symbiotic relationship, and can be used for treating diarrhea, constipation or irritable bowel syndrome.
Diarrhea and constipation caused by unbalanced intestinal flora, and the existing treatment methods mainly comprise antibiotic adjustment, traditional Chinese medicine adjustment and probiotic preparation adjustment, but have certain defects. First, antibiotics regulate. The antibiotics are taken for a long time, so that dysbacteriosis is more easily caused, and even double infection such as pseudomembranous enteritis, acute hemorrhagic enteritis, candida infection and the like, and acute hemorrhagic enteritis and the like are caused. Second, traditional Chinese medicine is regulated. The traditional Chinese medicine for treating constipation and diarrhea has a plurality of side effects after long-term administration, for example, the traditional Chinese medicine for treating constipation usually contains traditional Chinese medicines such as rhubarb, senna leaf, polygonum multiflorum and the like, and contains small molecular components such as sennoside, emodin, anthraquinone and the like, and the traditional Chinese medicine has quick response, but can cause further dysbacteriosis, aggravate constipation, even produce abdominal pain, black bowel disease, liver injury and other side effects after long-term administration. Third, probiotic preparation modulation. At present, the probiotic preparation is used for regulating diarrhea and constipation, most of the products are multi-strain mixed products, and the effect of regulating diarrhea and constipation at the same time is not obvious. The main components of the multi-strain mixed product, such as the Xinyi pefeikang, are bifidobacterium longum, lactobacillus acidophilus and enterococcus faecalis, are used for treating diarrhea and abdominal distension caused by intestinal dysbacteriosis, and are mainly used for treating diarrhea and poor constipation. A single strain product, such as Lizhu Changle, contains Bifidobacterium adolescentis as main ingredient, and is used for treating intestinal dysfunction caused by intestinal dysbacteriosis, such as acute diarrhea, chronic diarrhea, constipation, etc., but has insignificant overall bidirectional regulation effect.
Chinese patent publication No.: CN112210516a, publication date: 2021, 1 and 12 days, a lactobacillus helveticus L1258 with intestinal tract regulating function and a composition thereof are disclosed. The composition mainly comprises an A component (lactobacillus helveticus L1258, lactobacillus acidophilus La28 and lactobacillus plantarum LP 45) and a B component (citrus fiber, inulin, lactitol and stachyose). 150 people are selected for clinical trial taking, and the age is 18-60 years; the composition meets the Roman III standard of constipation, the total effective rate of the composition is 66.67-80% after being taken for 7 days, and the total effective rate of the composition is 73.33-96.67% after being taken for 14 days. In addition, diarrhea was treated with the E.coli EAEC infection diarrhea model, and 100 mice (5 weeks old) were randomly grouped into 10 groups, with reduced average time to cure the first diarrhea and reduced incidence of re-diarrhea. The above patent publications only studied the human constipation and diarrhea model of escherichia coli infection in mice on lactobacillus helveticus L1258, lactobacillus acidophilus La28 and lactobacillus plantarum LP45 compositions, and did not study lactobacillus helveticus L1258 alone and comprehensively for constipation and diarrhea.
The invention mainly adopts a single strain, namely lactobacillus helveticus OPB102, and simultaneously carries out bidirectional regulation on diarrhea and constipation, and through searching, no related patent publication document for carrying out bidirectional regulation on diarrhea and constipation by using the single strain lactobacillus helveticus is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea and application thereof.
The technical scheme adopted for solving the technical problems is as follows:
lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea is named: OPB102, class name: lactobacillus helveticus (Lactobacillus helveticus), accession number: CGMCC No.26806, date of preservation: 2023, 2, 14, deposit unit: china general microbiological culture Collection center (CGMCC) of the China Committee for culture Collection of microorganisms, beijing, chaoyang area, north Chenxi Lu No. 1, 3.
Further, the lactobacillus helveticus OPB102 has the following biological properties:
(1) Characteristics of the cells: gram-positive, non-sporulating, non-motile bacteria;
(2) Colony characteristics: the convex part is smooth and neat;
(3) Growth characteristics: culturing in MRS culture medium at 37deg.C for 11.5 hr to end of logarithmic phase;
(4) Has strong tolerance to simulated gastrointestinal fluid.
Further, the lactobacillus helveticus OPB102 has a function of relieving constipation and diarrhea.
Use of lactobacillus helveticus OPB102 as described above for the preparation of a general food and/or a special functional food and/or a health food and/or a medicament for alleviating constipation and/or diarrhea.
Further, the food or drug has at least one of the following effects:
improving constipation, in particular shortening defecation time, increasing fecal quantity and quality and increasing small intestine propulsion rate;
alternatively, improving constipation, specifically restoring damaged colon tissue, improving villus density and increasing goblet cell number, restoring normal colon tissue, providing a better colonization environment for the flora;
or improving constipation, in particular improving intestinal flora environment, promoting beneficial bacteria such as lactobacillus and the like to reproduce, and inhibiting harmful flora from reproducing;
alternatively, the diarrhea is ameliorated, specifically the diarrhea loose stool rate, loose stool grade, and diarrhea index.
Further, the lactobacillus helveticus OPB102 is a live strain, a strain metabolite or an inactivated strain;
further, the medicament also contains a pharmaceutically acceptable carrier, which comprises: one or more of a filler, diluent, binder, disintegrant, and lubricant.
Further, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talcum powder or silicon dioxide.
The application of lactobacillus helveticus OPB102 in preparing fermented food, metazoan and ferment is described.
Further, the application comprises using lactobacillus helveticus OPB102 as a fermenting microorganism, and fermenting with food material.
The invention has the advantages and positive effects that:
1. the lactobacillus helveticus OPB102 can resist the gastrointestinal environment, improve the survival rate of the gastrointestinal tract, is beneficial to the field planting of strains, and can be used for preparing functional microbial agents, foods and medicines for relieving constipation or diarrhea; the lactobacillus helveticus OPB102 can simultaneously carry out bidirectional regulation of diarrhea and constipation.
2. The lactobacillus helveticus OPB102 disclosed by the invention has the advantages that the experiment on toxic and side effects shows that the effect on mice is not obvious, the weight is not obviously changed, the death phenomenon is not generated, and obvious pathological symptoms are not generated.
3. The lactobacillus helveticus OPB102 can accelerate the defecation time of a constipation mouse, increase the number of fecal grains and the quality of the feces, and promote the small intestine to advance; at the same time, the damaged colon tissue is restored, the villus density is improved, the goblet cell number is increased, and the normal colon tissue is restored.
4. Through genome analysis of intestinal flora, the invention can effectively change the structure of intestinal flora of a constipation mouse, improve the intestinal environment, simultaneously increase the abundance of various probiotics, reduce pathogenic bacteria and effectively improve the structure of the intestinal flora by taking single strain lactobacillus helveticus OPB 102. Can reduce the loose stool rate, loose stool grade and diarrhea index of diarrhea mice.
Drawings
FIG. 1 is a colony morphology of Lactobacillus helveticus OPB102 according to the invention;
FIG. 2 is a microscopic photograph of Lactobacillus helveticus OPB102 according to the invention;
FIG. 3 is a graph showing the histological characteristics of Lactobacillus helveticus OPB102 versus constipation mice colon according to the invention; wherein, (a) H & E staining (200 x magnification); (B) AB-PAS staining (200-fold magnification);
FIG. 4 is a Venn diagram showing the change of Lactobacillus helveticus OPB102 against intestinal flora in constipation mice according to the invention;
FIG. 5 is a bar graph of the composition of Lactobacillus helveticus OPB102 versus the family horizontal colony of constipation mice intestinal flora change in accordance with the present invention;
FIG. 6 is a bar graph of the composition of Lactobacillus helveticus OPB102 according to the invention for a horizontal colony of a constipation mouse with a change in intestinal flora;
FIG. 7 is a visual circle of the change of Lactobacillus helveticus OPB102 in constipation mice intestinal flora according to the invention;
FIG. 8 is a graph of BLAST results versus tree for the Lactobacillus helveticus OPB102 genomic sequence according to the invention;
lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea is named: OPB102, class name: lactobacillus helveticus (Lactobacillus helveticus), accession number: cgmccno.26806, date of preservation: 2023, 2, 14, deposit unit: china general microbiological culture Collection center (CGMCC) of the China Committee for culture Collection of microorganisms, beijing, chaoyang area, north Chenxi Lu No. 1, 3.
Detailed Description
The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified, the methods used in the invention are conventional methods in the art unless otherwise specified, and the mass of each substance used in the invention is conventional.
Lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea is named: OPB102, class name: lactobacillus helveticus (Lactobacillus helveticus), accession number: cgmccno.26806, date of preservation: 2023, 2, 14, deposit unit: china general microbiological culture Collection center (CGMCC) of the China Committee for culture Collection of microorganisms, beijing, chaoyang area, north Chenxi Lu No. 1, 3.
Preferably, the lactobacillus helveticus OPB102 has the following biological properties:
(1) Characteristics of the cells: gram-positive, non-sporulating, non-motile bacteria;
(2) Colony characteristics: the convex part is smooth and neat;
(3) Growth characteristics: culturing in MRS culture medium at 37deg.C for 11.5 hr to end of logarithmic phase;
(4) Has strong tolerance to simulated gastrointestinal fluid.
Preferably, the lactobacillus helveticus OPB102 has the function of relieving constipation and diarrhea.
Use of lactobacillus helveticus OPB102 as described above for the preparation of a general food and/or a special functional food and/or a health food and/or a medicament for alleviating constipation and/or diarrhea.
Preferably, the food or medicament has at least one of the following effects:
improving constipation, in particular shortening defecation time, increasing fecal quantity and quality and increasing small intestine propulsion rate;
alternatively, improving constipation, specifically restoring damaged colon tissue, improving villus density and increasing goblet cell number, restoring normal colon tissue, providing a better colonization environment for the flora;
or improving constipation, in particular improving intestinal flora environment, promoting beneficial bacteria such as lactobacillus and the like to reproduce, and inhibiting harmful flora from reproducing;
alternatively, the diarrhea is ameliorated, specifically the diarrhea loose stool rate, loose stool grade, and diarrhea index.
Preferably, the lactobacillus helveticus OPB102 is a live strain, a strain metabolite or an inactivated strain;
preferably, the medicament further comprises a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier comprising: one or more of a filler, diluent, binder, disintegrant, and lubricant.
Preferably, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talcum powder or silicon dioxide.
The application of lactobacillus helveticus OPB102 in preparing fermented food, metazoan and ferment is described.
Preferably, the use comprises fermenting the food material with lactobacillus helveticus OPB102 as a fermenting microorganism.
Specifically, the relevant preparation and detection steps are as follows:
example 1: screening of Lactobacillus helveticus OPB102
Isolation and screening of Lactobacillus
(1) 0.1ml of yoghurt sample (self-brewed yoghurt in the home of the herdsman in Kaisha of Xinjiang) is inoculated into a skim milk culture medium and placed in a constant temperature incubator at 37 ℃ for 48 hours. A small amount of bacterial liquid is picked by an inoculating loop, streaked and inoculated in an MRS agar culture medium plate.
(2) After 48 hours, different colonies are picked up by an inoculating loop according to the shape, size, color and the like of the colonies, and streaked separation and purification are carried out.
(3) Gram-positive bacteria and catalase-negative bacteria were retained by gram staining and catalase analysis.
(4) Obtaining thallus and colony characteristics of the final strain: gram-positive, non-sporulating, non-motile bacteria (fig. 1); has bulges and is smooth and neat (figure 2).
Molecular biological identification of Lactobacillus
(1) Single genome extraction
(A) Culturing the screened lactobacillus overnight;
(B) Using a Soxhaust bacteria genome DNA extraction kit, the product number is 1600, taking 1ml of the bacterial suspension cultured overnight, centrifuging in a 1.5ml centrifuge tube at 12000r/min for 1min, and sucking out the supernatant as much as possible;
(C) Adding 200ul of lysozyme with a final concentration of 20mg/ml into thalli, and treating for 40min at 37 ℃;
(D) Adding solution A (10 mmol/L, pH =8.0 Tris-HCl and 1mmol/L EDTA mixed solution, SDS with concentration of 0.08g/mL and NaCl with concentration of 1.5 mol/L) into the mixed solution obtained in the step (C), blowing by a pipette to fully suspend the thalli, adding 20ul rNaseA (10 mg/mL) into the suspension, fully reversing and uniformly mixing, and standing at room temperature for 30min;
(E) Adding 20ul proteinase K into the tube, mixing well, and digesting at 55deg.C for 60min; inverting the centrifuge tube for several times during digestion until the sample is clear and viscous, and proving that the sample is completely digested;
(F) Adding 200ul of solution B (mixed solution of phenol and isoamyl alcohol=25:24:1 by volume ratio) into the inquiry tube, fully inverting and uniformly mixing, if white precipitation appears, standing at 75 ℃ for 15-30 min, wherein the precipitation can disappear;
(E) Adding 200ul of absolute ethyl alcohol into a tube, fully and uniformly mixing, wherein flocculent precipitate can be generated at the moment without influencing DNA extraction, adding the solution and the flocculent precipitate into an adsorption column, and standing for 2min;
(F) Centrifuging at 12000rpm for 2min, discarding the waste liquid, and placing the adsorption column into a collecting pipe;
(G) Adding 600ul of rinsing liquid (75% ethanol) into the adsorption column, centrifuging at 12000rpm for 1min, discarding the waste liquid, placing the adsorption column into a collecting pipe, and repeating for one time;
(H) Centrifuging at 12000rpm for 2min, and placing the adsorption column in a 50 deg.C incubator for several minutes;
(I) Placing the adsorption column into a sterilized 1.5ml centrifuge tube, suspending and dripping 100ul of the washing liquid preheated by a water bath at 65 ℃ into the center of the adsorption film, standing for 5min at room temperature, and centrifuging for 1min at 12000 rpm;
(J) And adding the eluent obtained by centrifugation into an adsorption column, standing for 2min at room temperature, and centrifuging for 2min at 12000rpm to obtain the genome DNA of the lactobacillus.
The 16S rDNA gene sequence of the Lactobacillus helveticus OPB102 is as follows:
GAATGGCGGGCGTGCTAATACATGCAAGTCGAGCGAGCAGAACCAGCAGATTTACTTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACAGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGACCTTACCAGTCTTGACATCTAGTGCCATCCTAGAGATAGAGTTCCCTTCGGGACGCTAGACAGTGTGCATGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGGAAATTGCTGCTTAGACGGCTCCTTCCCGAAGGTTAGGCCACCGGCTTTGGGCATTGCAGACTTCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTTCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTCCTAATCTCTTAGGATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGGACACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCAGTACTACTCTATCGTTCTCACACACAGAGCTTTACGATCCGAAACTTCTCACTCACGCGCG。
（2）16S r DNA PCR
bacterial 16S r DNA 50. Mu. LPCR reaction System: 24 mu L of Taq enzyme; dd H2O: 18. Mu.L; 27F, 2. Mu.L; 1492R, 2. Mu.L; and (3) a template: 4. Mu.L.
PCR conditions: 94℃for 5min (i.e.step 1); 94℃for 30s (i.e.step 2); 55℃for 30s (i.e.step 3); 30s at 72 ℃ (i.e. step 4); step2 to step4 (30×); and at 72℃for 5min.
Preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;
the PCR products were sent to a professional sequencing company, and the sequencing results obtained were compared with searches and similarities performed in Gen Bank using BLAST (see FIG. 8), and identified as strains of Lactobacillus helveticus, and stored at-80 ℃.
Example 2: lactobacillus helveticus OPB102 has good tolerance to simulated gastrointestinal fluids
In vitro simulated gastric fluid: the pH value of the 0.1mol/L potassium phosphate buffer solution is respectively adjusted to 2.5, pepsin (10 g/L) is added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, so that simulated gastric juice can be obtained.
In vitro simulated intestinal juice: the pH value of the 0.1mol/L potassium phosphate buffer solution is regulated to 6.8, pancreatin (10 g/L) and pig bile salt (3 g/L) are added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, thus obtaining the simulated intestinal juice.
Gastric digestion was simulated:
taking culture solution of 3.0 mL Lactobacillus helveticus OPB102, centrifuging for 3min, collecting mycelium, placing in 3.0 mL of pH 2.5 simulated gastric juice environment, and simulating gastric juice digestion under anaerobic condition of 37deg.C and 90 r/min. Samples were taken at 0, 1.0, 2.0, 3.0 and h, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.
Intestinal digestion was simulated:
centrifuging culture solution of Lactobacillus helveticus OPB102 at 3.0 mL for 3min, collecting mycelium, and placing in 3.0 mL simulated intestinal juice environment with pH value of 6.8 at 37deg.C under anaerobic condition of 120 r/min to simulate intestinal juice digestion. Samples were taken at 0, 2.0, 4.0, 6.0, 8.0 and h, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.
The survival rate (%) was calculated as the ratio of the number of viable bacteria at the time of sampling to the number of viable bacteria at the time of 0h in the culture solution, expressed as%. The experimental results are shown in tables 1 and 2, and the results show that the lactobacillus helveticus OPB102 has better tolerance to artificial simulated gastrointestinal fluid.
Example 3: lactobacillus helveticus OPB102 has no toxic and side effects on SFP-class Balb/c mice
Suspending Lactobacillus helveticus OPB102 in 100g/L skim milk solution to give 4.0X10-g concentration 9 CFU/mBacterial suspension of L. 40 SPF-class Balb/c mice are bred in the environment of the light irradiation time of 12 h/12 h, wherein the male body mass is 18-20 g, the temperature is 20-25 ℃, the relative humidity is 50+/-5%. The group was divided into Lactobacillus helveticus OPB102 and control group. The group of Lactobacillus helveticus OPB102 was given 0.3mL of this concentrated bacterial suspension once daily for gavage, and the control group was gavaged with the same volume of 100g/L skim milk solution without Lactobacillus helveticus OPB102, and the death and weight were recorded for one week.
The results of these tests are shown in Table 3. These results indicate that the feeding concentration is 1X 10 9 CFU/Lactobacillus helveticus OPB102 has no obvious effect on mice, no obvious change of body weight and no death phenomenon. The appearance of the mice has no obvious pathological symptoms.
Example 4: measurement of the time to stool, the number of stool particles and the stool quality of constipation mice by lactobacillus helveticus OPB 102.
30 SPF-class Balb/c mice are bred in the environment of the light irradiation time of 12 h/12 h, wherein the male body mass is 18-20 g, the temperature is 20-25 ℃, the relative humidity is 50+/-5%. 30 mice were randomly divided into 3 groups of 10 mice each, and the groups were a blank control group, a model control group and an experimental group. Model control group and experimental group mice were perfused with loperamide hydrochloride (5 mg/(kg·bw)), and were continuously perfused with stomach for 7d each time in the morning and evening, to construct constipation models. The blank group was filled with an equal amount of distilled water. After successful modeling, mice in the blank control group and the model control group are respectively filled with 0.3mL of distilled water, mice in the experiment group are filled with 0.3mL of gastric probiotic suspension (1.0 g/(kg.bw), the mice are fed normally during the test once a day, and are continuously fed for 14 days, the mice are fed in a single cage, filter paper is filled under the cage, and the defecation amount of the mice in 6 hours is recorded after 14 days.
Example 5: effect of Lactobacillus helveticus OPB102 on constipation mice intestinal ink propulsion experiments
30 SPF-class Balb/c mice are bred in the environment of the light irradiation time of 12 h/12 h, wherein the male body mass is 18-20 g, the temperature is 20-25 ℃, the relative humidity is 50+/-5%. The animals were randomly divided into 3 groups of 10 animals each, and the three groups were a blank control group, a model control group, and a probiotic experimental group. Model control group and experimental group mice were perfused with loperamide hydrochloride (5 mg/(kg·bw)), and were continuously perfused with stomach for 7d each time in the morning and evening, to construct constipation models. The blank control group and the model control group were respectively filled with 0.3mL of distilled water, 0.3mL of probiotic suspension (1.0 g/(kg. Bw), fed normally once a day during the test, fed continuously for 14 days, fasted for 24 hours after 14 days, the blank control group was given an equal volume of distilled water, after molding for 30 minutes, each group was filled with ink (0.1 mL/10 g), the mice were sacrificed after 20 minutes by cervical vertebra removal, the small intestine was opened, the upper end to the pylorus was cut, the lower end to the intestinal canal of the ileocecum, gently pulled into a straight line, the ink advancing length and the total length of the small intestine were measured, and the ink advancing rate was calculated, and the test results are shown in Table 5.
Ink advance (%) = (ink advance length/total length of small intestine) ×100%
After each group of test mice was continuously perfused with the corresponding probiotic suspension for 7 and 14 days, the molding was successful. And the ink propulsion rate test of the intestinal tracts of the mice is carried out, and the ink propulsion rate of the intestinal tracts of the mice in each group is shown in table 5. As can be seen from Table 5, the ink push rate of the model group was significantly lower than that of the blank group, indicating that the constipation model was established in the mice. Compared with the model group, the small intestine ink propulsion rate of the probiotics group is higher, the difference is obvious, the probiotics group can promote small intestine peristalsis, meanwhile, the small intestine propulsion rate of 14 days is higher than the small intestine propulsion rate of 7 days, and the probiotics group is more beneficial to improving defecation conditions after long-term administration.
Example 6: effect of Lactobacillus helveticus OPB102 on pathological changes of colon tissue of model mice
For example 5, clean colon tissue samples were cut with sterile surgical scissors 2 cm, placed in 10% formalin solution for overnight fixation, and then prepared into paraffin sections of colon tissue. Sections were stained with hematoxylin-eosin (HE staining), alisxin blue-periodate schiff stain (AB-PAS staining). The colon histopathological changes were observed under an optical microscope and stored by photographing (fig. 3).
As shown in fig. 3, the normal colon tissue morphology is normal, the intestinal glands of the tissue mucous membrane layer are orderly arranged, the structure is clear, and the villi are compact and orderly. In contrast, the colon muscle layer of the model group was thinned, the villi was broken loose, the submucosa was damaged, and the intestinal gland was disordered. The colon muscle layer of the probiotics group is thickened, the intestinal gland arrangement is close to the normal level, the damage of submucosa is reduced, the tissue morphology is improved compared with that of the model group, and the probiotics group has a certain protection effect on intestinal tracts. By observing the ratio of the villus length to the crypt depth of panel A in FIG. 3, the model group has the shortest villus, and the villus length after feeding with Lactobacillus helveticus OPB102 is recovered, thus showing that Lactobacillus helveticus OPB102 can improve the colon villus length to some extent.
After AB-PAS staining, a large number of aligned goblet cells were observed in the normal group, whereas fewer goblet cells were observed in the model group. The lactobacillus helveticus OPB102 can restore the ordered arrangement of the goblet cells, increase the number of the goblet cells, restore the damaged colon tissues, increase the number of the goblet cells, restore the normal level state of the colon tissues, is more beneficial to the colonial growth and improves constipation.
Example 7: effect of Lactobacillus helveticus OPB102 on constipation mice intestinal flora
30 SPF-class Balb/c mice are bred in the environment of the light irradiation time of 12 h/12 h, wherein the male body mass is 18-20 g, the temperature is 20-25 ℃, the relative humidity is 50+/-5%. The random number of the bacteria was 4 groups, each group was 10, and the bacteria were a blank group (NC), a model group (MC), a probiotic group (YS) (Lactobacillus helveticus OPB102, 50 hundred million CFU/g) and a mixed bacteria positive group (YX) (Lactobacillus plantarum: lactobacillus bifidus: bifidobacterium bifidus: lactobacillus acidophilus: lactobacillus paracasei mixed bacteria, the mass ratio of which was 1:1:1:1:1, and the bacteria were commercially available, 50 CFU/g). The model group, the probiotic group and the mixed bacteria positive group mice were perfused with loperamide hydrochloride (5 mg/(kg.bw)), and were continuously perfused with stomach for 7d each time in the morning and evening, to construct a constipation model. The blank group and the model control group were respectively filled with 0.3mL of distilled water, the probiotic group and the mixed bacteria positive group were filled with 0.3mL of probiotic suspension (1.0 g/(kg. Bw) of the conversion amount was dissolved in 0.3 mL), and the test period was normally fed once a day, continuously fed for 14 days, and fasted for 24 hours after 14 days. The colon content of the mice is collected in a sterile freezing tube, the operation is rapid, the pollution is avoided as much as possible in the process, all samples are immediately placed in liquid nitrogen after being collected, and the dry ice is stored and transferred to Shanghai Meiji biological medicine science and technology Co for sequencing after the sampling is finished. The PCR reaction parameters of the formal test are as follows: (a) denaturation at 95℃for 3 min; (b) Cycle number (denaturation at 95℃30 s; annealing temperature 30 s; extension at 72℃45 s); (c) extending at 72 ℃ for 10 min, and preserving heat at 10 ℃. The PCR products were detected using 2% agarose gel electrophoresis. Sequences were clustered into Operational Taxonomies (OTUs) using the USEARCH11 usperse algorithm (version 7.0.1090 http:// drive5.Com/uparse /), with a similarity of 97%. Samples are further analyzed based on the minimum number of sample sequences extracted. Data analysis was performed using IBM SPSS Statistics software and GraphPad Prism 8.0.2 software.
1. Analysis of sample species composition
The Venn diagram can be used for counting the number of common and unique species (such as OTU) in different groups, and can intuitively represent the species (such as OTU) composition uniqueness and overlapping condition of colon content samples. We analyzed the number of OTUs in four groups of mice (blank, model, positive control, lactobacillus helveticus) and plotted Venn's chart, see fig. 4. The figure shows that there are unique OUT among different samples, 551 OUT in total, and 407 OUT overlaps among blank group, model group, positive control group and lactobacillus helveticus group. The blank (NC), model (MC), positive control (YX), lactobacillus helveticus (YS) had 13, 10, 5 and 3 unique OUT, respectively. The results showed that the number of intestinal flora OTUs increased after administration in the positive control group, the lactobacillus helveticus group, and that the number of OTUs in both the YS group and the YX group was higher than that in the MC group, and more similar to that in the NC group.
2. Colony composition analysis
According to the OTU classification analysis result, species composition conditions among different groups can be known. To further more intuitively present the different dominant species of each group at a certain taxonomic level (e.g. domain, kingdom, phylum, class, order, family, genus, species, OTU, etc.), and to further elucidate the effect of lactobacillus helveticus on the composition of the intestinal flora, we analyzed the composition of the intestinal flora at the family and genus level, and the results are shown in fig. 5 and 6. As can be seen from fig. 5, lactobacillaceae, muribaculaceae and Lachnospiraceae are three dominant bacterial groups in all groups at the family level. The percentage of Muribaculaceae, lactobacilliaceae and Lachnospiraceae in the whole flora was approximately 70%, and the three beneficial bacteria in the model group were relatively reduced by only 60% compared to the blank group. After lactobacillus helveticus treatment, the total number of Lactobacillaceae, muribaculaceae and Lachnospiraceae all tended to rise, especially the rising of Lactobacillaceae and Muribaculaceae was most obvious. Furthermore, eggerthellac is the most pathogenic bacteria in the current study that promote enteritis, and is highest in the model group, and the blank group, the positive control group and the probiotic group are relatively low. Prevoltellaceae is a commensal intestinal bacterium involved in intestinal mucosal mucus formation and SCFA production, and is more abundant in the NC, YX and YS groups than in the MC group. The increase in Prevotellaceae contributes to the formation of intestinal mucosa. This is consistent with the results of the colon section staining study above.
As can be seen from fig. 6, the MC group lactic acid bacteria content is significantly reduced at the genus level compared to the blank group, which may be related to the side effects of loperamide hydrochloride. The lactic acid bacteria content of the YX and YS groups is obviously increased, and the levels of the YX and YS groups are close. The results show that lactobacillus helveticus increases the content of lactobacillus.
3. Relationship between sample and species
Differences in bacterial genus between groups were investigated by analyzing the relationship between samples and species. As shown in FIG. 7, the distribution ratio of each dominant species in different groups is shown by a visual circle chart. Each group of flora consists essentially of Lactobacillus, norank_f __ Muribaculaceae, norank_f __ lachnospirace, unclassified_f __ lachnospirace and bacterioides. Lactobacillus accounts for 32% of the blank and 19% of the model. Compared with the model group, the Lactobacillus content of each treatment group is obviously increased, and the Lactobacillus content after YX and YS treatment is 25% and 24%, respectively. The improvement in the YX and YS treatment groups is particularly significant. Lactobacillus inhibits intestinal pathogenic bacteria by enhancing mucosal barrier, stimulates goblet cells of intestinal epithelium, and generates a large amount of mucus-inhibiting pathogenic bacteria to invade intestinal epithelium. In addition, lactobacillus tightly binds to the intestinal epithelium and increases the intercellular integrity of the tight junction at the tip to maintain intestinal permeability. Norank_f __ Muribaculaceae represents 24% and 22% respectively in the blank and model groups. After YX and YS treatments, the norank_f __ Muribaculaceae accounted for 25% and 29%, respectively. The proportion of norank_f __ Muribaculaceae in the model group was decreased compared to the blank group, while the proportion of YX and YS treatments was increased, approaching the blank group.
Example 8: effect of Lactobacillus helveticus OPB102 on the rare rate, rare grade and diarrhea index of diarrhea mice
40 SPF-class Balb/c mice are bred in the environment of the light irradiation time of 12 h/12 h, wherein the male body mass is 18-20 g, the temperature is 20-25 ℃, the relative humidity is 50+/-5%. The cells were randomly divided into 4 groups of 10 cells, and the cells were selected from a blank control group, a model group, a probiotic test group and a positive control group (Yimengding). The mice in the blank control group were given 10 mL/kg of the vehicle (3.2% Tween-80 aqueous solution), the mice in the model group were given 10 mL/kg of the vehicle (3.2% Tween-80 aqueous solution), the mice in the probiotic experimental group were given 1.0g/kg of the probiotic (3.2% Tween-80 aqueous solution as the vehicle), and the mice in the positive control group were given 10 mg/kg of Yimengting (3.2% Tween-80 solution as the vehicle) 1 time/d for 7 d. After the last administration of 1 h, the mice in the model group, the probiotics experiment group and the positive control group are subjected to gastric administration, castor oil 10 mL/kg is administered, the mice are immediately placed in a mouse cage, the normal stool number, the loose stool number and the loose stool level of the mice at each interval of 1 h in 5h are observed and recorded, and the diarrhea index is calculated according to the loose stool rate and the loose stool level. The calculation formula of the loose stool rate is as follows: rare stool rate = number of rare stools discharged in a certain time/total number of bowel stools diarrhea index calculation formula: diarrhea index = runny bowel rate x runny bowel grade. Compared with the control group, the probiotic bacteria and the diarrhea index of the mice with diarrhea after the easy Mongolian group are acted are reduced. Meanwhile, the diarrhea index of the probiotics group is slightly lower than that of the easy Mongolian group. As can be seen from table 6, the probiotic preparation can regulate intestinal flora, effectively improve castor oil-induced diarrhea in mice, and has an antidiarrheal effect similar to that of a positive control easy-to-stop group.
Comparative example 1:
the specific embodiment is the same as in example 5, except that Lactobacillus helveticus OPB102 is replaced by Lactobacillus helveticus LH-90 (commercially available strain of Jia Yi in the middle, and the 14-day propulsion rate of the small intestine ink of the constipation mice is measured, and the result shows that the propulsion rate of the Lactobacillus helveticus LH-90 to the small intestine ink of the constipation mice is 30.32+/-0.66%, which is obviously lower than the propulsion rate of the probiotics group 42.43 +/-1.74%. It can be seen that lactobacillus helveticus OPB102 has a significant effect on constipation mice intestinal thrust. Compared with the propulsion rate of the Lactobacillus helveticus LH-90, the Lactobacillus helveticus OPB102 relieves constipation and promotes small intestine peristalsis, which is obviously superior to the Lactobacillus helveticus LH-90.
Comparative example 2
The specific embodiment is the same as example 8, except that Lactobacillus helveticus OPB102 is replaced by Lactobacillus helveticus LH-90 (commercially available strain of Jia Yi in the middle, and the loose stool rate, loose stool grade and diarrhea index of diarrhea mice are measured, and the result shows that the Lactobacillus helveticus LH-90 has no alleviation effect on the loose stool rate, loose stool grade and diarrhea index of diarrhea mice compared with the diarrhea model group (0.82+ -0.07, 1.23+ -0.21, 1.01+ -0.13) of the diarrhea mice, wherein the loose stool rate, loose stool grade and diarrhea index of diarrhea mice are respectively 0.78+ -0.12, 1.21+ -0.09, 1.0+ -0.03.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (8)
1. A lactobacillus helveticus OPB102 capable of relieving constipation and diarrhea, characterized in that: the classification names are: lactobacillus helveticus（Lactobacillus helveticus）The preservation number is: CGMCC No.26806, date of preservation: 2023, 2, 14, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.
2. Use of lactobacillus helveticus OPB102 as claimed in claim 1 for the preparation of a medicament for alleviating constipation and diarrhea.
3. The use according to claim 2, characterized in that: the medicament has at least one of the following effects:
improving constipation, in particular shortening defecation time, increasing fecal quantity and quality and increasing small intestine propulsion rate;
alternatively, improving constipation, specifically restoring damaged colon tissue, improving villus density and increasing goblet cell number, restoring normal colon tissue, providing a better colonization environment for the flora;
or, improving constipation, in particular improving intestinal flora environment, promoting beneficial bacteria reproduction and inhibiting harmful bacteria reproduction;
alternatively, the diarrhea is ameliorated, specifically the diarrhea loose stool rate, loose stool grade, and diarrhea index.
4. The use according to claim 2, characterized in that: the lactobacillus helveticus OPB102 is a live strain.
5. The use according to claim 2, characterized in that: the medicament further comprises a pharmaceutically acceptable carrier, which comprises: one or more of a filler, diluent, binder, disintegrant, and lubricant.
6. The use according to claim 5, characterized in that: the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrating agent is one or more of sodium carboxymethyl starch, crosslinked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearate fumarate, talcum powder or silicon dioxide.
7. Use of lactobacillus helveticus OPB102 as claimed in claim 1 for the preparation of fermented food, metazoan, ferments.
8. The use according to claim 7, characterized in that: the application comprises using Lactobacillus helveticus OPB102 as a fermentation microorganism, and fermenting with food material.
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| CN117100773B (en) * | 2023-10-25 | 2024-02-09 | 山东威曼宠物食品有限公司 | Lactobacillus plantarum King07 metazoan preparation and application thereof |
| CN118165891B (en) * | 2024-05-09 | 2024-07-05 | 善恩康生物科技（苏州）有限公司 | Lactobacillus helveticus capable of relieving diarrhea and application thereof |
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