CN117903995A - Lactobacillus plantarum YM-4-3 and application thereof - Google Patents
Lactobacillus plantarum YM-4-3 and application thereof Download PDFInfo
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Abstract
The invention discloses a lactobacillus plantarum YM-4-3 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2023253 in 2023 and 03 and 06 days. The invention has the advantages that: lactobacillus plantarum YM-4-3 can effectively improve constipation, has good acid resistance, bile salt resistance and gastrointestinal tract adhesion capability, has high survival rate in gastrointestinal tract environment, can remarkably inhibit reproduction of gastrointestinal pathogenic bacteria, in particular staphylococcus aureus and salmonella typhimurium, and can be used for preparing products for preventing or improving constipation.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum capable of improving constipation and application thereof.
Background
Constipation is a complex disorder caused by abnormal movement of large intestine, and the prevalence of constipation is obviously increased by up to one third compared with the past year. Studies have shown that probiotics help to improve constipation. And compared with cathartic medicines, the probiotics has little side effect. The probiotics promote the growth and colonization of beneficial bacteria in vivo by reducing the pH value of the microenvironment in vivo, and adhere to intestinal mucosa and pathogenic bacteria competition colonization sites, so that the pathogenic bacteria are inhibited to a certain extent, and the function of regulating intestinal flora is achieved. Therefore, the screening of probiotics capable of effectively improving constipation has wide market prospect and value.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum capable of effectively improving constipation.
The invention provides lactobacillus plantarum YM-4-3, wherein the lactobacillus plantarum YM-4-3 (Lactobacillusplantarum YM-4-3) is preserved in China center for type culture Collection (CCTCC NO: M2023253) in the year 2023, namely 03 and 06.
The invention provides a microbial inoculum comprising lactobacillus plantarum YM-4-3.
The invention provides a fermentation product obtained by fermenting lactobacillus plantarum YM-4-3 or a microbial inoculum.
The invention provides an application of lactobacillus plantarum YM-4-3 or a microbial inoculum or a fermentation product in preparing a product for preventing or improving constipation.
The product is food, health product or medicine.
The invention has the beneficial effects that:
The lactobacillus plantarum YM-4-3 separated from the traditional anaerobic fermented soybean in Yimen county of Yuxi city of Yunnan province can effectively improve constipation, has good acid resistance, bile salt resistance and gastrointestinal tract adhesion capacity, has high survival rate in gastrointestinal tract environment, can obviously inhibit the reproduction of gastrointestinal tract pathogenic bacteria, in particular to staphylococcus aureus and salmonella typhimurium, and can be used for preparing products for preventing or improving constipation.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum YM-4-3;
FIG. 2 is a diagram showing a partial differential metabolite of fermented soybean milk and unfermented soybean milk obtained by fermentation using Lactobacillus plantarum YM-4-3;
FIG. 3 is a display of the portion of the metabolites most significantly up-and down-regulated by the differential metabolites.
Detailed Description
The following describes the embodiments of the present invention further with reference to the drawings. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention provides a lactobacillus plantarum YM-4-3 which is separated from traditional anaerobic fermentation fermented soybeans in Yimen county and De of Yumen City of Yunnan province, wherein the lactobacillus plantarum YM-4-3 (Lactobacillus plantarum YM-4-3) is preserved in the China Center for Type Culture Collection (CCTCC) on the year 03 and the day 06, and the preservation number is M2023253; the preservation address is: eight paths of Lopa nationality mountain in Wuhan city of Hubei province; contact phone: 027-68754052.
The invention provides a microbial inoculum comprising lactobacillus plantarum YM-4-3 and other auxiliary materials.
The invention provides a fermentation product obtained by fermenting lactobacillus plantarum YM-4-3 or a microbial inoculum.
The invention provides an application of lactobacillus plantarum YM-4-3 or a microbial inoculum or a fermentation product in preparing a product for preventing or improving constipation. The product is food, health product or medicine.
The Lactobacillus plantarum YM-4-3 and its application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the invention.
Example 1
Separation, purification, identification and preservation of lactobacillus plantarum YM-4-3
1. Isolation and purification of strains
Lactobacillus plantarum YM-4-3 is isolated from traditional anaerobic fermented soybean in Yimen county of Yuxi city of Yunnan province, and the method comprises the following steps: 100g of a traditional anaerobic fermentation fermented soybean sample in Yimen county and De of Yuxi city of Yunnan province is taken for standby. Weighing 1.0g of fermented soybean sample, placing in 10mL of sterile physiological saline, fully washing and desorbing microorganisms, grinding the sample, inoculating the ground sample into 5mL of MRS broth culture medium, and standing and anaerobic culturing for 24h at 37 ℃; then, the mixture is subjected to gradient dilution by using sterile physiological saline, the diluted solution is coated on MRS agar culture medium, and the mixture is placed at 37 ℃ for anaerobic culture for 48 to 72 hours. After single colony grows out, repeatedly streaking the single colony on an MRS agar culture medium, and separating and purifying strains. Single colonies are selected according to the morphological characteristics of the colonies, such as morphology, color, size, whether the colonies are raised, transparency, whether edges are regular, and the like. Single colonies were inoculated into 5mL of sterilized MRS broth medium, and after stationary culture at 37℃for 48 hours, they were kept in a-80℃refrigerator with 50% glycerol for use.
The formula of the MRS broth culture medium is as follows: 10g of protein wine, 8g of beef powder, 4g of yeast powder, 20g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of tri-ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 1L of distilled water, wherein the pH value is 6.2+/-0.2.
2. Morphological identification of strains
The isolated Lactobacillus plantarum YM-4-3 strain was subjected to gram staining and microscopic identification and catalase test. The result shows that the strain is short bacillus with gram-positive staining bacteria and negative catalase test; colonies on the MRS agar medium plate are milky white, smooth in surface, obvious in bulges, neat in edges, opaque and glossy. The colony morphology of Lactobacillus plantarum YM-4-3 is shown in FIG. 1.
3. Molecular biology identification of strains
The isolated and purified single colonies were sent to the biological engineering (Shanghai) Co., ltd, and the primers were identified as follows: 27F:5'-AGAGTTTGATCMTGGCTCAG-3';1492R:5'-GGTTACCTTGTTACGACTT-3'. The sequencing was analyzed using BLAST program in NCBI, and the result showed that YM-4-3 strain was Lactobacillus plantarum among lactic acid bacteria, which had a homology of 99.95% with known Lactobacillus plantarum in GeneBank database, designated Lactobacillus plantarum (Lactobacillusplantarum YM-4-3).
4. Preservation of strains
Lactobacillus plantarum YM-4-3 (Lactobacillus plantarum YM-4-3) was deposited with the China center for type culture Collection, address: eight paths of Lopa nationality mountains in Wuchang district of Wuhan, hubei province with the preservation number of CCTCCNO: M2023253, telephone: 027-68754052.
Example 2
Acid and bile salt resistance analysis of lactobacillus plantarum
1. Acid resistance test
After culturing Lactobacillus plantarum YM-4-3 in MRS broth for 24h at 37℃1mL of the bacterial liquid was taken out in a sterilized centrifuge tube of 1.5 mL. Treatment with MRS broth pH 3.0. 3 samples are repeated in parallel, 1mL of bacterial liquid is taken in 0h and 3h respectively, after a certain concentration is diluted, the viable count is measured by a viable count plate method: the diluted sample to be tested is evenly coated on a sterilized and cooled MRS agar plate, and the number of viable bacteria is measured after the culture is carried out for 24 hours at 37 ℃, wherein the number of viable bacteria in each plate is effective between 20 and 200. The strain survival rate was calculated as follows:
wherein N1 is the number of viable bacteria after 3 hours of culture in a culture medium with a pH of 3.0; n0 is the initial viable count of the strain.
The calculation result shows that: the survival rate of Lactobacillus plantarum YM-4-3 after 3h of acid treatment with pH 3.0 was 90.5%.
Example 3
Test for bile salt resistance
After culturing Lactobacillus plantarum YM-4-3 in MRS broth for 24h at 37℃1mL of the bacterial liquid was taken out in a sterilized centrifuge tube of 1.5 mL. Treated with porcine bile salt at a concentration of 0.3%. 3 samples are repeated in parallel, 1mL of bacterial liquid is taken respectively during the culture for 0h and 3h, and the viable count is measured by a viable count plate method after a certain concentration is diluted: the diluted sample to be tested is evenly coated on a sterilized and cooled MRS agar plate, the viable bacteria number is counted after the culture is carried out for 24 hours at 37 ℃, and the viable bacteria number in each plate is effective between 20 and 200. The strain survival rate was calculated as follows:
Wherein N1 is the number of viable bacteria after culturing YM-4-3 strain for 3 hours by adopting a culture medium containing bile salts; n0 is the initial viable count of YM-4-3 strain.
The calculation result shows that the survival rate of the lactobacillus plantarum YM-4-3 after being treated by the pig bile salt culture medium with the concentration of 0.3 percent is 85 percent. Experimental results show that the lactobacillus plantarum YM-4-3 has strong acid resistance and cholate resistance.
Example 4
Simulated artificial gastrointestinal fluid tolerance test
Preparation of artificial gastric juice: naCl 0.2g/100mL, pepsin 0.35g/100mL, pH 2.0,2.5,3.0,4.0 with 1mol/L HCl, filtering and sterilizing (0.22 um film), and making into artificial gastric juice with different pH values.
Preparation of artificial intestinal juice: naHCO 3 1.1.1 g/100mL, naCl 0.2g/100mL, trypsin 0.1g/100mL, pig bile salt 1.8g/100mL, adjusting pH value to 8.0, filtering, sterilizing for standby, and obtaining the artificial intestinal juice.
And adding 0.5mL of the test bacterial liquid into 4.5mL of artificial gastric juice, shaking for 10 seconds, then placing into anaerobic culture at 37 ℃, sampling after treatment for 0h, 1h, 2h and 3h, and measuring the number of living bacteria. Then 0.5mL of the 3h artificial gastric juice reaction solution is added into 4.5mL of artificial intestinal juice, the mixture is placed into anaerobic culture at 37 ℃ after shaking for 10s, and sampling is carried out for 0h, 3h, 8h and 24h of treatment, and the number of viable bacteria is measured.
N0: the number of viable bacteria before treatment;
n1: number of viable bacteria treated with artificial gastric juice or artificial gastrointestinal fluid.
The test result shows that the lactobacillus plantarum YM-4-3 can tolerate gastrointestinal fluid transportation with pH value of 4.0 and 3.0, and the survival rate is higher than 90% at the end of the test; has certain tolerance to gastrointestinal fluid transportation with pH of 2.5, the survival rate is 85.58% after gastric fluid treatment for 3 hours, and the survival rate is 72.57% after intestinal fluid treatment for 24 hours; for gastrointestinal fluid transport at pH2.0, the survival rate is reduced to 35.86% after gastric fluid treatment for 3 hours, the survival rate is kept almost unchanged after intestinal fluid treatment for 8 hours, and no viable bacteria are detected after treatment for 24 hours. It is demonstrated that Lactobacillus plantarum YM-4-3 is tolerant to gastrointestinal fluid transport at pH4.0, 3.0 and 2.5, and that stomach pH is maintained between 3.0 and 5.0 after normal human feeding. Thus, lactobacillus plantarum YM-4-3 can survive in the gastrointestinal tract of humans.
Example 4
Adhesion test of Lactobacillus plantarum YM-4-3 to intestinal epithelial cells
The test steps are as follows:
(1) Lactobacillus plantarum YM-4-3 is inoculated into 200mL of liquid culture medium according to the inoculation amount of 1%, the culture is carried out for 48 hours at 37 ℃, and physiological saline is used for adjusting the bacterial suspension to 10 7 cfu/mL for later use.
(2) HT-29 cells were cultured in DMEM medium, incubated overnight in a 5% CO 2 incubator at 37℃and the medium was changed the next day, cells were passaged with 0.2% pancreatin until the cell proliferation rate reached about 80%, and logarithmic growth of cells were taken for experiments at a ratio of 1:3.
(3) Co-culturing HT-29 cells with a probiotic strain: the cultured HT-29 is transferred into a 6-well plate for overnight culture, the cultured lactobacillus plantarum YM-4-3 is washed by PBS and resuspended in DMEM culture medium, and the resuspension is added into the HT-29 cell well plate for 3 hours according to the same volume. After washing the cells with PBS, they were fixed with methanol for 30min, and were observed by gram staining.
The adhesion rate of the counted YM-4-3 on HT-29 cells is 79%, which shows that the Lactobacillus plantarum YM-4-3 has stronger capability in the aspect of cell colonization.
Example 5
Bacteriostasis test
① Experimental materials
The probiotic strain YM-4-3, food-borne escherichia coli O157: H7, salmonella typhimurium and staphylococcus aureus are screened. Listeria monocytogenes is stored in the application microbiology laboratory of the university of kunming university student's life sciences and technology.
Culture medium
MRS agar medium;
LB medium: 10.0g of peptone, 5.0g of yeast powder, 5.0g of sodium chloride and 1.0g of glucose, adjusting the pH value to 7.1+/-0.1 (25 ℃), adding distilled water to a constant volume to 1L, and sterilizing for 15min at 121 ℃;
BHI medium: 10.0g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride and 1.0g of glucose, adjusting the pH value to 7.4+/-0.1 (25 ℃), adding distilled water to a constant volume to 1L, and sterilizing at 121 ℃ for 15min.
② Test method
The bacteriostasis capacity of the strain is measured by using an agar pore diffusion method, and the specific method comprises the following steps: firstly pouring a thin layer of MRS agar culture medium into a flat plate, solidifying, then pouring the thin layer of MRS agar culture medium into an oxford cup, pouring the thin layer of MRS agar culture medium into a second layer of MRS agar culture medium, taking out the oxford cup after solidifying, adding 100 mu L of YM-4-3 with the concentration of 10 9 cfu/mL which is activated in advance into a hole, placing the mixture into a culture box for culturing for 24 hours at 37 ℃, diluting food-borne escherichia coli O157H 7, salmonella typhimurium, staphylococcus aureus and single cell listeria monocytogenes to 10 6 cfu/mL, respectively adding the mixture into sterilized LB and BHI culture mediums cooled to about 45 ℃ in the amount of 1:100, shaking the mixture uniformly, quantitatively adding 20 mL/flat plate, cooling to prepare a bacteria-containing flat plate, culturing at the temperature of 37 ℃ for 48 hours, and measuring the size of a bacteriostasis zone of YM-4-3 aiming at different pathogenic bacteria by using a vernier caliper, wherein the measurement results are shown in the table below:
the results show that the lactobacillus plantarum YM-4-3 has certain inhibition capability on 4 common pathogenic bacteria of escherichia coli, salmonella typhimurium, staphylococcus aureus and listeria, wherein the inhibition capability on the escherichia coli and the staphylococcus aureus is the best, and the lactobacillus plantarum has the least inhibition capability on the salmonella typhimurium and the listeria monocytogenes.
Example 6
Metabolomic analysis of fermented soy milk
And (3) regulating the bacterial concentration of the activated YM-4-3, washing for 2 times by using normal saline, adding the bacterial concentration into 200mL of sterilized soybean milk, enabling the final bacterial concentration to be 1 x10 8 cfu/mL of soybean milk, and fermenting for 48 hours to obtain the fermented soybean milk. Samples were taken for metabolome analysis.
The metabolites of the fermented soybean milk were analyzed and compared with those of the soybean milk before fermentation by non-targeted metabonomics, and 937 positive ion-mode metabolites and 452 negative ion-mode metabolites were identified in total. Screening for differential metabolites was performed according to VIP >1.0, fc >1.2 criteria. 92 positive ion pattern metabolites differed significantly in the l48.vs.l24 comparison group, with 16 metabolites up-regulated and 76 metabolites down-regulated; there were 34 anion pattern metabolites that differed significantly, with 12 metabolites up-regulated and 22 metabolites down-regulated. The partial metabolites were displayed as shown in FIG. 2. Metabolites that were most significantly up-and down-regulated were selected for display based on log2FC size of the differential metabolites, as shown in figure 3.
The metabolome results show that the content of acidic amino acid in the fermented soybean milk is obviously up-regulated compared with the content of acidic amino acid in the unfermented soybean milk, the acidic amino acid can regulate the balance of intestinal microbiota in intestinal tracts, enhance the number of beneficial bacteria, inhibit the growth of harmful bacteria, and promote healthy intestinal tract environment. In addition, the acidic amino acid can promote intestinal peristalsis and reduce constipation risk. In addition, the active probiotics can increase lactic acid and short-chain fatty acid in the intestinal environment, further reduce the pH value of the intestinal environment, promote intestinal peristalsis, reduce the running time of the intestinal canal and improve constipation; metabolites of probiotics have important regulatory effects on intestinal functions such as nerve conduction and intestinal motility.
Example 7
Improvement effect of lactobacillus plantarum YM-3-4 fermented soybean milk on constipation model mice
1. Experimental animal
The invention selects 48 SPF-class Kunming mice with age of 8 weeks, which are male mice, the mice are purchased from Kunming medical university, and the mice are fed into animal houses with air filtered. All mice were randomly divided into four groups after 3-5 days of adaptive feeding, with 12 mice per group, namely: healthy control group (I), constipation model group (II), probiotic fermented soybean milk group (III) and positive drug feeding group (constipation+positive drug feeding) (IV). The animals are circulated for 12 hours in the room, the temperature is controlled at 25+/-2 ℃, the relative humidity is kept at 50% -70%, the noise in the room is low, the animals can eat and drink water freely, and the mice are fed with sterilized feed treated by radiation and drinking water sterilized by high-pressure steam. Mice body weight, food intake were recorded daily during the test period, and mice faeces were collected daily.
2. Test method
The healthy control group (I) and the constipation model group (II) are filled with 0.2ml of PBS every day, the positive medicine feeding group needs to be filled with 0.2ml of vegetable digestion powder every day at the same time besides 0.2ml of PBS every day, the probiotic fermented soybean milk group is filled with 0.2ml of probiotic fermented soybean milk every day, and the positive medicine feeding group is fed with the same diet and drinking water. After 7 days of gastric lavage, each group of mice was fasted without water for 16 hours and the mice were subjected to a stool test on day 9. After the defecation experiment was completed, the mice were continuously fasted for 16 hours without water control, and intestinal tract propulsion experiments were performed on day 16.
(1) Defecation experiment: after 7 days of gastric lavage, the mice in the healthy control group are fed with PBS, the mice in the next day are fed with 0.2mL of loperamide hydrochloride solution (10 mg/kg. W), after 30min, the mice in each group are fed with 0.2mL of ink by gastric lavage, normal drinking and feeding are recovered, and single-cage feeding is performed. And (5) starting to time when the ink filling is finished, and recording the first black excrement discharge time of the mice.
(2) Intestinal tract propulsion experiment: after the defecation experiment is completed, the mice in the blank control group are fasted without water control for 16 hours, PBS is given to the mice in the next day, 0.2mL of loperamide hydrochloride solution (10 mg/kg b.w) is given to the mice in the other groups, after 30 minutes, the stomach ink is respectively infused into the mice in each group, the mice are killed after 30 minutes, the abdominal cavity is opened, the upper end is cut from the pylorus, the lower end to the cecum, the total length of the small intestine is measured to be the total length of the small intestine, the front edge from the pylorus to the ink is the push length of the ink, and the push rate of the small intestine is calculated according to the following formula.
Small intestine propulsion (%) = (ink propulsion length (cm))/(small intestine total length (cm)) ×100%
The first black discharge time statistics measured are shown in the following table:
The results of the intestinal tract propulsion experiments are shown in the following table:
The mouse defecation experiment shows that the loperamide hydrochloride solution can effectively induce constipation, and the result shows that: the first stool discharge time of the constipation model group is obviously longer than that of the blank control group, and the stool quantity is obviously lower than that of the blank control group, so that the constipation model is successfully modeled. The first defecation time of mice with the probiotic fermented soybean milk filled in the stomach for 7 days is obviously lower than that of a constipation model group, and the fecal quantity is also obviously higher than that of the model group, so that the probiotic fermented soybean milk can be proved to be effective in improving the constipation of the mice. The first defecation time of the mice with 7 days of stomach lavage by the probiotic soybean milk is not greatly different from that of the positive control group, and the effect that the probiotic soybean milk can effectively relieve constipation of the mice can be also demonstrated. After the loperamide hydrochloride solution induces constipation, the small intestine propulsion rate of mice in the constipation model group is significantly lower than that of mice in the blank group. The probiotic fermented soybean milk intervention group can obviously improve intestinal peristalsis and propulsion rate. The result shows that the probiotic fermented soybean milk has good effect of improving constipation.
The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (5)
1. A lactobacillus plantarum YM-4-3, which is characterized in that: the lactobacillus plantarum YM-4-3 (Lactobacillusplantarum YM-4-3) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2023253 in 2023 and 03 month and 06 days.
2. A microbial inoculum comprising the Lactobacillus plantarum YM-4-3 according to claim 1.
3. A fermentation product obtained by fermentation of lactobacillus plantarum YM-4-3 of claim 1 or a microbial inoculum of claim 2.
4. Use of lactobacillus plantarum YM-4-3 according to claim 1 or a microbial inoculum according to claim 2 or a fermentation product according to claim 3 for the preparation of a product for preventing or ameliorating constipation.
5. The use according to claim 4, wherein the product is a food, a health product or a pharmaceutical product.
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