CN112625983B

CN112625983B - Lactobacillus casei L.Casei21 with treatment effect on diarrhea and application thereof

Info

Publication number: CN112625983B
Application number: CN202110242478.5A
Authority: CN
Inventors: 潘玉林; 司书锋; 曹维超; 李翠华; 潘仕城; 侯建亮; 孙川; 周文浩
Original assignee: Shandong Zhongke Jiayi Bio Engineering Co ltd
Current assignee: Minsheng Zhongke Jiayi (Shandong) Biotechnology Co.,Ltd.
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2021-05-28
Anticipated expiration: 2041-03-05
Also published as: CN112625983A

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus casei L.Casei21 with a treatment effect on diarrhea and application thereof, wherein the classification name of the lactobacillus casei L.Casei21 is lactobacillus caseiLactobacillus caseiThe preservation number is CGMCC NO.21373, the preservation date is 14 days 12 months 2020, the preservation unit is the China general microbiological culture Collection center, the preservation unit address is No. 3 of No. 1 Xilu-Shih-Taiyang district, Beijing, and the lactobacillus casei L.Casei21 can be used for preparing products for treating diarrhea. After the product prepared from the lactobacillus casei L.Casei21 is taken, thalli can enter the intestinal tract through the alimentary canal, inhibit pathogenic bacteria and colonize the intestinal tract, keep the balance of environmental flora, improve diarrhea symptoms and reduce diarrhea recurrence risk.

Description

Lactobacillus casei L.Casei21 with treatment effect on diarrhea and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus casei L.Casei21 with a treatment effect on diarrhea and application thereof.

Background

Diarrhea is a common symptom, which refers to frequent defecation obviously exceeding the frequency of ordinary daily habit, thin feces, increased moisture, more than 200g of feces per day, or containing undigested food or purulent blood, mucus. Diarrhea is often accompanied by symptoms such as a sense of urgency, anal discomfort, incontinence, etc. Diarrhea can be treated by taking medicine. Although the effect of the drug treatment is obvious, the defects are also obvious, for example, the drug containing antibiotics can kill pathogenic bacteria and beneficial bacteria together, temporarily relieve symptoms, be not beneficial to later recovery, and lead pathogenic bacteria to generate drug resistance after long-term administration. In addition, if the patient is not fully informed, other diseases may be induced by the drug therapy.

Based on the above, in order to solve diarrhea symptoms, avoid drug resistance and adverse reactions caused by using medicaments and reduce diarrhea recurrence risk, the invention provides lactobacillus casei L.Casei21 with a treatment effect on diarrhea and application thereof.

Disclosure of Invention

Aiming at the problems of drug resistance and adverse reaction possibly caused by drug treatment, the invention provides lactobacillus casei L.Casei21 with a treatment effect on diarrhea and application thereof, and the preserved strain lactobacillus casei L.Casei21 is utilized to prepare an ingestible product, after the oral administration, thalli can enter into intestinal tracts through digestive tracts, inhibit pathogenic bacteria and colonize the intestinal tracts, keep the balance of environmental flora, improve diarrhea symptoms and reduce diarrhea recurrence risk.

In a first aspect, the invention provides a lactobacillus casei L.Casei21, and the classification of the lactobacillus casei L.Casei21 is named as lactobacillus caseiLactobacillus caseiThe preservation number is CGMCC number 21373, the preservation date is 2020, 12 and 14 days, the preservation unit is the China general microbiological culture Collection center, and the preservation unit address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, North Chen, China.

In a second aspect, the invention provides the use of lactobacillus casei l.casei21 in the manufacture of a product for the treatment of diarrhoea.

Further, the product comprises lactobacillus casei powder, and the lactobacillus casei powder is prepared according to the following preparation method:

inoculating lactobacillus casei L.Casei21 into an MRS liquid culture medium according to the inoculation amount of 1% of the volume fraction, and performing centrifugation and vacuum freeze drying on the cultured L.Casei21 bacterial liquid to obtain lactobacillus casei bacterial powder.

Further, the culture temperature of lactobacillus casei l.casei21 was 37 ℃.

Further, the MRS liquid culture medium comprises the following components in percentage by weight:

10g of peptone, 5g of beef powder, 4g of yeast powder and K₂HPO₄•7H₂O2 g, triammonium citrate 2g, CH₃COONa•3H₂O5 g, glucose 20g, Tween 801 mL, MgSO₄•7H₂O 0.2g、MnSO₄•4H₂0.05g of O and 1000mL of distilled water.

Further, the product also comprises isomaltooligosaccharide.

Furthermore, the viable count of the lactobacillus casei L.Casei21 in the product is 50 hundred million/g, 100 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g and 1000 hundred million/g.

The beneficial effect of the invention is that,

the lactobacillus casei21 provided by the invention has good gastric acid resistance and cholate resistance, and also has strong capacity in the aspect of cell colonization, the lactobacillus casei L21 can greatly reduce the adhesion effect of pathogenic bacteria, and metabolites of the lactobacillus casei L21 also have good bacteriostatic effect on three pathogenic bacteria of diarrhea, so that the occurrence of diarrhea can be effectively inhibited. The product prepared from lactobacillus casei L.Casei21 for treating diarrhea also has obvious positive treatment effect on diarrhea patients.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a graph comparing the adhesion rate of strains to Caco-2 cells.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 isolation and purification of bacterial species

Carry out excrement and urine sampling to the lactation baby of Guangxi Bama Yao nationality autonomous county no family genetic disease history, the disinfection operation is worn to the gloves in the sampling process, avoids contacting the sample in order to prevent polluting. The samples were taken out and immediately stored in a refrigerated box and numbered.

Ten times of gradient dilution is carried out on the sample liquid, 100 mu L of each gradient is uniformly coated on an MRS solid plate culture medium, and the components and the content of the MRS solid plate culture medium are as follows:

10g of peptone, 5g of beef powder, 4g of yeast powder and K₂HPO₄•7H₂O2 g, triammonium citrate 2g, CH₃COONa•3H₂O5 g, glucose 20g, Tween 801 mL, MgSO₄•7H₂O 0.2g、MnSO₄•4H₂0.05g of O, 15g of agar and 1000mL of distilled water.

Then taking out and placing in an anaerobic jar, carrying out anaerobic culture at 37 ℃ overnight, and observing that bacterial colonies with different shapes and sizes grow on the flat plate the next day, wherein the bacterial colonies are milky white, convex, irregular in edge and slightly transparent.

Selecting characteristic bacterial colonies of a plurality of lactic acid bacteria for second-generation streak purification treatment, carrying out plate anaerobic activation for 1 day to obtain a culture, and then carrying out strain identification.

Example 2 species identification

Identification unit: biometrics (Shanghai) Ltd.

The primer sequence is as follows: 27F (5 '-AGAGTTTGATCMTGGCTCAG-3'), 1492R (5'-GGTTACCTTGTTACGACTT-3').

Strain sequence: TGCAAGTCGAACGAGTTTTGGTCGATGAACGGTGCTTGCACTGAGATTCGACTTAAAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGCAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAAATTTTCACAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTGAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCT are provided.

And (3) identification result: this strain was identified as Lactobacillus casei. The classification unit is as follows: (ii) Bacteria; firmicutes; bacillus; lactobacillus; lactobacillus acid; lactobacillus.

EXAMPLE 3 product preparation

A product for treating diarrhea comprises lactobacillus casei powder and isomaltooligosaccharide, wherein the lactobacillus casei powder is prepared by the following preparation method:

(1) inoculating lactobacillus casei L.Casei21 into an MRS liquid culture medium according to the inoculation amount of 1 percent of volume fraction for culture, wherein the culture temperature is 37 ℃, and the components and the content of the MRS liquid culture medium are as follows:

10g of peptone, 5g of beef powder, 4g of yeast powder and K₂HPO₄•7H₂O2 g, triammonium citrate 2g, CH₃COONa•3H₂O5 g, glucose 20g, Tween 801 mL, MgSO₄•7H₂O 0.2g、MnSO₄•4H₂0.05g of O and 1000mL of distilled water;

(2) centrifuging and vacuum freeze-drying the cultured L.Casei21 bacterial liquid to obtain lactobacillus casei powder;

regulating the ratio of lactobacillus casei powder to isomaltooligosaccharide, and respectively preparing products with the viable count of lactobacillus casei L.Casei21 of 50 hundred million/g, 100 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g and 1000 hundred million/g.

Example 4 Lactobacillus casei L.Casei21 gastric acid resistance test

Several samples of 100 hundred million/g viable lactobacillus casei l.casei21 prepared in example 3, each 1g in weight, were transferred to a preheated test tube containing 9mL of simulated gastric juice and treated at 37 ℃ and 80r/min for 0.5h, 1.5h and 2h, respectively, each 3 times.

The simulated gastric acid is prepared according to the following method: diluting 9.5% hydrochloric acid 16.4mL with distilled water to pH 2.0, adding pepsin 1.0g per 100mL, mixing, filtering with 0.22 μm sterile filter membrane, and making into preparation.

The experimental procedure was the same as for l.casei21, taking the four lactobacillus strains ATCC334, ATCC11578, ATCC393, ATCC15008 as controls.

And (4) performing gradient dilution on the treated sample solution and the control strain solution, and counting the viable bacteria by using a pouring method. The survival rate data of the strain in the in-vitro simulated gastric acid environment is shown in table 1, and the survival rate of the lactobacillus casei L.Casei21 in the in-vitro simulated gastric acid environment is obviously higher than that of other tested strains, and the cholate resistance is stronger.

TABLE 1 in vitro simulation gastric acid environment survival rate data table for each strain

Treatment time strains	0.5h	1.5h	2h
				L.Casei21	92%	87%	80%
ATCC334	72%	54%	38%
				ATCC11578	53%	46%	23%
ATCC393	71%	37%	22%
				ATCC15008	68%	57%	50%

Example 5 Lactobacillus casei L.Casei21 test for its ability to resist bile salts

Several samples of 100 hundred million/g viable lactobacillus casei l.casei21 prepared in example 3 were weighed, each sample weighed 1g, transferred to a test tube containing 9mL of simulated intestinal fluid, and treated at 37 ℃ and 80r/min for 0.5h, 1.5h and 2h, respectively, each treatment being repeated for 3 times.

The simulated intestinal juice is prepared according to the following method: mixing 6.8g KH₂PO₄Dissolving in 500mL of distilled water, adding 3g of cholate and 10g of trypsin, adjusting the pH value of the solution to 6.8 by using a NaOH solution with the concentration of 4g/L, diluting to 1L by using distilled water, mixing uniformly, and filtering by using a sterile filter membrane with the diameter of 0.22 mu m, wherein the cholate and the trypsin are used for preparation.

And (4) performing gradient dilution on the treated sample solution and the control strain solution, and counting the viable bacteria by using a pouring method. The survival rate data of the strain in the in-vitro simulated intestinal fluid environment is shown in table 2, and the survival rate of the lactobacillus casei L.Casei21 in the in-vitro simulated intestinal fluid environment is obviously higher than that of other test strains, and the cholate resistance is stronger.

TABLE 2 in vitro simulation gastric acid environment survival rate data table for each strain

Treatment time strains	0.5h	1.5h	2h
				L.Casei21	93%	85%	81%
ATCC334	62%	49%	36%
				ATCC11578	49%	30%	25%
ATCC393	57%	45%	32%
				ATCC15008	55%	49%	38%

Example 6 experiment on adhesion rate of Lactobacillus casei L.Casei21 to Caco-2 cells

Caco-2 cells were placed in DMEM medium (purchased from Gibco) containing 10% heat-inactivated newborn bovine serum and diabodies (penicillin concentration 100U/mL, streptomycin 1.0. mu.g/mL) at 37 ℃ with 5% CO₂And incubating in a carbon dioxide incubator with relative humidity of 95%, changing the culture solution for 1 time every day, carrying out passage for 1 time for 3-4 days, and inoculating the cells after 18 daysIn 24-well culture plates, the inoculation density is about 5X 10⁵CFU/mL, adhesion test after cell growth to monolayer.

The activated L.Casei21 was inoculated in MRS liquid medium (same as example 3) at a volume fraction of 1%, and cultured at 37 ℃ for 12 hours.

The cells were collected by centrifugation (4000 g, 4 ℃ C., 10 min), washed 3 times with sterile PBS buffer, and resuspended in sterile PBS buffer to a concentration of 2X 10⁸CFU/mL of L.Casei21 bacterial suspension and PBS buffer solution are prepared according to the following method:

KH₂PO₄ 0.27g、Na₂HPO₄1.42g, NaCl 8g and KCl 0.2g, adding about 800mL of deionized water, fully stirring and dissolving, adding concentrated hydrochloric acid to adjust the pH value to 7.4, adding deionized water to fix the volume to 1L, and sterilizing at 121 ℃ for 20min to prepare PBS buffer solution with the pH =7.4 for later use.

Caco-2 cells that had grown into monolayers were rinsed 2 times with sterile PBS buffer, 0.5mL L.Casei21 suspension and 0.5mL fresh DMEM medium were added to each well, and 5% CO was added at 37 deg.C₂Incubated in a carbon dioxide incubator at 95% relative humidity for 1h, and then the cells were rinsed 5 times with sterile PBS buffer to remove non-adherent bacteria. Each treatment was done in 3 replicates.

After rinsing, the number of bacteria adhered to 100 cells in 20 random fields was counted under an inverted microscope, fixed with formaldehyde, stained with gram, and examined under a microscope. Cell culture without inoculum was used as a control. The average number of bacteria per cell adhesion was calculated.

A comparison graph of the adhesion rate of the strain to Caco-2 cells is shown in figure 1, and it can be seen that Lactobacillus casei L.Casei21 has strong capacity in the aspect of cell colonization.

Example 7 Lactobacillus casei L.Casei21 inhibition of pathogen adhesion test

Caco-2 cells were placed in DMEM medium (purchased from Gibco) containing 10% heat-inactivated newborn bovine serum and diabodies (penicillin concentration 100U/mL, streptomycin 1.0. mu.g/mL),at 37 ℃ with 5% CO₂And incubating in a carbon dioxide incubator with relative humidity of 95%, changing culture solution 1 time per day, carrying out passage 1 time for 3-4 days, inoculating cells in a 24-hole tissue cell culture plate after 18 days, wherein the inoculation density is about 5 multiplied by 10⁵CFU/mL, adhesion test after cell growth to monolayer.

The cells were collected by centrifugation (4000 g, 4 ℃ C., 10 min), washed 3 times with sterile PBS buffer (same as example 6), and resuspended in sterile PBS buffer to a concentration of 2X 10⁸L.Casei21 bacterial suspension of CFU/mL.

3 strains of pathogenic bacteria (Clostridium difficile CICC22951, Salmonella typhimurium CICC21484 and Shigella sonnei CICC21535 which are all purchased from China Industrial microorganism culture Collection center) are respectively inoculated in a TSB liquid culture medium and anaerobically cultured for 24 hours at 37 ℃. The three pathogenic bacteria liquid was centrifuged to collect the bacteria (4000 g, 4 ℃, 10 min), washed 3 times with sterile PBS buffer (same as example 6), and the bacteria was resuspended in sterile PBS buffer to obtain a concentration of 2X 10⁸CFU/mL of three pathogenic bacteria suspensions. The TSB liquid culture medium comprises the following components in percentage by weight:

17g of tryptone, 3g of soybean peptone, 5g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose and 1000mL of distilled water.

Group l.casei 21: rinsing Caco-2 cells grown into a monolayer with sterile PBS buffer solution for 2 times, adding three pathogenic bacteria solutions into different wells according to the addition of 0.5mL to make each well only contain one pathogenic bacteria, and adding 0.5mL of fresh DMEM culture solution (purchased from Gibco company) at 37 ℃ and 5% CO₂And incubating for 1h in a carbon dioxide incubator with the relative humidity of 95%. Adding 2X 10 of the mixture into each hole⁸CFU/mL of L.Casei21 bacterial suspension and 0.5mL of fresh DMEM medium at 37 ℃ with 5% CO₂And incubating for 1h in a carbon dioxide incubator with the relative humidity of 95%.

Blank group: rinsing Caco-2 cells with sterile PBS buffer solution for 2 times to obtain three kinds of pathogenic bacteria0.5mL of the solution was added to each well, and 0.5mL of fresh DMEM medium (purchased from Gibco) was added at 37 ℃ with 5% CO₂And incubating for 1h in a carbon dioxide incubator with the relative humidity of 95%. 0.5mL of sterile water and 0.5mL of fresh DMEM medium were added to each well at 37 ℃ with 5% CO₂And incubating for 1h in a carbon dioxide incubator with the relative humidity of 95%.

After washing 3 times with PBS buffer for both blank and l.casei21 groups, cells were lysed by adding 1mL PBS buffer containing 0.5% volume fraction of polyethylene glycol octyl phenyl ether X-100 (purchased from modesty and chemical ltd, southwest). All cell fluids after dissolution were taken out, diluted with an appropriate gradient, and cultured in BHI medium (purchased from Oxoid) in an incubator at 37 ℃ for 48 hours, and then the number of pathogenic bacteria attached to Caco-2 in the wells was counted.

The statistical result of the adhesion number of pathogenic bacteria in each hole is shown in the following table 3, and it can be seen that the adhesion number of the pathogenic bacteria can still be greatly reduced after the Caco-2 cells are colonized by the pathogenic bacteria by the lactobacillus casei21, so that the diarrhea can be effectively inhibited.

TABLE 3 adhesion number scale of pathogenic bacteria (unit:. times.10)³CFU/mL）

Experimental group pathogenic bacteria	Blank space	L.Casei21	ATCC334	ATCC11578	ATCC393	ATCC15008
							Salmonella typhimurium	72	11	35	50	37	61
Shigella sonnei	31	4.0	12	27	15	26
							Clostridium difficile	60	5.1	49	55	37	46

Example 8 Lactobacillus casei L.Casei21 metabolite inhibition of pathogenic bacteria experiment

Inoculating activated L.Casei21 into MRS liquid medium (same as example 3) according to the inoculation amount of 1% of volume fraction, culturing at 37 ℃ for 24h, centrifuging L.Casei21 bacterial liquid at 4 ℃ at 10000r/min for 30min to obtain L.Casei21 bacterial liquid supernatant, and filtering and sterilizing the supernatant by using a 0.22 mu m microporous filter membrane.

The pathogenic bacteria (clostridium difficile CICC22951, salmonella typhimurium CICC21484 and Shigella sonnei CICC23875 which are purchased from China Industrial microorganism culture Collection) are inoculated into a TSB liquid culture medium (same as example 7), and L.Casei21 bacterial liquid supernatant with the volume fraction of 10 percent is added into the TSB liquid culture medium for anaerobic culture at 37 ℃ for 24 hours.

A blank control is set, and the experimental method is to replace 10% of L.Casei21 bacteria liquid supernatant with 10% of sterile water.

The light absorption values of the experimental groups at the wavelength of 600nm are respectively measured, the bacteriostatic conditions of different lactobacillus supernatants on pathogenic bacteria are analyzed according to the light absorption values, and the results are shown in the following table 4, and the lactobacillus casei L.Casei21 metabolite has good bacteriostatic effects on three pathogenic bacteria.

TABLE 4 bacteriostatic condition (OD) of supernatant of bacterial liquid against pathogenic bacteria₆₀₀）

Experimental group pathogenic bacteria	Blank control	L.Casei21	ATCC334	ATCC11578	ATCC393	ATCC15008
							Salmonella typhimurium	0.76	0.35	0.65	0.53	0.56	0.71
Shigella sonnei	0.68	0.24	0.54	0.58	0.43	0.61
							Clostridium difficile	0.45	0.11	0.39	0.34	0.30	0.37

Example 9 therapeutic Effect of Lactobacillus casei L.Casei21 on patients with diarrhea

100 patients with symptoms of abdominal pain, diarrhea, frequent defecation, cold limbs and the like are selected and divided into a control group and a test group, wherein each group comprises 50 patients, and the test is carried out for 30 days. During the test period, the daily dietary habits of each patient were unchanged, and the test group took 1g of the product of lactobacillus casei L.Casei21 prepared in example 3 with the number of 150 hundred million/g after taking the trametes maleate tablets once a day according to the medical advice. After 30 days, the curative effect is observed and counted, and the statistical result is shown in the following table 5, so that lactobacillus casei L.Casei21 and the products thereof have obvious positive treatment effect on diarrhea patients.

TABLE 5 statistical treatment of diarrhea patients (Unit: person)

Group treatment effect	Recovery method	Show effect	Is effective	Invalidation	Total effective number
						Test group	9	18	19	4	46
Control group	4	10	21	15	35

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

<110> Shandong Zhongke Jiayi bioengineering Co., Ltd

<120> Lactobacillus casei L.Casei21 with therapeutic effect on diarrhea and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.5

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aaacagatgc taataccgca taaatccaag aaccgcatgg ttcttggctg aaagatggcg 180

caagctatcg cttttggatg gacccgcggc gtattagcta gttggtgagg taacggctca 240

ccaaggcgat gatacgtagc cgaactgaga ggttgatcgg ccacattggg actgagacac 300

ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg caagtctgat 360

ggagcaacgc cgcgtgagtg aagaaggctt tcgggtcgta aaactctgtt gttggagaag 420

aatggtcggc agagtaactg ttgtcggcgt gacggtatcc aaccagaaag ccacggctaa 480

ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg 540

taaagcgagc gcaggcggtt ttttaagtct gatgtgaaag ccctcggctt aaccgaggaa 600

gcgcatcgga aactgggaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg 660

gtgaaatgcg tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac 720

tgacgctgag gctcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc 780

cgtaaacgat gaatgctagg tgttggaggg tttccgccct tcagtgccgc agctaacgca 840

ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaaa attttcacag 960

gtcttgacat cttttgatca cctgagagat caggtttccc cttcgggggc aaaatgacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080

gcaaccctta tgactagttg ccagcattga gttgggcact ctagtaagac tgccggtgac 1140

aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200

acgtgctaca atggatggta caacgagttg cgagaccgcg aggtcaagct aatctcttaa 1260

agccattctc agttcggact gtaggctgca actcgcctac acgaagtcgg aatcgctagt 1320

aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380

caccatgaga gtttgtaaca cccgaagccg gtggcgtaac ccttttaggg agcgagccgt 1440

ct 1442

Claims

1. The lactobacillus casei L.Casei21 is characterized in that the classification of the lactobacillus casei L.Casei21 is named as lactobacillus caseiLactobacillus caseiThe preservation number is CGMCC number 21373, the preservation date is 2020, 12 and 14 days, the preservation unit is the China general microbiological culture Collection center, and the preservation unit address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, North Chen, China.

2. Use of lactobacillus casei l.casei21 as claimed in claim 1 in the manufacture of a product for the treatment of diarrhoea.

3. The use of claim 2, wherein the product comprises a powder of lactobacillus casei, which is prepared according to the following method of preparation:

inoculating lactobacillus casei L.Casei21 into MRS liquid culture medium for culture, and centrifuging and vacuum freeze-drying the cultured L.CASEI21 bacterial liquid to obtain lactobacillus casei powder.

4. The use of claim 3, wherein the lactobacillus casei l.casei21 is inoculated in an amount of 1%.

5. Use according to claim 3, wherein the culture temperature of Lactobacillus casei L.Casei21 is 37 ℃.

6. The use according to claim 3, wherein the MRS liquid medium comprises the following components in percentage by weight:

7. The use of claim 2, wherein the product further comprises isomaltooligosaccharides.

8. The use according to claim 2, wherein the viable count of lactobacillus casei l.casei21 in the product is 50, 100, 150, 250, 500 or 1000 billion/g.