CN117384797B - Rhamnose cheese bacillus LRPerfectus158 and application thereof - Google Patents
Rhamnose cheese bacillus LRPerfectus158 and application thereof Download PDFInfo
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- CN117384797B CN117384797B CN202311514525.2A CN202311514525A CN117384797B CN 117384797 B CN117384797 B CN 117384797B CN 202311514525 A CN202311514525 A CN 202311514525A CN 117384797 B CN117384797 B CN 117384797B
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- lactobacillus rhamnosus
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Abstract
The invention provides a Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) LRPerfectus158 and application thereof, and relates to the technical field of functional microorganisms. The lactobacillus rhamnosus LRPerfectus158 is deposited with the cantonese province microorganism strain collection under the deposit number gdmccno.62937. The detected lactobacillus rhamnosus LRPerfectus158 has the functions of improving exercise endurance and recovering functions, and has good prevention and treatment effects on gastrointestinal damage and colpitis caused by low immunity of organisms. Preparing a product for preventing or treating gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria; and/or a product for preventing or treating vaginal inflammation; and/or an antimicrobial product; and/or anti-inflammatory products; and/or improving exercise endurance and function recovery products have wide application prospects.
Description
Technical Field
The invention relates to the technical field of functional microorganisms, in particular to a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) LRPerfectus158 and application thereof.
Background
Lactobacillus rhamnosus Lacticaseibacillus rhamnosus, the great name lactobacillus rhamnosus Lactobacillus rhamnosus, is one of intestinal resident strains of people, and is a probiotic with important commercial significance. Studies have shown that the lactobacillus rhamnosus has the effects of regulating intestinal flora, enhancing immunity, reducing blood sugar and lipid, preventing and treating osteoporosis, preventing and treating hyperuricemia and the like. For example: lactobacillus rhamnosus GG (ATCC 53103) is the first strain of lactobacillus rhamnosus to obtain patents, and is widely reported to have the efficacy of relieving gastroenteritis, the existing mechanism research of the lactobacillus rhamnosus GG is mainly focused on immune research on monocytes and macrophages, and the inhibition effect of the lactobacillus rhamnosus on neutrophil aggregation caused by pathogenic bacteria infection is rarely reported.
With a great increase in health and fitness awareness, more exercise nutrition is selected for daily intake to improve energy and exercise endurance, wherein the probiotic products have a small product ratio. Meanwhile, gastrointestinal disorders are also common in endurance athletes, such as changes in intestinal mucosa permeability or up-regulation of the proportion of endogenous opportunistic pathogens, so that some organism inflammatory reactions are triggered, and most common dietary supplements in sports nutrition have no effect of preventing and treating gastrointestinal damage, so that probiotics are expected to solve the problem.
In addition, excessive exercise can reduce immunity, so that the human body is more easily infected by exogenous pathogenic bacteria, and a series of inflammatory diseases are caused. Wherein:
listeria monocytogenes is a food-borne pathogen that causes febrile gastroenteritis in healthy subjects, systemic infection in immunocompromised individuals, and abortion, premature birth or stillbirth in pregnant individuals.
Adhesive Invasive Escherichia Coli (AIEC) is one of seven main pathogenic escherichia coli, and can destroy the tight junction function and the intestinal barrier function of intestinal epithelial cells, so that the incidence rate of intestinal inflammation can be improved, more serious inflammatory enteritis can be induced under the condition of intestinal flora disorder, and researches show that the AIEC prevalence rate of crohn's disease and ulcerative colitis is 38.9% and 37.5%, respectively.
Clostridium difficile is an anaerobic gram-positive bacillus capable of forming spores, colonizing the human large intestine, and can cause different clinical manifestations ranging from asymptomatic to mild diarrhea to pseudomembranous colitis, with increasing incidence and severity of clostridium difficile-associated diarrhea or clostridium difficile infection in the past decade, resulting in long-term hospitalization of patients, and placing a serious burden on the health care system.
Bacterial vaginitis and vulvovaginal candida are common vaginitis, and clinical researches show that the key pathogenic bacteria of the bacterial vaginitis and the vulvovaginal candida are gare and candida albicans respectively. The mixed infection of two or more kinds of vaginitis is common in clinic and has rising trend, the prevalence is up to 50 percent, the susceptibility in women of childbearing age and pregnant women is higher, and the pregnant women suffering from the vaginitis easily transmit pathogenic bacteria to infants, thereby causing thrush and other diseases.
Therefore, research and development of a microbial agent which not only has the functions of improving exercise endurance and recovering functions, but also has good prevention and treatment effects on gastrointestinal damage and colpitis becomes necessary and urgent.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide lactobacillus rhamnosus LRPerfectus158 and a microbial inoculum prepared from the same, and application of the lactobacillus rhamnosus LRPerfectus158 and the microbial inoculum prepared from the same in preparing a product for preventing or treating gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria,/or preparing a product for preventing or treating colpitis,/or preparing an antibacterial product and/or preparing an anti-inflammatory product and/or preparing a product for improving exercise endurance and function recovery.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the invention provides a lactobacillus rhamnosus LRPerfectus158, wherein the lactobacillus rhamnosus LRPerfectus158 is preserved in the Guangdong province microorganism strain collection center, and the preservation number is GDMCCNO.62937.
The lactobacillus rhamnosus LRPerfectus158 provided by the invention is preserved in the microorganism strain collection of Guangdong province at the year 2022, 11 and 10, and the preservation address is as follows: building 5, no. 59, of the university of Mitsui 100, guangzhou City, post code 510070, accession number GDMCC No.62937, classified name: lacticaseibacillus rhamnosus.
The lactobacillus rhamnosus LRPerfectus158 not only has the functions of improving exercise endurance and recovering functions, but also has good prevention and treatment effects on gastrointestinal damage and colpitis caused by low immunity of organisms.
The studied detection specific body shows that:
(1) The lactobacillus rhamnosus LRPerfectus158 does not cause erythrocyte hemolysis and is sensitive to antibiotics, and is safe for oral administration and intraperitoneal injection in a mouse test, and meets the safety standard of food-borne raw materials.
(2) The lactobacillus rhamnosus LRPerfectus158 has strong gastrointestinal fluid tolerance, can reach orally and stay in the intestinal tract for at least 48 hours, can not become 'passion way bacteria', and has the foundation of interaction with a host to play a role. Its gastrointestinal fluid tolerance capacity is comparable to that of the well-known commercial strain lactobacillus rhamnosus GG.
(3) The lactobacillus rhamnosus LRPerfectus158 can remarkably improve exercise endurance of young healthy mice, improve liver glycogen storage capacity of the mice, effectively remove urea level in vivo, can provide reference for developing food-borne lactobacillus rhamnosus raw materials for improving exercise endurance of athletes/young healthy groups in the future, and has important significance for development and utilization of probiotic resources.
(4) The live lactobacillus rhamnosus LRPerfectus158 can obviously inhibit the aggregation of neutrophils induced by listeria monocytogenes/clostridium difficile/Adhesion Invasive Escherichia Coli (AIEC) infection, and effectively inhibit gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria.
(5) The anti-infective effect of the live lactobacillus rhamnosus 158 on listeria monocytogenes/clostridium difficile/Adhesion Invasive Escherichia Coli (AIEC) is superior to that of lactobacillus rhamnosus GG, so that references are provided for developing and optimizing food-borne lactobacillus rhamnosus raw materials or live bacteria products for preventing and treating gastroenteritis.
(6) The live lactobacillus rhamnosus LRPerfectus158 can obviously inhibit the vaginal inflammation caused by mixed infection of gardnerella vaginalis and candida albicans, and provides a reference for developing the raw material of the healthy food-borne lactobacillus rhamnosus for women.
(7) The fermented supernatant of Lactobacillus rhamnosus LRPerfectus158 has strong antibacterial activity against Listeria monocytogenes, while Lactobacillus rhamnosus GG does not.
Further, the 16S rDNA sequence of the Lactobacillus rhamnosus LRPerfectus158 is shown in SEQ ID NO. 1.
The specific SEQ ID NO.1 sequence is as follows: CGAGTTCTGATTATTGAAAGGTGCT TGCATCTTGATTTAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCC.
Further, the molecular target nucleotide sequence of the lactobacillus rhamnosus LRPerfectus158 is shown as SEQ ID NO. 2.
The specific SEQ ID NO.2 sequence is as follows: GTTGATTATTGCGGTTTAGTTTAATGTCCTGATTCTTAACAATGATATTTTTAAATATCCAAACATGATTTTTTGTTAGTCGTGTACTTGTTGAGGCAACAATATATGGAGTGGTATTTATCCTGGTATAAAATATTTTTTTGCGTTGGTAGACATTGTAACGAATCGTCATCACCATGGTAGTTCTTTCAGAAACTGTTAACATGTAATGCTTCTTTGCGATAGTTATAGGTATAGGACGAGAATCGTACATGACACTTGTTGGAACTTCACCACCCGTTGATAAACATGTTTCATGTGTCGATAGCAGAAGCATGAGTAAGCTCAGACCTAAAGCGCTGTAGAGTGCTTTTTTTGTCCAGCTTTTTGTTTTCCTTGTTGTTTGTCCTATGTAGCCAACATTCAAAAGCATAAAAGCGCTACAGAGGAACCAAAAACAAGCTAGTGATGTTTGAAGGAGTACCCAATTGGACGTCAATAGAATGCCGATGAAAAGCAGTAAGAATAGTGTAAGAGAGAAAGTACGCTTGAATTTGTTTTTCATATTTCATCTCACTTCAAGTTAAAAAGCCGAGGAAAATTTTACTTTAACTACAGATGACGATCAATAAATTGTTGAAAGGCTTT。
Still further, the detection primer set of the molecular target nucleotide sequence of lactobacillus rhamnosus LRPerfectus158 comprises:
forward primer: 5'-TGCGGTTTAGTTTAATGTCCTG-3'
Reverse primer: 5'-GCGTACTTTCTCTCTTACACT-3'
The invention provides an application of lactobacillus rhamnosus LRPerfectus158 in preparing a product for preventing or treating gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria;
and/or preparing a product for preventing or treating vaginal inflammation;
and/or preparing an antimicrobial product;
and/or preparing an anti-inflammatory product;
and/or, preparing a product for improving exercise endurance and function recovery;
is provided.
Further, the application in preparing the gastrointestinal tract injury product caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria refers to:
lactobacillus rhamnosus LRPerfectus158 inhibits listeria monocytogenes/clostridium difficile/Adhesion Invasive Escherichia Coli (AIEC) -induced neutrophil aggregation.
Further, the use in the preparation of a product for preventing or treating vaginal inflammation refers to:
lactobacillus rhamnosus LRPerfectus158 inhibits mixed infection with gardnerella vaginalis and candida albicans, and infection causes neutrophil aggregation at the site of inflammation.
Further, in the application, the lactobacillus rhamnosus LRPerfectus158 exists in the form of a microbial inoculum or a fermentation supernatant stock solution.
Further, the preparation method of the microbial inoculum comprises the following steps:
fermenting and culturing the strain-activated lactobacillus rhamnosus LRPerfectus158 to obtain a culture solution, concentrating the culture solution, drying and crushing to obtain a lactobacillus rhamnosus LRPerfectus158 microbial inoculum, wherein the number of viable bacteria of the microbial inoculum raw powder is more than or equal to 3000 hundred million CFU/g.
Further, the fermentation parameters of the fermentation culture are as follows: inoculating at least two generations of activated Lactobacillus rhamnosus LRPerfectus158 into freshly prepared sterile MRS broth culture medium or complete nutrition culture medium at a bacterial concentration of 2% -10%, and culturing at 37.0+ -1.0deg.C for 12-18 hr to fermentation end point, namely OD 600nm ≥1.6。
Compared with the prior art, the invention has the beneficial effects that:
the lactobacillus rhamnosus LRPerfectus158 provided by the invention is preserved in the microorganism strain collection of Guangdong province at the year 2022, 11 and 10, and the preservation address is as follows: building 5, no. 59, qingzhou Miao 100, qingzhou, post code 510070, accession number GDMCC No.62937. The lactobacillus rhamnosus LRPerfectus158 not only has the functions of improving exercise endurance and recovering functions, but also has good effects on gastrointestinal damage and colpitis.
The lactobacillus rhamnosus LRPerfectus158 provided by the application can be widely applied to the preparation of products for preventing or treating gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria, and/or the preparation of products for preventing or treating colpitis, and/or the preparation of antibacterial products, and/or the preparation of anti-inflammatory products, and/or the preparation of products for improving exercise endurance and function recovery.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a macroscopic morphology of Lactobacillus rhamnosus LRPerfectus158 according to example 1 of the present invention;
fig. 2 is a microscopic morphology of lactobacillus rhamnosus LRPerfectus158 provided in example 1 of the present invention;
FIG. 3 is a 16srDNA sequence alignment of Lactobacillus rhamnosus LRPerfectus158 according to example 1 of the present invention;
FIG. 4 is a diagram showing the pheS sequence alignment of Lactobacillus rhamnosus LRPerfectus158 according to example 1 of the present invention;
FIG. 5 is a bacterial whole genome map of Lactobacillus rhamnosus LRPerfectus158 according to example 2 of the present invention;
FIG. 6 is an electrophoresis chart of the PCR amplification primer according to example 3 of the present invention;
FIG. 7 is a graph showing the dissolution profile of the real-time quantitative PCR of the specific primers provided in example 3 of the present invention;
FIG. 8 is an amplification plot of real-time quantitative PCR for specific primers provided in example 3 of the present invention;
FIG. 9 is a real-time fluorescence quantitative PCR standard curve of Lactobacillus rhamnosus LRPerfectus158 provided in example 3 of the present invention;
fig. 10 is a hemolysis test chart of lactobacillus rhamnosus LRPerfectus158 provided in example 4 of the present invention;
FIG. 11 is a graph showing the results of an in vitro artificial gastrointestinal fluid test provided in example 6 of the present invention;
FIG. 12 is a chart showing the fluorescence value of live bacteria in intestinal lumen of zebra fish according to example 7 of the present invention;
FIG. 13 is a graph showing the statistics of the load swimming time of the mice according to example 10 of the present invention;
FIG. 14 is a graph showing the measurement of serum lactic acid content in the swimming of mice without load provided in example 10 of the present invention;
FIG. 15 is a graph showing the measured serum urea levels in the swimming of a mouse without load provided in example 10 of the present invention;
FIG. 16 is a graph showing the measurement of liver glycogen content in a swimming state without load of a mouse according to example 10 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 isolation and characterization of Lactobacillus rhamnosus LRPerfectus158
1. Isolation of Lactobacillus rhamnosus LRPerfectus158
Taking 1g of feces sample of 4-month-old healthy male infant, adding 10mL of sterile physiological saline, mixing, centrifuging at 800rpm for 5min to remove large impuritiesTaking 1mL of supernatant suspension, carrying out 10-time gradient dilution with sterile physiological saline, and respectively taking 10 -4 ～10 -7 The diluted solution is coated on a YCFA complete nutrition culture medium flat plate and is placed in an anaerobic incubator for culturing for 24-48 hours at 37 ℃. White opaque circular colonies were picked with the inoculating loop and streaked 3-5 times consecutively until purified to single colonies. And (3) performing staining and microscopic examination according to the specification of the gram staining kit, and performing strain identification after confirming gram positive bacteria. Pure colonies were enriched and mixed with sterile glycerol (final concentration of 20% glycerol) and stored at-80 ℃.
2. Identification of Lactobacillus rhamnosus LRPerfectus158
The identification of Lactobacillus rhamnosus LRPerfectus158 was performed according to the method of multiphase identification detection of bacteria by FMIC-QO01-001-2015 microbiology. The results were as follows:
(1) Morphological observation:
a. macroscopic morphology: as shown in FIG. 1, lactobacillus rhamnosus LRPerfectus158 was cultured in MRS medium, anaerobically cultured at 37℃for 24 hours, and the colonies were white, nearly circular, moist, opaque, and irregular in edge.
b. Microscopic morphology: as shown in FIG. 2, the cells were rod-shaped under a microscope. (0.4-0.6 μm) x (1.0-2.9 μm), the cells are in single, paired or chain form, no spores are formed, and gram staining is positive.
(2) 16S rDNA identification and pheS sequence identification
The growth of Lactobacillus rhamnosus LRPerfectus158 in MRS and YCFA liquid media is similar, so after 12-18h incubation with MRS culture at 37℃the DNA is extracted and the genomic DNA is subjected to polymerase chain reaction using 16s rDNA and conventional primers of pheS (Polymerase Chain Reaction, PCR).
The gene sequence of the strain 16S rDNA claimed by the patent is shown as SEQ ID NO. 1.
Sequence alignment and phylogenetic analysis were performed using the national center for biotechnology information (National Center for Biotechnology Information, NCBI) database, the results of which are shown in fig. 3.
From the results of 16S rDNA, it was found that the similarity to Lactobacillus rhamnosus Lacticaseibacillus rhamnosus JCM 1136T (BALTO 1000058) reached 100%, and it was basically judged as Lactobacillus rhamnosus. To further confirm the accuracy of their type, an alignment was performed by the pheS sequence.
The results are shown in FIG. 4, where the rate of similarity of the Lactobacillus rhamnosus LRPerfectus158 to Lactobacillus rhamnosus Lacticaseibacillus rhamnosus LMG 6400T (AM 087716) is 98.4%, and the combined morphological observation, 16S rDNA identification and pheS sequence identification, the Lactobacillus rhamnosus LRPerfectus158 was identified as Lactobacillus rhamnosus.
EXAMPLE 2 Whole genome sequencing of Lactobacillus rhamnosus LRPerfectus158
Thawing a lactobacillus rhamnosus LRPerfectus158 glycerol tube preserved at the temperature of minus 80 ℃, inoculating to an MRS culture medium, culturing for 12-18 hours at the temperature of 37+/-1 ℃ for 2 times, centrifuging a thallus culture at the end of logarithmic growth in a sterile EP centrifuge tube, centrifuging at 8000rpm for 5min (4 ℃) to collect thalli, discarding supernatant, quick-freezing with liquid nitrogen, freezing the thalli thoroughly, transferring to a refrigerator at the temperature of minus 80 ℃, and mailing to a detection mechanism for carrying out second-generation and third-generation sequencing work. The second generation adopts a DNBSEQ sequencing platform, the third generation adopts a Nanopore sequencing platform, library construction and on-machine sequencing are respectively carried out according to the library building requirements of the sequencing platform, and after the data are qualified through quality inspection and filtration, the assembly of the whole genome of the bacteria is completed after the third generation data are calibrated by the second generation.
The results showed that the lactobacillus rhamnosus LRPerfectus158 genomic DNA was circular, plasmid-free, with a genome size of 2.96mb and a gc content of 46.8%, a total of 15 ribosomal RNAs (rRNA), 59 transfer RNAs (tRNA) and 7 small RNAs (sRNA) and 2,841 coding sequences (CDS) were detected.
FIG. 5 is a whole genome map. Based on the obtained genomic sequence alignment with a putative NT database, it was determined to be lactobacillus rhamnosus from the whole genome level.
Example 3 specific molecular target mining and validation of Lactobacillus rhamnosus LRPerfectus158
1. Mining of molecular targets specific for lactobacillus rhamnosus LRPerfectus158
The data obtained in example 2 were subjected to bioinformatics analysis in combination with NCBI database, and a specific gene fragment of Lactobacillus rhamnosus LRPerfectus158 was obtained by screening, wherein the nucleotide sequence of the gene fragment is shown as SEQ ID NO. 2.
A specific PCR amplification primer set is designed according to a gene fragment sequence SEQ ID NO.2 specific to the Lactobacillus rhamnosus LRPerfectus158 strain:
the forward primer is shown as SEQ ID NO. 3.
The specific SEQ ID NO.3 sequence is as follows: 5'-TGCGGTTTAGTTTAATGTCCTG-3'
The reverse primer is shown in SEQ ID NO. 4.
The specific SEQ ID NO.4 sequence is as follows: 5'-GCGTACTTTCTCTCTTACACT-3'
Specific information is shown in Table 1.
TABLE 1 PCR primer information for detecting specific gene fragment sequences
2. Validation of the effectiveness of the lactobacillus rhamnosus LRPerfectus158 specific molecule recognition target.
The effectiveness of the specific molecular target sequence of lactobacillus rhamnosus LRPerfectus158 was verified by polymerase chain reaction (Polymerase Chain Reaction, PCR) and agarose electrophoresis, and the detection template was bacterial DNA. The DNA was extracted using the extraction kit 2x Rapid Taq Master Mix from Nanjinouzan Biotechnology Co., ltd, and the procedure was as described. The PCR reaction system and the reaction conditions are as follows:
after completion of PCR, 10. Mu.l of the PCR product was subjected to 1.0% agarose electrophoresis. If a single specific band can appear in the middle of the corresponding position of 500-700 bp of the DNA Marker, the lactobacillus rhamnosus LRPerfectus158, and other non-target bacteria have no bands, the target has good recognition effect. The strains and the detection results are shown in Table 2, wherein "+" in the numbered columns is the target strain, "-" is the negative control group without the DNA template, serial numbers 1-18 are the control strains, "+" in the detection results columns indicates positive, and "+" in the detection results column indicates negative.
Table 2 list of strains detected with primers specific for Lactobacillus rhamnosus Lacticaseibacillus rhamnosus LRPerfectus158 and results
As shown in FIG. 6, in the gel results of the PCR amplification products: m is DL1000 DNA standard marker, and no specific amplification product is formed by the control strain except for the specific amplification product formed at about 518bp after the DNA of the target strain Lactobacillus rhamnosus LRPerfectus158 is amplified by the primer. The results demonstrate that the PCR amplification primer is an LRPerfectus158 specific molecular target primer.
3. Specificity verification of Lactobacillus rhamnosus LRPerfectus158 specific primer
The specificity of primers recognizing the specific sequence of lactobacillus rhamnosus LRPerfectus158 and the potential for use in quantitative determination were verified using Real-time fluorescent quantitative determination (Real-time Polymerase Chain Reaction, qPCR). The detection template is bacterial DNA, and the DNA extraction is the same as in the step 2 of the example 3. The qPCR amplification reaction system and reaction conditions are as follows:
the dissolution curve showed a single peak as shown in FIG. 7, no impurity peak appeared, indicating good primer specificity, while the amplification curve (FIG. 8) appeared flat within 35 cyclesStage, the amplification condition is proper; in addition, the real-time fluorescence quantitative determination was performed on DNA of different copy numbers diluted 10 times under the same conditions, and the results (FIG. 9) show the correlation coefficient R of the standard curve 2 >0.99, and combining the above results shows that the pair of primers can be used for quantitative determination of lactobacillus rhamnosus LRPerfectus 158.
EXAMPLE 4 Lactobacillus rhamnosus LRPerfectus158 phenotype and genome safety evaluation 1. Drug susceptibility test
The test was performed according to EFSA 5206-Guidance on the characterization of microorganisms used as feed additives or as production or ganisms-2.2 and the results show that Lactobacillus rhamnosus LRPerfectus158 is sensitive to all antibiotics, as shown in Table 3.
TABLE 3 Lactobacillus rhamnosus LRPerfectus158 MIC values and results of susceptibility testing
2. Hemolytic test
The strain hemolysis was tested according to SH-QO01-008-2019 microbiology test method for detecting hemolysis of microorganism strain. After inoculating the blood plate by puncture, no transparent ring appears around the puncture site of lactobacillus rhamnosus LRPerfectus158, and the hemolysis effect of lactobacillus rhamnosus LRPerfectus158 is similar to that of a negative control bacterium, namely, listeria english-and-no (CICC 10417), and is remarkably different from that of a positive control bacterium, namely staphylococcus aureus (CICC 10473) (fig. 10). The test results show that the lactobacillus rhamnosus LRPerfectus158 does not cause hemolysis, and the result is negative.
3. Bacterial pathogenicity test
Bacterial pathogenicity test is carried out according to annex A method of 'inspection and evaluation of bacterial safety for health food raw materials'.
Table 4 shows that the intraperitoneal injection of 1 time of bacterial suspension of Lactobacillus rhamnosus LRPerfectus158 into mice of both sexes was carried out, and the amount of the injected bacteria was 1.06X10 per mouse 7 CFU (bacterial concentration 5.28X10) 7 CFU/mL, injection volume of 0.2 mL/dose), the animal was normal during 21d observed, with no abnormalities such as toxic signs and death.
Table 4 results of bacterial pathogenicity (intraperitoneal injection) test of mice by LRPerfectus158
Table 5 results show that mice of both sexes were continuously perfused with a suspension of Lactobacillus rhamnosus LRPerfectus158 strain for 3 days at a respective gastric lavage dose of 5.16X10 9 CFU/kg.BW and 2.52X10 10 CFU/kg.BW (bacterial suspension concentrations 2.58X10 respectively) 8 CFU/mL and 1.26X10 9 CFU/mL, and lavage volume calculated as 20mL/kg·bw based on the actual weight of the mice), the animal was normal during 21d period observed, with no abnormalities such as toxic signs and death.
TABLE 5 bacterial pathogenicity (oral gavage) test results of mice with LRPerfectus158
According to the bacterial pathogenicity test method for the health food raw material, the bacterial pathogenicity test result of the lactobacillus rhamnosus LRPerfectus158 mouse is negative, which shows that the lactobacillus rhamnosus LRPerfectus158 has no pathogenicity in animal tests.
4. Lactobacillus rhamnosus LRPerfectus158 virulence factor, drug resistance factor and plasmid analysis
The genomic sequences measured in example 2 were aligned with the latest versions of the internationally recognized virulence gene database (VFDB) and antibiotic resistance gene database (CARD), and the results showed that lactobacillus rhamnosus LRPerfectus158 did not contain virulence factors (coverage >60% and similarity > 75%) and resistance genes. Meanwhile, the LRPerfectus158 contains no plasmid, and the stability and the safety of the genome of the LRPerfectus158 of the Lactobacillus rhamnosus are reflected.
The analysis of virulence genes and drug resistance genes combined with the test results of drug sensitivity test, hemolytic test and mouse bacterial pathogenicity test shows that the lactobacillus rhamnosus LRPerfectus158 has no drug resistance to common antibiotics, the hemolysis reaction is negative, no pathogenicity exists, and the gene analysis shows no virulence genes and transferable drug resistance genes. In conclusion, lactobacillus rhamnosus LRPerfectus158 is a safe strain, and meets the safety requirement of human bodies.
Example 5 preparation of lactobacillus rhamnosus LRPerfectus158 powder.
The method comprises the following specific steps:
1. seed activation: and (3) taking 1 ampoule tube of the lactobacillus rhamnosus LRPerfectus158 strain powder for resuscitation, and then obtaining fermentation seed liquid through primary culture amplification and secondary culture amplification.
2. Fermentation culture: the fermentation seed liquid is inoculated into a5 ton fermentation tank filled with 2 tons of fermentation liquid for fermentation for 8.5 hours at 37+/-2 ℃.
3. And (3) concentration of thalli: starting the centrifugal machine, and after the centrifugal machine stably operates, enabling the materials to enter the centrifugal machine for centrifugal separation.
4. Drying and crushing: and freeze-drying the bacterial sludge obtained by centrifugal concentration, and obtaining the lactobacillus rhamnosus LRPerfectus158 bacterial powder after drying. The results of the measurement of the moisture content, the water activity and the viable count were shown in Table 6.
TABLE 6 production results of Lactobacillus rhamnosus LRPerfectus158 powder
In conclusion, the lactobacillus rhamnosus LRPerfectus158 can be industrialized, and provides feasibility for realizing the application of the strain.
EXAMPLE 6 evaluation of Artificial gastrointestinal fluids tolerance of Lactobacillus rhamnosus LRPerfectus158
In the embodiment, the star strain lactobacillus rhamnosus GG is selected for comparison, and the test method is as follows:
commercial artificial gastric juice (ph=1.5) and artificial intestinal juice (pH 6.8) products were purchased, and after the pH of the artificial gastric juice was adjusted to 2.5, the artificial gastric juice was sterilized by filtration through a 0.22 μm filter membrane.
Mixing seed solution grown to late stage in logarithmic growth phase, separating into equal amount of 3 tubes, centrifuging to remove supernatant, washing with sterile physiological saline for 2 times, suspending the centrifuged strain with equal volume of physiological saline (blank group), artificial gastric juice (pH 2.5) and artificial intestinal juice (pH 6.8), incubating in incubator at 37+ -1deg.C for 3 hr, taking out, respectively performing 10-fold gradient dilution, and collecting 10 -5 ～10 -7 After 24 hours, the plates were counted to give a colony count in the range of 30-300, and the survival rate was finally counted, survival rate (%) = average viable count of experimental group/average viable count of blank group x 100%.
The test results are shown in FIG. 11. The lactobacillus rhamnosus LRPerfectus158 is incubated in artificial gastric juice (pH=2.5) and artificial intestinal juice (pH 6.8) for 3 hours, the survival rate can still reach 81% -93%, and the survival rate is not significantly different from that of the lactobacillus rhamnosus GG of a control strain, which indicates that the lactobacillus rhamnosus LRPerfectus158 has stronger gastrointestinal tract tolerance capability and is similar to that of the lactobacillus rhamnosus GG.
EXAMPLE 7 evaluation of adhesion colonization ability of Lactobacillus rhamnosus LRPerfectus158
(1) Experimental principle: zebra fish have structural and functional homology with the intestinal tract of mammals. CM-Dil is a living cell stain with strong and stable fluorescence that labels living cells by binding to lipid molecules of the membrane structure, and the dye passes into daughter cells with cell division but does not enter adjacent cells, suitable for monitoring cell movement and cell localization analysis. Bacteria that cannot adhere to the intestinal tract are gradually expelled from the intestinal lumen as the zebra fish is stopped feeding. Therefore, the CM-Dil dye is used for marking bacteria, and the transparent characteristic of zebra fish embryos is utilized, so that the fluorescence intensity change of living bacteria in the intestinal cavity of the zebra fish after feeding is stopped can be observed to judge the adhesion and colonization capacity of the living bacteria in the intestinal tract.
(2) The experimental method comprises the following steps: the test selects the star strain lactobacillus rhamnosus GG for comparison, and the test method is as follows:
selecting normal-development 5dpf zebra fish embryos, placing the zebra fish embryos into 12-well plates, adding 1mL of CM-Dil red fluorescent marked bacterial liquid into each well, culturing the zebra fish embryos in a 28 ℃ incubator for 18 hours, washing the zebra fish embryos with sterile embryo culture liquid for 2 times after incubation, transferring the zebra fish embryos into a new well plate, and replacing new embryo culture liquid every day. And respectively shooting intestinal fluorescence images of the zebra fish in 0h, 18h and 48h after the co-cultivation is finished. When the zebra fish is photographed, one side of the zebra fish faces downwards, the zebra fish is fixed by 4% methyl cellulose, the body is kept horizontal, photographing results of all the zebra fish are completed under the same instrument and environment, and the positions of the zebra fish are kept consistent.
The test results are shown in FIG. 12. And (3) injection: fig. 11 and 12: ns represents no significant difference in lactobacillus rhamnosus LRPerfectus158 compared to control strain GG.
According to FIG. 12, fluorescence of the Lactobacillus rhamnosus LRPerfectus158 and Lactobacillus rhamnosus GG was detected in the intestinal lumen of zebra fish after 48 hours of stopping feeding, wherein the average fluorescence intensity of Lactobacillus rhamnosus LRPerfectus158 after 18 hours of fasting was about 60% of the intake, and the average fluorescence intensity after 48 hours of fasting was about 23% of the intake, which was not significantly different from the control strain Lactobacillus rhamnosus GG in the intestinal lumen, indicating that Lactobacillus rhamnosus LRPerfectus158 could reach the intestinal site of zebra fish orally and had a strong adhesion capacity, providing a precondition for its interaction with the host for its beneficial host function.
Example 8 evaluation of the external bacteriostatic Capacity of the Lactobacillus rhamnosus LRPerfectus158 fermentation supernatant
3 pathogenic strains (Escherichia coli, listeria monocytogenes and Pseudomonas aeruginosa) are respectively inoculated in nutrient agar culture medium, cultured at 37deg.C and 180rpm at constant temperature overnight, and then pathogenic bacteria suspension is prepared. Cooling the high-temperature sterilized MRS solid culture medium to about 46+/-1 ℃, and uniformly mixing the MRS solid culture medium with pathogenic bacteria suspension in a certain proportion before solidification to ensure that the number of pathogenic bacteria viable count in a test system is 10 6 On the order of CFU/mL, then rapidly poured into a plate where oxford cups are placed in advance, after the medium cools and solidifies, the oxford cups are removed and 200. Mu.L of the sample solution to be tested (i.e., lactobacillus rhamnosus LRPerfectus158 and Rhamnus) is injected into each wellThe fermentation end point of the Lactobacillus saccharatum GG is cell-free supernatant, a blank control group is test solvent MRS broth culture medium), a plate is lightly covered and then is placed in a constant temperature incubator at 37 ℃, the culture is observed after proper time, and the diameter of a bacteriostasis ring is measured by a vernier caliper.
TABLE 7 antibacterial Activity of Lactobacillus rhamnosus LRPerfectus158 fermentation supernatant
As shown in table 7, the average diameter of the inhibition zone of the lactobacillus rhamnosus LRPerfectus158 fermentation supernatant stock solution is above 13mm, while the control strain lactobacillus rhamnosus GG fermentation supernatant stock solution does not have inhibition zone in the listeria monocytogenes group, which indicates that the lactobacillus rhamnosus LRPerfectus158 fermentation supernatant stock solution can effectively inhibit escherichia coli, pseudomonas aeruginosa and listeria monocytogenes, and has remarkable inhibition effect, wherein the inhibition effect on listeria monocytogenes is remarkably superior to that of the lactobacillus rhamnosus GG.
Example 9 evaluation of antibacterial anti-inflammatory Capacity of Lactobacillus rhamnosus LRPerfectus158 in vivo
The test selects the star strain lactobacillus rhamnosus GG for comparison, and the test method is as follows:
(1) Test principle: the transgenic neutrophils adopted in the embodiment can emit green fluorescence under the action of fluorescence microscope excitation light. As a first line of defense in innate immunity, neutrophils are recruited and aggregated to the site of inflammation after inflammation occurs, and changes in neutrophil numbers can be used to assess the magnitude of inflammatory responses. Meanwhile, the pathogenic bacteria marked by CM-Dil red fluorescence can emit red fluorescence, and the principle is the same as that of the embodiment 7, so that the fluorescent light representing pathogenic bacteria (red) and neutrophils (green) can be observed on the same fish at the same time. And judging whether the intestinal tract of the zebra fish successfully infects the pathogenic bacteria or not according to the fluorescence intensity of pathogenic bacteria in each pathogenic bacteria model group (the blank group does not fluoresce). Whether inflammation and severity thereof were induced after infection of the pathogenic bacteria by zebra fish was evaluated based on the statistical difference in the number of neutrophils in each pathogenic bacteria model group and the blank group (a significant difference was indicated at p < 0.05).
(2) The experimental method comprises the following steps: adjusting the bacterial powders of Lactobacillus rhamnosus GG and Lactobacillus rhamnosus LRPerfectus158 to 5.8X10 with water for fish culture 7 CFU/mL and 2.9X10 8 Two concentrations of CFU/mL were used as samples to be tested. The 4dpf transgenic neutrophil green fluorescent MPX strain zebra fish was randomly selected in 6-well plates, and 30 zebra fish were treated in each well (experimental group). The samples were given in water-soluble form, and a model control group and a blank control group were simultaneously set at a capacity of 3mL per well. After treatment at 28℃for 18h, the solution was changed. After liquid exchange, each experimental group is injected with CM-Dil red fluorescence marked pathogenic bacteria (gardnerella vaginalis combined with candida albicans, listeria monocytogenes, clostridium difficile or escherichia coli) to the intestinal tract of the zebra fish, and a zebra fish intestinal infection model is established. After the treatment is continued for 3 hours at 28 ℃, 10 zebra fish are randomly selected from each experimental group, photographed under a fluorescence microscope, analyzed and data are collected by NIS-Elements D3.20 advanced image processing software, the fluorescence intensity (F) and the number (N) of neutrophils of pathogenic bacteria in intestinal tracts of the zebra fish are analyzed, and the anti-infection rate and the inflammation inhibition rate are calculated according to the following formula:
TABLE 8 anti-infective and anti-inflammatory effects of Lactobacillus rhamnosus LRPerfectus158 and Lactobacillus rhamnosus GG and results of the inhibiting inflammatory effects
Note that: in contrast to the normal group, # p＜0.05， ## p＜0.01， ### p is less than 0.001; in comparison with the control group of the model, * p＜0.05， ** p＜0.01， *** p＜0.001。
as is clear from Table 8, the rate of infection and the rate of inhibition of inflammation by Lactobacillus rhamnosus LRPerfectus158 against Gardnerella vaginalis in combination with Candida albicans were 36.77% and 67.88%, respectively, the rate of infection and the rate of inhibition of inflammation against Listeria monocytogenes were 48.10% and 103.70%, respectively, the rate of infection and the rate of inhibition of inflammation against Clostridium difficile were 31.00% and 73.64%, respectively, and the rate of infection and the rate of inhibition of inflammation against Adhesive Invasive E.coli (AIEC) were 77.41% and 71.21%, respectively. Wherein, the lactobacillus rhamnosus LRPerfectus158 has the best anti-Adhesion Invasive Escherichia Coli (AIEC) infection effect, the listeria monocytogenes and clostridium difficile have the highest inflammation inhibition rate on the listeria monocytogenes, and the gardnerella vaginalis combined with candida albicans and clostridium difficile, and the effect is better than that of the lactobacillus rhamnosus GG. In combination with the results of example 8, both the live cell and the metabolite of lactobacillus rhamnosus LRPerfectus158 were effective in inhibiting listeria monocytogenes, and the control strain lactobacillus rhamnosus GG live cell and metabolite had no inhibitory effect on listeria monocytogenes under the same conditions.
The in vivo bacteriostasis experiment of the organism in the mode can obtain that the lactobacillus rhamnosus LRPerfectus158 has certain efficacy in inhibiting colpitis caused by mixed infection of bacteria and fungi and various intestinal inflammations.
Specific intestinal inflammation may be: listeria monocytogenes induced gastroenteritis; clostridium difficile disease caused by clostridium difficile; intestinal inflammation such as Crohn's disease associated with Adherent Invasive Escherichia Coli (AIEC) and ulcerative colitis.
Example 10 evaluation of Lactobacillus rhamnosus LRPerfectus158 on improved exercise endurance
The experiment divided ICR inbred mice (18-22 g) of 4 weeks of age into a blank control group, a Lactobacillus rhamnosus LRPerfectus158 intervention group and a fructo-oligosaccharide intervention group, 13 animals each group were fed the same normal feed every day, and gastric lavage was timed 1 time. Wherein the blank isControl group gavage sterile saline, lactobacillus rhamnosus LRPerfectus158 intervention group gavage lactobacillus rhamnosus LRPerfectus158 bacterial suspension (viable bacterial dose 1.5×10) 10 CFU/kg·bw, dissolved with sterile saline), fructo-oligosaccharide intervenes in the group lavage of fructo-oligosaccharide solution (fructo-oligosaccharide dose 3g/kg·bw, dissolved with sterile saline), the volume of each lavage was calculated at 0.1mL/10g·bw, and the lavage was continued for 30 days.
After the last gastric lavage for 30 minutes, 6 mice are taken for each group to carry out a load swimming experiment, and the mice with tail root parts loaded with 10% weight lead skin are placed in a swimming box for swimming. The water depth is not less than 30cm, the water temperature is 25+/-1.0 ℃, and the time from the beginning of the swimming of the mice to the beginning of the sinking of the mouth and nose into the water surface is recorded for more than 5s, namely the load swimming time of the mice.
Meanwhile, after the last gastric lavage of 7 mice is carried out for 30 minutes, the mice are swim for 90 minutes in water with the temperature of 30+/-1.0 ℃ and without load, the mice are left for 60 minutes after swimming, eyeballs are taken for blood of about 0.5mL (without anticoagulant), the mice are placed in a refrigerator with the temperature of 4 ℃ for about 3 hours, and after blood coagulation, the mice are centrifuged for 15 minutes at 2000r/min, and serum is taken for standby. The livers of the mice are taken after the mice are killed, the mice are placed in liquid nitrogen for quick freezing, and then the mice are placed in a refrigerator at the temperature of minus 80 ℃ for standby. The serum sample collected above adopts full-automatic biochemical analyzer to measure serum urea and lactic acid content, and liver sample adopts detection kit to measure liver glycogen content.
The detection results are as follows:
1. the load swimming time of the mice can reflect the exercise endurance of the mice.
The test results are shown in FIG. 13. Compared with the normal group, the interference group of the lactobacillus rhamnosus LRPerfectus158 has significant difference (p < 0.0001), and the interference group of the fructo-oligosaccharides has no significant difference, which indicates that the time of the strength training of healthy mice can be effectively improved by the lactobacillus rhamnosus LRPerfectus158, the improvement effect is more than 3 times, and the fructo-oligosaccharides have no significant effect.
2. Serum lactic acid, urea and liver glycogen content under no-load swimming of mice
When the body is in high-intensity exercise for a long time, protein decomposition in the body can be quickened, metabolism is converted into urea nitrogen, the urea nitrogen content in blood is increased, meanwhile, lactic acid in the body can be continuously increased, the continuous accumulation of the metabolites can lead to the reduction of exercise function, and reserved liver glycogen can continuously provide energy for exercise. Therefore, the serum lactic acid, serum urea and liver glycogen content of the mice at the same rest time after the same exercise intensity can reflect the recovery capability of the functions of the healthy mice after exercise, and can also indirectly reflect the exercise endurance of organisms.
The results are shown in fig. 14-16, and the serum lactate values of the lactobacillus rhamnosus LRPerfectus 158-interfered group and the fructo-oligosaccharide-interfered group are significantly different (p < 0.001) and similar compared with the normal group, which indicates that the lactobacillus rhamnosus LRPerfectus 158-interfered group and the fructo-oligosaccharide-interfered group at the test dose have equivalent lactate removal capacity. From serum urea results, the lactobacillus rhamnosus LRPerfectus158 live bacteria intervention group was significantly effective (p < 0.05), while the fructooligosaccharide intervention group was ineffective. In addition, liver tissue hepatic glycogen content was significantly increased in mice of the lactobacillus rhamnosus LRPerfectus158 live bacteria intervention group (p < 0.0001) and the fructooligosaccharide intervention group (p < 0.01).
Note that: fig. 13-16: in contrast to the blank, p <0.05, p <0.01, p <0.001, p <0.0001, ns represents no significant difference
The experimental results show that continuous administration of lactobacillus rhamnosus LRPerfectus158 can effectively improve exercise endurance of young healthy mice and can quickly restore functions after exercise: the method is characterized in that under the condition of being equivalent to the capacity of fructo-oligosaccharide in removing lactic acid, the liver glycogen storage capacity of mice is remarkably improved, the urea level in the body is effectively removed, and the metabolite level accumulated in exercise is reduced.
In conclusion, the lactobacillus rhamnosus LRPerfectus158 has the functions of improving exercise endurance and recovering functions, and has good prevention and treatment effects on gastrointestinal damage and colpitis caused by low body immunity.
The studied detection specific body shows that:
(1) The lactobacillus rhamnosus LRPerfectus158 does not cause erythrocyte hemolysis and is sensitive to antibiotics, and is safe for oral administration and intraperitoneal injection in a mouse test, and meets the safety standard of food-borne raw materials.
(2) The lactobacillus rhamnosus LRPerfectus158 has strong gastrointestinal fluid tolerance, can reach orally and stay in the intestinal tract for at least 48 hours, can not become 'passion way bacteria', and has the foundation of interaction with a host to play a role. Its gastrointestinal fluid tolerance capacity is comparable to that of the well-known commercial strain lactobacillus rhamnosus GG.
(3) The lactobacillus rhamnosus LRPerfectus158 can remarkably improve exercise endurance of young healthy mice, improve liver glycogen storage capacity of the mice, effectively remove urea level in vivo, can provide reference for developing food-borne lactobacillus rhamnosus raw materials for improving exercise endurance of athletes/young healthy groups in the future, and has important significance for development and utilization of probiotic resources.
(4) The live lactobacillus rhamnosus LRPerfectus158 can obviously inhibit the aggregation of neutrophils induced by listeria monocytogenes/clostridium difficile/Adhesion Invasive Escherichia Coli (AIEC) infection, and effectively inhibit gastrointestinal damage caused by exogenous pathogenic bacteria and opportunistic pathogenic bacteria.
(5) The anti-infective effect of the live lactobacillus rhamnosus 158 on listeria monocytogenes/clostridium difficile/Adhesion Invasive Escherichia Coli (AIEC) is superior to that of lactobacillus rhamnosus GG, so that references are provided for developing and optimizing food-borne lactobacillus rhamnosus raw materials or live bacteria products for preventing and treating gastroenteritis.
(6) The live lactobacillus rhamnosus LRPerfectus158 can obviously inhibit the vaginal inflammation caused by mixed infection of gardnerella vaginalis and candida albicans, and provides a reference for developing the raw material of the healthy food-borne lactobacillus rhamnosus for women.
(7) The fermented supernatant of Lactobacillus rhamnosus LRPerfectus158 has strong antibacterial activity against Listeria monocytogenes, while Lactobacillus rhamnosus GG does not.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (4)
1. Rhamnose cheese bacillusLacticaseibacillus rhamnosus) LRPerfectus158, wherein the lactobacillus rhamnosus LRPerfectus158 is deposited with the cantonese province microorganism strain collection under the accession number GDMCC No.62937.
2. A product of lactobacillus rhamnosus LRPerfectus158 according to claim 1 for the preparation of a medicament for preventing or treating gastrointestinal damage caused by exogenous pathogenic and opportunistic pathogenic bacteria;
and/or preparing an antimicrobial product;
and/or preparing an anti-inflammatory product;
and/or, preparing a product for improving exercise endurance and function recovery;
is used for the purposes of (a);
the gastrointestinal tract injury caused by the exogenous pathogenic bacteria and the opportunistic pathogenic bacteria is as follows: gardnerella vaginalis in combination with candida albicans, listeria monocytogenes, clostridium difficile, and escherichia coli;
the antibacterial bacteria are of the following species: gardnerella vaginalis, candida albicans, listeria monocytogenes, clostridium difficile or escherichia coli with adhesive aggression;
the anti-inflammatory is as follows: inhibit listeria monocytogenes, clostridium difficile or adhesion invasive escherichia coli-induced neutrophil aggregation.
3. The use according to claim 2, wherein lactobacillus rhamnosus LRPerfectus158 is present in the form of a bacterial agent.
4. The use according to claim 3, wherein the preparation method of the microbial inoculum comprises:
fermenting and culturing the activated lactobacillus rhamnosus LRPerfectus158 strain to obtain a culture solution, concentrating the culture solution, drying and crushing to obtain the lactobacillus rhamnosus LRPerfectus158 microbial inoculum, wherein the number of viable bacteria of the microbial inoculum raw powder is more than or equal to 3000 hundred million CFU/g.
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| KR102560091B1 (en) * | 2023-01-19 | 2023-07-28 | 주식회사 큐옴바이오 | Novel Scalp-derived Lacticaseibacillus rhamnosus sc.Q5 strain having antibacterial effect against hair loss-inducing harmful bacteria and hair loss prevention activity and use thereof |
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| KR102560091B1 (en) * | 2023-01-19 | 2023-07-28 | 주식회사 큐옴바이오 | Novel Scalp-derived Lacticaseibacillus rhamnosus sc.Q5 strain having antibacterial effect against hair loss-inducing harmful bacteria and hair loss prevention activity and use thereof |
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