CN112210507A - Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof - Google Patents
Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof Download PDFInfo
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- CN112210507A CN112210507A CN202010793756.1A CN202010793756A CN112210507A CN 112210507 A CN112210507 A CN 112210507A CN 202010793756 A CN202010793756 A CN 202010793756A CN 112210507 A CN112210507 A CN 112210507A
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- lactobacillus rhamnosus
- lra05
- lactobacillus
- shigella
- rhamnosus lra05
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Abstract
The invention discloses lactobacillus rhamnosus LRa05 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 1.12734; the shigella has good antagonistic action on shigella, and the antagonistic capability of the shigella is superior to that of common lactobacillus and commercial strain LGG of lactobacillus rhamnosus; and has good acid resistance and cholate resistance; animal tests and human test results of the health food prepared from the lactobacillus rhamnosus LRa05 show that the health food can effectively relieve symptoms such as abdominal pain, diarrhea, fever and the like caused by Shigella; in addition, the lactobacillus rhamnosus belongs to probiotics, is included in safe strains which can be applied to food lists, has excellent safety and can replace the traditional antibiotics.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a shigella-antagonizing lactobacillus rhamnosus LRa05, a screening method and application thereof.
Background
Shigella (Shigella Castellani) is a group of gram-negative bacillus pumilus, the most common pathogenic bacteria of human bacillary dysentery, belonging to the family enterobacteriaceae, divided into 4 groups: shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei. No spore, no capsule, no flagellum, and most of them have pili. Is an intestinal pathogen of humans and primates and causes bacillary dysentery. The number of worldwide shigella infections is as high as 2 billion per year, with millions of people requiring hospitalization each year, with a significant proportion of fatal cases occurring in developing countries. As many as fifty thousand people die each year. The dysentery caused by Shigella is mainly due to the destruction of the epithelial mucosa of the intestine (in particular the cecum and rectum). Some lines can produce enterotoxin and shiga toxin, similar to the escherichia coli bacterial exotoxin, with the most common symptoms being diarrhea (watery diarrhea), fever, nausea, vomiting, gastric cramps, flatulence and constipation.
Generally, for the diseases caused by Shigella, antibiotics are mainly used for treatment, such as ampicillin and chloramphenicol, but the use of antibiotics is very easy to cause the Shigella to generate drug resistance and destroy the normal balance of intestinal flora, and the use of the antibiotics is more and more paid attention and cautions by people along with the 'super bacteria' generated by the abuse of the antibiotics.
Intestinal micro-ecological therapy is a hot spot of recent research by many researchers, especially on lactic acid bacteria. Lactic acid bacteria are a general name of bacteria capable of fermenting carbohydrates to generate a large amount of lactic acid, are widely present in naturally fermented dairy products, fermented traditional foods and human intestinal tracts, can regulate the balance of intestinal flora, control endotoxin and inhibit the growth and reproduction of pathogenic bacteria in the intestinal tracts, so that beneficial effects are generated on the nutritional state, physiological function, drug effect, immune response and the like of organisms, belong to beneficial bacteria in the intestinal tracts, resist the growth of other pathogenic bacteria, have no toxic or side effect and do not generate drug resistance, but because the physiological and biochemical characteristics and the bacteriostatic characteristics of different bacterial strains are different, the screening, identification and application of the lactic acid bacteria specially aiming at resisting Shigella are not provided for the moment.
Disclosure of Invention
In order to solve the technical problems, the invention provides a lactobacillus rhamnosus LRa05 for antagonizing shigella, a screening method and application thereof.
The technical scheme adopted by the invention is as follows:
the invention provides a shigella-resistant Lactobacillus rhamnosus named LRa05, which is preserved in China general microbiological culture Collection center (CGMCC) at 20 days 7 and 2020, has a preservation number of CGMCC 1.12734, is classified and named as Lactobacillus rhamnosus (Lactobacillus rhamnosus), and has a preservation address of the institute of microbiology of China institute of academy of sciences No. 3, North China center of the sunward region, North Cheng-Lu-1, Beijing.
Further, the 16S rRNA gene sequence of the lactobacillus rhamnosus LRa05 is shown as SEQ ID NO: 1 is shown.
Further, the pheS gene sequence of lactobacillus rhamnosus LRa05 is shown as SEQ ID NO: 2, respectively.
The strain of lactobacillus rhamnosus LRa05 is characterized in that: gram-positive bacteria, rod-shaped, chain-shaped, flagellate-free, capsula-free, motionless, facultative anaerobe, the optimum temperature is 30-40 degrees generally, the colony is round, the color is milk white, the surface is smooth and opaque, the edge is neat.
The invention also aims to provide a screening method of the lactobacillus rhamnosus LRa05 for resisting Shigella, which comprises the steps of sequentially carrying out tests such as sample collection, separation, purification, identification, storage and the like, and finally screening the lactobacillus rhamnosus LRa05 for antagonizing Shigella. The method specifically comprises the following steps:
collecting samples, namely pretreating collected traditional fermented food, adding 25mL of samples into 225mL of sterilized physiological saline, and fully and uniformly mixing to obtain a bacterial suspension;
separating the strain, and diluting the bacterial suspension to 10 times of gradient dilution-3、10-4、10-5、10-6Then, coating on MRS agar plate culture medium containing cycloheximide (10-50ug/g, OXID company) and vancomycin (20-50ug/g, Zhejiang Haizhig pharmaceutical Co., Ltd.) with calcium carbonate on the surface, and anaerobically culturing at 30-37 deg.C for 48-72 hr; selecting the colony with larger calcium-dissolving ring, inoculating into a liquid culture medium (containing bile salt with concentration of 0.1-0.3%) of TPY, and performing anaerobic culture at 30-37 deg.C for 12-48 h; the concentration (OD) of the cultured bacteria in the second step600) Performing an antibacterial test of antagonistic Shigella by adopting an Oxford cup method on a higher colony liquid culture medium, performing anaerobic culture at 30-37 ℃ for 12-48h, selecting a single colony with the largest antibacterial zone, streaking and inoculating a colony culture onto an MRS agar culture medium, and performing anaerobic culture at 30-37 ℃ for 48-72 h.
Purifying the strain, selecting a single colony growing on a culture dish, continuously streaking and inoculating the single colony to an MRS agar culture medium, and carrying out anaerobic culture at the temperature of 30-37 ℃ for 48-72 h; continuously carrying out streak culture for three times to obtain pure lactobacillus rhamnosus LRa 05;
and identifying the strain, namely performing gram staining and microscopic observation on the lactobacillus rhamnosus LRa05 to show that the strain is gram-positive and rod-shaped, sending the strain to the common microorganism center of China Committee for culture Collection of microorganisms to identify the 16S rRNA gene sequence and the pheS gene sequence, and showing that the strain belongs to the lactobacillus rhamnosus.
And (3) storing the strain, namely placing the colony culture in sterile 20% glycerol for storage at-80 ℃, and simultaneously inoculating an MRS agar culture medium test tube inclined plane for temporary storage.
Further, the MRS agar plate culture medium consists of: 10.00g of peptone, 10.00g of beef extract, 5.00g of yeast extract, 2.00g of diammonium hydrogen citrate, 20.00g of glucose, 801mL of tween-801, 2.00g of dipotassium phosphate, 0.58g of manganese sulfate, 0.28g of magnesium sulfate and 1L of distilled water, wherein the pH value is 6.4 +/-0.1.
Further, the TPY enrichment medium comprises the following components: 10.0g of hydrolyzed casein peptone, 5.0g of soytone peptone, 2.0g of yeast powder, 5.0g of glucose, 0.5g of L-cysteine, 2.0g of dipotassium phosphate, 0.5g of magnesium chloride, 0.25g of magnesium sulfate, 0.15g of calcium chloride, 0.000001g of ferric chloride, 801.0g of tween and 1L of distilled water, wherein the pH value is 4.5 +/-0.5.
The invention also aims to provide an application of the lactobacillus rhamnosus LRa05 in resisting Shigella, in particular an application of the lactobacillus rhamnosus LRa05 in health-care food.
Further, the health food comprises the following components: calculated according to the mass percentage, the lactobacillus rhamnosus LRa 052-5%, the lactobacillus plantarum 1-4%, the lactobacillus acidophilus 1-5%, the bifidobacterium lactis 0.5-3%, the lactobacillus paracasei 0.5-5%, the inulin 20-50% and the polydextrose 20-60%.
Preferably, the health food comprises the following components: according to the mass percentage, the lactobacillus rhamnosus LRa 054%, the lactobacillus plantarum 1%, the lactobacillus acidophilus 2%, the bifidobacterium lactis 1%, the lactobacillus paracasei 2%, the inulin 30% and the polydextrose 60%.
Compared with the prior art, the technical scheme of the invention has the following advantages and beneficial effects:
the lactobacillus rhamnosus LRa05 provided by the invention has good antagonistic action on Shigella, and the antagonistic capability of the lactobacillus rhamnosus LRa05 is superior to that of common lactobacillus and commercial strain lactobacillus rhamnosus LGG; has good acid resistance and bile salt resistance; animal tests and human test results of the health food prepared from the lactobacillus rhamnosus LRa05 show that the health food can effectively relieve symptoms such as abdominal pain, diarrhea, fever and the like caused by Shigella; in addition, the lactobacillus rhamnosus belongs to probiotics, is included in safe strains which can be applied to food lists, has excellent safety and can replace the traditional antibiotics.
Drawings
FIG. 1 microscopic picture of morphological characteristics of Lactobacillus rhamnosus LRa05
FIG. 2 colony morphology feature map of Lactobacillus rhamnosus LRa05
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
It should be noted that the terms "comprises" and "comprising," and any variations thereof, in the description and claims of the present invention and the above-described drawings, are intended to cover a non-exclusive inclusion, such that a process, method, apparatus, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The Lactobacillus rhamnosus used in the following examples is named LRa05, which has been deposited in China general microbiological culture Collection center (CGMCC) at 20.7.2020, with the deposition number of CGMCC 1.12734, classified and named as Lactobacillus rhamnosus (Lactobacillus rhamnosus), and the deposition address is the institute of microbiology of national academy of sciences No. 3 of North Chen West Lu No. 1 of the sunward area, Beijing.
The materials and methods used in the following examples are, unless otherwise specified, conventional in the art; materials, reagents and the like used in examples are commercially available unless otherwise specified;
the quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1
Screening of Lactobacillus rhamnosus LRa05 Strain
Firstly, collecting samples
Collecting traditional fermented food (in the embodiment, selecting local traditional handmade yoghourt collected in autonomous state of Congo, Kyoho, Qinghai) for pretreatment to obtain a sample treatment solution, and taking 25mL of the sample treatment solution in 225mL of sterilized physiological saline to be fully and uniformly mixed to obtain a bacterial suspension;
second, strain isolation
Diluting the bacterial suspension to 10 times in a gradient way-3、10-4、10-5、10-6Four concentrations, respectively coating on MRS agar plate culture medium containing cycloheximide (cycloheximide concentration of 10-50ug/g, XIXID) and vancomycin (vancomycin 20-50ug/g, Zhejiang Hengzheng pharmaceutical Co., Ltd.), each gradient concentration is 10 plates, pouring calcium carbonate on the surface of MRS agar plate culture medium, performing anaerobic culture at 30-37 deg.C for 48-72h, and observing the size of calcium dissolving ring;
100 strains with calcium-dissolving rings (expressed by the diameter of the calcium-dissolving ring and unit mm) are selected, the number is 1-100, the number data of the first 20 strains with larger calcium-dissolving rings are selected, and the results are shown in Table 1:
TABLE 1 strains with caldolytic rings ranked top 20
| Strain numbering | Diameter of calcium dissolving ring (mm) | Strain numbering | Diameter of calcium dissolving ring (mm) |
| Number 2 | 19.62±0.04 | Number 72 | 18.88±0.04 |
| Number 6 | 16.88±0.03 | Number 78 | 16.48±0.04 |
| Number 13 | 13.75±0.06 | Number 83 | 23.90±0.02 |
| Number 16 | 12.99±0.03 | Number 85 | 14.05±0.06 |
| Number 29 | 18.78±0.06 | Number 86 | 17.26±0.04 |
| Number 41 | 14.25±0.05 | Number 88 | 19.47±0.04 |
| Number 57 | 13.52±0.08 | Number 90 | 17.11±0.05 |
| Number 61 | 10.21±0.07 | Number 93 | 13.37±0.03 |
| Number 63 | 22.45±0.02 | Number 95 | 14.67±0.04 |
| Number 69 | 17.25±0.04 | Number 97 | 12.54±0.04 |
Selecting the first 20 strains of the bacterial colony with a large calcium-dissolving ring, inoculating the bacterial colony into a TPY enrichment bacterial liquid culture medium, carrying out anaerobic culture at the temperature of 30-37 ℃ for 12-48h, and detecting the bacterial liquid concentration (OD value);
centrifuging the bacterial liquid with higher bacterial liquid concentration, collecting supernatant, performing an antibacterial test of antagonistic Shigella (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) by an Oxford cup method, performing anaerobic culture at 30-37 ℃ for 12-48h, numbering again for 1-50, selecting 10 strains with larger antibacterial zones, and numbering, wherein the results are shown in Table 2:
TABLE 2 bacteriostatic effect of mixed bacterial liquid on Shigella
Selecting the strain with the largest inhibition zone and the serial number of 25, streaking and inoculating the strain with the serial number of 25 to an MRS agar culture medium, and carrying out anaerobic culture at the temperature of 30-37 ℃ for 48-72h to obtain an antagonistic strain with stronger Shigella resistance.
Purification of the strains
Continuously streaking and inoculating the separated antagonistic strain to an MRS agar culture medium, and carrying out anaerobic culture at the temperature of 30-37 ℃ for 48-72 h; continuously carrying out streak culture for multiple times to obtain a pure antagonistic strain serving as the Lactobacillus rhamnosus LRa 05.
Preservation of the Strain
The lactobacillus rhamnosus LRa05 is placed in sterile 20% glycerol for storage at-80 ℃, and simultaneously, an MRS agar culture medium test tube slant is inoculated for temporary storage for later use.
Example 2
Identification of Lactobacillus rhamnosus LRa05
The lactobacillus rhamnosus LRa05 screened in the example 1 is subjected to 16S rRNA gene sequence and pheS gene sequence identification, and Blast comparison with a model strain shows that the lactobacillus rhamnosus LRa05 belongs to lactobacillus rhamnosus. Wherein the 16S rRNA gene sequence and the pheS gene sequence are as follows:
the 16S rRNA gene sequence of Lactobacillus rhamnosus LRa05 is as follows:
TATACATGCAGTCGAACGAGTTCTGATTATTGAAAGGTGCTTGCATCTT GATTTAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCT GCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAT CCAAAAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTG GATGGACCCGCGGCGTATTAACTAGTTGGTGAGGTAACGGCTCACCAAGG CAATGATACGTAACCCAACTGAAAGGTTGATCGGCCACATTGGGACTGAA ACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATG GACGCAAGTCTGATGGAACAACGCCCCGTGAGTGAAGAAGGCTTTCGGGT CGTAAAACTCTGTTGTTGGAAAAAAATGGTCGGCAGAATAACTGTTGTCG GCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCC GCGGTAATACGTAAGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCG AGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAA GAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAAGACAGTGGAA CTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCG AARGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTA GCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTA GGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATT CCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGA ACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCC TTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGT GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCC AGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAA GGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACG TGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAAT CTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGA AGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGA AGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAA TGATTAGGGTGAAGTCGTAAC
the pheS gene sequence of lactobacillus rhamnosus LRa05 is as follows:
GCTGATGCGGTCCAAACCAGCCCGATGCAGGCGCGGACGATGGAGAA ACATGACTTTACTAAGGGCCCGCTGAAAATGATCAGTCCAGGCGTGGTTTA TCGGCGTGATGATGATGATGCCACACATAGCCACCAGTTCCATCAGATGGA AGGCCTGGTGATCGACAAGCATATTACAATGGCTGATCTTAAGGGGACGTT ACTCGCCATGTGTCAGCATGTGTTTGGCGCTGATCGGACGATTCGCCTGCG CCCGAGCTATTTTCCGTTTACAGAACCGTCTGTAGAAGTGGACGTTTCCTG CTTCCGCTGCGGTGGCAAGGGCTGCCCTGTTTGCAAGTATACCGGCTGGAT TGAAGTTCTCGGTGCCGGCATGGTGCATCCGAATGTTTTACGTGCGGCTAA TATTGACGCCGATGTATATGGC
example 3
Morphological characteristics of Lactobacillus rhamnosus LRa05
Gram staining of Lactobacillus rhamnosus LRa05 followed by microscopic examination gave the results shown in FIG. 1, from which it can be seen that Lactobacillus rhamnosus LRa05 has the following characteristics: the thallus is rod-shaped, has round end, length of 4.0-10.0 μm and width of 0.4-1.0 μm, and exists in pairs, chains and short chains.
Example 4
Colony morphology characteristics of lactobacillus rhamnosus LRa05
After lactobacillus rhamnosus LRa05 is cultured in MRS culture medium for 18h at 37 ℃, dilution and plating are carried out, the obtained lactobacillus rhamnosus LRa05 is observed, and the result is shown in figure 2, and the colony morphology of the lactobacillus rhamnosus LRa05 is as follows: milky white colony, non-transparent, round, smooth surface, convex and diameter of about 1.3-2.0 mm.
Example 5
According to the traditional indexes of microorganism classification and the identification method of physiological and biochemical characteristics in Bergey's Manual of bacteria, the lactobacillus rhamnosus LRa05 is identified, and the cell morphology and the physicochemical experiment results of the lactobacillus rhamnosus LRa05 are shown in Table 3.
TABLE 3 cellular morphology and results of physicochemical experiments on Lactobacillus rhamnosus LRa05
As can be seen from Table 3, Lactobacillus rhamnosus LRa05 is a gram-positive, rod-shaped, catalase and oxidase test-negative strain. In the carbohydrate acid production profile, "+" indicates that fermentation is available and "-" indicates that fermentation is unavailable.
Example 6
Antibiotic sensitivity test of lactobacillus rhamnosus LRa05
TABLE 4 antibiotic sensitivity test results of Lactobacillus rhamnosus LRa05
As can be seen from table 4, lactobacillus rhamnosus LRa05 was sensitive to the 27 antibiotics tested.
Example 7
Bacteriostatic experiment of lactobacillus rhamnosus LRa05 on Shigella
The lactobacillus rhamnosus LRa05 separated and purified in the embodiment 1 is taken, activated, cultured and passaged for 3 times at 37 ℃ in an MRS liquid culture medium, inoculated in the MRS liquid culture medium, cultured for 18-24h at 37 ℃, centrifuged for 15min at 6500rpm and 4 ℃, collected, filtered by a 0.22um microfilm to obtain a lactobacillus rhamnosus LRa05 fermentation supernatant, and stored in a refrigerator at 4 ℃ for later use.
Taking Shigella dysenteriae (China Industrial microbial strains Collection center (CICC) number is 10983), Shigella flexneri (CICC 10865), Shigella bodyii (CICC 21680) and Shigella sonnei (CICC 21535) intestinal pathogenic bacteria as index bacteria, and performing activated culture on the index bacteria in NB liquid culture medium to obtain an index bacteria liquid for later use;
the NB liquid medium composition was: 10g/L of peptone, 3g/L of beef extract and 5g/L of sodium chloride, adjusting the pH to about 7.4, sterilizing at 121 ℃ for 15 min.
Indicating bacteriaDiluting the bacterial suspension to 106-107CFU/mL, at 1: adding the indicating bacterium liquid into a sterilized MRS culture medium at about 45 ℃ in a volume ratio of 100, shaking up, then pouring the plate, adding 15 mL/plate, and cooling to prepare a bacterium-containing plate; 4 small holes were punched evenly with an 8mm punch on each plate and the plate was marked with the bacterial number. 50-100. mu.L of Lactobacillus rhamnosus LRa05 fermentation supernatant was added per well, while fermentation supernatant of conventional lactic acid bacteria, commercial strain Lactobacillus rhamnosus LGG, was used as control. After all plates were diffused in a refrigerator at 4 ℃ for 12 hours, they were incubated at 37 ℃ for 48 hours, and the size of the zone of inhibition was observed, with the results shown in Table 5.
TABLE 5 bacteriostatic results of Lactobacillus rhamnosus LRa05 on Shigella
Among them, lactobacillus rhamnosus LGG was isolated from healthy human in 1983 by two american professors of north carolina state university (Gorbach and Goldin), has a very prominent property in the resistance to gastric acid and bile, can colonize and maintain activity in the human intestinal tract, has the effects of balancing and improving gastrointestinal tract function, enhancing the human autoimmune ability, preventing and helping to treat diarrhea, and thus is the third generation probiotic of the most global research.
As can be seen from Table 5, the diameter of the inhibition zone of the fermented supernatant of Lactobacillus rhamnosus LRa05 on Shigella is the largest, which indicates that the fermented supernatant of Lactobacillus rhamnosus LRa05 has a good inhibition effect on Shigella.
Example 8
Acid and bile salt resistance of lactobacillus rhamnosus LRa05
Acid resistance experiment: culturing a test strain of lactobacillus rhamnosus LRa05 in an MRS culture medium to the middle stage of logarithmic phase, culturing test strain suspensions in the MRS culture medium with pH2.0 and pH3.0 at 37 ℃ for 3 hours, and measuring the number of viable bacteria before and after culture by using a plate counting method to obtain the survival rate of the strain; other lactobacilli (see Table 6 for details) were used as control bacteria and subjected to acid resistance test, the results of which are shown in Table 6.
Bile salt resistance test: the test strain (Lactobacillus rhamnosus LRa05) was inoculated into MRS medium containing 0.3% (mass fraction) of bile salt at an inoculum size of 4%, cultured at 37 ℃, and the time required for the absorbance of the culture solution at a wavelength of 600nm to reach 0.3 unit was used as the criterion for the bile salt tolerance of the strain, with the results shown in Table 7.
TABLE 6 survival rate of Lactobacillus rhamnosus in acidic environment
TABLE 7 incremental time comparison of Lactobacillus rhamnosus under 0.3% bile salt conditions
Remarking: the shorter the time required, the stronger the bile salt resistance.
The results in tables 6 and 7 show that lactobacillus rhamnosus LRa05 has good acid and bile salt resistance compared to the other lactobacilli of the control group.
Example 9
Preparation method of health food containing Lactobacillus rhamnosus LRa05
The preparation method of the lactobacillus rhamnosus LRa05 health food (LRa 05 health food) comprises the following steps: fermenting Lactobacillus rhamnosus LRa05 in MRS culture medium to obtain fermentation liquid, centrifuging at 6500rpm for 20min to obtain bacterial mud, mixing bacterial mud with protective agent (10-25% trehalose, 1-5% sucrose, 10-15% maltodextrin) at a ratio of 1: 1-3, vacuum freeze drying or spray drying, pulverizing, adding corresponding adjuvants, stirring, and mixing.
The health food containing lactobacillus rhamnosus LRa05 mainly comprises the following components: lactobacillus rhamnosus LRa 054%, lactobacillus plantarum 1%, lactobacillus acidophilus 2%, bifidobacterium lactis 1%, lactobacillus paracasei 2%, inulin 30% and polydextrose 60%.
Example 10
Animal test containing Lactobacillus rhamnosus LRa05 health food
(1) Influence on intestinal lesions caused by Shigella
Constructing a shigella induced diarrhea model for rats, wherein unmodeled rats serve as normal control groups and normally drink and eat water; the rats successfully molded are divided into a model control group (n is 10) and a large-dose test LRa-H group (LRa 05 health food is diluted to 10)10CFU/mL, n 10), low dose test LRa-L group (LRa 05 health food diluted to 109CFU/mL, n 10). The colon lesions of rats were observed by gavage 1/d for each group with 1mL/100g of the corresponding preparation for 14 consecutive days, and the results are shown in tables 8 and 9 below.
TABLE 8 Effect of Lactobacillus rhamnosus LRa05 on Shigella-induced intestinal lesions
As can be seen from table 8, the rectal and lower colon histological score test groups were lower than the model control group, but not statistically significant; and the histologic score of the upper segment in the colon and the colonic ulcer index of the test group are lower than those of the model control group, and have significant difference, which shows that the lactobacillus rhamnosus LRa05 has a certain promotion effect on the improvement of intestinal lesions caused by Shigella.
TABLE 9 influence of Lactobacillus rhamnosus LRa05 on lymphocyte transformation efficiency and serum IL-8, TNF-alpha levels
As can be seen from Table 9, the T cell conversion rate of the model control group is significantly reduced compared with that of the normal control group (p <0.05), and the T cell conversion rate in the test group is somewhat increased but not significantly increased compared with that of the model control group. The serum IL-8 level of the model control group is obviously increased compared with that of the normal control group. The serum IL-8 and TNF-alpha levels of each treatment group are obviously reduced compared with the model control group; the lactobacillus rhamnosus LRa05 has obvious improvement effect on rat inflammatory factors caused by Shigella, improves the conversion rate of lymphocytes and improves immunity.
(2) Effect of Lactobacillus rhamnosus LRa05 on Shigella-induced diarrhea
Rats (48) were selected as study subjects and randomly divided into normal control, model and experimental groups, wherein the experimental groups were divided into two groups, low (L) and high (H) dose groups. Normal control group and model group were gavaged daily with normal saline, and experimental groups were gavaged with equal volumes of the product prepared in example 9; and on the eighth day, normal gavage normal saline is still performed on the control group, a certain amount of shigella bacterium liquid is perfused on the other groups after normal gavage is performed for 0.5h, diarrhea conditions of rats of each group are observed, and various indexes of experimental rats are detected, and the results are shown in tables 10 and 11.
TABLE 10 comparison of growth and diarrhea in the groups of rats
Note: different letters indicate significant difference (P <0.05), same letters indicate insignificant difference (P >0.05)
As can be seen from the data in Table 10, the average daily weight gain and the feed-to-weight ratio of the rats in each group were not significantly different between the initial time and the time before the administration of Shigella (P > 0.05). After the shigella is irrigated, the model group, the LRa-L group and the LRa-H group have loose stools with different degrees, but the significance (P >0.05) of the loose stool rate of the LRa-L group and the LRa-H group is lower than that of the model group, and the difference of the loose stool rate of the LRa-L group and the LRa-H group is not significant; the diarrhea index of 8 days shows obvious difference, wherein the lavage LRa-L group and LRa-H are respectively reduced by 12.5 percent and 18.5 percent compared with the model group, which indicates that the lactobacillus rhamnosus LRa05 has certain resistance effect on diarrhea caused by Shigella.
As can be seen from Table 11, the contents of endotoxin and D-lactic acid in the serum of the rats in the model group are significantly higher than those in the control group, and after the lactobacillus rhamnosus LRa05 is perfused, the contents of endotoxin and D-lactic acid in the serum are significantly reduced in both the LRa-L group and the LRa-H group, which indicates that the lactobacillus rhamnosus LRa05 is perfused to reduce the intestinal permeability of the rats with acute diarrhea.
TABLE 11 serum endotoxin and D-lactic acid content in various groups of rats
Note: different letters indicate significant difference (P <0.05), same letters indicate insignificant difference (P >0.05)
Example 11
Human test with Lactobacillus rhamnosus LRa05 health food
The volunteers were 60 people aged 25-45 years. 30 test groups and control groups respectively; before the test was started, the initial basic conditions of the test population were known, such as the degree of abdominal pain caused by Shigella, the frequency of diarrhea, the desire to eat, etc.
Using the Lactobacillus rhamnosus LRa05 health food prepared in example 8 as a test subject, the number of living bacteria in the test group was 1X 1010-2×1010CFU/g of the health food containing the lactobacillus rhamnosus LRa05, the test subjects take the health food containing the lactobacillus rhamnosus LRa05 once every morning and evening, and the abdominal pain degree, diarrhea frequency and appetite are counted and summarized after two weeks of use; the control group was administered maltodextrin under the same conditions. The results are shown in Table 12.
TABLE 12 volunteer test control sheet
As can be seen from Table 12, the test group taking Lactobacillus rhamnosus LRa05 health food can significantly improve the abdominal pain degree, diarrhea frequency and appetite of diet of people caused by Shigella.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> Jiangsu microbial science and technology Limited
<120> Shigella-antagonizing lactobacillus rhamnosus and application thereof
<130> 20200101
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1479
<212> DNA
<213> Lactobacillus rhamnosus
<400> 1
tatacatgca gtcgaacgag ttctgattat tgaaaggtgc ttgcatcttg atttaatttt 60
gaacgagtgg cggacgggtg agtaacacgt gggtaacctg cccttaagtg ggggataaca 120
tttggaaaca gatgctaata ccgcataaat ccaaaaaccg catggttctt ggctgaaaga 180
tggcgtaagc tatcgctttt ggatggaccc gcggcgtatt aactagttgg tgaggtaacg 240
gctcaccaag gcaatgatac gtaacccaac tgaaaggttg atcggccaca ttgggactga 300
aacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat ggacgcaagt 360
ctgatggaac aacgccccgt gagtgaagaa ggctttcggg tcgtaaaact ctgttgttgg 420
aaaaaaatgg tcggcagaat aactgttgtc ggcgtgacgg tatccaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaagtggc aagcgttatc cggatttatt 540
gggcgtaaag cgagcgcagg cggtttttta agtctgatgt gaaagccctc ggcttaaccg 600
aagaagtgca tcggaaactg ggaaacttga gtgcagaaga agacagtgga actccatgtg 660
tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaargcggc tgtctggtct 720
gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac cctggtagtc 780
catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt gccgcagcta 840
acgcattaag cattccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
caggtcttga catcttttga tcacctgaga gatcaggttt ccccttcggg ggcaaaatga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttatgactag ttgccagcat ttagttgggc actctagtaa gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200
cacacgtgct acaatggatg gtacaacgag ttgcgagacc gcgaggtcaa gctaatctct 1260
taaagccatt ctcagttcgg actgtaggct gcaactcgcc tacacgaagt cggaatcgct 1320
agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacaccatg agagtttgta acacccgaag ccggtggcgt aaccctttta gggagcgagc 1440
cgtctaaggt gggacaaatg attagggtga agtcgtaac 1479
<210> 2
<211> 426
<212> DNA
<213> Lactobacillus rhamnosus
<400> 2
gctgatgcgg tccaaaccag cccgatgcag gcgcggacga tggagaaaca tgactttact 60
aagggcccgc tgaaaatgat cagtccaggc gtggtttatc ggcgtgatga tgatgatgcc 120
acacatagcc accagttcca tcagatggaa ggcctggtga tcgacaagca tattacaatg 180
gctgatctta aggggacgtt actcgccatg tgtcagcatg tgtttggcgc tgatcggacg 240
attcgcctgc gcccgagcta ttttccgttt acagaaccgt ctgtagaagt ggacgtttcc 300
tgcttccgct gcggtggcaa gggctgccct gtttgcaagt ataccggctg gattgaagtt 360
ctcggtgccg gcatggtgca tccgaatgtt ttacgtgcgg ctaatattga cgccgatgta 420
tatggc 426
Claims (9)
1. The Lactobacillus rhamnosus LRa05 for antagonizing shigella is characterized in that the Latin name of the Lactobacillus rhamnosus LRa05 is Lactobacillus rhamnosus LRa05, the preservation unit is China general microbiological culture Collection center (CGMCC) at 7/20/2020, and the preservation number is CGMCC 1.12734.
2. The lactobacillus rhamnosus LRa05 of claim 1, wherein the 16S rRNA gene sequence of lactobacillus rhamnosus LRa05 is as shown in SEQ ID NO: 1 is shown.
3. The lactobacillus rhamnosus LRa05 of claim 1, wherein the pheS gene sequence of lactobacillus rhamnosus LRa05 is as shown in SEQ ID NO: 2, respectively.
4. A screening method of Lactobacillus rhamnosus LRa05 according to any of claims 1-3, characterized by comprising the following steps:
collecting a sample, pretreating the collected sample, putting 25mL of the sample into 225mL of sterilized physiological saline, and fully and uniformly mixing to obtain a bacterial suspension;
separating strains, performing gradient dilution on the bacterial suspension by 10 times, respectively coating the bacterial suspension on MRS agar plate culture medium containing cycloheximide and vancomycin and with calcium carbonate poured on the surface, performing anaerobic culture at 30-37 ℃ for 48-72h, and observing the size of a calcium dissolving ring; selecting a bacterial colony with a large calcium-dissolving ring, inoculating the bacterial colony into a TPY enrichment liquid culture medium, and carrying out anaerobic culture at the temperature of 30-37 ℃ for 12-48 h; measuring the concentration of the bacterial liquid; selecting a bacterium solution with a larger concentration, performing an antibacterial test of antagonistic Shigella by an Oxford cup method, performing anaerobic culture at 30-37 ℃ for 12-48h, and observing the size of an antibacterial zone; selecting a colony with the largest inhibition zone, streaking and inoculating a colony culture to an MRS agar culture medium, and carrying out anaerobic culture at 30-37 ℃ for 48-72 h;
purifying the strain, selecting a single colony growing on a culture dish, continuously streaking and inoculating the single colony to an MRS agar culture medium, and carrying out anaerobic culture at the temperature of 30-37 ℃ for 48-72 h; the streaking was repeated until a pure species of lactobacillus rhamnosus LRa05 was obtained.
5. The screening method of Lactobacillus rhamnosus LRa05 according to claim 4, wherein the MRS agar plate medium consists of: 10.00g of peptone, 10.00g of beef extract, 5.00g of yeast extract, 2.00g of diammonium hydrogen citrate, 20.00g of glucose, 801mL of tween-801, 2.00g of dipotassium phosphate, 0.58g of manganese sulfate, 0.28g of magnesium sulfate and 1L of distilled water, wherein the pH value is 6.4 +/-0.1.
6. The screening method of Lactobacillus rhamnosus LRa05 according to claim 4, wherein the TPY enrichment medium consists of: 10.0g of hydrolyzed casein peptone, 5.0g of soytone peptone, 2.0g of yeast powder, 5.0g of glucose, 0.5g of L-cysteine, 2.0g of dipotassium phosphate, 0.5g of magnesium chloride, 0.25g of magnesium sulfate, 0.15g of calcium chloride, 0.000001g of ferric chloride, 801.0g of tween and 1L of distilled water, wherein the pH value is 4.5 +/-0.5.
7. Use of lactobacillus rhamnosus LRa05 according to any of claims 1 to 3 wherein the lactobacillus rhamnosus LRa05 is for use in a health food.
8. The use of lactobacillus rhamnosus LRa05 according to claim 7 wherein the composition of the health food is: calculated according to the mass percentage, the lactobacillus rhamnosus LRa 052-5%, the lactobacillus plantarum 1-4%, the lactobacillus acidophilus 1-5%, the bifidobacterium lactis 0.5-3%, the lactobacillus paracasei 0.5-5%, the inulin 20-50% and the polydextrose 20-60%.
9. The use of lactobacillus rhamnosus LRa05 according to claim 8 wherein the composition of the health food is: according to the mass percentage, the lactobacillus rhamnosus LRa 054%, the lactobacillus plantarum 1%, the lactobacillus acidophilus 2%, the bifidobacterium lactis 1%, the lactobacillus paracasei 2%, the inulin 30% and the polydextrose 60%.
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