CN112266890A - Lactobacillus casei and application thereof - Google Patents
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Abstract
Lactobacillus casei and application thereof relate to the field of microorganisms, in particular to lactobacillus casei and application thereof. The problem that the inhibition effect of the existing lactic acid bacteria on escherichia coli is not ideal is solved. The strain is Lactobacillus casei (Lactobacillus casei) R9, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has the preservation date of 10 months and 14 days in 2020, and has the preservation number of CGMCC No. 20888. The inhibition effect of the lactobacillus casei R9 on pathogenic escherichia coli can reach more than 70%, which shows that the lactobacillus casei R9 has better antagonistic action on the pathogenic escherichia coli and lays a good foundation for the development of microbial agents or additives for regulating the ecological balance of intestinal flora of livestock. The invention is used for inhibiting pathogenic escherichia coli.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly relates to lactobacillus casei and application thereof.
Background
Pathogenic escherichia coli is one of important zoonosis pathogenic bacteria in public health, and is one of main reasons for human and animal infection and global food-borne outbreak. Statistically, more than 1.6 million cases and 100 million deaths occur worldwide each year. Infection of pathogenic E.coli in livestock not only causes significant economic losses, but also poses a threat to food safety and human health. Research shows that pathogenic Escherichia coli isolates from cattle, sheep and other livestock can be transmitted to human to cause diseases. Therefore, if the disease caused by escherichia coli in intestinal tract of livestock cannot be effectively prevented and treated, the health of human beings is possibly harmed along with the transmission of food chain.
According to research, the lactobacillus has treatment and health care effects on diseases caused by intestinal bacteria such as escherichia coli, organic acids such as lactic acid and acetic acid are generated through growth and reproduction of the lactobacillus, the pH value of an intestinal microenvironment is reduced, and meanwhile, substrates are competed with harmful microorganisms such as pathogenic escherichia coli in living environment, and the growth of the harmful microorganisms is inhibited. Therefore, the micro-ecological preparation which is developed by using the lactic acid bacteria for efficiently inhibiting the pathogenic escherichia coli and has the advantages of disease prevention and treatment, safety and no side effect has wide development potential and application prospect in the animal husbandry. However, the existing lactic acid bacteria have poor inhibition effect on escherichia coli, and have poor effect on preventing and treating diseases caused by escherichia coli.
Disclosure of Invention
The invention aims to solve the problem that the existing lactic acid bacteria have an unsatisfactory inhibition effect on escherichia coli, and provides lactobacillus casei and application thereof.
The Lactobacillus casei is Lactobacillus casei (Lactobacillus casei) R9 and is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing republic of Chaoyang district, the preservation date is 10 and 14 days in 2020 and the preservation number is CGMCC No. 20888.
The bacterial colony of the Lactobacillus casei R9 is circular, the size of the bacterial colony is 0.5-1.5 mm, the surface of the bacterial colony is raised, the edge is neat and moist, the color is milky white, and the taste is slightly sour. The thallus is in a short rod shape, generally in a single, paired or short chain shape by microscopic examination.
The lactobacillus casei R9 of the invention enters a logarithmic growth phase after being cultured for 6h, the OD value reaches the highest peak after 24h, then the strain almost stops growing after entering a stabilization phase, and the number of viable bacteria and the number of apoptosis are in a relative balance state. The pH value is 3.4 when the growth lasts for 24-48 h, and the acid production capacity is strong. The results of the catalase test, the nitrate reduction test, the indole test, the hydrogen sulfide test and the gelatin liquefaction test of the lactobacillus casei R9 are all negative,
the lactobacillus casei R9 has extremely high homology with lactobacillus casei through 16S rDNA sequence alignment analysis, and the homology is 99%. The strain R9 belongs to the genus Lactobacillus casei, which is determined by combining morphological characteristics, growth conditions and physiological and biochemical identification results of thalli.
The invention also provides application of the lactobacillus casei R9 in preparing a medicament for inhibiting escherichia coli.
Further, the Escherichia coli is an Escherichia coli standard quality control strain ATCC 25922.
Further, the conditions for inhibiting Escherichia coli are constant temperature culture at 30 ℃ for 72 h.
Further, the concentration of Escherichia coli was 105CFU/mL。
According to the principle of adaptability and specificity of microorganisms to a habitat, the lactobacillus is separated from the silage eaten by the livestock and is applied to the aspect of preventing and treating diseases of the livestock, and the lactobacillus can be colonized more quickly to form a dominant flora. Therefore, the lactobacillus is selected from the silage grass, and the lactobacillus strain which can effectively inhibit pathogenic escherichia coli is selected, so that the important guarantee for preventing and treating intestinal diseases of livestock caused by the escherichia coli is provided.
A large number of experiments prove that the inhibition effect of the lactobacillus casei R9 on pathogenic escherichia coli can reach more than 70%, which shows that the lactobacillus casei R9 has better antagonistic action on the pathogenic escherichia coli and lays a good foundation for the development of microbial agents or additives for regulating the ecological balance of intestinal flora of livestock.
The Lactobacillus casei (Lactobacillus casei) R9 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, the preservation date is 10 and 14 days in 2020, and the preservation number is CGMCC No. 20888.
Drawings
FIG. 1 is the colony morphology of Lactobacillus casei R9 strain on MRS medium;
FIG. 2 is an optical microscope photograph of Lactobacillus casei R9 strain;
FIG. 3 is a physiological curve and pH of the growth cycle of Lactobacillus casei R9 strain;
FIG. 4 is a molecular identification clustering map of Lactobacillus casei R9 strain;
FIG. 5 is a graph showing the inhibitory effect of Lactobacillus casei R9 strain on pathogenic Escherichia coli.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
Example 1: the Lactobacillus casei (Lactobacillus casei) R9 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing republic of south China Korean district, the preservation date is 10 and 14 days of 2020 and the preservation number is CGMCC No. 20888.
The method for obtaining the lactobacillus casei R9 comprises the following steps:
primary screening: weighing natural herba Leymi chinensis silage 5g in 15mL 0.9% normal saline by plate dilution coating method, oscillating at 120rpm for 20min, standing, taking supernatant, performing 10 times concentration gradient dilution, and respectively taking 10 times of the supernatant-1、10-2、10-3、10-4、10-5Each 100uL of dilution solution with the concentration is coated with 3 MRS medium plates in each gradient, the plates are placed upside down in an incubator at 37 ℃ for 3-5 days, and the growth condition of colonies is observed.
Re-screening: combining gram-positive staining, thallus morphology and microscopic separation to select more than 30 strains of lactobacillus, carrying out plate streaking, and screening single colonies to obtain pure separated strains. Selecting the strain to 10mL MRS liquid culture medium, culturing at 37 deg.C for 48h, and determining OD at intervals of 6h600And pH value, drawing growth curve and pH change curve, screening out bacteria with good acid-producing capacityThe strain is the strain R9 of the present embodiment.
Example 2: physicochemical characteristics and molecular identification of Lactobacillus casei R9
(1) Morphological characteristics of lactobacillus casei R9:
after lactobacillus casei R9 is cultured in an MRS culture medium (components g/L: 10g of peptone, 5g of yeast powder, 10g of beef extract, 20g of glucose, 801 mL of tween, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of diammonium citrate, 0.58/4 of 7-water magnesium sulfate, 0.25g of 7-water magnesium sulfate, 15g of agar, 1L of ultrapure water and pH 6.4-6.8) for 3-5 days, a colony with the diameter of 0.5-1.5 mm is formed, the colony is circular, the surface of the colony is raised, the edge is neat and moist, the color is milky white, and the taste is slightly sour. The thallus is in the shape of short rod, generally in the shape of single, paired or short chain, as shown in fig. 1 and fig. 2.
(2) Physiological and biochemical characteristics of lactobacillus casei R9:
inoculating lactobacillus casei R9 in MRS liquid culture medium (components g/L: 10g of peptone, 5g of yeast powder, 10g of beef extract, 20g of glucose, 801 mL of Tween, 2g of dipotassium phosphate, 5g of sodium acetate, 2g of diammonium citrate, 0.58/4 of 7-water magnesium sulfate, 0.25g of 0.58/4-water manganese sulfate and 1L of ultra-clean water), standing and culturing at 37 ℃ for 48h, and detecting bacterial liquid OD (origin-to-diameter) by using a microplate reader every 6h600. The growth curve detects that the lactobacillus casei R9 enters a logarithmic growth phase after 6h, the OD value reaches the highest peak after 24h, then the strain almost stops growing after entering a stable phase, and the number of live bacteria and the number of apoptosis are in a relative balance state. By detecting the pH value of the solution in different growth periods, the pH value is 3.4 in 24-48 h, and the acid production capacity is strong, as shown in FIG. 3 (the column in FIG. 3 shows OD600And the line graph shows pH).
(3) The identification result of lactobacillus casei R9 is as follows:
biochemical identification: according to the 'common bacteria system identification manual' and the 'Bergey system identification manual', the isolated strain R9 is subjected to a catalase test, a nitrate reduction test, an indole test, a hydrogen sulfide test and a gelatin liquefaction test, the results are negative, and the strain is preliminarily determined to be Lactobacillus.
And (3) molecular identification: a genome extraction Kit (FastDNA Spin Kit for Soil) is used for extracting a strain genome, lactobacillus casei R9 genome DNA is used as a template, bacterial 16S rRNA gene sequence primers (27F and 1492R) are used for PCR amplification, and after the amplification, a product is subjected to 1.5% agarose gel electrophoresis inspection, and an obvious characteristic band is obtained at 1500 bp. The amplification products were sequenced by the Biotechnology engineering (Shanghai) Kogyo Co. The length of the 16SrDNA sequence of the lactobacillus casei R9 is 1478bp, and the 16SrDNA sequence is shown as SEQ ID NO: 1 is shown. Blast was performed by NCBI for homology alignment with sequences in the GenBank and the MEGA5.1 software Neihbor-Joinhing was used to construct phylogenetic evolutionary trees, see FIG. 4. The R9 strain has the highest homology with Lactobacillus casei (Lactobacillus casei) up to 99 percent, and is further identified as Lactobacillus casei by combining morphological characteristics, physiological and biochemical characteristics and homology analysis identification, and named Lactobacillus casei R9.
Example 3: inhibition of growth of pathogenic E.coli by Lactobacillus casei R9.
Adopting double-layer plate confronting culture method, firstly introducing MRS culture medium into culture dish to make lower layer plate, drawing two lines with length of about 2cm on the lower layer plate with Lactobacillus casei R9, culturing in 37 deg.C constant temperature incubator for 24 hr, and culturing 5mL Escherichia coli with concentration of 105Pouring the CFU/mL PDA semisolid culture medium on a lower-layer flat plate, requesting to shake to enable the upper-layer culture medium to be uniformly distributed on the whole flat plate, horizontally placing the flat plate for about 20min, placing the flat plate in a 30 ℃ constant-temperature incubator for culturing for 72h when the flat plate is completely solidified, taking the lower-layer flat plate without lactobacillus as a control group, repeating the steps for 3 times for each group, checking a bacteriostatic circle around the lactobacillus after 72h, measuring the diameter of escherichia coli, and calculating the bacteriostatic rate.
The bacteriostatic ratio (%) was (control group escherichia coli diameter-lactobacillus group escherichia coli diameter)/control group escherichia coli diameter × 100%
The results show that Lactobacillus casei R9 pair has a concentration of 105The CFU/mL Escherichia coli has inhibiting effect (figure 5), the left side of figure 5 is the control group with the lower plate not connected with lactobacillus, the control group is full of Escherichia coli colony, the right side is the experimental group with lactobacillus casei R9, only the edge of the plate is connected with Escherichia coli, and there is no large intestine around lactobacillus caseiThe bacterial colony of bacillus, calculate the bacteriostatic rate through the above-mentioned formula, the bacteriostatic effect reaches more than 70% in many times of experiments.
Sequence listing
<110> institute of geography and agroecology of northeast China academy of sciences
<120> lactobacillus casei and application thereof
<160> 1
<210> 1
<211> 1478
<212> DNA
<213> Lactobacillus casei
<220>
<223> Lactobacillus casei R9 16SrDNA
<400> 1
aagcatgcga cgtgctatgc atggaagtcg tacgagttct cgttgatgat cggtgcttgc 60
accgagattc aacatgggaa cgagtggcgg acgggtgagt aacacgtggg taacctgccc 120
ttaagtgggg gataacattt ggaaacagat gctaataccg catagatcca agaaccgcat 180
ggttcttggc tgaaagatgg cgtaagctat cgcttttgga tggacccgcg gcgtattagc 240
tagttggtga ggtaatggct caccaaggcg atgatacgta gccgaactga gaggttgatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc tttcgggtcg 420
taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc gtgacggtat 480
ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600
agccctcggc ttaaccgagg aagcgcatcg gaaactggga aacttgagtg cagaagagga 660
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag cgaacaggat 780
tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag ggtttccgcc 840
cttcagtgcc gcagctaacg cattaagcat tccgcctggg gagtacgacc gcaaggttga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat caggtttccc 1020
cttcgggggc aaaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta tgactagttg ccagcattta gttgggcact 1140
ctagtaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200
ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgagaccgcg 1260
aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca actcgcctac 1320
acgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc 1380
cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccgaagccg gtggcgtaac 1440
ccttttaggg agcgagccgt ctaagttgaa ccaaaagt 1478
Claims (5)
1. The Lactobacillus casei is characterized in that the Lactobacillus casei is Lactobacillus casei R9 and is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 Xilu No. 1 of Beijing Korean district, the preservation date is 10 and 14 days of 2020 and the preservation number is CGMCC No. 20888.
2. Use of lactobacillus casei as claimed in claim 1 for the manufacture of a medicament for the inhibition of escherichia coli.
3. Use according to claim 2, characterized in that the E.coli is E.coli strain ATCC 25922.
4. The use according to claim 2, characterized in that the conditions for inhibiting Escherichia coli are incubation at 30 ℃ for 72 h.
5. Use according to claim 2, characterized in that the E.coli concentration is 105CFU/mL。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116948916A (en) * | 2023-08-11 | 2023-10-27 | 四川大学 | Lactobacillus casei and application thereof |
| CN118109373A (en) * | 2024-04-30 | 2024-05-31 | 山东中科嘉亿生物工程有限公司 | Lactobacillus casei JYLC-109 capable of synthesizing urolithin A and microbial inoculum and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116948916A (en) * | 2023-08-11 | 2023-10-27 | 四川大学 | Lactobacillus casei and application thereof |
| CN116948916B (en) * | 2023-08-11 | 2024-06-11 | 四川大学 | Lactobacillus casei and application thereof |
| CN118109373A (en) * | 2024-04-30 | 2024-05-31 | 山东中科嘉亿生物工程有限公司 | Lactobacillus casei JYLC-109 capable of synthesizing urolithin A and microbial inoculum and application thereof |
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