Disclosure of Invention
The invention solves the problems that the cost is high when the male prostate disease is improved, the curative effect is slow, and the traditional Chinese medicine or western medicine generates toxic and side effects on human bodies, aiming at solving the problem of the lack of probiotic resources for improving the prostate disease; the lactobacillus casei JYLC-374 has the functions of correcting the micro-ecological structure of a urinary system, reducing the number of prostate pathogenic bacteria, improving the immunity of a prostate system, improving the expression function of an immune balance factor and effectively improving male prostate diseases.
In view of the above problems, the invention aims to provide lactobacillus casei JYLC-374, the strain number of the lactobacillus casei is JYLC-374, and the preservation number is as follows: CGMCC NO.18096, the preservation unit is China general microbiological culture Collection center, the preservation date is 7 months and 8 days in 2019, and the preservation place isSite: west road No.1, north chen, chaoyang district, beijing, zip code: 100101 the classification of the strains is named as Lactobacillus caseiLactobacillus casei.The classification unit is as follows: lactobacillus casei.
Further, the lactobacillus casei JYLC-374 is separated from fermented yoghourt at home of farmers in the inner Mongolia Teqi.
Further, the second purpose of the invention is to apply the lactobacillus casei JYLC-374 in preparing food or/and medicines.
Further, the food or/and the medicine has a preservation number of: lactobacillus casei JYLC-374 with CGMCC NO. 18096.
Furthermore, the food or/and the medicine have the application of improving the prostate of men.
Furthermore, the food or/and the medicine have the function of improving immunity.
Further, the food contains lactobacillus casei JYLC-374 bacterial powder.
Furthermore, the viable count of the lactobacillus casei JYLC-374 bacterial powder is more than or equal to 50 hundred million/g.
Furthermore, the viable count of the lactobacillus casei JYLC-374 bacterial powder is 50 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g or 1000 hundred million/g.
Further, the preparation method of the lactobacillus casei JYLC-374 bacterial powder comprises the following steps: inoculating lactobacillus casei JYLC-374 to a lactobacillus casei culture medium plate, carrying out aerobic culture at 37 ℃ for 24h, selecting a single colony with the diameter of more than 1.5mm, washing the single colony for 3 times by using PBS buffer solution, centrifuging, discarding the supernatant, and drying to obtain lactobacillus casei JYLC-374 bacterial powder.
Further, the inoculation amount of lactobacillus casei JYLC-374 was 1% by volume.
Further, the lactobacillus casei culture medium comprises 10g of casein peptone, 10g of beef extract, 5g of yeast powder, 5g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 801 g of Tween and K2HPO4 2g、MgSO4.7H2O 0.2g、MnSO4.H2O 0.05g、CaCO320g of agar and 15g of agar, and steamingDistilled water 1.0L.
Furthermore, the food or/and the medicine containing lactobacillus casei JYLC-374 have the functions of correcting the micro-ecological structure of the urinary system and reducing the number of prostate pathogenic bacteria.
Furthermore, the food or/and the medicine containing the lactobacillus casei JYLC-374 have the functions of improving the immunity of a prostate system and improving the expression of an immune balance factor.
The invention has the beneficial effects that:
1. the lactobacillus casei JYLC-374 provided by the invention is obtained by separating and screening domestic fermented yoghourt of a farmer in the inner Mongolia Teqi, belongs to the genus lactobacillus casei by identification, is named as JYLC-374, and has the preservation number: CGMCC NO.18096, wherein the preservation unit is China general microbiological culture Collection center, the preservation date is 7/8/2019, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101 the classification of the strains is named as Lactobacillus caseiLactobacillus caseiMicroorganism (strain): lactobacillus casei (Lactobacillus casei) has classification units as follows: lactobacillus casei; the food or/and the medicine containing the lactobacillus casei JYLC-374 have the functions of correcting the micro-ecological structure of the urinary system, reducing the number of pathogenic bacteria of the prostate, improving the immunity of the prostate system and improving the expression of immune balance factors.
2. The food or/and the medicine containing lactobacillus casei JYLC-374 provided by the invention can effectively improve male prostate diseases, avoid the pain of patients caused by the current surgical treatment, simultaneously avoid the side effect caused by western medicine treatment, have good effect of improving the male prostate diseases, have no side effect and reduce the treatment cost.
The specific implementation mode is as follows:
for better understanding of the present invention, the technical solution of the present invention will be described in detail with specific examples, but the present invention is not limited thereto.
The lactobacillus casei JYLC-374 provided by the invention is obtained by separating and screening domestic fermented yoghourt of a farmer in the inner Mongolia Teqi, belongs to the genus lactobacillus casei by identification, is named as JYLC-374, and has the preservation number: CGMCC NO.18096, wherein the preservation unit is China general microbiological culture Collection center, the preservation date is 7/8/2019, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101 the classification of the strains is named as Lactobacillus caseiLactobacillus caseiMicroorganism (strain): lactobacillus casei (Lactobacillus casei) has classification units as follows: lactobacillus casei;
the preservation number of the model strain used in the invention is CICC-6117, and the model strain is purchased from China center for culture preservation and management of industrial microorganisms;
the bacterial genome extraction kit (DP 302) is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd, and has a commodity number of DP 302;
LIVE/DEAD BacLight bacterial cell activity assay kit purchased from seimer feishell science and technology (china) ltd, cat #: l7007;
the real-time fluorescent quantitative PCR Kit is purchased from Qiajie biotechnology Qiagen Quanntinova SYBR Green PCR Kit, Cat No.: 208054, respectively;
the components of the improved MRS culture medium plate are 10g of casein peptone, 10g of beef extract, 5g of yeast powder, 5g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 801 g of Tween and K2HPO4 2g、MgSO4.7H2O 0.2g、MnSO4.H2O 0.05g、CaCO320g of agar, 15g of agar, 1.0L of distilled water and hydrochloric acid for adjusting the pH value to 6.2 +/-0.2.
The liquid MRS culture medium comprises components of casein peptone 10g, beef extract 10g, yeast powder 5g, and,5g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 801 g of Tween and K2HPO4 2g、MgSO4.7H2O 0.2g、MnSO4.H2O 0.05g、CaCO320g, 1.0L of distilled water and hydrochloric acid for adjusting the pH value to 6.2 +/-0.2.
Gastric juice simulated medium was purchased from: PERFEMIKER, item number: PM 10004;
the lactobacillus casei culture medium comprises 10g of casein peptone, 10g of beef extract, 5g of yeast powder, 5g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 801 g of Tween and K2HPO4 2g、MgSO4.7H2O 0.2g、MnSO4.H2O 0.05g、CaCO320g of agar, 15g of agar and 1.0L of distilled water.
Example 1
The invention provides lactobacillus casei JYLC-374
1. Strain screening
1) Sample source
Selecting self-fermented yoghourt of inner Mongolia Teqi farmer
2) Strain preparation
Adding 0.7 percent of saline with the concentration of 0.9 percent into sample yoghourt according to the volume percentage, fully shaking and uniformly mixing to form mixed liquid, taking 1 percent of the mixed liquid according to the volume percentage under an anaerobic condition, streaking and inoculating the mixed liquid onto an improved MRS culture medium plate, carrying out anaerobic culture for 48 hours at 37 ℃, selecting a single bacterial colony with the diameter of 1.5mm, inoculating the single bacterial colony with the diameter of 1.5mm into the improved MRS culture medium plate, carrying out anaerobic culture for 72 hours at 37 ℃ until the character is stable, namely the physiological state of bacteria is stable, then picking the single bacterial colony to inoculate in a liquid MRS culture medium, carrying out anaerobic culture for 18 hours at 37 ℃, and obtaining a culture bacterial liquid, wherein the JYLC-374 name is a JYLC;
2. identification of strains
1) Taking 5ml of culture solution into an aseptic centrifuge tube, washing for 2 times by using PBS buffer solution, centrifuging, extracting genome DNA by using a bacterial genome extraction kit (DP 302) to obtain strain DNA, and averagely dividing the strain DNA into two parts, namely strain DNA-A and strain DNA-B;
2) using universal primers
27f :5'-AGAGTTTGATCCTGGCTCAG-3';
1492r :5-'CTACGGCTACCTTGTT-3';
3) Identification of species (systematics)
Adding 38 mu L of deionized water, 5 mu L of Buffer, 1 mu L of upstream primer, 1 mu L of downstream primer, 1 mu L of dNTP (10 mM), 0.5 mu L of Taq enzyme and 0.5 mu L of strain DNA-A into a 50 mu L LPCR amplification system, and uniformly mixing to form a PCR amplification solution;
setting PCR reaction parameters: A. pre-denaturation at 94 ℃ for 5 min; B. 30 cycles: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30 s; C. fully extending for 10min at 72 ℃, and finishing the reaction to obtain a PCR product I;
thirdly, the PCR product I is sent to the company Limited in the Biotechnology engineering (Shanghai) to be sequenced, the full length of 16S rDNA is detected by sequencing, the sequenced Gene sequence is subjected to blast comparison with related sequences in Gene Bank to carry out homology comparison, and the Gene sequence in the application is found to be compared with lactobacillus casei recorded by NCBILactobacillus caseiThe similarity of the sequences reaches 99 percent, and the strains are identified as lactobacillus casei homologous strains and are named as JYLC-374;
4) identification of species (Gene map)
RAPD amplification is carried out on the strain DNA-B by taking the strain DNA-B as a template and taking the bacterial universal primer 5'-GACGGATCAG-3' as an amplification primer to prepare an amplification product II, and then the amplification product II is detected by a QIAGEN QIAxcel capillary electrophoresis instrument;
wherein the result of RAPD amplification is shown in figure 1 by using the deposit number CICC-6117 as a model strain;
it is found from fig. 1 that: the lactobacillus casei JYLC-374 has a different genome structure from a model strain (CICC-6117) and belongs to different strains in the same bacterial species.
Example 2
Gastric acid barrier permeability of lactobacillus casei JYLC-374
Setting a lactobacillus casei JYLC-374 group and a CICC-6117 group, wherein the CICC-6117 group takes CICC-6117 as a control strain;
taking lactobacillus casei JYLC-374 as an example to prepare a lactobacillus casei JYLC-374 culture solution, the steps are as follows: inoculating 1mg of lactobacillus casei JYLC-374 into 10ml of gastric juice simulated culture medium, and culturing for 2 hours to obtain a lactobacillus casei JYLC-374 culture solution;
according to the preparation method of the lactobacillus casei JYLC-374 culture solution, CICC-6117 culture solution is prepared according to the method;
3ml of culture solution in each group is taken respectively, the cell activity of the culture solution in each group is measured and dyed by a LIVE/DEAD Baclight bacterial cell activity measuring kit, meanwhile, the dyed culture solution strain is observed and counted under an Olympus BX51 epifluorescence microscope, and DEAD cells and LIVE cells are recorded;
viability = viable cell count/initial viable cell count × 100%
Results and conclusions: the gastric acid barrier tolerance test result shows that the survival rate of the lactobacillus casei JYLC-374 in a gastric acid simulated culture medium with the pH value of 3.0 reaches 92.12 percent after 2 hours, and the survival rate of the lactobacillus casei JYLC-6117 is 71.9 percent, so that the survival rate of the lactobacillus casei JYLC-374 in the gastric acid barrier and intestinal tract is higher than that of the lactobacillus casei JYLC-6117.
Example 3
Auxiliary improvement effect of lactobacillus casei JYLC-374 on prostate cancer
The method is characterized in that drosophila melanogaster of prostate cancer mutants is used as a test sample to simulate prostatitis patients induced by prostatitis, after lactobacillus casei JYLC-374 viable bacteria are added into a culture medium, the promotion effect of lactobacillus casei JYLC-374 on the life of the drosophila melanogaster of the prostate cancer is detected through tests such as 'full life cycle life prolonging', 'tumor necrosis factor', 'inflammation promotion factor expression quantity', 'balanced immune factor expression detection', and the like, and the CICC-6117 is used as a control strain;
test samples: the drosophila melanogaster of the prostatic cancer mutant is purchased from the resource center of Indiana Blomointon drosophila melanogaster of America, RNAi is adopted to construct the mutant drosophila melanogaster and simulate MRGBP related to prostatic cancerNIG.13746RAn allelic mutation. Biological functions: a drosophila prostate cancer pathological model;
preparing a basal medium containing lactobacillus casei JYLC-374: uniformly mixing 2ml of improved MRS liquid culture medium and 20 mu g of lactobacillus casei JYLC-374 bacterial powder to prepare a basic culture medium containing lactobacillus casei JYLC-374, wherein the viable count of the lactobacillus casei JYLC-374 bacterial powder is 50 hundred million/g;
basic culture medium containing CICC-6117: uniformly mixing 2ml of improved MRS liquid culture medium and 20 mu g of CICC-6117 bacterial powder to prepare a basic culture medium containing CICC-6117, wherein the viable count of the CICC-6117 bacterial powder is 50 hundred million/g;
the test method comprises the following steps: the drosophila melanogaster with male prostate cancer is respectively placed in a drosophila melanogaster tube of 2ml of basic culture medium containing lactobacillus casei JYLC-374 and a drosophila melanogaster tube of basic culture medium containing CICC-6117, each tube has 100, and the culture conditions are as follows: the temperature is 25 ℃, the light and dark rhythm is alternated (namely, the culture is carried out by adopting 12h of visible light within one day and the culture is carried out in a dark place for 12 h), the humidity is 60 percent, the basic culture medium is replaced every 5 days, the death number of the fruit flies is observed and recorded every day, and simultaneously, after the fruit flies are fed for 60 days, the fluorescence quantitative PCR is carried out on the fruit flies by a real-time fluorescence quantitative PCR kit, wherein the gene name of a primer is eiger、Rp49、Upd1、bap180The primer sequence (see table 1) of (1), expression of related genes such as tumor necrosis factor and inflammation marker, and determining the life-span promoting effect of lactobacillus casei JYLC-374 on drosophila melanogaster with prostate cancer tumor, the results are shown in table 2, fig. 2 and fig. 3;
TABLE 1 fluorescent quantitative PCR detection of the genes involved and primer pairs
TABLE 2 Drosophila Life Curve Log-rank test under two probiotic feeding conditions
Results and conclusions: the lactobacillus casei JYLC-374 related to the invention can obviously increase the life span (Log-rank test) of the prostate cancer mutant drosophilap= 0.0096), can significantly improve the 60-day survival rate of mutant drosophila melanogaster compared to the control strain cic-6117. The strain can remarkably improve the organism immunity of female drosophila melanogasterThe expression of the balance factors improves the cancer inhibition effect of the prostate cancer mutant drosophila on the whole organism layer; female fruit fly fed with Lactobacillus casei JYLC-374 strain, proinflammatory factor, compared to control groupeigerExhibits 2-fold down-regulation of expression (p <0.01) to prove that the bacterial strain has good treatment or improvement effect on the aseptic prostatitis by activating the immunity of the organism while reducing the colonization of harmful bacteria.
Example 4
Function of lactobacillus casei JYLC-374 in improving prostatitis
Preparation before the test: preparation of the microbial inoculum: adopting lactobacillus casei JYLC-374 bacterial powder and CICC-6117 bacterial powder;
randomly selecting 100 chronic prostatitis patients (diagnosed as chronic prostatitis, the age is 30-35 years old), randomly dividing into 2 groups, and dividing each group into 50 persons, namely lactobacillus casei JYLC-374 group and CICC-6117 control group;
the test method comprises the following steps: the lactobacillus casei JYLC-374 group orally takes lactobacillus casei JYLC-374 bacterial powder 3mg daily for 50 days continuously;
CICC-6117 control group orally takes CICC-6117 bacterial powder every day, 3mg every day, and continuously takes 50 days;
recording nighttime toileting times of each group of test members, counting morning brix rate (visit every 10 days, data recording) by using morning brix self-test strips, and simultaneously carrying out rectal digital examination and B-ultrasonic examination (prostate fluid sample submission) on each group of test members 1 day before the test begins; the results of digital rectal examination and B-ultrasonic examination (prostate fluid sample presentation) on day 1 after the end of the test were shown in Table 3
TABLE 3 statistics of prostate fluid test data for 50 days after taking micro-ecological preparation
(1) Calculating the short-term diarrhea rate according to the statistical data of patients with mild diarrhea within five days after taking;
(2) the sperm morphology improvement rate is calculated by taking the number of double-headed sperms and the ratio of hemospermia as the basis;
(3) the removal rate of mixed bacteria is calculated according to the culture result of prostatic fluid bacteria;
results and conclusions: the data show that after the lactobacillus casei JYLC-374 strain is taken for 50 days as a microecological preparation of a core component, the prostate fluid detection index of a reference volunteer is obviously improved, the red and white blood cell content is obviously reduced, the expression level of inflammatory factors and markers is reduced, harmful bacteria are obviously reduced, and the sperm morphology is improved. Meanwhile, the living quality of the test volunteers is obviously improved, the frequent micturition and odynuria symptoms are greatly improved, and male physical signs such as morning bok and the like are better improved. The above investigation data all take the model strain CICC-6117 as reference, and the statistical test result shows that the recovery effect of taking JYLC-374 to the male prostate function is obviously higher than that of the model strain CICC-6117.
Example 5
Lactobacillus casei JYLC-374 effect on improving prostate gland in men
Test group 1: adopting lactobacillus casei JYLC-374 bacterial powder, wherein the viable count is 100 hundred million/g;
control group 1: tamsulosin hydrochloride sustained-release capsules and levofloxacin tablets (both the tamsulosin hydrochloride sustained-release capsules and the levofloxacin tablets are purchased from drugstores, the tamsulosin hydrochloride sustained-release capsule manufacturer is Hangzhou kang Enbei pharmaceutical Co., Ltd., the specification is 0.2mg, the levofloxacin tablet manufacturer is the first Sanshu pharmaceutical (Beijing) Co., Ltd., the specification is 0.5 mg)
Control group 2: levofloxacin tablets + pule ' an tablets (prostaglandins) (both levofloxacin tablets and pule ' an tablets are purchased from drugstores, the levofloxacin tablet manufacturer is the first three-component pharmaceutical (Beijing) company Limited and has the specification of 0.5mg, the pule ' an tablet manufacturer is Zhejiang Kangbei pharmaceutical company Limited and has the specification of 0.57g 60s (tablets) (OTC))
The number of the test persons in each group: 50 persons/group
Symptoms are: each group of patients is type I prostatitis patients with age of 30-35 years, and the clinical symptoms of type I prostatitis patients are fever, fatigue, weakness, frequent micturition and urgent micturition;
the test contents are as follows: test group 1 was taken half an hour after breakfast and supper every day at a dose of 2g/50kg with warm water;
the tamsulosin hydrochloride sustained-release capsules and the levofloxacin tablets in the control group 1 are taken according to the usage amount on the medicine box;
the pulean tablet (Qianlikang) and levofloxacin tablet in the control group 2 are taken according to the usage amount on the medicine box;
the test group 1, the control group 1 and the control group 2 are all continuously taken for 30 days, the cure rate in 10 days, 20 days and 30 days, the symptoms and the side effects of the patients are recorded respectively, and the details are shown in a table 4;
the calculation method of the cure rate comprises the following steps: cure rate = (number of cure/number of test) × 100%
Table 4: the detection indexes of the test group 1, the control group 1 and the control group 2 for improving the male prostate are determined
As can be seen from Table 4, the Lactobacillus casei JYLC-374 bacterial powder has obvious effects on improving and treating or improving the type I prostatitis and has no side effect.
Example 6
Lactobacillus casei JYLC-374 effect on improving prostate gland in men
Test group 2: adopting lactobacillus casei JYLC-374 bacterial powder, wherein the viable count is 100 hundred million/mL;
control group 3: tamsulosin hydrochloride sustained-release capsules and levofloxacin tablets (both the tamsulosin hydrochloride sustained-release capsules and the levofloxacin tablets are purchased from drugstores, the tamsulosin hydrochloride sustained-release capsule manufacturer is Hangzhou kang Enbei pharmaceutical Co., Ltd., the specification is 0.2mg, the levofloxacin tablet manufacturer is the first Sanshu pharmaceutical (Beijing) Co., Ltd., the specification is 0.5 mg)
Control group 4: levofloxacin tablets + pule ' an tablets (prostaglandins) (both levofloxacin tablets and pule ' an tablets are purchased from drugstores, the levofloxacin tablet manufacturer is the first three-component pharmaceutical (Beijing) company Limited and has the specification of 0.5mg, the pule ' an tablet manufacturer is Zhejiang Kangbei pharmaceutical company Limited and has the specification of 0.57g 60s (tablets) (OTC))
The number of the test persons in each group: 50 persons/group
Symptoms are: the clinical symptoms of the type III prostatitis of each group are fever, fatigue, weakness, frequent micturition, urgent micturition, increased nocturia, abnormal urination, premature ejaculation and impotence;
the test content is the same as that of the example 5, the cure rates of the test group 2, the control group 3 and the control group 4 at 10 days, 20 days and 30 days, the symptoms and the side effects of the medicine are recorded respectively, and the details are shown in a table 5;
the calculation method of the cure rate comprises the following steps: cure rate = (number of cure/number of test) × 100%
Table 5: the detection indexes of the test group 2, the control group 3 and the control group 4 for improving the male prostate are measured
As can be seen from Table 5, the Lactobacillus casei JYLC-374 bacterial powder has obvious effect on improving or treating the type III prostatitis and has no side effect.
Sequence listing
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