CN116004446B - Luo Yishi lactobacillus LL029 for improving immunity and application thereof - Google Patents
Luo Yishi lactobacillus LL029 for improving immunity and application thereof Download PDFInfo
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- 241000186660 Lactobacillus Species 0.000 title claims abstract description 21
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 21
- 241000186604 Lactobacillus reuteri Species 0.000 claims abstract description 30
- 229940001882 lactobacillus reuteri Drugs 0.000 claims abstract description 30
- 238000004321 preservation Methods 0.000 claims abstract description 7
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- 239000003814 drug Substances 0.000 abstract description 7
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- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 2
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- 239000000243 solution Substances 0.000 description 7
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- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940093761 bile salts Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
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- 235000018291 probiotics Nutrition 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
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- 238000012258 culturing Methods 0.000 description 2
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- 108010019160 Pancreatin Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
- 230000007413 intestinal health Effects 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 210000004400 mucous membrane Anatomy 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
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- 239000008188 pellet Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of microorganisms. The invention discloses a Luo Yishi lactobacillus LL029 for improving immunity, wherein the lactobacillus reuteri LL029 is preserved by the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 10 and 22 days of 2021, the preservation address is 1 # 3 of North Chen West Luo in the Korean region of Beijing city, and the preservation number is CGMCC No.23648. The lactobacillus reuteri LL029 has excellent gastrointestinal fluid resistance and cholate resistance, and can improve the immunity of a host. The invention also discloses an application of the Luo Yishi lactobacillus LL029 for improving immunity, which can be used for preparing foods or medicines for regulating the immunity of organisms.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Luo Yishi lactobacillus LL029 for improving immunity and application thereof.
Background
Probiotics are active microorganisms that are beneficial to a host by altering the flora composition of a part of the host in the human body. By regulating the immune function of host mucous membrane and system or regulating the balance of flora in intestinal tract, the effect of promoting nutrient absorption and maintaining intestinal health is achieved, so that single microorganism or mixed microorganism with definite composition beneficial to health is produced. Probiotics are generally characterized as follows: can endure gastrointestinal fluid and bile salts, can eliminate or reduce adhesion of pathogenic bacteria, is safe and has no pathogenicity.
With the acceleration of the life rhythm of people, the phenomena of weak constitution, malnutrition, low immunity and the like occur due to uneven diet, large working pressure, lack of exercise and the like. Treatment is usually carried out by supplementing various vitamins and amino acids, but after the treatment is stopped, the immunity is reduced, and especially for children, the physiological structure and function are not perfect, the children are more vulnerable to pathogenic bacteria, and food allergy is easy to occur.
Lactobacillus reuteri is an important functional probiotic in human body, can effectively induce lymphocyte production, change inflammatory factors in human body, improve allergic constitution and effectively improve host immunity. However, the function of Guan Luoyi Lactobacillus LL029 has not been studied.
Disclosure of Invention
The invention aims to provide Luo Yishi lactobacillus LL029 for improving immunity and application thereof, which have good gastrointestinal fluid and bile salt resistance, can effectively maintain the immune balance of a host organism and improve the immunity of the host.
In order to achieve the above purpose, the invention provides a Luo Yishi lactobacillus LL029 for improving immunity and application thereof, wherein the lactobacillus reuteri LL029 is preserved by the China general microbiological culture collection center (CGMCC) No.23648 at the 10 th month 22 of 2021, and the preservation number is the address of the preservation unit: microbial classification naming is carried out on Beijing city, chaoyang district, north Chen Xili No.1, 3: lactobacillus reuteri.
Preferably, the 16s rRNA of Lactobacillus reuteri LL029 has the sequence shown in SEQ ID NO. 1.
The invention also provides an application of the Luo Yishi lactobacillus LL029 for improving immunity in preparing foods or medicines for improving immunity.
Therefore, the Luo Yishi lactobacillus LL029 for improving the immunity and the application thereof have the following technical effects:
(1) Lactobacillus reuteri LL029 has good gastrointestinal fluid and bile salt resistance, and can improve the immunity of a host.
(2) Lactobacillus reuteri LL029 can be used for preparing food or medicine, and the prepared food or medicine also has the effect of improving the immunity of a host.
The technical scheme of the invention is further described in detail through examples.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be further described by examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples of the invention, which a person skilled in the art would have without inventive effort, fall within the scope of the invention.
The Luo Yishi lactobacillus LL029 of the invention is a lactobacillus, can effectively improve the immunity of a host, and can also be used as a component of food or medicine.
The new strain Luo Yishi lactobacillus LL029 is preserved by the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 10 and 22 days of 2022, and has a preservation address of No.1 and No. 3 of North Chenxi Lu in the Korean area of Beijing city and a preservation number of CGMCC No.23648.
The sequence of 16s rRNA of Lactobacillus reuteri LL029 is shown in SEQ ID NO. 1.
Example 1
Gastrointestinal tract resistance test of Lactobacillus reuteri LL029
Activating strains: lactobacillus reuteri LL029 was inoculated in MRS liquid medium and cultured at 37 ℃ and passaged 2 times at 1% of the inoculum size, and then preserved for later use.
Preparing gastric juice: the simulated gastric juice is prepared by NaCl and pepsin, the pH values are respectively adjusted to 2, 3 and 4 by a proper amount of hydrochloric acid, and the gastric juice is filtered by a microporous filter membrane with the thickness of 0.2 mu m for later use.
Preparing intestinal juice: dissolving potassium dihydrogen phosphate in water, and regulating pH with 0.1mol/L sodium hydroxide solution; dissolving pancreatin in water, mixing the two solutions, and diluting with water to obtain simulated intestinal juice.
Inoculating the test strain to MRS liquid culture medium, culturing at 37deg.C for 17-18h, centrifuging 10000g for 15min, removing supernatant, weighing wet weight of thallus, re-suspending thallus and physiological saline according to the ratio of 0.1g/ml as control group, respectively adding into simulated gastric fluid with pH value of 2, 3, 4 and simulated intestinal fluid with pH value of 7.5 according to the ratio of 1:10, mixing thoroughly, and culturing in a 37 deg.C constant temperature incubator.
As is clear from the table below, after 2 hours of cultivation, the survival rate of the live bacteria of Lactobacillus reuteri LL029 is about 10.84% when the pH value of gastric juice is 2; when the pH value of gastric juice is 3, the survival rate is about 52.37 percent; at a gastric juice pH of 4, the survival rate is about 96.84%.
After 4h of culture, the survival rate of lactobacillus reuteri LL029 is about 10.55 percent when the pH value of gastric juice is 2; when the pH value of gastric juice is 3, the survival rate is about 46.58 percent; at a gastric juice pH of 4, the survival rate is about 94.21%. Shows that the lactobacillus reuteri LL029 has stronger gastric juice resistance.
TABLE 1 Luo Yishi results of gastric acid resistance test of Lactobacillus LL029
After 2h of culture, lactobacillus reuteri LL029 had a survival rate of about 96.32% in simulated intestinal fluid, and after 4h of culture, the survival rate was about 91.32%. Shows that the lactobacillus reuteri LL029 has stronger intestinal juice resistance.
TABLE 2 results of Lactobacillus reuteri LL029 intestinal juice test
| Cultivation time (h) | Simulating the pH value of intestinal juice | Viable count (cfu/ml) | Survival (%) |
| 0 | ControlGroup of | 3.80*10 7 | / |
| 2 | 7.5 | 3.66*10 7 | 96.32±1.23 |
| 4 | 7.5 | 3.47*10 7 | 91.32±0.55 |
(II) Lactobacillus reuteri LL029 bile salt resistance test
The test strains are respectively inoculated into sterilized MRS liquid culture medium containing 0.1%, 0.2% and 0.3% bile salts, and are placed in a constant temperature incubator at 37 ℃ for 2 hours.
As is clear from the following table, after 2 hours of cultivation, lactobacillus reuteri LL029 had a survival rate of about 93.20% at a bile salt concentration of 0.1%; when the concentration of bile salt is 0.2%, the survival rate is about 39.88%; when the concentration of bile salt is 0.3%, the survival rate is about 2.75%.
After 4h of culture, the survival rate of lactobacillus reuteri LL029 is about 88.40 percent when the concentration of bile salts is 0.1 percent; when the concentration of bile salt is 0.2%, the survival rate is about 34.16%; when the concentration of bile salt is 0.3%, the survival rate is about 1.34%. Shows that lactobacillus reuteri LL029 has strong cholate resistance.
Table 3 Luo Yishi Lactobacillus LL029 cholate resistance test results
Example two
Test preparation
40 healthy mice with the age of 6-8 weeks are selected, and the mice are respectively and freely drunk and taken into feed in a room with the temperature of 25+/-2 ℃ and the humidity of 60+/-5% and the light and dark circulation time of 12 hours. The test personnel add water and feed in time.
Model establishment for low immunity
The mice were randomly divided into 4 groups of 10 mice each, a blank control group, a model control group, luo Yishi lactobacillus helveticus+cyclophosphamide group, and lactobacillus helveticus+cyclophosphamide group, respectively. The blank control group and the model control group were intragastrically filled with an equivalent amount of physiological saline for 14 consecutive days, 1 time a day, 0.2ml at a time. The remaining groups, except the blank, were injected with cyclophosphamide at a dose of 80mg/kg on days 7, 8, 9 of the trial. Mice of the lactobacillus reuteri+cyclophosphamide group started on day 7 with a bacterial solution concentration of 1 x 10 9 cfu/ml of Lactobacillus reuteri LL029 bacteria liquid was infused 1 time per day at a dose of 0.2ml. Mice of the lactobacillus helveticus + cyclophosphamide group started on day 7 with a bacterial solution concentration of 1 x 10 9 cfu/ml of Lactobacillus helveticus WHH2580 bacterial liquid is irrigated for 1 time a day, and the dosage is 0.2ml. There have been studies to confirm that lactobacillus helveticus WHH2580 has an effect of enhancing immunity.
Weight and organ ratio measurement
The body weight of each mouse before and after the test was recorded, the mice were sacrificed to remove spleen after the test was completed, the mice were placed in normal saline to wash out blood, and the water was sucked dry by filter paper, and after the mice were completely dried, they were weighed.
Organ ratio = spleen weight/body weight
The results are shown in the following table:
table 4 table of changes in body weight and organ ratio of mice
Mice fed Luo Yishi lactobacillus LL029 had significantly higher body weight and organ ratio than the model control group and similar body weight and organ ratio to mice fed lactobacillus helveticus WHH2580. The lactobacillus reuteri LL029 can obviously relieve the phenomenon of weight loss caused by low immunity and obviously relieve spleen injury.
(two) lymphocyte proliferation potency assay
Mice were sacrificed 14 days after the test, spleens were aseptically removed with forceps, crushed, filtered using a 200 mesh metal screen, and washed with Hank's solution, and centrifuged at 1000rpm for 5min. Cell lysates 5 times the cell volume were added for 5min, washed with Hank's solution, centrifuged at 1000rpm for 5min and the supernatant was removed. Washed with PRMI-1640 medium and centrifuged at 1000rpm for 5min. The pellet was resuspended in 5ml PRMI-1640 medium. Cell count, adjusted cell concentration to 1 x 10 7 cells/ml. OD at 570nm was measured. The results are shown in the following table:
TABLE 5 results of OD values of mice from different test groups
The OD value of the mice in the model control group is obviously reduced compared with that of the mice in the blank control group, which indicates that the immune system of the mice is destroyed by cyclophosphamide, and the immunity of the mice is low. The OD value of the mice of the Lactobacillus reuteri+cyclophosphamide group is higher than that of the mice of the model control group, and the lymphocytes are obviously increased, which indicates that after the mice are fed with Luo Yishi Lactobacillus LL029, the proliferation of the lymphocytes is promoted, and thus the immunity of the organism is improved. Furthermore, mice fed Luo Yishi lactobacillus LL029 had higher OD values than mice fed lactobacillus helveticus WHH2580 group.
(III) intestinal regulatory flora test
30 healthy mice with the age of 6-8 weeks are selected, and the mice are respectively and freely drunk and taken into feed in a room with the temperature of 25+/-2 ℃ and the humidity of 60+/-5% and the light and dark circulation time of 12 hours. The test personnel add water and feed in time.
The mice were randomly divided into 3 groups of 10 mice each, control group and experimental group one and experimental group two, respectively. The control group was fed normally, and the test group was fed with Lactobacillus reuteri LL029 at a dose of 0.2ml/10g each day; the experimental group II was perfused with Lactobacillus helveticus WH 2580 in a dose of 0.2ml/10g daily.
Before stomach irrigation, mouse feces are collected, stored at-20 ℃ and intestinal flora is detected.
After 14 days, feces from each mouse were collected under aseptic conditions, stored at-20℃and tested for intestinal flora. The bacterial flora was analyzed for diversity by Shannon index and Simpson index, with a larger Shannon index indicating high diversity and a smaller Simpson index indicating high diversity. The test results are shown in the following table:
TABLE 6 results of intestinal flora diversity in mice
After the test is finished, the Shannon index and the Simpson index of the intestinal flora of the control group mice have no obvious change. The Shannon index of the intestinal flora of the mice in the experimental group is obviously increased compared with that before the experiment, and the Simpson index is obviously reduced compared with that before the experiment, which shows that Luo Yishi lactobacillus LL029 effectively promotes the increase of the intestinal flora of the mice. And, the Shannon index of mice in experimental group one is greater than that in experimental group two and the Simpson index is less than that in experimental group two.
Therefore, the Luo Yishi lactobacillus LL029 for improving the immunity and the application thereof are adopted, and the lactobacillus reuteri LL029 has good gastrointestinal fluid resistance and bile salt resistance and can improve the immunity of a host. Lactobacillus reuteri LL029 can be used for preparing food or medicine, and the prepared food or medicine also has the effect of improving the immunity of a host.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (1)
1. The Luo Yishi lactobacillus LL029 for improving immunity is characterized in that the lactobacillus reuteri LL029 is preserved by the China general microbiological culture Collection center (CGMCC) in the 10 th month 22 of 2021, and the preservation number is CGMCC No.23648;
the sequence of the 16srRNA of lactobacillus reuteri LL029 is shown in SEQ ID NO. 1.
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| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| CN106520599A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus reuteri and applications |
| KR20180053499A (en) * | 2016-11-11 | 2018-05-23 | 대한민국(농촌진흥청장) | Lactobacillus reuteri induced from rat and composition for enhancement of immunity using the same |
| CN112358999A (en) * | 2020-11-26 | 2021-02-12 | 中国农业大学 | Lactobacillus reuteri and application thereof |
| KR102234835B1 (en) * | 2020-01-31 | 2021-04-01 | 우진 비앤지 주식회사 | Composition for enhancing immunity of companion animals comprising novel Lactobacillus Luteri LBR_C1 strain and novel Lactobacillus Ashdophilus LBA_C5 strain |
| CN114836358A (en) * | 2022-06-28 | 2022-08-02 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus reuteri SXDT-32 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200129571A1 (en) * | 2017-06-28 | 2020-04-30 | Moviscom S.r.l. | New strain of lactobacillus reuteri with probiotic characteristics, its use and compositions containing said strain |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219683A (en) * | 2015-11-04 | 2016-01-06 | 广东省农业科学院动物科学研究所 | One strain has L. reuteri strain and the application thereof of prebiotic characteristics |
| CN106520599A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus reuteri and applications |
| KR20180053499A (en) * | 2016-11-11 | 2018-05-23 | 대한민국(농촌진흥청장) | Lactobacillus reuteri induced from rat and composition for enhancement of immunity using the same |
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| CN112358999A (en) * | 2020-11-26 | 2021-02-12 | 中国农业大学 | Lactobacillus reuteri and application thereof |
| CN114836358A (en) * | 2022-06-28 | 2022-08-02 | 中国农业科学院北京畜牧兽医研究所 | Lactobacillus reuteri SXDT-32 and application thereof |
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