CN103421715B - Lactobacillus rhamnosus and application thereof - Google Patents
Lactobacillus rhamnosus and application thereof Download PDFInfo
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- CN103421715B CN103421715B CN201310335282.6A CN201310335282A CN103421715B CN 103421715 B CN103421715 B CN 103421715B CN 201310335282 A CN201310335282 A CN 201310335282A CN 103421715 B CN103421715 B CN 103421715B
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Abstract
The invention aims to provide lactobacillus rhamnosus having bacteriostatic and immunity enhancement function, with the preservation number: CCTCC NO: M 2013355. The lactobacillus rhamnosus provided by the invention can be applied in the biological products which are medicines, food or health products. The lactobacillus rhamnosus has very powerful bacteriostatic ability, can obviously improve the achroacyte conversion and HCso of a mouse, phagocytosis rate and index of macrophage and NK cell activity, can enhance normal mouse immunity, and therefore can be widely applied in the medicines, food or the health products.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of lactobacillus rhamnosus, and the application in antibacterial and strengthening immunity.
Background technology
In human body, intestinal microflora is huge, altogether about has 10
14individual, and of a great variety, nearly 400 various bacteria.Entero-bacte can be divided into probiotics, harmful bacteria and neutral bacterium.Harmful bacteria, to the harm of human body mainly their decomposing proteins and fat, produces the material that a large amount of ammonia, amine and imines, indoles, phenols etc. are harmful to health.These objectionable impuritiess can cause enteron aisle uncomfortable, increase the weight of burden of liver, cause immunity degradation, bring out cancer.And probiotics fermentable carbohydrates can generate organic acid, thus make enteron aisle keep subacidity, suppress the growth of harmful bacteria.When in human intestinal enteron aisle, harmful bacteria is preponderated, body immunity will decline; When in human intestinal enteron aisle, probiotics is preponderated, body immunity will improve.
Lactobacillus rhamnosus is that two scientists of 20 th century America obtain from healthy human body separation, and it can resist gastral poor environment, arrives at enteron aisle smoothly, efficiently surely grows in human intestinal.It can adsorption of aflatoxin, removal heavy metal cadmium.But lactobacillus rhamnosus antibacterial with strengthening immunity in but there is no correlation report.
Summary of the invention
The object of this invention is to provide a kind of antibacterial and lactobacillus rhamnosus that is strengthening immunity function of having, and the purposes of this bacterium in pharmaceutical compositions, food or healthcare products, thus make up the deficiencies in the prior art.
One aspect of the invention provides a kind of lactobacillus rhamnosus YJM-L01(Lactobacillus rhamnosus YJM-L01), be stored in the China typical culture collection center of Wuhan, China Wuhan University on July 23rd, 2013, deposit number is CCTCC NO:M 2013355.
Above-mentioned lactobacillus rhamnosus, for the preparation of bacterium powder preparation, is obtained by frozen dried by the fermentation culture bacterium liquid of lactobacillus rhamnosus.
The application of lactobacillus rhamnosus in the biological products preparing antibacterial and strengthening immunity of the present invention's screening, described biological products are medicine, food or healthcare products.
Lactobacillus rhamnosus provided by the invention has very strong bacteriostasis, and the lymphocyte transformation of mouse, half hemolysis value, the phagocytic rate of scavenger cell and phagocytic index, NK cytoactive can be significantly improved, strengthen the immunizing power of normal mouse, therefore can be widely used in medicine, food or healthcare products.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.For concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out conventional replacement according to existing technology and select, and be not limited only to the concrete record of the embodiment of the present invention.
The culture medium prescription selected in the embodiment of the present invention is as follows:
MRS liquid nutrient medium:
Peptone 10.0 g; Extractum carnis 10.0 g; Yeast extract paste 5.0 g; Diammonium hydrogen citrate 2.0 g; Glucose 20.0 g; Tween 80 1.0 mL; Sodium acetate 5.0 g; Dipotassium hydrogen phosphate 2.0 g; Magnesium sulfate 0.58 g; Manganous sulfate 0.25 g; Distilled water 1 000 mL; PH 6.2-6.4
Add the MRS solid medium of calcium carbonate:
Peptone 10.0 g; Extractum carnis 10.0 g; Yeast extract paste 5.0 g; Diammonium hydrogen citrate 2.0 g; Glucose 20.0 g; Tween 80 1.0 mL; Sodium acetate 5.0 g; Dipotassium hydrogen phosphate 2.0 g; Magnesium sulfate 0.58 g; Manganous sulfate 0.25 g; Agar 18.0 g; Calcium carbonate 5g; Distilled water 1 000 mL; PH 6.2-6.4
Calcium carbonate and agar powder have adjusted pH to add again, shakes up before a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.
The selection systems of embodiment 1 lactobacillus rhamnosus
Gather normal adults fresh excreta posterior segment, with sterile glass rod, ight soil is smashed to pieces, add 10 times of weight PBS damping fluids, the centrifugal 5min of concuss 30min, 2000g, collect supernatant liquor; By centrifugal for supernatant liquor 5000g 10min, collecting precipitation; 2 times are precipitated with PBS damping fluid repetitive scrubbing; Add a small amount of PBS damping fluid fully to suspend precipitation; By suspension in the MRS selective medium adding calcium carbonate, 37 DEG C of Anaerobic culturel 48h; Choosing colony is large, grow fast bacterial strain, purifying of ruling further.
The strain bacterium called after YJM-L01 obtained will be separated.The logical circular or similar round of bacterium colony, neat in edge, bacterium colony size is generally 2 ~ 3mm, oyster white.Be lactobacillus rhamnosus (Lactobacillus rhamnosus) from this bacterium of Physiology and biochemistry qualification result preliminary evaluation described in following table.
Table 1:YJM-L01 bacterial strain physio-biochemical characteristics qualification result
Adopt this bacterium 16S rRNA sequence of pcr amplification simultaneously, compare, the homology that result shows lactobacillus rhamnosus in this sequence and ncbi database reaches 99%, therefore be lactobacillus rhamnosus by this dientification of bacteria, called after lactobacillus rhamnosus YJM-L01(Lactobacillus rhamnosus YJM-L01), and the China typical culture collection center of Wuhan, China Wuhan University is preserved on July 23rd, 2013, deposit number is CCTCC NO:M 2013355.
Embodiment 2: acidproof, the resistance to biliary salts experiment of lactobacillus rhamnosus YJM-L01 of screening
2.1 acidproof experiments
The pH value of MRS liquid nutrient medium is adjusted to 2.0,3.0,4.0 respectively; 121 DEG C of sterilizing 15min; Be inoculated into according to 2% inoculum size in the substratum cooled by the lactobacillus rhamnosus of activation, be placed in 37 DEG C of constant incubators and cultivate, shake up once, sample respectively at when 0h, 1h, 2h every 30min, plate count measures viable count, the results are shown in Table 1.
Table 2: lactobacillus rhamnosus acid resistance is tested
As can be seen from Table 2, the present invention screens after the lactobacillus rhamnosus obtained cultivates 1h in the MRS substratum of pH2.0, viable bacteria rate is up to 80%, thus illustrate that this bacterial strain has stronger acid-fast ability, effectively can resist gastric juice, show lactobacillus rhamnosus of the present invention and there is the potential making oral preparation.
2.2 resistance to biliary salts experiments
Microorganism is that it can be survived at enteron aisle, grow and one of prerequisite playing effect for the resistance of cholate.The restraining effect of cholate to bacterial strain depends on the concentration of cholate and the characteristic of bacterial strain itself, and the content of different digestive tube positions cholate is different, and in human small intestine, cholate content fluctuates between 0.03% ~ 0.3%.Just may can survive in enteron aisle by the bacterial strain of metabolism and growth in normal physiological gallbladder salinity.
Oxgall powder is added in MRS liquid nutrient medium, its massfraction is made to be respectively 0.0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, lactobacillus rhamnosus of the present invention is inoculated according to 2% inoculum size, 37 DEG C of Anaerobic culturel, take purpurum bromocresolis as indicator, observe substratum colour-change, the results are shown in Table 2.
Principle: lactobacillus rhamnosus produces acid, and Medium's PH Value can be caused to decline, and indicator will become yellow from purple.
Table 3: lactobacillus rhamnosus bile tolerance is tested
| Gallbladder salinity (%) | Growing state |
| 0.0 | ++ |
| 0.1 | ++ |
| 0.2 | + |
| 0.3 | + |
| 0.4 | - |
| 0.5 | - |
Note: ++ be substratum variable color within 24h ,+be variable color within 48h ,-be substratum nondiscoloration
As can be seen from Table 3, lactobacillus rhamnosus of the present invention is still can survive in the environment of 0.3% at gallbladder salinity, and well-grown, make substratum variable color in 48 hours, thus illustrate that this bacterial strain has very strong tolerance to cholate, effectively surely can grow in small intestine condition.
Above-mentioned result shows that the lactobacillus rhamnosus that the present invention screens has good tolerance for internal milieu, can be used for preparing oral preparation.
Embodiment 3 lactobacillus rhamnosus suppresses colon bacillus 0157: H7 to test
Lactobacillus rhamnosus of the present invention is inoculated in MRS liquid nutrient medium, after cultivating 18h, by measuring OD
500value adjustment viable count is about 5 × 10
8cfu/mL.Intestinal bacteria are inoculated in nutrient broth simultaneously, after 37 DEG C of cultivation 18h, adjustment viable count to 1 × 10
6cfu/mL.Be cooled to by nutrient agar be poured in sterile petri dish when about 40 DEG C, after to be solidified, draw Escherichia coli bacteria liquid 1mL evenly spreadable at planar surface, 37 DEG C of fixing cultivation bacterium colony 30min, flat board are beaten the hole of diameter 4mm, hole depth 3mm.By adjusting the bacterium liquid note of concentration to expiring hole, cultivating 48h observationss in 37 DEG C of micro-aerobic environments, measuring inhibition zone size.
Lactobacillus rhamnosus to all antibacterial circle diameter of intestinal bacteria all at more than 10mm, average at more than 12.7mm, and general milk-acid bacteria is to colon bacillus 0157: the suppression loop diameter of H7 is at about 7 ~ 10mm, and test-results shows that the lactobacillus rhamnosus that the present invention screens acquisition can effectively suppress or kill Escherichia coli O 157: H7.
Strengthening immunity experiment in embodiment 4 body
Animal grouping and administration:
Laboratory animal uses healthy Kunming mouse 40, male and female half and half, 6 ~ 8 week age, body weight 18 ~ 22g, is provided by laboratory animal field, the jiangsu wuxi Hui Shan south of the River.Mouse is at room temperature raised, and free choice feeding is drunk water, after the adaptability of 3d is raised, be divided into 4 groups at random, often organize 10, set up basic, normal, high three dosage groups, be respectively 0.17,0.33,1g/kg, be equivalent to 5,10,30 times of human body recommended amounts respectively, separately establish distilled water control group.Every day 1 time, according to dosage design and feed 30d continuously, phase and experiment are weighed once latter stage respectively in an experiment.
Testing index: body weight, internal organs/body weight ratio (thymus gland/body weight ratio, spleen/body weight ratio), cellular immune function (mouse spleen lymphocyte conversion test), humoral immune function (antibody producing cells detect, serum hemolysin measure), monokaryon one macrophage function (chicken red blood cell experiment is engulfed in the experiment of mouse carbonic clearance, Turnover of Mouse Peritoneal Macrophages), NK cytoactive.
Criterion: in any two, result is positive in cellular immune function, humoral immune function, monokaryon one macrophage function, NK cytoactive 4, then can have strengthening immunity function by discriminating test sample.Measurement result is in table 4 and table 5.
Table 4: lactobacillus rhamnosus various dose is to Mouse Weight changing conditions
| Group | Original body mass (g) | Final body weight (g) | Weightening finish (g) |
| Blank group | 21±0.7 | 34±0.3 | 13±0.6 |
| Low dose group | 21±0.3 | 34±0.4 | 13±0.3 |
| Middle dosage group | 21±0.1 | 34±0.8 | 13±0.4 |
| High dose group | 21±0.8 | 34±0.2 | 13±0.2 |
As can be seen from Table 4, compared with control group, each dosage group Mouse Weight increment does not have significant difference, shows that the body weight of lactobacillus rhamnosus of the present invention to mouse has no significant effect.
Table 5: lactobacillus rhamnosus various dose is on the impact of mouse immune index
As can be seen from Table 5, each dosage group thymus index and spleen index all do not have significant difference, show that lactobacillus rhamnosus does not make significant difference to the thymus gland of mouse and spleen; And middle and high dosage group can significantly improve lymphocyte transformation, half hemolysis value, the phagocytic rate of scavenger cell and phagocytic index, NK cytoactive, judge to be the positive in the measurement result of cellular immune function, humoral immune function, monokaryon one macrophage function, NK cytoactive, show that lactobacillus rhamnosus of the present invention significantly can strengthen the immunizing power of normal mouse.
Lactobacillus rhamnosus of the present invention can be widely used in medicine, food or healthcare products, for suppressing the growth of harmful bacteria, and the immunizing power of enhancing body.
Claims (5)
1. a lactobacillus rhamnosus (
lactobacillus rhamnosus) YJM-L01, it is characterized in that, the deposit number of described lactobacillus rhamnosus strain is CCTCC NO:M 2013355.
2. lactobacillus rhamnosus strain according to claim 1 is preparing the application in bacteria preparation.
3. a bacterium powder preparation, is characterized in that, described bacterium powder preparation is obtained by frozen dried by the fermentation culture bacterium liquid of lactobacillus rhamnosus strain according to claim 1.
4. lactobacillus rhamnosus strain according to claim 1 is preparing the application in the biological products suppressing intestinal bacteria and strengthening immunity.
5. apply as claimed in claim 4, it is characterized in that described biological products are medicine, food or healthcare products.
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| CN104593307B (en) * | 2014-03-09 | 2018-06-22 | 黑龙江大学 | With the Lactobacillus rhamnosus and purposes for inhibiting angiotensins enzyme effect |
| CN103937708B (en) * | 2014-03-24 | 2016-05-25 | 华东理工大学 | Lactobacillus rhamnosus and application thereof |
| CN106511976A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Composition for relieving gynecologic inflammation and improving immunity |
| CN106520598A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus rhamnosus and application |
| CN106497847A (en) * | 2016-12-14 | 2017-03-15 | 曹书华 | A kind of lactobacillus rhamnosuss lyophilized powder and preparation method thereof |
| CN110122877B (en) * | 2018-02-09 | 2022-12-23 | 深圳华大基因农业控股有限公司 | Lactobacillus rhamnosus and application thereof |
| CN111944721A (en) * | 2020-08-17 | 2020-11-17 | 东北农业大学 | Lactobacillus rhamnosus with immunoregulation effect and application thereof |
| CN112940985A (en) * | 2021-03-31 | 2021-06-11 | 盐城维康生物科技有限公司 | Lactobacillus rhamnosus preparation for enhancing human immunity and preparation method thereof |
| CN114774315B (en) * | 2022-04-13 | 2023-12-19 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product |
| CN116333945B (en) * | 2023-05-12 | 2023-08-01 | 成都海若健康科技有限公司 | Helicobacter pylori resistant lactobacillus rhamnosus PUK09 and application, product and method thereof |
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