CN100591756C - Acidproof and bile-salt-resisting rhamnose lactobacillus strain with anti-enterovirus and antioxidant functions - Google Patents
Acidproof and bile-salt-resisting rhamnose lactobacillus strain with anti-enterovirus and antioxidant functions Download PDFInfo
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Abstract
Lactobacillus rhamnosus and the use for it and its metabolin are disclosed. The strain is sieved and obtained from excrement of long-lived old. It's safe, has excellent antibacterial, acid-resisting and antioxidant performances. It can be used in dairy process and functional food production.
Description
Technical field
The invention belongs to the microbiology field, more specifically, the present invention relates to a kind of acidproof bile tolerance, anti-enterovirus and resistance of oxidation lactobacillus rhamnosus strain of novelty.
Background technology
Milk-acid bacteria is can a ferment Kohlenhydrate and mainly generate the general name of the bacterium of lactic acid of a class.At present, milk-acid bacteria is carried out a large amount of research in the world, and in practice, milk-acid bacteria is applied in many fields.
Usually, milk-acid bacteria has avidity to the Alimentary epithelial cell that contains a large amount of Sugar receptorss.Thereby the milk-acid bacteria that obtains from external environment tends to be attached on this zone in vivo, and breeds thereon.And, dailyly be present in intravital milk-acid bacteria and on this attaching surface, form colony.
Be present in the lactobacillus on the epithelial cell, survive mutually with these epithelial cells, this characteristic is brought the healthy functions effect to the host.Milk-acid bacteria not of the same race can play different effects, and for example, (1) forms dominant microflora at cell surface, and pathogenic micro-organism is produced restraining effect; (2) bacterial strain secretion antibacterium material; (3) bacterial strain helps digestion active; (4) produce anti-cancer active matter etc.
Recent discovers that the probiotic bacterium that is used for the people preferably derives from the human body.But the many functional lactobacillus bacterial strains that obtain are from acquisitions such as other animal intestine, ight soil, food, environment, soil mostly now.Therefore, though lactic bacterium strains has the ability of sticking to small intestine and large intestine, when with them during as ingestion of food, many milk-acid bacterias can not tolerate hydrochloric acid in gastric juice and bile, thereby can not pass through stomach, arrive enteron aisle.They are nearly all killed by hydrochloric acid in gastric juice.
Therefore, press for problems such as the application that solves milk-acid bacteria and effect in the prior art.Therefore in human body, seek out the advantage milk-acid bacteria and be used,, will have more strong functions than the milk-acid bacteria in other source for keeping HUMAN HEALTH, promote the body normal function, delaying senility etc.
Summary of the invention
The object of the present invention is to provide a kind of new lactobacillus rhamnosus bacterial strain that derives from human body, this bacterial strain can adhere well on stomach and the intestinal mucosa, and can resist acid, bile, and pathogenic microbes (as Hp and intestinal bacteria) is had resistance, have anti-oxidant function simultaneously.
Another object of the present invention provides the purposes that contains described lactobacillus rhamnosus and/or its meta-bolites.
In a first aspect of the present invention, a kind of lactobacillus rhamnosus (Lactobacillusrhamnosus) is provided, the preserving number of described lactobacillus rhamnosus at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.1531.
In a second aspect of the present invention, the purposes of described lactobacillus rhamnosus and/or its meta-bolites is provided, be used to prepare food and/or the medicine that suppresses Hp, intestinal bacteria or the pathogenic bacterium of other enteron aisle.
In a third aspect of the present invention, the purposes of described lactobacillus rhamnosus or its meta-bolites is provided, be used to prepare the food and/or the medicine that prevent and/or treat bacillary digestive tract diseases.
In a preference of the present invention, described bacillary digestive tract diseases comprises: gastritis, stomach ulcer, duodenitis, enteritis, colitis or rectitis.
In a fourth aspect of the present invention, the purposes of described lactobacillus rhamnosus and/or its meta-bolites is provided, be used to prepare oxidation resistant food and/or medicine.
In a fifth aspect of the present invention, a kind of foodstuffs compositions is provided, wherein contain significant quantity (as 10
2-10
12Cfu/mL or 10
2-10
12Cfu/g, preferred as 10
5-10
10Cfu/g or 10
5-10
10Cfu/ml, preferred as 10
6-10
9Cfu/g or 10
6-10
9Cfu/ml) described lactobacillus rhamnosus and/or its meta-bolites, and acceptable carrier on the food of surplus.
In a preference of the present invention, described foodstuffs compositions is selected from solid, dairy products, solution goods, pulverulent product or suspension goods.
In a sixth aspect of the present invention, a kind of pharmaceutical composition is provided, wherein contain significant quantity (as 10
2-10
12Cfu/mL or 10
2-10
12Cfu/g, preferred as 10
5-10
10Cfu/g or 10
5-10
10Cfu/ml, preferred as 10
6-10
9Cfu/g or 10
6-10
9Cfu/ml) lactobacillus rhamnosus and/or its meta-bolites, and pharmaceutically acceptable carrier.
In a preference of the present invention, described medicine composition dosage form is selected from granule, capsule, tablet, powder agent, oral liquid, suspension or emulsion.
In another preference of the present invention, the prescription of described medicine or foodstuffs compositions is as follows:
Contain 10
2-10
12The lactobacillus rhamnosus of cfu/mL (or g) and/or its meta-bolites; And
Surplus is on the food or pharmaceutically acceptable carrier.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 a and 1b describe the situation of lactobacillus rhamnosus of the present invention behind gramstaining.
Fig. 2 describes the carbohydrate response analysis that uses API KIT 50CHL to carry out in the lactobacillus rhamnosus of the present invention, in 50 tubules (1-49 number), 0 expression contrast tubule, its interior carbohydrate of yellow pipe expression ferments from the purple stain Huang because of lactobacillus rhamnosus, and it is then black from purple stain that polychrom is measured (No. 25 pipes).
Fig. 3 describes the nucleotide sequencing result of the 16S rRNA in the Bacterium lacticum of the present invention.
Fig. 4 describes the nucleotide sequence of the 16S rRNA in the Bacterium lacticum of the present invention.
Fig. 5 describes the Auele Specific Primer (L73R and LC4F) that uses lactobacillus rhamnosus described Bacterium lacticum is carried out electrophoresis result behind the PCR.Swimming lane 1 is dna molecular amount marker λ-EcoT14I digest; Swimming lane 2 negative contrasts; Swimming lane 3 is bacterial strain of the present invention, swimming lane 4,5, and 6 are respectively lactobacillus rhamnosus 6012, lactobacillus rhamnosus 6013, lactobacillus rhamnosus grx05.
Fig. 6 describes the PCR qualification result of lactobacillus rhamnosus primer Rha1 of the present invention and Rha2; Swimming lane M is DL2000, and swimming lane 1 is blank, swimming lane 2 negative contrasts, and swimming lane 3 is the qualification result of lactobacillus rhamnosus of the present invention.
Fig. 7 describes and uses primer OPG4 amplification lactobacillus rhamnosus RAPD-PCR result, and swimming lane M is DL2000, and swimming lane 1 is a rhamnosyl breast bar 6012, and swimming lane 2 is lactobacillus rhamnosus grx01, and swimming lane 3 is lactobacillus rhamnosus grx02; Swimming lane 4 is lactobacillus rhamnosus grx03, and swimming lane 5 is bacterial strain of the present invention, and swimming lane 6 is lactobacillus rhamnosus grx05, and swimming lane 7 is lactobacillus rhamnosus grx07, and swimming lane 8 is lactobacillus rhamnosus grx09.
Fig. 8 describes can adhere to human colon adenocarcinoma cell line Caco-2 cells in vitro adhesiveness test characteristic.
Fig. 9 describes the inhibition effect of described Bacterium lacticum to Hp.
Figure 10 describes described Bacterium lacticum to colibacillary inhibition effect.
Embodiment
The inventor is through extensive and deep research and experiment, and (preserving number is: CGMCC No.1531), the inventor is with this bacterial strain called after " lactobacillus rhamnosus LV108 " finally to have found a kind of lactobacillus rhamnosus bacterial strain with excellent antibacterial characteristic.Multiple test shows that described lactobacillus rhamnosus LV108 has ability and the resistance of oxidation of harmful bacterium such as anti-Hp and intestinal bacteria, has the ability of low pH value of tolerance and bile salt, and has very strong digestive tube and stick ability.Finished the present invention based on this.
As used herein, term " lactobacillus rhamnosus of the present invention ", " lactobacillus rhamnosus bacterial strain LV108 " is used interchangeably, and refers to that all preserving number is the lactobacillus rhamnosus bacterial strain LV108 of CGMCC No.1531.
Lactobacillus rhamnosus bacterial strain LV108 of the present invention is by the following method, obtains through secular analysis and screening: (1) is isolating milk-acid bacteria from Yao Autonomous County of Bama, Guangxi and hotan area long lived elder ight soil; (2) to the strains of lactic acid bacteria more than 1000 that separate to obtain, select the specificity substratum for use, in conjunction with growth characteristics etc., tentatively determine the milk-acid bacteria monoid; (3) analyze general character advantage milk-acid bacteria, determine 3 kinds of advantage milk-acid bacterias; (4) from the preliminary advantage milk-acid bacteria of determining, the processing and the growth characteristics that compare them, filter out have excellent antibacterial characteristic, the safe milk-acid bacteria of acidproof and bile salt ability, excellent antioxidant ability, thereby can be widely used in diary processing, functional food production, salubrious additive, new drug development etc.
The present invention adopts following approach that described lactobacterium strain is identified: (1) utilizes physiology, biochemical character to analyze; (2) use API KIT 50 CHL to carry out the sugar-fermenting analysis; (3), the bacterial strain of lactobacterium strain of the present invention and standard is compared, so that definitely confirm described bacterial isolates by carrying out the 16SrRNA sequential analysis.(4) carry out RAPD polymerase chain reaction (PCR), so that gained result and conventional bacterial strain are compared.As a result, identifying described Bacterium lacticum is exactly lactobacillus rhamnosus.
Particularly, use API KIT 50 CHL sugar-fermentings, analyze and use specific program, confirm that 99% tolerance range belongs to lactobacillus rhamnosus.By using 16S rRNA order-checking and nucleotide sequence BLAST to analyze, described bacterial strain is proved with the tolerance range more than 95% and belongs to lactobacillus rhamnosus.In addition, when use is carried out PCR to the specific primer of lactobacillus rhamnosus, all found the similar fragment of about 300bp in the lactobacillus rhamnosus of bacterial strain of the present invention and empirical tests, this has proved that this bacterial strain of the present invention belongs to lactobacillus rhamnosus.
On the other hand, when 16SrDNA order-checking relatively as a result the time, bacterial strain of the present invention shows and exists some different with lactobacillus rhamnosus MCRF-412 and MCRF-271 strain.
When using when the special L73R of lactobacillus rhamnosus and LC4F primer carried out the polymerase chain reaction, have in the lactobacillus rhamnosus of test strain and other empirical tests in the fragment of 730bp and 448bp, other band is then different.Yet, when using when the special Rha1 of lactobacillus rhamnosus and Rha2 primer carried out PCR, in this bacterial strain, find the fragment of about 150bp and 448bp, and when carrying out PCR, have notable difference at 850bp place and other bacterial strain with the OPG4 primer.Therefore utilize Rha1 and Rha2, and the OPG4 primer can be used for Screening and Identification and go out bacterial strain of the present invention, thereby provable this bacterial strain has its oneself special feature.
In order to confirm the security of described bacterial isolates, select to use the ICR mouse as laboratory animal.In first embodiment of the invention, inoculation was cultivated 24 hours (about 5 * 10 in 10% skimming milk
9Cfu/mL), get 20 of healthy mices, male and female half and half, body weight 20 ± 2g, breeding observing one day, movable normal, feed is tested preceding fasting and is weighed after 12 hours in order, gavages sample by 0.4mL/10g respectively, 2 times/day, after the administration promptly to the mental status of animal, hair color, autonomic activities, breathe, diet, two just, general situation such as mouth and nose secretory product and death condition are observed, and feed continuously 7 days, detect its security after 2 months, find that organ keeps normal outward appearance, comprise that the digestion organs of the digestion organs of stomach mucous membrane and mucous membrane of small intestine and control group are similar.
On the other hand, lactobacillus rhamnosus of the present invention also has opposing acid and cholate ability.In order to study antiacid, the bile ability of bacterial strain, the inventor is adjusted to pH2.0MRS substratum, pH3.0MRS substratum, pH4.0MRS substratum with mixing acid respectively with the MRS substratum, respectively adds 2.0%, 3.0%, 4.0%, 5.0% bile salt in pH2.0MRS substratum, pH3.0MRS substratum.The result shows that bacterial strain of the present invention can be placed 5 hours and still have viable bacteria to detect in pH2,3.0% bile acide, explanation can have strong opposing acid and cholate dual capability.
On the other hand, lactobacillus rhamnosus bacterial strain of the present invention has stronger adhesive capacity.In order to detect the adhesive capacity of bacterial strain, the inventor has carried out human colon adenocarcinoma cell line Caco-2 cells in vitro adhesiveness test.Measured the adhesive capacity of bacterial strain respectively and the adherent competitiveness of pathogenic colon bacillus has been sticked restraining effect, and contrasted with other lactobacillus rhamnosus bacterial strain.Confirm that by experiment bacterial strain has the enteron aisle adhesive capacity.
On the other hand, lactobacillus rhamnosus bacterial strain of the present invention has the effect of the Hp of inhibition.Hp after the freeze thawing is drawn on a small quantity in the SkirrowShi media surface, after pushing open slightly, placed 37 ℃ of cultivations of sealed can, create little oxygen environment in the sealed can, be evacuated filling CO
2Gas and N
2Gas.Elute with the bacterium colony of physiological saline, carry out determining the bacterium number, make its concentration be controlled at 10 than turbid with Maxwell standard opacity tube with the culture dish surface
5Cfu/mL.Draw bacterium liquid 0.5mL on corresponding solid medium flat board, use aseptic paint daubs, logical sterile air in Bechtop is placed liquid-solid dull and stereotyped back, surface punching, the diameter 4mm in hole, the hole depth 3mm of fixing on of bacterium that 1h treats the surface.The lactic acid bacterial liquid of adjusting concentration is annotated to expiring the hole, can not overfolw hole outside, cultivate the 48h observations and measure antibacterial circle diameter in the proper growth environment; Use simultaneously with other lactobacillus rhamnosus strain and do contrast.Confirmed the restraining effect that this bacterial strain is stronger to Hp.
On the other hand, lactobacillus rhamnosus bacterial strain of the present invention has the colibacillary effect of inhibition.Adopt individual layer nutrient agar plate diffusion process (AWDA method) to carry out qualitative test, found that isolating lactic bacterium strains has restraining effect to intestinal bacteria.
On the other hand, lactobacillus rhamnosus bacterial strain of the present invention has oxidation resistant ability.Utilize the ICR mouse, male and female half and half, body weight 20g ± 2.0g irritates stomach strain culture (about 1 * 10
9Cfu/g) and positive reference substance V
E, continuous 8 days, 1h behind the last filling stomach plucked eyeball and gets blood, separation of serum test SOD vigor and MDA content, and this strain culture has the anti-oxidative ability of mice of significantly improving as a result, reduces the generation of body lipid superoxide.
The present invention also provides and has contained as the lactobacillus rhamnosus of the present invention of effective constituent or the foodstuffs compositions of its meta-bolites or described antibacterium material.This bacterial strain or the foodstuffs compositions that contains bacterial strain can play the effect of enteritis, colitis and the rectitis etc. that prevent and/or treat the gastritis, stomach ulcer and the duodenitis that are caused by Hp and caused by intestinal bacteria and other enteron aisle pathogenic bacteria.
In addition, the present invention also provides the lactobacillus rhamnosus that can be used as activeconstituents or the functional food or the medicine of its meta-bolites or described antibacterium material.Described food or medicine can play the effect of enteritis, colitis and the rectitis etc. that prevent and/or treat the gastritis, stomach ulcer and the duodenitis that are caused by Hp and caused by intestinal bacteria and other pathogen enterobacteria.
More specifically, described food can be fermented milk or other food that contains described lactobacillus rhamnosus, such as cultivating the various powdery products that the product that obtains is prepared into by the lactobacillus rhamnosus that lives and/or by lactobacillus rhamnosus, add the lactobacillus rhamnosus alive or the various frozen prodss of its meta-bolites, various fermented milk prods through the lactobacillus rhamnosus fermentation, add the various beverages that active lactobacillus rhamnosus is made, added the various beverages of extract of lactobacillus rhamnosus and frozen prods or the like.
In addition, the present invention's various edible dosage form of the various cultures of the lactobacillus rhamnosus that contains lactobacillus rhamnosus alive or inactivation or liquid that meta-bolites is made, pressed powder etc. also being provided and containing the antibacterium material of described milk-acid bacteria extract and purifying.
Among the present invention, term " contains " the various compositions of expression and can be applied to together in mixture of the present invention or the composition.Therefore, term " mainly by ... form " and " by ... composition " be included in during term " contains ".
Among the present invention, the composition of " pharmaceutically acceptable " or " acceptable on the food " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is promptly arranged.
Among the present invention, " pharmaceutically acceptable carrier " or " acceptable carrier on the food " is acceptable solvent, suspension agent or the vehicle pharmaceutically or on the food that is used for lactobacillus rhamnosus of the present invention or its meta-bolites are sent to the animal or human.Carrier can be a liquid or solid, preferably can higher degree keeps the carrier of lactobacillus rhamnosus or its metabolite activity.
The present invention also comprises medicine and/or foodstuffs compositions, and it comprises to the lactobacillus rhamnosus of significant quantity on administration medicine or the food or its meta-bolites.Composition of the present invention can be used for preventing and/or treating gastritis, stomach ulcer and the duodenitis that is caused by Hp, enteritis, colitis and the rectitis etc. that are caused by intestinal bacteria and other pathogen enterobacteria; And can be used for anti-oxidant.
When lactobacillus rhamnosus of the present invention or its meta-bolites are used for such use; they can with one or more food on or pharmaceutically acceptable carrier or mixed with excipients; as solvent, thinner etc., and can be Orally administered with following form: tablet, capsule, dispersible powder, particle or suspension (containing 0.05%-5% suspension agent according to appointment), syrup (containing 10%-50% sugar according to appointment).For example, these preparations can contain 10
2-10
12Cfu/g or 10
2-10
12Cfu/ml (special, can contain 10
5-10
10Cfu/g or 10
5-10
10Cfu/ml more particularly, can contain 10
6-10
9Cfu/g or 10
6-10
9Cfu/ml) activeconstituents that active rhamnosyl breast bar and/or fermentation produce.
In the present invention, " significant quantity " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.Such as, in the present invention, can prepare and contain 10
2-10
12Cfu/g or 10
2-10
12Cfu/ml (special, can contain 10
5-10
10Cfu/g or 10
5-10
10Cfu/ml more particularly, can contain 10
6-10
9Cfu/g or 10
6-10
9Cfu/ml) the rhamnosyl breast bar and/or the preparation of its meta-bolites.
When being used for pharmaceutical compositions, the used lactobacillus rhamnosus or the effective dose of its meta-bolites can change with the severity of pattern of using and disease to be treated.Be applicable to dosage form for oral administration, comprise and solid-state or liquid pharmaceutically acceptable carrier intimately mixed about 10
2-10
12Cfu/g or 10
2-10
12Cfu/ml (special, can contain 10
5-10
10Cfu/g or 10
5-10
10Cfu/ml more particularly, can contain 10
6-10
9Cfu/g or 10
6-10
9Cfu/ml) activeconstituents that active rhamnosyl breast bar or fermentation produce.Can regulate this dosage replys so that optimal treatment to be provided.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.
Described lactobacillus rhamnosus or its meta-bolites can give by approach such as oral.Solid-state carrier comprises: starch, lactose, Lin Suanergai, Microcrystalline Cellulose, sucrose and white bole, and liquid carrier comprises: substratum, polyoxyethylene glycol, nonionic surface active agent and edible oil (as Semen Maydis oil, peanut oil and sesame oil), as long as be fit to lactobacillus rhamnosus or its meta-bolites characteristic and required specific administration mode.Normally used adjuvant also can advantageously be comprised in pharmaceutical compositions, for example seasonings, pigment, sanitas and antioxidant such as vitamin-E, vitamins C, BHT and BHA.
From being easy to prepare the position with administration, preferred pharmaceutical composition is a solid-state composition, and especially tablet and solid are filled or the capsule of liquid filling.Oral administration is preferred.
Major advantage of the present invention is:
(a) lactobacillus rhamnosus of the present invention has excellent antibacterial characteristic, has excellent acidproof and bile salt ability and has excellent resistance of oxidation.
(b) lactobacillus rhamnosus of the present invention has gastritis, stomach ulcer and duodenitis that prevention and treatment cause by Hp and the function of the enteritis, colitis and the rectitis that are caused by intestinal bacteria and other enteron aisle pathogenic bacteria.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to " microorganism: laboratory manual " (James Cappucc ino and Natalie Sherman volume, Pear son Education press) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the source of sample and collection
Bacterium source of the present invention also is the world the 4th and the 5th Changshou village in 2 Changshou villages of China---Yao Autonomous County of Bama, Guangxi and hotan area long lived elder ight soil.
The Ba Ma county is positioned at the northwestward in Guangxi, belongs to the mountain and hill area.Mean sea level is at 400-600 rice, and negative oxygen ion is many, and is with fresh air.Locals's polydispersion is lived in mountain area, river valley, forest zone, noiselessness.Crust horse located in subtropical zone, luxuriant growth, evergreen all the year round, help vision protection.Nearly more than 2000 kinds, quantity of precipitation and water resources are abundant for floristics.Drinking-water all is the hard water of certain degree, contains a large amount of various trace elements.In rain same season of heat, the cool autumn in spring is refreshing, the Rhizome of Decumbent Corydalis heat, and the winter is warmed up no severe cold.Advantageous physical environment provides good natural condition for crust horse people longevity.
Be positioned at Xinjiang southernmost with the field, Gu claims in being full of, and hides the words meaning and is " producing the place of jade ".It is the important city on Nandao, the Silk Road.Hotan Prefecture's total area is 24.8 ten thousand square kilometres, population 1,500,000.Wherein the Uygur nationality accounts for more than 97% of total population.As far back as period in merchant week, ancient in just be full of and in original material exchange of crossing.B.C. 68 years, Supreme Being Chinese a surname " send defend department protect all states to the west of the Shan " formally included under the governing of the central government of Han dynasty in being full of.It is Buddhism centers the earliest, the Western Regions that Gu Yu is full of, and abundant Buddhist cultural heritage is arranged; When many famous eminent monks such as Shanxi when Faxian, Tang Xuanzang all once set foot in and the field.And strong, unique folk customs landscape more make the people fresh and new, the full foreign lands' elegance of leading.Warm with the field weather, special product is abundant, and The people are simple and honest, and is plain so that " township of the nation of gold and jade, the storehouse of grain and cotton, the Silk Road, melon and fruit is world-famous for.And enjoy great prestige at home and abroad with traditional produce such as jade, carpet, silks.The wonderful scenery that the second in the world nefud is arranged here, bimillennium history the silk historic site, and the places of cultural interest such as the oasis created of people and the Nature fight, a thousand li grape gallery.
Sample collecting of the present invention divides 3 times, and wherein Guangxi is 2 times, Xinjiang 1 time.The ight soil of two the long-lived family of typical case different generations and local long-lived typical village safety village, first seal character township, Yao Autonomous County of Bama crust dish small head of the stationary troops longevity old man's ight soil are taked in Guangxi, object comprises 23 of the good health and a long life old man of crust horse more than 90 years old, 6 of above long lived elders wherein at the age of one hundred years old.
Xinjiang takes to be primarily aimed in Hotan Prefecture's periphery 50 kilometer range, is distributed in 18 long lived elders more than 90 years old in 3 districts.
Embodiment 2: isolate milk-acid bacteria from long lived elder ight soil
Bacterial screening has carried out 5 years (2000-2005) altogether, immerses the MRS broth culture from Guangxi crust horse and 54 faecal samples of hotan long lived elder (more than 90 years old), cultivates 24 hours 37 ℃ of anaerobism.This culture is coated on the MRS agar plate, cultivated 3 days at 37 ℃ then.After the cultivation, be collected in the bacterium colony that occurs on the nutrient agar, and be further purified, utilize the colonial morphology screening to obtain the acid-producing bacteria of strain more than 1000 altogether, obtain milk-acid bacteria by its product is measured, according to growth characteristic, metabolic characteristics, and select the specificity substratum for use, obtain the advantage milk-acid bacteria.From 3 kinds of advantage milk-acid bacterias,, obtain one strain of good characteristic milk-acid bacteria according to its characteristic.Described lactobacillus rhamnosus has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preserving number is CGMCC No.1531, and preservation day is on November 10th, 2005.
With its bacterial detection feature, comprise biochemical reactions, and gramstaining as mentioned below, sugar-fermenting ability etc. (the CHARACTERISTICS IDENTIFICATION method is as mentioned below).
Isolated strains adds the broth culture of 10% skimming milk, cultivation, freeze-drying, is kept at-18 ℃, is used for the various token tests of bacterial detection then.
Embodiment 3: the lactic bacterium strains gramstaining
Adopt gram staining method to detect the feature of bacterial strain.The result as shown in Figure 1, bacterial strain of the present invention is gram-positive microorganism, and is bar-shaped for no gemma, size is 1.0-1.5 μ m, this is the characteristic feature of lactobacillus.
Then, study this milk-acid bacteria, to illustrate its cultural characteristic.The result is, this bacterial classification grows up to the grey bacterium colony that diameter is 2-3mm on the MRS agar plate, and optimum culturing temperature is at 37 ℃.
Embodiment 4: use API KIT 50CHL that bacterial strain is analyzed
At 37 ℃, under anaerobic this bacterial strain was cultivated 48 hours on the MRS agar plate, with the suspension culture base gained culture is suspended then, the turbidity of suspension is identical with McFarland's 2.Then, this culturing cell is inoculated in the API 50CHL reagent strip (available from French Biomerieux SA), layer covers the aseptic paraffin of one deck thereon, so that detect the fermentation capacity to 49 kinds of carbohydrate.
Use API identification software program (French Biomerieux SA) to analyze the carbohydrate fermentation capacity.The results are shown in Figure 2 and table 1.In the table 1,0 expression contrast tubule, the carbohydrate of the various confessions of GLY-5KG (totally 49 kinds) expression lactobacillus rhamnosus fermentation of the present invention, correspond respectively to (the positive findings: of each tubule among Fig. 2 if fermentable carbohydrates, because contained purpurum bromocresolis indicator acid production is from the purple stain Huang in the culture, it is then black from purple stain that polychrom is measured (No. 25 pipes)), the software analysis result shows that the sugar-fermenting ability of bacterial strain of the present invention is similar to the lactobacillus rhamnosus of other empirical tests, and similarity is more than 99%.Meaning of each abbreviation of used carbohydrate GLY-5KG can provide referring to Co., Ltd among the biological Mei Liai of France in the table 1 operational manual---" API Bacteria Identification gold standard ", 122-125 page or leaf.
Table 1
Embodiment 5: the extracting of total genomic dna
To the degreasing milk medium activation, plate streaking separates (MRS nutrient agar) with bacterial classification inoculation of the present invention, and picking list colony inoculation liquid nutrient medium places CO
2Thermostat container deep layer anaerobism is cultivated (37 ℃ of concentration 10% of temperature), collects the lactic-acid bacteria cells of test tube bottom after 48 hours, is used for the extracting of genomic dna.Get the 1.5mL cell culture fluid in centrifuge tube, 6000g is centrifugal, and 5min abandons supernatant; Precipitation is washed somatic cells 3-5 time with 3mL physiological saline (85%NaCl); Sedimentation cell adds 1200 μ L extraction buffers, and makes it to suspend 250 μ L10%SDS and 750 μ L Benzyl Chlorides.The vibration mixing, 60 ℃ of shaking baths insulation 40min, its at interval 10min put upside down mixing for several times, but the proper extension time to 1h, better effects if; Ultrasonication 10min; The NaOAc (pH5.0) that adds 750 μ L3M, ice bath 20min; The centrifugal 10min of 8000g collects supernatant, adds the centrifugal 10min precipitation of Virahol 10000g of 0.8 times of volume; The DNA that obtains is dissolved in the TE damping fluid of 150 μ L with 70% washing with alcohol, adds RNase and further removes RNA at 37 ℃ of digestion 1h.
With the milk-acid bacteria genome DNA sample that extracts, respectively get 10uL and carry out gel electrophoresis with 1% agarose, EB observes the integrity of purity assay and genomic dna after dyeing on gel imaging system.Then the milk-acid bacteria genome DNA sample that extracts is measured OD260nm and OD280nm on ultraviolet spectrophotometer, DNA concentration is carried out quantitatively.
Embodiment 6:16S rRNA sequencing analysis
Because for bacterium, 16s rRNA is more stable, so the sequential analysis of 16s rRNA is the fine method of Bacteria Identification.
The genomic dna of the experimental strain that extracts is used for polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification 16S-rDNA sequence as template.
The primer that is used for the 16S-rDNA sequencing analysis of milk-acid bacteria of the present invention is:
Upstream primer (SEQ ID NO:2):
5 '-AGAGT TTGAT YMTGG CTCAG-3 ' (wherein, Y, M represent degenerate primer, and Y represents (CT), and M represents (AC))
Downstream primer (SEQ ID NO:3):
5’-ACGGT TACCT TGTTA CGACTT-3’
The PCR reaction system consists of: the cumulative volume 50 μ L of PCR reaction, wherein contain 10 * PCR damping fluid, 5 μ L, 2.5mmol/L dNTPs 4.0 μ L, 5U/ μ L Tag enzyme 2.0 μ L, 50pmol/ μ L upstream primer 4.0 μ L, 50pmol/ μ L downstream primer 4.0 μ L, 20ng/ μ L template DNA 5.0 μ L, ddH2O26 μ L.
Response procedures is: 95 ℃ of pre-sex change 5min, and 55 ℃ of annealing of 95 ℃ of sex change 1min 50s, 72 ℃ are extended 30 circulations of 2min; 55 ℃ of annealing of 95 ℃ of sex change 1min 50s, 72 ℃ are extended 1 circulation of 2min; Extend 1 circulation of 10min after 72 ℃.
Amplified production is electrophoretic separation on 1.0% sepharose, ethidium bromide staining with 0.5 μ g/mL, behind observation analysis on the gel imaging system, reclaim the dna fragmentation of 1500bp size with gel extraction agent box (available from Shanghai lottery industry bio-engineering corporation), and be cloned in the pEGM-T carrier (available from Shen energy betting office), by the method transformed into escherichia coli that provides on the molecular cloning handbook, at the dull and stereotyped enterprising row filter of the IPTG/X-galLB that contains penbritin, the picking positive colony is measured the 16S-rDNA sequence after identifying.Sequencing result is seen Fig. 3, and the 16S-rDNA sequence is seen Fig. 4 (being SEQ ID NO:1).
Embodiment 7: use BLAST analysis of nucleotide sequences data
The 16S-rRNA sequence of said determination is used blast program, and (U.S.'s bioinformation center website, NCBI www.ncbi.nlm.nih.gov) carry out homology relatively with known multiple lactobacillus rhamnosus, according to the distance value that obtains, set up genealogical tree.
Found that the 16S-rRNA sequence of lactobacillus rhamnosus of the present invention is neither together with the corresponding sequence of present known other all lactobacillus rhamnosus, with the homology the highest (reaching more than 95%) of lactobacillus rhamnosus.
Embodiment 8: Auele Specific Primer is identified lactobacillus rhamnosus
The inventor has designed a kind Auele Specific Primer according to lactobacillus rhamnosus 16S rRNA and 23S rRNA, as table 2.
Table 2 primer
| | Sequence |
| LC4F | |
| 5′-AGGGTGAAGTCGTAACAAGT-3′(SEQ ID NO：4) | |
| |
5′-GCCAACAAGCTATGTGTTCGCTTGC-3′(SEQ ID NO：5) |
The inventor adopts 20 μ l PCR reaction systems (the PCR condition is the same), found that when L73R and LC4F primer carry out the polymerase chain reaction, fragment at 730bp and 448bp is being tested strain (swimming lane 3) and other lactobacillus rhamnosus ( swimming lane 4,5,6 are respectively lactobacillus rhamnosus 6012, lactobacillus rhamnosus 6013, lactobacillus rhamnosus grx05, all available from Yangzhou University) in have, other band is then different.The results are shown in Figure 5.
Embodiment 9: multiple RAPD pcr analysis
Genomic dna to lactobacillus rhamnosus bacterial strain among the present invention and known lactobacillus rhamnosus bacterial strain carries out random polymorphism amplification (Random Amplified Polymorphism DNA, RAPD) analyze, with relatively this bacterial strain and other lactobacillus rhamnosus bacterial strain in the difference of genomic dna.Other lactobacillus rhamnosus bacterial strain comprises in the present embodiment: swimming lane 1 is lactobacillus rhamnosus 6012, and swimming lane 2 is lactobacillus rhamnosus grx01, and swimming lane 3 is lactobacillus rhamnosus grx02; Swimming lane 4 is lactobacillus rhamnosus grx03, and swimming lane 5 is bacterial strain of the present invention, and swimming lane 6 is lactobacillus rhamnosus grx05, and swimming lane 7 is lactobacillus rhamnosus grx07, and swimming lane 8 is lactobacillus rhamnosus grx09.(lactobacillus rhamnosus 6012, lactobacillus rhamnosus grx02; Lactobacillus rhamnosus grx03, lactobacillus rhamnosus grx05, lactobacillus rhamnosus grx07, lactobacillus rhamnosus grx09 is all available from Yangzhou University).
Carry out the extraction of milk-acid bacteria genomic dna according to method noted earlier, get the 100ng genome DNA sample behind integrity and the concentration analysis and carry out the RAPD analysis as follows.The sequence of synthetic random primer is as shown in table 1.Adopt RAPD amplification reaction system: the cumulative volume 25 μ L of reaction through optimizing, wherein contain 10 * PCR damping fluid, 2.5 μ L, 2.5mmol/L dNTP 2.0 μ L, 5U/ μ L Tag enzyme 1.0 μ L, 50pmol/ μ L random primer 2.0 μ L, 20ng/ μ L template DNA 5.0 μ L, ddH2O 12.5 μ L.Response procedures is: 93 ℃ of 2min, 36 ℃ of 1min, 1 circulation of 72 ℃ of 2min; 93 ℃ of 1min, 36 ℃ of 1min, 72 ℃ of 2min, 40 circulations; 93 ℃ of 1min, 36 ℃ of 1min, 72 ℃ of 1 circulations of 10min (in the screening of carrying out random primer,, need carry out suitable adjustment) to annealing temperature at different primers.Amplification finishes, and on 1.4% sepharose, adds and contains EB0.5 μ g/mL electrophoretic separation, and film making is analyzed on gel imaging system.This experiment from 30 random primers (sequence is shown in Table 3), screen 1 plant and the bacterial strain level have good resolving ability primer OPG4.
The result shows at 850bp place and other bacterial strains to have notable difference as shown in Figure 7, and the OPG4 primer can be used for Screening and Identification and go out bacterial strain of the present invention.
Employed random primer during table 3 RAPD analyzes
| Numbering | Sequence | SEQ ID NO： | Numbering | Sequence | SEQ ID NO： |
| OPG-1 | 5’-AATCAACTTC-3’ | 6 | OPG-16 | 5’-CCGCAGCCAA-3’ | 21 |
| OPG-2 | 5’-TTCTTTAATT-3’ | 7 | OPG-17 | 5’-AACGCGCAAC-3’ | 22 |
| OPG-3 | 5’-TTTAACAACT-3’ | 8 | *OPG-18 | 5-GTGGATGCGA-3 | 23 |
| OPG-4 | 5’-CTGCGCTGGA-3’ | 9 | OPG-19 | 5’-GTGACGTAGG-3’ | 24 |
| OPG-5 | 5’-ACGTATCTGC-3’ | 10 | OP6-20 | 5’-CAATCGCCGT-3’ | 25 |
| OPG-6 | 5’-GCGATCCCCA-3’ | 11 | OPG-21 | 5’-GGGAGGCAAA-3’ | 26 |
| OPG-7 | 5’-GTAGACCCGT-3’ | 12 | OPG-22 | 5’-CCAAGAGGCT-3’ | 27 |
| OPG-8 | 5’-GTTTCCGCCC-3’ | 13 | OPG-23 | 5’-TCCCCAGGAG-3’ | 28 |
| OPG-9 | 5’-CTGCTGGGAC-3’ | 14 | OPG-24 | 5’-AGTGCACACC-3’ | 29 |
| OPG-10 | 5’-TGCCGAGCTG-3’ | 15 | *OPG-25 | 5’-AGGCATCGTG-3’ | 30 |
| OPG-11 | 5’-GGGAACGTGT-3’ | 16 | OPG-26 | 5’-ACGCGACAGA-3’ | 31 |
| OPG-12 | 5’-AGGCGGGAAC-3’ | 17 | OPG-27 | 5’-AGTATGGCGG-3’ | 32 |
| OPG-13 | 5’-CAGCACCCAC-3’ | 18 | OPG-28 | 5’-GGAAGTCCTG-3’ | 33 |
| OPG-14 | 5’-AGGTTGCAGG-3’ | 19 | OPG-29 | 5’-CCAGGCTGAC-3’ | 34 |
| OPG-15 | 5’-AGGTGACCGT-3’ | 20 | OPG-30 | 5’-GGTCGGGTCA-3’ | 35 |
Annotate: * represents to have the specific random primer of bacterial strain level through screening
In the test in front, thereby the inventor produces the specific mark of bacterial strain by adopting multiple RAPD-PCR technology to screen random primer.By the combination that random primer is different to 30, the product segment is between 0.6-2.2kb.Primer 5 '-find in the PCR product analysis of ACGAGGCAC-3 ' and 5 '-ACGCGCCCT-3 ' combination, have a bacterial strain specific marker at the 613bp place, can be in order to identify lactobacillus rhamnosus.This product is reclaimed cloning and sequencing, and is cloning pulsating sequence tip designs primer (table 4):
Table 4 primer
| | Sequence |
| Rha1 | |
| 5′-CTATTTAGTAATCACAGAAAAC-3′(SEQ ID NO：36) | |
| |
5′-TAACAGCAGTCTCCAAATGG-3′(SEQ ID NO：37) |
The results are shown in Figure 6, in this bacterial strain, find the fragment of about 150bp and 448bp, different with other lactobacillus rhamnosus, confirm that this bacterial strain has its distinctive characteristic.
Embodiment 10: bacterial strain of the present invention and lactobacillus rhamnosus DNA are carried out nucleotide sequence relatively
The 16s-rDNA nucleotide sequence of measuring bacterial strain of the present invention and lactobacillus rhamnosus MCRF-412, lactobacillus rhamnosus MCRF-271 compares, bacterial strain of the present invention is G at the 545th Nucleotide, and is C among lactobacillus rhamnosus MCRF-412, the lactobacillus rhamnosus MCRF-271.
Embodiment 11: bacterial strain LV108 of the present invention is to the security of ICR mouse
In order to detect the security of LV108, select to use the ICR mouse.Specifically be that the LV108 inoculation is cultivated 24h (about 5 * 10 in 11% skimming milk
9Cfu/mL), get 20 of healthy mices, male and female half and half, body weight 20 ± 2g, breeding observing one day is movable normal, feed in order, fasting was weighed after 12 hours before the experiment, gavaged sample by 0.4mL/10g respectively, 2 times/day, after the administration promptly to the mental status of animal, hair color, autonomic activities, breathing, diet, two just, general situation such as mouth and nose secretory product and death condition observe, fed 7 days continuously, detect its security after 2 months, the result is as shown in table 5.
The security of table 5 milk-acid bacteria of the present invention
By table 5 as seen, mouse is 80mL/kg to the maximum tolerated dose of this fermented liquid.Malaise symptoms does not appear in mouse after the administration, and the mouse activity reduces to some extent, and complementary basis is originally normal, good in the whole viewing duration mental status, hair color is bright and clean, activity freely, no abnormal secretory product in the eupnea, mouth and nose, the animal diet followed amount is normal, and is just soft, and it is no abnormal to urinate.After observing a week, weigh, all body weight gain rates are: 25.1%.Took off cervical vertebra and put to death animal after 2 months, and all animals are performed an autopsy on sb, internal organs such as the visual inspection heart, liver, spleen, lung, kidney there is no any unusual.
Mucus in stomach, small intestine and the large intestine of administration mouse is applied on the MRS agar plate,, from the administration group, isolates a large amount of milk-acid bacteria LV108 strains, but in control group, do not detect in 37 ℃ of cultivations 48 hours.
Embodiment 12: the antiacid and anti-cholate ability of bacterial strain LV108 of the present invention
Studied the antiacid and anti-cholate ability of lactobacillus rhamnosus LV108, on the MRS substratum, with mixing acid the MRS substratum is adjusted to pH2.0MRS substratum, pH3.0MRS substratum, pH4.0MRS substratum respectively, in pH2.0MRS substratum, pH3.0MRS substratum, respectively adds 2.0%, 3.0%, 4.0%, 5.0% bile salt.
In different substratum, the nutrient solution of the inoculum size inoculation test bacterium by 2% is put 37 ℃ and is cultivated 1h, 2h, 3h, 4h, 5h, 6h, 16h respectively, is contrast with 0h, the detection of active lactic acid bacteria number.
The result shows that bacterial strain of the present invention can be placed 5 hours, still had the living lactic acid bacteria number to detect in pH2,3.0% bile acide.Survival rate is higher than the survival rate among the pH2,3.0% in pH3,3.0%.Prove that bacterial strain of the present invention has stronger antiacid and anti-cholate ability.
Embodiment 13: lactobacillus rhamnosus LV108 is to human colon adenocarcinoma cell line Caco-2 cells in vitro adhesiveness test
In order to detect the adhesive capacity of bacterial strain of the present invention, the inventor has carried out the external adhesiveness test (see figure 8) of human colon adenocarcinoma cell line Caco-2 cell (available from the biochemical institute in Chinese Academy of Sciences Shanghai), this test strain in human colon adenocarcinoma cell line Caco-2 cell surface tight adhesion a large amount of bacterial strains of the present invention, found through experiments bacterial strain of the present invention and on average reach 18.7 ± 5.92/cell, many than other milk-acid bacteria, average general lactobacillus rhamnosus exceeds about 6.0/cell.
Measured bacterial strain of the present invention simultaneously to the adherent competitive inhibition of pathogenic colon bacillus, and contrasted with lactobacillus rhamnosus 6012, lactobacillus rhamnosus 6013, the competitive adherence inhibition effect of bacterial strain of the present invention is strong.
Embodiment 14: the resistance of oxidation of lactobacillus rhamnosus LV108
The inventor has studied the influence of lactobacillus rhamnosus LV108 to experiment mice (body weight: 20 ± 2g is available from Jiangning, Nanjing Tang Shan Green Dragon Experimental Animal Center for ICR mouse, male and female half and half) resistance of oxidation.Adopt normal mouse to irritate stomach respectively and give lactobacillus rhamnosus fermented-milk (about 1 * 10
9Cfu/g), set up (establishing high and low two dosage groups), other establishes control group and positive reference substance (vitamin-E 9mg/kg), and every group of 10 mouse are pressed 40mLkg
-1D
-1Deng capacity not isoconcentration irritate stomach and be subjected to test product and positive reference substance vitamin-E, continuous 8 days, control group was irritated stomach physiological saline simultaneously.Last is irritated behind the stomach and to be plucked eyeball in 1 hour and get blood, separation of serum test SOD vigor and MDA content.Press xanthine oxidase (the SOD test kit is available from the poly-Lik-Sang Tetramune company in Nanjing) and measure the SOD content in the serum; Press thiobarbituricacid method (the MDA test kit is available from the poly-Lik-Sang Tetramune company in Nanjing) and measure the MDA in the serum.
The results are shown in Table 6.The lactobacillus rhamnosus fermented-milk with according to product vitamin-Es the SOD vigor is increased the positive, the reduction of MDA content is with control group comparing difference highly significant.Illustrate that lactobacillus rhamnosus LV108 can strengthen the resistance of oxidation of mouse in various degree, reduce the generation of body lipid peroxidation product.
Table 6 lactobacillus rhamnosus fermented-milk anti-oxidant function test determination result
Compare * P＜0.05, * * P＜0.01 with control group
Embodiment 15: lactobacillus rhamnosus LV108 is to colibacillary restraining effect
Escherichia coli O 157: H7 (available from Yangzhou University) is inoculated in the nutrient broth, behind 37 ℃ of cultivation 18h, colony counting method meter viable count.Control its viable count about 10
6Cfu/mL, this concentration is easily caused a disease.Draw bacterium liquid 0.5mL on corresponding solid medium flat board, use aseptic paint daubs, logical sterile air in Bechtop is placed liquid-solid dull and stereotyped back, surface punching, the diameter 4mm in hole of fixing on of bacterium that 1h treats the surface.Hole depth 3mm annotates the lactic acid bacterial liquid of adjusting concentration to expiring the hole, can not overfolw hole outside, cultivate the 48h observations, measure antibacterial circle diameter in growing environment separately; Use lactobacillus rhamnosus 6012 (available from Yangzhou University) to do contrast simultaneously.
Bacterial strain of the present invention is 12.6mm (Figure 10) to the antibacterial circle diameter of Escherichia coli O 157: H7, and lactobacillus rhamnosus 6012 inhibition zones are 12.0mm, shows that lactobacillus rhamnosus LV108 has stronger restraining effect.
Embodiment 16: lactobacillus rhamnosus LV108 is to the restraining effect of Hp
With the Hp after the freeze thawing (available from Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an).Draw on a small quantity in the SkirrowShi media surface, after pushing open slightly, place 37 ℃ of cultivations of sealed can, create little oxygen environment (85%N in the sealed can
2, 10%CO
2, 5%O
2), be evacuated filling CO
2Gas and N
2Gas.Elute with the bacterium colony of physiological saline, carry out determining the bacterium number, make its concentration be controlled at 10 than turbid with Maxwell standard opacity tube with the culture dish surface
5Cfu/mL.Draw bacterium liquid 0.5mL on corresponding solid medium flat board, use aseptic paint daubs, logical sterile air in Bechtop is placed liquid-solid dull and stereotyped back, surface punching, the diameter 4mm in hole of fixing on of bacterium that 1h treats the surface.Hole depth 3mm annotates the lactic acid bacterial liquid of adjusting concentration to expiring the hole, can not overfolw hole outside, cultivate the 48h observations and measure antibacterial circle diameter in the suitable growth environment; Use lactobacillus rhamnosus 6012 (available from Yangzhou University) to do contrast simultaneously.
Bacterial strain of the present invention is 12.7mm (Fig. 9) to the Hp antibacterial circle diameter, and lactobacillus rhamnosus 6012 only is 9.5mm, shows the lactobacillus rhamnosus LV108 restraining effect stronger to Hp.
Embodiment 17: the foodstuffs compositions that contains lactobacillus rhamnosus
Proportioning raw materials such as table 7.
Table 7
| Raw material | Mass percent (%) |
| Fresh milk | 92.5 |
| White sugar | 7.5 |
By above-mentioned formula rate, fresh milk is heated to more than 50 ℃, add white sugar and be stirred to fully, be preheating to 60-65 ℃, 20Mpa pressure homogeneous, 90 ℃ of left and right sides sterilizations 5-10 minute are cooled to 40-43 ℃, inoculate.Inoculation quantity is: every 1g said mixture inoculation thermophilus streptococcus 6035 (available from Yangzhou University) 1-100 * 10
6Cfu, lactobacillus bulgaricus 6032 (available from Yangzhou University) 1-100 * 10
6Cfu, lactobacillus rhamnosus LV108 1-50 * 10
6Cfu.
40-42 ℃ ferment to the pH value for 4.2-4.5, stir, filling, the sour milk that contains active lactobacillus rhamnosus is promptly made in refrigeration.
Embodiment 18: the pharmaceutical composition that contains lactobacillus rhamnosus
Proportioning raw materials such as table 8.
Table 8
| Raw material | Mass percent (%) |
| Skim-milk | 10 |
| Lactose | |
| 5％ |
| Yeast powder | 0.5 |
| Peptone | |
| 1％ | |
| Pure water | 83.5％ |
Proportionally skim-milk, lactose, yeast powder, peptone are mixed with pure water, be preheating to 60-65 ℃, 20Mpa pressure homogeneous, 90 ℃ of left and right sides sterilizations 20-30 minute are cooled to 36-38 ℃, insert lactobacillus rhamnosus viable bacteria (1-50 * 10
6Cfu/mL), 36-38 ℃ ferment to the pH value for 4.2-4.5, centrifugal, lyophilize to water content less than 3%, promptly be prepared into lactobacillus rhamnosus lyophilize thing.Take by weighing and incapsulate after 0.5 gram lactobacillus rhamnosus lyophilize thing and the maltodextrin balanced mix, promptly make the pharmaceutical composition that contains lactobacillus rhamnosus.
Culture presevation
Lactobacillus rhamnosus of the present invention (Lactobacillus rhamnosus) LV108 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preserving number is CGMCC No.1531, and preservation day is on November 10th, 2005.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉unify Investment Co., Ltd of enterprise (China)
Kunshan Research and Development Center, Uni-President Enterprises (China) Co.,
＜120〉a kind of acidproof bile tolerance of novelty, anti-enterovirus and resistance of oxidation lactobacillus rhamnosus strain
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<170>PatentIn version 3.3
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tgcatgcctg caggtcgact ctagactcga gggatccaga tctccagtct tacggttacc 60
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tacgccaccg gcttcgggtg ttacaaactc tcatggtgtg acgggcggtg tgtacaaggc 180
ccgggaacgt attcaccgcg gcgtgctgat ccgcgattac tagcgattcc gacttcgtgt 240
aggcgagttg cagcctacag tccgaactga gaatggcttt aagagattag cttgacctcg 300
cggtctcgca actcgttgta ccatccattg tagcacgtgt gtagcccagg tcataagggg 360
catgatgatt tgacgtcatc cccaccttcc tccggtttgt caccggcagt cttactagag 420
tgcccaacta aatgctggca actagtcata agggttgcgc tcgttgcggg acttaaccca 480
acatctcacg acacgagctg acgacaacca tgcaccacct gtcattttgc ccccgaaggg 540
gaaagctgat ctctcaggtg atcaaaagat gtcaagacct ggtaaggttc ttcgcgttgc 600
ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc 660
aaccttgcgg tcgtactccc caggcggaat gcttaatgcg ttagctgcgg cactgaaggg 720
cggaaaccct ccaacaccta gcattcatcg tttacggcat ggactaccag ggtatctaat 780
cctgttcgct acccatgctt tcgagcctca gcgtcagtta cagaccagac agccgccttc 840
gccactggtg ttcttccata tatctacgca tttcaccgct acacatggag ttccactgtc 900
ctcttctgca ctcaagtttc ccagtttccg atgcacttcc tcggttaagc cgagggcttt 960
cacatcagac ttaaaaaacc gcctgcgctc gctttacgcc caataaatcc ggataacgct 1020
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gattccctac tgctgcctcc cgtaggagtt tgggccgtgt ctcagtccca atgtggccga 1260
tcaacctctc agttcggcta cgtatcattg ccttggtgag ccgttacctc accaactagc 1320
taatacgccg cgggtccatc caaaagcgat agcttacgcc atctttcagc caagaaccat 1380
gcggttcttg gatttatgcg gtattagcat ctgtttccaa atgttatccc ccacttaagg 1440
gcaggttacc cacgtgttac tcacccgtcc gccactcgtt caaaattaaa tcaagatgca 1500
agcacctttc aataatcaga actcgttcga cttgcatgta ttaggcacgc cgccagcgtt 1560
catcctgagc cagaatcaaa ctctagacct ggtctgcagg cggccgccca tgggatatca 1620
tcgatcatat gtcgccctat agtgagtcgt attacggtac cgagct 1666
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Claims (9)
1. a lactobacillus rhamnosus is characterized in that, the preserving number of described lactobacillus rhamnosus at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.1531.
2. the purposes of the described lactobacillus rhamnosus of claim 1 is characterized in that, is used for preparation and suppresses Hp or colibacillary food and/or medicine.
3. the purposes of the described lactobacillus rhamnosus of claim 1, it is characterized in that, be used to prepare the food and/or the medicine that prevent and/or treat bacillary digestive tract diseases, wherein said bacillary digestive tract diseases is selected from down group: the gastritis, stomach ulcer or the duodenitis that are caused by Hp; And the enteritis, colitis or the rectitis that cause by intestinal bacteria.
4. the purposes of the described lactobacillus rhamnosus of claim 1 is characterized in that, is used to prepare oxidation resistant food and/or medicine.
5. a foodstuffs compositions is characterized in that, wherein contains the described lactobacillus rhamnosus of claim 1 of significant quantity, and acceptable carrier on the food of surplus.
6. foodstuffs compositions as claimed in claim 5 is characterized in that, described foodstuffs compositions is selected from solid, dairy products, solution goods, pulverulent product or suspension goods.
7. a pharmaceutical composition is characterized in that, wherein contains the described lactobacillus rhamnosus of claim 1 of significant quantity, and pharmaceutically acceptable carrier.
8. pharmaceutical composition as claimed in claim 7 is characterized in that described medicine composition dosage form is selected from granule, capsule, tablet, powder agent, oral liquid, suspension or emulsion.
9. as claim 5 or 7 described foodstuffs compositions or pharmaceutical compositions, it is characterized in that the prescription of described composition is as follows:
10
2-10
12Cfu/mL or 10
2-10
12The lactobacillus rhamnosus of cfu/g; And
Surplus is on the food or pharmaceutically acceptable carrier.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200510111588A CN100591756C (en) | 2005-12-16 | 2005-12-16 | Acidproof and bile-salt-resisting rhamnose lactobacillus strain with anti-enterovirus and antioxidant functions |
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| CN200510111588A CN100591756C (en) | 2005-12-16 | 2005-12-16 | Acidproof and bile-salt-resisting rhamnose lactobacillus strain with anti-enterovirus and antioxidant functions |
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| CN1982437A CN1982437A (en) | 2007-06-20 |
| CN100591756C true CN100591756C (en) | 2010-02-24 |
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| CN200510111588A Active CN100591756C (en) | 2005-12-16 | 2005-12-16 | Acidproof and bile-salt-resisting rhamnose lactobacillus strain with anti-enterovirus and antioxidant functions |
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Families Citing this family (16)
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| CN101407835B (en) * | 2007-10-12 | 2011-08-24 | 统一企业(中国)投资有限公司 | Genetic marker and method for detecting rhamnose bacterium lacticum |
| CN101328470B (en) * | 2008-07-09 | 2010-06-09 | 扬州大学 | Rhamnose bacterium lacticum grx10 having cholesterol lowering and antibacterial functionsand use thereof |
| CN104365863B (en) * | 2014-09-28 | 2017-09-12 | 中国农业大学 | Lactobacillus rhamnosus and its application and cheese and preparation method thereof |
| CN108410761B (en) * | 2018-03-06 | 2020-03-20 | 山东凤凰生物有限公司 | Lactobacillus rhamnosus with nitrite reducing and oxidation resisting functions and screening and separating method |
| CN109136131A (en) * | 2018-08-27 | 2019-01-04 | 南昌大学 | One plant has effects that alleviate the Lactobacillus rhamnosus of colitis and its application |
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| CN109536415B (en) * | 2018-12-26 | 2021-12-07 | 江西仁仁健康产业有限公司 | Lactobacillus rhamnosus and application thereof |
| CN109662976B (en) * | 2019-02-14 | 2023-08-08 | 西南大学 | Application of lactobacillus rhamnosus in preparation of medicine for preventing ulcerative colitis |
| CN110129220B (en) * | 2019-04-30 | 2021-06-22 | 石河子大学 | Lactobacillus bulgaricus BSTS6-4 and application thereof |
| CN110106119B (en) * | 2019-05-28 | 2021-07-06 | 北京科拓恒通生物技术股份有限公司 | Lactobacillus rhamnosus M9 separated from breast milk and application thereof |
| CN110938563B (en) * | 2019-12-05 | 2020-09-29 | 北京伯恩世纪科技发展有限公司 | Lactobacillus BJ-REBORN001 and application thereof in preparation of helicobacter pylori inhibiting fermentation broth |
| KR102166461B1 (en) * | 2020-03-10 | 2020-10-15 | 주식회사 종근당바이오 | Lactobacillus rhamnosus strain having intestinal immunomodulatory function and preventive or therapeutic activity for inflammatory bowel disease |
| CN111607538B (en) * | 2020-05-29 | 2021-03-02 | 江南大学 | Lactobacillus rhamnosus and application thereof in inhibiting helicobacter pylori |
| CN112210507B (en) * | 2020-08-10 | 2022-09-06 | 江苏微康生物科技有限公司 | Shigella-antagonistic lactobacillus rhamnosus LRa05, screening method and application thereof |
| CN116747246A (en) * | 2023-06-16 | 2023-09-15 | 广东南芯医疗科技有限公司 | Application of lactobacillus rhamnosus NX-2 in preparing antioxidant and anti-aging products |
| CN117586927B (en) * | 2024-01-18 | 2024-04-23 | 山东中科嘉亿生物工程有限公司 | Lactobacillus rhamnosus JYLR-127 for improving difficult defecation after fracture, microbial inoculum and application thereof |
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2005
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