CN109810912A - One lactobacillus plantarum LH-511 and its application - Google Patents
One lactobacillus plantarum LH-511 and its application Download PDFInfo
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Abstract
This application discloses a lactobacillus plantarum LH-511 and its applications.The lactobacillus plantarum LH-511 of the application, deposit number are CGMCC No.14512.The lactobacillus plantarum LH-511 of the application, alpha-glucosidase activity inhibiting rate with higher can adjust blood glucose, have excellent prebiotic effect, also, have very strong acidproof and bile tolerance ability, being capable of long-term preservation；It can be used for preparing various food, health care product, food additives or the drugs for adjusting blood glucose, provide a kind of new efficient raw material to adjust blood glucose.
Description
Technical field
This application involves lactobacillus plantarum fields, more particularly to a lactobacillus plantarum LH-511 and its application.
Background technique
With the development of the social economy, the disease incidence of the metabolic syndromes such as diabetes and patient populations are in worldwide
In increasing trend year by year, it has also become endanger the third-largest chronic disease of human life and health.Diabetes are a kind of generations of multi-pathogenesis
Thanking property disease, clinical symptoms are hyperglycemia, glucose tolerance reduction and abnormal serum insulin concentration etc..Main cause is that pancreas is thin
Postprandial hyperglycemia caused by the insulin of intracrine is reduced or insulin sensitivity reduces, and hyperglycemia inhibits pancreatic secretion
Insulin reduces insulin and adjusts peripheral tissues to the absorbability of glucose, lacks so as to cause because of insulin secretion and effect
Fall into caused sugar, fat and protein metabolism disorder.
Diabetes are divided by the World Health Organization by the diabetes cause of disease and pathogenesis: the I type glycosuria of insulin-dependent
Disease, the type-2 diabetes mellitus of non-insulin-depending type, specific type and four seed type of gestational diabetes mellitus, wherein type 2 diabetes patient
Account for about 90% or more.Type-2 diabetes mellitus main feature shows as insulin resistance, i.e., cell can not be right after generating insulin self
It reacts, and can be with multiple complications, such as coronary heart disease, atherosclerosis, renal lesions, neuropathy, retina
Lesion and foot lesion etc. there is also typical " three-many-one-little " symptom, i.e., more drinks, diuresis, more foods and weight loss are existing
As.
Currently, there are mainly four types of the oral hypoglycemic drugs for the treatment of type-2 diabetes mellitus: (1) alpha-glucosidase (α-
Glucosidase) inhibitor: such as acarbose, voglibose；(2) Drugs Promoting Insulin Secretion: such as sulfonylurea, Rui Ge
How column are with Nateglinide etc.；(3) inhibit biguanides and the thiazolidinedione etc. of liver output glucose；(4) alleviate insulin to support
Anti- glitazone etc..Wherein, alpha-glucosidase restrainer can reversibly occupy the complexing position of alpha-glucosidase and sugar
Point inhibits alpha-glucosaccharase enzymatic polysaccharide to be degraded to monosaccharide, to delay the absorption of enteron aisle carbohydrate.Alpha-glucosaccharase
Enzyme inhibitor is the activity by inhibiting glucuroide, slows down the generation of glucose and absorption, reduces postprandial hyperglycemia, is adjusted
Whole blood glucose level improves the sensibility of insulin to reduce stimulation of the hyperglycemia to pancreas, effectively prevents and improve glycosuria
The generation of complications makes patient blood glucose maintain certain level, stabilizing blood sugar index.Alpha-glucosidase restrainer has effect
The advantages that mild lasting, nontoxic or Small side effects, by the concern of lot of domestic and international researcher, and its source also becomes and grinds
Study carefully bright spot.People are initially the glucosidase inhibitors extracted from actinomyces and chain enzyme bacteria, with going deep into for research, α-Portugal
The every field of ambient enviroment has been goed deep into the source of polyglycoside enzyme inhibitor.Wherein, the alpha-glucosidase suppression of originating in lactic acid bacterium
The important indicator that system is also screened at probiotics function of polysaccharide.Therefore, what screening acquisition was new there is high alpha-glucosidase to inhibit
The exploitation of the probiotics of effect, treatment and its novel drugs or health care product for type-2 diabetes mellitus is of great significance.
Summary of the invention
The purpose of the application is to provide one plant of new lactobacillus plantarum LH-511 and its application.
The application uses following technical scheme:
The one side of the application discloses a lactobacillus plantarum (Lactobacillus plantarum) LH-511,
Deposit number is CGMCC No.14512.
It should be noted that the deposit number of the application is the lactobacillus plantarum LH-511 of CGMCC No.14512, it is newly to send out
Existing one plant has the good bacterial strain for adjusting blood glucose function, and the research through the application confirms that the bacterial strain all has in vivo, in vitro
Have a good blood sugar decreasing effect, and have good tolerance to acid and bile, which means that the application lactobacillus plantarum
LH-511 can be eaten or take orally it is medicinal, to adjust blood glucose.Therefore, specially by the lactobacillus plantarum LH-511 of the application in
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the address of depositary institution on August 10th, 2017
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.14512, are protected
Hide entitled lactobacillus plantarum LH-511.
It should also be noted that, the lactobacillus plantarum LH-511 of the application is different from being in for existing other lactobacillus plantarums
In, the study found that the lactobacillus plantarum LH-511 of the application is greater than 33% to the external activity inhibiting rate of alpha-glucosidase, and
Lactic acid bacteria generally 10% once, have once in a while it is high be also no more than 20%, this explains the lactobacillus plantarum of application from principle
The blood sugar decreasing effect of LH-511.
The lactobacillus plantarum LH-511 that the another side of the application discloses the application adjusts food, the health care of blood glucose in preparation
Application in product, food additives, drug or composition.
The another side of the application discloses a kind of composition, and the composition contains the lactobacillus plantarum LH-511 of the application.
The composition of the application refer in addition to food, health care product, food additives, drug it is other types of contain this
Edible or Pharmaceutical composition, the composition of the lactobacillus plantarum LH-511 of application can be used for adjusting blood glucose.
It should be noted that the lactobacillus plantarum LH-511 due to the application has good function of blood sugar reduction, and can
With edible, therefore, various food, health care product, food additives or drug can be made into completely, for adjust blood glucose or
Play health-care effect.
The another side of the application discloses a kind of food, the lactobacillus plantarum LH-511 in the food containing the application.
Preferably, also containing acceptable additive or auxiliary material in bromatology in the food.
It is furthermore preferred that the food of the application is lactic acid drink or soya-bean milk drink.
It should be noted that the lactobacillus plantarum LH-511 of the application can be made various as existing lactobacillus
Food, such as lactic acid drink, soya-bean milk drink etc., prepared food mainly plays health-care effect, therefore, to the plant cream in food
Bacillus LH-511 activity bacterium amount or intake are not specifically limited, can be according to actual state flexible choice in practical application.
The studies have shown that of the application daily intakes 1.0 × 109The active plant lactobacillus LH-511 of CFU unit, can be substantially reduced dynamic
Blood-sugar content in object blood, the active bacteria dosage can be used as food, health care product, food additives or drug reference dosage or
With reference to intake.
In addition it is also necessary to which explanation, the food of the application, are sensu lato esculents existing in any form
Product are not limited in lactic acid drink or soya-bean milk drink, such as can also be fermented food, and fermented food also includes animal feed
Deng.
The another side of the application discloses a kind of health care product, the lactobacillus plantarum LH- in the health care product containing the application
511。
It, therefore, can be with it should be noted that the lactobacillus plantarum LH-511 of the application has the function of good adjusting blood glucose
It is added in existing various health care products, it is made to have the good healthcare function for adjusting blood glucose, as long as other groups of health care product
Divide between lactobacillus plantarum LH-511 without mutually inhibition or adverse side effect.
The another side of the application discloses a kind of food additives, the plant cream bar in the food additives containing the application
Bacterium LH-511.
It should be noted that the lactobacillus plantarum LH-511 of the application can be matched with common food material.Example
Such as, cereal and potato, cereal include rice, face, coarse cereals, and potato includes potato, sweet potato etc.；Animal food, including meat, fowl,
Fish, milk, egg etc.；Beans and its product, including soybean and other dry beans；Vegetable and fruit class, including fresh beans, rhizome, leaf vegetables, eggplant
Fruit etc.；Pure thermal energy food, including vegetable and animals oils, starch, table sugar and drinks etc.；Therefore, the lactobacillus plantarum LH- of the application
511 can play the guarantor for adjusting blood glucose individually as directly eating in food additives or the various food materials of modulator addition
Strong effect.
The another side of the application discloses a kind of drug, the lactobacillus plantarum LH-511 in the drug containing the application.
Preferably, the drug of the application adjusts blood glucose and specifically includes the enzyme for inhibiting alpha-glucosidase for adjusting blood glucose
Activity in one day dosage of drug, contains at least 1.0 × 109The active plant lactobacillus LH-511 of CFU unit.
Preferably, medically acceptable carrier or auxiliary material are also contained in drug.
Preferably, carrier or auxiliary material be selected from glucose, lactose, sucrose, starch, mannitol, dextrin, fatty glyceride,
Polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water and
At least one of normal saline solution.
It is appreciated that drug when specific preparation is made, usually requires addition carrier or auxiliary material, only load to be added
Without mutually inhibition or adverse side effect between body or auxiliary material and lactobacillus plantarum LH-511.
It is furthermore preferred that drug is in tablet, granule, powder, capsule, solution, suspending agent, emulsion and lyophilized preparation
At least one；
It is furthermore preferred that lactobacillus plantarum piece.
It is furthermore preferred that including lactobacillus plantarum LH-511, dietary fiber, D-sorbite, crystallite fibre in lactobacillus plantarum piece
Dimension element and magnesium stearate.
It include the lactic acid bacteria freeze drying of lactobacillus plantarum LH-511 in a kind of preferred embodiment of the application, in lactobacillus plantarum piece
20% parts by weight of powder, 60% parts by weight of dietary fiber, 10% parts by weight of D-sorbite, 5% parts by weight of microcrystalline cellulose and tristearin
Sour 5% parts by weight of magnesium, after being mixed uniformly, tablet is made in tabletting.
It should be noted that the drug of the application, due to the lactobacillus plantarum LH-511 containing the application, have
The good function of adjusting blood glucose；The drug of the application can be the various of the lactobacillus plantarum LH-511 of independent active ingredient
Preparation can also be used cooperatively with other active ingredients, as long as not influencing activity between each other.
It is appreciated that the application lactobacillus plantarum LH-511 as active bacteria drug, as long as not influencing its strain activity,
Drug can use existing various dosage forms.And in the drug of the application, it further can also include normal in drug or dosage form
Auxiliary material, such as stabilizer, wetting agent, emulsifier, adhesive, isotonic agent etc..
Lactobacillus plantarum LH-511 activity bacterial content or pharmaceutical quantities are not particularly limited in the drug of the application, practical
It, can be according to the health status flexible choice of administration object in.But the studies have shown that of the application daily intake 1.0 ×
109The active plant lactobacillus LH-511 of CFU unit, the blood-sugar content that can be substantially reduced in animal blood, this dosage can be made
It is referred to for lactobacillus plantarum LH-511 activity bacterial content in drug or dosage.
As long as the lactobacillus plantarum LH-511 of the application and other probiotics or prebiotic combination of materials are in use, each component
Between there is no inhibiting effect or adverse reaction, it will be understood that best situation is that can have function between each component
Complementary or facilitation, such as compound probiotic piece can be made in the lactobacillus plantarum LH-511 of the application, by with it is other
Probiotic combinations reach more preferable or more active function, such as in addition to adjusting blood glucose, with other probiotic combinations, may be used also
Adjust functions of intestines and stomach to play, improve the functions such as immunity, specifically, can depending on each component of compound probiotic piece,
It is not limited here.
The beneficial effects of the present application are as follows:
The lactobacillus plantarum LH-511 of the application, alpha-glucosidase activity inhibiting rate with higher can adjust blood
Sugar has excellent prebiotic effect, also, has very strong acidproof and bile tolerance ability, being capable of long-term preservation；It can be used for making
Standby various food, health care product, food additives or the drugs for adjusting blood glucose provides a kind of new efficient former material to adjust blood glucose
Material.
Detailed description of the invention
Fig. 1 is the lactobacillus plantarum LH-511 growth curve chart that anti-cholate is tested in vitro in the embodiment of the present application.
The lactobacillus plantarum LH-511 of the application, classification naming are lactobacillus plantarum Lactobacillus plantarum,
Preservation, depositary institution were carried out in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 08 10th, 2017
Address be Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, deposit number CGMCC
No.14512。
Specific embodiment
The lactobacillus plantarum LH-511 of the application is the activity with well-tuned blood glucose function of one plant of new separation discovery
Bacterium.The lactobacillus plantarum LH-511 of the application is in spherical bacillus form, diameter about 1 in MRS culture medium under an optical microscope
μm, no gemma, cell wall structures are complete；Without plasmid, metabolin does not generate the noxious materials such as D-ALPHA-Hydroxypropionic acid, indoles, does not express nitro
Reductase does not have the ability of generation amine yet；Gentamicin,Kanamycin,Streptomycin,Tetracycline,
Erythromycin、Clindamycin、Chloramphenicol、Amplicilin、Neomycin、Trimethoprim、
The minimal inhibitory concentration of 13 kinds of antibiotic such as Ciprofoxacin, Rifampicin, Vancomycin also all meets European Food
The regulation of drug surveilance office (abbreviation EFSA).
The lactobacillus plantarum LH-511 bacterial strain of the application is good to the resistance of simulation simulated gastric fluid environment, and external, internal
Alpha-glucosidase activity inhibiting rate all with higher, blood sugar decreasing effect are obvious；As it can be seen that the application newly separates the plant of discovery
Lactobacillus LH-511 has the necessary condition as medicinal fungus or edible mushroom, can be used in developing new drug, food, health care product, food
Product additive or other edible or Pharmaceutical compositions.
The application is described in further detail below by specific embodiment.Following embodiment only to the application carry out into
One step explanation, should not be construed as the limitation to the application.
Embodiment one
This example is sample as research object using traditional fermented food, therefrom the doubtful bacterium colony of separation screening lactic acid bacteria, then
It is identified using doubtful bacterial strain of the 16s DNA cloning to picking.It is as follows in detail:
(1) culture medium prescription
This example has prepared the MRS solid medium and MRS culture solution of optimization.
MRS solid medium, abbreviation MRS culture medium, the formula of 1L are as follows: casein peptone 10.0g/L, beef extract 10.0g/
L, yeast extract 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, Tween 80 1.0g/L, Triammonium citrate 2.0g/
L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, agar 17.5g.
MRS broth bouillon, the i.e. formula of MRS culture solution do not add agar only compared with MRS culture medium, remaining is identical.
The culture medium and culture solution of configuration adjust pH to 6.5, and 121 DEG C of sterilizing 20min, then 4 DEG C of refrigerations are spare.
(2) source of lactobacillus plantarum LH-511 bacterial strain and identification
Using Inner Mongol farmers' Yoghourt fermentation food as sample, 10 times of gradient dilutions are carried out with sterile saline, by sample
It is diluted to 10-3, it is coated on the MRS media surface of this example preparation, is placed in 37 DEG C of incubators and cultivates 24-48h, observes bacterium
Fall morphological feature.Therefrom the doubtful lactic acid bacteria of picking but bacterium colony scribing line separation on new MRS culture medium, 37 DEG C are incubated overnight
Afterwards, scribing line is carried out again to isolate and purify.Using sterile toothpick from several single colonies of picking on the second isolated culture medium of scribing line,
It is numbered respectively.It chooses wherein 20 plants of single colonies and carries out 16s DNA sequencing.
The primer sequence of this example 16s DNA cloning sequence as shown in SEQ ID NO.1 and SEQ ID NO.2.
SEQ ID NO.1:5 '-AGAGTTTGATCATGGCTCAG-3 '
SEQ ID NO.2:5 '-TAGGGTTACCTTGTTACGACTT-3 '
Primer is synthesized by BGI-Shenzhen.
PCR reaction system be include: 1 μ L of 10mM dNTPs, 10 × buffer, 5 μ L, 10mM in the reaction solution of 50 μ L
The 1 μ L of Taq enzyme of each 1 μ L of upstream and downstream primer, 1 μ L of bacterium solution pcr template, 5U/ μ L supplement ddH2O is supplemented to 50 μ L.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min are recycled: 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C subsequently into 35
1min, after circulation terminates, 72 DEG C of extension 5min, 4 DEG C standby.
Gel extraction is carried out to pcr amplification product, and the PCR product of recycling is sequenced.This example uses kit
TaKaRa MiniBEST agarose GeL DNA Extraction kit carries out gel extraction, and detailed step is referring to kit
Specification.The PCR product of this example is sequenced by BGI-Shenzhen.
Sequencing result shows that the bacterial strain that number is LH-511,16s rDNA sequencing result is sequence shown in SEQ ID NO.3
Column.
SEQ ID NO.3:
5’-GAGGGCGCAGCTATACATGCAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGAT
TGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACC
TGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCT
GGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGG
GTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGAC
GCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTG
GGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAA
TACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAG
CCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTA
GCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTC
GAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGT
TTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAG
AATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA
CATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGTGTGCATGATGTCGTCAGCTCGTG
TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTAT-3’
It is compared in ncbi database with BLAST tool, comparison result is shown, LH-511 bacterial strain and lactobacillus plantarum
Lactobacillus plantarum affiliation is nearest, and similitude reaches 99%, therefore, identifies that LH-511 bacterial strain is plant
Lactobacillus is named as lactobacillus plantarum LH-511, the i.e. lactobacillus plantarum of this example screening acquisition.
The single colonie of lactobacillus plantarum LH-511 is transferred in MRS culture solution and carries out pure culture, 37 DEG C of culture 18h, with
Standby subsequent use.
In addition, in MRS culture medium under optical microscopy observation of plant lactobacillus LH-511, the results show that plant is newborn
Bacillus LH-511 meets the form knot of lactobacillus plantarum in spherical bacillus form, about 1 μm of diameter, complete without gemma, cell wall structures
Structure.
The lactobacillus plantarum LH-511 that this example screening obtains was preserved in Chinese microorganism strain on 08 10th, 2017
Preservation administration committee common micro-organisms center, the address of depositary institution are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
Institute of microbiology, the academy of sciences, deposit number are CGMCC No.14512.
(3) bacterium safety
1) antibiotics sensitivity is tested
This example test lactobacillus plantarum LH-511 to Gentamicin, Kanamycin, Streptomycin,
Tetracycline、Erythromycin、Clindamycin、Chloramphenicol、Amplicilin、Neomycin、
The resistance of 13 kinds of antibiotic such as Trimethoprim, Ciprofoxacin, Rifampicin, Vancomycin.Specific test side
Method is as follows:
13 kinds of antibiotic are first made into a series of 2 doubling dilution respectively, having respectively obtained concentration is 64 μ g/mL, 32 μ g/
The diluent of mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL
Object.
Diluted antibiotic is separately added into 96 orifice plate corresponding apertures, then corresponding aperture is added lactobacillus plantarum LH-511's
MRS culture solution, after mixing, 37 DEG C of culture 48h observe cultivation results.This example is with the antibacterials minimal inhibitory concentration in each hole
(abbreviation MIC), characterizes the susceptibility of lactobacillus plantarum LH-511, test result is as shown in table 1.
1 lactobacillus plantarum LH-511 of table is to 13 kinds of antibiotic resistance measurement results
| Antibiotic | MIC(μg/mL) | EFSA provides safety value |
| Gentamicin | 2 | 16 |
| Kanamycin | 16 | 64 |
| Streptomycin | 16 | NR |
| Tetracycline | 8 | 32 |
| Erythromycin | 0.25 | 1 |
| Clindamycin | 0.25 | 2 |
| Chloramphenicol | 2 | 8 |
| Amplicilin | 0.125 | 2 |
| Neomycin | 0.5 | 8 |
| Trimethoprim | 64 | 128 |
| Ciprofoxacin | 16 | 64 |
| Rifampicin | 2 | 4 |
| Vancomycin | 8 | NR |
In table 1, NR indicate European Food drug surveilance office that is not required, table 1 the results show that this example separation obtain
Lactobacillus plantarum LH-511 to Gentamicin, Kanamycin, Tetracycline, Erythromycin,
Clindamycin、Chloramphenicol、Amplicilin、Neomycin、Trimethoprim、Ciprofoxacin、
The minimal inhibitory concentration of Rifampicin all meets the regulation of European Food drug surveilance office (abbreviation EFSA), and is much smaller than it
Defined safety value；Lactobacillus plantarum LH-511 is respectively 16 to the minimal inhibitory concentration of Streptomycin and Vancomycin
μ g/mL and 8 μ g/mL, although the two antibiotic do not require in EFSA, the value that this example measures is also all basic to be accorded with
It closes and is expected, it is seen then that the lactobacillus plantarum LH-511 of this example has preferable sensibility to antibiotic.
In addition, carrying out plasmid pumping using lactobacillus plantarum LH-511 of the small extraction reagent kit DP103 of TIANGEN plasmid to this example
It mentions, the results show that not finding that lactobacillus plantarum LH-511 carries any plasmid, shows lactobacillus plantarum LH-511 without gene level
Metastatic potential.
2) metabolin toxicity detection
The detection of lactic acid optical activity: it is detected using the D-/L- lactate acid detection kit of Irish Megazyme company.Knot
Fruit shows that the lactobacillus plantarum LH-511 of this example does not generate D-ALPHA-Hydroxypropionic acid.
Nitrate reductase activity detection: the lactobacillus plantarum LH-511 of this example of this example is transferred in MRS Liquid Culture
In base, for 24 hours in 37 DEG C of cultures, aseptically, activated bacterial strain is accessed in nitrate culture-medium by 3% bacterium amount that connects
After 37 DEG C of culture 5d, liquor kalii iodide and each 10 drop of starch solution, observation experiment result is added dropwise.Positive control test is done simultaneously.
Experimental result shows that bacterium solution does not become blue, is negative reaction, and positive control has change blue, illustrates the lactobacillus plantarum LH- of this example
511 do not express nitroreductase.
Indoles experiment: under aseptic condition, activated bacterial strain is linked into peptone water medium by 3% bacterium amount that connects
In, then 37 DEG C of culture 72h are added indole reagent 8~10 and drip, observation experiment result.Positive control test is done simultaneously.Experiment knot
Fruit shows that red annulus does not occur in bacterium solution, and positive control is displayed in red, and shows the lactobacillus plantarum LH-511 metabolism of this example
Edwardsiella hoshinae.
The detection of amino decarboxylase: the bacterium with amino acid decarboxylases, can decompose amino acid makes its decarboxylation generate amine
And carbon dioxide, so that culture medium is become alkalinity, such as lysine is decomposed into cadaverine, ornithine is decomposed into putrescine, and arginine decomposes
For spermine；Indicator, such as bromocresol purple is added dropwise, is feminine gender in yellow, it is purple for the positive.Therefore, this example is in aseptic condition
Under, activated bacterial strain is linked into peptone water medium, 37 DEG C of culture 72h by 3% bacterium amount that connects, bromine first is then added
Phenol violet 8~10 drips, observation experiment result.Titration results are in yellow, are feminine gender, illustrate that the lactobacillus plantarum LH-511 of this example does not have
The ability of standby generation amine.
Embodiment two
The lactobacillus plantarum LH-511 that this example obtains the separation of embodiment one has carried out external inhibition alpha-glucosidase enzyme
Test living.It is specific as follows:
It include: the PBS solution of 50 μ L concentration 0.1mol/L, pH=6.8 in the system of 205 μ L, 50 μ L concentration are 20mmol/
P-nitrophenol-α-D- pyrans Portugal glucoside (abbreviation PNPG, the be purchased from Sigma) solution of L and the sample to be tested of 25 μ L, will
Alpha-glucosaccharase enzyme solutions the reaction was continued the 20min of 30 μ L concentration 20U/mL is added in 37 DEG C of hatching 10min in mixture, then plus
Enter the Na that 50 μ L concentration are 1mol/L2CO3As reaction terminating liquid, reaction solution is surveyed into its light absorption value in 405nm, light absorption value with it is right
The free amount of nitrophenol PNP is directly proportional, and the PBS solution conduct that pH value is 0.1mol/L for 6.8 concentration is used in reaction system
The blank control of alpha-glucosaccharase enzyme solutions and sample to be tested calculates lactic acid bacteria and presses down to alpha-glucosidase enzyme activity after being reacted
Property processed, inhibiting rate calculation formula are thallus alpha-glucosidase enzyme activity inhibiting rate=[1- (A-B)/(C-D)] × 100%.
Wherein, A, B, C, D are respectively the light absorption value of four sample to be tested groups measurement, specifically, A is the extinction of positive group
Value, i.e. LH-511 containing lactobacillus plantarum phage solution and alpha-glucosaccharase enzyme solutions；B is to contain only lactobacillus plantarum LH-511
The light absorption value of the group measurement of phage solution；C is the light absorption value of control group, i.e., containing only alpha-glucosaccharase enzyme solutions；D is blank group
Light absorption value is also free of alpha-glucosaccharase enzyme solutions without lactobacillus plantarum LH-511 phage solution.
At the same time, this example measures it to phlorose using identical method using commercial strain 299V as control
The enzyme activity inhibiting rate of glycosides enzyme.
The results show that the alpha-glucosaccharase enzyme inhibition rate for the lactobacillus plantarum LH-511 that the separation of embodiment one obtains is
33.7%, inhibiting rate is very significant, and compareing commercial strain 299V is only 14.2%.As it can be seen that the plant that the separation of embodiment one obtains
Object lactobacillus LH-511 has good alpha-glucosidase activity inhibiting rate.
Embodiment three
This example tries external acidproof, the resistance to bile ability for the lactobacillus plantarum LH-511 that the separation of embodiment one obtains
It tests, specific as follows:
Simulate the gastric juice SGF:2.0g NaCl, 3.2g pepsin are prepared, the dense HCl of 7mL adds water to adjust to PH3.0, then
After 0.2 μm of filtering with microporous membrane, that is, the SGF liquid of this example is obtained, for use.
Picking lactobacillus plantarum LH-511 slant strains are in MRS culture solution, 37 DEG C of 16~18h of culture.Bacteria suspension 4
000r/min is centrifuged 15min, removes supernatant, weighs thallus weight in wet base, according to 0.1g/mL ratio again suspension thalline in physiological saline
In, it is added in SGF liquid according still further to the ratio of 1:10, mixes well to be placed in 37 DEG C of constant incubators and cultivate 2h, it is right respectively
It cultivates 0h and 2h and carries out cell count.Cell counts are as shown in table 2.
Tolerance count plate result of the 2 lactobacillus plantarum LH-511 of table to simulated gastric fluid SGF
| Bacterial strain | 0h is counted | 2h is counted | Survival rate |
| LH-511 | 2.56×109 | 2.55×109 | 99.6% |
Table 2 the results show that the lactobacillus plantarum LH-511 of embodiment one, viable count is maintained at a quantity after cultivating 2h
Grade, survival rate are up to 99.6%, and showing lactobacillus plantarum LH-511 in vitro has stronger acid-fast ability.
Further, the 10mL containing 0.3% cholate of the parts by weight bacterium solution of activation twice to be sterilized by the access of 1% amount
MRS culture solution is cultivated, and measures growth curve of the lactobacillus plantarum LH-511 in culture solution, and this example is repeated 4
Secondary test, as a result as shown in Figure 1.In Fig. 1, each curve is 4 duplicate growth curves respectively, the results show that in the training of cholate
Support base in lactobacillus plantarum LH-511 can normal growth, it is stronger to illustrate that the lactobacillus plantarum LH-511 of embodiment one has in vitro
Anti- bile ability.
Example IV
The lactobacillus plantarum LH-511 that this example obtains the separation of embodiment one adjusts cell factor experiment in vitro, specifically such as
Under:
Take growth conditions good, culture medium, waste liquid are drawn in the Human THP-1 cells system cell in logarithmic growth phase, centrifugation
It discards, appropriate complete medium is added, the cell for mixing removing is blown and beaten with pipettor, is prepared into cell suspension, cell count tune
Whole density is 5 × 105Cfu/mL draws on 0.9mL cell suspension inoculation to 24 orifice plates, above-mentioned spare bacteria suspension is drawn after 2h
It is added in corresponding aperture, makes the concentration 10 of lactic acid bacteria suspension in each hole6Cfu/mL concentration, three repetitions after culture for 24 hours, are inhaled
Supernatant is taken, 3000r/min is centrifuged 10min, takes supernatant, and test into ELISA: Interleukin -1β (IL-1 β), IL-6, tumour are bad
Necrosis factor-α (TNF-α) and IL-10, experimental result is as shown in table 3, wherein each group P < 0.05.
The cell in vitro cytokine regulatory test result of 3 lactobacillus plantarum LH-511 of table
| Group | IL-1β/(ng/mL) | IL-6/(ng/mL) | TNF-α(pg/mL) | IL-10/(ng/mL) |
| Blank control | 40.15 | 89.28 | 15.68 | 19.29 |
| LH-511 | 35.31 | 61.54 | 12.93 | 96.56 |
Table 3 the results show that the lactobacillus plantarum LH-511 of embodiment one to IL-1 β of THP-1 cell line, IL-6,
The proinflammatory factors such as TNF-α have downward effect, have up-regulation effect to anti-inflammatory factors such as IL-10, show lactobacillus plantarum LH-
511 have the function of anti-inflammatory really.
Embodiment five
The lactobacillus plantarum LH-511 that this example obtains the separation of embodiment one carries out internal study on the efficiency, and this example uses
5 week old rat of Sprague-Dawley system carries out in vivo studies, specific as follows:
1. the preparation of experimental strain
Activation lactobacillus plantarum LH-511 twice is inoculated in MRS fluid nutrient medium, in 37 DEG C of culture 18h,
6000r/m is centrifuged 10min, collects thallus after being washed with sterile saline.Then, 0.85% physiological saline is added, adjusts bacterium
Body number is to 1.0 × 109Then viable bacteria is sub-packed in 15mL centrifuge tube by daily usage amount by CFU/mL, taking dose is daily
2mL/ only, every group 10,6 groups, is fed 28 days totally.
2. experimental animal grouping and feeding manner
This example uses 5 week old rat of Sprague-Dawley system, induces SD system 5 using streptozotocin joint high lipid food
Week old rat completes hyperglycemic rat modeling, feeds to the 28th day, is equally divided into 6 groups, every group 10.Each group is respectively according to such as
Under type continues to feed:
Model group, the group feed high-calorie feed and distilled water；
Positive group, which feeds high-calorie feed and melbine, and melbine is supplied by 0.3mg/g weight；
LH-511 group, the group feed high-calorie feed and lactobacillus plantarum LH-511；
299v group, the group feed high-calorie feed and probiotics 299v；
Sc52 group, the group feed high-calorie feed and probiotics Sc52；
Normal group, this group of feeding standard feed and distilled water.
The above forage feed amount is identical, distilled water, melbine, lactobacillus plantarum LH-511, probiotics 299v and prebiotic
Bacterium Sc52 daily every 2mL stomach-filling in each group, continues 28 days.
Wherein, high-calorie feed is voluntarily prepared, and is that fat or high-fat object are added on the basis of the standard feed of purchase
Matter, specific formula are as follows: 78.8% standard feed, 10% lard, 10% yolk powder, l% cholesterol, 0.2% cholate.Standard feed
It speeds the muroid special feed of experimental animal feed factory to purchase in Henan day.Probiotics 299v is commercial strain, commercially available purchase；
Probiotics Sc52 is purchased from CGMCC, and deposit number CGMCCNo.11027 is recorded in patent CN 201510581420.8.
3. sample acquisition is tested with analysis
Before formal test and blood was collected the 28th day period.Blood-sampling method is after going on a hunger strike a night, to adopt in rat femoral vein
Blood, 4000r/m is centrifuged 10min and separates serum after blood coagulation.Detect fasting blood-glucose, 2h-plasma glucose, glycosylated hemoglobin, adiponectin
With the content of TNF-α and the 28th day measurement hepatic tissue SOD value.
4. experimental result
Experimental result is as shown in table 4, wherein each group P < 0.05.
4 animal test results of table
Table 4 the results show that the 28th day empty stomach blood glucose target of LH-511 group rat oral gavage is reduced than model group
The 28th day postprandial blood sugar index of 24.0%, LH-511 group reduces 37.1% than model group, shows that lactobacillus plantarum LH-511 has
There is internal antidiabetic function, and other indexs show lactobacillus plantarum LH-511 to internal inflammatory environment caused by hyperglycemia
Also there is relaxation effect.Also, the lactobacillus plantarum LH-511 of this example, function and other control strain 299v (P > 0.05) and
Sc52 (12.6%, P < 0.05) is more preferable compared to effect.
Embodiment six
Respectively common drug and food is made in the lactobacillus plantarum LH-511 that the separation of embodiment one obtains by this example, specifically
It is as follows:
(1) lactobacillus plantarum tablet
Formula: CFU109The freeze-dried powder 20% of lactobacillus plantarum LH-511, dietary fiber 60%, D-sorbite 10%,
Microcrystalline cellulose 5%, magnesium stearate 5%；The above component is parts by weight, and after mixing by each component, tablet is made in tabletting.
Wherein, the freeze-dried powder of lactobacillus plantarum LH-511 is that its bacteria suspension is freezed vacuum drying in an aseptic environment using freeze dryer
It forms.
(2) lactobacillus plantarum fermentation cultured milk
Milk powder and water are mixed, homogeneous, sterilized with 121 DEG C of ultra-high temperature sterilization 300s, is cooled to 42 DEG C, access activation
Good leavening: access amount is parts by weight 0.4% in total for lactobacillus bulgaricus and lactobacillus thermophilus bacterium powder.It ferments at 42 DEG C
10h, it is 10 that parts by weight 1%CFU is added after cooling8Lactobacillus plantarum LH-511, stirring, it is filling.
Wherein, milk powder is conventional use of milk powder in Yoghourt preparation.
(3) pharmacodynamic test of the above drug and food
This example is using the identical method of embodiment five to the sour ox of lactobacillus plantarum tablet and lactobacillus plantarum fermentation of this example
Cream carries out hypoglycemic test in vivo.
Wherein, suspension made of sterile water, adjustment bacterium number to 1.0 × 10 are added after lactobacillus plantarum tablet is smashed to pieces9CFU/mL。
Lactobacillus plantarum fermentation cultured milk equally adds suspension made of sterile water, adjustment bacterium number to 1.0 × 109CFU/mL.It is replaced with this
Viable bacteria suspension in embodiment five.
Feeding method and subsequent blood sampling, measurement method are all identical as embodiment five.
The results show that drawing blood after being fed high lipid food rat 28 days with the drug of lactobacillus plantarum LH-511 and food respectively
The rat of lactobacillus plantarum fermentation cultured milk is fed in detection, discovery, its blood glucose has dropped 24.5%, P < 0.05 compared with the 0th day,
0th day fasting plasma glucose concentration is 4.7mmol/L, and the fasting plasma glucose concentration after 28 days is 3.55mmol/L；Feed plant cream bar
The rat of bacterium piece is suitable with the lactobacillus plantarum fermentation rat effect of cultured milk is fed, and the fasting plasma glucose concentration after 28 days has dropped
About 24.8%, P < 0.05.As it can be seen that the drug and food of addition lactobacillus plantarum LH-511, lactobacillus plantarum LH-511 is big
It is colonized in mouse body and has played effect, show that the drug for adding lactobacillus plantarum LH-511 and truly having for food reduce blood glucose in vivo
The effect of.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made.
SEQUENCE LISTING
<110>Shenzhen Hua Da three lives garden Science and Technology Ltd.
<120>one lactobacillus plantarum LH-511 and its application
<130> 17I25288
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agagtttgat catggctcag 20
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
tagggttacc ttgttacgac tt 22
<210> 3
<211> 1110
<212> DNA
<213>the 16s rDNA sequencing result of lactobacillus plantarum LH-511
<400> 3
gagggcgcag ctatacatgc agtcgaacga acttccgtta attgattatg acgtacttgt 60
actgattgag attttaacac gaagtgagtg gcgaacgggt gagtaacacg tgggtaacct 120
gcccagaagt aggggataac acctggaaac agatgctaat accgtataac agagaaaacc 180
gcatggtttt cttttaaaag atggctctgc tatcacttct ggatggaccc gcggcgtatt 240
agctagttgg tgaggtaaag gctcaccaag gcagtgatac gtagccgacc tgagagggta 300
atcggccaca ttgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360
cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420
tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt aactgtttac ccagtgacgg 480
tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540
aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtctttta agtctaatgt 600
gaaagccttc ggctcaaccg aagaagtgca ttggaaactg ggagacttga gtgcagaaga 660
ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720
cgaaggcggc tgtctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag 780
gattagatac cctggtagtc catgccgtaa acgatgatta ctaagtgttg gagggtttcc 840
gcccttcagt gctgcagcta acgcattaag taatccgcct ggggagtacg accgcaaggt 900
tgaaactcaa aagaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960
agctacgcga agaaccttac caggtcttga catcttctga cagtctaaga gattagaggt 1020
tcccttcggg gacagaatga cagtgtgcat gatgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctat 1110
Claims (10)
1. a lactobacillus plantarum LH-511, deposit number is CGMCC No.14512.
2. lactobacillus plantarum LH-511 according to claim 1, it is characterised in that: the lactobacillus plantarum LH-511 is to α-Portugal
The maximum inhibition of polyglycoside enzyme is greater than 33%.
3. food, health care product, food that lactobacillus plantarum LH-511 according to claim 1 or 2 adjusts blood glucose in preparation
Application in additive, drug or composition.
4. a kind of composition, it is characterised in that: the composition contains lactobacillus plantarum LH-511 of any of claims 1 or 2.
Preferably, the composition can be used for adjusting blood glucose.
5. a kind of food, it is characterised in that: contain lactobacillus plantarum LH-511 of any of claims 1 or 2 in the food.
Preferably, also containing acceptable additive or auxiliary material in bromatology in the food；
Preferably, the food is lactic acid drink or soya-bean milk drink.
6. a kind of health care product, it is characterised in that: contain lactobacillus plantarum LH- of any of claims 1 or 2 in the health care product
511。
7. a kind of food additives, it is characterised in that: contain plant cream of any of claims 1 or 2 in the food additives
Bacillus LH-511.
8. a kind of drug, it is characterised in that: contain lactobacillus plantarum LH-511 of any of claims 1 or 2 in the drug.
9. drug according to claim 8, it is characterised in that: the drug is used to adjust blood glucose, the drug one day
In dosage, contain at least 1.0 × 109The active plant lactobacillus LH-511 of CFU unit.
10. drug according to claim 8 or claim 9, it is characterised in that: also contain medically acceptable load in the drug
Body or auxiliary material；
Preferably, the carrier or auxiliary material be selected from glucose, lactose, sucrose, starch, mannitol, dextrin, fatty glyceride,
Polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water and
At least one of normal saline solution；
Preferably, the drug is in tablet, granule, powder, capsule, solution, suspending agent, emulsion and lyophilized preparation
It is at least one；
It is furthermore preferred that the drug is lactobacillus plantarum piece, it include lactobacillus plantarum LH-511, meals in the lactobacillus plantarum piece
Eat fiber, D-sorbite, microcrystalline cellulose and magnesium stearate.
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| CN111304134A (en) * | 2020-03-26 | 2020-06-19 | 上海理工大学 | Lactobacillus plantarum capable of effectively relieving diabetes |
| CN111387290A (en) * | 2020-03-03 | 2020-07-10 | 宁波大学 | Functional yogurt product with α -glucosidase and tyrosinase activity inhibiting effect and its preparation method |
| CN111733111A (en) * | 2020-07-20 | 2020-10-02 | 广东南芯医疗科技有限公司 | Lactobacillus plantarum NX-1 and application thereof in preparation of hypoglycemic drugs |
| CN112852657A (en) * | 2019-11-27 | 2021-05-28 | 大江生医股份有限公司 | Application of lactobacillus plantarum and/or metabolite thereof in preparing weight-losing composition |
| CN113234639A (en) * | 2021-06-16 | 2021-08-10 | 广东海天创新技术有限公司 | Lactobacillus plantarum ZF632 and application thereof |
| CN113462613A (en) * | 2021-08-03 | 2021-10-01 | 浙江大学 | Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof |
| CN113881604A (en) * | 2021-11-10 | 2022-01-04 | 深圳大学 | Lactobacillus plantarum MM89 and polysaccharide and application thereof |
| CN114752529A (en) * | 2022-04-29 | 2022-07-15 | 科郦有限公司 | Lactobacillus plantarum HOM3201 strain, viable bacteria preparation thereof, preparation method and application |
| CN115851500A (en) * | 2022-09-23 | 2023-03-28 | 四川大学 | Lactobacillus plantarum and application thereof |
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| CN112852657A (en) * | 2019-11-27 | 2021-05-28 | 大江生医股份有限公司 | Application of lactobacillus plantarum and/or metabolite thereof in preparing weight-losing composition |
| CN111387290A (en) * | 2020-03-03 | 2020-07-10 | 宁波大学 | Functional yogurt product with α -glucosidase and tyrosinase activity inhibiting effect and its preparation method |
| CN111387290B (en) * | 2020-03-03 | 2023-01-31 | 宁波大学 | Functional yogurt product with alpha-glucosidase and tyrosinase activity inhibition function and preparation method thereof |
| CN111304134A (en) * | 2020-03-26 | 2020-06-19 | 上海理工大学 | Lactobacillus plantarum capable of effectively relieving diabetes |
| CN111733111B (en) * | 2020-07-20 | 2022-04-01 | 广东南芯医疗科技有限公司 | Lactobacillus plantarum NX-1 and application thereof in preparation of hypoglycemic drugs |
| CN111733111A (en) * | 2020-07-20 | 2020-10-02 | 广东南芯医疗科技有限公司 | Lactobacillus plantarum NX-1 and application thereof in preparation of hypoglycemic drugs |
| CN113234639A (en) * | 2021-06-16 | 2021-08-10 | 广东海天创新技术有限公司 | Lactobacillus plantarum ZF632 and application thereof |
| CN113462613A (en) * | 2021-08-03 | 2021-10-01 | 浙江大学 | Lactobacillus plantarum ZJUIDS04 capable of reducing blood sugar and application thereof |
| CN113881604A (en) * | 2021-11-10 | 2022-01-04 | 深圳大学 | Lactobacillus plantarum MM89 and polysaccharide and application thereof |
| CN113881604B (en) * | 2021-11-10 | 2023-02-28 | 深圳大学 | Lactobacillus plantarum MM89 and polysaccharide and application thereof |
| CN114752529A (en) * | 2022-04-29 | 2022-07-15 | 科郦有限公司 | Lactobacillus plantarum HOM3201 strain, viable bacteria preparation thereof, preparation method and application |
| CN114752529B (en) * | 2022-04-29 | 2023-12-19 | 科郦有限公司 | Lactobacillus plantarum HOM3201 strain and viable bacteria preparation, preparation method and application thereof |
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| CN115851500B (en) * | 2022-09-23 | 2024-04-02 | 四川大学 | Lactobacillus plantarum and application thereof |
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