CN110141583A - A kind of application of Kefir grains lactobacillus M3 in active constituent antibacterial and as treatment type II diabetes medicament - Google Patents

A kind of application of Kefir grains lactobacillus M3 in active constituent antibacterial and as treatment type II diabetes medicament Download PDF

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CN110141583A
CN110141583A CN201910422020.0A CN201910422020A CN110141583A CN 110141583 A CN110141583 A CN 110141583A CN 201910422020 A CN201910422020 A CN 201910422020A CN 110141583 A CN110141583 A CN 110141583A
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lactobacillus
kefir grains
group
grains lactobacillus
diabetes
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CN110141583B (en
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孟利
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Heilongjiang University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A kind of application of Kefir grains lactobacillus M3 in active constituent antibacterial and as treatment type II diabetes medicament.The present invention solves the problems, such as that existing lactic acid bacteria surface hydrophobic is low and lactic acid bacteria bacteriostasis is poor.Kefir grains lactobacillus Lactobacillus kefiri M3 of the present invention has apparent inhibiting effect, good antimicrobial effect to Escherichia coli, staphylococcus aureus.Kefir grains lactobacillus Lactobacillus kefiri M3 and its bacteria preparation of the present invention all have excellent surface hydrophobic and coherency, there is good adhesive capacity to enteron aisle, so that it has good blood sugar regulation ability in the application of type II diabetes, Lactobacillus kefiri M3 of the present invention has apparent relaxation effect to type II diabetes illness.

Description

A kind of Kefir grains lactobacillus M3 is in work antibacterial and as treatment type II diabetes medicament The application of property ingredient
Technical field
The present invention relates to a kind of applications of Kefir grains lactobacillus.
Background technique
Bacillus acidi lactici is the fungal component in animal intestinal tract, can be helped digest, and the health of human body intestinal canal is helped, containing active The food of Bacillus acidi lactici is often considered as healthy food, and usual Bacillus acidi lactici addition is in the fermented foods such as Yoghourt, to human body people Colony balance is adjusted in class enteron aisle.It (may be a kind of protein or lipid that enterocyte, which can secrete certain substance, in vivo Teichoic acid), be conducive to the probiotics such as Bacillus acidi lactici and be colonized on intestinal mucosa, to form mycoderm barrier, repels pathogenic bacteria in intestines Adherency on wall.Meanwhile Bacillus acidi lactici can generate lactic acid, acetic acid and bacteriocin, inhibit the intrusion of various pathogens.Therefore it applies It is important in current food and feed industry that Bacillus acidi lactici replaces antibiotic to prevent the intestines problem of the mankind or animal from having become Research field.2019, YANG research discovery Lactobacillus rhamnosus GG can also increase the permeability of intestine of young pigs, and pass through regulation The secretion of antibacterial peptide, cell factor and chemotactic factor (CF) is to improve the immunization barrier function of enteron aisle.
The adhesion characteristics of Bacillus acidi lactici are one of the important indicators for evaluating the prebiotic function of Bacillus acidi lactici, and Colonization is lactic acid Bacillus plays the premise and basis of physiological function in enteron aisle.There is scholar research shows that microbial cell surface hydrophobicity and automatic Ability of aggregation plays main effect to the adhesiveness of bacterial strain, and hydrophobicity and the higher Bacillus acidi lactici of autohemagglutination ability are in enteron aisle Higher adhesiveness is presented, i.e. phage surface hydrophobicity and autohemagglutination ability is directly proportional to the adhesion strength of intestinal epithelial cell to it.
Currently, the function that lactobacillus potentially prevents and treats type II diabetes obtained in animal body and the confirmation of clinical trial and It is widely recognized as.When body takes in suitable lactobacillus, energetic supersession can be effectively adjusted, metabolism of lipid and cholesterol accumulation is reduced, has Intestinal flora caused by effect supplement type II diabetes lacks, and establishes new enteron aisle balance stable state, intestinal microflora of getting well, Gut permeability is reduced, gut barrier effect is improved, inhibits inflammatory reaction, mitigates glucose intolerance and improves glucose tolerance And insulin sensitivity.The surface hydrophobic of existing ordinary lactic acid bacteria is low, and poor adhesion, blood sugar regulation ability is poor, anti- It is poor to control type II diabetes application effect, in addition, Bacillus acidi lactici also needs to generate pathogenic bacteria inhibiting effect, its tune of competence exertion The effect of whole blood glucose.Now with ordinary lactic acid bacteria bacteriostasis it is poor, limit lactic acid bacteria in the application of type II diabetes.
Summary of the invention
The present invention is in order to solve the problems, such as that existing lactic acid bacteria surface hydrophobic is low and lactic acid bacteria bacteriostasis is poor.And it mentions Supply a kind of Kefir grains lactobacillus Lactobacillus kefiri M3 in work antibacterial and as treatment type II diabetes medicament The application of property ingredient.
Application of the Kefir grains lactobacillus Lactobacillus kefiri M3 of the present invention on antibacterial.
Activity of the Kefir grains lactobacillus Lactobacillus kefiri M3 of the present invention as treatment type II diabetes medicament Then the application of ingredient is re-used as treatment II type sugar wherein bacteria preparation is made by frozen dried in the Kefir grains lactobacillus Sick active constituent is urinated to be applied.
Kefir grains lactobacillus passes through the method that bacteria preparation is made in frozen dried are as follows: Kefir grains lactobacillus Lactobacillus Kefiri M3 is centrifuged after cultivating in MRS culture medium to logarithmic growth phase, and supernatant is gone to stay thallus, is then added into thallus de- Rouge cream, sucrose, sodium alginate mixing, obtain Kefir grains lactobacillus bacteria preparation by frozen dried；Wherein, thallus and degreasing Cream, sucrose, sodium alginate weight ratio are 100:6~7:1~2:1~2.
Above-mentioned Kefir grains lactobacillus Lactobacillus kefiri M3 is improving patients with NIDDM enteric microorganism Application in flora.
Kefir grains lactobacillus (Lactobacillus kefiri) M3 of the present invention is preserved in Chinese Typical Representative culture Collection (CCTCC), preservation address are Wuhan Universitys, and the deposit date is on January 24th, 2019, deposit number was CCTCC NO: M2019079。
Lactobacillus kefiri M3 of the invention derives from tibet koumiss, and thalli morphology is Gram-positive Bacterium, middle long bacillus, colonial morphology are that milky, circle, surface are smooth.
Kefir grains lactobacillus Lactobacillus kefiri M3 of the present invention has Escherichia coli, staphylococcus aureus Apparent inhibiting effect, good antimicrobial effect.Kefir grains lactobacillus Lactobacillus kefiri M3 and its bacteria preparation of the present invention Excellent surface hydrophobic and coherency are all had, has good adhesive capacity to enteron aisle, so that its answering in type II diabetes There is good blood sugar regulation ability in, while reducing the level of the malonaldehyde in serum and kidney, mitigates caused by diabetes Oxidative damage, while having good inhibiting effect to pathogenic bacteria (Escherichia coli, staphylococcus aureus), the present invention is to II The illness of patients with type Ⅰ DM has apparent relaxation effect；In addition, Kefir grains lactobacillus Lactobacillus kefiri of the present invention M11 improves significantly to patients with NIDDM intestinal microflora tool.
Detailed description of the invention
Fig. 1 is the colony characteristics of Lactobacillus kefirii M3；
Fig. 2 is the thallus feature of Lactobacillus kefirii M3；
Fig. 3 is the total DNA PCR amplification figure of Lactobacillus kefirii M3；
Fig. 4 is Kefir grains lactobacillus Lactobacillus kefirii M3 Phylogenetic Analysis figure；
Fig. 5 is Bulgarian bacteriumLactobacillus bulgaricusThe antibacterial result figure of ATCC11842；
Fig. 6 is the antibacterial result figure of Kefir grains lactobacillus Lactobacillus kefirii M3；
Fig. 7 is Lactobacillus kefirii M3 and Lactobacillus kefirii M3-20 DEG C of items of bacteria preparation Influence of the storage time to viable count under part；
Fig. 8 is Lactobacillus kefirii M3 tolerability results figure in simulated gastric fluid；
Fig. 9 is Lactobacillus kefirii M3 tolerability results in simulated intestinal fluid；
Figure 10 is lactobacillus and bacteria preparation type II diabetes mouse experiment；
Figure 11 is Kefir grains lactobacillus bacteria preparation to type II diabetes mice serum oxidative damage result；
Figure 12 is Kefir grains lactobacillus bacteria preparation to type II diabetes mouse kidney oxidative damage result；
Figure 13 is the influence of Kefir grains lactobacillus Lactobacillus kefirii M3 mouse intestinal flora.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment Any combination.
Specific embodiment 1: present embodiment Kefir grains lactobacillus Lactobacillus kefiri M3 is on antibacterial Application.
Specific embodiment 2: present embodiment Kefir grains lactobacillus Lactobacillus kefiri M3 is as treatment The application of the active constituent of type II diabetes medicament.
Specific embodiment 3: present embodiment is unlike specific embodiment two: Kefir grains lactobacillus is by freezing Bacteria preparation is made in dry-cure, and the active constituent for being then re-used as treatment type II diabetes medicament apply, the tool of bacteria preparation The production method of body are as follows: Kefir grains lactobacillus Lactobacillus kefiri M3 is cultivated in MRS culture medium to logarithmic growth It is centrifuged after phase, supernatant is gone to stay thallus, skimmed milk, sucrose, sodium alginate mixing are then added into thallus, is by frozen dried Obtain Kefir grains lactobacillus bacteria preparation；Wherein, thallus and skimmed milk, sucrose, sodium alginate weight ratio are 100:6~7:1~2:1 ~2.Other are identical with embodiment two.
Specific embodiment 4: present embodiment is unlike specific embodiment three: frozen dried method is thallus 1~5h is freezed under the conditions of -20 DEG C with the mixture of skimmed milk, sucrose and sodium alginate, is then less than -40 in condenser temperature DEG C, vacuum degree be greater than 200mtorr under conditions of vacuum freeze drying, that is, complete frozen dried.Other and specific embodiment Three is identical.
Specific embodiment 5: present embodiment is unlike specific embodiment three: centrifugal condition is centrifugal rotational speed For 5000r/min, centrifugation time 10min.Other are the same as the specific implementation mode 3.
Specific embodiment 6: present embodiment is unlike specific embodiment three: thallus and skimmed milk, sucrose, Sodium alginate weight ratio is 100:6.67::1.67:1.67.Other are the same as the specific implementation mode 3.
Specific embodiment 7: present embodiment Kefir grains lactobacillus Lactobacillus kefiri M3 is improving II Application in diabetes mellitus type intestinal microflora.
Present embodiment is being made for Kefir grains lactobacillus Lactobacillus kefiri M11 by frozen dried For improving patients with NIDDM intestinal microflora after bacteria preparation.Bacterium is made by frozen dried in Kefir grains lactobacillus Preparation, specific method are as follows: Kefir grains lactobacillus Lactobacillus kefiri M11 is cultivated in MRS culture medium to logarithm It is centrifuged after growth period, supernatant is gone to stay thallus, skimmed milk, sucrose, sodium alginate mixing are then added into thallus, at freeze-drying Reason obtains Kefir grains lactobacillus bacteria preparation；Wherein, thallus and skimmed milk, sucrose, sodium alginate weight ratio are 100:6~7:1 ~2:1~2.
1 Kefir grains lactobacillus Lactobacillus kefiri M3 of embodiment separation, purifying and identification:
One, Xizang Linggu Fungus grain culture
1, Xizang Linggu Fungus grain activates: Xizang Linggu Fungus grain is inoculated into sterilizing Wanda Mountain plain chocolate by 5% inoculum concentration, 21 DEG C of constant temperature incubations for 24 hours, by the tibet koumiss fermentation liquid after culture by sterilising filtration net filtration, retain Xizang Linggu Fungus grain, It repeats one week；Wherein Xizang Linggu Fungus grain was acquired in 2017 from microorganism key lab, Heilongjiang University.
2, Xizang Linggu Fungus grain passes on: the Xizang Linggu Fungus grain after activation is expanded culture to sterilizing according to 5% ratio In cow's milk, 21 DEG C of constant temperature incubation 48h retain Tibet spirit by the tibet koumiss fermentation liquid after culture by sterilising filtration net filtration Mushroom grain；It is primary every 48h passage, it passes on 4 times altogether；
Two, lactobacillus separation, purifying
Xizang Linggu Fungus grain after passing in the clean step one of 2g sterile saline is mixed with 1mL sterile saline It is put into mortar, being ground to particle diameter is 1-3mm；Xizang Linggu Fungus grain obtained is transferred to the centrifuge tube of 10ml several times In be settled to 10ml, the concussion that is vortexed is uniform；10 are pressed respectively to bacteria suspension4、105、106Gradient is diluted.In MRS solid culture There is lactic acid bacteria colonial morphology (5 to 10 bacterium colonies of usual each sample) to carry out Gram's staining, choosing for 37 DEG C of culture selections on base It selects gram-positive bacterium and is viewed as rod-shaped typical lactobacillus thalli morphology under an electron microscope, trained in MRS solid It supports and is purified on base, separation obtains one plant of lactobacillus, number M3.Three generations is purified to M3 bacterial strain.
Three, the lactobacillus M3 bacterial strain in step 2 after purification is identified
1, the extraction of DNA of bacteria
Lactobacillus is extracted according to the Ezup pillar DNA of bacteria extraction agent box operating instruction of the raw work biology Co., Ltd in Shanghai The total DNA of M3 bacterial strain.
2, the PCR amplification of lactobacillus M3 total DNA
PCR amplification primer: 5'-TACCTTGTTAGCACTT-3'
5'-AGAGTTTGATCCTGGCTCAG-3'
PCR reaction system (50 μ L): 10 × PCR buffer, 10 μ L, each 5 μ L, dNTP MIX of primer pair (10 μm of ol/L) (100mmol/L) 4 μ L, Taq archaeal dna polymerase (5U/ μ L) 2.5 μ L, dd H2O 26.5 μ L, 2 μ L of template DNA.
PCR reaction condition: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 1min, 52 DEG C of annealing 2min, 72 DEG C of extension 2min, into It is saved at row circulation 35,72 DEG C of extension 10min, 4 DEG C.
3, agarose gel electrophoresis
PCR product is subjected to agarose gel electrophoresis, weighs 1g agarose, with 1 × TAE buffer preparation, 1.0% fine jade Lipolysaccharide running gel, ethidium bromide is added when not solidifying makes its final concentration reach 1 μ g/mL, glue.Electrophoresis is added in electrophoresis tank Buffer makes liquid level not have glue surface.5 μ L PCR products are mixed with 1 μ 6 × loading of L buffer respectively, point sample, wherein one DNA marker is added in hole.Electrophoresis 30min, ultraviolet gel imager observe electrophoresis result.
4, the 16S rDNA sequencing of lactobacillus M3
Length is sent to the raw work in Shanghai for the PCR product of 1000bp or so and is surveyed after selecting agarose gel electrophoresis to be imaged Sequence.Use the blast search for being directed to GenBank DNA database (www.ncbi.nlm.nih.gov/Genbank/) (http://www.ncbi.nlm.nih.gov.blast) identifies the lactobacillus separated from Xizang Linggu Fungus grain.Use software MEGA 5.1 constructs chadogram, carries out Phylogenetic Analysis, as a result as shown in Figure 4.
The colony characteristics of present embodiment M3 bacterial strain are as shown in Figure 1；The thallus feature of M3 bacterial strain as shown in Fig. 2, from Fig. 1 and Shown in Fig. 2, the M3 bacterial strain thalli morphology of present embodiment is gram-positive bacteria, middle long bacillus, and colonial morphology is milky, circle Shape, surface are smooth.
The PCR amplification result of the total DNA of present embodiment M3 bacterial strain is as shown in Figure 3, wherein M maker, 1 is M3 bacterium Strain；From the figure 3, it may be seen that the gene order length for the present embodiment M3 bacterial strain isolated from tibet koumiss is in 1000bp or so, The genetic fragment of extraction is complete, and no degradation can be sequenced.
Determine that present embodiment M3 bacterial strain is Kefir grains cream bar according to molecular biological analysis and bacterium colony, thallus feature Bacterium is named as Lactobacillus kefirii M3.
Application of the Lactobacillus kefiri M3 bacterium of the present invention of embodiment 2 in antibacterial
Strain to be tested: Lactobacillus kefiri M3 bacterium of the invention, Bulgarian bacterium (Lactobacillus bulgaricusATCC11842)；
Specific test method:
One, the fermentation liquid for taking thallus to be measured wants that being centrifuged 12min goes precipitating to stay supernatant in 3500r/min condition；
Two, the preparation of the double-deck detection plate
The element agar (2%) that 20mL heating and melting is poured into sterilized petri dishes (d=90mm), is fully cooled solidification to it Afterwards, neat n sterilized Oxford cups (d-7mm) are put in a certain order.The finger that the 30mL being sub-packed in triangular flask is melted Show that bacterium semisolid culturemedium is cooled to 50 DEG C or so, is added 105The fresh bacteria suspension 1mL of cfu/mL indicator bacteria (B, subtilis), It is uniformly mixed rapidly, pours into plate.After cooling, Oxford cup, the as double-deck detection plate of bacteriostatic test are taken out with aseptic nipper.
Three, the preparation of sample to be tested
First group: the pH for the supernatant that the step of preparing one obtains being adjusted to neutrality with 0.25mol/L NaOH solution； Second group of supernatant not adjust pH in step 1；Third group is blank control, i.e., will connect the MRS culture medium of bacterium with lactic acid PH is adjusted to identical as the pH of the supernatant of step 1；4th group: with 0.25mol/L NaOH solution by the supernatant in step 1 PH be adjusted to neutrality, then be added trypsase (1.5mg/L) mixing；
Four, the detection of Substance
1, the culture medium for detecting plate is respectively Escherichia coli culture medium and golden yellow grape bacterium culture medium, Escherichia coli training Feeding base (LB (Luria-Bertani) culture medium) ingredient is as shown in table 1, and Escherichia coli culture medium is wanted to sterilize in 121 DEG C of conditions 20min；Golden yellow grape bacterium culture medium be beef-protein medium (culture bacterium use) as shown in table 2, it is used to be Beef-protein medium (culture bacterium use), wants the 20min that sterilizes in 121 DEG C of conditions.
2, first group, second group, the sample of third group are taken in 100 μ l step 3 respectively, and it is flat that detection is added with sterile working In the Oxford cup aperture of plate, 37 DEG C of culture 15h；For 4th group of sample after 37 DEG C of isothermal holding 1h, boiling water bath 3min makes trypsase Then inactivation is added detection plate Oxford cup aperture and makees bacteriostatic test；
3, it sees whether to generate inhibition zone and with vernier caliper measurement antibacterial circle diameter (mm).
1 Escherichia coli culture medium of table (LB (Luria-Bertani) culture medium)
Peptone 10g
Yeast extract 5g
Nacl 10g
Distilled water 1000mL
pH 7.0
2 beef-protein medium of table (culture bacterium use)
Beef extract 3g
Peptone 10g
Nacl 5g
Agar 15-20g
Water 1000mL
pH 7.0-7.2
Fungistatic effect is as shown in Figure 5, Figure 6, Fig. 5 be Bulgaria (Lactobacillus bulgaricus ATCC11842) the antibacterial result figure of bacterium, Fig. 6 are the antibacterial result figure of Lactobacillus kefiri M3 bacterium of the invention, In figure, it is Escherichia coli culture medium that gold, which is staphylococcus aureus culture medium, E,；Guarantor be Bulgarian bacterium (Lactobacillus bulgaricusATCC11842), M3 is Lactobacillus kefiri M3 bacterium；1: the first group of hole sample to be tested, hole 2: the Two groups of samples to be tested, hole 3:: third group sample to be tested, hole 4:: third group sample to be tested.
From figs. 5 and 6, it can be seen that Lactobacillus kefiri M3 bacterium of the invention and lactobacillus bulgaricus Compare, the Lactobacillus kefiri M3 bacterium of the application has stronger suppression to Escherichia coli and golden light color staphylococcus Production is used, and after adjusting pH, fungistatic effect weakens, but when the culture medium for not connecing bacterium being adjusted to fermentation liquid pH with lactic acid, protect plus The bacteriostasis of Leah lactobacillus does not have the effect of M3 bacterial strain obvious, this illustrates exist in M3 to Escherichia coli and Staphylococcus aureus The inhibited substance of bacterium, but the substance is not lactic acid, and the fungistatic effect of the substance is influenced by pH；And pass through pancreas egg After white enzymatic treatment, the fungistatic effect of the bacterial strain weakens, and illustrates that the substance may be protide or peptide constituents.
The measurement of 3 surface hydrophobic of embodiment, the measurement of self-solidifying ability and the measurement with the copolymerized ability of pathogenic bacteria
One, the measurement of surface hydrophobic
Sample to be tested are as follows: Kefir grains lactobacillus (Lactobacillus kefirii) M3 bacterial strain (gymnobacteria) of the invention is opened Fei Er lactobacillus (Lactobacillus kefirii) M3 bacteria preparation, lactobacillus bulgaricus (Lactobacillus bulgaricusATCC11842) bacterial strain (gymnobacteria) and lactobacillus bulgaricus (Lactobacillus bulgaricus ATCC11842) bacterium powder.Wherein before the experiment of Kefir grains lactobacillus of the invention (Lactobacillus kefirii) M3 bacteria preparation It is placed in -20 DEG C of environment and saves 12 months.
Specific measuring method is as follows:
1, sample to be tested is centrifuged under conditions of revolving speed is 5000r/min in 37 DEG C of growth 18h in MRS culture solution 5min, cell precipitation are washed twice with phosphate buffer (pH6.8)；
2, the precipitating after washing in step 1 is resuspended in PBS buffer solution and adjusts to cell density (2 × 108CFU/mL)；
3, the mixed liquor of 3.0mL vortex mixed cell suspension and 1.0mL dimethylbenzene is added into step 2, is incubated at 30 DEG C 10min obtains of short duration vortex mixed object；
4, the of short duration vortex mixed object of step 3 is incubated for 1h at 30 DEG C and is mutually separated with reaching, and measures after removing upper strata aqueous phase Its absorbance at 600nm of remainder.
Surface hydrophobic (%) be original suspension with mix after water phase absorbance reduction percentage.
The results are shown in Table 3 for the surface hydrophobic of sample to be tested.
3 surface hydrophobic testing result of table
As shown in Table 1, the surface hydrophobic of Lactobacillus kefirii M3 of the invention and its bacteria preparation is high InLactobacillus bulgaricus。
Two, self-solidifying ability measures
Strain to be tested are as follows: Kefir grains lactobacillus (Lactobacillus kefirii) M3 bacterial strain (gymnobacteria) of the invention is protected Add Leah lactobacillus (Lactobacillus bulgaricusATCC11842) bacterial strain.
Specific assay method is as follows:
Strain to be tested is cultivated for 24 hours in MRS culture solution, and centrifugation 10min collects bacterium under the conditions of revolving speed is 5000r/min Body is resuspended in PBS after being washed 2 times using PBS (pH6.8), bacteria concentration is adjusted to 108The cell bacterium of CFU/mL, 4mL equivalent Suspension is fitted into the centrifuge tube of 5mL, is stood at room temperature after mixing.The time of autohemagglutination measurement is 1,2,3,4,5h, is taken every time The top bacteria suspension of 0.5mL, is added in the PBS of 1.5mL, mixes, and the light absorption value under 600nm is measured, using PBS as blank pair According to.
Agglutination rate (A%) calculation formula are as follows: A (%)=(A0- At)/A0×100
In formula: A0Absorbance when for initial time under 600nm；
AtFor the absorption photometric value of different time
Self-solidifying ability measurement result is as shown in table 4.
Three, Bacillus acidi lactici and the copolymerized ability of pathogenic bacteria (Escherichia coli) measure
Strain to be tested are as follows: Kefir grains lactobacillus (Lactobacillus kefirii) M3 bacterial strain (gymnobacteria) of the invention is protected Add Leah lactobacillus (Lactobacillus bulgaricusATCC11842) bacterial strain.
Specific assay method is as follows:
Bacillus acidi lactici is cultivated for 24 hours in MRS culture medium, and centrifugation 10min collects bacterium under the conditions of revolving speed is 5000r/min Body is resuspended in PBS after being washed 2 times using PBS (pH6.8), bacterium number is adjusted to 108cfu/mL.Meanwhile pathogenic bacteria are using same The method of sample, is resuspended in PBS, and adjusting bacterium number is 108cfu/mL.2mL bacterium bacteria suspension to be measured and pathogenic bacteria are drawn respectively In test tube, whirlpool shakes 10s and mixes bacteria suspension, measures its light absorption value in the case where wavelength is 600nm in 1,2,3,4,5h respectively. The agglutination rate of pathogenic bacteria is calculated by following equation:
A (%)=(A0- Amix)/A0×100
In formula: AmixIt is the light absorption value of pathogenic bacteria and bacterium mixed liquor to be measured in various time points；
A0The light absorption value of mixed liquor when being initial time.
Bacillus acidi lactici and the copolymerized ability of pathogenic bacteria (Escherichia coli) measurement are as shown in table 4.
4 self-solidifying ability of table and with the copolymerized ability measurement result with pathogenic bacteria
As shown in Table 4, the autohemagglutination ability and Escherichia coli copolymerized ability of Lactobacillus kefiri M3 of the invention It is better thanLactobacillus bulgaricus。
Lactobacillus kefiri M3 bacterium of the invention and its bacteria preparation have excellent surface hydrophobic, simultaneously Lactobacillus kefiri M3 bacterium of the present invention also has good cumulative power and Escherichia coli copolymerized ability, Lactobacillus kefiri M3 bacterium and its bacteria preparation have good adhesiveness in enteron aisle.
The preparation method of 4 Kefir grains lactobacillus bacteria preparation of embodiment
Kefir grains lactobacillus bacteria preparation the preparation method comprises the following steps: above-mentioned Kefir grains lactobacillus (Lactobacillus kefiri) M3 is cultivated in MRS culture medium to logarithmic growth phase, and centrifugation goes supernatant to stay thallus, and skimmed milk, sucrose, seaweed are added into thallus Sour sodium mixing, obtains Lactobacillus kefiri M3 bacteria preparation by frozen dried；Wherein, thallus and skimmed milk, sugarcane Sugar, sodium alginate weight ratio are 100:6.67:1.67:1.67.
The cultivation temperature of Kefir grains lactobacillus (Lactobacillus kefiri) M3 of present embodiment is 37 DEG C.
The centrifugal condition of present embodiment is that centrifugal rotational speed is 5000r/min, centrifugation time 10min.
Present embodiment frozen dried method are as follows: the mixture of thallus and skimmed milk, sucrose and sodium alginate is in -20 DEG C of items 1~5h is freezed under part, then vacuum freeze drying under conditions of condenser temperature is less than -40 DEG C, vacuum degree is greater than 200mtorr, Complete frozen dried.Wherein, thickness is less than or equal to 20mm after mixture freezing.
The Lactobacillus kefiri M3 bacteria preparation and Lactobacillus kefiri M3 of present embodiment (gymnobacteria) under the conditions of -20 DEG C influence of the storage time to viable count as shown in fig. 7, in figureFor Lactobacillus Kefiri M3 bacteria preparation,For Lactobacillus kefiri M3 gymnobacteria, from figure 7 it can be seen that 1,2,3,4,5, 8, measurement bacteria preparation viable count is respectively 1.0 × 10 after 12 weeks9CFU/mL、4.6×108CFU/mL、2.6×108CFU/mL、 1.3×108CFU/mL、9.4×107CFU/mL、9.3×107CFU/mL、4.9×107CFU/mL, viable count after storing 12 weeks It remains to reach 107CFU/mL.And Lactobacillus kefiri M3 gymnobacteria saves one week at -20 DEG C, viable count 0CFU/ mL。
5 Kefir grains lactobacillus of embodiment and its bacteria preparation gastro-intestinal Fluid tolerance
One, artificial gastro-intestinal Fluid configuration: artificial gastro-intestinal Fluid is prepared referring to " Chinese Pharmacopoeia "；
1, simulated gastric fluid configures: taking dilute hydrochloric acid 16.4mL, the water of 800mL and the pepsin of 10g is added, adjusts pH after mixing Value adds water to 1.3 and is settled to 1L；
2, simulated intestinal fluid is prepared: taking dipotassium hydrogen phosphate 6.8g, 500mL water is added and makes it dissolve, with 0.1mol/L NaOH tune PH value obtains dipotassium hydrogen phosphate solution to 6.8；Weighing 10g trypsase again adds suitable quantity of water to dissolve to obtain trypsin solution, will After dipotassium hydrogen phosphate solution and trypsin solution mixing, water is added to be settled to 1L；
Two, it the detection of Lactobacillus kefiri M3 bacteria preparation tolerance in gastro-intestinal Fluid: weighs Lactobacillus kefiri bacteria preparation is separately added into the simulated gastric fluid of 10mL and artificial according to the ratio that volume ratio is 1:10 It in intestinal juice liquid, mixes well, measures viable count every 1h, observe 6h；
Three, the detection of Lactobacillus kefiri M3 gymnobacteria bacterium mud tolerance in gastro-intestinal Fluid: Lactobacillus kefiri M3 bacterium mud is centrifuged 10min in 5000r/min, removes supernatant, then molten with the PBS buffering of sterilizing It liquid washing precipitating 3 times, is added separately in simulated gastric fluid and simulated intestinal fluid liquid according to the ratio that volume ratio is 1:10, it is sufficiently mixed It is even, viable count is measured every 1h, observes 6h.
Testing result is as shown in Fig. 8~9, wherein as can be seen from Figure 8 Lactobacillus kefiri M3 bacterium and its Bacteria preparation all has good gastro-intestinal Fluid tolerance, and 1g Lactobacillus kefiri M3 bacteria preparation is in 10mL simulated gastric fluid And retains 6h viable count in intestinal juice and remain to reach 106Lactobacillus kefiri M3 bacteria preparation after CFU/g or more, i.e. 6h It remains to reach 1.64 × 10 in the viable count of simulated gastric fluid6CFU/g, as can be seen from Figure 9 Lactobacillus kefiri M3 Bacteria preparation reaches 1.01 × 10 in simulated intestinal fluid6CFU/g；Lactobacillus kefiri M3 gymnobacteria bacterium mud carries out stomach and intestine Liquid tolerance test, as the result is shown after 6h, Lactobacillus kefirii M3 viable count exists gastro-intestinal Fluid tolerance test Simulated gastric fluid reaches 1.68 × 105CFU/g, reach 5.04 × 10 in simulated intestinal fluid5CFU/g。
Lactobacillus kefiri M3 bacteria preparation viable count in artificial gastro-intestinal Fluid is apparently higher than The viable count of Lactobacillus kefiri M3 bacterium gymnobacteria bacterium mud, Lactobacillus kefiri M3 bacteria preparation enhance stomach Intestinal juice tolerance.
6 type II diabetes mouse experiment of embodiment
One, influence of the Kefir grains Lemonal to type II diabetes mouse blood sugar
Referring to the method in " health food is examined and assessment technique enforcement of regulations handbook ", test is male using 21 age in days of Kunming Property mouse carry out.Mouse starts to model in adaptive feeding in animal culturing room after 1 week, randomly selects 12 and is only used as normal feminine gender Control group (normal group).For 24 hours (free water), ALX is injected intraperitoneally in 130mg/kg dosage to model group mouse for fasting before modeling.Note Fasting 4h docking takes blood after penetrating 10 days, measures fasting plasma glucose concentration with blood glucose meter, blood sugar concentration is small between 10-25mmol/L Mouse is that type II diabetes models successfully mouse.
It chooses 36 type II diabetes and models successfully mouse and be divided into 3 groups (T2B2 group, M3 group and BJLY groups), every group 12 Only, T2B2 group is blank control group, type II diabetes intragastric administration on mice tap water, given low 10mL/kgd, continuous gavage 5 Week；M3 group type II diabetes intragastric administration on mice Lactobacillus kefiri M3 bacteria preparation, 0.1gLactobacillus The phosphate buffer dissolution that kefiri M3 1mL sterilizes, given low 10mL/kgd, continuous gavage 5 weeks；BJLY group Type II diabetes intragastric administration on mice Bulgaria bacterium powder, 0.1g Bulgaria (Lactobacillus bulgaricus ATCC11842 the phosphate buffer dissolution that) bacterium powder 1mL sterilizes, given low 10mL/kgd, continuous gavage 5 weeks.Blood Sugar value test result is as shown in Figure 10, as shown in Figure 10, it will be seen that and obviously normal group of T2B2 group mouse blood sugar, and M3 group mouse blood sugar Significantly lower than T2B2 group, Lactobacillus kefiri M3 bacteria preparation of the invention has type II diabetes mouse blood sugar value Reduced effect.The reduction blood glucose effect of Lactobacillus kefiri M3 bacteria preparation of the invention is significantly better than Bao Jiali Sub- lactobacillus.
Two, influence of the Kefir grains lactobacillus bacteria preparation to type II diabetes oxidative damage
Malonaldehyde (Maloondialdehyde, MDA) is the common counter of a judgement oxidative damage, is usually utilized to sentence Determine oxidative damage degree.Experiment mice is put to death after the end of the experiment in step 1, four groups of test mices (normal group, T2B2 groups, M3 Group and BJLY group mouse) take serum and renal tissue to be detected respectively；Specific method is limited referring to the prosperous happy biotechnology in Shanghai The mice plasma lipopolysaccharides kit specification that company provides, and operated in strict accordance with specification, take serum or renal tissue 0.10g is simultaneously added 0.90mL extracting solution and is sufficiently homogenized, and 3000r/min is centrifuged 20min, measures malonaldehyde in serum or kidney and contains Amount.
The mda content result measured in each group mice serum is as shown in figure 11, as can be seen from Figure 11 T2B2 group mouse Serum malondialdehyde content is apparently higher than normal group, and M3 group mice serum mda content is low, Lactobacillus kefiri M3 bacteria preparation has reduction effect to type II diabetes mice serum mda content.
It is as shown in figure 12 to measure mda content result in each group mouse kidney.T2B2 group Mouse Kidney as can be seen from Figure 12 Dirty mda content is apparently higher than normal group, and M3 group mouse kidney mda content is low, Lactobacillus kefiri M3 Bacteria preparation has reduction effect to type II diabetes mouse kidney mda content.M3 group kidney mda content is significantly lower than BJLY Group.
Lactobacillus kefiri M3 bacteria preparation of the invention acts on type II diabetes remission most bright It is aobvious.
Three, influence of the Kefir grains lactobacillus bacteria preparation to type II diabetes mouse intestinal flora
1, sample pre-treatments
Weigh each experimental mice in 200mg step 1 (normal group, T2B2 group be that blank control group, II type of M3 group are sugared Urinate disease intragastric administration on mice Lactobacillus kefiri M3 bacteria preparation, BJLY group type II diabetes intragastric administration on mice Bulgaria bacterium Powder) fresh excreta, be respectively put into the 2mL centrifuge tube of sterilizing, add 1mL70% ethyl alcohol, concussion mixes, 10000r/min Room temperature is centrifuged 3min, throws aside supernatant liquid.PBS solution is added, concussion mixes, and 10000r/min room temperature is centrifuged 3min, throws aside Layer liquid is inverted 2mL pipe in 1min on blotting paper, until flowing out without liquid.Sample cell is put into 55 DEG C of baking oven 10min, is made Residual alcohol volatilizees completely, guarantees subsequent experimental operation.
2, intestinal flora variance analysis
The above-mentioned excrement that processing terminate, V3-V4 area of the Sheng Gong bio-engineering corporation to intestinal flora 16S rDNA in Shanghai Classification examining order is carried out, the diversity and abundance to each group mouse intestinal flora carry out horizontal point of different taxonomies Analysis carries out correlation analysis using 17.0 software of SPSS.
Lactobacillus kefiri M3 bacteria preparation is multifarious on mouse intestinal flora to be influenced as shown in table 3.Each group The sequence number of sample is above 35000, and coverage rate reaches 99%, this shows that 99% intestinal flora of mouse has been sequenced.With Normal group is compared, and T2B2 group mouse intestinal flora diversity reduces 12.11%, compared with T2B2 group, M3, B group mouse intestinal Bacterial diversity increases 9.44%, 2.91%.As shown in Table 5, type II diabetes mouse intestinal flora (normal group) diversity It reduces；M3 group, BJLY group have the ability for restoring type II diabetes mouse intestinal flora to normal level, M3 group intestinal flora Alpha index close and closer to normal group, the results showed that Lactobacillus kefiri M3 bacteria preparation is to II type glycosuria Sick mouse intestinal flora diversity adjustment effect is best.
5 intestinal flora alpha index of table
LEfSe (LDA Effect Size) analysis, can be used for the comparison between two or more groupings, to find There are the species of significant difference between group.T2B2 intestinal flora compared with normal group is analyzed by LEfSe (LDA Effect Size) Changed, wherein clostridium guiding principle (Clostridia) Prey irrigates the nocuousness such as Pseudomonas (Prevotellaceae), Alistipes Bacterium increases.Normal group works in Bacteroidetes (Bacteroides)；T2B2 group clostridium guiding principle in Firmicutes (Clostridia) it plays an important role；M3 group plays an important role at lactobacillus (Lactobacillus)；BJLY group is becoming Shape bacterium door (Proteobacteria) plays an important role.The result shows that Lactobacillus kefiri M3 bacteria preparation can make Beneficial bacterium increases in type II diabetes mouse intestinal flora.
Each processing group belongs to horizontal variation: high-flux sequence find normal mouse intestinal flora by Barnesiella, Bacteroides、Alloprevotella、Helicobacter、Alistipes、Clostridium XlVa、 Parabacteroides、Escherichia/Shigella、Coprobacter、Lactobacillus、Holdemanella、 Odoribacter composition.Wherein Barnesiella, Bacteroides, Alloprevotella are the advantage in intestinal flora Pseudomonas.
Each processing group category level difference: normal group, T2B2 group, M3 group, Barnesiella in BJLY group mouse intestinal flora Relative abundance be respectively 14692,10883.75,13833,15906.5, compared with normal group, T2B2 group Barnesiella drop Low 46.15%, compared with T2B2 group, M3, BJLY group increase separately 27.10%, 46.15%；In mouse intestinal flora The relative abundance of Bacteroides is respectively 20781.25,3687.5,2975,1203.75,3062.5,13631, with normal group It compares, T2B2 group Bacteroides reduces by 463.56%, and compared with T2B2 group, BJLY group increases by 269.65%, M3 to be reduced respectively 19.32%；Lactobacillus relative abundance in each group mouse intestinal flora is respectively 595.5,260.25,1287.75, 819.5,269.5,674.5, compared with normal group, T2B2 group Lactobacillus relative abundance reduces by 128.82%, with T2B2 Group is compared, and M3, BJLY group Lactobacillus relative abundance increase separately 394.81%, 159.17%.In mouse intestinal flora The relative abundance of Alloprevotella is respectively 4746,6913.75,5345,4203.75,4764.25,11267.75, and just Normal group is compared, and T2B2 group Alloprevotella increases by 31.35%, and compared with T2B2 group, BJLY group increases by 62.98%, M3 drop Low 22.69%.The influence of normal group, T2B2, M3 group, BJLY group mouse intestinal flora is analyzed, analysis result is as shown in figure 13, from Figure 13 can be seen that Barnesiella and bacteroides abundance in type II diabetes mouse intestinal flora and reduce, Lactobacillus kefiri M3 makes Barnesiella and bacteroides in type II diabetes mouse intestinal flora rich It spends to normal level and restores；T2B2 group relative abundance reduces, and Lactobacillus kefiri M3 bacteria preparation makes II type glycosuria Sick mouse Lactobacillus increases most.M3 group Lactobacillus relative abundance highest, with surface hydrophobic, self-solidifying energy The measurement result of power and the result of gastro-intestinal Fluid tolerance are consistent, Lactobacillus kefiri M3 adhesiveness of the invention Most preferably, it was demonstrated that Lactobacillus kefiriM3 bacteria preparation can be adhered on enteron aisle.In normal group intestinal flora Alloprevotella abundance increases, and Lactobacillus kefiri M3 bacteria preparation makes type II diabetes mouse intestinal bacterium Alloprevotella abundance is restored to normal level in group；It follows that Lactobacillus kefiri M3 bacteria preparation energy So that beneficial bacterium increases in type II diabetes mouse intestinal, harmful bacteria is reduced.

Claims (7)

1. a kind of application of Kefir grains lactobacillus M3 on antibacterial.
2. a kind of application of Kefir grains lactobacillus M3 as the active constituent for the treatment of type II diabetes medicament.
3. Kefir grains lactobacillus M3 according to claim 2 is answered as the active constituent for treating type II diabetes medicament With, it is characterised in that bacteria preparation, specific method is made by frozen dried in Kefir grains lactobacillus are as follows: Kefir grains lactobacillus Lactobacillus kefiri M3 is centrifuged after cultivating in MRS culture medium to logarithmic growth phase, goes supernatant to stay thallus, then Skimmed milk, sucrose, sodium alginate mixing are added into thallus, obtains Kefir grains lactobacillus bacteria preparation by frozen dried；Its In, thallus and skimmed milk, sucrose, sodium alginate weight ratio are 100:6~7:1~2:1~2.
4. Kefir grains lactobacillus M3 according to claim 3 is answered as the active constituent for treating type II diabetes medicament With, it is characterised in that frozen dried method are as follows: the mixture of thallus and skimmed milk, sucrose and sodium alginate is under the conditions of -20 DEG C 1~5h is freezed, then vacuum freeze drying under conditions of condenser temperature is less than -40 DEG C, vacuum degree is greater than 200mtorr, i.e., complete At frozen dried.
5. Kefir grains lactobacillus M3 according to claim 3 is answered as the active constituent for treating type II diabetes medicament With, it is characterised in that centrifugal condition be centrifugal rotational speed be 5000r/min, centrifugation time 10min.
6. Kefir grains lactobacillus M3 according to claim 3 is answered as the active constituent for treating type II diabetes medicament With, it is characterised in that thallus is 100:6.67:1.67:1.67 with skimmed milk, sucrose, sodium alginate weight ratio.
7. Kefir grains lactobacillus M3 is improving the application in patients with NIDDM intestinal microflora.
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