CN110184214B - Lactobacillus kefir and bacterial preparation thereof - Google Patents
Lactobacillus kefir and bacterial preparation thereof Download PDFInfo
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- CN110184214B CN110184214B CN201910422107.8A CN201910422107A CN110184214B CN 110184214 B CN110184214 B CN 110184214B CN 201910422107 A CN201910422107 A CN 201910422107A CN 110184214 B CN110184214 B CN 110184214B
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
A kefir lactobacillus and a bacterial preparation thereof relate to a lactobacillus and a bacterial preparation thereof. The invention provides a kefir lactobacillus and a bacterial preparation thereof, which solve the problems of low surface hydrophobicity of the existing lactobacillus and reduction of the number of viable bacteria of the lactobacillus after freeze-drying. The Lactobacillus kefir is Lactobacillus kefir (Lactobacillus kefir) M11; the bacterial preparation is prepared by the following steps: lactobacillus kefir M11 was cultured in MRS medium and then mixed with cryoprotectant for lyophilization. The Lactobacillus kefir (Lactobacillus kefir) M11 and Lactobacillus kefir (Lactobacillus kefir) M11 bacterial preparations of the invention both have excellent surface hydrophobicity and cohesiveness and have good adhesion capability to intestinal tracts. The effective viable count of the Lactobacillus kefir M11 prepared into the microbial inoculum is large and can reach more than 90 percent.
Description
Technical Field
The invention relates to lactic acid bacteria and a bacterial preparation thereof.
Background
The lactobacillus is symbiotic bacteria in animal intestinal tracts, can help digestion and is beneficial to the health of human intestinal tracts, and food containing active lactobacillus is often regarded as healthy food and is usually added into fermented food such as yoghourt and the like for the balance adjustment of flora in human intestinal tracts. Intestinal epithelial cells in vivo can secrete a certain substance (possibly protein or lipid teichoic acid) which is beneficial to the colonization of probiotics such as lactobacillus on intestinal mucosa, thereby forming a biofilm barrier and rejecting the adhesion of pathogenic bacteria on the intestinal wall. Meanwhile, the lactobacillus can generate lactic acid, acetic acid and bacteriocin to inhibit the invasion of various pathogenic bacteria. The use of lactobacilli instead of antibiotics to prevent intestinal diseases in humans or animals has therefore become an important research area in the current food and feed industry. In 2019, YANG research finds that Lactobacillus rhamnosus GG can also increase the permeability of intestinal tracts of piglets, and can improve the immune barrier function of the intestinal tracts by regulating and controlling the secretion of antibacterial peptides, cytokines and chemotactic factors.
The adhesion property of lactobacillus is one of important indexes for evaluating the probiotic function of lactobacillus, and the adhesion colonization is the premise and the basis for the physiological function of lactobacillus in intestinal tract. Studies of researchers show that the cell surface hydrophobicity and the self-aggregation capability of microorganisms play a main role in the adhesion of strains, and lactobacillus with higher hydrophobicity and self-aggregation capability also shows higher adhesion in intestinal tracts, namely the cell surface hydrophobicity and the self-aggregation capability are in direct proportion to the adhesion of lactobacillus to intestinal epithelial cells.
At present, the potential function of lactobacillus for preventing and treating type II diabetes is proved and widely accepted by animal in vivo and clinical experiments. When a proper amount of lactobacillus is ingested by a organism, the lactobacillus can effectively regulate energy metabolism, reduce fat and cholesterol accumulation, effectively supplement intestinal flora loss caused by type II diabetes, establish a new intestinal balance steady state, recover a healthy intestinal flora structure, reduce intestinal permeability, improve intestinal barrier function, inhibit inflammatory reaction, relieve glucose intolerance and improve glucose tolerance and insulin sensitivity. The existing common lactobacillus has low surface hydrophobicity and poor adhesiveness, and the probiotic function of the existing common lactobacillus is influenced. In addition, the use is convenient, the lactic acid bacteria are prepared into bacteria powder for use, the bacteria powder is generally prepared by adopting a freezing treatment method, the number of effective viable bacteria of the lactic acid bacteria after the freezing treatment can be reduced, and the use effect of the lactic acid bacteria is greatly influenced.
Disclosure of Invention
The invention aims to solve the problems that the surface hydrophobicity of the existing lactobacillus is low and the viable count of the freeze-dried lactobacillus is reduced. And provides a lactobacillus kefir and its bacterial preparation.
The Lactobacillus kefir is Lactobacillus kefir M11, is preserved in China center for type culture Collection, and has a preservation number of CCTCC NO: m2019080.
The Lactobacillus kefir M11 is derived from Tibetan mushroom, the thallus forms of the Lactobacillus kefir M11 are gram-positive bacteria and medium-long-rod bacteria, and the colony forms are milky white, round and smooth in surface.
The Lactobacillus kefir preparation is prepared by culturing Lactobacillus kefir (Lactobacillus kefir) M11 in an MRS culture medium until logarithmic growth phase, centrifuging, removing supernatant to retain thallus, adding skim milk, sucrose and sodium alginate into the thallus, mixing, and lyophilizing to obtain Lactobacillus kefir M11 preparation; wherein the weight ratio of the thalli to the skim milk to the sucrose to the sodium alginate is 100: 6-7: 1-2: 1 to 2.
Through the determination of surface hydrophobicity and self-coagulation capability, the Lactobacillus kefir M11 and Lactobacillus kefir M11 bacterial preparations have excellent surface hydrophobicity and coagulation property, and have good adhesion capability to intestinal tracts.
The number of effective viable bacteria of the Lactobacillus kefir M11 prepared into the microbial inoculum is 1.41 multiplied by 108The survival rate reaches more than 90 percent, the live bacteria can still keep the effective viable count after being stored for 12 weeks at the temperature of minus 20 DEG CHolder 108cfu/mL。
The Lactobacillus kefir (Lactobacillus kefir) M11 is Lactobacillus and is preserved in China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan university, the preservation date is 2019, 1 and 24 days, and the preservation number is CCTCC NO: m2019080.
Drawings
FIG. 1 is a characteristic of a colony of Lactobacillus kefir M11;
FIG. 2 shows the bacterial characteristics of Lactobacillus kefir M11;
FIG. 3 is a diagram of total DNA PCR amplification of Lactobacillus kefir M11;
FIG. 4 shows the effect of storage time at-20 ℃ on viable count of Lactobacillus kefir M11 and Lactobacillus kefir M11 bacterial preparations;
FIG. 5 is a graph showing the results of the tolerance of Lactobacillus kefir M11 in artificial gastric juice;
FIG. 6 shows the results of tolerance of Lactobacillus kefir M11 in artificial intestinal fluid;
FIG. 7 shows the mouse experiment for type II diabetes mellitus with Lactobacillus and bacterial preparation;
FIG. 8 shows the result of oxidative damage of the serum of type II diabetic mice by Lactobacillus kefir preparation;
FIG. 9 shows the results of oxidative damage of Lactobacillus kefir preparation to kidney of type II diabetic mice;
FIG. 10 is a phylogenetic analysis diagram of Lactobacillus kefiri M11;
FIG. 11 is the effect of the intestinal flora of Lactobacillus kefiri M11 mouse.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the Lactobacillus kefir of the embodiment is Lactobacillus kefir (Lactobacillus kefir) M11, which is preserved in the chinese typical culture collection with a preservation number of CCTCC NO: m2019080.
The separation, purification and identification of Lactobacillus kefir M11 of the embodiment:
first, culture of Tibetan mushroom fungus
1. Activation of Tibetan mushroom bacterium particles: inoculating Tibetan mushroom fungus granules into sterilized Dada pure milk according to the inoculation amount of 5%, culturing at constant temperature of 21 ℃ for 24h, filtering the cultured Tibetan mushroom fermentation liquor through a sterilized filter screen, retaining the Tibetan mushroom fungus granules, and repeating for one week; wherein the Tibetan mushroom mycelia are collected from the microorganism focus laboratory of the university of Heilongjiang in 2017.
2. And (3) carrying out bacterium particle passage on Tibetan mushroom: enlarging and culturing the activated Tibetan mushroom fungus granules into sterilized milk according to the proportion of 5%, culturing at constant temperature of 21 ℃ for 48h, filtering the cultured Tibetan mushroom fermentation liquor through a sterilized filter screen, and reserving Tibetan mushroom fungus granules; passage is carried out once every 48h for 4 times;
secondly, separating and purifying lactobacillus
2g of sterile normal saline is cleaned, the Tibetan mushroom bacterium grains after passage in the step one are mixed with 1mL of sterile normal saline and put into a mortar, and the mixture is ground until the diameter of the particles is 1-3 mm; transferring the prepared Tibetan mushroom fungus granules into a 10ml centrifuge tube for many times, fixing the volume to 10ml, and uniformly whirling and shaking; the bacterial suspensions are respectively treated according to the proportion of 104、105、106The dilution was performed with a gradient. Selecting a Lactobacillus colony morphology (usually 5 to 10 colonies per sample) on MRS solid medium at 37 deg.C for gram staining, selecting gram positive bacteria and observing the typical Lactobacillus thallus morphology as rod under electron microscope, purifying on MRS solid medium, and isolating to obtain a Lactobacillus strain with number M11. The M11 strain was purified for three generations.
Thirdly, identifying the lactobacillus M11 strain purified in the second step
1. Extraction of bacterial DNA
Total DNA of the Lactobacillus M11 strain was extracted according to the protocol of the Ezup column type bacterial DNA extraction kit of Shanghai Biotechnology Ltd.
2. PCR amplification of Lactobacillus M11 Total DNA
PCR amplification primers: 5'-TACCTTGTTAGCACTT-3'
5'-AGAGTTTGATCCTGGCTCAG-3'
PCR reaction (50. mu.L): 10 XPCR buffer 10. mu.L, primer pairs (10. mu. mol/L) each 5. mu.L, dNTP MIX (100mmol/L) 4. mu.L, Taq DNA polymerase (5U/. mu.L) 2.5. mu.L, dd H2O 26.5.5. mu.L, template DNA 2. mu.L.
And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 52 deg.C for 2min, extension at 72 deg.C for 2min, circulating for 35 times, extension at 72 deg.C for 10min, and storing at 4 deg.C.
3. Agarose gel electrophoresis
And (3) carrying out agarose gel electrophoresis on the PCR product, weighing 1g of agarose, preparing 1.0% agarose electrophoresis gel by using 1 XTAE buffer solution, adding ethidium bromide when not solidified to enable the final concentration to reach 1 mu g/mL, and preparing the gel. Adding electrophoresis buffer solution into the electrophoresis tank to ensure that the liquid surface is over the glue surface. mu.L of each PCR product was mixed with 1. mu.L of 6 × loading buffer, spotted, and one well was added with DNA marker. And (5) carrying out electrophoresis for 30min, and observing an electrophoresis result by using an ultraviolet gel imager.
4. 16S rDNA sequencing of Lactobacillus M11
And (3) selecting a PCR product with the length of about 1000bp after agarose gel electrophoresis imaging, and sending the PCR product to Shanghai workers for sequencing. Lactobacillus isolated from C.tibetana grain was identified using a BLAST search (http:// www.ncbi.nlm.nih.gov.blast) against the GenBank DNA database (www.ncbi.nlm.nih.gov/Genbank /). The phylogenetic analysis was performed by constructing an evolutionary tree using the software MEGA 5.1, and the results are shown in FIG. 10.
The colony characteristics of the M11 strain of the present embodiment are shown in FIG. 1; the cell characteristics of the M11 strain are shown in FIG. 2, and as shown in FIGS. 1 and 2, the M11 strain of the present embodiment is a gram-positive bacterium or a medium-long-rod bacterium, and the colony is milky white, round and smooth in surface.
The PCR amplification result of the total DNA of the M11 strain of the present embodiment is shown in FIG. 3, wherein M is marker, and 1 is M11 strain; as can be seen from FIG. 3, the length of the gene sequence of the M11 strain of the present embodiment isolated from Pleurotus tibetans was about 1000bp, and the extracted gene fragment was complete, free of degradation, and capable of sequencing.
According to the molecular biological analysis and the characteristics of colonies and thalli, the M11 strain is determined to be Lactobacillus kefir, and is named as Lactobacillus kefir M11.
The second embodiment is as follows: a kefir lactobacillus preparation, the preparation method thereof comprises the following steps: culturing Lactobacillus kefir M11 in MRS culture medium, centrifuging to logarithmic phase, removing supernatant to obtain thallus, adding skimmed milk, sucrose and sodium alginate, mixing, and lyophilizing to obtain Lactobacillus kefir M11 strain preparation; wherein the weight ratio of the thalli to the skim milk to the sucrose to the sodium alginate is 100: 6-7: 1-2: 1 to 2.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: the culture temperature of Lactobacillus kefir (Lactobacillus kefir) M11 was 37 ℃. Other steps and parameters are the same as those in the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the first or second embodiment in that: the centrifugation conditions are that the centrifugation rotating speed is 5000r/min and the centrifugation time is 10 min. Other steps and parameters are the same as those in the second or third embodiment.
The fifth concrete implementation mode: the fourth difference between this embodiment and the specific embodiment is that: the freeze-drying treatment method comprises the steps of freezing a mixture of the thalli, skim milk, cane sugar and sodium alginate for 1-5 hours at the temperature of-20 ℃, and then carrying out vacuum freeze-drying at the temperature of a cold trap being less than-40 ℃ and the vacuum degree being more than 200mtorr, so that freeze-drying treatment is completed. Other steps and parameters are the same as those in the fourth embodiment.
The thickness of the mixture of the present embodiment after freezing is 20mm or less.
The sixth specific implementation mode: the fifth embodiment is different from the fifth embodiment in that: the weight ratio of the thalli to the skim milk to the sucrose to the sodium alginate is 100: 6.3-6.8: 1.2-1.8: 1.2 to 1.8. Other steps and parameters are the same as those in the fifth embodiment.
The seventh embodiment: the sixth embodiment is different from the sixth embodiment in that: the weight ratio of the thalli to the skim milk to the sucrose to the sodium alginate is 100:6.67: 1.67: 1.67. other steps and parameters are the same as those in the fifth embodiment.
The specific implementation mode is eight: the preparation method of the lactobacillus kefir preparation of the embodiment comprises the following steps: culturing Lactobacillus kefir M11 in MRS culture medium to logarithmic phase, centrifuging to remove supernatant and retain thallus, adding skimmed milk, sucrose and sodium alginate into the thallus, mixing, and lyophilizing to obtain Lactobacillus kefir M11 strain preparation; wherein the weight ratio of the thalli to the skim milk to the sucrose to the sodium alginate is 100:6.67: 1.67: 1.67.
the culture temperature of Lactobacillus kefir (Lactobacillus kefir) M11 in this embodiment was 37 ℃.
The centrifugation conditions in this embodiment are a centrifugation rotation speed of 5000r/min and a centrifugation time of 10 min.
The freeze-drying treatment method of the embodiment is that the mixture of the thalli and the skim milk, the sucrose and the sodium alginate is frozen for 1-5 hours at the temperature of-20 ℃, and then the mixture is subjected to vacuum freeze-drying at the temperature of a cold trap less than-40 ℃ and the vacuum degree of more than 200mtorr, so that the freeze-drying treatment is completed.
FIG. 4 shows the effect of the storage time of the Lactobacillus kefiri M11 bacterial preparation and Lactobacillus kefiri M11 (naked bacteria) at-20 ℃ on the viable count of the bacteria in this embodiment, wherein
Is a Lactobacillus kefiri M11 bacterial preparation,
the number of viable bacteria of the bacterial preparation was 3.92X 10 after 0, 1, 2, 3, 4, 5, 8 and 12 weeks, respectively, as seen from FIG. 4, in a case of Bacillus kefiri M11 naked bacteria9CFU/mL、1.41×109CFU/mL、9.89×109CFU/mL、5.12×108CFU/mL、2.27×108CFU/mL、1.881×108CFU/mL、1.87×108CFU/mL、4.85×107CFU/mL, viable count of 10 after 12 weeks of storage7CFU/mL. And the Lactobacillus kefiri M11 naked bacterium is preserved for one week at the temperature of minus 20 ℃, and the viable count is 0 CFU/mL.
Example 1 measurement of surface hydrophobicity, measurement of self-coagulating ability and copolymerization ability with pathogenic bacteria-measurement of surface hydrophobicity
The samples to be tested were: the Lactobacillus kefir (Lactobacillus kefir) M11 strain (naked bacteria), the Lactobacillus kefir (Lactobacillus kefir) M11 strain preparation, the Lactobacillus bulgaricus (Lactobacillus bulgaricus ATCC11842) strain (naked bacteria) and the Lactobacillus bulgaricus (Lactobacillus bulgaricus ATCC11842) powder of the invention. Wherein the Lactobacillus kefir (Lactobacillus kefir) M11 strain preparation is preserved for 12 months at-20 ℃ before experiment.
The specific measurement method is as follows:
1. growing a sample to be detected in MRS culture solution at 37 ℃ for 18h, centrifuging for 5min under the condition of the rotating speed of 5000r/min, and washing cell sediment twice by using phosphate buffer solution (pH6.8);
2. the pellet washed in step one was resuspended in PBS buffer and adjusted to cell density (2X 10)8CFU/mL)；
3. Adding a mixed solution of 3.0mL of vortex mixed cell suspension and 1.0mL of xylene into the second step, and incubating for 10min at 30 ℃ to obtain a transient vortex mixture;
4. the brief vortexed mixture of step three was incubated at 30 ℃ for 1h to achieve phase separation, and the remaining portion was measured for absorbance at 600nm after removal of the upper aqueous phase.
Surface hydrophobicity (%) is the percentage of absorbance decrease of the original suspension and the aqueous phase after mixing.
The results of surface hydrophobicity of the samples to be tested are shown in table 1.
TABLE 1 results of surface hydrophobicity measurements
As can be seen from Table 1, the surface hydrophobicity of Lactobacillus kefiri M11 and its bacterial preparation of the present invention is higher than that of Lactobacillus bulgaricus.
Second, self-coagulation ability measurement
The strains to be tested are as follows: the Lactobacillus kefir (Lactobacillus kefir) M11 strain (naked bacterium) and the Lactobacillus bulgaricus (Lactobacillus bulgaricus ATCC11842) strain are disclosed.
The specific determination method is as follows:
culturing the strain to be tested in MRS culture solution for 24h, centrifuging at 5000r/min for 10min to collect thallus, washing with PBS (pH6.8) for 2 times, resuspending in PBS, and adjusting the concentration of the thallus to 108CFU/mL, 4mL of the same amount of cell suspension was put into a 5mL centrifuge tube, mixed well and left to stand at room temperature. The time for self-agglutination determination is 1, 2, 3, 4, 5h, 0.5mL upper bacterial suspension is taken each time, added into 1.5mL PBS, mixed evenly, and the absorbance value under 600nm is determined, and the PBS is used as a blank control.
The agglutination ratio (A%) is calculated by the formula of A (%) ═ A0－At)/A0×100
In the formula A0Absorbance at 600nm at initial time;
Atas absorbance values at different times
The results of the self-setting ability measurement are shown in Table 2.
Thirdly, measuring the copolymerization capability of the lactobacillus and the pathogenic bacteria (escherichia coli)
The strains to be tested are as follows: the Lactobacillus kefir (Lactobacillus kefir) M11 strain (naked bacterium) and the Lactobacillus bulgaricus (Lactobacillus bulgaricus ATCC11842) strain are disclosed.
The specific determination method is as follows:
culturing lactobacillus in MRS culture medium for 24h, centrifuging at 5000r/min for 10min to collect thallus, washing with PBS (pH6.8) for 2 times, re-suspending in PBS, and adjusting the number of bacteria to 108cfu/mL. Meanwhile, the pathogenic bacteria are resuspended in PBS by the same method, and the number of the bacteria is adjusted to 108cfu/mL. Respectively sucking 2mL of bacterial suspension to be detected and pathogenic bacterial suspension into a test tube, uniformly mixing by vortex oscillation for 10s, and respectively measuring the light absorption value of the bacterial suspension to be detected and the pathogenic bacterial suspension under the wavelength of 600nm in 1, 2, 3, 4 and 5 h. The agglutination ratio for pathogenic bacteria was calculated by the following formula:
A(％)＝(A0－Amix)/A0×100
in the formula AmixFor the mixture of pathogenic bacteria and test bacteria at various time pointsA light absorption value;
A0-is the absorbance of the mixture at the initial time.
TABLE 2 measurement results of self-setting ability
As is clear from Table 2, the self-polymerization ability and the Escherichia coli copolymerization ability of Lactobacillus kefiri M11 of the present invention were both better than those of Lactobacillus bulgaricus.
The Lactobacillus kefiri M11 strain and the bacterial preparation thereof have excellent surface hydrophobicity, and meanwhile, the Lactobacillus kefiri M11 strain also has good polymerization capacity and escherichia coli copolymerization capacity, and the Lactobacillus kefiri M11 strain and the bacterial preparation thereof have good adhesion in intestinal tracts.
Example 2 gastrointestinal fluid tolerance of Lactobacillus kefir and bacterial preparations thereof
Firstly, preparing artificial gastrointestinal fluids: preparing artificial gastrointestinal fluid according to Chinese pharmacopoeia;
1. preparing artificial gastric juice: taking 16.4mL of dilute hydrochloric acid, adding 800mL of water and 10g of pepsin, mixing, adjusting the pH value to 1.3, and adding water to a constant volume of 1L;
2. preparing artificial intestinal juice: taking 6.8g of dipotassium phosphate, adding 500mL of water to dissolve the dipotassium phosphate, and adjusting the pH value to 6.8 by using 0.1mol/L NaOH to obtain a dipotassium phosphate solution; weighing 10g of trypsin, adding a proper amount of water to dissolve the trypsin to obtain a trypsin solution, mixing the dipotassium hydrogen phosphate solution and the trypsin solution, and adding water to a constant volume of 1L;
secondly, the detection of the tolerance of the Lactobacillus kefiri M11 bacterial preparation in gastrointestinal fluid: weighing Lactobacillus kefiri preparation, adding into 10mL artificial gastric juice and artificial intestinal juice respectively according to the volume ratio of 1:10, mixing well, measuring viable count every 1h, and observing for 6 h.
Thirdly, detecting the tolerance of the Lactobacillus kefiri M11 naked bacterium mud in gastrointestinal fluid: centrifuging Lactobacillus kefiri M11 bacterial mud at 5000r/min for 10min, removing supernatant, washing precipitate with sterilized PBS buffer solution for 3 times, adding into artificial gastric juice and artificial intestinal juice respectively at a volume ratio of 1:10, mixing well, measuring viable count every 1 hr, and observing for 6 hr.
The detection results are shown in fig. 5-6, wherein it can be seen from fig. 5 and 6 that the Lactobacillus kefiri M11 bacterium and the bacterial preparation thereof have good gastrointestinal fluid tolerance, and the number of viable bacteria of 1g of the Lactobacillus kefiri M11 bacterium preparation can still reach 10 after 6h of viable bacteria are reserved in 10mL of artificial gastric juice and intestinal fluid6More than CFU/g, the viable count of L.kefiri M11 can still reach 1.44 x 10 in the artificial gastric juice6CFU/g, reaching 2.77 x 10 in artificial intestinal juice6CFU/g; the Lactobacillus kefiri M11 naked bacterium mud is subjected to gastrointestinal fluid tolerance experiment, and the result of the gastrointestinal fluid tolerance experiment shows that after 6 hours, the viable count of Lactobacillus kefiri M11 reaches 5.02 multiplied by 10 in artificial gastric juice5CFU/g, up to 5.01X 10 in artificial intestinal juice5CFU/g。
The viable count of the Lactobacillus kefiri M11 bacterial preparation in the artificial gastrointestinal fluid is obviously higher than that of the Lactobacillus kefiri M11 naked bacterial sludge, and the Lactobacillus kefiri M11 bacterial preparation enhances the tolerance of the gastrointestinal fluid.
Example 3 type II diabetic mouse experiment
Influence of Lactobacillus kefir preparation on blood sugar of type II diabetic mice
According to the method in the implementation manual of health food inspection and evaluation technical specifications, male mice of 21 days old Kunming are used for the test. The mice were subjected to adaptive breeding in an animal culture room for 1 week, and then the modeling was started, and 12 mice were randomly selected as a normal negative control group (normal group). Model group mice were fasted for 24h (free drinking) before modeling and were injected intraperitoneally with ALX at a dose of 130 mg/kg. After 10 days of injection, fasting is carried out for 4 hours, blood is taken after tail breaking, fasting blood glucose concentration is measured by a glucometer, and a mouse with the blood glucose concentration between 10 mmol/L and 25mmol/L is a mouse successfully modeled for type II diabetes.
Selecting 36 mice with successful type II diabetes modeling, averagely dividing the mice into 3 groups (T2B2 group, M11 group and BJLY group), wherein each group comprises 12 mice, the T2B2 group is a blank control group, the type II diabetes mice are perfused with tap water, the perfusion dosage is 10 mL/kg. d, and the continuous perfusion lasts for 5 weeks; m11 group II type diabetes mice lavage Lactobacillus kefir M11 bacterial preparation, 0.1g Lactobacillus kefir M11 with 1mL sterile phosphate buffer solution dissolved, the lavage dose is 10mL/kg d, continuous lavage for 5 weeks; BJLY group II type diabetes mice gavage Bulgaria bacteria powder, 0.1g Bulgaria (Lactobacillus bulgaricus ATCC11842) bacteria powder with 1mL sterile phosphate buffer solution dissolved, the gavage dose is 10mL/kg d, continuous gavage for 5 weeks. The blood sugar test results are shown in fig. 7, and it can be seen from fig. 7 that the blood sugar of the mice in the T2B2 group is obviously normal, while the blood sugar of the mice in the M11 group is obviously lower than that of the mice in the T2B2 group, and the Lactobacillus kefir M11 strain preparation of the invention has the effect of reducing the blood sugar of the mice with type ii diabetes. The Lactobacillus kefir M11 bacterial preparation of the invention has obviously better blood sugar reducing effect than Lactobacillus bulgaricus.
Second, the influence of Lactobacillus kefiri preparation on the oxidative damage of type II diabetic mice
Malondialdehyde (MDA) is a common indicator for determining oxidative damage, and is often used to determine the degree of oxidative damage. In the first step, the experimental mice are sacrificed after the experiment is finished, and serum and kidney tissues of four groups of experimental mice (mice of a normal group, a T2B2 group, an M11 group and a BJLY group) are respectively taken for detection; the specific method refers to the specification of a mouse plasma lipopolysaccharide kit provided by Hixin le Biotechnology Limited, and strictly operates according to the specification, 0.10g of serum or kidney tissue is taken, 0.90mL of extracting solution is added to the serum or kidney tissue for fully homogenizing, the serum or kidney tissue is centrifuged at 3000r/min for 20min, and the content of malondialdehyde in the serum or kidney is measured.
The result of measuring the malondialdehyde content in the serum of each group of mice is shown in fig. 8, and it can be seen from fig. 8 that the malondialdehyde content in the serum of the T2B2 group mice is obviously higher than that in the normal group, while the malondialdehyde content in the serum of the M11 group mice is low, and the Lactobacillus kefiri M11 strain preparation has a reducing effect on the malondialdehyde content in the serum of the type II diabetic mice.
The results of measuring the malondialdehyde content in the kidney of each group of mice are shown in FIG. 9. From fig. 9, it can be seen that the kidney malondialdehyde content of the mice in the T2B2 group is significantly higher than that of the normal group, while the kidney malondialdehyde content of the mice in the M11 group is low, the Lactobacillus kefiri M11 bacterial preparation has a reducing effect on the kidney malondialdehyde content of the mice with type ii diabetes, and the kidney malondialdehyde content of the mice in the M11 group is significantly lower than that of the BJLY group.
The Lactobacillus kefiri M11 bacterial preparation has the most obvious effect of relieving the II-type diabetes.
Influence of Lactobacillus kefiri preparation on intestinal flora of type II diabetic mice
1. Sample pretreatment
Weighing 200mg of fresh excrement of each experimental group of mice (a normal group, a T2B2 group as a blank control group, an M11 group II type diabetes mellitus mice gavage Lactobacillus kefiri M11 strain preparation and a BJLY group II type diabetes mellitus mice gavage Bulgaria strain powder) in the step one, respectively putting the fresh excrement into a sterilized 2mL centrifuge tube, adding 1mL 70% ethanol, shaking and mixing uniformly, centrifuging at 10000r/min for 3min at room temperature, and discarding the upper layer liquid. Adding PBS solution, shaking and mixing, centrifuging at 10000r/min room temperature for 3min, discarding the upper layer liquid, and inverting 2mL tube on absorbent paper for 1min until no liquid flows out. And (3) putting the sample tube into an oven with the temperature of 55 ℃ for 10min to completely volatilize the residual alcohol and ensure the subsequent experimental operation.
2. Differential analysis of intestinal flora
The feces after the treatment are classified and sequenced in the V3-V4 region of the intestinal flora 16S rDNA by Shanghai Biotech engineering company, the diversity and abundance of the intestinal flora of each group of mice are analyzed at different biological taxonomic levels, and the SPSS 17.0 software is used for correlation analysis.
The effect of the Lactobacillus kefiri M11 bacterial preparation on the diversity of the mouse intestinal flora is shown in Table 3. The sequence number of each group of samples exceeds 35000, and the coverage rate reaches 99%, which indicates that 99% of intestinal flora of mice are sequenced. Compared with the normal group, the diversity of intestinal flora of mice in the T2B2 group is reduced by 10.8%, and compared with the T2B2 group, the diversity of intestinal flora of mice in the M11 and B groups is increased by 6.30% and 2.91%. As can be seen from Table 3, the diversity of intestinal flora (normal group) was reduced in type II diabetic mice; the M11 group and the BJLY group have the capability of restoring the intestinal flora of type II diabetic mice to a normal level, the alpha index of the intestinal flora of the M11 group is similar and is closer to that of the normal group, and the result shows that the Lactobacillus kefiri M11 bacterial preparation has the best regulation effect on the intestinal flora diversity of the type II diabetic mice.
TABLE 3 intestinal flora alpha index
Lefse (lda Effect size) analysis, which can be used for comparison between two or more groups to find species with significant differences between groups. Analysis of T2B2 by lefse (lda Effect size) revealed a change in intestinal flora compared to the normal group, in which harmful bacteria such as prevotella (Prevotellaceae) and Alistipes of the class Clostridia (clostridium) were increased. The normal group functions in bacteroidetes (Bacteroides); the T2B2 group plays an important role in the firmicutes intermediate Clostridia (clostridium); group M11 plays an important role in Lactobacillus (Lactobacillus); the BJLY group plays an important role in Proteobacteria. The result shows that the Lactobacillus kefiri M11 bacterial preparation can increase beneficial bacteria in intestinal flora of type II diabetic mice.
Variation in the levels of the genera for each treatment group: high throughput sequencing found that normal mouse intestinal flora consisted of Barnesiella, Bacteroides, Alloprovellula, Helicobacter, Alisipes, Clostridium XlVa, Parabacteroides, Escherichia/Shigella, Coprobacter, Lactobacillus, Holdemanella, and Odoribacter. Wherein Barnesiella, Bacteroides and Alloprevella are dominant genera in the intestinal flora.
Differences in levels for each treatment group: the relative abundance of Barnesiella in intestinal flora of mice in the normal group, the T2B2 group, the M11 group and the BJLY group is 14692, 10883.75, 11328.5 and 15906.5 respectively, compared with the normal group, the Barnesiella in the T2B2 group is reduced by 46.15 percent, and compared with the T2B2 group, the M11 and the BJLY group are increased by 4.09 percent and 46.15 percent respectively; relative abundance of Bacteroides in intestinal flora of mice is 20781.25, 3687.5, 2975, 1203.75, 3062.5 and 13631 respectively, compared with a normal group, Bacteroides in a T2B2 group is reduced by 463.56%, compared with a T2B2 group, BJLY group is increased by 269.65%, and M11 is reduced by 19.32%; the relative abundance of Lactobacillus in intestinal flora of mice in each group is 595.5, 260.25, 1287.75, 819.5, 269.5 and 674.5, compared with the normal group, the relative abundance of Lactobacillus in the T2B2 group is reduced by 128.82%, and compared with the T2B2 group, the relative abundance of Lactobacillus in the M11 and BJLY groups is increased by 214.89% and 159.17%, respectively. Relative abundance of Alloprevotella in intestinal flora of mice was 4746, 6913.75, 5345, 4203.75, 4764.25, 11267.75, respectively, and Alloprevotella in T2B2 group was increased by 31.35% compared to normal group, BJLY group was increased by 62.98% compared to T2B2 group, and M11 was decreased by 39.20%. Analyzing the influence of the intestinal flora of the mice in the normal group, the T2B2 group, the M11 group and the BJLY group, wherein the analysis result is shown in figure 11, as can be seen from figure 11, the abundance of Barnesiella and bacteroides in the intestinal flora of the mice with type II diabetes is reduced, and the abundance of Barnesiella and bacteroides in the intestinal flora of the mice with type II diabetes is recovered to the normal level by Lactobacillus kefir M11; the relative abundance of the T2B2 group was reduced, and the Lactobacillus kefiri M11 bacterial preparation increased the most in type II diabetic mice. The relative abundance of Lactobacillus in group M11 was consistent with the results of surface hydrophobicity and self-coagulation measurements and gastrointestinal fluid resistance, and the Lactobacillus kefiri M11 of the present invention showed good adhesion, thus demonstrating that the Lactobacillus kefiri M11 bacterial preparation could adhere to the intestinal tract. The abundance of Alloprevotella in the intestinal flora of the normal group is increased, and the preparation of the Lactobacillus kefiri M11 bacterium can restore the abundance of Alloprevotella in the intestinal flora of the type II diabetic mice to the normal level; therefore, the Lactobacillus kefiri M11 bacterial preparation can increase beneficial bacteria and reduce harmful bacteria in intestinal tracts of type II diabetic mice.
Claims (6)
1. Lactobacillus kefir (L.) KuntzeLactobacillus kefiri) M11, deposited in China center for type culture Collection with a collection number of CCTCC NO: m2019080.
2. Lactobacillus kefir (L.kefir)Lactobacillus kefiri) M11 bacterial preparation, characterized by the bacterium Lactobacillus kefir (L.), (Lactobacillus kefiri) M11 preservation number is CCTCC NO: m2019080, the bacterial preparation is obtained by the following method: lactobacillus kefir (L.kefir) ((R))Lactobacillus kefiri ) M11 is cultured in MRS culture medium to logarithmic phase, centrifuged, supernatant is removed, and skimmed milk, sucrose and sodium alginate are added into thallus and mixedMixing, and lyophilizingLactobacillus kefiriM11 bacterial preparation; wherein, Lactobacillus kefir: (Lactobacillus kefiri) The weight ratio of the M11 bacteria to the skim milk to the sucrose to the sodium alginate is (100: 6) - (7: 1) - (2): 1 to 2.
3. Lactobacillus kefir according to claim 2 (L.), (Lactobacillus kefiri) M11 bacterial preparation, characterized by the bacterium Lactobacillus kefir (L.), (Lactobacillus kefiri) The culture temperature of M11 was 37 ℃.
4. Lactobacillus kefir (L.) according to claim 2 or 3, (B)Lactobacillus kefiri) The M11 bacterial preparation is characterized in that the freeze-drying treatment method comprises the following steps: freezing the mixture of the thalli, skim milk, cane sugar and sodium alginate for 1-5 h at-20 ℃, and then carrying out vacuum freeze drying at a cold trap temperature of less than-40 ℃ and a vacuum degree of more than 200mtorr to complete freeze drying treatment.
5. Lactobacillus kefir according to claim 4 (L.), (Lactobacillus kefiri) The M11 bacterial preparation is characterized in that the freeze-drying treatment conditions are as follows: freezing the mixture of the thalli, the skim milk, the sucrose and the sodium alginate for 1-5 h at-20 ℃, and then carrying out vacuum freeze drying under the conditions that the temperature of a cold trap is less than-40 ℃ and the vacuum degree is more than 200mtorr, thus completing freeze-drying treatment.
6. Lactobacillus kefir (L.) according to claim 2, 3 or 5, (b) aLactobacillus kefiri) The M11 bacterial preparation is characterized in that the weight ratio of the bacterial to the skim milk, the sucrose and the sodium alginate is 100:6.67: 1.67: 1.67.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001321163A (en) * | 2000-05-17 | 2001-11-20 | Bioneer Corp | Microorganism for treatment of obesity or diabetes mellitus, and medicinal composition containing the same |
| WO2004076615A2 (en) * | 2003-02-27 | 2004-09-10 | Bioneer A/S | Immunomodulating probiotic compounds |
| WO2017137920A2 (en) * | 2016-02-12 | 2017-08-17 | Probiotical S.P.A. | Use of bacterial strains belonging to the species lactobacillus kefiri in paediatrics to generate and/or mantai n a state of homeostasis |
| CN107927168A (en) * | 2017-12-17 | 2018-04-20 | 石家庄君乐宝乳业有限公司 | A kind of acidified milk containing Kefir grains lactobacillus and preparation method thereof |
| CN108883140A (en) * | 2016-01-14 | 2018-11-23 | 英特瑞克斯顿阿克图比奥帝克斯有限公司 | The composition and method for treating type 1 diabetes |
| CN109310719A (en) * | 2016-06-08 | 2019-02-05 | 索发股份公司 | The new medical application of probiotics |
| CN110184214A (en) * | 2019-05-21 | 2019-08-30 | 黑龙江大学 | A kind of Kefir grains lactobacillus and its bacteria preparation |
-
2019
- 2019-05-21 CN CN201910422107.8A patent/CN110184214B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001321163A (en) * | 2000-05-17 | 2001-11-20 | Bioneer Corp | Microorganism for treatment of obesity or diabetes mellitus, and medicinal composition containing the same |
| WO2004076615A2 (en) * | 2003-02-27 | 2004-09-10 | Bioneer A/S | Immunomodulating probiotic compounds |
| CN108883140A (en) * | 2016-01-14 | 2018-11-23 | 英特瑞克斯顿阿克图比奥帝克斯有限公司 | The composition and method for treating type 1 diabetes |
| WO2017137920A2 (en) * | 2016-02-12 | 2017-08-17 | Probiotical S.P.A. | Use of bacterial strains belonging to the species lactobacillus kefiri in paediatrics to generate and/or mantai n a state of homeostasis |
| CN108779433A (en) * | 2016-02-12 | 2018-11-09 | 波比奥泰克股份公司 | Belong to application of the bacterium bacterial strain of Kefir grains lactobacillus species in the paediatrics for generating and/or maintaining homeostasis state |
| CN109310719A (en) * | 2016-06-08 | 2019-02-05 | 索发股份公司 | The new medical application of probiotics |
| CN107927168A (en) * | 2017-12-17 | 2018-04-20 | 石家庄君乐宝乳业有限公司 | A kind of acidified milk containing Kefir grains lactobacillus and preparation method thereof |
| CN110184214A (en) * | 2019-05-21 | 2019-08-30 | 黑龙江大学 | A kind of Kefir grains lactobacillus and its bacteria preparation |
Non-Patent Citations (5)
| Title |
|---|
| Selecting probiotics with the abilities of enhancing GLP-1 to mitigate the progression of type 1 diabetes in vitro and in vivo;Wei, Shih-Hung等;《JOURNAL OF FUNCTIONAL FOODS》;20151031;第18卷;473-486 * |
| 开菲尔中半乳糖霉菌的分离鉴定及其与开菲尔乳杆菌相互作用研究;汤华成;《中国博士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20180115(第1期);B024-11 * |
| 开菲尔乳杆菌KL22的鉴定及其益生潜力评价;赵彤;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20190215(第2期);B024-162 * |
| 开菲尔菌粒及其发酵乳的研究进展;姜志辉等;《肠外与肠内营养》;20180619(第4期);65-69 * |
| 藏灵菇发酵乳生产工艺优化及其功能特性;张飞飞等;《乳业科学与技术》;20200301(第2期);18-23 * |
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