CN114752529B - Lactobacillus plantarum HOM3201 strain and viable bacteria preparation, preparation method and application thereof - Google Patents
Lactobacillus plantarum HOM3201 strain and viable bacteria preparation, preparation method and application thereof Download PDFInfo
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- CN114752529B CN114752529B CN202210472568.8A CN202210472568A CN114752529B CN 114752529 B CN114752529 B CN 114752529B CN 202210472568 A CN202210472568 A CN 202210472568A CN 114752529 B CN114752529 B CN 114752529B
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- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum HOM3201 strain, a viable bacteria preparation containing the same, and a preparation method and application thereof. The preservation number of the lactobacillus plantarum HOM3201 strain is CGMCC No.22700. The lactobacillus plantarum HOM3201 strain can obviously reduce the fasting blood glucose level of rats with insulin resistance glucose/lipid metabolic disorder models, increase the tolerance of organisms to glucose and reduce the insulin resistance index.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum (Lactobacillus plantarum) HOM3201 strain, a viable bacteria preparation containing the same, and a preparation method and application of the viable bacteria preparation.
Background
The World Health Organization (WHO) defines probiotics as active microorganisms that are beneficial to the host when consumed in sufficient quantities. Only strains that have been found to be beneficial to health through scientific research can be referred to as "probiotics". The core characteristics of probiotics are a sufficient number, a viable bacterial status and a beneficial health function. Lactobacillus plantarum (Lactobacillus plantarum) is a ubiquitous probiotic and is often separated from fermented foods such as pickled vegetables and pickle and human bodies. Lactobacillus plantarum has beneficial effects in improving intestinal barrier, diabetes, hypertension, obesity, anti-infection, mental diseases, etc.
The incidence of type 2 diabetes is rising year by year, with china in the leading part of the world. The etiology and pathogenesis of type 2 diabetes mellitus are extremely complex, and the defects of insulin resistance and insulin secretion of peripheral tissues mainly caused by genetic and environmental factors are considered to cause relative or absolute deficiency of insulin of organisms, so that the utilization of glucose is reduced, and hyperglycemia is caused to cause diabetes mellitus.
Current studies on probiotics to regulate blood glucose levels are incomplete and systematic.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a lactobacillus plantarum (Lactobacillus plantarum) HOM3201 strain, a live bacteria preparation containing the same, a preparation method and application.
In order to achieve the above object, the present invention provides the following technical solutions:
in one aspect, the invention provides a lactobacillus plantarum (Lactobacillus plantarum) HOM3201 strain, wherein the preservation number of the lactobacillus plantarum HOM3201 strain is CGMCC No.22700.
Preferably, the lactobacillus plantarum HOM3201 strain comprises the 16srDNA sequence represented by SEQ ID No. 1.
In another aspect, the invention provides a live bacterial formulation comprising a lactobacillus plantarum HOM3201 strain according to the description above.
Preferably, the live bacterial formulation comprises up to 4.0-8.0X10 11 CFU/g viable bacteria.
Preferably, the live bacteria preparation further comprises an auxiliary material.
In yet another aspect, the invention provides a food or nutraceutical comprising a viable bacterial formulation as described above.
In yet another aspect, the invention provides the use of lactobacillus plantarum HOM3201 strain as described above for the manufacture of a medicament for assisting in lowering blood glucose.
The lactobacillus plantarum HOM3201 strain is used for reducing the fasting blood glucose level of a rat with an insulin resistance glucose metabolism disorder/lipid metabolism disorder model, increasing the tolerance of an organism to glucose and reducing the insulin resistance index.
Preferably, the lactobacillus plantarum HOM3201 strain is used for the treatment of diabetes.
In yet another aspect, the present invention provides a method of preparing a viable bacterial preparation as described above, comprising the steps of: expanding the lactobacillus plantarum HOM3201 strain of claim 1 in an optimized liquid medium; collecting thalli; adding a protective agent for resuspension, vacuum freeze-drying, and crushing to obtain the active microbial inoculum.
The invention has the following advantages:
(1) The lactobacillus plantarum HOM3201 disclosed by the invention can obviously reduce the fasting blood glucose level of a rat with an insulin resistance glucose/lipid metabolic disorder model, increase the tolerance of an organism to glucose and reduce the insulin resistance index.
(2) The lactobacillus plantarum provided by the invention has an excellent in-vitro probiotics function. The strain has excellent resistance to artificial gastrointestinal fluids, bacteriostasis, antioxidation activity and the like.
(3) The active microbial inoculum has the advantages of simple production process parameters, short period, high viable count, good stability and long-term efficacy.
Drawings
FIG. 1 shows a graph of RAPD cluster analysis of Lactobacillus plantarum HOM3201 strain constructed based on UPGMA method.
FIG. 2 shows the inhibition of DPP-4 and alpha-glucosidase by Lactobacillus plantarum HOM3201 strain of the invention.
FIG. 3 shows the effect of Lactobacillus plantarum HOM3201 strain of the invention on glucose tolerance in rats, model of insulin-resistant glucose/lipid metabolism disorder.
Fig. 4 shows a process flow diagram of the present invention.
Description of the preservation of microorganisms
The lactobacillus plantarum (Lactobacillus plantarum) HOM3201 strain of the invention is preserved in China general microbiological culture Collection center (CGMCC) for culture Collection of microorganisms at 2021, 6 and 11 days, and the preservation address is as follows: the national institute of microbiology, national institute of sciences, 1 st, g.d.3, north Chen, west, chat, of the Chao, of Beijing, city; the preservation number is CGMCC No.22700.
Detailed Description
The invention discloses strains, characteristics and application, and one skilled in the art can refer to the content of the invention to properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Dipeptidyl peptidase 4 inhibitors (i.e., DPP-4 inhibitors) are a novel class of drugs for the treatment of type 2 diabetes, such as sitagliptin, vildagliptin, and the like. DPP-4 inhibitors can inhibit the activity of DPP-4, thereby stimulating the secretion of incretins [ glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) ]. GLP-1 and GIP can inhibit glucagon release, increase insulin secretion, reduce gastric emptying, and lower blood glucose levels.
The alpha-glucosidase inhibitor is an oral hypoglycemic agent for type 2 diabetes mellitus, and is mainly acarbose and voglibose which are commonly used. The mechanism of reducing blood glucose of the alpha-glucosidase inhibitor is that the speed of decomposing starch into glucose is slowed down by inhibiting alpha-glucosidase on intestinal mucosa, and the absorption of glucose by small intestine is reduced and delayed, so that the blood glucose is reduced, and the effect on postprandial hyperglycemia is obvious. Alpha-glucosidase inhibitors do not stimulate insulin secretion, and such drugs alone typically do not induce hypoglycemia, and thus can help reduce fluctuations in blood glucose.
The lactobacillus plantarum HOM3201 disclosed by the invention can obviously reduce the fasting blood glucose level of a rat with an insulin resistance glucose/lipid metabolic disorder model, increase the tolerance of an organism to glucose and reduce the insulin resistance index. The hypoglycemic mechanism is that lactobacillus plantarum HOM3201 is grown and metabolized to produce DPP-4 inhibitor and alpha-glucosidase inhibitor. DPP-4 inhibitors stimulate the body to produce glucagon-like peptide-1 (GLP-1) by inhibiting DPP-4, and promote insulin secretion; the alpha-glucosidase inhibitor can slow down the rate of converting starch into glucose by inhibiting alpha-glucosidase, thereby achieving the purpose of reducing blood glucose.
In order to make the technical problems to be solved, the technical scheme adopted and the advantages of the invention more clear, the invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The experimental methods used in the present invention are all conventional methods unless otherwise specified.
The reagents and materials used in the present invention, unless otherwise specified, are formulated conventionally or commercially available.
Kimchi used in the present invention:
the kimchi sample used in the present invention for separating lactobacillus plantarum HOM3201 strain was from home-made hand kimchi in all households of the province of Sichuan. The manufacturing method comprises the following steps: cleaning vegetables (green turnip, white turnip, carrot, cabbage, cowpea, green pepper, etc.), cutting into strips, and air drying; cleaning pickle jar, air drying, and pouring a little high-degree white spirit for disinfection; pouring clear water into an oilless pot, adding cortex Cinnamomi Japonici, herba Pelargonii Graveolentis, fructus Zanthoxyli, fructus Anisi Stellati, crystal sugar, and rhizoma Zingiberis recens, boiling, thoroughly cooling, pouring into a pickle jar, and pouring Zanthoxylum piperitum juice, jowar wine and vegetables. After the cover is buckled, clean water is poured into the water tank to seal the jar. Fermenting in a cool and ventilated place for 10 days to obtain the pickle used in the invention.
The commercial strain of Lactobacillus plantarum used in the present invention was isolated from a commercial product containing the desired strain, as described in example 1.
EXAMPLE 1 isolation and identification of Lactobacillus plantarum HOM3201 Strain
(1) Lactobacillus plantarum strain screening culture medium formula
Improved MRS solid medium:
MRS medium (OXOID, CM 1163) was added with 0.05g bromocresol green (Shanghai Biotechnology) and double distilled water 1L, and the mixture was stirred well. Adjusting pH to 5.5, sterilizing at 121deg.C for 20 min.
(2) Isolation and screening of Lactobacillus plantarum Strain
Isolation and screening of HOM Lactobacillus plantarum Strain
1g of kimchi was weighed and 9mL of 0.9% physiological saline solution was used to prepare kimchi juice. Sucking 1mL of pickle juice, diluting the sample by using a 10-fold dilution method, wherein the dilution is 10 -3 -10 -5 . The diluted kimchi juice was coated on the above modified MRS plate and anaerobically cultured at 35℃for 72 hours. Single colonies with wet and smooth surfaces, neat edges, milky yellow or milky white colonies and yellow circumferences are selected, streaked, cultured and purified. And simultaneously gram staining and microscopic examination are carried out to observe colony morphology. Transferring single colony into MRS liquid culture medium for pure culture, and culturing with glycerol to obtain 5 lactobacillus plantarum strains, which are named HOM3201, HOM3202, HOM3203, HOM3204, and HOM3205.
b. Isolation and screening of commercial strains of Lactobacillus plantarum
The lactobacillus plantarum commercial strain used in the invention is separated from commercial probiotics products, and the strain and product information are as follows: lactobacillus plantarum Lp-115 (isolated from Yineng 300), lactobacillus plantarum LP-ONLLY (isolated from Ondelia probiotic powder), lactobacillus plantarum P-8 (isolated from Yitanou), lactobacillus plantarum CCFM8661 (isolated from Yitanou and Mei), and Lactobacillus plantarum ST-III (isolated from Guangming Leun fermented milk).
Isolation method of commercial strains of lactobacillus plantarum: 1g of a commercial strain powder or fermented milk containing Lactobacillus plantarum was weighed and resuspended in 9mL of 0.9% physiological saline. 1mL of the sample is sucked, the sample is diluted by a 10-fold dilution method, 2-3 proper gradient dilutions are selected to be coated on a modified MRS plate, and the sample is cultured for 72 hours at 35 ℃. Single colonies with wet and smooth surfaces, neat edges, milky yellow or milky white colonies and yellow circumferences are selected, streaked, cultured and purified. And simultaneously gram staining and microscopic examination are carried out to observe colony morphology. Transferring single colony into MRS liquid culture medium for pure culture, and preserving glycerol.
(3) Identification of HOM Lactobacillus plantarum Strain
The strains are inoculated into MRS liquid culture medium, cultured for 48 hours at 35 ℃, bacterial total DNA is extracted from each bacterial liquid, 16S rDNA amplification is carried out, PCR amplification and agarose gel electrophoresis are carried out by utilizing universal primers 27F and 1492R, and the sequencing (Shanghai) is carried out after gel cutting recovery. Then, the above 5 strains HOM3201, HOM3202, HOM3203, HOM3204 and HOM3205 were identified as Lactobacillus plantarum by BLAST tool in NCBI database (Lactobacillus plantarum). Wherein the 16S rDNA of HOM3201 is shown as SEQ ID NO. 1.
The sequence of the 16S rDNA of HOM3201 was determined as follows:
CATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCAGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTA
(4) Molecular biological characterization of Lactobacillus plantarum strains
The resulting Lactobacillus plantarum HOM3201 strain was subjected to genotyping comparison studies with Lactobacillus plantarum HOM3202, HOM3203, HOM3204 and HOM3205 strains, and with the commercial strains Lactobacillus plantarum Lp-115, LP-ONLLY, P-8, CCFM8661 and ST-III strains, using the random amplified polymorphic DNA marker (RAPD) method to determine the specificity of the resulting strains.
The genomic DNA of the strain was randomly amplified by selecting primers OPA-02, OPA-18, OPL-07, OPL-16 and OPM-05 as shown in Table 1 below. The amplification conditions were as follows: the template and primers were first kept at 95℃for 5min, then cooled to 56℃and the reaction mixture was added to amplify 45 cycles as follows: denaturation at 94℃for 1min, annealing at 30℃for 1min and extension at 72℃for 2min.
TABLE 1
Primer(s) | Sequence(s) | SEQ ID NO |
OPA-02 | 5’-TGCCGAGCTG-3’ | 2 |
OPA-18 | 5’-AGGTGACCGT-3’ | 3 |
OPL-07 | 5’-AGGCGGGAAC-3’ | 4 |
OPL-16 | 5’-AGGTTGCAGG-3’ | 5 |
OPM-05 | 5’-GGGAACGTGT-3’ | 6 |
27F | 5’-AGTTTGATCMTGGCTCAG-3’ | 7 |
1492R | 5’-GGTTACCTTGTTACGACTT-3’ | 8 |
mu.L of the PCR amplification product was detected by 2% agarose gel electrophoresis, followed by imaging in a gel imager. The RAPD patterns were clustered using Bionumerics version 6.6.6 software based on UPGMA method. The results are shown in FIG. 1.
It is generally considered that strains having a similarity of more than 90% in phylogenetic tree have the possibility of homozygotic strains. The phylogenetic tree results of this experiment show that the HOM3201 strain is not on one branch with other lactobacillus plantarum, the similarity is lower than 60%, and the significant difference exists. Thus, HOM3201 strain has genotype specificity and uniqueness compared to HOM3202, HOM3203, HOM3204 and HOM3205 strains, and commercial strains Lp-115, LP-ONLLY, P-8, CCFM8661 and ST-III strains.
Example 2 gastrointestinal trafficability test
(1) Isolation and activation of strains
Three commercial strains of lactobacillus plantarum 299V (isolated from the Jarrow probiotic product by the same method as in example 1 b. lactobacillus plantarum commercial strain isolation method), and the strains lactobacillus plantarum Lp-115 and lactobacillus plantarum ST-III isolated from the commercial probiotic product of example 1 were selected as positive controls in this experiment. Each strain was inoculated into MRS liquid medium, cultured at 37℃for 24 hours, and activated twice for use.
(2) Preparation of artificial gastric juice
16.4mL of dilute hydrochloric acid and 10g of pepsin are taken, about 800mL of water is added, the pH is regulated to 3.0 after shaking, water is added to a constant volume of 1L, and the mixture is filtered by a microporous filter membrane with the thickness of 0.22 mu m for later use.
(3) Preparation of artificial intestinal juice
Taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve the monopotassium phosphate, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution; and dissolving 10g of pancreatin in a proper amount of water, mixing the two solutions, diluting to 1000mL with water, and filtering with a 0.22 μm sterile filter membrane under a sterile environment for later use.
(4) Evaluation of viability of Strain in simulated Artificial gastrointestinal fluids
Taking 1mL of bacterial liquid of each strain, centrifuging, collecting bacterial bodies, adding the bacterial liquid into 10mL of artificial gastric juice, uniformly mixing, immediately counting the number of viable bacteria, and marking as T 0 Culturing in a 37 deg.C incubator for 3 hr, counting viable bacteria, and counting as T 1 . Centrifuging the sample, adding 10mL of artificial intestinal juice, mixing, culturing in a 37 ℃ incubator for 3h to count viable bacteria, and recording as T 2 . The survival rate was calculated using the following formula:
wherein T is 0 The viable count CFU/mL of the test strain is 0h before the untreated; t (T) 1 The viable count of the tested strain after being treated by artificial gastric juice for 3 hours; t (T) 2 Is the viable count of the tested strain after 3h of treatment by artificial gastric juice and artificial intestinal juice.
TABLE 2 survival of Lactobacillus plantarum in simulated artificial gastrointestinal fluids
* : significant differences compared to HOM3201 group (p < 0.05)
* *: significant differences compared to HOM3201 group (p < 0.01)
As can be seen from table 2: after 3 hours of simulated artificial gastric juice treatment, the survival rate of the lactobacillus plantarum HOM3201 strain can reach 118.87%, and after the strain is continuously treated by 3 hours of simulated artificial intestinal juice treatment, the survival rate of the lactobacillus plantarum HOM3201 strain can still reach 122.59%, which indicates that the lactobacillus plantarum HOM3201 strain has higher survival rate in the gastrointestinal tract.
Lactobacillus plantarum HOM3201 is more resistant to gastric juice than the commercial lactobacillus plantarum 299V, LP-115, ST-III strains, and has a significant or very significant difference compared to the commercial lactobacillus plantarum 299V, LP-115, ST-III strains.
Example 3 test for the ability to inhibit common pathogenic bacteria
(1) Pathogenic bacteria activation
Five pathogenic bacteria are selected in the test: coli (e.coli) ATCC8739, salmonella typhimurium (Salmonella typhimurium) ATCC14028, staphylococcus aureus (Staphylococcus aureus) ATCC6538, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC9027, listeria monocytogenes (Listeria monocytogenes) ATCC19111. These pathogenic strains were purchased from ATCC (american type culture collection ).
Pathogenic strains were inoculated into nutrient agar medium, respectively, and cultured at 37℃with shaking at 200rpm for 12 hours. The indicator bacteria were conditioned with fresh medium to od=0.1 for use.
(2) Lactic acid bacteria strain activation
In this experiment, commercial strains Lactobacillus plantarum 299V (isolation method is the same as examples 1 and 2), lp-115 (isolation method is the same as example 1), ST-III strain (isolation method is the same as example 1) and Lactobacillus rhamnosus (Lactobacillus rhamnosus) LGG strain was selected as control strain, wherein LGG strain was isolated from Kang Cui Le powder product, isolation method is the same as example 1.
And (3) inoculating the strains into an MRS liquid culture medium respectively, standing and culturing for 24 hours at 37 ℃, and activating twice to obtain strain fermentation liquor. Centrifugation at 11000rpm for 10min, taking supernatant for antibacterial test, and taking MRS liquid culture medium as negative control. Annotation: lactic acid bacteria are a general generic term comprising lactobacillus and bifidobacterium.
(3) Flat plate preparation
The sterilized nutrient agar is cultured and poured into a culture dish, 100 mu L of indicator bacteria liquid is added into a culture medium, and after uniform mixing, standing and solidification are carried out.
(4) Bacteriostasis experiment
The oxford cup was gently placed on the plate with sterile forceps and the holes were kept a distance apart. 150. Mu.L of fermentation supernatant was added to each well, and after diffusion in a refrigerator at 4℃for 12 hours, the mixture was cultured in an incubator at 37℃for 18 hours, and the diameter of the zone of inhibition was observed and measured.
TABLE 3 inhibitory Effect of Lactobacillus plantarum HOM3201 Strain on pathogenic bacteria
Note that: "-" has no antibacterial activity, < 11mm; the "+" is 11mm or less and the bacteriostasis area is 16mm or less; the "++" is 16mm or less and the bacteriostasis area is less than 23mm; "+". ++'s more than or equal to 23mm
As shown in Table 3, lactobacillus plantarum HOM3201 has inhibitory effect on all 5 pathogenic bacteria. Compared with commercial strains of lactobacillus plantarum 299V, lp-115, ST-III strain and lactobacillus rhamnosus LGG strain, the antibacterial capability is equivalent.
EXAMPLE 4 antibiotic susceptibility test
Drug susceptibility testing the K-B agar method recommended by the American clinical standards Committee (NCCLS) was followed as follows:
(1) As a control strain, a commercial strain Lactobacillus plantarum 299V, lp-115, ST-III strain (same as in example 3) was selected. The strains were inoculated into MRS liquid medium, cultured at 37℃for 24 hours, and activated continuously for 3 generations.
(2) 1mL of the bacterial liquid (1.5X10) 8 CFU/mL), 15mL of MRS solid culture medium are placed in a sterilization culture dish, uniformly mixed, stuck with standard antibiotic drug sensitive paper sheets after the flat plate is solidified, cultured for 24-48 hours at 37 ℃, and then the diameter of a bacteriostasis ring is measured and recorded. The results are shown in Table 4, interpreted according to the CLSI decision criteria.
TABLE 4 determination of the sensitivity of lactic acid bacterial strains to 9 antibiotics
S (susceptible) is sensitive; i (intermediate), medium; r (resistance) represents drug resistance.
As can be seen from Table 4, HOM3201 strain exhibited resistance to vancomycin, gentamicin, streptomycin, kanamycin, clindamycin, and was sensitive to chloramphenicol, tetracycline, ampicillin, and erythromycin. In contrast to the commercial strain Lactobacillus plantarum 299V, lp-115, ST-III strain, HOM3201 did not find abnormal resistance.
Example 5 antioxidant Activity assay
(1) Lactobacillus plantarum Strain activation
Commercial strains Lactobacillus plantarum Lp-115, ST-III strain (isolation procedure as in example 1) were selected as positive controls. Inoculating each strain into MRS culture medium, standing at 37deg.C for 24 hr, activating for 3 generations, centrifuging, and adjusting strain concentration to 3×10 10 CFU/mL for use.
(2) Antioxidant Activity assay
The indicators include total antioxidant capacity assay (T-AOC), hydroxyl radical scavenging assay (OH), DPPH radical scavenging assay. The first two indexes are measured according to the operation instruction by using a kit purchased by Nanjing built company. The DPPH free radical scavenging test adopts a colorimetric method, and the principle is that the free radical scavenger provides a lone pair electron pairing of an electron and DPPH free radical, so that the self purple color is changed into yellow, the absorbance at the wavelength of 517nm is reduced, the variation degree and the free radical scavenging degree are in a linear relation, namely, the stronger the scavenging capability of the free radical scavenger is, the smaller the absorbance is. The results are shown in Table 5.
TABLE 5 measurement of in vitro antioxidant Activity of Lactobacillus plantarum Strain
* : significant differences compared to HOM3201 group (p < 0.05)
* *: significant differences compared to HOM3201 group (p < 0.01)
As is clear from Table 5, lactobacillus plantarum HOM3201 strain showed remarkable effects in terms of total antioxidant capacity, hydroxyl radical scavenging and DPPH radical scavenging. In terms of T-AOC, HOM3201 strain performed excellently and had very significant differences compared to Lp-115 strain; no obvious difference exists among the three groups in terms of hydroxyl radical scavenging; in DPPH radical scavenging, HOM3201 strain scavenging rate is higher compared with Lp-115 strain, and has significant difference; there was no significant difference compared to the ST-III strain.
EXAMPLE 6 preparation Process of active bacterial powder of Lactobacillus plantarum HOM3201 Strain
(1) Culture of strains
Lactobacillus plantarum HOM3201 strain is inoculated in MRS liquid culture medium, cultured at 37 ℃ for 24 hours, and activated for 2 generations. Inoculating into fermentation medium (shown in Table 6), culturing at 37deg.C, and culturing at viable count of 2×10 10 CFU/mL or more.
TABLE 6 fermentation Medium formulation
(2) Preparation of freeze-drying protective agent
The protective agent containing 100g/L skimmed milk powder, 50g/L trehalose, 3g/L vitamin C and 5g/L L-sodium glutamate is prepared by mixing sterile water with protective agent raw materials.
(3) Freeze drying
Centrifuging fermentation broth of Lactobacillus plantarum HOM3201 strain to collect bacterial mud, washing bacterial mud with 0.9% sterile physiological saline, mixing bacterial mud with the protective agent to make bacterial liquid concentration reach 10 10 Freeze drying the above CFU/mL in a freeze dryer, and pulverizing the bacterial cake with a fine grinder to obtain the freeze-dried bacterial powder with viable count higher than 6.0X10 11 CFU/g。
Example 7 in vitro hypoglycemic efficacy and mechanism exploration of Lactobacillus plantarum HOM3201 Strain
(1) MRS liquid culture medium with improved configuration
MRS culture medium (OXOID), adding 0.05% L-cysteine hydrochloride based on the final culture medium, stirring, and sterilizing at 121deg.C for 20 min.
(2) Strain activation and sample processing
Commercial strains Lactobacillus plantarum 299V, lp-115, ST-III (isolation procedure same as example 1), and Lactobacillus reuteri (Lactobacillus reuteri) ADR1 strain were selected as positive controls, and ADR-1 strain was isolated from a Glycometole product by the same method as example 1.
The strains are respectively inoculated into fresh modified MRS liquid culture medium with the inoculum size of 1 percent, and are subjected to standing anaerobic culture for 24 hours at 37 ℃ and activated for 3 generations. The bacterial solution was centrifuged at 8000rpm for 10min and washed three times with Phosphate Buffered Saline (PBS). The number of viable bacteria is regulated to 4 multiplied by 10 10 CFU/mL, anaerobic culture for 8h, centrifuging at 8000rpm for 10min, and collecting supernatant.
(3) DPP-4 inhibitory and alpha-glucosidase inhibitory screening
In vitro screening for hypoglycemic function was performed using DPP-4 inhibitor screening kit (Abnova#KA1311) and alpha-glucosidase inhibitor screening kit (Biovision#K938). The inhibition ratio of the lactobacillus samples to the above two enzymes was calculated (see fig. 2 for the results).
TABLE 7 strains of the respective strainsInhibition rate of DPP-4 and inhibition rate of alpha-glucosidase
* : significant differences compared to HOM3201 group (p < 0.05)
As can be seen from Table 7, the HOM3201 strain has high inhibition rate on DPP-4 and alpha-glucosidase. In the DPP-4 inhibition assay, the HOM3201 strain group was higher than the other groups, and the HOM3201 group had a significant difference (P < 0.05) compared with the ADR-1, 299V strain group. In the α -glucosidase inhibition assay, the HOM3201 strain group was lower than the ADR-1 group, but all were higher than the other groups. Clinical literature studies have shown that lactobacillus reuteri ADR-1 strain has hypoglycemic efficacy. Taken together, HOM3201 strain achieves the goal of lowering blood glucose levels by producing higher levels of DPP-4 inhibitor and alpha-glucosidase inhibitor (fig. 2).
Example 8 Lactobacillus plantarum HOM3201 Strain stimulates GLP-1 secretion by NCI-H716 cells
(1) Culture of NCI-H716 cells
The experiment selects NCI-H716 cells (purchased from the cell bank of the national academy of sciences) and NCI-H716 cells were grown in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Hyclone), 1% double antibody (penicillin and streptomycin, hyclone), 37℃and 5% CO 2 Cells were grown in suspension in the incubator of (C).
(2) Culture of lactic acid bacteria strains
Three commercial strains (lactobacillus reuteri ADR-1, lactobacillus rhamnosus LGG and bifidobacterium lactis (Bifidobacterium lactis) CECT 8145) were selected as positive controls. The bifidobacterium lactis CECT8145 strain was isolated from ADM corporation donor meal.
The separation method of bifidobacterium lactis CECT8145 comprises the following steps: 1g of the bacterial powder was weighed and resuspended in 9mL of 0.9% physiological saline. 1mL of the sample was aspirated, diluted by 10-fold dilution, and 2-3 suitable gradient dilutions were selected and plated on TOS (Merck, 1.00043.0500) plates for anaerobic incubation at 37℃for 72h. Selecting single milky colony with wet and smooth surface, regular edge, streaking culture and purification. And simultaneously gram staining and microscopic examination are carried out to observe colony morphology. Single colonies were transferred to mrs+0.5% cysteine hydrochloride (Sigma, 1161509) liquid medium for pure culture and glycerol stock.
Respectively inoculating lactobacillus plantarum HOM3201, lactobacillus reuteri ADR-1 and lactobacillus rhamnosus LGG into MRS liquid culture medium, standing at 37 ℃ for culturing for 24 hours, and activating for 3 generations; inoculating bifidobacterium lactis CECT8145 into MRS+0.5% cysteine hydrochloride liquid culture medium, standing at 37 ℃ for anaerobic culture for 24 hours, and activating for 3 generations; each broth was centrifuged at 8000rpm for 10min and washed three times with Krebs buffer (Sigma). The number of viable bacteria is regulated to be 1 multiplied by 10 10 CFU/mL, ready for use.
(3) GLP-1 endocrine experiments
NCI-H716 cells were grown at 1.5X10 6 Density of individual/well in matrigel coated 24 well plate (Corning), added to endocrine differentiation medium, 37 ℃,5% CO 2 Is cultured for 2 days, and an endocrine differentiation experiment is performed. Endocrine differentiation medium is high sugar DMEM (Gibco) medium containing 10% fetal bovine serum, 1% diabody.
After 2 days, the DMEM medium was replaced with Krebs-Ringer buffer containing 1X 10 each 10 After culturing the CFU/mL probiotic strain for 2 hours, centrifuging at 8000rpm for 10min, and collecting the supernatant. To the supernatant, 50. Mu.g/mL of phenylmethylsulfonyl fluoride (Roche) and 10. Mu.g/mL of sitagliptin (Sigma) were added, and then the concentration of GLP-1 was detected using ELISA kit (Raybiotech).
TABLE 8 stimulation of GLP-1 production by NCI-H716 cells by the strains
* : significant differences compared to HOM3201 group (P < 0.05)
* *: significant differences compared to HOM3201 group (P < 0.01)
Three commercial strains (lactobacillus reuteri ADR-1, lactobacillus rhamnosus LGG and bifidobacterium lactis CECT 8145) all have literature-demonstrated related studies with hypoglycemic activity.
As can be seen from Table 8, the HOM3201 strain stimulated NCI-H716 cells to secrete high levels of GLP-1 at concentrations of 1890.21 + -158.38 pg/mL. HOM3201 strain has very significant differences (p < 0.01) compared to lactobacillus reuteri ADR-1; in contrast to lactobacillus rhamnosus LGG and bifidobacterium lactis CECT8145, HOM3201 strain had significant differences (p < 0.05). The results indicate that lactobacillus plantarum HOM3201 strain has the effect of reducing blood sugar, and the sugar reducing mechanism is probably through stimulating intestinal canal L cells to produce high-content GLP-1.
Example 9 Effect of Lactobacillus plantarum HOM3201 Strain on insulin resistance by Tetraoxypyrimidine on rat blood glucose in a model of disorder of glucose/lipid metabolism
(1) Beijing Wanghua Fukang Biotechnology Co., ltd. [ license number: SCXK- (Beijing) 2019-0008] bred 140g-160g healthy SPF grade SD male rats, 46 experimental animals were bred in SPF grade animal room of health food function detection center of applied culture and application university of Beijing association university, and the use license number of the experimental animals: SYXK (Beijing) 2017-0038. Experiments were performed in two batches. Experiment a batch of 10 experiments were performed on the effects of fasting blood glucose in normal rats; experiment two 36 rats with the model of insulin resistance and glucose/lipid metabolism disorder induced by tetraoxypyrimidine were subjected to fasting glucose experiment; sugar tolerance experiments; serum triglyceride and total cholesterol experiments; serum insulin experiments. Wherein the formula of the high heat energy feed comprises: 52.6% of basic feed, 15% of sucrose, 15% of yolk powder, 10% of lard, 5% of casein, 1.2% of cholesterol, 0.2% of sodium cholate, 0.6% of calcium bicarbonate and 0.4% of stone powder.
(2) Normal rat fasting blood glucose effect experiment: the common feed is suitable for 3 days of feeding, fasted for 4 hours, blood is taken, after standing for 10 minutes, serum is separated by centrifugation for 10 minutes at 3000r/min, and the blood sugar value before glucose is given (namely 0 hour) and the blood sugar value after BW glucose is given by 2.5g/kg for 0.5 hour and 2 hours are measured by a biochemical analyzer to be used as the basic value of animals in the batch. Setting up a normal controlGroup (0 CFU/just) and HOM3201 group (5 x 10) 10 CFU/kg BW); each group of 5 rats was fasted for 4 hours after the end of the test, blood was collected from the inner canthus venous plexus, and the fasting blood glucose level was measured using a biochemical analyzer, and the body weight of the rats at the beginning and end was measured.
TABLE 9 influence of Lactobacillus plantarum HOM3201 Strain on normal rat body weight
As can be seen from table 9, the body weights of normal rats before and after the experiment were compared between the two groups, and the difference was not significant (P > 0.05).
TABLE 10 Effect of Lactobacillus plantarum HOM3201 Strain on fasting blood glucose in normal rats
As can be seen from table 10, there was no significant difference in blood glucose values between the two groups before and after the experiment (P > 0.05).
(3) Tetraoxypyrimidine-induced insulin resistant glucose/lipid metabolism disorder model rat fasting glucose assay: establishing a blank control group, a model group and a HOM3201 strain group, wherein each group comprises 12 mice, and the gastric lavage dosage of the HOM3201 strain group is 5 x 10 10 CFU/kg BW. The test substance was administered by the gavage method in an amount of 0.5mL per animal, and the blank control group and the model control group were administered with the same volume of physiological saline, and were gavaged once daily. After the basal feed is fed for one week, the model control group and the HOM3201 strain group are replaced by high-heat feed, and after the basal feed is fed for three weeks, the model control group and the HOM3201 strain group are fasted for 24 hours, and the tetraoxapyrimidine is fed by intraperitoneal injection for 105mg/kg BW, and the injection quantity is 1mL/100g body weight. The high heat feed was fed for 5 days after injection. At the end of the trial, rats of each group fasted4h, blood is taken, and fasting blood glucose, glucose tolerance, serum insulin, total cholesterol and triglyceride levels are detected.
TABLE 11 rat body weight measurement/>
As can be seen from table 11, there was no significant difference in body weight between the rats before and after the experiment in the model control group compared to the blank control group.
TABLE 12 blood sugar and glucose tolerance in rat models of glycolipid metabolism disorders
* *: has extremely significant difference compared with the blank control group (p < 0.01)
TABLE 13 blood lipid and insulin resistance index of rat glycolipid metabolism disorder model
* : compared with the blank control group, the difference is significant (p < 0.05)
As can be seen from tables 12 and 13, the model control group has very significant differences (P < 0.01) between the blood glucose values of 0h and 0.5h after glucose administration, and the blood glucose value of 0.5h of the model control group is not less than 10mmol/L, which indicates that the model sugar metabolism disorder is established. Comparing the model control group with the blank control group, wherein the serum total cholesterol rise has a significant difference (P < 0.05), and judging that the lipid metabolism disorder model is established; the model control group is compared with the blank control group, the insulin resistance index is obviously reduced (P is less than 0.05), and the comprehensive judgment of the insulin resistance glucose/lipid metabolism disorder model is successful.
TABLE 14 influence of HOM3201 Strain on model rat body weight
TABLE 15 influence of HOM3201 Strain on fasting blood glucose in model rats
* : compared with the model control group, the difference is significant (p < 0.05)
TABLE 16 influence of HOM3201 Strain on sugar tolerance in model rats
* : compared with the model control group, the difference is significant (p < 0.05)
As can be seen from tables 15 and 16, there was a significant difference in blood glucose levels between the two groups at 0h (p < 0.05), and there was a trend of decrease in blood glucose in HOM3201 group at 0.5h, but no significant difference (p > 0.05). At 2h, there was no significant difference between the two groups. This suggests that HOM3201 probiotics significantly reduced fasting blood glucose in the model rats, decreased postprandial blood glucose levels at 0.5h, and improved blood glucose tolerance (fig. 3).
TABLE 17 HOM3201 Strain versus model rat serum cholesterol and TriglyceridesInfluence of
As can be seen from table 17, there was no significant difference in serum cholesterol and triglycerides in HOM3201 strain group (P > 0.05) compared to model control group.
TABLE 18 influence of HOM3201 on serum insulin resistance index of model rats
As can be seen from Table 18, the HOM3201 strain group had a decrease in serum insulin resistance index, but the difference was not significant (P > 0.05) compared to the model control group.
Fig. 4 shows a flow chart of the process of isolation, screening, preparation of formulations, in vitro and hypoglycemic effect in animal experiments of lactobacillus plantarum HOM3201 strain of the present invention.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Although embodiments of the present invention have been disclosed above, it is not limited to the details of the description and the examples set forth, which are well suited to various fields of use, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and details shown and described herein, without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (10)
1. A lactobacillus plantarum (Lactobacillus plantarum) HOM3201 strain, wherein the lactobacillus plantarum HOM3201 strain has a preservation number of CGMCC No.22700, and the lactobacillus plantarum HOM3201 strain is capable of producing a DPP-4 inhibitor and an alpha-glucosidase inhibitor, stimulating cells to secrete GLP-1, and has antioxidant activity.
2. The lactobacillus plantarum HOM3201 strain according to claim 1, wherein the lactobacillus plantarum HOM3201 strain comprises the 16s rDNA sequence represented by SEQ ID No. 1.
3. A live bacterial formulation comprising the lactobacillus plantarum HOM3201 strain of claim 1.
4. A viable bacteria preparation according to claim 3, characterized in that the viable bacteria preparation comprises up to 4.0-8.0 x 10 11 CFU/g viable bacteria.
5. The viable bacteria preparation of claim 3 or 4, further comprising an adjuvant.
6. A food or health product comprising the viable bacteria preparation according to any one of claims 3 to 5.
7. Use of a lactobacillus plantarum HOM3201 strain according to claim 1 in the manufacture of a medicament for assisting in lowering blood glucose.
8. The use according to claim 7, wherein lactobacillus plantarum HOM3201 strain is used for reducing fasting blood glucose levels, increasing glucose tolerance in the body and reducing insulin resistance index in rats with insulin resistant glucose metabolism disorder/lipid metabolism disorder models.
9. The use according to claim 7, characterized in that the lactobacillus plantarum HOM3201 strain is used for the treatment of diabetes.
10. A method of preparing the live bacterial formulation according to any one of claims 3 to 5, comprising the steps of:
expanding the lactobacillus plantarum HOM3201 strain of claim 1 in an optimized liquid medium;
collecting thalli;
adding a protective agent for resuspension, vacuum freeze-drying, and crushing to obtain the active microbial inoculum.
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