CN117327604A - Live bacterial preparation containing lactobacillus rhamnosus strain and application thereof - Google Patents
Live bacterial preparation containing lactobacillus rhamnosus strain and application thereof Download PDFInfo
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- CN117327604A CN117327604A CN202310744266.6A CN202310744266A CN117327604A CN 117327604 A CN117327604 A CN 117327604A CN 202310744266 A CN202310744266 A CN 202310744266A CN 117327604 A CN117327604 A CN 117327604A
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- lactobacillus rhamnosus
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- hom1213
- live bacterial
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of microorganisms, in particular to a live bacteria preparation containing lactobacillus rhamnosus (Lactobacillus rhamnosus) strain and application thereof. The preservation number of the lactobacillus rhamnosus strain is CGMCC No.25682. The live bacteria preparation containing lactobacillus rhamnosus strain can generate DPP-4 and alpha-glucosidase inhibitor and stimulate NCI-H716 cells to generate GLP-1, and has the potential of regulating blood sugar; has good free radical scavenging, oxidation stress state coping, oxidation resistance and aging resistance; can increase the level of cytokine TNF-alpha and/or IL-6, and regulate immunity.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a live bacteria preparation containing lactobacillus rhamnosus strain and application thereof.
Background
Diabetes, particularly type II diabetes, is one of the health problems of worldwide concern at present, and is a metabolic disease that is manifested as hyperglycemia symptoms caused by insufficient insulin secretion. Currently, the blood glucose level of diabetics can be controlled by drugs such as α -glucosidase inhibitors and dipeptidyl peptidase 4 inhibitors (i.e. DPP-4 inhibitors).
Studies have shown that oxidative damage to pancreatic beta cells is one of the causes of diabetes. Excessive free radicals attack insulin-specific receptors on the liver cell membrane, thereby causing insulin resistance, and the body's antioxidant defense system's ability to scavenge free radicals is greatly reduced, resulting in a series of oxidative damage.
The hypoimmunity or lack of immunity often causes various local or systemic infections, light cold, secondary infection of hepatitis B and hepatitis C virus to damage liver and cause liver cancer, and the worst infection of HIV to lose the anti-pathogenic ability of human body. The hypoimmunity can also cause pharyngitis, gastritis, enteritis, pneumonia, bronchitis, rhinitis, otitis media, hepatitis, mastitis, cancer, skin infection and other diseases.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) is an anaerobic, acid-tolerant, non-spore-forming gram-positive bacterium that is present in the intestinal tract of humans and animals. Most strains have the biological characteristics of acid resistance, bile salt resistance and multiple antibiotic resistance.
Currently, studies on live bacterial preparations comprising lactobacillus rhamnosus strains are inadequate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a live bacteria preparation containing lactobacillus rhamnosus strain, which has excellent performances in reducing blood sugar, resisting oxidation, resisting aging and regulating immunity.
In one aspect, the invention provides a live bacterial preparation comprising a lactobacillus rhamnosus (Lactobacillus rhamnosus) strain, wherein the lactobacillus rhamnosus strain has a preservation number of CGMCC No.25682.
In one example, the live bacterial formulation comprises up to 3.0X10 11 CFU/g viable bacteria.
In another example, the live bacteria formulation comprises an adjunct.
In another aspect, the invention provides a food or health product comprising a viable bacteria formulation as described above.
In another example, the food product is selected from one or more of a milk powder, a solid beverage, a fermented milk product, a milk-containing beverage, and cheese.
Preferably, the fermented dairy product is selected from fermented cow dairy products and fermented oat dairy products.
Preferably, the fermented dairy product is a fermented cow dairy product, wherein the content of active lactobacillus rhamnosus in the fermented cow dairy product is higher than 6.0×10 8 CFU/mL。
Preferably, the fermented product is fermented oat dairy product, wherein the viable count of lactobacillus rhamnosus in the fermented oat dairy product is higher than 5.0x10 8 CFU/mL。
In another aspect, the present invention provides the use of a live bacterial preparation comprising a lactobacillus rhamnosus strain as described above for the manufacture of a medicament for the production of DPP-4 and an alpha-glucosidase inhibitor.
The invention also provides a use of a live bacterial preparation comprising a lactobacillus rhamnosus strain as described above in the manufacture of a medicament for stimulating the production of GLP-1 by NCI-H716 cells.
In another example, the viable bacteria formulation is used to assist in lowering blood glucose.
The invention also provides application of the live bacteria preparation containing the lactobacillus rhamnosus strain in preparing medicines for scavenging free radicals, coping with oxidative stress, resisting oxidation and resisting aging.
In one example, the live bacterial formulation is used to increase the levels of superoxide dismutase, glutathione peroxidase, and glutathione, and/or decrease the level of malondialdehyde.
In one example, the live bacterial formulation is used to scavenge excess free radicals, increasing hydroxyl radical and DPPH radical scavenging rates.
The invention also provides the use of a live bacterial preparation comprising a lactobacillus rhamnosus strain as described above for the manufacture of a medicament for modulating immunity, optionally for increasing the level of cytokines TNF- α and/or IL-6.
In another aspect, the present invention also provides a method for preparing a viable bacterial preparation as described above, comprising the steps of:
expanding the lactobacillus rhamnosus strain as described above in an optimized liquid medium;
collecting thalli;
adding a protective agent for resuspension, vacuum freeze-drying, and crushing to obtain the active microbial inoculum.
Advantageous effects
The live bacteria preparation containing lactobacillus rhamnosus strain can generate DPP-4 and alpha-glucosidase inhibitor and stimulate NCI-H716 cells to generate GLP-1, and has the potential of regulating blood sugar; has good free radical scavenging, oxidation stress state coping, oxidation resistance and aging resistance, can improve the levels of superoxide dismutase, glutathione peroxidase and glutathione, reduce the level of malondialdehyde, and improve the scavenging rate of hydroxyl free radicals and DPPH free radicals; can increase the level of cytokine TNF-alpha and/or IL-6, and regulate immunity. The viable bacteria preparation of the invention has simple production process parameters, easy control and short period, ensures the high survival rate of the lactobacillus rhamnosus, and the obtained product can be stored for a long time and has stable quality.
Drawings
FIG. 1 shows RAPD cluster analysis graphs of Lactobacillus rhamnosus HOM1213 strain and a portion of commercial Lactobacillus rhamnosus strain constructed based on the UPGMA method.
FIG. 2 shows the inhibition of DPP-4 and alpha-glucosidase by Lactobacillus rhamnosus HOM1213 strain of the invention.
FIG. 3 shows the in vitro antioxidant capacity of the Lactobacillus rhamnosus HOM1213 strain of the present invention.
FIG. 4 shows the effect of Lactobacillus rhamnosus HOM1213 strain of the present invention on in vivo antioxidant and anti-aging indicators in mice with oxidative damage models.
Fig. 5 shows a process flow diagram of the present invention.
Description of the preservation of microorganisms
The lactobacillus rhamnosus (Lactobacillus rhamnosus) HOM1213 strain is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (CGMCC) at the 9 th year 2022, and the preservation address is: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC No.25682.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) HOM1213 strain of the present invention was identified by the institute of microbiology, national academy of sciences, at month 5 of 2023.
The detection and identification results are as follows: under the laboratory condition, according to the comprehensive analysis of experimental data such as cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, pheS gene sequence and the like of the strain to be inspected, reference is made to the research papers related to Bonji system bacteriology handbook, international Journal of Systematic and Evolutionary Microbiology, and the identification result of the strain to be inspected (strain number: HOM 1213) is as follows: lacticaseibacillus rhamnosus (Lactobacillus rhamnosus). Synonyms of the same kind: lactobacillus rhamnosus (Lactobacillus rhamnosus).
The cell morphology of the strain is: a rod shape; the physiological and biochemical characteristics are as follows: gram positive, contact enzyme negative (-), oxidase negative (-); the 16S rRNA gene sequence is shown as SEQ ID NO. 1, and the pheS gene sequence is shown as SEQ ID NO. 9.
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.
In order to further illustrate the technical means and effects of the present invention for achieving the intended purpose, the following description will further illustrate the technical scheme of the present invention with reference to the specific embodiments, but is not limited thereto. All modifications and equivalent substitutions to the technical proposal of the invention are included in the protection scope of the invention without departing from the spirit and scope of the technical proposal of the invention. The following experimental procedures, in which specific conditions are not noted, were carried out according to methods and conditions conventional in the art.
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.
As identified above, lactobacillus rhamnosus (Lactobacillus rhamnosus) is homonymous with lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), and thus lactobacillus rhamnosus (Lactobacillus rhamnosus) and lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) are used interchangeably in the present invention.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain HOM1213 used in the present invention, isolated from healthy infant feces, physicochemical properties: the growth temperature is 37 ℃, the metabolites are rich, and the feed has the characteristics of acid resistance and bile salt resistance, antibacterial property, immunoregulation, blood sugar regulation, oxidation resistance and anti-aging activity.
In order to further understand the present invention, the lactobacillus rhamnosus provided by the present invention and its uses are described in detail below with reference to examples, and the scope of protection of the present invention is not limited by the following examples.
Example 1 isolation and characterization of Lactobacillus rhamnosus Strain HOM1213
(1) Preparation of culture Medium
MRS liquid medium:
MRS medium (OXOID, CM 1163) and double distilled water 1L were stirred well. Sterilizing at 121deg.C for 20 min.
Improved MRS solid medium:
MRS medium (OXOID, CM 1163), bromocresol green (Shanghai Biotechnology) 0.05g, double distilled water 1L, and stirred well. Adjusting pH to 5.5, sterilizing at 121deg.C for 20 min.
(2) Isolation of strains
1g of healthy infant feces is weighed, 9mL of 0.9% physiological saline is fully and evenly mixed in an oscillating way, a ten-fold dilution method is utilized to carry out gradient dilution on a sample, two proper dilutions are selected, 100 mu L of the sample is taken and coated in an improved MRS solid flat plate, and anaerobic culture is carried out for 72 hours at 37 ℃. Picking single colony with smooth surface, milky white or milky yellow colony and yellow periphery, streaking culturing and purifying. And observing colony morphology by microscopic examination. Single colonies were picked into MRS broth for pure culture, glycerol stock and number HOM1213.
(3) Identification of strains
The strain of the selected strain is inoculated into MRS liquid culture medium, cultured for 24 hours at 37 ℃, bacterial total DNA of the bacterial liquid is extracted, 16S rRNA gene amplification is carried out, PCR amplification and agarose gel electrophoresis are carried out by using universal primers 27F and 1492R, gel cutting and recovery are carried out, and sequencing (Shanghai process) is carried out. Then, alignment was performed in NCBI database using BLAST tool, HOM1213 strain was identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus). The 16S rRNA gene of HOM1213 strain has the sequence shown in SEQ ID NO. 1.
The sequence of the 16S rRNA gene of the HOM1213 strain was determined as follows:
TGCAGTCGAACGAGTTCTGATTATTGAAAGGTGCTTGCATCTTGATT
TAATTTTGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCC
CTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAATCC
AAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGG
ATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
CAATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGA
GACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAA
TGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTC
GGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGT
TGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCA
GCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCG
TAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCT
TAACCGAGGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGG
ACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA
CACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCG
AAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAA
ACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCT
AACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTC
AAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT
CGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCT
GAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGT
TGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG
CAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACT
GCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCC
CCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTG
CGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGAC
TGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGG
ATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG
TCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTT
AGGGAGCGAGCCGTCT
(4) Random Amplified Polymorphic DNA (RAPD) strain identification
Random polymorphic DNA analysis (RAPD) was performed on the isolated Lactobacillus rhamnosus HOM1213, which indicated that HOM1213 was specific to a portion of the commercial Lactobacillus rhamnosus strain.
Extracting DNA from the preserved strain, and using the strain DNA as a template, and respectively using 5 primers OPA-02: TGC CGA GCT G; OPA-18: AGG TGA CCG T; OPL-07: AGG CGG GAA C; OPL-16: AGG TTG CAG G; OPM-05: GGG AAC GTG T DNA fragments capable of showing polymorphism were amplified by PCR, and different DNA differences were exhibited after gel electrophoresis, and analyzed by cluster analysis software, the results of which are shown in FIG. 1. As shown in FIG. 1, the RAPD cluster analysis plot of the Lactobacillus rhamnosus HOM1213 strain is unique from that of the commercial Lactobacillus rhamnosus strain.
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 |
Example 2 preparation Process of active bacterial powder
(1) Culture of strains
The strain was inoculated into MRS liquid medium, cultured at 37℃for 24 hours, and activated for 2 passages. Inoculating in MRS culture medium, culturing at 37deg.C, and the viable count can reach 1×10 10 CFU/mL or more.
(2) Freeze drying
Centrifuging lactobacillus rhamnosus HOM1213 strain fermentation broth to collect bacterial mud, washing the bacterial mud with 0.9% sterile physiological saline, mixing the bacterial mud with the protective agent (100 g/L skimmed milk powder), freeze-drying 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 3.0X10 11 CFU/g。
Example 3 gastrointestinal trafficability test
(1) Isolation and activation of strains
The commercial strain lactobacillus rhamnosus GG was selected as a control and purchased from ATCC (american type culture collection ). 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; another 10g of pancreatin, 3g of oxgall (BD Difco) and a proper amount of water are taken and dissolved, after the two solutions are mixed, the mixture is diluted to 1000mL by adding water and filtered by a sterile filter membrane with the thickness of 0.22 mu m under a sterile environment for standby.
(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 recording 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.
As can be seen from table 2: after 3 hours of simulated artificial gastric juice treatment, the survival rate of lactobacillus rhamnosus HOM1213 can reach more than 98%, and after the bacterial liquid is continuously treated by 3 hours of simulated intestinal juice treatment, the survival rate of lactobacillus rhamnosus HOM1213 can still reach more than 80%, which indicates that lactobacillus rhamnosus HOM1213 has higher survival rate in intestinal tracts.
TABLE 2 survival of the strains in simulated gastrointestinal fluids
EXAMPLE 4 antibiotic sensitivity
Drug susceptibility testing the K-B agar method recommended by the American clinical standards Committee (NCCLS) was followed as follows:
(1) The commercial strain lactobacillus rhamnosus GG was selected as a control strain. HOM1213 and GG were inoculated into MRS liquid medium, respectively, and cultured at 37℃for 24 hours, followed by continuous activation 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 48 hours at 37 ℃, and then the diameter of a bacteriostasis ring is measured and recorded.
The results are shown in Table 3, interpreted according to the CLSI judgment standard. Lactobacillus rhamnosus GG served as a commercial strain control, and the results showed that HOM1213 was resistant to vancomycin, gentamicin, kanamycin, ampicillin, moderate to streptomycin resistance, and sensitive to the remaining four antibiotics. Lactobacillus is naturally resistant to vancomycin.
TABLE 3 determination of sensitivity of Lactobacillus rhamnosus HOM1213 to nine antibiotics
S (susceptible) is sensitive; i (intermediate), medium; r (resistance) represents drug resistance
Example 5 test for the ability to repress 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. 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) Strain activation
The commercial strain lactobacillus rhamnosus GG was selected as a control strain for this experiment. Commercial strain GG and HOM1213 strain isolated in example were inoculated into MRS liquid medium at an inoculum size of 3%, respectively, and were subjected to stationary culture at 37℃for 24 hours, followed by activation twice to obtain strain fermentation broth. Centrifugation at 11000rpm for 10min, taking supernatant for antibacterial test, and taking MRS liquid culture medium as negative control.
(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 inhibition zone was observed and measured, and the results are shown in Table 4.
TABLE 4 inhibition of pathogenic bacteria by groups
Note that: "-" has no antibacterial activity, <11mm; the "+"11mm is less than or equal to the bacteriostasis ring <16mm; the "++"16mm is less than or equal to the bacteriostasis ring <23mm;
“+++”≥23mm
as can be seen from Table 4, HOM1213 has inhibitory effects on all 5 pathogens. HOM1213 has a greater bacteriostatic ability than commercial strain GG, and is shown in the inhibition of salmonella and listeria monocytogenes.
EXAMPLE 6 lactic acid and short chain fatty acid content
(1) Preparation of Strain supernatant
The commercial strain lactobacillus rhamnosus GG was selected as a control strain. The strain HOM1213 and commercial strain GG isolated in example 1 were inoculated in 3% inoculum size respectively to MRS liquid medium, cultured at 37 ℃ for 24 hours, activated for 3 passages continuously, centrifuged at 11,000rpm at 4 ℃ and the supernatants were taken for use.
(2) Determination of lactic acid, acetic acid and formic acid content
Taking 4mL of supernatant, adding 2 drops of phenolphthalein, regulating to slight color change by using KOH solution, fixing the volume to 5mL by using deionized water, and centrifuging at 11,000rpm and 4 ℃ for 10 minutes, wherein the sample is ready for use. Respectively taking lactic acid, acetic acid, formic acid and samples with different concentrations in different 10mL headspace sample injection bottles, adding 2mL of phosphate buffer solution, 2mL of derivatization reagent and 1mL of acetone, capping and sealing, performing water bath reaction at 100 ℃ for 40min, cooling to room temperature, taking the derivatized samples in a 2mL centrifuge tube, adding an equal volume of isooctane for vortex extraction, taking an isooctane layer, and performing Gas Chromatography (GC) analysis, wherein the results are shown in Table 5.
TABLE 5 determination of lactic acid, acetic acid and formic acid content
* Comparison of p <0.01 with MRS control group
As can be seen from Table 5, both strains HOM1213 and GG produced a higher content of lactic acid and a certain content of acetic acid. Lactic acid and acetic acid can inhibit pathogenic bacteria proliferation, and help to maintain good intestinal flora.
Example 7 in vitro cytokine secretion test
The commercial strain lactobacillus rhamnosus GG was selected as a control strain. Lactobacillus rhamnosus strain HOM1213 isolated in example 1 and commercial strain GG were inoculated in 3% inoculum size respectively in MRS liquid medium, fermented at 37 ℃ for 24 hours, and activated continuously for three generations. Centrifugation was carried out at 11000rpm for 10min, and after the collection of the cells, the cells were adjusted to a working concentration (about 5.0X10) 5 CFU/mL). Mouse macrophage RAW264.7 (about 5X 10) 5 Individual cells/mL) was added to 24-well plates at 1mL per well. After 2h adherence, the medium was discarded and 1mL of the bacteria-containing medium was added to each well. A blank control group was set and 1mL of DMEM medium was added. Cell supernatants were collected after 24h co-culture.
The levels of TNF- α and IL-6 in the cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.
TABLE 6 HOM1213 secretion of cytokines by mouse macrophages
Note that: * : comparison to the blank group p <0.05,: comparison with the blank group p <0.01
The results are shown in Table 6. Lactobacillus rhamnosus HOM1213 was able to significantly increase TNF- α and IL-6 secretion by RAW264.7 cells. It can be seen that lactobacillus rhamnosus has potential immunomodulatory capacity.
Example 8 in vitro hypoglycemic efficacy and mechanism exploration
(1) Improved MRS liquid culture medium
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
The commercial strain Lactobacillus rhamnosus GG was selected as a positive control, and the HOM1213 strain and commercial strain GG isolated in example 1 were inoculated into fresh modified MRS liquid medium at an inoculum size of 1% of the total amount of the medium, and subjected to anaerobic culture at 37℃for 24 hours, followed by activation for 3 generations. The bacterial solution was centrifuged at 11000rpm 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, centrifuge at 11000rpm for 10min, and collect supernatant for later use.
(3) DPP-4 inhibitory and alpha-glucosidase inhibitory assays
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 rates of the above two enzymes were calculated for each sample, and the results are shown in Table 7 and FIG. 2.
TABLE 7 inhibition of DPP-4 and alpha-glucosidase by groups
* *: significant differences compared to HOM1213 group (p < 0.01)
As can be seen from Table 7 and FIG. 2, the HOM1213 strain has higher inhibition rate to DPP-4 and alpha-glucosidase. In both of the above criteria, HOM1213 group was higher than GG group. Taken together, HOM1213 strains have the potential to regulate blood glucose, which may result from the production of higher levels of DPP-4 and α -glucosidase inhibitors.
Example 9HOM1213 Strain stimulated GLP-1 secretion by NCI-H716 cells
(1) Culture of NCI-H716 cells
NCI-H716 cells (from the China academy of sciences cell Bank) were selected for this experiment. NCI-H716 cells in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Hyclone), 1% diabody (penicillin and streptomycin, hyclone) at 37℃and 5% CO 2 Is grown in suspension in an incubator.
(2) Strain activation and sample processing
The commercial strain lactobacillus rhamnosus GG was selected as a positive control. Lactobacillus rhamnosus HOM1213 isolated in example 1 and commercial strain GG were inoculated into MRS liquid medium at 3% inoculum size, and subjected to stationary culture at 37deg.C for 24h, and activated for 3 generations. Each broth was centrifuged at 11000rpm 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 into endocrine differentiation medium, 37 deg.C, 5% CO 2 Is cultured in an incubator 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), and the results are shown in Table 8.
TABLE 8 stimulation of GLP-1 production by NCI-H716 cells by the strains
* *: significant differences compared to HOM1213 group (p < 0.01)
As can be seen from Table 8, the HOM1213 strain stimulated NCI-H716 cells to secrete high levels of GLP-1 at concentrations of 1781.83 + -22.22 pg/mL. HOM1213 strain had very significant differences (p < 0.01) compared to lactobacillus rhamnosus commercial strain GG. The results indicate that lactobacillus rhamnosus HOM1213 strain has the potential to reduce blood glucose, possibly by stimulating intestinal L cells to produce high levels of GLP-1.GLP-1 is an incretin, promotes insulin secretion by islet beta cells and inhibits glucagon secretion by alpha cells in a glucose concentration-dependent manner, has a good postprandial blood glucose reducing effect, does not stimulate insulin secretion when the glucose concentration is lower than a normal value, and thus prevents occurrence of hypoglycemia.
Example 10 in vitro antioxidant Capacity test
(1) Strain activation
Lactobacillus rhamnosus HOM1213 and GG strains are respectively inoculated into an MRS liquid culture medium, fermented and cultured for 24 hours at 37 ℃, activated for three generations continuously, centrifuged for 10min at 11000rpm, washed by PBS buffer solution, re-suspended and centrifuged, and repeated three times. Regulating the bacterial count to 2 x 10 10 CFU/ml was ready for use.
(2) Detection of antioxidant indicators
The index includes total antioxidant capacity assay (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), hydroxyl radical scavenging assay (OH) and DPPH radical scavenging assay. 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. Other indexes are measured according to the operation instruction by using a kit purchased by Nanjing built company. The antioxidant indices of HOM1213 and GG bacterial suspensions were determined using 0.05% vitamin C as a positive control, and the results are shown in table 9 and fig. 3.
TABLE 9 detection of antioxidant capacity in vitro
Note that: ND: undetected,: has very significant differences (p < 0.01) compared to HOM1213 group
As can be seen from Table 9 and FIG. 3, lactobacillus rhamnosus HOM1213 has high activity of T-AOC, SOD, GSH-Px, has high hydroxyl radical and DPPH radical scavenging rate, and particularly has outstanding SOD enzyme activity, hydroxyl radical scavenging and DPPH radical scavenging. This indicates that HOM1213 has excellent antioxidant activity.
Example 11 increasing antioxidant level in mice and delaying aging in mice
(1) Mouse origin
30 female Kunming healthy SPF-class mice of 22-26g were bred by Beijing Fukang Biotechnology Co., ltd. The experimental animals were raised in SPF class animal room at the applied university of Beijing-incorporated applied culture center for health food function detection, and the maintenance feed was produced by Beijing Fukang biosciences Co.
(2) D-galactose oxidation injury model
30 mice were divided into 3 groups of 10 mice each. Wherein, D-galactose prepared by physiological saline is injected subcutaneously into the neck and back of two groups of mice at the dosage of 300mg/kg BW, the injection amount is 0.1mL/10g, the molding is carried out 1 time a day, the molding is carried out continuously for 6 weeks, and the level of malondialdehyde is measured by taking blood. Randomly divided into 2 groups based on malondialdehyde levels, including model control and HOM1213 groups. The other group is a blank control group, and the equivalent amount of physiological saline is injected into the cervical and the back of the human body in a daily injection mode during the blank control group modeling period.
(3) Dose design
After successful molding, the samples were given orally once daily, and the mice were perfused with 10mL/kg BW. HOM1213 group was given 1×10 9 Sterile water for CFU bacterial liquid, blank control group and model control groupInstead of the samples, the daily lavage volumes were the same as the sample group. Each group was given maintenance feed. After 45d of continuous gastric lavage, various indexes in the mice are measured. The HOM1213 group and the model control group continued to be injected with the same dose of D-galactose while the samples were administered, and the blank control group was injected with an equal amount of physiological saline.
According to the specific experimental method, according to the technical specification of health food inspection and evaluation (2012 edition), the statistical analysis is carried out on experimental result data by adopting SPSS software.
(4) Index measurement
The kit required in the test is purchased from Nanjing to build the bioengineering institute.
A. Measurement of Malondialdehyde (MDA) content in lipid peroxide-serum
Blood was collected from the inner canthus venous plexus of the mouse in an amount of 0.5mL, centrifuged at 3000r/min for 10min, and the content of malondialdehyde in serum was measured by using the kit, and the results are shown in Table 12.
B. Protein carbonyl (Protein carbonyl) content determination of liver tissue Protein oxidation product
A certain amount of liver tissue is taken, rinsed in ice-cold physiological saline to remove blood stains on the surface, and the filter paper is wiped dry. Protein carbonyl content was determined using protein carbonyl kit (A087-1-2, nanjing's as bioengineering). The specific method comprises the following steps: adding the first reagent according to the weight-volume ratio of 1:9, mechanically homogenizing under ice bath condition, centrifuging the homogenate for 10min by using a centrifuge of 3000r/min, and taking the supernatant, namely 10% liver tissue homogenate for measurement. Taking 450 mu L of 10% liver tissue homogenate, adding 50 mu L of reagent II, standing at room temperature for 10min, centrifuging for 10min at a rotating speed of 11000r/min, and taking supernatant to be measured. The results are shown in Table 13.
C. Antioxidant enzyme
Blood was collected from the canthus venous plexus of an aged mouse at 0.5mL and centrifuged at 3000r/min for 10min, and the activity of superoxide dismutase (SOD) in serum was measured by using the superoxide dismutase activity kit, and the results are shown in Table 14.
10 mu L of blood is taken from the canthus venous plexus of the old mice, distilled water is added to 1mL, and 1:99 of blood is prepared. Glutathione peroxidase (GSH-Px) activity was measured using glutathione peroxidase kit and the results are shown in Table 15.
D. Antioxidant substance-reduced Glutathione (GSH) assay
Shearing a certain amount of liver, washing with physiological saline, drying, weighing, shearing, adding cold physiological saline into a glass homogenizer, and homogenizing to obtain 10% tissue homogenate. Centrifugation was performed at 2500r/min for 10min, and the supernatant was taken and assayed for GSH content using GSH kit, and the results are shown in Table 16.
TABLE 10 serum malondialdehyde content of mice after molding
* *: comparison of model control with blank control p <0.01
As can be seen from table 10, after 6 weeks of injection of D-galactose by subcutaneous injection into mice, the model groups were randomly divided into model control group and sample group HOM1213 group according to the serum malondialdehyde content of mice, and HOM1213 group was compared with model control group, without significant difference (p > 0.05), indicating that the serum malondialdehyde content of mice was more balanced among model groups; the serum malondialdehyde content was increased with significant differences (p < 0.01) compared to the model control and the blank control; the method shows that the D-galactose oxidative damage model is successfully established.
TABLE 11 weight changes in mice before and after administration of samples
As can be seen from table 11, the body weight of the mice before and after the oral administration of the samples was compared between the model control group and the blank control group (p > 0.05), and the sample group was compared with the model control group (p > 0.05). I.e. the sample had no adverse effect on the body weight of the mice.
TABLE 12 influence of HOM1213 group on mouse serum malondialdehyde
* *: comparison of sample group with model control group p <0.01
As can be seen from table 12, the HOM1213 group serum malondialdehyde content was reduced compared to the model control group, with a very significant difference (p < 0.01) after 45d of the oral administration of the mouse sample. From this, HOM1213 group samples can significantly reduce malondialdehyde content in the serum of mice model of D-galactose oxidative damage. Malondialdehyde is the major product of the peroxidation reaction of polyunsaturated fatty acids. Malondialdehyde content has been widely used to determine the level of oxidative damage in vivo.
TABLE 13 influence of HOM1213 group on carbonyl content of mouse liver tissue protein
Protein carbonylation is one of the oxidative damage of proteins and refers to the conversion of amino acid residue side chains to carbonyl products after attack by oxygen radicals. Carbonyl modification causes structural changes in proteins, resulting in cellular and tissue lesions, involved in aging and apoptosis in the body. As can be seen from Table 13, after 45d of the mice were given orally, the carbonyl content of the liver tissue protein was reduced in HOM1213 group compared with the model control group, but the differences were not significant (p > 0.05).
TABLE 14 influence of HOM1213 group on the SOD Activity of mouse serum superoxide dismutase
* : comparison of sample group with model control group p <0.05
As can be seen from table 14, the HOM1213 group serum SOD activity was increased with a significant difference (p < 0.05) compared to the model control group after 45d of the orally administered mouse sample. HOM1213 may increase body SOD levels to counteract oxidative stress levels.
TABLE 15 influence of HOM1213 group on the glutathione peroxidase activity of mouse Whole blood
* *: comparison of sample group with model control group p <0.01
As can be seen from Table 15, 45D of the mice samples were orally administered, the whole blood glutathione peroxidase activity was increased in HOM1213 group, with a very significant difference (p < 0.01), i.e., HOM1213 group was able to increase the whole blood glutathione peroxidase activity of D-galactose oxidative damage model mice to resist oxidative stress level.
TABLE 16 influence of HOM1213 group on reduced glutathione content in liver tissue of mice
* : comparison of sample group with model control group p <0.05
As can be seen from Table 16, after 45d of the oral administration of the mice samples, the HOM1213 group had elevated levels of reduced glutathione (p < 0.05) compared to the model control group. Namely HOM1213 can raise the reduced glutathione content of the liver tissue of the mouse model of the D-galactose oxidative damage.
According to the method for judging the antioxidant animal experiment of the health-care food function inspection and evaluation method (2022 edition), three indexes of lipid oxidation products, protein oxidation products, antioxidant enzymes and antioxidant substances are positive, the tested sample can be judged to be beneficial to the positive of the antioxidant animal experiment result. This demonstrates that lactobacillus rhamnosus HOM1213 has antioxidant and anti-aging functions.
FIG. 4 shows the effect of the Lactobacillus rhamnosus HOM1213 strain of the present invention on the in vivo antioxidant and anti-aging index of mice with oxidative damage model. After 45 days of orally administered mice samples, the HOM1213 group serum superoxide dismutase (SOD) activity was increased with a significant difference (p < 0.05), whole blood glutathione peroxidase (GSH-Px) activity was increased with a very significant difference (p < 0.01), liver tissue reduced Glutathione (GSH) levels were all increased with a significant difference (p < 0.05), serum Malondialdehyde (MDA) levels were reduced with a very significant difference (p < 0.01), liver tissue protein carbonyl levels were reduced but the difference was not significant (p > 0.05), thus it was seen that HOM1213 group samples could significantly increase D-galactose oxidative damage model mice serum and GSH-Px activity, GSH levels, significantly reduced MDA levels.
The overall process of the present invention is shown in the process flow diagram of fig. 5.
EXAMPLE 12 fermented cow milk test
Weighing 12g of skim milk powder, 2g of glucose and 2g of yeast powder, supplementing double distilled water to 100mL, uniformly stirring, homogenizing (60 ℃ and 22 MPa) by a high-pressure homogenizer, sterilizing at 95 ℃ for 10min, and cooling to 37 ℃ for later use.
Lactobacillus rhamnosus HOM1213 is inoculated into MRS liquid culture medium, cultured for 18 hours at 37 ℃, and passaged for 2 times according to the method, so as to obtain high-activity bacterial liquid for later use.
The bacterial liquid is used for 0.9 percent of rawSaline eluting and re-suspending to 1×10 7 Inoculating CFU/mL inoculum size into cow milk, mixing well, standing at 37deg.C, fermenting for 16 hr to obtain fermented dairy product. The content of active lactobacillus rhamnosus in the fermented milk is higher than 6.0X10 8 CFU/mL, pH can reach 4.50, and the taste is mellow, sweet and sour.
Example 13 fermented oat milk test
120g of enzymolysis oat flour, 20g of yeast powder and double distilled water are added to 1000mL, homogenizing is carried out at 200bar, sterilization is carried out at 90 ℃ for 10min, and cooling is carried out to 37 ℃ for standby.
Inoculating lactobacillus rhamnosus HOM1213 seed solution into MRS liquid culture medium, culturing at 37deg.C for 18h, subculturing for 2 times to obtain high-concentration bacterial solution, and preserving at low temperature for use.
The bacterial solution was resuspended by eluting with 0.9% physiological saline to 1X 10 7 Inoculating CFU/mL inoculum size into oat pulp, uniformly mixing, standing at a constant temperature of 37 ℃ and fermenting for 6 hours to obtain fermented oat milk. The viable count of lactobacillus rhamnosus in the fermented milk is higher than 5.0X10 8 CFU/mL, pH can be reduced to 4.35, and the taste is rich.
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.
Claims (13)
1. A live bacterial preparation comprising a lactobacillus rhamnosus (Lactobacillus rhamnosus) strain, characterized in that the lactobacillus rhamnosus strain has a preservation number of CGMCC No.25682.
2. The viable bacteria formulation of claim 1, wherein the viable bacteria formulation comprises up to 3.0 x 10 11 CFU/g viable bacteria.
3. A food or health product comprising the viable bacteria formulation of claim 1 or 2.
4. A food or health product according to claim 3, wherein the food is selected from one or more of milk powder, solid beverage, fermented milk product, milk-containing beverage and cheese.
5. The food or health product of claim 4, wherein the fermented dairy product is selected from the group consisting of fermented cow dairy products and fermented oat dairy products.
6. Use of a live bacterial formulation comprising a lactobacillus rhamnosus strain according to claim 1 in the manufacture of a medicament for the production of DPP-4 and alpha-glucosidase inhibitors.
7. Use of a live bacterial formulation comprising a lactobacillus rhamnosus strain according to claim 1 in the manufacture of a medicament for stimulating the production of GLP-1 by NCI-H716 cells.
8. The use according to claim 6 or 7, wherein the live bacterial preparation is for assisting in lowering blood glucose.
9. Use of a live bacterial formulation comprising a lactobacillus rhamnosus strain according to claim 1 for the preparation of a medicament for scavenging free radicals, coping with oxidative stress conditions, anti-oxidation, anti-aging.
10. Use according to claim 9, wherein the live bacterial preparation is used for increasing the level of superoxide dismutase, glutathione peroxidase and glutathione and/or for decreasing the level of malondialdehyde.
11. The use according to claim 9, wherein the live bacterial formulation is used to scavenge excess free radicals and to increase the scavenging rate of hydroxyl radicals and DPPH radicals.
12. Use of a live bacterial preparation comprising a lactobacillus rhamnosus strain according to claim 1 for use in a medicament for modulating immunity, optionally for increasing the level of cytokines TNF- α and/or IL-6.
13. A method of preparing the viable bacteria formulation of claim 1 or 2, comprising the steps of:
expanding the lactobacillus rhamnosus strain according to claim 1 or 2 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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