CN116179441A - Pediococcus acidilactici JYPA-16 for preventing, improving and treating inappetence symptoms of pet cats and application thereof - Google Patents
Pediococcus acidilactici JYPA-16 for preventing, improving and treating inappetence symptoms of pet cats and application thereof Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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Abstract
The invention relates to the technical field of microorganisms, in particular to Pediococcus acidilactici JYPA-16 for preventing, improving and treating inappetence symptoms of pet cats and application thereof. Pediococcus acidilactici (Pediococcus acidilactici) JYPA-16 is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 9 th month of 2021, and has a preservation address of Hospital No.1 and 3 of North Chenxi of the Korean area of Beijing city and a preservation number of CGMCC No.23352. The pediococcus acidilactici JYPA-16 provided by the invention has stronger gastrointestinal survival rate and intestinal tract colonization capability, and can synthesize vitaminsB 6 Improving inappetence.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Pediococcus acidilactici JYPA-16 for preventing, improving and treating inappetence symptoms of pet cats and application thereof.
Background
Cats have long been a domestic pet, and modern people often choose to feed pet cats with cat food for convenience of preservation and use as the pace of life increases. When the pet cat suffers from inappetence, people usually consider that the cat is tired of a cat food, so that another cat food can be purchased to feed the pet cat, the pet cat can increase the appetite of a bit in a short time, but the method is not a long-term method, after a period of time, the cat still suffers from inappetence, only symptoms of inappetence can be cured, and the symptoms of inappetence can not be cured radically.
Some cats also have a certain dosage of drugs incorporated into their food to treat anorexia in their pet cats. Gentian sodium bicarbonate tablet is a common medicine for treating inappetence, but long-term feeding can cause vomiting symptoms of pet cats. Lactic acid bacteria can also be added into cat food to regulate and maintain intestinal flora balance, thereby realizing the relief and treatment of symptoms of inappetence. The acid-resistant and bile-salt-resistant products with the effect of treating inappetence on the market are poor in acid resistance and bile-salt resistance, and are usually coated, but the intestinal tracts of cats are short, the digestion time is limited, and the coated products are difficult to rapidly exert the effect in a short time.
Therefore, it is necessary to provide a product which can solve the problem of inappetence of pet cats, avoid harm to the bodies of the pet cats caused by medication, and can exert efficacy in a short time.
Disclosure of Invention
Aiming at the technical problem that the coated bacterial powder is difficult to exert efficacy in a short time when the intestinal tract of a cat is short, the invention provides the Pediococcus acidilactici JYPA-16 for preventing, improving and treating the inappetence symptom of the pet cat and the application thereof, the Pediococcus acidilactici JYPA-16 has stronger gastrointestinal tract survival rate and intestinal tract colonization capability and can synthesize vitamin B 6 Improving inappetence.
In a first aspect, the present invention provides a pediococcus acidilactici (Pediococcus acidilactici) JYPA-16 for preventing, improving and treating inappetence symptoms of pet cats, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 9/2/2021, wherein the preservation address is 1/3 of North West Lu in the Yangyang area of Beijing city, and the preservation number is CGMCC No. 23152.
Furthermore, the gene sequence of the pediococcus acidilactici JYPA-16 is shown as SEQ ID NO. 1.
In a second aspect, the invention provides a method for synthesizing vitamin B by using the pediococcus acidilactici JYPA-16 6 Is used in the field of applications.
In a third aspect, the invention provides an application of the Pediococcus acidilactici JYPA-16 in preparing a product for preventing, improving or treating anorexia of pet cats.
Further, the dosage form of the product for preventing, ameliorating or treating anorexia in pet cats is in powder form.
Further, the preparation method of the product for preventing, improving or treating the anorexia symptom of the pet cat comprises the steps of inoculating the Pediococcus acidilactici JYPA-16 into an MRS culture medium, and freeze-drying after the culture is finished.
Further, the product for preventing, improving or treating the poor appetite symptom of the pet cat contains 10-100 hundred million live bacteria of Pediococcus acidilactici JYPA-16.
Further, adult cats are drenched daily with 5g of product to prevent, ameliorate or treat the symptoms of anorexia in pet cats.
The invention has the beneficial effects that:
the pediococcus acidilactici JYPA-16 provided by the invention has strong gastrointestinal survival rate and intestinal tract colonization capability, and can synthesize vitamin B 6 Preventing and improving and treating inappetence.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is vitamin B 6 Standard curve of concentration versus absorbance.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1 isolation and identification of strains
1. Bacterial screening
The sample used in the experiment is taken from the county of the King of Guangxi Bama Yao nationality in 2018, 9 and 15 days, 1mL of the sample is taken and diluted with sterile 0.85% physiological saline, and the mixture is uniformly mixed to prepare 10 -1 And the sample is taken as a tube 1 to be subjected to gradient dilution until the sample reaches 10 -8 Take 10 -5 、10 -6 、10 -7 、10 -8 Four gradients containing 1.5% CaCO 3 Coating on MRS solid culture medium, and placing into anaerobic bagIn the above step, the mixture was placed in a constant temperature incubator at 37℃and cultured for 48 hours. Selecting strains with obvious calcium dissolving rings, continuously streaking and culturing on an MRS solid culture medium for 3 times to obtain purified strains, and preserving the purified strains at-80 ℃ by adopting a glycerol preservation method.
2. Identification of species
(1) Gram staining the strain obtained in the last step, observing that the strain is gram-positive, and observing that the colony is milky white, round, smooth and opaque and has regular edges and spherical distribution under a microscope; the strain is subjected to physiological and biochemical identification according to a common bacterial system identification manual and a Berger bacterial identification manual, the nitrate reduction test result is negative, the gelatin liquefaction test result is negative, the catalase test result is negative, the starch hydrolysis test result is positive, and the sugar fermentation test result shows that the strain can ferment fructose, lactose, sucrose, galactose and glucose, and does not produce acid or gas, and the identification result is shown in table 1.
TABLE 1 physiological and biochemical identification
(2) The single colony separated and purified in the previous step is sent to China center for identification of industrial microorganism strain collection, the primer sequences are 27F and 1492R, and the strain is identified as Pediococcus acidilactici. The identified strain is named as Pediococcus acidilactici JYPA-16, and the gene sequence is as follows:
AACGAACTTCCGTTAATTGATTATGACGTGCTTGCACTGAATGAGATTTTAACACGAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGCAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCCTGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATGCGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTGAGAGTAACTGTTCACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAAACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACC。
the Pediococcus acidilactici JYPA-16 is sent to China general microbiological culture Collection center for preservation, the preservation date is 2021, 9 and 2 days, the preservation address is North Chen Xili No.1 and 3 in the Korean area of Beijing city, the preservation number is CGMCC No. 2352, and the classification is Pediococcus acidilactici.
Example 2 preparation of a product for preventing, ameliorating or treating anorexia in a pet cat
Pediococcus acidilactici is inoculated into MRS culture medium according to different inoculation amounts, and freeze-dried powder with the bacterial count of 10 hundred million/g, 50 hundred million/g and 100 hundred million/g is obtained after the culture is finished.
EXAMPLE 3 Pediococcus acidilactici JYPA-16 stress resistance experiment
1. Acid resistance test
The pH value of gastric juice of cat is between 1 and 2, and the Pediococcus acidilactici JYPA-16 is respectively inoculated into MRS culture medium with pH value of 1.5 according to the inoculum size of 5 percent, and is cultured for 24 hours at 37 ℃, so that the acid resistance of the cat is measured.
2. Test for bile salt resistance
Pediococcus acidilactici JYPA-16 was inoculated into MRS medium with a bile salt concentration of 0.3% (w/v) at an inoculum size of 5%, and cultured at 37℃for 24 hours, and the bile salt resistance was measured.
3. Survival results
The treated sample solution was subjected to gradient dilution, viable bacteria were counted by a pouring method, and the survival rate was calculated according to the formula of survival rate= (treated bacteria count/original bacteria count) ×100%, and the results are shown in table 2.
TABLE 2 data sheet for JYPA-16 survival in stress-resistance experiments
| Survival rate | Acid resistance test | Experiment of bile salt resistance |
| 0h | 100％ | 100％ |
| 1h | 99.5％ | 99.4％ |
| 2h | 97.4％ | 98.6％ |
| 3h | 95.1％ | 96.7％ |
| 4h | 92.7％ | 96％ |
The table shows that the survival rate of the Pediococcus acidilactici JYPA-16 is still over 90 percent after 4 hours of culture in an in vitro simulated gastrointestinal environment, which indicates that the Pediococcus acidilactici JYPA-16 has strong stress resistance and can play a role in survival in intestines and stomach.
Example 4 Pediococcus acidilactici JYPA-16 adhesion control experiment to Caco-2
Caco-2 cells were placed in DMEM medium (available from Gibco Co.) containing 10% heat-inactivated new born calf serum and double antibody (penicillin concentration 100U/mL, streptomycin 1.0. Mu.g/mL) at 37℃and 5% CO 2 Incubating in a carbon dioxide incubator with a relative humidity of 95%, changing culture solution 1 time a day, passaging for 1 time 3-4 days, inoculating cells into 24-well culture plate after 18 days, and inoculating at a density of about 5×10 5 CFU/mL, and adhesion test is carried out after the cells grow to a monolayer.
The activated JYPA-16 was inoculated into MRS liquid medium at an inoculum size of 1% by volume and cultured for 12h at 37 ℃. Centrifuging to collect thallus (4000 g, 4 deg.C, 10 min), washing with sterile PBS buffer for 3 times, and suspending the thallus in sterile PBS buffer to obtain 2×10 concentration 8 CFU/mL JYPA-16 bacterial suspension and PBS buffer solution are prepared according to the following method:
KH 2 PO 4 0.27g、Na 2 HPO 4 1.42g, 8g NaCl and 0.2g KCl are added with about 800mL of deionized water, fully stirred and dissolved, added with concentrated hydrochloric acid to adjust the pH to 7.4, added with deionized water to fix the volume to 1L, sterilized at 121 ℃ for 20min, and finally PBS buffer with pH=7.4 is prepared for standby.
Rinsing Caco-2 cells growing into monolayer with sterile PBS buffer for 2 times, adding 0.5mL JYPA-16 bacterial suspension and 0.5mL fresh DMEM culture solution into each well, and culturing at 37deg.C and 5% CO 2 Incubating in a carbon dioxide incubator with a relative humidity of 95% for 1h, rinsing the cells with sterile PBS buffer for 5 times to remove non-adherent bacteria, and subjecting the treated sample solution to gradient dilution using decantationViable bacteria count was made by the method, and the adhesion rate was calculated according to the formula of adhesion rate= (initial bacteria number-bacteria number under rinsing)/initial all belong to x 100%. Each treatment was done in 3 replicates.
After rinsing, the cells were fixed with formaldehyde, stained with gram, microscopic examined, and the number of bacteria adhering to 100 cells in 20 random fields was counted under an inverted microscope. Cell culture without bacteria liquid is used as a control. The average number of bacteria per cell adhesion was calculated.
The adhesion rate of the Pediococcus acidilactici JYPA-16 to Caco-2 cells is 82% and the number of cell adhesion is 634, so that the Pediococcus acidilactici JYPA-16 has strong capability in the aspect of cell field planting.
EXAMPLE 5 Pediococcus acidilactici JYPA-16 vitamin B synthesis 6 Determination of Capacity
1. Preparation of bacterial liquid
Inoculating the Pediococcus acidilactici JYPA-16 into MRS liquid culture medium according to the inoculation amount of 2%, culturing for 24 hours at 37 ℃, sub-packaging into a sterile centrifuge tube, centrifuging for 10min at 3000r/min to remove supernatant, adding sterile physiological saline for resuspension, continuing centrifuging, repeating for 3 times, finally adding 20-30mL of sterile physiological saline, and adjusting turbidity to a certain range by a spectrophotometer to obtain bacterial liquid.
2. Production of standard curve
Accurately weighing vitamin B 6 0.015g of standard substance is dissolved by 0.1mol/L HCl and is fixed in a 100mL brown volumetric flask, and the solution is 150mg/L vitamin B6 standard solution.
Accurate aspiration of microorganism B 6 Standard solutions 4.0, 6.0, 8.0, 10.0, 12.0mL were diluted to scale with 0.1mol/L HCl solution in 100mL brown volumetric flasks, respectively, all performed in a dark environment. Taking HCl solution with concentration of 0.1mol/L as blank, measuring absorbance value under the condition of wavelength of 290nm, and using vitamin B 6 Concentration is on the abscissa, OD 290 A standard curve (as shown in fig. 1) is plotted on the ordinate.
3. Vitamin B in sample 6 Content determination
1mL of the bacterial liquid is taken and put into a 250mL conical flask, 72mL of H with the concentration of 0.22mol/L is added 2 SO 4 Is arranged at 12Hydrolyzing at 1deg.C for 4 hr, cooling in ice water, regulating pH to 4.5, transferring the solution into 100mL volumetric flask, constant volume with distilled water, filtering with filter paper, measuring absorbance at 290nm, and calculating vitamin B according to standard curve 6 The mass concentration of (C) is 33.56+/-0.15 mg/100mL.
Example 6 Pediococcus acidilactici JYPA-16 drenching experiment
50 adult cats aged 5 years with symptoms of anorexia were selected and divided into 1 control group and 4 experimental groups (experimental group 1, experimental group 2, experimental group 3 and experimental group 4), the control group and the experimental group were fed the same food, and the cats of the 4 experimental groups were filled with 5g of the Pediococcus acidilactici JYPA-16 powder (50 hundred million/g) of example 2 daily, and after 10 days of filling, the appetite and defecation amount of the cats were observed, while a pet locator was mounted on each cat, and the exercise amount within 1 day was checked. The results are shown in Table 3.
Table 3JYPA-16 drenching test results
| Grouping | Appetite stimulating | Average defecation times | Average exercise amount (km) |
| Control group | Difference of difference | 0.3 | 2.59 |
| |
Good (good) | 1.6 | 7.88 |
| Experiment group 2 | Good (good) | 1.8 | 8.76 |
| Experiment group 3 | Good (good) | 1.6 | 8.54 |
| Experiment group 4 | Good (good) | 1.9 | 9.87 |
Vitamin B6 is an important component for synthesizing a plurality of coenzymes in the body, and participates in a plurality of metabolic reactions, especially the metabolism of amino acids, and when the vitamin B6 is lacking in the body of a pet, the situation that the appetite of the cat is poor and the food utilization rate is low can be caused. By combining the data, after the cats are drenched with the pediococcus acidilactici JYPA-16 bacterial powder for 10 days, the mental state and appetite of the experimental group are greatly recovered compared with those of the control group, the defecation times and the exercise amount of each day are increased, and the pediococcus acidilactici JYPA-16 has obvious effects of treating and preventing the inappetence of the pet cats.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. The Pediococcus acidilactici JYPA-16 for preventing, improving and treating the inappetence symptom of pet cats is characterized in that Pediococcus acidilactici (Pediococcus acidilactici) JYPA-16 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 9 th month of 2021, and the preservation address is North Chen West Lu No.1 and 3 in the Korean region of Beijing city, and the preservation number is CGMCC No.23352.
2. The pediococcus acidilactici JYPA-16 as defined in claim 1, wherein the gene sequence of the pediococcus acidilactici JYPA-16 is shown in SEQ ID NO. 1.
3. A method for synthesizing vitamin B from Pediococcus acidilactici JYPA-16 as defined in claim 1 6 Is used in the field of applications.
4. Use of pediococcus acidilactici JYPA-16 as described in claim 1 for the preparation of a product for preventing, improving or treating anorexia in pet cats.
5. The use according to claim 4 wherein the product for preventing, ameliorating or treating anorexia in a pet cat is in the form of a powder.
6. The use according to claim 4, wherein the product for preventing, ameliorating or treating anorexia in pet cats is prepared by inoculating Pediococcus acidilactici JYPA-16 into MRS medium, and lyophilizing after culturing.
7. The use according to claim 4, wherein the product for preventing, ameliorating or treating anorexia in a pet cat comprises from 10 to 100 million live bacteria of Pediococcus acidilactici JYPA-16 per gram.
8. The use according to claim 4 wherein the adult cat is drenched daily with 5g of the product for preventing, ameliorating or treating anorexia in the pet cat.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467581A (en) * | 2023-12-22 | 2024-01-30 | 山东威曼宠物食品有限公司 | Pediococcus acidilactici King73 capable of improving canine immunity and application thereof |
| CN117568242A (en) * | 2024-01-15 | 2024-02-20 | 山东中科嘉亿生物工程有限公司 | Pediococcus acidilactici JYPA-30 for improving female mastitis, and microbial inoculum and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117467581A (en) * | 2023-12-22 | 2024-01-30 | 山东威曼宠物食品有限公司 | Pediococcus acidilactici King73 capable of improving canine immunity and application thereof |
| CN117467581B (en) * | 2023-12-22 | 2024-03-08 | 山东威曼宠物食品有限公司 | Pediococcus acidilactici King73 capable of improving canine immunity and application thereof |
| CN117568242A (en) * | 2024-01-15 | 2024-02-20 | 山东中科嘉亿生物工程有限公司 | Pediococcus acidilactici JYPA-30 for improving female mastitis, and microbial inoculum and application thereof |
| CN117568242B (en) * | 2024-01-15 | 2024-04-16 | 山东中科嘉亿生物工程有限公司 | Pediococcus acidilactici JYPA-30 for improving female mastitis, and microbial inoculum and application thereof |
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