CN117887607A - Pediococcus acidilactici and application thereof - Google Patents
Pediococcus acidilactici and application thereof Download PDFInfo
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- CN117887607A CN117887607A CN202310721540.8A CN202310721540A CN117887607A CN 117887607 A CN117887607 A CN 117887607A CN 202310721540 A CN202310721540 A CN 202310721540A CN 117887607 A CN117887607 A CN 117887607A
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- pediococcus acidilactici
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses pediococcus acidilactici and application thereof, and relates to the field of microorganisms. The invention provides Pediococcus acidilactici CLP03, the discovery of the strain enriches microbial resources which can be practically applied, the strain is derived from the feces of China garden cats, has good antibacterial effect, is acid-resistant, cholate-resistant, artificial intestinal gastric juice-resistant, high-temperature-resistant, strong in adhesiveness, good in antioxidant capacity, free of drug-resistant genes, good in probiotics potential, capable of promoting the development of a host immune system, improving immunity, capable of preparing a probiotic preparation without toxic or side effect and residues, and applied to disease prevention and treatment and health care of pets.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to pediococcus acidilactici and application thereof.
Background
Antibiotics play an important role in animal husbandry history and are used as feed additives to improve gastrointestinal diseases. However, in recent years, the use of antibiotics in animals has been gradually prohibited due to the residue of antibiotics and the increase in bacterial resistance. Several studies have reported that antibiotics induce undesirable gastrointestinal symptoms in companion animals (anti-inflammatory-associated gastrointestinal signs, AAGS). Today, companion animal diets have shifted from self-feeding and simple feeding to a diet pattern that pursues scientificalness, refinement, and health, and companion animal food development has progressed toward nutrient balance and assurance of animal health. The probiotic preparation without toxic and side effects and residues has wider application in treating, preventing and protecting the diseases of companion animals.
Researchers have suggested using probiotics from the intestinal tract of host species to be optimal, however, bacterial species from feline sources are still less studied and used for most companion animal probiotic products on the market today that are not host-derived.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide pediococcus acidilactici and application thereof.
The invention is realized in the following way:
in a first aspect, an embodiment of the present invention provides a pediococcus acidilactici, wherein the pediococcus acidilactici (Pediococcus acidilactici) CLP03 is deposited with the chinese collection of typical cultures, with the deposit number: cctccc NO: m2023402.
In a second aspect, an embodiment of the present invention provides a method for producing pediococcus acidilactici, including: pediococcus acidilactici as described in the previous examples was cultured.
In a third aspect, an embodiment of the present invention provides a microbial inoculum, which includes the pediococcus acidilactici described in the previous embodiment.
In a fourth aspect, embodiments of the present invention provide an additive, the active ingredients of which include: the Pediococcus acidilactici and/or the metabolite thereof described in the previous examples or the microbial inoculum described in the previous examples.
In a fifth aspect, embodiments of the present invention provide a food product comprising the additive of the previous embodiments.
In a sixth aspect, the embodiment of the present invention provides an application of pediococcus acidilactici and/or a metabolite thereof described in the previous embodiment or the microbial inoculum described in the previous embodiment in preparing food.
In a seventh aspect, the present embodiment provides an application of pediococcus acidilactici and/or a metabolite thereof described in the previous embodiment or the microbial inoculum described in the previous embodiment in feeding animals.
In an eighth aspect, the present embodiment provides an application of the pediococcus acidilactici and/or the metabolite thereof described in the previous embodiment or the microbial inoculum described in the previous embodiment in preparing a product with at least one of the functions (1) to (3): (1) enhancing immunity; (2) preventing, treating or adjunctively treating diarrhea; (3) antioxidant.
In a ninth aspect, the embodiment of the present invention provides an application of pediococcus acidilactici and/or a metabolite thereof described in the previous embodiment or the microbial inoculum described in the previous embodiment in preparing a bacteriostatic product.
The invention has the following beneficial effects:
(1) Has good antibacterial effect, can effectively inhibit various diarrhea bacteria, and can be used for preventing and treating diarrhea;
(2) The high temperature resistance is strong, the survival rate is still higher under the condition of exceeding 70 ℃, the technical barrier that probiotics cannot be sprayed onto the surface of pet daily ration or functional food at high temperature can be solved, the probiotics can be directly sprayed into the pet food, the processing flow is simplified, and the processing cost is reduced;
(3) Acid resistance, bile salt resistance, artificial intestinal juice resistance, strong adhesiveness, good probiotics potential and good colonization ability in intestinal tracts;
(4) The feed has good oxidation resistance, can obviously reduce MDA content in blood of fed mice, improves SOD activity, can relieve organ injury caused by animal stress, delays aging, and prolongs the service life;
(5) The CLP03 can be fed to obviously improve the thymus organ index of mice, promote the development of the immune system of a host, improve the immunity and have the potential of preventing diseases.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing colony morphology and gram staining of Pediococcus acidilactici CLP 03;
FIG. 2 shows the Pediococcus acidilactici CLP03 evolution tree;
FIG. 3 is a graph showing the growth and acid production rate of Pediococcus acidilactici CLP 03;
FIG. 4 shows the tolerance of Pediococcus acidilactici CLP03 to high temperatures;
FIG. 5 shows the tolerance of Pediococcus acidilactici CLP03 to acidity;
FIG. 6 shows the survival rate of Pediococcus acidilactici CLP03 in artificial gastrointestinal fluids;
FIG. 7 shows the survival of Pediococcus acidilactici CLP03 in acid and various bile salt concentrations;
FIG. 8 shows the results of the CLP03 adhesion of Pediococcus acidilactici;
FIG. 9 shows the result of hemolysis of Pediococcus acidilactici CLP 03;
FIG. 10 shows growth and feeding status of mice fed CLP 03;
FIG. 11 is organ index of clP03 fed mice;
FIG. 12 shows serum biochemical markers of mice fed CLP 03;
FIG. 13 is a graph showing serum antioxidant index of mice fed CLP 03;
FIG. 14 is a circle diagram of the strain CLP03 genome;
FIG. 15 is a diagram of functional annotation of strain CLP03 genome COG;
FIG. 16 is a functional annotation of strain CLP03 genome GO;
FIG. 17 is a functional annotation of strain CLP03 genome KEGG.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In one aspect, the embodiment of the invention provides a pediococcus acidilactici with a preservation number of: cctccc No. M2023402. The taxonomic name was Pediococcus acidilactici (Pediococcus acidilactici) CLP03, which was deposited at the China center for type culture Collection, at the university of Wuhan, 2023, 24.
CLP03 is Pediococcus of China field cat origin, the discovery of the strain enriches microbial resources which can be practically applied, has good antibacterial effect, is acid-resistant, cholate-resistant, artificial intestinal juice-resistant, high-temperature-resistant, strong in adhesiveness, good in antioxidant capacity, free of drug-resistant genes, good in probiotics potential, capable of promoting development of a host immune system, improving immunity, capable of preparing probiotics preparation without toxic and side effects and residues, and applied to disease prevention and treatment and health care of animals (such as pets).
In some embodiments, the nucleotide sequence of 16sRNA of Pediococcus acidilactici CLP03 is shown in SEQ ID NO. 1.
On the other hand, the embodiment of the invention also provides a production method of Pediococcus acidilactici, which comprises the following steps: pediococcus acidilactici as described in the previous examples was cultured.
In some embodiments, the culturing process of Pediococcus acidilactici CLP03 can be referred to other existing methods for culturing Pediococcus acidilactici.
Specifically, pediococcus acidilactici CLP03 can grow on common culture media such as MRS, TSA, LB and the like, can grow at 20-50 ℃ and pH of 4-7, and can grow in aerobic and anaerobic environments under proper growth conditions with pH of 6.2-6.5.
On the other hand, the embodiment of the invention also provides a microbial inoculum, which comprises the pediococcus acidilactici described in the previous embodiment.
In another aspect, the embodiment of the invention also provides an additive, which comprises the following active ingredients: the Pediococcus acidilactici and/or the metabolite thereof according to any one of the preceding embodiments or the microbial inoculum according to any one of the preceding embodiments.
In some embodiments, the additive may further include adjuvants, diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents, and adsorption carriers, and the like.
In another aspect, embodiments of the present invention also provide a food product comprising the additive of any of the previous embodiments.
In some embodiments, the food product is an animal food product. The animals may include humans and mammals.
In some embodiments, the animal comprises a pet.
In some embodiments, the pet comprises: a cat.
In some embodiments, the food product further comprises: a basal feed.
On the other hand, the embodiment of the invention also provides the application of the pediococcus acidilactici and/or the metabolite thereof in any embodiment or the microbial inoculum in any embodiment in preparing food.
In some embodiments, the food product is an animal food product. The animals are the same as those in the corresponding embodiments described above, and will not be described in detail.
On the other hand, the embodiment of the invention also provides the application of the pediococcus acidilactici and/or the metabolite thereof in any embodiment or the microbial inoculum in any embodiment in the feeding of animals.
In some embodiments, the animal is as described in the corresponding examples above, and will not be described in detail.
On the other hand, the embodiment of the invention also provides the application of the pediococcus acidilactici and/or the metabolite thereof in any embodiment or the microbial inoculum in any embodiment in the preparation of products with at least one of the functions (1) to (3): (1) enhancing immunity; (2) preventing, treating or adjunctively treating diarrhea; (3) antioxidant.
In some embodiments, the subject to which the product is administered comprises an animal. The animals are the same as those in the corresponding embodiments described above, and will not be described in detail.
In addition, the embodiment of the invention also provides the application of the pediococcus acidilactici and/or the metabolite thereof in any embodiment or the microbial inoculum in any embodiment in preparing antibacterial products.
By "bacteriostatic" herein is understood that inhibits or prevents the growth, reproduction, colonization and/or activity of bacteria.
In some embodiments, the bacteriostatic comprises: inhibiting any one or more of E.coli, salmonella, staphylococcus aureus, pseudomonas aeruginosa, and Listeria monocytogenes.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
1. Isolation and identification of Pediococcus catus
1.1 Medium for isolation: MRS medium, medium composition is as follows.
Sterilizing at pH 6.2-6.5 and 121 for 15min.
1.2 separation method
(1) Sample collection and microorganism separation: fecal samples were collected from adult chinese rural cats in the chinese Qingdao i.e. the ink farm laboratory. Fecal samples were collected by rectal sampling with sterile swabs and placed into 50mL sterile screw capped tubes containing 10mL of saline. Transported to Qingdao university animal nutrition laboratory under refrigeration condition, immediately subjected to gradient dilution, three plates per gradient, separated and cultured by pouring method, and cultured in 37 inverted manner.
(2) And (3) separating and purifying: taking the MRS flat plate with various bacteria, selecting single colony of lactic acid bacteria, and separating and purifying by adopting a continuous streaking method. Single colonies with different colony morphologies were purified. Repeating for 3-4 times, and performing microscopic examination until pure seeds are obtained.
(3) Bacterial colony characteristics were observed, and bacterial morphology was observed using gram staining and microscopy.
(4) Bacterial DNA was extracted according to the beijing tiangen biology company bacterial genome extraction kit instructions, using the general primers for bacteria: f (5'-AGAGTTTGATCCTGGCTCAG-3') and R (5'-TACGGCTACCTTGTTACGACTT-3') were amplified and sequenced, primer synthesis and sequencing were performed by Beijing, biotechnology, inc., and 16S rRNA sequences were compared for homology. The amplification system for 16S rDNA was as follows, and the total volume of the amplification system was 50. Mu.l:
| ddH 2 O | to 50μl |
| DNA plate | 2μl |
| Primer F | 2μl |
| Primer R | 2μl |
| 2xEs Taq MasterMix (with dye) | 25μl |
PCR reaction procedure: 94 pre-denaturation for 5min,94 denaturation for 45s,56 annealing for 30s,72 extension for 45s, total 35 cycles, and finally 72 extension for 10min, and preservation at 4 ℃.
1.3 isolation screening identification of Pediococcus acidilactici:
morphological observation, microscopic examination, enzymatic analysis and carbohydrate utilization experiments, combined with 16S rRNA sequencing alignment, identified as Pediococcus acidilactici, designated as CLP03, 16S rRNA gene sequences are shown below. After the bacterial strain is cultured for 24 hours, the middle of the bacterial colony is convex, the edge is neat, and the surface is smooth; facultative anaerobic, gram positive, globular. Colony morphology of CLP03 is shown in fig. 1 a, and microscopic morphology is shown in fig. 1B.
1.3.1 The 16S rRNA gene sequence (5 '-3') is shown as SEQ ID NO:1 is shown as follows:
AGTCGAACGAACTTCCGTTAATTGATCATGAGGTGCTTGCACTGAATGAGATTTTAACATGAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGCAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCCTGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGATGCGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTGAGAGTAACTGTTCACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAACCTAAGAGATTAGGCGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAAACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCT。
1.3.2 evolutionary tree
Phylogenetic analysis was performed on the 16S rRNA genes of CLP03 to determine their close relationship with Pediococcus acidilactici, and the evolutionary tree was shown in FIG. 2.
2. Growth characteristics
2.1 physiological and Biochemical Properties
And taking the separated bacteria liquid after the activation culture for 18 hours, and carrying out the identification of the physiological and biochemical characteristics of the lactic acid bacteria by referring to the Bojie's bacteria identification manual, the common bacteria system identification manual and the lactic acid bacteria classification identification and experimental method, and carrying out the identification of the biochemical characteristics such as a gelatin test, a V-P test, a Methyl Red (MR) test, a sugar fermentation test and the like.
2.2 determination of growth Property and acid production Property
Inoculating the separated bacteria solution after activation culture for 18 hr into MRS culture medium with 2.0% of inoculum size, culturing at 37 constant temperature for 24 hr, and controllingThe group is a liquid culture medium without strain liquid. The absorbance OD values at 600nm of the culture solutions were sampled and measured at 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 48 hours, and the pH values were sampled and measured at 1, 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 48 hours. Time is taken as an abscissa, and pH and OD of the culture solution are taken as 600 The values are on the ordinate, and the growth curve and the acid production curve are plotted.
2.3 high temperature resistance test
And (3) taking the separated bacterial liquid after the activation culture for 18 hours, respectively treating the bacterial liquid in water baths at 50 ℃,60 ℃,70 ℃,80 ℃ and 90 ℃ for 5min, and immediately placing the treated bacterial liquid in an ice box. The viable count was calculated by plate counting by applying 100. Mu.L of the culture medium after gradient dilution, and the survival rate was calculated by taking the viable count of 0h as a control.
Survival surviving (%) =t treatment 1 /T initial 1 ×100％；
Wherein T is initial 1 And T treatment 1 The number of surviving bacteria (log CFU/mL) was 0min and 5min, respectively.
2.4 acid resistance test
The separated bacteria liquid after the activation culture for 18 hours is inoculated into MRS liquid culture media with initial pH of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 respectively by an inoculum size of 2.0 percent, and the culture is carried out at a constant temperature of 37. OD was measured at 3, 6, 9, 12, 18, 24, 30, 36, 42h, respectively 600 Values, 3 in parallel per group. And after the measurement is completed, taking 3 groups of parallel test results to calculate an average value, and drawing a curve according to the calculated results.
2.5 gastrointestinal tract colonization ability
2.5.1 test for resistance to intestinal juice of Artificial stomach
The preparation of the artificial gastric juice and the artificial intestinal juice is referenced in 2010 edition of Chinese pharmacopoeia.
The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 4,10000 rpm for 10min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), resuspended in artificial gastric juice and incubated at 37,200 rpm with stirring for 1.5h. After incubation, bacterial pellet was washed twice with sterile neutral PBS buffer (pH 7.0), centrifuged at 10000rpm and 4 for 10min, resuspended in artificial intestinal fluid and incubated for 2h with stirring at 37, 200 rpm. Bacterial strain viability was calculated by plate count dilution before and after incubation in artificial gastric juice and intestinal juice and repeated 3 times. Survival was calculated using the following formula:
Survivability(％)＝T treatment 2 /T initial 2 ×100％；
wherein T is initial 2 And T treatment 2 The number of surviving bacteria (log CFU/mL) before and after incubation in artificial gastric or intestinal fluid, respectively.
2.5.2 acid and bile salt resistance test
The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 4,10000 rpm for 10min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), and resuspended in sterile PBS buffer (pH 2.5). After incubation for 0 and 3h at 37, serial dilutions of the cultures were plated on MRS agar plates and then incubated for 24h at 37, and the test repeated 3 times.
The isolated bacterial solution after 18h of activation culture was taken, centrifuged at 4,10000 rpm for 10min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), and resuspended in PBS buffer containing 0.3% and 0.5% (w/v) pig bile salt (Solarbio, china). After incubation for 0 and 4h at 37, serial dilutions of the cultures were plated on MRS agar plates and then incubated for 24h at 37, and the test was repeated 3 times. Survival was calculated using the following formula:
Survivability(％)＝T treatment 3 /T initial 3 ×100％；
wherein T is initial 3 And T treatment 3 The number of surviving bacteria (log CFU/mL) before incubation (0 h) and after incubation (3 h/4 h), respectively.
2.5.3 determination of surface hydrophobicity
Bacterial hydrocarbon compound adhesion (bacterium adhesion to hydrocarbons, BATH) was used to react strain tables by affinity of lactic acid bacteria for hydrocarbon compoundsHydrophobic properties of the facets. Washing the separated bacterial liquid (10000×g,10min, 4) after activation culture for 18 hr with sterile neutral PBS buffer solution (pH 7.0) for 3 times, and regulating bacterial liquid OD 600 The value of the catalyst is 0.25+/-0.05 (A) 1 ) And (5) standby. Then, xylene and chloroform were added in a ratio of 1:1, vortexed for 3min, and then incubated at 37℃for 3h. Finally, the aqueous phase was carefully absorbed and the OD was measured 630 Value (A) 2 ). The test was repeated 3 times. The formula for calculating the hydrophobicity is as follows:
CSH(％)＝(1-A 2 /A 1 )×100％；
wherein CHS is the hydrophobicity, A 1 And A 2 Absorbance before and after incubation for 3h, respectively.
2.5.4 self-cohesiveness determination
The isolated strain broth (10000 Xg, 10min, 4) cultured for 18h was washed 2 times with sterile neutral PBS buffer (pH 7.0) and then resuspended in the same volume of sterile neutral PBS buffer (pH 7.0) to measure OD 600 Value (A) 3 ) After 10s vortex shaking, 37 incubated for 8h, stirring was avoided, the upper part was carefully absorbed, and absorbance was measured at 600nm (a 4 ) The test was repeated 3 times.
The automatic aggregation rate is calculated as follows:
Autoaggregation ability(％)＝(1-A 4 /A 3 )×100％；
wherein A is 3 And A 4 Absorbance before and after 8h incubation, respectively.
2.5.5 cell adhesion test
2.5.5.1 cell climbing tablet
Caco-2 cells were cultured in a cell culture flask containing MEM broth (HyClone, USA) of 1.0% (w/v) nonessential amino acids and 20% (v/v) fetal bovine serum (Gibco, USA), and 100. Mu.g penicillin and 100. Mu.g streptomycin (Sigma, USA) were added. When the cells are spread in the space of about 90% of the cell bottle, 1mL of 0.25% trypsin-EDTA digestion solution (Solarbio, china) 37 is added for digestion for 5min, and the mixture is added with culture medium and blown to be uniformly prepared into a cell suspension (5×10) 4 CFU/mL), spreading on 24-well plate with glass climbing plate, culturing to compact monolayer, discarding culture solution, and sterilizingAfter 3 times washing with neutral PBS buffer (pH 7.0), 1mL of the isolated bacterial liquid (the bacterial liquid concentration is adjusted to 1X 10) was added to each well for 18 hours of activation culture 8 CFU/mL) and 1mL of cell culture broth, 37 for 2h. The mixed liquid in the wells was aspirated, washed 3 times with sterile neutral PBS buffer (pH 7.0), and then an appropriate amount of paraformaldehyde was added to each well for fixation for 30min. The paraformaldehyde in the wells was aspirated, washed 3 times with sterile neutral PBS buffer (pH 7.0), and then gram stained and observed under a microscope.
2.5.5.2 plate coating method
Digesting Caco-2 cells cultured in the flask to prepare 5×10 cells 5 The cell suspension of/mL is inoculated into a 24-hole cell culture plate to be cultured to form monolayer cells. After the cells which had grown into monolayers were washed 2 times with MEM, 100. Mu.L of the bacterial suspension (1X 10) was added to each well 8 CFU/mL) and 900uL MEM broth (without antibiotics) were placed in 37, 5% CO 2 Culturing in a carbon dioxide incubator with RH90% for 90min. After the end of the incubation, the monolayer of cells was washed 5 times with PBS to remove non-adherent bacteria. 0.5mL of PBS solution containing 0.05% (v/w) of triton X-100 was added, and the cell lysate containing the cells after 20min treatment of 37 lysed cells was spread on MRS agar plates after appropriate dilution, and counted after 37 culture for 48 hours. 3 replicates were run for each experiment and adhesion was calculated according to the following formula:
adhesion rate (CFU/100 cells) =number of adherent bacteria (CFU)/number of adherent cells.
2.6 bacteriostasis test
The antibacterial performance of the strain is measured by an oxford cup agar diffusion method. Taking fresh bacterial liquid cultured for 18 hours, centrifuging at 4,8000 rpm for 10min, taking supernatant for later use, and respectively taking 200 mu L of escherichia coli ATCC25922; salmonella ATCC14028; staphylococcus aureus ATCC25923; pseudomonas aeruginosa ATCC27853; the fresh bacterial liquid of the listeria monocytogenes ATCC19115 is uniformly coated on the LB solid medium. After the bacterial liquid is completely absorbed, 3 oxford cups are placed on each flat plate in a shape like a Chinese character 'ji', 200 mu L of supernatant is added into each oxford cup, the flat plates are placed in a 37 constant temperature incubator for culture, after 48 hours, whether a bacteriostasis ring is formed is observed, and the diameter of the bacteriostasis ring is accurately measured.
2.7 antibiotic susceptibility test
The antibiotic susceptibility of the strains was determined by means of a paper sheet diffusion method. According to the recommendations for evaluating the safety of probiotics in combination with antibiotics common on the market, 30 antibiotics were selected for testing in the form of 6mm paper sheets (BIO-KONT, china): penicillin, oxacillin, ampicillin, piperacillin, cefalexin, cefazolin, cefuroxime sodium, ceftazidime, ceftriaxone, cefoperazone, imipenem, vancomycin, streptomycin, gentamicin, amikacin, kanamycin, tetracycline, chloramphenicol, minocycline, doxycycline, compound neonomine, azithromycin, lincomycin, polymyxin B, florfenicol, erythromycin, clindamycin, norfloxacin, ciprofloxacin, and levofloxacin.
The isolated bacterial solution (10000 Xg, 10min, 4) after 18h of activation culture was taken, washed 2 times with sterile neutral PBS buffer (pH 7.0) and then resuspended in sterile neutral PBS buffer (pH 7.0) to obtain a bacterial solution in logarithmic growth phase (0.5 McFarland suspension). 100uL of McFarland suspension of the isolated strain was plated on MRS agar plates. And finally, sticking an antibiotic paper sheet on the surface of an MRS agar plate within 15min, culturing the plate at a constant temperature of 37 ℃ for 48h, observing whether a bacteriostasis ring is formed or not, and measuring the diameter of the bacteriostasis ring by using a vernier caliper.
2.8 free radical scavenging test
The DPPH removing capacity of the isolated strain is determined by using a Soxhlet DPPH detection kit, and according to the operation of the specification, in short, 100 mu L of the isolated strain bacterial liquid cultured for 18 hours is sucked, 900 mu L of the extracting solution is added, vortex oscillation and mixing are carried out, centrifugation is carried out at 10000rpm at room temperature for 10min, 25 mu L of the supernatant is taken and added into 975 mu L of the DPPH working solution, vortex mixing is carried out, and the mixture is kept at rest for 30min at room temperature in a dark place. Absorbance was recorded at 515nm on a microplate reader.
The DPPH radical scavenging rate was calculated as follows:
wherein: a is that assay 1 Absorbance of the sample to be measured; a is that control 1 Absorbance of the supernatant and absolute ethanol mixture for isolation of the strain; a is that blank 1 Absorbance is the absorbance of the mixture of the extract and the working solution.
2.9 safety test
2.9.1 hemolysis experiments
The bacterial isolates after 18h of activation culture were streaked onto Tryptic Soy Agar (TSA), containing 5.0% (w/v) sheep blood (Oxoid, germany), the plates were incubated at 37 for 48h and the blood agar plates were checked for signs of beta-hemolysis (clear area around colonies), alpha-hemolysis (green area around colonies) or gamma-hemolysis (no area around colonies). Staphylococcus aureus (ATCC 25923) strain was used as a positive control.
2.9.2 safety test of mice
Grown healthy, body weight-approximated Kunming mice 80 were randomly divided into 4 groups of 20 male and female halves. The control group (CK) mice are filled with 0.2mL of sterile physiological saline, and the other three groups of mice are respectively filled with 0.2mL of separated bacterial solutions with different concentrations, wherein the separated bacterial solutions are respectively as follows: 1X 10 9 (LC03),1×10 10 (MC 03) and 1×10 11 (HC 03) CFU/mL. Body weight and feed intake were measured every 3d and the health status of mice was recorded. After a duration of 27d, the mice were fasted for 12h and then anesthetized with 1% sodium pentobarbital (50 mg/kg). The abdominal aorta was sampled to collect blood and serum was collected by centrifugation (4000 rpm,4 ℃,10 min) for further analysis. After the mice were sacrificed, visceral toxicity was anatomically observed in each group of mice, and organs such as heart, liver, kidney, spleen and thymus were collected and weighed, and the organ index was calculated as organ weight/body weight×100.
Aspartic acid Aminotransferase (AST), alanine Aminotransferase (ALT), total Bilirubin (TBIL), direct Bilirubin (DBIL), urea nitrogen (BUN), malondialdehyde (MDA) and superoxide dismutase (SOD) levels in mouse serum were determined using commercial ELISA kits (south kyo bioengineering limited, china). Measured by an enzyme-labeled instrument (Oriental, china) or a biochemical automatic analyzer (Selectra-E, netherlands).
2.10 Whole genome sequencing
Combined sequencing was performed using the Illumina high throughput sequencing platform NovaSeq 6000, and genome assembly was performed using Unicycler (version: 0.5.0, https:// gitub. Com/rrwick/Unicycler). The assembled genome was predicted for the coding gene by Prokka (version: 1.14.6, https:// gitsub.com/tseemann/Prokka) software. Predicted gene sequences were analyzed using COG, KEGG, uniProt and RefSeq blast+ (version: 2.11.0 +) and compared to these functional databases to obtain gene function annotation results. Functional annotation was performed using software Hmmer (version: 3.3.2) based on Pfam and TIGERFAMs databases, and genome loop maps were drawn using R packages.
3. Results and analysis
3.1 Growth characteristics
3.1.1 physiological and Biochemical Properties
The results of the partial physiological and biochemical identification of strain CLP03 are shown in table 1. Pediococcus acidilactici CLP03 may utilize glucose, cellobiose, galactose, maltose, fructose, arabinose and xylose. The MR test was positive, and the remaining test results were negative.
TABLE 1 physiological and biochemical characteristics of Pediococcus acidilactici CLP03
Note that: "+" represents positive; "-" represents negative.
3.1.2 determination of growth Property and acid production Property
As can be seen from FIG. 3, the growth is slow and is in a growth retardation region 0-3h after inoculation; after 3 hours, bacterial liquid OD 600 The value rises rapidly, and enters the logarithmic growth phase; bacterial liquid OD after 20 hours 600 The value is gradually flattened, the strain reaches the growth stabilization period, the strain activity is strong, and the number of viable bacteria reaches a peak. The pH change trend of the culture solution is consistent with the growth curve, and the pH of the culture solution slowly drops within 3 hours after the pediococcus acidilactici CLP03 is inoculated; after 3h, the phase enters into logarithmic phase, and the pH value decreases at a rateAccelerating, the pH change of the bacterial liquid after 20 hours is gradually gentle, and the bacterial liquid is stabilized at about 3.68.
3.1.3 resistance of strains to high temperatures
As can be seen from fig. 4, the survival rate below 60 can be more than 70%, the survival rate of 70 is close to 50%, the survival rate of 80 is more than 40%, and the survival rate of 90 is more than 30%, which indicates that the pediococcus acidilactici CLP03 has better high temperature resistance.
3.1.4 resistance of strains to acids
As can be seen from FIG. 5, the strain CLP03 was completely inhibited from growing at pH 3.0 or less, and OD was increased with the time of cultivation 600 The values were unchanged. At ph=4.0, the strain grew slowly, but OD 600 Values significantly below ph=5.0, 6.0, 7.0. At pH not less than 5.0, the strain CLP03 can grow normally, but OD when reaching the stationary phase 600 There is a certain difference in value, in which OD 600 Ph=7.0>pH＝6.0>ph=5.0, viable count and OD 600 The value change trend is consistent.
3.2 colonization ability of strains in the gastrointestinal tract
3.2.1 tolerance of Strain to Artificial gastrointestinal fluids
As can be seen from FIG. 6, pediococcus acidilactici CLP03 has strong survival ability in artificial gastrointestinal fluids. The survival rate of the strain after being inoculated in the artificial gastric juice for 1.5 hours is 54.81 percent, and the number of viable bacteria is about 3.93 multiplied by 10 4 CFU/mL; the survival rate of the strain after being inoculated in the artificial intestinal juice for 2 hours is 93.65 percent, and the number of viable bacteria is about 5.67 multiplied by 10 7 CFU/mL, survival rate is 52.45% after artificial gastrointestinal fluid is digested for 3.5h, and viable count is about 5.24X10 4 CFU/mL。
3.2.2 tolerance of strains to acids and bile salts
As can be seen from FIG. 7, the strain CLP03 has a strong tolerance to bile salts and acids. The survival rate of the strain after 3 hours of inoculation in MRS liquid culture medium with pH=2.5 is 59.75%, and the number of viable bacteria is about 3.77×10 5 CFU/mL. The survival rate of the strain after being inoculated in MRS liquid culture medium with 0.3 percent of bile salt concentration for 4 hours is 95.78 percent, and the number of viable bacteria is about 9.31 multiplied by 10 7 CFU/mL; the survival rate of the strain after being inoculated in MRS liquid culture medium with 0.5 percent of bile salt concentration for 4 hours is 88.12 percent, and the number of viable bacteria is about 8.59 multiplied by 10 6 CFU/mL。
3.2.3 surface hydrophobicity and self-condensing Property of Strain
Bacteria are generally set to be highly hydrophobic by a surface hydrophobicity definition standard with CSH% >50%, moderately hydrophobic between 20% and 50% and non-hydrophobic with CSH% < 20%. Self-agglomerating ability is generally divided into: low (16% -35%), medium (35% -50%) and high (more than 50%). As can be seen from Table 2, pediococcus acidilactici CLP03 has high hydrophobicity and high self-coagulation performance.
TABLE 2 surface hydrophobicity and self-aggregation Rate of Pediococcus acidilactici CLP03
3.2.4 adhesion ability of Strain to intestinal epithelial cells
The staining results are shown in FIG. 8, and the adhesion results of the plating method show that the adhesion of Pediococcus acidilactici CLP03 to cells is 102.67 cells/cell, which is stronger than that of general lactic acid bacteria.
3.3 antibacterial Properties of Strain
The diameter of the oxford cup is 8mm, so that the diameter of the bacteriostasis ring is set to be more than 9mm and is not more than 9mm, and the oxford cup is regarded as having no bacteriostasis. From Table 3, the fermentation supernatant of the strain CLP03 has good inhibition effect on escherichia coli, salmonella and staphylococcus aureus, and has the strongest antibacterial activity on staphylococcus aureus and listeria monocytogenes, and the diameter of a bacteriostasis ring is larger than 21mm; secondly, salmonella, the diameter of the inhibition zone is larger than 17mm; the diameter of the colibacillus inhibition zone is larger than 16mm; the diameter of the pseudomonas aeruginosa is also about 14 mm.
TABLE 3 antibacterial Activity of Pediococcus acidilactici CLP03
Note that: diameter of zone of inhibition (mm): +++:21-26mm; ++:15-20mm; +:9-14mm; -: there is no inhibition zone.
3.4 antibiotic sensitivity of Strain
The diameter of the drug sensitive tablet is 6mm, so that the antibacterial effect is realized when the diameter of the antibacterial ring is larger than 7mm, and the antibacterial effect is not realized when the diameter is smaller than or equal to 7 mm. As can be seen from Table 4, the strain CLP03 has different sensitivities to various common antibiotics. The sensitivity to imipenem is strong, and the diameter of the inhibition zone is larger than 23mm; is sensitive to penicillin, ampicillin, cefazolin, ceftazidime, ceftriaxone, cefoperazone, minocycline, erythromycin, azithromycin, chloramphenicol, clindamycin, doxycycline and florfenicol, and the diameter of a bacteriostasis ring is larger than 15mm; lincomycin has weak sensibility, and the diameter of the inhibition zone is in the range of 7-14mm; the composition is insensitive to oxacillin, piperacillin, cefalexin, cefuroxime sodium, amikacin, gentamicin, kanamycin, streptomycin, tetracycline, norfloxacin, ciprofloxacin, vancomycin, polymyxin B, compound neonomine and levofloxacin.
TABLE 4 sensitivity of Pediococcus acidilactici CLP03 to commonly used antibiotics
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Note that: diameter of zone of inhibition (mm): +++:23-30mm; ++:15-22mm; +:7-14mm; -: there is no inhibition zone.
3.5 free radical scavenging ability of Strain
The experiment tests the DPPH scavenging ability of the strain CLP03 thallus, and the DPPH free radical scavenging rate of the strain CLP03 is 53.55 percent according to the conversion of the measurement result, which shows that the strain CLP03 has better oxidation resistance.
3.6 safety test results
3.6.1 hemolysis experiment results
The positive control (left half of FIG. 9) was treated with Staphylococcus aureus, and apparent beta-hemolysis (clear area around colony) was observed, but CLP03 was gamma-hemolysis (no area around colony, right half of FIG. 9), and it was possible to preliminarily determine whether the strain CLP03 was safe and nonpathogenic.
3.6.2 feeding mice experimental results
All mice are clinically observed to have good mental condition, can eat and drink water normally, have no abnormal feces, have no diarrhea, and have no death during the test. After the test is finished, the viscera of the strain CLP03 are observed through sectioning, and no abnormal pathological and histological changes and bacterial translocation are found, so that the strain CLP03 is safe and has no toxic or side effect on mice.
3.6.2.1 effects of probiotic CLP03 fed on growth and feeding of mice
Body weight, food intake and organ index are common indicators for assessing animal health, and are used to assess the safety of isolated strains. First, the effect of CLP03 supplementation on mouse body weight and food intake was observed (fig. 10). The results show that the addition of CLP03 has no adverse effect on the weight and the food intake of mice, as shown by no significant differences among the weights, average Daily Gain (ADG) and Average Daily Food Intake (ADFI) of mice supplemented with different doses of CLP03 and the CK groups.
3.6.2.2 effects of probiotic CLP03 fed on organ index in mice
Figure 11 shows the effect of feeding probiotic CLP03 on organ index in mice. There were no significant differences in cardiac, liver, spleen and kidney indices between groups except thymus. The thymus index was significantly higher in both HC03 and MC03 groups than in CK group (P < 0.05). Thymus index is a marker of immune system development, and is significantly improved in HC03 and MC03 mice, which suggests that the strain CLP03 may promote development of the host immune system, thereby having health and probiotic effects on animals.
3.6.2.3 influence of probiotic CLP03 fed to mice on serum Biochemical index
Figure 12 shows the effect of CLP03 feeding on the biochemical index of blood in mice. The AST, ALT, T-BIL, I-BIL and D-BIL contents of the groups are not obviously different. But the BUN content was significantly lower in HC03 group than in CK group (P <0.05, C in fig. 12).
3.6.2.4 influence of probiotic CLP03 fed to mice on antioxidant index
Figure 13 shows the effect of feeding probiotic CLP03 on the antioxidant index of mice. The serum SOD levels were significantly higher in mice in HC03 group than in CK group (P <0.01, a in fig. 13). Furthermore, HC03 and MC03 groups mice had significantly lower serum MDA levels than the CK group (P <0.001, B in fig. 13). These results indicate that the supplementation of CLP03 has no negative effect on the liver and kidney function index of mice, and has an important role in reducing the body damage caused by oxidative stress.
3.7 results of full genome sequencing of Strain CLP03
To understand the characteristics of probiotics and explore the probiotic potential of CLP03, whole genome sequencing was performed. The results of the complete circular genomic profile of CLP03 are shown in fig. 14. The complete genome of CLP03 consists of a 2.07Mbp circular chromosome and a circular plasmid, with (g+c) contents of 42.20% and 40.1%, respectively, and genome sizes of 2,014,515bp, belonging to a medium-sized genome, these bacteria are generally very metabolic, tolerant and adaptable. GOC, GO and KEGG results of the strain CLP03 genome are shown in FIGS. 15-17.GOC annotation results show that CLP03 is mainly enriched in carbohydrate transport metabolism, translation, ribosome structure and biosynthesis, and the main functions are predicted in transcription and amino acid transport metabolism; GO annotation results show that the biological process (biological process) is enriched with the translation, transcriptional regulation and phosphoenolpyruvate dependent sugar phosphotransferase system, respectively; the first three of the cellular components (cellular component) are membrane integral components, cytoplasmic and cytoplasmic membranes, respectively; the first three of the molecular functions (molecular function) are ATP binding, DNA binding and hydrolase activity, respectively; KEGG notes the first five signal paths: global and overview (Global and overview maps, 391), carbohydrate metabolism (Carbohydrate metabolism, 185), transmembrane transport (Membrane transport, 104), transcription (Transcription, 79) and nucleotide metabolism (Nucleotide metabolism, 67). In terms of drug resistance genes, no CLP03 strain was found to have drug resistance genes, indicating that feeding the strain was not at risk of drug resistance gene transfer to the host, further indicating that the strain was safe.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Pediococcus acidilactici, characterized in that Pediococcus acidilactici (Pediococcus acidilactici) CLP03 is deposited with the China center for type culture Collection with the deposit number: cctccc NO: m2023402.
2. A method for producing pediococcus acidilactici, comprising: culturing Pediococcus acidilactici according to claim 1.
3. A microbial agent comprising the pediococcus acidilactici of claim 1.
4. An additive characterized in that the active ingredients thereof comprise: pediococcus acidilactici and/or the metabolite thereof according to claim 1 or the microbial inoculum according to claim 3.
5. A food comprising the additive according to claim 4.
6. The food product according to claim 5, characterized in that the food product is an animal food product;
optionally, the food product further comprises: a basal feed.
7. Use of pediococcus acidilactici and/or a metabolite thereof according to claim 1 or a microbial agent according to claim 3 for the preparation of a food product;
optionally, the food is an animal food;
optionally, the animal comprises a pet;
optionally, the pet comprises: a cat.
8. Use of pediococcus acidilactici and/or a metabolite thereof according to claim 1 or a microbial agent according to claim 3 in feeding animals;
optionally, the animal comprises a pet;
preferably, the pet comprises: a cat.
9. Use of pediococcus acidilactici and/or a metabolite thereof as defined in claim 1 or a microbial inoculum as defined in claim 3 for the preparation of a product with at least one of the functions (1) to (3): (1) enhancing immunity; (2) preventing, treating or adjunctively treating diarrhea; (3) antioxidant;
optionally, the subject to which the product is administered comprises an animal;
optionally, the animal comprises a pet;
optionally, the pet comprises: a cat.
10. Use of pediococcus acidilactici and/or a metabolite thereof according to claim 1 or a fungicide according to claim 3 for the preparation of a bacteriostatic product;
optionally, the bacteriostasis includes: inhibiting any one or more of E.coli, salmonella, staphylococcus aureus, pseudomonas aeruginosa, and Listeria monocytogenes.
Priority Applications (1)
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| CN116590172A (en) * | 2023-03-28 | 2023-08-15 | 青岛农业大学 | Pediococcus acidilactici GLP06, application and product thereof |
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