CN115747111A - Pediococcus pentosaceus and application thereof - Google Patents
Pediococcus pentosaceus and application thereof Download PDFInfo
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- CN115747111A CN115747111A CN202211489854.1A CN202211489854A CN115747111A CN 115747111 A CN115747111 A CN 115747111A CN 202211489854 A CN202211489854 A CN 202211489854A CN 115747111 A CN115747111 A CN 115747111A
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- pediococcus pentosaceus
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- pentosaceus
- pediococcus
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Abstract
The invention discloses Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 and application thereof. The strain is separated from traditional fermented food (vinegar pepper) collected from Lizhai, riping county, guizhou province, has good growth on an MRS agar culture medium, no hemolysis phenomenon, sensitivity to various common types of antibiotics, better tolerance to acid, bile salt, artificial gastric juice and artificial intestinal juice, strong adhesion to human colon cancer cells HT-29, stronger acetic acid and lactic acid production capacity, certain potential for reducing blood sugar and cholesterol, stronger inhibition capacity to Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli, better prevention and treatment effects on DSS-induced mouse colitis models and obviously reduced inflammation indexes. Therefore, the strain can be applied to the field of functional foods of human and animals.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to pediococcus pentosaceus with strong antibacterial and anti-inflammatory effects, and particularly relates to the field of production and development of functional lactic acid bacteria and products thereof.
Background
Intestinal microorganisms are considered "important organs to be ignored". The organism provides a suitable living environment for a wide variety of intestinal microorganisms, and the intestinal microorganisms can regulate the metabolism and the immune function of a host, so that the intestinal microorganisms and the host become a coordinated whole. Probiotics refer to a class of active microorganisms that can regulate the microenvironment of the intestinal tract of a host, produce probiotic substances, and maintain the health of the organism. Lactic Acid Bacteria (LAB) are a widely recognized probiotic bacterium that can utilize fermentable carbohydrates to produce large amounts of lactic acid, primarily in rod, pellet, chain or palisade forms. Lactic acid bacteria in nature are various in types and widely distributed. In recent years, research on lactic acid bacteria has been enriched, and attention has been paid to the field of disease control in humans and animals. Inflammatory Bowel Disease (IBD) is a chronic intestinal disease threatening the health of modern human beings and animals, and the cause of IBD is complex and mainly caused by factors such as genetics, environment and flora. Common clinical manifestations of the disease include weight loss, diarrhea, abdominal pain and bloody stools, with an increased potential for cancer. Because the occurrence reason is complex, the development process is difficult to search, and the radical cure cannot be achieved. As an adjunct to traditional medicines, probiotic preparations are gaining increasing attention in the prevention and treatment of inflammatory bowel disease. Currently, active development of novel probiotic bacterial strains has become a worldwide trend.
The fermented food is always a valuable source for developing novel probiotics, the food source lactic acid bacteria not only have higher safety, but also have better properties than other sample sources, and the fermented food utilizes microorganisms to be attached to the surface of vegetables so as to carry out natural fermentation. In China, fermented foods have a long eating tradition, and vegetables, meat, milk products and the like in human daily food can be fermented by utilizing lactic acid bacteria to produce foods with special flavor and probiotic characteristics. Bacterial activity directly affects the final quality and flavor of the fermented food product. Although various microorganisms are involved in food fermentation, studies have shown that lactic acid bacteria are the main role of the microorganisms. The number and kind of lactic acid bacteria are small in the early stage of fermentation, and they are dominant over time. The lactobacillus can generate organic acid in the growth and metabolism to regulate the surrounding environment and reduce the pH value of intestinal microecology, thereby inhibiting the growth of pathogenic bacteria. In addition, the produced antimicrobial active substances such as bacteriocin can also inhibit the growth of saprophytic bacteria and pathogenic bacteria. Some lactic acid bacteria can bring health efficacy to a host by generating functional active factors, regulating intestinal flora and regulating the brain and intestinal axis.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a pediococcus pentosaceus strain with strong antibacterial and anti-inflammatory effects and application thereof.
The technical scheme of the invention is as follows: a Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620, which is preserved in China general microbiological culture Collection center (CGMCC) 10/13/2022, with the address: no. 3 of Xilu No.1 of Beijing, chaoyang, the institute of microbiology of the Chinese academy of sciences, the collection number is CGMCC No.25906.
Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 for inhibiting Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni or Campylobacter coli for non-disease treatment purposes.
Use of Pediococcus pentosaceus GZ0620 for the manufacture of a medicament for the treatment of a disease caused by Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni or Campylobacter coli.
Application of Pediococcus pentosaceus GZ0620 in preparing anti-enteritis medicine is provided.
Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 in a functional food for modulating the intestinal flora of a human or an animal.
An application of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 in preparing medicines for reducing blood sugar or cholesterol is provided.
The pediococcus pentosaceus GZ0620 is separated from traditional fermented food (hot and sour pepper) of fruit Lizhai in Riping county, guizhou province. The pediococcus pentosaceus GZ0620 grows well on an MRS agar culture medium, the bacterial colony is milky white, the surface is smooth, the edge is neat and opaque, the bacterial morphology is subjected to microscopic examination, and the gram stain is purple and spherical. A target gene sequence consisting of 1010 base pairs (bp) is obtained by using 27F/1492R of bacterial universal primers 16S rRNA to perform PCR amplification on the total genome DNA of the bacterial universal primers as a template, and the sequence is shown as SEQ ID No. 1. The gene sequence obtained by sequencing is input into an NCBI database for comparison, the similarity rate of the gene sequence and a standard strain Pediococcus pentosaceus strain FB181 in GenBank reaches 99.43 percent, and the strain can be preliminarily identified as Pediococcus pentosaceus.
Acid-resistant, cholate-resistant and adhesion tests show that the pediococcus pentosaceus GZ0620 has better tolerance to acid and cholate and stronger adhesion to human colon cancer cells HT-29.
The pediococcus pentosaceus GZ0620 has strong inhibitory activity on various pathogenic bacteria, and can inhibit the growth of Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli.
Animal experiments show that the pediococcus pentosaceus GZ0620 has better prevention and treatment effects on a DSS mouse colitis model, obviously reduces the levels of various inflammatory factors, and has better regulation effect on intestinal flora.
Compared with the prior art, the invention has the following beneficial effects:
1. the pediococcus pentosaceus GZ0620 provided by the invention is separated and screened from Guizhou acid peppers, grows well on an MRS agar culture medium, has certain tolerance capacity to acid and bile salt, and has strong adhesion capacity to human colon cancer cells HT-29.
2. Pediococcus pentosaceus GZ0620 has strong bacteriostatic ability on common pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli.
3. Animal experiments show that the pediococcus pentosaceus GZ0620 has better prevention and treatment effects on a DSS mouse colitis model, and the inflammation index is obviously reduced. Therefore, the method is applied to the field of functional foods, not only has actual production value, but also has very important significance on human and animal health.
Preservation information:
pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620, deposited in the China general microbiological culture Collection center at 10 months and 13 days 2022, address: no. 3 of Xilu No.1 of Beijing, chaoyang, the institute of microbiology of the Chinese academy of sciences, the collection number is CGMCC No.25906.
Drawings
FIG. 1 phylogenetic relationship of Pediococcus pentosaceus GZ0620 with other strains;
FIG. 2 colony morphology (A) and gram stain (B) of Pediococcus pentosaceus GZ0620 strain on MRS agar medium;
FIG. 3 hemolytic experiment of Pediococcus pentosaceus GZ0620 strain;
FIG. 4 is a chromatogram of total ion current measured by GC-MS of short-chain fatty acid of fermentation liquor of Pediococcus pentosaceus GZ0620 strain;
FIG. 5 shows spleen index measurement of DSS-induced colitis in mice, where A is the prevention phase and B is the treatment phase;
figure 6DSS induced colitis mouse intestinal flora diversity (a) and species composition (B).
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
The standard strain LGG in the present invention is Lactobacillus rhamnosus ATCC53103.
Example 1 isolation, identification and safety assessment of pediococcus pentosaceus GZ 0620:
1.1 isolation and identification of Pediococcus pentosaceus GZ 0620: the Guizhou pickled Chinese cabbage sample is collected from Lizhai fruit in Riping county of Guizhou province; a small amount of vinegar-pepper seed sample is taken and put into 50mL of MRS broth, and the mixture is fully shaken and mixed evenly and is placed into a constant temperature shaking table at 37 ℃ for 24h. The mixture is diluted by 10 times of gradient, spread and inoculated in MRS agar medium, cultured for 24h at 37 ℃, then single colony is picked up and purified for 3 times continuously. The purified strain is inoculated in 600 mu L of MRS broth culture medium, shake-cultured for 18h at 37 ℃, added with 400 mu L of sterile glycerol with the concentration of 50% (V/V) and frozen in a super-low temperature refrigerator at-80 ℃ for standby.
Performing activation culture on the cryopreserved strain, performing amplification culture by using an MRS broth culture medium, extracting strain DNA by using a Tiangen bacterium genome DNA extraction kit, and performing 16S rRNA amplification by adopting a colony PCR technology, wherein PCR primers are 16S rRNA universal primers 27F and 1492R and have the following sequences:
27F：5-AGAGTTTGATCMTGGCTCAG-3，1492R：5-GGTTACCTTGTTACGACTT-3
the sequencing of the PCR product is completed by the biological engineering (Shanghai) company Limited, and the 16S rRNA sequence is shown as SEQ ID No. 1. The sequence is compared with BLAST in NCBI, the similarity rate with a standard strain Pediococcus pentosaceus strain FB181 reaches 99.43 percent, the strain can be preliminarily identified as Pediococcus pentosaceus (Pediococcus pentosaceus), and the strain is named GZ0620. The phylogenetic relationship between the strain and other strains is shown in figure 1. The strain is preserved in China general microbiological culture Collection center at 10 months and 13 days in 2022, address: the collection number of the microbial research institute of Chinese academy of sciences is CGMCC No.25906, no. 3 of Xilu No.1 of Beijing, chaoyang, and Beijing.
The pediococcus pentosaceus GZ0620 is inoculated on an MRS agar medium and cultured for 24 hours at 37 ℃, and the morphology of a single colony is observed and recorded. Gram staining is carried out on the pediococcus pentosaceus GZ0620 by a kit method, and the stained bacteria form is observed and recorded under a microscope. Pediococcus pentosaceus GZ0620 grows well on the MRS agar medium, and the colony morphology is milky white, round and convex, the edge is flat, and the surface is smooth, as shown in A in figure 2. After gram staining, the cells were spherical and purple under an optical microscope, and the staining characteristics of gram-positive bacteria were consistent (fig. 2, B).
1.2 hemolytic and antibiotic resistance
The sensitivity of the strain to common antibiotics is tested by adopting a paper agar diffusion method. Activating and culturing Lactobacillus paracasei GZ0613 strain, and adjusting the concentration of the strain to 1 × 10 6 CFU/mL, uniformly smearing the bacteria liquid on the surface of an MRS culture medium flat plate by using a sterile cotton swab, placing drug sensitive paper sheets after 10min at room temperature, culturing for 24h at 37 ℃, measuring the diameter of a bacteriostatic ring around each drug sensitive paper sheet by using a vernier caliper, repeating each antibiotic for 3 times, judging the drug sensitivity of the strain according to the standard of the American clinical laboratory standards Committee (NCCLS) according to the test result, and expressing the result by sensitivity (S), medium (I) and drug resistance (R).
The results of the hemolytic experiment showed that no hemolysis occurred around the colonies (FIG. 3); the 6 antibiotics tested, tetracycline, ampicillin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol, were all shown to be sensitive (S) or intermediate (I), indicating the safety of the P.pentosaceus GZ0620 strain (Table 1).
TABLE 1 sensitivity of Pediococcus pentosaceus GZ0620 strain to 6 antibiotics
| Numbering | Tetracycline derivatives | Ampicillin | Ceftriaxone | KelinMycin | Clarithromycin | Chloromycetin |
| GZ0620 | I | I | I | I | S | S |
Example 2 detection of major fermentation characteristics of Pediococcus pentosaceus GZ0620
2.1, acid and bile salt resistance assessment: resuscitating and activating strain to be detected, namely Pediococcus pentosaceus GZ0620 on MRS agar plate for 3 generations, and adjusting the initial concentration of bacterial liquid to 1 × 10 6 CFU/mL. MRS broth medium was prepared with acidity (pH = 3.0) and bile salt concentration (0.3%) using hydrochloric acid and swine bile salt, respectively. 1mL of a test strain of Pediococcus pentosaceus GZ0620 was inoculated into a broth medium with pH =3.0 and cultured at 37 ℃ for 18 hours. After the culture, the centrifuge tube is shaken properly, 20 mul of the bacterial liquid is absorbed and spread on MRS agar plate culture medium, 3 times of the culture are set, and the culture is carried out for 24 hours at 37 ℃. And observing whether colonies grow on the surface of the MRS plate after culture. Similarly, 1mL of the strain GZ0620 bacterial liquid of the strain to be tested is inoculated into an MRS broth with 0.3% of cholate concentration, and after the strain is cultured for 18h at 37 ℃, the strain is coated on a flat plate and cultured for 24h at 37 ℃, and the growth condition of the bacterial colony is checked.
Acid resistance test results show that normal colonies can still grow on an MRS agar plate after the pediococcus pentosaceus GZ0620 is treated in an MRS broth with pH =3.0 for 18 hours; the results of the salt tolerance test show that the pediococcus pentosaceus GZ0620 can still grow normal colonies on an MRS agar plate after being tolerant for 18 hours in an MRS broth culture medium with 0.3 percent of bile salt concentration; the pediococcus pentosaceus GZ0620 has strong acid resistance and bile salt resistance.
2.2, cell adhesion test: making pentose tabletThe cocci GZ0620 are inoculated into MRS broth culture medium after being recovered and cultured for 24 hours at the constant temperature of 37 ℃. After the culture is finished, the mixture is placed at the temperature of minus 4 ℃ and centrifuged for 10min at the speed of 5000r/min, and is washed by sterile PBS buffer solution for a plurality of times. Adjusting the concentration of the bacterial suspension to 1 × 10 6 CFU/mL for use. Recovering human colon cancer cell HT-29, inoculating to cell culture plate, adding DMEM complete culture medium, and placing at 37 deg.C under CO 2 Culturing in 5% carbon dioxide incubator, and replacing culture solution every other day. When the cell attachment state reached 80%, digestion was performed using 0.25% trypsin-EDTA, and subcultured. After the culture, the cells were counted by a cell-blood counting plate, and the cell concentration was adjusted to 5X 10 6 one/mL. 1mL of the cell suspension was added to one of the culture wells of a six-well cell culture dish and placed in an incubator for culture. After the cells in the plates grew to a monolayer, the DMEM medium was discarded and each well was washed 3 times with sterile PBS. Adding 1mL of prepared bacterial suspension into the cell hole, slightly shaking the cell culture plate, sucking a small amount of bacterial liquid in the hole for flat plate counting, and taking the result as the initial viable count in the bacterial suspension. The cell plates were incubated at 37 ℃ for 2h, the medium was discarded and washed 3 times with sterile PBS buffer. Digesting the cells with 0.7mL0.25% trypsin-EDTA for 10min, adding 0.3mLDMEM culture medium to stop digestion after the cells completely fall off, collecting the culture medium after the adhesion experiment is finished, and counting by plate to obtain the number of adhesion viable bacteria. And the standard strain LGG was used as a control.
Adhesion rate (%) = number of lactic acid bacteria at end stage/number of initial lactic acid bacteria inoculation × 100%
The results are shown in Table 2. The adhesion rate of Pediococcus pentosaceus GZ0620 to human colon cancer cells HT-29 was 18.55%, which is higher than that of LGG standard strain.
TABLE 2 adhesion ratio (%)
| Bacterial strains | Repetition of 1 | Repetition 2 | Repetition 3 | Mean value |
| Pediococcus pentosaceus GZ0620 | 17.79 | 19.46 | 18.41 | 18.55 |
| Standard strain LGG | 15.13 | 16.42 | 15.14 | 15.56 |
2.3, simulated gastric fluid and intestinal fluid tolerance experiment: simulated gastric and intestinal fluids were purchased from Shanghai leaf Biotech, inc. The simulated liquid of the artificial gastric juice comprises dilute hydrochloric acid, pepsin and sodium chloride, and the final pH value is 2.5; the artificial intestinal juice simulant comprises potassium dihydrogen phosphate and trypsin, and the final pH is 6.8. Reviving and activating Pediococcus pentosaceus GZ0620 strain, and adjusting the concentration of the strain to 1 × 10 8 CFU/mL, adding 1mL of bacterial liquid into 9mL of simulated artificial gastric juice, performing gradient dilution by 10 times, and sucking 20 mu L of viable count of a flat-coating meter as an initial viable count of the resistant artificial gastric juice; after the inoculated simulated gastric juice is cultured for 3 hours at 37 ℃, the viable count is again coated on a flat plate to be used as the end viable count of the tolerance artificial gastric juice. Similarly, 1mL of the solution was added at a concentration of 1X 10 8 Adding the CFU/mL Pediococcus pentosaceus GZ0620 fermentation broth into 9mL simulated intestinal fluid, counting viable bacteria, culturing at 37 deg.C for 6h, counting viable bacteria again, and countingThe survival rate. Survival = number of viable cells at end/number of viable cells at start 100%.
The results show that the pediococcus pentosaceus GZ0620 strain has good tolerance capability for artificially simulating gastric and intestinal juice, the survival rate of the pediococcus pentosaceus is 90.91% after 3 hours in the artificial gastric juice, and the survival rate of the pediococcus pentosaceus is 76.27% after 6 hours in the artificial intestinal juice (Table 2).
TABLE 2 tolerance of Pediococcus pentosaceus GZ0620 strain to artificially simulated gastric and intestinal fluids (%)
Example 3: pediococcus pentosaceus GZ0620 acid production performance determination
3.1 GC-MS (gas chromatography-Mass Spectrometry) determination of short-chain fatty acid content of pediococcus pentosaceus GZ0620 fermentation liquor
Preparing fermentation liquor: activating and culturing the preserved strain of pediococcus pentosaceus GZ0620 for 24h, sucking 4ul of bacterial liquid, adding into 4mL of broth culture medium, and culturing at 37 ℃ for 24h for later use.
Detection of short-chain fatty acids: the detection instrument is a gas chromatograph-mass spectrometer (GCMS-QP 2010 Plus) of Shimadzu corporation, japan, and the chromatographic column is an Rtx-5 fused silica capillary column (30 m × 0.25mm × 0.25 um) of RESTEK corporation, USA. The GC ramp was initiated at 40 ℃ for 5min, 5 ℃ to 150 ℃ per minute, 10 ℃ to 280 ℃ per minute for 2min. The carrier gas is high-purity helium (purity is more than 99.999%), and the flow rate is as follows: 1.0mL/min. MS conditions: the ionization mode is EI; the temperature is 200 ℃, the interface temperature is 220 ℃, and the mass scanning range m/z is 33-500. Taking 4mL of fermentation liquor, adding 10ul of 2-ethyl butyric acid internal standard solution with the concentration of 200ug/mL, injecting 1 mu L of sample in a split mode 1. The concentrations of 5 short-chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, isocaproic acid) were calculated by the internal standard method.
GC-MS detection results show that the content of acetic acid in the fermentation liquor of the pediococcus pentosaceus GZ0620 is the highest and is 10.287ug/mL, which is close to that of an LGG standard strain. In addition, the composition also contains three kinds of butyric acid, i-butyric acid, 2-methyl butyric acid and isovaleric acid. Recent studies have found that acetic acid not only increases IgA production in the colon (immunoglobulin a is the most abundant immunoglobulin in mammals, a component that maintains mucosal surface homeostasis), but also alters IgA' ability to bind to specific microorganisms, including enterobacteria. Thus, acetic acid produced by intestinal microorganisms is thought to have the effect of regulating IgA production to maintain mucosal homeostasis.
TABLE 3 Pediococcus pentosaceus GZ0620 fermentation broth short chain fatty acid content (ug/mL)
| Strain number | Name of strain | Acetic acid | N-butyric acid | Isobutyric acid | Isovaleric acid | 2-methyl butyric acid |
| GZ0620 | Pediococcus pentosaceus | 10.287 | 0.107 | 0.12 | 0.259 | 0.111 |
| LGG | Lactobacillus rhamnosus | 13.08 | -- | 0.18 | 0.41 | 0.23 |
3.2, measuring the lactic acid content of the fermentation liquor of pediococcus pentosaceus GZ 0620:
lactic acid is closely related to sugar metabolism, ester metabolism, protein metabolism, and metabolism of intracellular energy, and lactic acid content is an important index for evaluating glycogen metabolism and aerobic metabolism. The determination of the lactic acid content was carried out strictly according to the instructions of the lactic acid determination kit (Beijing Sorbao science and technology Co., ltd.). Activating and culturing Pediococcus pentosaceus GZ0620 strain, inoculating to MRS broth, culturing at 37 deg.C for 24h, adding 1mL of extract I into 100ul of culture solution, centrifuging at 4 deg.C for 10min at 12000g, collecting 0.8mL of supernatant, adding 0.15mL of extract II, centrifuging at 12000g for 10min, collecting supernatant, and measuring OD at 570nm of microplate reader.
The determination result shows that the pediococcus pentosaceus GZ0620 strain has stronger lactic acid producing capacity, and the concentration of lactic acid in fermentation liquor reaches 76.91umol/mL. Lactic acid is closely related to sugar metabolism, ester metabolism, protein metabolism, and metabolism of intracellular energy, and lactic acid content is an important index for evaluating glycogen metabolism and aerobic metabolism.
TABLE 4 lactic acid content (umol/mL) of Pediococcus pentosaceus GZ0620 fermentation broth
| Strain number | Bacterial name | Repetition of 1 | Repetition 2 | Repetition of 3 | Average out |
| GZ0620 | Pediococcus pentosaceus | 73.37 | 77.44 | 79.92 | 76.91 |
Example 4 evaluation of bacteriostatic Activity of Pediococcus pentosaceus GZ0620 Strain
Escherichia coli (Escherichia coli CMCCB 44102), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), pseudomonas aeruginosa (Pseudomonas aeruginosa CMCCB 10104), campylobacter jejuni (Campylobacter jejuni ATCC 33291) and Campylobacter coli (Campylobacter coli ATCC 43478) were inoculated into the nutrient agar medium, respectively, and were recovered and activated 3 times. Sucking proper amount of trypticase soy peptone liquid culture medium to a centrifuge tube, inoculating the activated pathogenic bacteria to the meat soup culture medium, and regulating the concentration of the bacteria liquid to 1 × 10 8 CFU/mL. Sucking 1mL of the mixed solution of the pathogenic bacteria and the broth, adding the mixed solution into 500mL of nutrient agar medium (the temperature is cooled to about 40 ℃) which is not solidified temporarily after sterilization, fully mixing the mixture uniformly, and subpackaging the mixture into culture dishes according to the amount of 20mL per dish. After the culture medium is cooled and solidified, a puncher with the diameter of 6mm is used for punching on the flat plate to prepare a pathogenic bacteria agar plate, each plate corresponds to one strain of bacteria, and three holes are arranged for repeating. Resuscitating and activating the strain to be detected, and adjusting the concentration of the cultured bacterial liquid to 1 × 10 8 CFU/mL. 50 mu L of bacterial liquid of the bacteria to be detected is absorbed and added into the holes of the pathogenic bacteria agar plate, and the bacteria are cultured for 24h at 37 ℃. After the culture, the diameter of the zone of inhibition around the point of perforation was measured with a vernier caliper and recorded. Subjecting standard bacteriaThe strain LGG is used as a control strain, and the experimental operation is carried out simultaneously with the strain to be tested.
The results show that the fermentation broth of the pediococcus pentosaceus GZ0620 strain has stronger inhibitory activity on the growth of pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli and the like, and the effect is basically equivalent to that of an LGG standard strain (Table 4).
TABLE 4 evaluation of the antibacterial Activity of Pediococcus pentosaceus GZ0620 Strain (diameter: mm)
Example 5 test of preventive and therapeutic Effect of Pediococcus pentosaceus GZ0620 on mouse colitis model
5.1 animal experiments
Animal grouping and treatment: a total of 27 mice were randomly divided into 9 cages of 3 mice each. The groups were randomly divided into 3 groups of 3 cages each, including a blank control group (CK), a natural recovery group (DSS) and a GZ0620 treatment group. The experimental treatment is shown in table 5.
TABLE 5DSS Induction of colitis in mice animal Experimental treatment
Dextran sulfate sodium salt solution (DSS): DSS aqueous solution with concentration of 4% (w/v) was prepared with dextran sulfate sodium salt. Preparation of bacterial suspension: the standard strains LGG and pediococcus pentosaceus GZ0620 were revived and activated for 3 generations. Centrifuging the bacterial solution at-4 deg.C and 6000r/min for 5min, and removing supernatant. Resuspending the cells in sterile PBS buffer to adjust the concentration of the cells to 5X 10 9 CFU/mL, and storing at low temperature for later use. During the experiment, the activity, the fecal status and the bloody stool were observed every day.
At the end of the prevention (14 days) and treatment (7 days) periods, weights were weighed, 3 mice per group were sacrificed at random and blood was drawn for serum inflammatory factor detection. Collecting fresh blood sample with sterile centrifuge tube, centrifuging at 3000r/min for 10min to obtain serum. Serum inflammatory factors (IL-1. Beta., IL-6, IL-8 and TNF-. Alpha.) were measured using ELISA kit (Brilliant America Biotech, inc., jiangsu) and the procedures were performed according to the kit instructions. Spleen index was determined by immediately dissecting out the spleen after sacrifice, rinsing with physiological saline, blotting with filter paper, and weighing. Spleen index = spleen mass (g)/mouse body weight (g) × 100%. At the end of the preventive phase, fresh stool samples were collected and sent to sequencing companies for 16S rRNA gene V3-V4 hypervariable region sequencing, and the sequences were analyzed on the QIIME2 platform.
5.2 analysis of results:
the weight change indicated that the DSS-treated group lost 9.7% and 28.8% of body weight during the prevention and treatment periods, respectively, significantly higher than the pediococcus pentosaceus GZ0620 feeding group (table 6); the spleen index in the DSS-treated group was also significantly higher than the control group and the pediococcus pentosaceus GZ 0620-fed group (fig. 5A-B), indicating severe inflammation in the DSS-treated group; the serum inflammatory factor assay results are shown in table 7. As can be seen from Table 7, in the prevention period and the treatment period, the indexes of the four inflammatory factors, namely IL-1 beta, IL-6, IL-8 and TNF-alpha, in the GZ0620 treatment group are obviously lower than those in the DSS group, which indicates that the GZ0620 strain fed with pediococcus pentosaceus has obvious remission effect on the DSS-induced colitis in mice.
TABLE 6 weight change of mice (unit: g)
TABLE 7 mouse serum inflammatory factor assay results (pg/ml)
Intestinal flora 16S rRNA sequencing results showed that pediococcus pentosaceus GZ0620 fed group had higher flora abundance and uniformity (table 8, a in fig. 6), and the number of unique flora was higher than the control group and DSS treated group (B in fig. 6). Further analysis revealed that the gut flora of GZ0620 group contained very abundant lactobacilli (12.60%) and bifidobacteria (6.23%) which are globally recognized as animal gut probiotics compared to the control and DSS groups.
TABLE 8 diversity analysis of intestinal flora Alpha for DSS-induced colitis experiments in mice
In conclusion, the pediococcus pentosaceus GZ0620 fed with the composition can effectively regulate the intestinal flora imbalance caused by DSS, can obviously alleviate the symptoms of DSS-induced colitis, is applied to the field of functional foods, has wide prospect, and is very beneficial to human health.
Example 6 in vitro assay for hypoglycemic and hypocholesterolemic Activity of Pediococcus pentosaceus GZ0620
6.1 inhibitory Effect of Pediococcus pentosaceus YJ0202 on alpha-glucosidase:
the alpha-glucosidase participates in the decomposition and utilization of carbohydrate, and researches show that the inhibition of the activity of the alpha-glucosidase can reduce the blood sugar level in human blood. Restoring and culturing a strain preserved by Pediococcus pentosaceus GZ0620 for 24 hours, inoculating the strain into an MRS liquid culture medium according to the inoculation amount of 2%, culturing for 24 hours at 37 ℃, centrifuging a bacterial liquid for 15 minutes at 4 ℃ under 4000r/min, and filtering a supernatant through a 0.22 mu m filter membrane to obtain a fermentation supernatant (CFS) for later use; collecting supernatant 25 μ L, adding PBS buffer (pH = 6.8) 50 μ L, 20mmol/L PNPG solution 50 μ L, and water bathing at 37 deg.C for 10min; adding 30 mu L of 20U/ml alpha-glucosidase solution, and continuing to react for 10min at 37 ℃; 50 μ L of 1mol/L Na was added 2 CO 3 Terminating the reaction by the solution; performed in 96-well plates (365 μ Ι _), with 3 replicates per set; and measuring the light absorption value at 405nm of an enzyme labeling instrument, and calculating to obtain the alpha-glucosidase inhibition rate (%). The calculation formula is as follows:
wherein group A is alpha-glucosidase; group B is PBS buffer solution; c is a sample group to be detected, which contains alpha-glucosidase and a sample to be detected; group D contained only the samples to be tested.
The detection result shows that the fermentation supernatant of the pediococcus pentosaceus GZ0620 has the activity of inhibiting alpha-glucosidase, the inhibition rate is 16.44 percent, and the inhibition activity is higher than that of the LGG of the standard strain lactobacillus rhamnosus, and the pediococcus pentosaceus GZ0620 preparation possibly has the potential of reducing the blood sugar level of animals and human beings (Table 9).
TABLE 9 inhibition ratio (%)
6.2 degradation of Cholesterol by Pediococcus pentosaceus GZ0620
Research shows that some lactic acid bacteria can adsorb or absorb cholesterol, and the cholesterol in animals can be reduced by being discharged from the bodies. Therefore, a certain amount of bile salt and cholesterol are added into a cholesterol-MRS culture medium (MRS-CHOL culture medium), and the degradation capability on the cholesterol can be calculated by measuring the change of the concentration of the cholesterol after culture. The specific operation method comprises the following steps:
preparation of the cholesterol solution: 0.06g of cholesterol, 0.12g of ox bile salt, 0.06g of sucrose fatty acid ester, 5ml of glacial acetic acid and 0.6ml of tween, oscillating by ultrasonic until the components are completely dissolved, and filtering by using a 0.22 mu m filter membrane under the aseptic condition for later use;
cholesterol-MRS medium (MRS-CHOL medium) preparation: adding 5mL of cholesterol solution and 12mL of 6mol/L NaOH solution into 300mL of MRS liquid culture medium;
inoculating and culturing: activating and culturing Pediococcus pentosaceus GZ0620 strain for 24h, inoculating the strain in MRS-CHOL liquid culture medium at an inoculation amount of 2%, culturing at 37 ℃ for 48h, and using another strain without inoculation as a blank control.
Standard cholesterol working solutions: 0.05g of cholesterol, using glacial acetic acid to fix the volume to 50mL, and preparing a cholesterol standard solution with the mass concentration of 1 mg/mL; diluting with glacial acetic acid by 10 times to obtain standard cholesterol working solution;
and (3) measuring the content of cholesterol: respectively putting 500 mu L of the bacterial suspension and the blank control into a 5mL test tube, slowly adding 4.5mL of absolute ethyl alcohol, standing for 10min, and centrifuging for 15min at 3000r/min; taking 0.5ml of the supernatant, adding 0.2ml of 1mg/ml o-phthalaldehyde and 4.3ml of mixed acid into a test tube, shaking up, and standing for 30min; measuring the absorbance at a wavelength of 550 nm; the cholesterol content in the sample was calculated from the standard curve:
wherein C0 is the OD value of the blank control group; c1 is the OD value of the experimental group;
the experimental results show that pediococcus pentosaceus GZ0620 has weak capacity of degrading cholesterol, the average degradation rate is 6.98%, and the pediococcus pentosaceus GZ0620 has certain potential of reducing cholesterol in animals and human bodies (Table 10).
TABLE 10 degradation of Cholesterol by Pediococcus pentosaceus GZ0620
Claims (6)
1. A Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620, which is preserved in China general microbiological culture Collection center (CGMCC) 10/13/2022, with the address: no. 3 of Xilu No.1 of Beijing, chaoyang, beijing, and the institute of microbiology of Chinese academy of sciences, with the collection number of CGMCC No.25906.
2. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 according to claim 1 for inhibiting Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni or Campylobacter coli for non-disease treatment purposes.
3. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 according to claim 1 for the preparation of a medicament for the treatment of diseases caused by Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, or Campylobacter coli.
4. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 according to claim 1 for the preparation of an anti-inflammatory medicament.
5. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 according to claim 1 in a functional food for modulating the intestinal flora of a human or an animal.
6. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) GZ0620 according to claim 1 for the preparation of a medicament for lowering blood glucose or cholesterol.
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