Pediococcus and culture method and application thereof

Abstract

The invention discloses pediococcus as well as a culture method and application thereof. The pediococcus is pediococcus 003W, and the preservation number of the pediococcus is CCTCC (China Center for Type Culture Collection) NO: M 2024479. The pediococcus 003W can inhibit growth of escherichia coli and staphylococcus aureus, remove DPPH free radicals and superoxide free radicals and inhibit expression of inflammation-related genes, so that occurrence and development of inflammation are inhibited, and the pediococcus 003W is applicable to preparation of drugs or skin care products and the like with antibacterial, antioxidant or anti-inflammatory functions and can also be prepared into food preservatives, so that the pediococcus 003W has a wide application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a Pediococcus strain and a culture method and application thereof. Background Art

[0002] The production process of many chemically synthesized fungicides and antioxidants involves environmental pollution, including the discharge of chemical waste and resource consumption, which may have a negative impact on the ecological environment. In addition, some synthetic preparations may contain chemicals that are harmful to human health, such as benzopyrene, dimethyl sulfide, etc., which can cause health problems if used for a long time. Some synthetic fungicides or antioxidants are difficult to degrade in the environment and will accumulate in the ecosystem, which will have an adverse effect on the ecological balance. Chemically synthesized antibacterial and anti-inflammatory drugs have side effects such as drug resistance, drug side effects or drug allergies, and the production costs are often high.

[0003] Compared with synthetic preparations, microbial preparations have the following obvious advantages: (1) Microbial preparations are usually derived from natural microorganisms, have natural and non-toxic properties, and are more compatible with human physiology; (2) Microbial preparations tend to have good biodegradability, have less impact on the environment, will not accumulate in the ecosystem, and help protect the environment; (3) The production of microbial preparations usually adopts fermentation or biosynthesis, can use cheap raw materials for production, and have high sustainability.

[0004] Lactic acid bacteria are a type of spore-free, Gram-positive bacteria whose main product of fermented sugars is lactic acid. They have a variety of probiotic properties. Some lactic acid bacteria strains have antibacterial properties and can be used as antibacterial agents or drugs. Some lactic acid bacteria strains have unique antioxidant activity and can be used as food preservatives, skin care products, etc. Studies have also shown that some lactic acid bacteria can inhibit the development of inflammation and have the potential to be used as anti-inflammatory and anti-inflammatory drugs or products. Literature 1 reports a strain of Pediococcus acidilactici, which has antibacterial and antioxidant properties, but its ability to scavenge DPPH free radicals is weak and does not show anti-inflammatory properties (LiC, Wang S, Chen S, et al. Screening and Characterization of Pediococcus acidilactici LC-9-1 toward Selection as a Potential Probiotic for Poultry with Antibacterial and Antioxidative Properties. Antioxidants (Basel, Switzerland). 2023, 12 (2): 215.). Document 2 reports the screening of five lactic acid bacteria strains and points out that they have antioxidant and anti-inflammatory properties, but their antioxidant properties are weak and they do not show antibacterial properties (Kim KT, Kim JW, Kim SI, et al. Antioxidant and Anti-Inflammatory Effect and Probiotic Properties of Lactic Acid Bacteria Isolated from Canine and Feline Feces. Microorganisms. 2021, 9 (9): 1971.). Document 3 reports a Pediococcus preparation that has anti-inflammatory effects and can fight against Salmonella infection, but the strain or its preparation does not show antioxidant properties (Wu H, Ding C, Ma X, et al. Microencapsulate Probiotics (MP) Promote Growth Performance and Inhibit Inflammatory Response in Broilers Challenged with Salmonella typhimurium. Probiotics Antimicrob Proteins. 2024, 16 (2): 623-635.). In summary, there are currently no literature reports on lactic acid bacteria that have antibacterial, antioxidant and anti-inflammatory activities. Summary of the invention

[0005] The invention provides a Pediococcus sp. and a culture method and application thereof. The strain has an antibacterial effect on common pathogenic bacteria Escherichia coli and Staphylococcus aureus, and also has the ability to scavenge free radicals and inhibit the expression of inflammation-related genes.

[0006] The inventor isolated and purified a lactic acid bacteria from a naturally fermented kimchi sample in Nanjing, Jiangsu Province, which was identified as Pediococcus sp. by molecular biology and named Pediococcus 003W. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on March 14, 2024, with the deposit number CCTCC NO: M 2024479, and the deposit address is Wuhan University, Wuhan, Hubei Province, China.

[0007] The present invention also provides a method for culturing the above-mentioned Pediococcus 003W, which specifically comprises the following steps:

[0008] Pediococcus 003W was inoculated into a culture medium for culture, the pH of the culture medium was 5.5-6.0, and the culture temperature was 30-35°C.

[0009] Preferably, the composition of the culture medium is: glucose 15-25 g / L, peptone 7-13 g / L, yeast powder 3-8 g / L, beef extract 3-8 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, Tween-80 1 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, calcium carbonate 5-15 g / L, Congo red 0.05 g / L, nystatin 50 μg / mL, pH 5.5-6.0.

[0010] Furthermore, the present invention provides the use of the Pediococcus 003W in the preparation of an antibacterial agent.

[0011] Specifically, the antibacterial agent inhibits at least one of Escherichia coli and Staphylococcus aureus.

[0012] The present invention also provides the use of the Pediococcus 003W in the preparation of antioxidant products.

[0013] Specifically, the antioxidant product is an antioxidant skin care product, food or medicine.

[0014] In addition, the present invention provides the use of the Pediococcus 003W in the preparation of an anti-inflammatory agent.

[0015] Specifically, the anti-inflammatory agent is an anti-inflammatory skin care product, food or medicine.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention isolates for the first time a Pediococcus 003W having antibacterial, antioxidant and anti-inflammatory activities, which can be used as a microbial preparation for preparing skin care products, foods or medicines having antibacterial, antioxidant and anti-inflammatory activities, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of Pediococcus 003W;

[0019] Figure 2 This is a microscopic image of Pediococcus 003W;

[0020] Figure 3 is the phylogenetic tree of Pediococcus 003W constructed using the maximum likelihood method;

[0021] Figure 4 This is an analysis chart of the antibacterial effect of Pediococcus 003W;

[0022] Figure 5 This is the analysis chart of the DPPH free radical scavenging results of Pediococcus 003W;

[0023] Figure 6 This is the analysis chart of the superoxide free radical scavenging results of Pediococcus 003W;

[0024] Figure 7 This is an analysis chart of the results of Pediococcus 003W inhibiting the expression of inflammatory factors. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific embodiments and drawings so that those skilled in the art can more fully understand the present invention, but the present invention is not limited in any way.

[0026] In the following examples, unless otherwise specified, the reagents used are all commercially available, and the methods used are all methods routinely used by those skilled in the art.

[0027] In the following examples, the composition of the liquid culture medium used is: glucose 18g / L, peptone 8g / L, yeast powder 4g / L, beef extract 6g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, ammonium citrate 2g / L, Tween-80 1g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, calcium carbonate 6g / L, Congo red 0.05g / L, dissolved in pure water, adjusting the pH to 5.5; sterilized at 121°C for 30 minutes and then cooled naturally, and nystatin was added at a final concentration of 50μg / mL.

[0028] Solid culture medium: Add 20g / L agar to the liquid culture medium.

[0029] Example 1

[0030] Isolation and identification of Pediococcus 003W.

[0031] 1. Isolation of Pediococcus 003W

[0032] The Pediococcus 003W of the present invention is isolated from naturally fermented kimchi in Nanjing, Jiangsu Province, and the specific isolation method is as follows:

[0033] (1) Strain isolation: Samples from different pickled vegetables were divided into 5 groups. 1 g of sample was mixed with a small amount of juice from each group and inoculated into liquid culture medium. The culture temperature was 30°C and the shaking was 200 rpm for 2 days for enrichment. Then 1 mL of bacterial solution was taken from each group of conical flasks and inoculated into new liquid culture medium again. The culture was carried out under the same conditions for 2 days and enriched again. Then 1 mL of enriched bacterial solution was taken from each group and added to 9 mL of sterile water. The mixture was mixed to make a bacterial suspension. A 10-fold gradient dilution was performed and 10 -6 -10 -4 The diluted bacterial suspension was spread on solid culture medium and cultured at 37°C.

[0034] (2) Strain purification: The coated plate was cultured for 2 days. After the colonies grew, a single colony was picked according to the morphological characteristics and streaked on a new solid culture medium. The culture was cultured at 30°C. After the colonies grew, a single colony was picked and streaked again. This process was repeated 3-4 times until the colonies grown on the culture dish were all single-morphological colonies and the strain was completely purified. Then, colonies with white color and smooth edges were selected and inoculated into liquid culture medium respectively. The culture was cultured at 200 rpm and 30°C for 24 hours.

[0035] (3) Add 500-1000 μL of sterile saline to a sterile centrifuge tube, carefully pick up a single colony with a pipette tip, and dissolve it in sterile saline for 10 -4 The culture medium was diluted 10,000 times and 100,000 times, and then plated on an MRS plate and cultured at 35°C for two days. If the culture medium becomes basically transparent, it is considered to be the target strain, and further purified and cultured, diluted 10,000 times and 100,000 times, and cultured at 35°C for 2 days.

[0036] 2. Identification of strains

[0037] (1) Morphological characteristics: After culturing the strain at 35°C on MRS medium for 2 days, the colonies were round, white, with neat edges, smooth surface and soft texture.

[0038] (2) Physiological and biochemical characteristics: Gram-positive, facultative anaerobic, and produces acid by fermentation using glucose as the carbon source.

[0039] (3) Using the genomic DNA of strain 003W as a template and the universal primers for PCR amplification of the 16S rRNA gene (SEQ ID NO.1) as primers, PCR amplification was performed to determine its full sequence, and an online homology comparison was performed with the sequences in the GenBank database. The results showed that the sequence of strain 003W and Pediococcus acidilactici had a 99% similarity.

[0040] Based on the morphology, physiological and biochemical tests and molecular biological analysis of strain 003W, the strain 003W was identified as Pediococcus and named Pediococcus 003W.

[0041] Example 2

[0042] Study on the antibacterial properties of Pediococcus 003W.

[0043] This example uses Escherichia coli and Staphylococcus aureus as pathogenic experimental strains to verify the antibacterial ability of Pediococcus 003W. The specific process is as follows:

[0044] (1) Escherichia coli and Staphylococcus aureus were inoculated on LB medium, and mature strains were selected and cultured in the corresponding medium at 37°C for 12 hours. The number of viable cells was adjusted to 1×10 with PBS. 8 CFU / mL;

[0045] (2) Using the agar well diffusion method, evenly spread 100 μL of pathogenic bacteria suspension on the corresponding indicator bacteria (Escherichia coli, Staphylococcus aureus) culture medium plate;

[0046] (3) Use a sterile hole puncher to punch out antibacterial wells with a diameter of 7 mm on the plate. Punch three wells in a triangular pattern on each plate as a parallel control. Use tweezers to carefully remove the agar blocks in the wells.

[0047] (4) Use sterile saline to adjust the concentration of the bacterial suspension of Pediococcus 003W to OD 600 =0.5, 1.0, 2.0;

[0048] (5) Add 200 μL of bacterial suspension to each well of the plate, and use physiological saline as a blank control. After standing at room temperature for half an hour, place it in a 37°C incubator for 24 hours, and determine the antibacterial ability of lactic acid bacteria by measuring the size of the inhibition zone.

[0049] The results are as follows Figure 4 As shown, from Figure 4 It can be seen that the bacterial suspension of Pediococcus 003W significantly inhibited the growth of pathogenic bacteria, and with the increase of bacterial liquid concentration, the diameter of the inhibition zone increased significantly. The above results show that Pediococcus 003W has significant antibacterial properties and can be made into antibacterial agents, disinfectants and other products.

[0050] Example 3

[0051] Study on the antioxidant properties of Pediococcus 003W strain.

[0052] 1. DPPH free radical scavenging ability determination

[0053] (1) Test group: Weigh 6 mg of DPPH and dissolve it in anhydrous ethanol, dilute to 40 mL, and prepare a 0.4 mM DPPH solution. Use sterile saline to adjust the concentration of the bacterial suspension of Pediococcus 003W to OD 600 =2.0, 1.5, 1.0, 0.5, and respectively aspirate 0.5 mL of bacterial suspension and add it into 0.5 mL of DPPH solution. React for 30 min in the dark at room temperature (25°C), centrifuge at 8000 g for 10 min, take the supernatant, and measure the absorbance at 517 nm, which is recorded as A1.

[0054] (2) Control group: DPPH solution was replaced with 0.5 mL of anhydrous ethanol. Other operations were the same as those of the test group. The absorbance was detected to be A2.

[0055] (3) Blank group: The bacterial suspension was replaced with 0.5 mL of anhydrous ethanol. Other operations were the same as those of the test group. The absorbance was detected as A0.

[0056] (4) Based on the absorbance data of the three groups, the free radical scavenging rate was calculated according to the following formula:

[0057]

[0058] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that the physiological saline suspension of Pediococcus 003W has the ability to scavenge DPPH free radicals, and with the increase of the concentration of the bacterial suspension, the free radical scavenging effect shows an upward trend.

[0059] 2. Superoxide free radical scavenging experiment

[0060] (1) Test group: 3.4 mL of Tris-HCl solution (50 mM, pH 8.2) and 0.5 mL of pyrogallol solution (50 mM) were taken. The concentration of the bacterial suspension of Pediococcus 003W obtained in Example 3 was adjusted to OD 600 =2.0, 1.5, 1.0, 0.5, add the bacterial suspension into the above system, mix well and place in an incubator at 25°C for 4 minutes, then add 0.1mL of HCl solution (8M) to terminate the reaction, and measure the absorbance A1 at 320nm.

[0061] (2) Control group: The bacterial solution was replaced with 1 mL of saline. Other operations were the same as those of the test group. The absorbance value was measured as A0.

[0062] (3) The calculation formula for superoxide anion radical scavenging rate is:

[0063]

[0064] The results are as follows Figure 6 As shown, from Figure 6 It can be seen that the physiological saline suspension of Pediococcus 003W has the ability to scavenge superoxide free radicals, and with the increase of the concentration of the bacterial suspension, the free radical scavenging effect shows a significant upward trend. 600 The maximum value is reached when =2.0.

[0065] Combining the results of the above two experiments, it can be found that the bacterial suspension of Pediococcus 003W has significant antioxidant properties and has the potential to be prepared into antioxidants or preservatives.

[0066] Example 4

[0067] Study on the anti-inflammatory properties of Pediococcus 003W strain.

[0068] In this example, mouse peritoneal macrophage cell line J774A.1 was used as the research object. After the cells were treated with bacterial lipopolysaccharide (LPS) and inactivated 003W bacterial solution, qRT-PCR was used to analyze the expression of inflammation-related genes. The specific process is as follows:

[0069] (1) Mouse peritoneal macrophages in the logarithmic growth phase were obtained, digested with trypsin, and resuspended in DMEM complete medium at 4×10 5 / well of a 24-well plate, and wait for 4 hours for the cells to adhere;

[0070] (2) Take the fermentation broth of Pediococcus 003W strain, collect the cells by centrifugation, and adjust the OD using liquid culture medium. 600 =1.0, sterilize in an autoclave at 121°C for 30 minutes;

[0071] (3) Collect the cells by centrifugation at 6000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in an equal volume of DMEM complete medium;

[0072] (4) LPS was added at a final concentration of 5 μg / ml, 250 μl of inactivated bacteria solution was added to the experimental group, and an equal amount of DMEM complete medium was added to the control group;

[0073] (5) Culture in a 37°C incubator for 12 hours;

[0074] (6) Cell mRNA was extracted using a kit, and cDNA was obtained after reverse transcription. The expression changes of inflammation-related genes IL-1β and IL-6 were detected by qRT-PCR.

[0075] The final result is Figure 7 As shown, from Figure 7 It can be seen that LPS significantly induced the gene expression of pro-inflammatory factors in mouse peritoneal macrophages, while the addition of inactivated Pediococcus 003W bacterial suspension significantly reversed the upregulation of related genes, indicating that it has the function of inhibiting the development of inflammation and has the potential to become an anti-inflammatory drug or preparation.

Claims

1. Pediococcus Pediococcus sp.)003W, the deposit number is CCTCC NO: M 2024479.

2. The method for culturing Pediococcus 003W according to claim 1, It is characterized in that The specific steps are: Pediococcus 003W was inoculated into a culture medium for culture, the pH of the culture medium was 5.5-6.0, and the culture temperature was 30-35°C.

3. The culture method according to claim 2, It is characterized in that The composition of the culture medium is: glucose 15-25 g / L, peptone 7-13 g / L, yeast powder 3-8 g / L, beef extract 3-8 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, Tween-80 1 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, calcium carbonate 5-15 g / L, Congo red 0.05 g / L, nystatin 50 μg / mL, pH 5.5-6.

0.

4. Use of Pediococcus 003W as claimed in claim 1 in the preparation of an antibacterial agent.

5. The use according to claim 4, It is characterized in that The antibacterial agent inhibits at least one of Escherichia coli and Staphylococcus aureus.

6. Use of Pediococcus 003W as claimed in claim 1 in the preparation of antioxidant products.

7. The use according to claim 6, It is characterized in that The antioxidant product is an antioxidant skin care product, food or medicine.

8. Use of Pediococcus 003W as claimed in claim 1 in the preparation of anti-inflammatory agents.

9. The use according to claim 8, It is characterized in that The anti-inflammatory agent is an anti-inflammatory skin care product, food or medicine.

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