A kind of probiotic Pediococcus acidilactici and its application

Abstract

A probiotic lactic acid bacteria, classified as Pediococcus acidilactici ( Pediococcus acidilactici ) XJ‑24, deposited in China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.30078, and the deposit date is March 21, 2024. During the fermentation process, the lactic acid bacteria XJ‑24 of the present invention not only grows and metabolizes to produce metabolites that inhibit the growth of Listeria monocytogenes, but also, when co-fermented with Listeria monocytogenes, it is not only not inhibited by Listeria monocytogenes, but the bacteria can also adhere to the surface of Listeria monocytogenes, effectively exerting an in situ inhibitory effect, and finally reducing the number of viable bacteria of Listeria monocytogenes to 0, thereby killing Listeria monocytogenes.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to probiotic Pediococcus acidilactici and applications thereof. Background Art

[0002] Listeria monocytogenes is a common foodborne pathogen that can cause severe listeriosis, especially for people with weakened immunity, such as pregnant women, infants and the elderly, which may lead to serious invasive diseases such as bacteremia, meningitis, pneumonia, endocarditis, sepsis, etc. Dairy products are easily contaminated with Listeria monocytogenes, causing health problems. Although proper pasteurization can eliminate Listeria monocytogenes in dairy products, there are still reports of listeriosis associated with dairy products.

[0003] Traditional physical and chemical methods can effectively inhibit the growth of Listeria monocytogenes, but these methods may have an adverse effect on the quality of dairy products. It has been reported in the prior art that lactic acid bacteria are widely used as starting or auxiliary cultures in the production of dairy products and other fermented products, and can inhibit the growth of Listeria monocytogenes by competing for nutrients and producing antibacterial substances such as organic acids, hydrogen peroxide, and bacteriocins. Lactic acid bacteria can also play a probiotic function in the host, reduce the impact of gastrointestinal invasion by Listeria monocytogenes and immunomodulate Listeria monocytogenes infection. Therefore, more and more studies are focused on the use of lactic acid bacteria and their metabolites to inhibit Listeria monocytogenes in dairy products. Lactic acid bacteria inhibiting Listeria monocytogenes is a complex process. At present, most studies focus on the functional study of antibacterial substances in cell-free supernatants. However, there are few studies on the interaction between lactic acid bacteria and Listeria monocytogenes during co-culture, and it is unknown whether lactic acid bacteria can tolerate gastrointestinal fluid and play a probiotic role. Summary of the invention

[0004] The present invention aims to provide a probiotic lactic acid bacteria that inhibits Listeria monocytogenes. The probiotic lactic acid bacteria is Pediococcus acidilactici, and in addition to its metabolites being able to inhibit the growth of Listeria monocytogenes, its bacterial bodies can adhere to the surface of Listeria monocytogenes and destroy the cell morphology of Listeria monocytogenes, thereby achieving the effect of reducing the number of viable Listeria monocytogenes to 0 and killing Listeria monocytogenes.

[0005] Another object of the present invention is to improve the application of the above-mentioned Pediococcus acidilactici.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A probiotic lactic acid Pediococcus, characterized in that: the classification name of the lactic acid Pediococcus is Pediococcus acidilactici ( Pediococcus acidilactici)XJ-24, deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.30078, and the deposit date is March 21, 2024.

[0008] The present invention isolated the strain XJ-24 from Xinjiang yogurt pimple samples, conducted homology analysis and constructed a phylogenetic tree, and combined with morphological observation, confirmed that the isolated strain XJ-24 was Pediococcus acidilactici. After incubation in simulated gastric fluid for 3 h, the number of surviving live bacteria exceeded 10 after incubation in simulated intestinal fluid for 8 h. 7 CFU / mL, with good gastrointestinal fluid tolerance.

[0009] The bile salt tolerance test found that XJ-24 had excellent bile salt tolerance at a bile salt concentration of 0.3% (w / v).

[0010] When Pediococcus acidilactici XJ-24 and Listeria monocytogenes are inoculated into pasteurized milk at the same time, the live count of Listeria monocytogenes is well inhibited without inhibiting its own growth. It has a strong in situ inhibition effect and can reduce the live count of Listeria monocytogenes to 0.

[0011] Application of Pediococcus acidilactici XJ-24 in inhibiting Listeria monocytogenes.

[0012] Furthermore, the Pediococcus acidilactici XJ-24 and Listeria monocytogenes inhibit Listeria monocytogenes through co-culture.

[0013] The present invention has the following technical effects:

[0014] The Pediococcus acidilactici XJ-24 of the present invention not only grows and metabolizes to produce metabolites that inhibit the growth of Listeria monocytogenes during the fermentation process, but also is not inhibited by Listeria monocytogenes during co-existence and fermentation with Listeria monocytogenes. In addition, the bacterial body can adhere to the surface of Listeria monocytogenes, effectively exerting an in-situ inhibitory effect, and ultimately reducing the number of live bacteria of Listeria monocytogenes to 0, thereby killing Listeria monocytogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Screening results of lactic acid bacteria inhibiting Listeria monocytogenes.

[0016] Figure 2 : Phylogenetic tree of 16S rDNA corresponding to lactic acid bacteria.

[0017] Figure 3 : Survival rates of lactic acid bacteria in simulated gastric fluid (A) and intestinal fluid (B).

[0018] Figure 4: The effect of lactic acid bacteria on in situ inhibition of Listeria monocytogenes in pasteurized milk (A); The growth of Listeria monocytogenes in pasteurized milk pre-fermented with lactic acid bacteria (B).

[0019] Figure 5 : Scanning electron micrograph of the co-culture solution of 5 lactic acid bacteria and Listeria monocytogenes.

[0020] Figure 6 : P. acidilactici Scanning electron micrograph of the co-culture fluid of XJ-24 and Listeria monocytogenes. DETAILED DESCRIPTION

[0021] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0022] The yogurt lumps used in the present invention were purchased from production sites in different regions of Xinjiang, with a total of 9 yogurt lump samples, of which 4 samples were from Altay City, 2 samples were from Yili Prefecture, 2 samples were from Changji Prefecture, and 1 sample was from Shache County. The preservation numbers of Listeria monocytogenes used for testing were ATCC 19115.

[0023] MRS medium and TSB medium were purchased from Hangzhou Best Biotechnology Co., Ltd.; PALCAM agar medium was purchased from Beijing Luqiao Co., Ltd.; pepsin and trypsin were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; ox bile salt was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; whole-fat pasteurized milk was purchased from a local supermarket.

[0024] Example 1

[0025] Strain isolation, screening and identification

[0026] Nine yogurt pimple samples from different regions of Xinjiang were collected. A small piece of each yogurt pimple sample was placed in a sterile test tube, homogenized after adding sterile PBS, and the homogenized sample was enriched and cultured in liquid MRS medium at 37 °C for 24 h, then diluted 10 times in a gradient, and 100 μL of the appropriate gradient was evenly spread on an MRS plate containing 2% calcium carbonate, cultured at 37 °C for 48 h, and colonies with a calcium-dissolving ring, a round shape, a smooth or slightly rough surface, and a white or milky color were selected for further streaking and purification on the MRS plate, and Gram staining was performed. A total of 26 lactic acid bacteria were isolated.

[0027] Finally, 16S rDNA sequencing was used to further identify the obtained strains. By PCR amplification of the 16S rDNA of lactic acid bacteria, sequencing of the PCR, and comparison with the NCBI database, it was confirmed that these strains included 10 strains of Pediococcus acidilactici, 7 strains of Lactobacillus acidophilus, 3 strains of Lactobacillus helveticus, 2 strains of Lactobacillus plantarum, 2 strains of Lactobacillus mucilaginosus, 1 strain of Lactobacillus pentosus, and 1 strain of Lactobacillus rhamnosus.

[0028] Then, use L. monocytogenes ATCC 19115 was used as an indicator bacteria to screen these lactic acid bacteria for their inhibitory activity against Listeria monocytogenes. Figure 1 -A showed that a total of 13 lactic acid bacteria showed antibacterial effects, of which 5 had an antibacterial diameter of less than 15 mm, and 8 had an antibacterial circle diameter between 15 and 20 mm. The five strains with the best antibacterial effects were XJ-24, XJ-47, XJ-4, XJ-14 and XJ-7. Figure 1 -B.

[0029] The information obtained after sequencing the five strains with the best antibacterial effect was analyzed and compared using BLAST, and the sequences with high similarity were downloaded and aligned using MEGA 11.0 to construct a phylogenetic tree. The constructed phylogenetic tree is shown in Figure 2 As shown, strains XJ-7 and XJ-14 were P. acidilactici FCP-1 is the closest relative, and strain XJ-24 is closely related to P. acidilactici NGRI 0510Q is the closest, while strains XJ-4 and XJ-47 are L. reuteri LQ2 is more closely related. Combined with morphological observation, strains XJ-7, XJ-14 and XJ-24 were confirmed to be Pediococcus acidilactici, and strains XJ-4 and XJ-47 were confirmed to be Lactobacillus reuteri. The probiotic potential, safety and in situ inhibition of Listeria monocytogenes in dairy products of these five strains were further evaluated.

[0030] In the present invention, the lactic acid Pediococcus XJ-24 was biologically preserved and preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 30078, and the preservation date is March 21, 2024.

[0031] Example 2

[0032] Simulated gastrointestinal fluid tolerance test

[0033] Tolerating the environment in the gastrointestinal tract is a basic requirement for potential probiotics, otherwise they will not be able to survive and function in the host. P. acidilacticiThe survival rate of XJ-24 in simulated gastrointestinal fluid was tested.

[0034] Simulated gastric fluid: add pepsin into sterile PBS (0.1 mol / L, pH 2.5) to a final concentration of 3 g / L; simulated intestinal fluid: add trypsin into sterile PBS (0.1 mol / L, pH 8.0) to a final concentration of 1 g / L.

[0035] After activating two generations of lactic acid bacteria overnight culture, they were washed twice with sterile saline (0.85% NaCl, w / v) and resuspended, and then inoculated into simulated gastric fluid to make the final concentration of the bacterial solution 108 CFU / mL. Incubate at 37 °C for 3 h, take the bacterial solution at 0 and 3 h, dilute it 10 times with sterile saline, take 100 μL of the appropriate gradient dilution and spread it on the MRS plate, and calculate the number of viable bacteria after incubation at 37 °C for 48 h. Then 1 mL of the culture was inoculated into 9 mL of simulated intestinal fluid, incubated at 37 °C for 8 h, take the bacterial solution at 0, 2, 4, and 8 h, dilute it 10 times with sterile saline, take 100 μL of the appropriate gradient dilution and spread it on the MRS plate, and calculate the number of viable bacteria after incubation at 37 °C for 48 h. The survival rate of lactic acid bacteria in the simulated human gastrointestinal tract was calculated according to the following formula:

[0036]

[0037] Where: Nt, the number of live lactic acid bacteria at different times after treatment with simulated gastrointestinal fluid; N0, the initial number of live lactic acid bacteria.

[0038] The results are as follows Figure 3 As shown, after 3 hours of incubation in simulated gastric fluid, P. acidilactici The survival rate of the XJ-24 dropped to 73.30%. P. acidilactici XJ-24 can still survive in simulated gastric acid. It is worth noting that the acid resistance of lactic acid bacteria is related to specific strains and species. In this test, the five strains screened above were all tested accordingly. The results showed that the survival rate of the three strains of Pediococcus acidilactici in simulated gastric fluid was significantly lower than that of the two strains of Lactobacillus reuteri (P<0.05). After incubation in simulated gastric fluid for 3 hours, the lactic acid bacteria were transferred to simulated intestinal fluid and incubated for 8 hours. The test results are as follows Figure 3 -B shows that among the 5 strains of lactic acid bacteria, L. reuteri The survival rate of XJ-4 dropped to 0, while the survival rates of the other four lactic acid bacteria decreased to varying degrees with the extension of incubation time. L. reuteri XJ-47 showed the strongest activity in simulated intestinal fluid, and the survival rate remained above 90% after 8 h. P. acidilactici XJ-7, P. acidilactici XJ-24, P. acidilactici The activity of XJ-14 in simulated intestinal fluid was slightly lower, with the survival rates at 8 h being 88.75%, 80.43%, and 75.08%, respectively. L. reuteri XJ-4 and the remaining four strains had more than 10 surviving cells after incubation in simulated intestinal fluid for 8 h. 7 CFU / mL.

[0039] Bile Salt Tolerance Test:

[0040] After activating two generations of lactic acid bacteria overnight, they were inoculated in MRS liquid medium containing 0.3% ox bile salts at a 2% inoculation rate. The control group was inoculated in MRS liquid medium without ox bile salts and incubated at 37 °C for 4 h. The incubated bacterial solution was diluted 10 times with sterile saline, and 100 μL of the dilution was selected to be spread on the MRS plate. After culturing at 37 °C for 48 h, the number of viable bacteria was calculated. The bile salt tolerance of lactic acid bacteria was calculated according to the following formula:

[0041]

[0042] Where: N 1 , the number of viable lactic acid bacteria inoculated in MRS medium containing ox bile salts; N 2 , the number of viable lactic acid bacteria in the control group.

[0043] Bile salts affect the survival rate of cells by destroying the cell membrane structure of lactic acid bacteria. In the human intestine, the concentration of bile salts varies with digestion time, and 0.3% (w / v) is the average concentration of bile salts in the intestine. Therefore, we evaluated the tolerance of 5 lactic acid bacteria under 0.3% (w / v) bile salt concentration conditions. The results are shown in Table 1.

[0044] Table 1: Tolerance of lactic acid bacteria to 0.3% (w / v) bile salt concentration

[0045]

[0046] After 4 h of incubation, all five lactic acid bacteria strains were able to tolerate bile salts at a concentration of 0.3% (w / v). P. acidilactici XJ-24, P. acidilactici XJ-7, L. reuteri The tolerance of XJ-47 was 89.72%, 87.80% and 86.81% respectively. L. reuteri XJ-4 and P. acidilactici The tolerance of XJ-14 was significantly lower than that of the above three lactic acid bacteria, which were 80.14% and 78.45% respectively. This indicates that among the five isolated lactic acid bacteria, P. acidilacticiXJ-24 has excellent tolerance at 0.3% (w / v) bile salt concentration.

[0047] Example 3

[0048] Co-cultivation of lactic acid bacteria and Listeria monocytogenes in pasteurized milk

[0049] Pasteurized milk is inoculated with Listeria monocytogenes (about 10 2 -10 3 CFU / mL and lactic acid bacteria (10 7 CFU / mL, and the control group was inoculated with Listeria monocytogenes (about 10 2 -10 3 CFU / mL and lactic acid bacteria (10 7 CFU / mL), and cultured at 37 °C for 24 h. PALCAM agar plates and MRS agar plates were used to determine the viable counts of Listeria monocytogenes and lactic acid bacteria at 0, 6, 12, 18, and 24 h, respectively.

[0050] According to the above method, the 5 selected lactic acid bacteria were tested. The 5 lactic acid bacteria were inoculated with Listeria monocytogenes in pasteurized milk. The growth of Listeria monocytogenes was as follows: Figure 4 -A. The L. monocytogenes in the control group showed rapid growth, with the viable count reaching a maximum level of 7.31 Log CFU / mL within 24 h. Compared with the control group, starting from 12 h, all five lactic acid bacteria could significantly reduce the viable count of L. monocytogenes (P<0.05). At 24 h, the viable count of L. monocytogenes in the five different lactic acid bacteria groups decreased by 0.99-1.71 Log CFU / mL compared with the control group. P. acidilactici The XJ-24 group showed the best in situ inhibition effect, and the number of viable L. monocytogenes bacteria decreased by 1.71 LogCFU / mL. L. reuteri The XJ-47 group had the worst effect, only reducing 0.99 Log CFU / mL. Overall, within 24 h, all five lactic acid bacteria could reduce the viable count of Listeria monocytogenes, but could not completely inhibit its growth compared with the initial viable count.

[0051] The growth of 5 lactic acid bacteria and Listeria monocytogenes inoculated simultaneously into pasteurized milk is shown in Table 2.

[0052] Table 2: Growth of lactic acid bacteria co-cultured with Listeria monocytogenes in pasteurized milk

[0053]

[0054] At 24 h of co-culture, compared with the control group, P. acidilacticiThe growth of XJ-24 was not affected, and its viable count only decreased by 0.02 Log CFU / mL. However, the viable counts of the other four lactic acid bacteria decreased to varying degrees compared with the control group, ranging from 0.34 to 0.77 Log CFU / mL, which was caused by the competition for nutrients between Listeria monocytogenes and the four lactic acid bacteria.

[0055] Example 4

[0056] Pre-fermentation of pasteurized milk with lactic acid bacteria followed by inoculation with Listeria monocytogenes

[0057] Lactobacillus (10 7 CFU / mL) were inoculated into pasteurized milk and pre-fermented at 37 °C for 12 h before inoculation with Listeria monocytogenes (about 10 2 -10 3 CFU / mL), and continue to culture for 12 h, and measure the number of viable L. monocytogenes every 3 h. After the unfermented pasteurized milk was left to stand at 37°C for 12 h, only L. monocytogenes (about 10 2 -10 3 CFU / mL) was used as a control.

[0058] The above method was used to test the in situ inhibitory effect of 5 lactic acid bacteria on Listeria monocytogenes after 12 h of pre-fermentation of pasteurized milk. Figure 4 -B. In the control group, the number of viable bacteria of Listeria monocytogenes increased from 3.48 Log CFU / mL at 0 h to 7.68 Log CFU / mL at 12 h. However, the experimental groups of pasteurized milk pre-fermented with different lactic acid bacteria showed different in situ antibacterial effects. Starting from 3 h, all five lactic acid bacteria could significantly inhibit the growth of Listeria monocytogenes (P<0.05). Compared with the control group at 12 h, L. reuteri XJ-4, P. acidilactici XJ-7, P. acidilactici XJ-14, L. reuteri The live bacterial counts of Listeria monocytogenes in the XJ-47 fermented milk group were 3.61, 3.86, 3.14, and 3.48 Log CFU / mL lower than those in the control group, but the live bacterial counts were higher than the original live bacterial counts. P. acidilacticiThe XJ-24 fermented milk group showed a strong in situ inhibition effect. The number of live Listeria monocytogenes in this group showed a downward trend compared with the initial stage. At the 9th hour, the number of live bacteria dropped to 0, that is, Listeria monocytogenes was killed. Since lactic acid bacteria and Listeria monocytogenes were inoculated into pasteurized milk at the same time, the growth of lactic acid bacteria was inhibited, and the antibacterial metabolites released by growth were relatively small. The above 5 lactic acid bacteria were pre-fermented in pasteurized milk for 12 hours, which promoted their growth and released a large number of antibacterial metabolites. Therefore, compared with the pasteurized milk inoculated with lactic acid bacteria and Listeria monocytogenes at the same time, they showed a more significant in situ inhibition effect.

[0059] Given that P. acidilactici XJ-24 showed excellent in situ inhibition of Listeria monocytogenes in pasteurized milk. P. acidilactici The state of XJ-24 when co-cultured with Listeria monocytogenes, to study its mechanism of reducing the number of viable Listeria monocytogenes to 0, we collected the co-cultured bacterial solution and observed it under scanning electron microscopy P. acidilactici The interaction mode between XJ-24 and Listeria monocytogenes. The specific steps are as follows:

[0060] The activated P. acidilactici The concentrations of XJ-24 and Listeria monocytogenes were adjusted to 1×10 8 CFU / mL, then mixed in a 1:1 ratio, inoculated into 10 mL TSB medium with a 1% inoculum, and cultured overnight at 37°C. Next, centrifuge at 8000 rmp for 5 min at 4°C, wash the bacteria twice with sterile PBS, resuspend them in 2.5% glutaraldehyde, and fix them overnight at 4°C. Subsequently, wash the bacteria three times with sterile PBS, dehydrate the bacteria in a series of ethanol solutions (30%, 50%, 70%, 85%, 90%) for 15 min, and then elute them twice with 100% ethanol solution for 15 min each time, dry them, and then plate them with gold for observation under a scanning electron microscope. At the same time, the co-culture fluid of the other 4 strains of lactic acid bacteria and Listeria monocytogenes was also observed, and the results are as follows Figure 5 As shown. A in the figure is the untreated L. monocytogenes , B is L. reuteri XJ-4 With L. monocytogenes Co-culture; C is P. acidilactici XJ-7 With L. monocytogenes Co-culture; D is P. acidilactici XJ-14 and L. monocytogenes Co-culture; E is P. acidilactici XJ-24 and L. monocytogenes Co-culture; F is L. reuteri XJ-47 and L. monocytogenesCo-culture (the red arrow points to Listeria monocytogenes). It can be seen that among A, B, C, D, E, and F, except for E, the surface of Listeria monocytogenes in the other pictures is smooth and the structure is intact. P. acidilactici The surface of Listeria monocytogenes cells adhered to XJ-24 was rough and uneven, with concave and deformed shapes, such as Figure 6 As shown, it can be clearly seen that the cell morphology of Listeria monocytogenes was severely damaged, and even cell rupture was observed. P. acidilactici XJ-24 adhered to the surface of Listeria monocytogenes and destroyed its cell morphology, thus achieving better antibacterial effect. The other four lactic acid bacteria adhered to the surface of Listeria monocytogenes but did not destroy its cell morphology. P. acidilactici XJ-24 has an excellent inhibitory effect on the growth of Listeria monocytogenes through the inhibitory effect of metabolites and the adhesion of bacteria to the surface of Listeria monocytogenes to destroy its cell morphology. The dual effects work synergistically, achieving a killing effect on Listeria monocytogenes.

Claims

1. A probiotic Pediococcus acidilactici, characterized in that: The classification name of the lactic acid Pediococcus is Pediococcus acidilactici ( Pediococcus acidilactici )XJ-24, deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.30078, and the deposit date is March 21, 2024.

2. Use of Pediococcus acidilactici XJ-24 as claimed in claim 1 in inhibiting Listeria monocytogenes for non-therapeutic purposes.

3. The use according to claim 2, characterized in that The Pediococcus acidilactici XJ-24 and Listeria monocytogenes inhibit Listeria monocytogenes through co-culture.

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