CN104611255B - One plant height coherency Pediococcus pentosaceus and its application in purifying water body in vibrio parahemolyticus - Google Patents

Abstract

The invention belongs to the technical field of microbes, and in particular relates to a Pediococcus pentosaceae screened from fermented bean products with high aggregation activity and its application in purifying Vibrio parahaemolyticus in water. The Pediococcus pentosaceus F28-8 (Pediococcus pentosaceus) of the present invention was preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on November 13, 2014, and the preservation number is: CGMCC No.9956. The Pediococcus pentosaceae of the present invention has high agglutinating activity, can be used for the bacteria reduction and purification treatment of Vibrio parahaemolyticus in the water body, and avoids the use of chemical bacteriostatic agents and antibiotics to carry out the water body bacteria reduction and purification treatment, and is useful for breeding and The biological control of Vibrio parahaemolyticus in temporary water provides a new means, and at the same time helps to prevent the spread of aquatic animal diseases and foodborne diseases caused by Vibrio parahaemolyticus.

Description

A strain of highly agglomerative Pediococcus pentosaceus and its growth in Vibrio parahaemolyticus in purified water Applications

technical field

The invention belongs to the technical field of microbes, and in particular relates to a Pediococcus pentosaceae screened from fermented bean products with high aggregation activity and its application in purifying Vibrio parahaemolyticus in water.

Background technique

Vibrio parahaemolyticus, widely distributed in coastal oceans and river estuaries, is an important pathogenic bacterium that causes food poisoning from seafood and an important pathogenic factor of bacterial diseases in aquatic animals. At present, the important means to prevent and control vibriosis in aquatic animals and reduce the load of Vibrio parahaemolyticus in water is to use antibiotics and chemical agents, but this leads to problems such as the emergence of drug-resistant bacteria and antibiotic residues, which has a major impact on food safety , so it is urgent to develop new means to reduce bacteria and purify water.

Contents of the invention

The technical problem to be solved by the present invention is to provide a strain of Pediococcus pentosaceae with high agglomeration activity, which can be used for bacteria reduction and purification treatment of Vibrio parahaemolyticus in water, while avoiding the use of chemical bacteriostatic agents and antibiotics to carry out water treatment. Bacteria reduction and purification treatment provide new means for the biological control of Vibrio parahaemolyticus in cultured and temporary water bodies, and at the same time help prevent the spread of aquatic animal diseases and foodborne diseases caused by Vibrio parahaemolyticus.

The present invention isolates a strain of Pediococcus pentosaceus F28-8 (Pediococcuspentosaceus) from the brine of fermented soybean products, and preserves it in the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee (CGMCC), address: No. 1 Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is: CGMCCNo.9956, the preservation date is November 13, 2014, and the classification of the strain is named Pediococcus pentosaceus.

Pediococcus pentosaceae F28-8 in the present invention has the following biological characteristics:

(1) Morphological characteristics: Gram staining of Pediococcus pentosaceae F28-8 is positive, the cells are spherical, arranged in pairs or quadruples, without spores, capsules, and flagella.

(2) Colony characteristics: Pediococcus pentosaceae F28-8 grows well on MRS solid medium, and the colony shape is round, milky white, smooth, raised, with neat edges and opaque.

(3) Physiological and biochemical characteristics: Catalase is negative, does not produce indole, hydrogen sulfide and ammonia, does not reduce nitrate, does not hydrolyze arginine, can ferment glucose, does not ferment sorbose, melibiose, and xylitol , sucrose, lactose, melezitose, arabinose, xylose, rhamnose, raffinose, sorbitol and mannitol; , Fermentation of mannose to produce acid.

(4) It has strong coagulation and antibacterial properties against Vibrio parahaemolyticus.

The present invention isolates Pediococcus pentosaceae isolates from Pediococcus pentosaceae in fermented soybean products, and screens Pediococcus pentosaceae with high cohesion by measuring its antibacterial properties and coagulation ability against Vibrio parahaemolyticus , and observe the surface microstructure of Pediococcus pentosaceae strains with different cohesion through electron microscopy, and measure its effect on the survival rate of pathogenic Vibrio parahaemolyticus in water to investigate its purification effect on Vibrio parahaemolyticus in water, Specifically include the following steps:

(1) Recovery culture of bacterial strains Pediococcus pentosaceae F28-8 was inoculated with 5 mL of MRS liquid medium, and tdh gene-positive pathogenic Vibrio parahaemolyticus ATCC33847 was inoculated with LBS liquid medium for later use.

(2) Determination of antibacterial ability The antibacterial ability was detected by measuring the size of the antibacterial zone of the cell-free extract of Pediococcus pentosaceae strain on Vibrio parahaemolyticus on the LBS solid plate by using the agar well diffusion method.

(3) Determination of coagulation ability Adjust the concentration of Pediococcus pentosaceae bacterial suspension, mix the adjusted concentration of Pediococcus pentosaceae suspension with the corresponding Vibrio parahaemolyticus suspension, measure the absorbance value, record For Amix, use the Vibrio parahaemolyticus suspension without mixing Pediococcus pentosaceus as a control, according to the following formula: {[(Ap+Av)/2]-Amix}/(Ap+Av)/2*100 Calculate the bacterial coagulation rate, where Ap and Av are the A values measured at 600 nm for the suspensions of Pediococcus pentosaceae and Vibrio parahaemolyticus respectively.

(4) Electron microscope observation of condensed cells The unmixed bacterial suspension sample of Pediococcus pentosaceae taken from coagulation for 2 hours was subjected to scanning electron microscopy, and the mixed bacterial suspension of Pediococcus pentosaceae and Vibrio parahaemolyticus co-condensed for 2 hours was drawn The samples were subjected to scanning electron microscopy to observe the structure and aggregation characteristics of the cells.

(5) Determination of surviving cells of Vibrio parahaemolyticus during the coagulation process Mix the mixed bacteria suspension of Pediococcus pentosaceae and Vibrio parahaemolyticus at 37°C for 2 h, 12 h, 24 h, 60 h, and 96 h, then pipette The upper layer and the lower layer solution in the sample were used to measure the bacterial concentration in the system by selective TCBS agar plate.

The beneficial effects of the present invention are:

Pediococcus pentosaceus, a species of lactic acid bacteria, is widely distributed in traditional fermented foods such as vegetables, cheese, and sausages, and is recognized as a safe microorganism. The bacteria metabolize sugars to produce lactic acid and produce Class IIa pediocins, which have a strong inhibitory effect on pathogenic bacteria and spoilage microorganisms. Some of these strains have probiotic properties such as improving animal natural immunity, promoting health, and resisting the invasion of pathogenic bacteria. Pediococcus saccharomyces and its metabolites have potential value and broad prospects for the development of new antibacterial agents and microecological agents for the purification control of Vibrio in food and the environment and the biological control of vibriosis.

Pediococcus pentosaceae F28-8 of the present invention has stronger antibacterial property, coagulation ability to pathogenic Vibrio parahaemolyticus, and its antibacterial activity is all insensitive to catalase, protease and high temperature condition, therefore can Wide range of applications. Electron microscopy showed that the bacterium has a unique cell surface structure and cell cohesion mode, which is easy to form large clusters by self-aggregation, and forms a tight coagglomerate with Vibrio parahaemolyticus, thus preventing the contamination of Vibrio parahaemolyticus in water. Bacteria reduction and purification treatment. After coagulation of Vibrio parahaemolyticus in water by Pediococcus pentosaceae F28-8, the number of cultivable cells in the supernatant suspension and condensate was significantly reduced.

Description of drawings

Figure 1: Scanning electron microscope results of Pediococcus pentosaceae cells with different cohesion.

Figure 2: Effect of coaggregation of Pediococcus pentosaceae on the number of cultureable cells of Vibrio parahaemolyticus in water.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the medium and test conditions used in the examples of the present invention are conventional medium and tests in the art. Unless otherwise specified, the reagents used in the examples of the present invention are commercially available.

Embodiment 1 bacterial strain recovery culture

Thaw the seed preservation solution of Pediococcus pentosaceus at 0°C, aseptically draw 200 μL of the seed preservation solution and inoculate it into 10 mL of MRS liquid medium, culture it under anaerobic conditions at 37°C for 24 h, and take the culture to streak and inoculate in On the MRS solid plate, cultured anaerobically at 37°C for 24 h, then picked a single colony and inoculated in 5 mL of MRS liquid medium, cultured under anaerobic conditions at 37°C for 24 h, and set aside.

Inoculate the tdh gene-positive pathogenic Vibrio parahaemolyticus ATCC33847 in LBS liquid medium, shake and culture at 37°C for 16 h; take the culture to streak on the TCBS plate, culture at 37°C for 20 h, pick a single colony and inoculate 5 mL of LBS liquid medium, cultivated at 37°C for 12 h under constant temperature and shaking conditions, and set aside.

Embodiment 2 Determination of antibacterial ability

Comparative determination of antibacterial ability of Pediococcus pentosaceae F28-8, Y27-4, Y45-30, H13, H6, H10 and F3:

Centrifuge the culture of Pediococcus pentosaceae at 12,000g for 15 min at different temperatures of 70°C, 100°C, and 121°C respectively, take the supernatant, and sterilize it through a 0.22 µm microporous membrane to obtain a cell-free culture of Pediococcus pentosacea. extract. Use the agar well diffusion method to measure the antibacterial ability of the cell-free extract of the strain: Dilute the bacterial suspension of Vibrio parahaemolyticus to 10 ⁸ CFU/mL with sterile physiological saline, pipette 1 mL of the bacterial suspension in LBS On a solid plate, coat it evenly, and dry it in an ultra-clean workbench for 15 minutes. Use a hole puncher to evenly punch small holes with a diameter of 10 mm in the plate, and add 200 μL of the cell-free extract of Pediococcus pentosaceae culture and Treat with 9 mg/mL catalase, 1 mg/mL proteinase K, and 1 mg/mL trypsin. The strains treated at three different conditions of 70°C, 100°C, and 121°C were made in parallel in 3 wells, diffused at room temperature for 5 h, and then cultured at 37°C overnight, measured with a vernier caliper and recorded the diameter of the inhibition zone. The pH of the MRS liquid medium was adjusted with lactic acid to be the same as that of the cell-free extract of the test strain as a control, and the antibacterial effects of different treatment groups were compared.

The metabolites of seven strains of Pediococcus pentosaceae in this study showed a strong inhibitory effect on pathogenic Vibrio parahaemolyticus ATCC33847, and the diameter of the inhibition zone was between 21.0 mm and 25.5 mm, which was the same pH as the lactic acid control 1.1 to 1.3 times that of F28-8, which has the strongest antibacterial effect. After the culture supernatant was treated with catalase, trypsin, proteinase K and heat treatment, the antibacterial effect was reduced to varying degrees, but the residual antibacterial effect was still between 75-96%. The organic acid produced is the main factor to inhibit the growth of Vibrio parahaemolyticus, while the production of hydrogen peroxide and protein antibacterial components such as bacteriocin have relatively little effect.

Example 3 Determination of Co-agglomeration Ability

Comparative determination of coagulation ability of Pediococcus pentosaceae F28-8, Y27-4, Y45-30, H13, H6, H10 and F3:

Centrifuge the culture of Pediococcus pentosaceae strain at 5000g for 10 min, collect the cells, wash twice with normal saline, adjust the concentration of the suspension of the tested strain with normal saline, so that the absorbance value at 600 nm wavelength is about 0.4, that is, A600 =0.4. Mix 2 mL of the adjusted Pediococcus pentosaceae suspension and the corresponding Vibrio parahaemolyticus suspension in a 15 mL graduated test tube, vortex for 120 s, let stand at 37°C for 2 h, and draw 1 mL The absorbance value of the upper layer solution was measured at 600 nm, which was recorded as Amix. Each mixed solution was repeated in 3 tubes in parallel. The suspension of Vibrio parahaemolyticus without Pediococcus pentosaceae was used as a control to calculate the bacterial coagulation rate. Bacterial coagulation rate is calculated according to the following formula: {[(Ap+Av)/2]-Amix}/(Ap+Av)/2*100, where Ap and Av are Pediococcus pentosaceus and Vibrio parahaemolyticus respectively The A value of the suspension was measured at 600 nm.

The test results showed that Pediococcus pentosaceae has the ability to significantly promote the aggregation of pathogenic Vibrio parahaemolyticus, but the coagulation ability has significant strain differences. The co-aggregation rate of different Pediococcus pentosaceae and Vibrio parahaemolyticus ATCC33847 is between 14.8-37.6%, and the order of co-aggregation rate is: F28-8, Y27-4 > Y45-30 > H13, H6, H10 > F3 , all strains were significantly higher than the control group without Pediococcus pentosaceae ( P <0.01), and the co-aggregation rates of F28-8 and Y27-4 were the highest, respectively 37.5% and 36.9%, which were significantly higher than other strains ( P < 0.01), thus providing candidate strains for the purification and reduction of Vibrio parahaemolyticus in water.

Example 4 Electron Microscopic Observation of Condensed Cells

Comparative determination of coagulation of Pediococcus pentosaceae strains F28-8 and H13 on Vibrio parahaemolyticus ATCC33847:

The unmixed Vibrio parahaemolyticus suspension samples of Pediococcus pentosaceae collected from coagulation for 2 hours were subjected to scanning electron microscopy, and the mixed bacteria of Pediococcus pentosaceae F28-8 or H13 and Vibrio parahaemolyticus were drawn and coagulated for 2 hours The suspension sample was subjected to scanning electron microscopy to observe the structure and aggregation characteristics of the cells respectively. The steps were as follows: draw 1 mL of the lower layer solution from the coagulation or co-aggregation bacterial suspension sample, add an equal volume of 5% glutaraldehyde solution to fix overnight; double distilled water Wash 3 times, 15 min each time; dehydrate with 50%, 70%, 80%, 90%, 100% ethanol gradient; mix ethyl acetate and absolute ethanol for 30 min, centrifuge and discard the supernatant; After 30 min, the supernatant was discarded by centrifugation; the precipitate was resuspended in ethyl acetate solution, and the sample was added to the spotting plate, dried; coated, and observed with a microscope.

The test results are shown in Figure 1. The bacterial cells of the highly cohesive strain F28-8 are firmly bonded together and are easy to form large bacterial clusters, as shown in Figure 1 (A), while the cells of the low cohesive strain H13 Scattered, only a small number of cells stick together, see Figure 1 (B), large bacterial clusters in the field of view are relatively rare. In terms of cell aggregation, the surface of H13 cells is relatively smooth, without protruding structures, and the cells are mainly connected by four-terminals of quadruplets or doublets, which are easy to form chains or sheets, while the surface of F28-8 cells is rougher, adherent There are protruding structures, and the cell aggregation is not only through the four-terminal bonding, but also through the side stacking connection, forming a large three-dimensional cluster, see Figure 1 (C), (D). The scanning results of co-condensates of strains F28-8 and H13 against Vibrio parahaemolyticus ATCC33847 are shown in Figure 1 (E) and (F). In co-agglomerates, F28-8 makes ATCC33847 tightly packed together, and the formed aggregates are tight and firm, see Figure 1 (E), while H13 aggregates ATCC33847 cells are relatively loose, see Figure 1 (F), easy to Resuspend. The results showed that the coagulation ability of Pediococcus pentosaceae to Vibrio parahaemolyticus was related to the surface structure of the strain and the tightness of cell aggregation. Previous reports suggest that the coagulation-promoting effect of different species of lactic acid bacteria on pathogenic bacteria may be related to the polysaccharides and proteins on the cell surface, but the mechanism of the difference in the coagulation-promoting ability among strains within the species is still unclear. In this study, by comparing the high The cell morphology of cohesive strains and low cohesive strains indicated that the diversity of surface microstructure and cohesion modes of strains may lead to differences in coagulation-promoting ability.

Example 5 Determination of Vibrio parahaemolyticus surviving cells in the coagulation process

Comparative determination of Vibrio parahaemolyticus surviving cells during aggregation of Pediococcus pentosaceae strains F28-8 and H13:

Prepare Pediococcus pentosaceae and Vibrio parahaemolyticus with an initial concentration of 9.1 Log _{10 CFU/mL of Pediococcus pentosaceae suspension and 5.5 Log 10 CFU} /mL of Vibrio parahaemolyticus according to the coagulation assay method of lactic acid bacteria and pathogenic bacteria For the mixed bacterial suspension, the suspension of Vibrio parahaemolyticus without Pediococcus pentosaceus was used as the control, and the mixed bacterial suspension of Pediococcus pentosaceus and Vibrio parahaemolyticus was left standing at 37°C for 2 h, 12 h, 24 h, After 60h and 96h, draw 1 mL each of the upper and lower layers of the sample, dilute it 10 times with sterile normal saline, and spread it on a TCBS plate. Take 200 μL for each dilution and spread it on three plates. Put the TCBS plate at 37°C Cultivate for 24 h. After the end of the culture, select the dilution with the number of colonies between 30-300, and use the average value of the number of colonies on the three plates to calculate the bacterial concentration in the system.

The test results are shown in Figure 2, the number of culturable cells in the control group decreased slowly with the prolongation of the storage time, and the coagulation treatment of strains F28-8 and H13 significantly aggravated the decline in the number of culturable cells in the system, see Attached Figure 2, Figures 2 (A) and (B) respectively show the changes in the number of cultivable cells in the upper suspension and the lower aggregate after coagulation of Vibrio parahaemolyticus with an initial concentration of 9.1 Log ₁₀ CFU mL ^-1 ; Figures 2 (C) and (D) show the changes in the number of cultivable cells in the upper layer suspension and lower layer condensate of Vibrio parahaemolyticus with an initial concentration of 5.5 Log ₁₀ CFU mL ^-1 respectively; the data points in the figure are 3 Repeated results are expressed as mean ± standard deviation.

After the Vibrio parahaemolyticus suspension with an initial concentration of 9.1 Log ₁₀ CFU mL ^-1 was agglomerated with F28-8 for 2 h, the number of culturable cells in the supernatant suspension was 1.8 Log ₁₀ CFU mL ^-1 lower than that of the control group. 24 h lower than the control group 2.7 Log ₁₀ CFU mL ^-1 , the most decreased at 60 h, lower than the control 3.1 Log ₁₀ CFU mL ^-1 ; and the low aggregation strain H13 after 2 h, 24 h, and 60 h respectively decreased In the control group at 0.9, 2.0 and 2.5 Log ₁₀ CFU mL ^-1 , the bacterial reduction effect was significantly lower than that of F28-8 ( P <0.05), but there was no significant difference in the bacterial reduction effect of F28-8 and H13 after 96 h ( P > 0.05), all lower than the control 2.6 Log ₁₀ CFU mL ^-1 , see Figure 2 (A). The Vibrio parahaemolyticus suspension with an initial concentration of 5.5 Log ₁₀ CFU mL ^-1 treated with Pediococcus pentosacea also led to a significant reduction in cultivable cells, and the downward trend was consistent with that of the high-concentration suspension, but the bacterial reduction effect Weaker, after 24 hours of action, the F28-8 treatment group decreased 1.3 Log ₁₀ CFU mL ^-1 compared with the control group, while the H13 treatment decreased 0.8 Log ₁₀ CFU mL ^-1 , see Figure 2 (C). The analysis results of Vibrio parahaemolyticus in the lower layer suspension (including agglutinate) showed that the number of residual cells in the agglutinate was also significantly lower than that of the control group, and the high concentration suspension was significantly lower than the control group after 12 hours, see Figure 2 (B); while the low-concentration suspension was significantly lower than that of the control group after 60 h, see Figure 2 (D).

The results show that in the coagulation system, Pediococcus pentosaceae not only can coagulate the Vibrio parahaemolyticus cells suspended in the water to form large flocs that can be removed by sedimentation, but also the pentoses in the flocs Pediococcus can also produce antibacterial substances to inactivate Vibrio parahaemolyticus, achieving the bacteria reduction effect of Vibrio parahaemolyticus in water. Although the antibacterial and bactericidal effects of the metabolites formed by lactic acid bacteria in the medium have been reported in previous studies, due to the limitation of nutrients in the water environment, the types and quantities of metabolites produced by lactic acid bacteria are limited, and the formation of aggregates is beneficial The antibacterial substances produced by lactic acid bacteria work at close range, and Pediococcus pentosaceae reduces the distance between cells through coagulation, which may be an important way to play a bactericidal effect in the water environment.

Claims (2)

1. one plant of Pediococcus pentosaceus (Pediococcus pentosaceus) F28-8, China is deposited on November 13rd, 2014 Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.9956.

2. the application in vibrio parahemolyticus of the bacterial strain described in claim 1 in purifying water body.