Screening method of psychrophilic bacteria for producing heat-resistant protease and application of psychrophilic bacteria

Abstract

The invention discloses a method for screening psychrophilic bacteria for producing heat-resistant protease and application thereof, and belongs to the technical field of food safety. In order to solve the problems that the research on psychrophilic bacteria heat-resistant protease at present is limited to a single strain or one type of species and the protease activity cannot be effectively detected, the invention provides a method for high-throughput screening of psychrophilic bacteria for producing heat-resistant protease, and the method comprises two parts, the psychrophilic bacteria are respectively primarily screened by an agar diffusion method to produce protease and secondarily screened by an azocasein method to produce heat-resistant protease. The screening method provided by the invention is simple to operate, high in accuracy and low in cost, can comprehensively cover all identified psychrophilic bacteria, provides an effective strategy for high-throughput screening of psychrophilic bacteria producing heat-resistant protease, and lays a foundation for targeted screening of key psychrophilic bacteria and deep excavation of influence of heat-resistant protease on milk quality.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety, and in particular relates to a method for screening psychrophilic bacteria producing thermostable protease and application thereof. Background Art

[0002] As a food rich in protein, fat, minerals, etc., cow's milk has become a staple food in China's diet. Raw milk is a key factor in its quality assurance. Although raw milk is considered sterile before leaving the cow's udder, it is extremely susceptible to microbial contamination during milking, storage and transportation, most of which are psychrophilic bacteria. Psychrophilic bacteria themselves have limited damage to milk quality, but the heat-resistant extracellular enzymes they produce still have residual enzyme activity after pasteurization or ultra-high temperature sterilization, and continue to hydrolyze protein and fat in milk, resulting in sourness, flavor changes or aging gelation of finished milk, causing serious economic losses. Psychrophilic bacteria cover many different species, including both Gram-negative and Gram-positive bacteria, but the number of species reported to be involved in dairy product spoilage is extremely limited. Therefore, the screening of psychrophilic bacteria that produce heat-resistant enzymes is of great significance for exploring the spoilage effects of psychrophilic bacteria on milk and dairy products.

[0003] Most of the thermostable proteases produced by psychrophilic bacteria are alkaline metalloproteases with strong heat resistance. Under high-temperature heat treatment conditions of 140°C, at least 300 seconds are required to reduce the residual enzyme activity to less than 10%. In addition, proteases have strong recovery activity in milk. After high-temperature heat treatment, although some proteases have been unfolded and lost their activity, their structure has not been destroyed. During low-temperature storage, proteases in this state will refold into their natural conformation, gradually restore their activity, and continue to act on proteins in dairy products, causing the quality of dairy products to deteriorate. At present, research on thermostable proteases of psychrophilic bacteria is limited to a single strain or a species, so it is urgent to fully understand the psychrophilic bacteria that produce thermostable proteases.

[0004] According to existing research, the main methods for detecting extracellular proteases include formaldehyde method, ninhydrin method, 2,4,6-trinitrobenzenesulfonic acid (TNBS) method, o-phthalaldehyde (OPA) method, Folin-phenol method and polyacrylamide gel electrophoresis (SDS-PAGE). These methods detect by detecting protein hydrolysis substrates, and some have disadvantages such as low detection sensitivity or complicated operation process. Therefore, considering the hydrolysis characteristics of psychrophilic bacteria and the size of the workload, a simpler and more economical screening scheme is urgently needed. Summary of the invention

[0005] In view of the problem that the current research on thermostable proteases of psychrophilic bacteria is limited to a single strain or a type of species, and the protease activity cannot be effectively detected, the present invention provides a method for high-throughput screening of thermostable protease-producing psychrophilic bacteria. The screening method includes two parts, namely, using an agar diffusion method using a skim milk culture medium to initially screen the thermostable protease-producing psychrophilic bacteria and using an azocasein method to rescreen the thermostable protease-producing psychrophilic bacteria.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for screening psychrophilic bacteria producing heat-resistant proteases, the method comprising the steps of using a skim milk culture medium to perform primary screening on psychrophilic bacteria producing proteases by an agar diffusion method, and using an azocasein method to perform secondary screening on psychrophilic bacteria producing heat-resistant proteases.

[0008] In one embodiment of the present invention, the screening method further comprises the steps of isolating, culturing and identifying psychrophilic bacteria from the sample before the initial screening.

[0009] In one embodiment of the present invention, the primary screening comprises the following steps:

[0010] (1) Cultivating psychrophilic bacteria to obtain bacterial liquid;

[0011] (2) preparing a skim milk solid culture medium and punching holes in the culture medium;

[0012] (3) 100 μL of the bacterial solution obtained in step (1) was inoculated into the wells of the skim milk solid culture medium in step (2), and LB liquid culture medium was added to one of the wells as a blank control group. After culturing, whether a protein transparent hydrolysis zone was formed around the pore diameter was observed. If a hydrolysis zone was formed, the protease production capacity of the strain was calculated.

[0013] In one embodiment of the present invention, the culturing in step (1) is to inoculate the psychrophilic bacteria into LB liquid culture medium and culture at 25° C. and 150 r / min with shaking for 24 hours.

[0014] In one embodiment of the present invention, the preparation method of the skim milk solid culture medium in step (2) is to dissolve 30g skim milk powder and 15g agar in 1000mL distilled water, sterilize at 105°C for 15min, and pour the plate into a skim milk solid culture medium.

[0015] In one embodiment of the present invention, the culture in step (3) is cultured at 7°C for 10 days or at 25°C for 24 hours; the calculation formula for the protease production capacity of the strain is as follows:

[0016] R = Dd;

[0017] Where D is the diameter of the hydrolysis zone of protease / lipase, in cm, and d is the colony diameter, in cm.

[0018] In one embodiment of the present invention, the re-screening is to culture the protease-producing psychrophilic bacteria obtained in the initial screening to obtain a bacterial liquid, centrifuge, take the supernatant containing extracellular protease, take four portions of 100 μL of the supernatant, three of which are pasteurized in a 65°C water bath for 30 minutes, pasteurized in a 72°C water bath for 15 seconds, and ultra-high temperature instantaneous sterilization in a 140°C oil bath for 5 seconds; the protease activity of the supernatant is determined by the azocasein method, the heat resistance of the protease after heat treatment is evaluated by the residual enzyme activity rate, and the thermostable protease-producing psychrophilic bacteria are re-screened.

[0019] In one embodiment of the present invention, the culturing is culturing at 7°C for 10 days or culturing at 25°C for 24 hours.

[0020] In one embodiment of the present invention, the azocasein method for determining the protease activity of the supernatant is to mix 100 μL of the supernatant with 100 μL of a 1.5% azocasein solution, react at 37°C for 120 min, add 500 μL of 20% TCA to terminate the reaction, stand at room temperature for 30 min, centrifuge, take 500 μL of the supernatant, add 500 μL of 1M NaOH, and use a microplate reader to measure OD 345 The absorbance value is in nm.

[0021] In one embodiment of the present invention, the preparation method of the azocasein solution with a mass fraction of 1.5% is to dissolve 0.15g of azocasein in 10mL of PBS buffer (50mM, pH 7.2), fully mix and stand for 2-3 days for standby use; the preparation method of the TCA solution with a mass fraction of 20% is to dissolve 2g of TCA in 10mL of water for standby use.

[0022] In one embodiment of the present invention, the enzyme activity of the protease is defined as one enzyme activity unit when the absorbance value changes by 0.01 per milliliter of protease solution per hour at 345 nm.

[0023] In one embodiment of the present invention, the residual enzyme activity (RA) is calculated according to the following formula:

[0024]

[0025] RA: residual enzyme activity (%);

[0026] At: enzyme activity after heat treatment (U);

[0027] A0: untreated enzyme activity (U).

[0028] The second object of the present invention is to provide the application of the above screening method in high-throughput screening of psychrophilic bacteria producing thermostable proteases.

[0029] In one embodiment of the present invention, the application is to use the screened psychrophilic bacteria that produce thermostable proteases to further explore the harm of thermostable proteases of psychrophilic bacteria to milk quality and to develop and establish corresponding prevention and control measures by studying the enzymatic properties of the dominant thermostable proteases of dominant psychrophilic bacteria.

[0030] Beneficial effects of the present invention:

[0031] 1) The present invention combines the agar diffusion method for primary screening and the azocasein method for secondary screening to screen psychrophilic bacteria that produce thermostable proteases. The screening method is simple to operate and can comprehensively cover all identified psychrophilic bacteria, thus solving the problem that the current research on thermostable proteases of psychrophilic bacteria is limited to a single strain or a type of species.

[0032] 2) The screening method provided by the present invention has high accuracy and low cost, and provides an effective strategy for high-throughput screening of psychrophilic bacteria producing thermostable proteases, laying a foundation for targeted screening of key psychrophilic bacteria and in-depth exploration of the effects of thermostable proteases on milk quality.

[0033] 3) The psychrophilic bacteria producing thermostable proteases screened out by the screening method provided by the present invention can be used to further explore the harm of thermostable proteases of psychrophilic bacteria to milk quality and develop and establish corresponding prevention and control measures through the study of the enzymatic properties of the dominant thermostable proteases of dominant psychrophilic bacteria, thereby providing a theoretical basis for solving the problem of thermostable proteases of psychrophilic bacteria destroying milk quality from the root, and providing strategies for reducing production costs of enterprises and avoiding economic losses. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the results of the protease production capacity test of the psychrophilic bacteria that were screened out from the 74 psychrophilic bacteria that have been identified so far;

[0035] Figure 2 The figure shows the results of protease activity assay of 10 psychrophilic bacteria with strong protease activity;

[0036] Figure 3 This is a statistical chart showing the residual rate of protease activity of 10 psychrophilic bacteria with strong protease production activity after different heat treatments. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. It should be noted that the embodiments in this application and the features in the embodiments can be combined with each other without conflict. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work.

[0038] The experimental methods used in the following examples are all conventional methods unless otherwise specified. If no specific conditions are specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels by those skilled in the art.

[0039] Example 1: Isolation and identification of psychrophilic bacteria

[0040] (1) Isolation of psychrophilic bacteria

[0041] Take 1 mL of raw milk sample, add 9 mL of sterile peptone saline solution, vortex mix, then take 1 mL of sample from the test tube and add 9 mL of sterile peptone saline solution, and continuously dilute the sample to 10 -5 . Take 10 respectively -1 Up to 10 -5 100 μL of the sample of the dilution gradient was spread on the MPC solid medium, and three parallels were set for each gradient. The inverted medium was cultured at 7°C aerobic conditions for 7 to 10 days. Then, colonies with different morphological characteristics were randomly picked from the plates with 30 to 300 colonies of each sample and inoculated into sterile LB liquid medium, cultured at 25°C with shaking (150 r / min) for 24 hours, and purified and cultured with the corresponding solid medium and then subcultured to obtain pure culture isolates. A part of the pure culture isolates was used for subsequent genomic DNA extraction, and a part was mixed evenly with 80% glycerol at a ratio of 1:4 and stored at -80°C.

[0042] (2) Identification of psychrophilic bacteria

[0043] ①DNA extraction

[0044] A one-step method was used to extract genomic DNA from the isolated and purified strains. 1 mL of LB bacterial suspension of the isolated strain was pipetted into an EP tube and centrifuged at 12000 r / min for 2 min to collect the precipitated bacterial sludge. 300 μL of DNA extraction solution was added to the bacterial sludge, and the mixture was vortexed and heated in a 56°C water bath for 30 min and 100°C for 10 min, respectively. The supernatant was then centrifuged at 12000 r / min for 2 min to collect the DNA solution.

[0045] ②PCR amplification

[0046] The extracted single colony DNA was PCR amplified using universal primers 27F and 1492R. The primer sequences are shown in Table 1. The PCR amplification system was 27 μL ddH2O, 6 μL 2×Taq PCR MasterMix, 5 μL DNA template, and 1 μL each of upstream and downstream primers. The PCR amplification conditions were 94°C for 5 min; 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, 35 cycles; 72°C for 10 min. The qualified PCR amplification products were stored at low temperature and sent to Shanghai Shenggong Bioengineering Technology Service Co., Ltd. for purification and bidirectional sequencing. The DNAMAN 5.29 software was used to cut off the fragments with poor sequencing results at both ends, and the sequence obtained by sequencing was spliced. The spliced ​​gene sequence of the psychrophilic bacteria strain was submitted to the NCBI database for BLAST comparison. The taxonomic status of the strain was determined by selecting a sequence similarity of more than 99% with the reference strain as the identification threshold. The sequencing results are shown in Table 2.

[0047] Table 1 Primer sequence information

[0048]

[0049] Table 2 Names and sequencing results of isolated psychrophilic bacteria

[0050]

[0051]

[0052] Example 2: Initial screening of psychrophilic bacteria producing thermostable proteases

[0053] (1) Activation and cultivation of psychrophilic bacteria

[0054] The 74 strains of psychrophilic bacteria obtained in Example 1 preserved in 80% glycerol were inoculated into LB liquid culture medium at a 2% inoculum, and cultured in a shaker at 25°C (150 r / min). After culturing for 24 h, three zones were drawn on LB nutrient agar, and then placed in a shaker, cultured at 25°C (150 r / min) for 24 h, and subcultured for two generations.

[0055] (2) Agar diffusion method to screen psychrophilic bacteria with strong protease production ability

[0056] Prepare skim milk culture medium: 3% skim milk powder and 1.5% agar are dissolved in 1000mL distilled water, sterilized at 105℃ for 15min, poured into plates to make solid culture medium, and holes are punched on the solid culture medium with a hole diameter of 8mm. Take 100μL of activated bacterial solution and inoculate it into the holes of the skim milk culture medium plate, and add LB culture medium as a blank control group. Put the skim milk plate in an incubator and culture it at 7℃ for 10d. Observe the formation of a transparent protein hydrolysis circle around the hole diameter. For strains that have formed a hydrolysis circle, the difference between the hydrolysis circle diameter and the colony diameter is used as the hydrolysis radius (R) to indirectly reflect the ability of each strain to produce protease. The formula is as follows:

[0057] R=Dd

[0058] D: diameter of the hydrolysis zone of protease / lipase (cm);

[0059] d: colony diameter (cm).

[0060] The initial screening results are as follows Figure 1 As shown, only 22 of the 74 psychrophilic bacteria had the ability to produce proteases, among which 10 strains had strong protease production ability, including Pseudomonas azotoformans, Pseudomonas gessardii, Pseudomonas brenneri, Microbacterium maritypicum, Acinetobacter parvus, Epilithonimonas vandammei, Chryseobacterium manosquense, Acinetobacterjohnsonii, Staphylococcus epidermidis, and Enterococcus gallinarum.

[0061] Example 3: Rescreening of psychrophilic bacteria producing thermostable proteases

[0062] The 10 psychrophilic bacteria with strong protease-producing activity obtained in Example 2 were inoculated into LB liquid culture medium with an inoculation amount of 2%, and cultured at 7°C for 10 days or at 25°C for 24 hours to obtain a bacterial solution. The bacterial solution was placed at a low temperature of 4°C, centrifuged at 15000×g for 20 minutes, and filtered using a 0.45 μm filter membrane to harvest the supernatant. Three portions of 100 μL of the supernatant were taken and subjected to simulated pasteurization at a water bath of 65°C for 30 minutes, a pasteurization at a water bath of 72°C for 15 seconds, and an ultra-high temperature instantaneous sterilization at an oil bath of 140°C for 5 seconds; the protease activity of the supernatant was determined by the azocasein method, and the thermostable protease-producing psychrophilic bacteria were screened again.

[0063] The reagent preparation method used in the azocasein method to determine the protease activity of the supernatant is as follows:

[0064] The preparation method of the azocasein solution with a mass fraction of 1.5% is as follows: 0.15 g of azocasein is dissolved in 10 mL of PBS buffer (50 mM, pH 7.2), mixed thoroughly and allowed to stand for 2-3 days for use.

[0065] Preparation method of 20% trichloroacetic acid (TCA) solution: dissolve 2 g TCA in 10 mL water and set aside.

[0066] The specific method for determining the protease activity of the supernatant using the azocasein method is as follows:

[0067] 100 μL of supernatant was mixed with 100 μL of 1.5% azocasein solution, reacted at 37°C for 120 min, 500 μL of 20% TCA solution was added to terminate the reaction, allowed to stand at room temperature for 30 min, centrifuged at 12000 r / min for 5 min, 500 μL of supernatant was added with 500 μL of 1M NaOH, and OD was measured using an enzyme-labeled instrument. 345nm The blank control in the azocasein method was as follows: 100 μL of supernatant was added with 500 μL of 20% TCA solution to terminate the reaction, and then mixed with 100 μL of 1.5% azocasein solution, allowed to stand at room temperature for 30 min, centrifuged at 12000 r / min for 5 min, 500 μL of supernatant was added with 500 μL 1M NaOH, and OD was measured using an enzyme-labeled instrument. 345nm The definition of protease activity is that at 345 nm, the absorbance change of 0.01 per milliliter of protease solution per hour is one enzyme activity unit (1U).

[0068] The results of protease activity determination of the supernatant of 10 psychrophilic bacteria are as follows Figure 2 As shown by Figure 2It can be seen that the enzyme activities of these 10 strains are mostly between 20-40U, and Epilithonimonas vandammei has the highest protease activity.

[0069] Determination of heat resistance of proteases after heat treatment:

[0070] The heat resistance of protease is expressed as the residual enzyme activity rate, and the residual enzyme activity rate (RA) is calculated according to the following formula:

[0071]

[0072] RA: residual enzyme activity (%);

[0073] At: enzyme activity after heat treatment (U);

[0074] A0: untreated enzyme activity (U);

[0075] The enzyme activity was determined using the azocasein method described above.

[0076] The results of heat resistance test of protease after heat treatment are as follows Figure 3 As shown by Figure 3 It can be seen that the ability to produce heat-resistant proteases varies among strains. Some strains have strong protease production ability but poor heat resistance, such as Epilithonimonas vandammei. The proteases produced by some typical psychrophilic bacteria such as Pseudomonas azotoformans, Pseudomonas gessardii, Acinetobacterparvus, Chryseobacterium manosquense, Acinetobacter johnsonii, and Staphylococcusepidermidi are all very heat-resistant, and some even have residual enzyme activity of up to 85% after treatment at 140°C.

[0077] The above-screened psychrophilic bacteria producing thermostable proteases can be used to further explore the harm of thermostable proteases of psychrophilic bacteria to milk quality, and to develop and establish corresponding prevention and control measures through the study of the enzymatic properties of dominant thermostable proteases of dominant psychrophilic bacteria, so as to provide a theoretical basis for solving the problem of thermostable proteases of psychrophilic bacteria destroying milk quality from the root, and to provide strategies for reducing enterprise production costs and avoiding economic losses.

[0078] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for screening psychrophilic bacteria producing thermostable protease, characterized in that: The method comprises the steps of using agar diffusion method to preliminarily screen the psychrophilic bacteria producing protease using skim milk culture medium, and using azocasein method to rescreen the psychrophilic bacteria producing heat-resistant protease.

2. The screening method according to claim 1, characterized in that The screening method also includes isolating, culturing and identifying psychrophilic bacteria from the sample before the initial screening.

3. The screening method according to claim 1, characterized in that The initial screening includes the following steps: (1) Cultivating psychrophilic bacteria to obtain bacterial liquid; (2) preparing a skim milk solid culture medium and punching holes in the culture medium; (3) Take 100 μL of the bacterial solution obtained in step (1) and inoculate it into the wells of the skim milk solid culture medium in step (2), and use the wells added with LB liquid culture medium as blank controls. After culturing, observe whether a protein transparent hydrolysis zone is formed around the wells. If a hydrolysis zone is formed, calculate the protease production capacity of the strain.

4. The screening method according to claim 3, characterized in that The culture in step (1) is to inoculate the psychrophilic bacteria into LB medium and culture at 25° C. and 150 rpm with shaking for 24 hours.

5. The screening method according to claim 3, characterized in that The method for preparing the skim milk solid culture medium in step (2) is to dissolve 30 g skim milk powder and 15 g agar in 1000 mL distilled water.

6. The screening method according to claim 3, characterized in that The culture in step (3) is carried out at 7°C for 10 days or at 25°C for 24 hours; the calculation formula for the protease production capacity of the strain is as follows: R = Dd; Where D is the diameter of the hydrolysis zone of protease / lipase, and d is the colony diameter.

7. The screening method according to claim 1, characterized in that The rescreening is to culture the protease-producing psychrophilic bacteria obtained in the initial screening to obtain a bacterial liquid, centrifuge, take three portions of 100 μL of the supernatant, and respectively perform pasteurization in a 65°C water bath for 30 minutes, pasteurization in a 72°C water bath for 15 seconds, and ultra-high temperature instantaneous sterilization in a 140°C oil bath for 5 seconds; use the azocasein method to determine the protease enzyme activity of the supernatant, evaluate the heat resistance of the protease after heat treatment with the enzyme activity residual rate, and rescreen the psychrophilic bacteria that produce heat-resistant protease.

8. The screening method according to claim 7, characterized in that The method for determining the protease activity of the supernatant is to mix 100 μL of the supernatant with 100 μL of a 1.5% azocasein solution, react at 37° C. for 120 min, add 500 μL of 20% TCA to terminate the reaction, stand at room temperature for 30 min, centrifuge, take 500 μL of the supernatant, add 500 μL of 1M NaOH, and measure the OD using an enzyme marker. 345nm The absorbance value.

9. The screening method according to claim 8, characterized in that The enzyme activity of the protease is defined as one enzyme activity unit when the absorbance value changes by 0.01 per milliliter of protease solution per hour at 345 nm.

10. Use of the screening method according to any one of claims 1 to 9 in high-throughput screening of psychrophilic bacteria producing thermostable proteases.

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