Bacillus cereus, quorum sensing inhibitor related to protease production of bacillus cereus, preparation method of quorum sensing inhibitor and application of curcumin

Abstract

The invention discloses bacillus cereus, a quorum sensing inhibitor related to protease production of the bacillus cereus, a preparation method of the quorum sensing inhibitor and application of curcumin, and belongs to the technical field of biology. The invention provides bacillus cereus, and the bacillus cereus D8 is preserved in the Guangdong Microbial Culture Collection Center on June 28, 2024, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO.64815. The bacillus cereus D8 has been preserved in the Guangdong Microbial Culture Collection Center on June 28, 2024. The bacillus cereus protease-producing inhibitor is prepared by providing bacillus cereus with a LuxS / AI-2 type quorum sensing system and inhibiting quorum sensing related to protease production of the bacillus cereus by using curcumin as an active inhibiting component, so that the protease activity of the bacillus cereus can be effectively inhibited; under the condition of not influencing the growth of bacteria, the protease activity is reduced by interfering a LuxS / AI-2 quorum sensing system existing in the bacillus cereus, so that the effect of inhibiting the protease activity of the bacillus cereus is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to Bacillus cereus, a quorum sensing inhibitor related to protease production by Bacillus cereus, a preparation method thereof, and application of curcumin. Background Art

[0002] In raw milk, Bacillus cereus is one of the common foodborne pathogens. It can produce biofilms and toxins, contaminate dairy products, and thus pose a threat to consumers' health. During the growth of Bacillus cereus, proteases, lipases, amylases, and phospholipases are produced. Among them, the heat-resistant enzymes mainly composed of proteases are one of the main extracellular enzymes related to the corruption of milk and dairy products. Due to the heat resistance of the protease produced by Bacillus cereus, it is difficult to inactivate it by conventional heat treatment. In addition, the protease may be resistant to low temperatures and will continue to exist during the production and sales of dairy products. The protease hydrolyzes κ-casein, β-casein, and α-casein in milk, resulting in protein precipitation and bitterness in dairy products, which seriously affects the product quality and shortens its shelf life. Therefore, the development of new inhibitors of Bacillus cereus protease to solve the problem that it is difficult to inactivate it by conventional heat treatment technology has an important impact on ensuring the quality and safety of dairy products and extending the shelf life. Summary of the invention

[0003] The purpose of the present invention is to provide a quorum sensing inhibitor related to Bacillus cereus and Bacillus cereus protease production, a preparation method and the application of curcumin, and to develop new inhibitors of Bacillus cereus protease to solve the problem that conventional heat treatment technology is difficult to inactivate it, which has an important impact on ensuring the quality and safety of dairy products and extending the shelf life.

[0004] The present invention is achieved through the following technical solutions:

[0005] On the one hand, the present invention provides a Bacillus cereus, wherein the Bacillus cereus D8 (Bacillus cereusD8) has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 28, 2024, with a deposit number of GDMCC NO.64815.

[0006] Furthermore, in the Bacillus cereus, the Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

[0007] In another aspect, the present invention provides the use of curcumin in the preparation of a quorum sensing inhibitor associated with protease production by Bacillus cereus, wherein the Bacillus cereus includes Bacillus cereus D8.

[0008] In another aspect, curcumin is used to inhibit the activity of Bacillus cereus proteases, including extracellular proteases produced by Bacillus cereus D8.

[0009] In another aspect, curcumin is used to inhibit the signaling molecules of quorum sensing associated with protease production by Bacillus cereus, wherein the protease produced by Bacillus cereus includes the extracellular protease produced by Bacillus cereus D8.

[0010] On the other hand, curcumin is used in the preparation of a signal molecule preparation for inhibiting quorum sensing associated with protease production by Bacillus cereus, wherein the protease produced by Bacillus cereus includes an extracellular protease produced by Bacillus cereus D8.

[0011] Furthermore, the quorum sensing signal molecule is AI-2.

[0012] On the other hand, the present invention provides a quorum sensing inhibitor related to protease produced by Bacillus cereus, wherein the quorum sensing inhibitor comprises curcumin; the protease produced by Bacillus cereus comprises an extracellular protease produced by Bacillus cereus D8, and the Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

[0013] Furthermore, in the quorum sensing inhibitor related to protease production by Bacillus cereus, the concentration of curcumin is 0.1 μg / mL to 400 μg / mL.

[0014] In another aspect, the present invention provides a method for preparing the above-mentioned quorum sensing inhibitor related to protease production by Bacillus cereus, comprising:

[0015] The activated Bacillus cereus D8 is inoculated into the first culture medium, cultured at a temperature of 25 to 45° C. for 10 to 15 hours, then inoculated into the second culture medium containing curcumin at an inoculum amount of 1% to 2%, and cultured at 25 to 45° C. for 10 to 15 hours at 50 to 200 r / min and shaking.

[0016] Further, in the method for preparing the quorum sensing inhibitor related to protease production by Bacillus cereus, the first culture medium is LB broth medium;

[0017] Preferably, the second culture medium is LB broth medium;

[0018] Preferably, the concentration of curcumin in the second culture medium is 0.1 μg / mL to 400 μg / mL;

[0019] Preferably, the preparation of the second culture medium containing curcumin comprises: dissolving curcumin in an organic solvent, preserving the solution at -20°C, and adding the preserved curcumin solution to the second culture medium in different amounts to prepare curcumin solutions of different concentrations.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] The invention provides Bacillus cereus with a LuxS / AI-2 quorum sensing system, and uses curcumin as an inhibitory active ingredient to inhibit quorum sensing related to protease production of Bacillus cereus to prepare a Bacillus cereus protease inhibitor. The protease activity of Bacillus cereus can be effectively inhibited. Without affecting bacterial growth, the LuxS / AI-2 quorum sensing system present in Bacillus cereus is interfered with to reduce the enzymatic activity of the protease, thereby achieving the effect of inhibiting the protease activity of Bacillus cereus.

[0022] The curcumin provided by the present invention is used as an inhibitory active ingredient to inhibit the protease activity of Bacillus cereus, has the characteristics of not being easy to produce drug resistance and having a good inhibitory effect, and can effectively inhibit the protease activity of Bacillus cereus to ensure the quality and safety of dairy products. The curcumin provided by the present invention effectively inhibits the protease activity of Bacillus cereus in a non-thermal treatment manner, that is, interferes with the LuxS / AI-2 type quorum sensing system of Bacillus cereus, and can also effectively avoid the thermal damage caused by heat treatment to the biologically active substances in the matrix where Bacillus cereus is located. The curcumin provided by the present invention is a natural compound with the characteristics of high efficacy and low toxicity, and has good development prospects in inhibiting Bacillus cereus protease as an inhibitory active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. 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 those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0024] Figure 1 is the enzyme activity of protease produced by Bacillus cereus D8;

[0025] Figure 2 is the thermal stability of the protease produced by Bacillus cereus D8; a) and b) are the relative enzyme activities of the protease at 65℃ for 30min and 140℃ for 4s, respectively;

[0026] Figure 3 This is a diagram showing the detection results of the luxS gene of Bacillus cereus;

[0027] Figure 4 This is the liquid chromatogram of AI-2 standard product; where X: time / min; Y: light absorption intensity / mAU;

[0028] Figure 5 This is the standard curve of AI-2 standard product;

[0029] Figure 6 This is the liquid chromatography diagram of Bacillus cereus D8 AI-2; where X: time / min; Y: light absorption intensity / mAU;

[0030] Figure 7 It is a graph showing changes in protease activity and signal molecule concentration at different culture temperatures; different letters in the same index indicate significant differences (p < 0.05);

[0031] Figure 8 is a graph showing changes in protease activity and signal molecule concentration under different osmotic pressures; different letters in the same index indicate significant differences (p < 0.05);

[0032] Fig. 9 It is a graph showing the changes in protease activity and signal molecule concentration at different pH values; different letters in the same index indicate significant differences (p < 0.05);

[0033] Fig.10 It is a graph showing the changes in protease activity and signal molecule concentration under different nutritional conditions and a graph showing bacterial density; among them, a) is the protease activity and signal molecule concentration under different nutritional conditions, b) is the strain concentration, different letters indicate significant differences (p < 0.05); different letters in the same indicator indicate significant differences (p < 0.05);

[0034] Fig.11 The growth curve and protease activity diagram of Bacillus cereus D8 with exogenous addition of AI-2; a) is the growth curve, b) is the protease activity under different concentrations of AI-2; different letters in the same index indicate significant differences (p < 0.05);

[0035] Fig.12 is the growth curve of Bacillus cereus D8 at different curcumin concentrations;

[0036] Fig.13 Graph showing AI-2 concentration and protease activity at different curcumin concentrations; a) and b) are AI-2 concentration and protease activity at different curcumin concentrations, respectively; different letters in the same index indicate significant differences (p < 0.05). DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with examples and drawings. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0038] On the one hand, the present invention provides a Bacillus cereus, wherein the Bacillus cereus D8 (Bacillus cereusD8) has been deposited in Guangdong Provincial Microbiological Culture Collection Center on June 28, 2024, with a deposit number of GDMCC NO.64815.

[0039] Furthermore, in the Bacillus cereus, the Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

[0040] As a communication mechanism between cells, quorum sensing (QS) can regulate related gene expression and physiological activities, such as biofilm formation, virulence factor production and extracellular protease production, through a series of reactions. Therefore, the present invention uses quorum sensing inhibitors (QSI) to block the QS system of Bacillus cereus to inhibit protease activity without affecting bacterial growth, which is a potential strategy with important research value.

[0041] In another aspect, the present invention provides the use of curcumin in the preparation of a quorum sensing inhibitor associated with protease production by Bacillus cereus, wherein the Bacillus cereus includes Bacillus cereus D8.

[0042] As a plant extract, curcumin is a natural QSI that can effectively inhibit QS-related physiological activities without affecting the growth of the strain. Therefore, using curcumin as a QSI to inhibit protease activity can provide theoretical support for controlling the protease activity of Bacillus cereus.

[0043] In another aspect, curcumin is used to inhibit the activity of Bacillus cereus proteases, including extracellular proteases produced by Bacillus cereus D8.

[0044] In another aspect, curcumin is used to inhibit the signaling molecules of quorum sensing associated with protease production by Bacillus cereus, wherein the protease produced by Bacillus cereus includes the extracellular protease produced by Bacillus cereus D8.

[0045] On the other hand, curcumin is used in the preparation of a signal molecule preparation for inhibiting quorum sensing associated with protease production by Bacillus cereus, wherein the protease produced by Bacillus cereus includes an extracellular protease produced by Bacillus cereus D8.

[0046] Furthermore, the quorum sensing signal molecule is AI-2.

[0047] On the other hand, the present invention provides a quorum sensing inhibitor related to protease produced by Bacillus cereus, wherein the quorum sensing inhibitor comprises curcumin; the protease produced by Bacillus cereus comprises an extracellular protease produced by Bacillus cereus D8, and the Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

[0048] Further, in the quorum sensing inhibitor related to Bacillus cereus protease production, the concentration of curcumin is 0.1 μg / mL to 400 μg / mL, and the concentration can be 0.1 μg / m, 0.5 μg / m, 1 μg / m, 5 μg / m, 10 μg / m, 50 μg / m, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, etc., but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0049] In another aspect, the present invention provides a method for preparing the above-mentioned quorum sensing inhibitor related to protease production by Bacillus cereus, comprising:

[0050] The activated Bacillus cereus D8 is inoculated into the first culture medium and cultured at a temperature of 25°C to 45°C for 10 h to 15 h. The culture temperature can be 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, 45°C, etc., but is not limited to the listed values, and other values ​​not listed within the range are equally applicable; the culture time can be 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, etc., but is not limited to the listed values, and other values ​​not listed within the range are equally applicable. The inoculation amount is 1-2% and inoculated into the second culture medium containing curcumin. The inoculation amount can be 1.1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc., but is not limited to the listed values. Other values ​​not listed in this range are also applicable; the culture is shaken at 50r / min-200r / min for 10h-15h at a temperature of 25°C-45°C, and the culture temperature can be 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, 40°C, 42°C, 45°C The rotation speed may be 50r / min, 80r / min, 100r / min, 120r / min, 140r / min, 150r / min, 160r / min, 180r / min, 200r / min, etc., but is not limited to the values ​​listed, and other values ​​not listed in the range are also applicable; the culture time may be 10h, 11h, 12h, 13h, 14h, 15h, etc., but is not limited to the values ​​listed, and other values ​​not listed in the range are also applicable.

[0051] Further, in the method for preparing the quorum sensing inhibitor related to protease production by Bacillus cereus, the first culture medium is LB broth medium;

[0052] Preferably, the second culture medium is LB broth medium;

[0053] Preferably, the concentration of curcumin in the second culture medium is 0.1 μg / mL to 400 μg / mL, and the concentration may be 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, etc., but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0054] Preferably, the preparation of the second culture medium containing curcumin comprises: dissolving curcumin in an organic solvent, preserving the solution at -20°C, and adding the preserved curcumin solution to the second culture medium in different amounts to prepare curcumin solutions of different concentrations.

[0055] In order to further illustrate the present invention, a Bacillus cereus, a quorum sensing inhibitor related to protease production by Bacillus cereus, a preparation method and the application of curcumin provided by the present invention are described below in combination with examples. However, it should be understood that these examples are implemented on the premise of the technical scheme of the present invention, and detailed implementation methods and specific operating processes are given only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the present invention, and the protection scope of the present invention is not limited to the following examples.

[0056] Example 1: Isolation and identification of Bacillus cereus D8 with strong heat resistance and high protease activity

[0057] 1.1 Isolation and identification of Bacillus cereus D8

[0058] After diluting the raw milk (collected from a ranch in Tianjin) several times with PBS, take samples of appropriate dilution and apply them to MYP medium and culture them at 30℃ for 24-48h. Colonies that produce a slightly pink color and are surrounded by a light pink precipitation ring on the MYP medium are regarded as suspicious colonies. After the isolated suspicious colonies are sequenced by 16S, their sequences are compared.

[0059] The strain was identified as Bacillus cereus D8 and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 28, 2024, with the deposit number GDMCC NO.64815.

[0060] 1.2 Evaluation of extracellular protease capacity of Bacillus cereus D8

[0061] The bacterial solution was centrifuged at 10000rpm for 10min, the supernatant was filtered through a 0.25μm filter membrane, 0.5ml of the supernatant was mixed with 0.5ml of 0.8% azocasein solution (dissolved in 0.1M Tris-HCl buffer (pH 8.0)), and the mixture was incubated in a water bath at 40°C for 2h. 1ml of 10% ice TCA solution was added to terminate the reaction, and the mixture was allowed to stand on ice for 30min. The mixture was then centrifuged at 4°C and 10000rpm for 10min to remove the precipitate. 600μl of the reactant was added with 700μl of NaOH (1M), and its absorbance at 440nm was measured. Control group: Mix 1 ml of 10% ice TCA solution with 0.5 ml of 0.8% azocasein solution (dissolved in 0.1 M Tris-HCl buffer (pH 8.0)), incubate in a water bath at 40°C for 2 h, add 0.5 ml of the supernatant of the bacterial solution, let stand on ice for 30 min, then centrifuge at 4°C 10000 rpm for 10 min to remove the precipitate, take 600 μl of the reaction and add 700 μl of NaOH (1 M).

[0062] The definition of protease activity unit is: the absorbance change of the liquid after reaction at 440nm per hour is 0.01 as one enzyme activity unit. The extracellular protease activity of Bacillus cereus D8 was determined, and the results are shown in Figure 1 described.

[0063] from Figure 1 The results show that the enzyme activity of Bacillus cereus D8 reaches 25.5U / mL, indicating that the protease activity of this strain is high.

[0064] 1.3 Evaluation of the heat resistance of protease produced by Bacillus cereus D8

[0065] The activated strain was inoculated into LB liquid culture medium at 2%. After 12 hours, the bacterial solution was centrifuged at 10000rpm for 10 minutes. After the supernatant was filtered through a 0.25μm filter, it was heat-treated by simulating pasteurization (65℃ water bath for 30 minutes) and ultra-high temperature instantaneous sterilization (140℃ oil bath for 4 seconds). After treatment, it was immediately placed in ice water and cooled to room temperature. The treated supernatant was tested for residual protease activity as described in 1.2 of Example 1. The relative enzyme activity of Bacillus cereus D8 protease under heat treatment conditions of 65℃ for 30 minutes and 140℃ for 4 seconds was tested. The results are shown in Table 1. Figure 2 shown.

[0066] The relative enzyme activity (RA) was expressed as the percentage of the residual enzyme activity after heat treatment ((Ct)) and the enzyme activity of the control group without heat treatment (C0). The calculation formula is as follows:

[0067]

[0068] Wherein: RA: relative enzyme activity (%);

[0069] Ct: residual enzyme activity after heat treatment (U / mL);

[0070] C0: enzyme activity of control group (U / mL);

[0071] from Figure 2 It can be seen that under the heat treatment conditions of 65°C for 30 min and 140°C for 4 s, the heat resistance and relative enzyme activity of Bacillus cereus D8 protease are 86.9% and 47.3%, respectively. Considering that Bacillus cereus with high protease activity and strong heat resistance exists in raw milk for a long time and will damage subsequent products, seriously affecting the health of consumers. Therefore, it is necessary for the present invention to select Bacillus cereus D8 as a research object.

[0072] Example 2: Detection of Bacillus cereus D8 in the presence of LuxS / AI-2QS system

[0073] 1.1 Detection of luxS gene

[0074] The strain was streaked on LB nutrient agar medium, and a single colony was picked into LB broth. After shaking culture at 37°C and 150 r / min overnight, DNA was extracted using a bacterial genomic DNA extraction kit.

[0075] The Bacillus cereus luxS gene (Gene ID=72451455) was searched according to NCBI, and primers were designed and sent to Shanghai Biotechnology Co., Ltd. for primer synthesis.

[0076] The primer sequences were luxS-F: 5′-ATGCCATCAGTAGAAAGCTTTGAA-3′ and luxS-R: 5′-TTATCCAAATACTTTCTCAAGTTC-3′.

[0077] The polymerase chain reaction (PCR) reaction system (25 μl) was as follows: 2×Taq Master Mix 12.5 μl, ddH2O 9.5 μl, upstream and downstream primers 1 μl each, DNA 1 μl. The reaction system was as follows: 94°C pre-denaturation for 10 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. The amplified products were separated by agarose gel electrophoresis, and the electrophoresis separation results were qualitatively analyzed by the gel imaging system. The results are shown in Figure 3 shown.

[0078] The luxS gene was amplified using the above primer sequences luxS-F and luxS-R. Figure 3 The results showed that this was consistent with the size of the luxS gene of Bacillus cereus FORC_047 on NCBI (Gene ID: 72451455), both of which were 474 bp in size, with clear bands and no other bands, indicating that the Bacillus cereus D8 used in this study had the luxS gene.

[0079] 1.2 Detection of AI-2 signaling molecules

[0080] Using liquid chromatography, first detect the AI-2 standard to determine the time of appearance of its characteristic peak, and then detect the sample. The specific method is as follows: After the activated strain is cultured for 12 hours, the strain is centrifuged at 8000×g for 10 minutes, the supernatant is filtered through a 0.25μm filter membrane, 600μl of the supernatant is mixed with equal volumes of DAN solution (0.2mg DAN dissolved in 1mL 0.1M HCl), mixed for 2 minutes, reacted at 90℃ for 40 minutes, then cooled at 4℃ for 10 minutes, filtered through a 0.45μm filter membrane, and excess impurities were removed. 50μl was taken for liquid chromatography detection, and the ultraviolet wavelength was 268nm. Liquid chromatography mobile phase A: 0.1% formic acid, mobile phase B: acetonitrile, the program is as follows: t = 0 min, 70% A, 30% B; t = 4 min, 70% A, 30% B; t = 12 min, 35% A, 65% B; t = 20 min, 35% A, 65% B; t = 24 min, 70% A, 30% B; t = 27 min, 70% A, 30% B. The detection results of AI-2 signal molecules are shown in Figure 4 .

[0081] Since DPD is a precursor of AI-2, it cannot be detected directly. It can be reacted with 2,3-diaminonaphthalene (DAN) to form a derivative 1-(3-methyl-benzo[g]quinoxaline-2-yl)-ethane-1,2-diol, which is usually detected by HPLC-UV. Therefore, the present invention indirectly indicates the content of AI-2 by measuring the DPD derivatization product. Figure 4 It can be seen that a peak value appeared between 6 and 7 minutes, indicating that this is the peak time of the DPD derivative product. According to the difference in peak area of ​​AI-2 standard products with different concentrations, the standard curve formula of the standard product is y=3×10 6 x+10 6 , the standard curve is as follows Figure 5 As shown, the concentration of AI-2 under different culture conditions can be calculated based on the standard curve. Figure 6 As shown, a certain amount of derivatives was detected in the sterile supernatant, indicating that Bacillus cereus D8 can produce AI-2 signal molecules.

[0082] Combined with the above test results, it is proved that the Bacillus cereus D8 provided by the present invention has the LuxS / AI-2 quorum sensing system.

[0083] Example 3 Detection of the relationship between LuxS / AI-2QS signal molecule AI-2 and protease in Bacillus cereus D8

[0084] The preparation process of protease produced by Bacillus cereus under different conditions includes:

[0085] The activated Bacillus cereus D8 was inoculated into the first culture medium, cultured at 37°C for 12 hours, then inoculated into the second culture medium at a 2% inoculation amount, and cultured under different conditions for 12 hours to obtain Bacillus cereus protease.

[0086] Among them, the first and second culture media of part 1.1.1 of Example 3 are LB broth culture media, and the culture conditions are shaking culture at 150r / min for 12h at 4°C, 25°C, 37°C and 45°C; the first culture medium of part 1.1.2 of Example 3 is LB broth culture media, and the second culture medium is LB broth culture media containing different contents of NaCl (0%, 0.5%, 1%, 2% and 3% NaCl), and the culture conditions are shaking culture at 37°C for 12h at 150r / min; the first culture medium of part 1.1.3 of Example 3 is LB broth culture media, and the second culture medium is pH 5, pH 5.5, pH 6, pH 7, pH 8 and pH 9 LB broth, the culture conditions are 37 ° C and 150r / min shaking culture for 12h; the first culture medium in part 1.1.4 of Example 3 is LB broth culture medium, and the second culture medium is LB culture medium containing 20%, 40%, 60%, 80% and 100% broth, and the culture conditions are 37 ° C and 150r / min shaking culture for 12h.

[0087] 1.1 Relationship between Bacillus cereus AI-2 and protease under different culture conditions

[0088] 1.1.1 Effect of culture temperature on concentration and protease activity of strain AI-2

[0089] The activated Bacillus cereus D8 was inoculated into LB broth, cultured at 37°C for 12 hours, and then inoculated into LB broth at 2%, and cultured at 4°C, 25°C, 37°C and 45°C with shaking at 150 r / min for 12 hours. The protease activity detection was referred to Section 1.2 of Example 1, and the signal molecule detection was referred to Section 1.2 of Example 2. The results are shown in Figure 7 shown.

[0090] like Figure 7 As shown, with the increase of temperature, the concentration of signal molecule AI-2 and the activity of protease both showed a trend of first increasing and then decreasing, and the two were positively correlated.

[0091] 1.1.2 Effect of osmotic pressure on concentration and protease activity of strain AI-2

[0092] The activated Bacillus cereus D8 was inoculated into LB broth, and after culturing at 37°C for 12 h, the strain was inoculated into LB broth medium containing 0%, 0.5%, 1%, 2% and 3% NaCl at a rate of 2%. The protease activity detection was referred to Section 1.2 of Example 1, and the signal molecule detection was referred to Section 1.2 of Example 2. The results are shown in Figure 8 shown.

[0093] like Figure 8 As shown in the figure, when the NaCl concentration in the LB broth medium was 0%, the concentration of the signal molecule in this group was the highest, which was 0.157 ng / mL. As the mass fraction of NaCl increased, the production of AI-2 by Bacillus cereus D8 was inhibited. At the same time, the protease activity in this group was the highest, which was 13.4 U / mL. However, as the osmotic pressure increased, the protease activity decreased and was significantly lower than that of the control group (p<0.05). Both the concentration of the signal molecule AI-2 and the protease activity decreased with the increase of osmotic pressure, and the two were positively correlated.

[0094] 1.1.3 Effect of pH on concentration and protease activity of strain AI-2

[0095] The activated Bacillus cereus D8 was inoculated into LB broth, and after culturing at 37°C for 12 h, the strain was inoculated at 2% into LB broth medium at pH 5, pH 5.5, pH 6, pH 7, pH 8, and pH 9. The protease activity detection was referred to Section 1.2 of Example 1, and the signal molecule detection was referred to Section 1.2 of Example 2. The results are shown in Fig. 9 shown.

[0096] like Fig. 9 As shown, with the continuous increase of pH, the change trend of protease activity is the same as that of AI-2 concentration, and the two are positively correlated.

[0097] 1.1.4 Effects of nutritional conditions on concentration and protease activity of strain AI-2

[0098] The activated Bacillus cereus D8 was inoculated into LB broth, and after culturing at 37°C for 12 h, the strain was inoculated at 2% into LB medium containing 20%, 40%, 60%, 80% and 100% broth. The protease activity detection was referred to Section 1.2 of Example 1, and the signal molecule detection was referred to Section 1.2 of Example 2. The results are shown in Fig.10 shown.

[0099] like Fig.10As shown in the figure, with the change of nutritional conditions, the two showed a negative correlation. The strain density under different nutritional conditions was analyzed to further understand the relationship between the two, indicating that nutritional deficiency would affect the normal growth of bacteria, which might be the main reason for the negative correlation between AI-2 concentration and protease activity.

[0100] 1.2 Effects of exogenous addition of AI-2 on the growth and protease activity of Bacillus cereus

[0101] 1.2.1 Effect of exogenous addition of different concentrations of AI-2 on strain growth

[0102] AI-2 was added to LB medium to a final concentration of 100, 300, 500, 800, 1500, 2000, 2500, and 3000 ng / mL. The activated strain was inoculated into the culture medium at different concentrations at 2%, and 200 μL of the bacterial solution was added to a 96-well plate. The OD595 was measured in the growth curve. The results are shown in Fig.11 a).

[0103] 1.2.2 Effect of exogenous addition of different concentrations of AI-2 on protease activity

[0104] The method of exogenous addition of AI-2 was as described in Section 1.2.1 of Example 3. The activated strain was inoculated into culture fluids of different concentrations at 2% and cultured for 12 hours. The protease activity was detected as described in Section 1.2 of Example 1. The results are shown in Fig.11 b).

[0105] Depend on Fig.11 a) It can be seen that exogenous addition of AI-2 will not affect the growth of the strain. After excluding the effect on growth, experiments were conducted to add different concentrations of AI-2. The results are as follows Fig.11 As shown in Figure b), with the increase in the concentration of exogenously added AI-2, the protease activity first showed an upward trend. When the AI-2 concentration was 2000 ng / mL, the protease activity reached the maximum, which was 15.8 U / mL, significantly higher than the protease activity when AI-2 was not added (p<0.05), indicating that AI-2 regulates the production of Bacillus cereus protease. After that, when the concentration of AI-2 continued to increase, the protease activity showed a downward trend, which may be because the signal molecule concentration cannot play an effective role after exceeding a certain range.

[0106] Based on the results of Example 3, it is shown that there is a certain correlation between the concentration of AI-2 and the protease activity in Bacillus cereus D8, and within a certain range of signal molecule concentrations, AI-2 regulates the production of Bacillus cereus protease.

[0107] Example 4 Effect of curcumin on Bacillus cereus AI-2 and protease

[0108] A method for preparing a quorum sensing inhibitor related to protease production by Bacillus cereus, comprising:

[0109] The activated Bacillus cereus D8 was inoculated into LB broth medium, cultured at 37°C for 12 hours, and then inoculated into LB broth medium containing curcumin at an inoculum amount of 2%, and cultured at 37°C with shaking at 150 r / min for 12 hours.

[0110] 1.1 Effects of different concentrations of curcumin on strain growth

[0111] Dissolve 10 mg of curcumin in 1 mL of 95% ethanol and store at -20°C until use. Add the prepared curcumin to LB liquid culture medium with final concentrations of 100, 200, 300, 400, 500, 800 and 1000 μg / mL, respectively, with 0 μg / mL as the control group. After inoculating the activated strain into the culture medium of each concentration of curcumin at 2%, take 200 μL of bacterial solution and add it to a 96-well plate, and measure OD595 in the growth curve. Among them, LB broth with various concentrations of curcumin without bacteria was set as a color control, and LB broth was used as a sterile control. The results are shown in Fig.12 shown.

[0112] As the concentration of curcumin increases, the growth of Bacillus cereus will be inhibited. Therefore, it is necessary to select an appropriate concentration of curcumin to eliminate the effect on the growth of Bacillus cereus. Fig.12 It can be seen that when the concentration of curcumin is 100 μg / mL and 200 μg / mL, there is no difference in the growth of the strain compared with the strain without curcumin. When the concentration of curcumin reaches 300 and 400 μg / mL, the growth of the strain is inhibited. When the concentration of curcumin reaches 500 μg / mL, the strain no longer grows. Therefore, the present invention selects curcumin concentrations of 0, 100, 200, 300, and 400 μg / mL for subsequent detection.

[0113] 1.2 Effects of different concentrations of curcumin on AI-2 concentration and protease activity

[0114] On the basis of 1.1 of Example 4, curcumin concentrations of 100, 200, 300, and 400 μg / mL were selected for subsequent testing, and 0 μg / mL was used as a control group.

[0115] The protease activity detection was carried out as described in Section 1.2 of Example 1, and the signal molecule detection was carried out as described in Section 1.2 of Example 2. Fig.13 shown.

[0116] exist Fig.13a), when the concentration of curcumin was 100 μg / mL, the concentration of AI-2 did not change significantly compared with the control group without curcumin (control group 0 μg / mL). When the concentration of curcumin was 200 μg / mL, the concentration of AI-2 decreased significantly (p<0.05), and the concentration of AI-2 was 0.92 ng / mL. As the concentration of curcumin continued to increase, the concentration of AI-2 also decreased. When the concentration of curcumin was 400 μg / mL, the growth of the strain was already restricted, and the concentration of AI-2 was only 0.66 ng / mL.

[0117] exist Fig.13 b), the protease activity decreased with the increase of curcumin concentration. Since the growth of strains was inhibited at curcumin concentrations of 300 and 400 μg / mL, the protease activity was extremely low. At a curcumin concentration of 200 μg / mL, the protease activity decreased by about 70%, which was significantly lower than the protease activity when curcumin was not added (p<0.05).

[0118] Based on the description in Examples 3 and 4, the protease inhibitor provided by the present invention achieves the purpose of inhibiting the protease activity of Bacillus cereus by interfering with the LuxS / AI-2QS system of Bacillus cereus without affecting the growth of bacteria.

[0119] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus cereus, characterized in that The Bacillus cereus D8 ( Bacillus cereus D8) was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 28, 2024, with the deposit number GDMCC NO.64815.

2. The Bacillus cereus according to claim 1, characterized in that The Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

3. The use of curcumin in the preparation of a quorum sensing inhibitor related to protease production by Bacillus cereus, characterized in that: The Bacillus cereus includes Bacillus cereus D8.

4. The application of curcumin in inhibiting the protease activity of Bacillus cereus, characterized in that: The Bacillus cereus protease includes an extracellular protease produced by Bacillus cereus D8.

5. The use of curcumin in inhibiting the quorum sensing signal molecule associated with protease production by Bacillus cereus, characterized in that: The protease produced by Bacillus cereus includes the extracellular protease produced by Bacillus cereus D8.

6. The use according to claim 5, wherein the quorum sensing signal molecule is AI-2.

7. A quorum sensing inhibitor related to protease production by Bacillus cereus, characterized in that: The quorum sensing inhibitor includes curcumin; the protease produced by Bacillus cereus includes an extracellular protease produced by Bacillus cereus D8, and the Bacillus cereus D8 has a LuxS / AI-2 type quorum sensing system.

8. The quorum sensing inhibitor related to protease production by Bacillus cereus according to claim 7, characterized in that The concentration of the curcumin is 0.1µg / mL to 400µg / mL.

9. A method for preparing a quorum sensing inhibitor related to protease production by Bacillus cereus according to claim 7 or 8, characterized in that: include: The activated Bacillus cereus D8 was inoculated into the first culture medium, cultured at 25°C~45°C for 10h~15h, and then inoculated into the second culture medium containing curcumin at an inoculum amount of 1%~2%, and cultured at 25°C~45°C and shaking at 50r / min~200r / min for 10h~15h.

10. The method for preparing a quorum sensing inhibitor related to protease production by Bacillus cereus according to claim 9, characterized in that: The first culture medium is LB broth medium; preferably, the second culture medium is LB broth medium; Preferably, the concentration of curcumin in the second culture medium is 0.1µg / mL to 400µg / mL; Preferably, the preparation of the second culture medium containing curcumin comprises: dissolving curcumin in an organic solvent, preserving the solution at -20°C, and adding the preserved curcumin solution to the second culture medium in different amounts to prepare curcumin solutions of different concentrations.

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