Application of piperazine derivative in bacteriostasis of putrefying bacteria

Abstract

The invention relates to the technical field of microorganisms, and discloses a putrefying bacterium bacteriostasis determination method of a piperazine derivative, which comprises the following steps: S1, taking endophyte bacillus coagulans of cultured pseudosciaena crocea as a research object, obtaining 9 cyclic dipeptide small molecule compounds from 30L of fermentation liquor of the endophyte bacillus coagulans, the cyclic (proline-valine) has bacteriostatic activity, the cyclic (proline-valine) has bacteriostatic activity, and the cyclic (proline-valine) has bacteriostatic activity; performing structural modification screening by taking a piperazine ring as a parent nucleus; s2, screening a solvent, washing and activating the cultured three bacteria to obtain a bacterial suspension, and culturing and observing the bacterial suspension; s3, determining the maximum solubility of the compound by using a dynamic method; s4, completing a compound sensitivity experiment in a sterile environment; and S5, determining the minimum inhibitory concentration by using a broth dilution method. The research on the antibacterial structure-function relationship of the diketopiperazine compound further finds that the 1-Boc-4 (piperidine-4-yl)-piperazine and the 1-cyclopropyl methyl piperazine have good antibacterial activity on the three indicator bacteria, and have the potential value of being developed into preservatives and preservatives.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to application of piperazine derivatives in inhibiting spoilage bacteria. Background Art

[0002] Since the 19th century, microbial preservatives have gradually attracted widespread attention and in-depth research from scientific researchers due to their many advantages, including natural origin, high safety, broad antibacterial spectrum, no toxic side effects, and strong antibacterial properties. This type of preservative is widely used in food, medicine, cosmetics and other fields, becoming an important choice to replace traditional chemical preservatives. Compared with chemical synthetic preservatives, microbial preservatives have better biocompatibility and environmental friendliness, and will not cause drug resistance problems during use, so they show great development potential in the market.

[0003] Among many microorganisms, lactic acid bacteria have become an ideal target for the development of microbial preservatives due to their unique biological characteristics and food-grade safety recognized by the scientific community. Lactic acid bacteria can not only produce a variety of metabolites with antibacterial activity, but also have a variety of biological functions such as antioxidant and immunomodulatory, which makes them have broad application prospects in the field of food preservation and safety. Existing studies have shown that lactic acid bacteria can have a significant inhibitory effect on a variety of pathogens and spoilage bacteria by producing lactic acid, hydrogen peroxide and polypeptide antibacterial substances.

[0004] However, although a lot of progress has been made in the research of lactic acid bacteria antimicrobial products, only one lactic acid bacteria metabolite, nisin, has been widely used in commercial applications. As a natural antimicrobial peptide, nisin has been widely used in the food industry, especially for its inhibitory effect on Gram-positive bacteria. Unfortunately, nisin has a very poor inhibitory effect on Gram-negative bacteria, which greatly limits its application in the field of broad-spectrum antimicrobial, especially in the field of food preservation.

[0005] Gram-negative bacteria are widely present in food and the environment, and are one of the main pathogens that cause food spoilage and foodborne diseases. How to effectively inhibit the growth of Gram-negative bacteria has become a difficult problem that needs to be solved in the field of food preservation and antiseptic. In this context, the present invention studies and develops a class of novel piperazine derivatives with broad-spectrum antibacterial activity, especially with significant inhibitory effect on Gram-negative bacteria, in view of the deficiencies of the prior art. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides the use of piperazine derivatives in inhibiting spoilage bacteria, which solves the problem that the active molecule nisin has extremely poor antibacterial effect on Gram-negative bacteria, limiting its application scope in the field of preservation.

[0007] To achieve the above object, the present invention is implemented by the following technical scheme: a method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria, comprising the following steps:

[0008] S1. Taking the endophytic bacteria Bacillus coagulans of farmed large yellow croaker as the research object, 9 cyclic dipeptide small molecule compounds were obtained from 30L fermentation broth of the bacteria, and the ring (proline-valine) with antibacterial activity was structurally modified to screen out piperazine derivatives 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine;

[0009] S2, screening of solvents, washing and activating the cultured 3 bacterial strains to obtain bacterial suspensions, and culturing and observing the bacterial suspensions;

[0010] S3. Determination of the maximum solubility of the compound using a dynamic method;

[0011] S4. Complete compound sensitivity experiments under sterile conditions;

[0012] S5. Determination of minimum inhibitory concentration: using the broth dilution method, the minimum inhibitory concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine against three spoilage bacteria were determined respectively;

[0013] S6. In vitro cytotoxicity test: the inhibition rate of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine on four types of cells (including human normal liver cells HL-7702) at different concentrations was determined by MTT method;

[0014] S7. Hemolysis experiment: The biosafety of 1-Boc-4(piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine was evaluated by collecting eye blood from healthy BALB / c mice using the hemolysis method.

[0015] Preferably, the step S2 comprises the following steps: washing the activated cultured three bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Shewanella putrefaciens) with sterile saline to obtain a bacterial suspension, and adjusting the absorbance of the bacterial suspension to OD 600 =0.5, dilute the bacterial suspension 10 times, take 100 μL of bacterial solution to coat the plate, use 7 μL of dimethyl sulfoxide solution with different concentration gradients, drop it on a sterile filter paper with a diameter of 6 mm, place it in a 37°C constant temperature incubator and culture it for 24 h. According to the above steps, repeat 3 groups of parallel experiments for each concentration to observe whether an inhibition zone is produced, among which normal saline is the blank control group and 0.7 mg / mL norfloxacin solution is the positive control.

[0016] Preferably, in step S3, 10 mg of the compound is added successively to 1 mL of 5% and 10% dimethyl sulfoxide solutions respectively until the solute reaches equilibrium, i.e., maximum solubility.

[0017] Preferably, the S4 step includes the following steps: in a sterile environment, using a pipette to draw 7 μL of a saturated concentration of the compound solution, dripping it on a sterile filter paper with a diameter of 6 mm, sticking the two compounds on a plate, using different concentrations of dimethyl sulfoxide solutions as blank controls (using a 10% dimethyl sulfoxide solution for Escherichia coli and Pseudomonas aeruginosa, and a 5% dimethyl sulfoxide solution for Shewanella putrefaciens), and using a 0.7 mg / mL norfloxacin solution as positive controls, and culturing in a 37°C constant temperature incubator for 24 hours. According to the above steps, three parallel experiments are repeated for each indicator bacterium to observe whether an inhibition zone is produced, and the antibacterial effect is described by the average diameter of the inhibition zone.

[0018] Preferably, the step S5 comprises the following steps:

[0019] S501, prepare two groups of 10 / 9 sterilized small test tubes respectively, mark them as No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10 (Group 2 to No. 9), use a pipette to transfer 4 mL of broth into test tube No. 1, and then use a pipette to transfer 2 mL of broth into test tubes No. 2-10 respectively;

[0020] S502, weigh 600 mg of 1-Boc-4 (piperidin-4-yl) -piperazine and dissolve it in 4 mL of 50% methanol solution to prepare a saturated concentration solution, marked as test tube No. 1; in the same way, weigh 509 mg of 1-cyclopropylmethylpiperazine and dissolve it in test tube No. 1 (test tube No. 1 contains 4 mL of LB nutrient broth), prepare a concentration of 1, and shake well;

[0021] S503, draw 2 mL of solution from test tube No. 1 and add it to test tube No. 2, mix well, draw 2 mL of solution from test tube No. 2 and add it to test tube No. 3, and so on, dilute by multiples;

[0022] S504, prepare two groups of 36 / 33 25mL sterilized conical bottles, respectively, and mark them as 1a, 1b, 1c, 2a, 2b, 2c, and so on, until 10a, 10b, 10c (the second group to 9a, 9b, 9c), blank a, blank b, blank c, control a, control b, control c, and add 10mL broth to each of them;

[0023] S505, after sterilization, aspirate 600 μL of broth (except blank and control) and add 100 μL OD 600 The value is 0.5 bacterial solution, 500 μL of the solution in the test tubes marked 1 to 10 (groups 2 to 9) are pipetted in turn and added to the corresponding conical flasks; for the control group, 100 μL of broth is pipetted and 100 μL of bacterial solution is added;

[0024] S506. Place the culture medium in a light-shaking incubator, set the temperature to 37°C, and the rotation speed to 180 r / min. Take it out after a certain period of time, and measure the absorbance at a wavelength of 600 nm using a UV-visible spectrophotometer. Cultivate Escherichia coli and Pseudomonas aeruginosa for 4 hours, and culture Shewanella putrefaciens for 5 hours.

[0025] Preferably, the step S6 comprises the following steps: inoculating HepG2 cells, A549 cells, HeLa cells and HL-7702 cells (normal human liver cells) in the logarithmic growth phase into a 96-well plate, with 8×10 3 After culturing cells in an incubator for 24 h, 1-Boc-4 (piperidin-4-yl) -piperazine and 1-cyclopropylmethylpiperazine were added at concentrations of 20, 50, 100, 200, and 500 μg / mL, respectively. After incubation with the cells for 48 h, 50 μL of MTT was added to each well and the incubation continued for 4 h. Then, 100 μL of DMSO was added to each well to dissolve the crystals, and the absorbance was measured at 570 nm with a microplate reader.

[0026] Preferably, the S7 step includes the following steps: taking mouse blood from the eyeball and placing it in a tube with sodium heparin, centrifuging the whole mouse blood at 2000rpm for 5min, washing with physiological saline until the supernatant is free of red, and removing the supernatant. The lower layer of red blood cells is prepared into a 4% red blood cell suspension according to the volume ratio for standby use. Take 1.5mL centrifuge tubes and number them, and add 0.5mL of 4% red blood cell suspension to each centrifuge tube. The sample group is added with 0.5mL of different concentrations (25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 500ug / mL) prepared with physiological saline; the negative control group is added with 0.5mL of physiological saline; and the positive control group is added with 0.5mL of water. The above-numbered groups are incubated at 37°C for 4h, centrifuged at 2000rpm for 5min, and then the supernatant is added to a 96-well culture plate, and the OD value of hemoglobin in the supernatant at 540nm is measured by an enzyme marker.

[0027] Preferably, in the step S6, cell inhibition rate = (average OD value of the negative group - average OD value of the experimental group) / (average OD value of the negative group - average OD value of the blank group) × 100%.

[0028] Preferably, in the step S7,

[0029] In addition, the present invention also provides the use of piperazine derivatives in inhibiting spoilage bacteria. The piperazine derivatives also have anti-tumor activity and have inhibitory effects on HepG2 cells, A549 cells and HeLa cells in in vitro experiments.

[0030] The present invention provides the use of piperazine derivatives in inhibiting spoilage bacteria. It has the following beneficial effects:

[0031] 1. The present invention uses a paper diffusion method to perform an inhibition zone experiment to screen out a solvent suitable for drug dissolution and an optimal solvent concentration, discloses a method for determining the inhibition of piperazine derivatives on three main microorganisms that cause spoilage, namely, Escherichia coli (E.co li), Pseudomonas aeruginosa (P.aerugi nosa) and Shewanella spp., and prepares different concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine solution and 1-cyclopropylmethylpiperazine by a broth dilution method. It is measured that the minimum inhibitory concentrations that can inhibit the normal growth and reproduction of the three spoilage bacteria are 3.75 mg / mL and 6.36 mg / mL;

[0032] 2. This experiment can preliminarily determine that 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine have a certain antibacterial effect on spoilage bacteria, with good safety. It also provides new ideas and references for the further development and utilization of antibacterial preservatives. The endophytic bacteria Bacillus coagulans of farmed large yellow croaker were used as the research object, and 9 cyclic dipeptide small molecule compounds were obtained from 30L fermentation broth of the bacteria;

[0033] 3. The antibacterial ring (proline-valine) was structurally modified to study the antibacterial structure-activity relationship of diketopiperazine compounds. Further screening revealed that 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine had good antibacterial activity against three indicator bacteria and had the potential to be developed as preservatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 1-Boc-4 (piperidin-4-yl)-piperazine Escherichia coli 4h OD 600 value;

[0035] Figure 2 OD of 1-Boc-4 (piperidin-4-yl)-piperazine Pseudomonas aeruginosa 4h in the present invention 600 value;

[0036] Figure 3 OD of 1-Boc-4 (piperidin-4-yl)-piperazine Shewanella putrefaciens in the present invention at 5h 600 value;

[0037] Figure 4 1-cyclopropylmethylpiperazine Escherichia coli 4h OD 600 value;

[0038] Figure 5 1-cyclopropylmethylpiperazine Pseudomonas aeruginosa 4h OD 600 value;

[0039] Figure 6 1-cyclopropylmethylpiperazine Shewanella putrefaciens 5h OD 600 value;

[0040] Figure 7 This is a diagram of the cytotoxicity test in the present invention (four types of cells were selected, A: human liver cancer cell HepG2; B: human lung cancer cell A549; C: human cervical cancer cell HeLa; D: human normal liver cell HL-7702);

[0041] Figure 8 This is a hemolysis rate test diagram of the present invention (A: hemolysis of 1-Boc-4 (piperidin-4-yl) -piperazine at different concentrations; B: 1-cyclopropylmethylpiperazine);

[0042] Fig. 9 It is the molecular formula of the present invention. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The embodiment of the present invention provides a method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria, comprising the following steps:

[0045] S1. Taking the endophytic bacteria Bacillus coagulans of farmed large yellow croaker as the research object, 9 cyclic dipeptide small molecule compounds were obtained from 30L fermentation broth of the bacteria, and the ring (proline-valine) with antibacterial activity was structurally modified to screen out piperazine derivatives 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine;

[0046] S2, screening of solvents, washing and activating the cultured 3 bacterial strains to obtain bacterial suspensions, and culturing and observing the bacterial suspensions;

[0047] S3. Determination of the maximum solubility of the compound using a dynamic method;

[0048] S4. Complete compound sensitivity experiments under sterile conditions;

[0049] S5. Determination of minimum inhibitory concentration: using the broth dilution method, the minimum inhibitory concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine against three spoilage bacteria were determined respectively;

[0050] S6. In vitro cytotoxicity test: the inhibition rate of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine on four types of cells (including human normal liver cells HL-7702) at different concentrations was determined by MTT method;

[0051] S7. Hemolysis experiment: The biosafety of 1-Boc-4(piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine was evaluated by collecting eye blood from healthy BALB / c mice using the hemolysis method.

[0052] Example 1: Screening of solvents

[0053] The activated cultured 3 bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Shewanella putrefaciens) were washed with sterile saline to obtain a bacterial suspension, and the absorbance of the bacterial suspension was adjusted to OD 600 =0.5, dilute the bacterial suspension 10 times, take 100 μL of bacterial solution to coat the plate, use 7 μL of dimethyl sulfoxide solution with different concentration gradients, drop it on a sterile filter paper with a diameter of 6 mm, place it in a 37°C constant temperature incubator and culture it for 24 h. According to the above steps, repeat 3 groups of parallel experiments for each concentration to observe whether an inhibition zone is produced, among which normal saline is the blank control group and 0.7 mg / mL norfloxacin solution is the positive control.

[0054] The results of solvent screening are shown in the following table: From Table 1 and Table 2, the dimethyl sulfoxide solution with a minimum concentration of 10% has no antibacterial effect on Escherichia coli and Pseudomonas aeruginosa, and the dimethyl sulfoxide solution with a minimum concentration of 5% has no antibacterial effect on Shewanella putrefaciens.

[0055] Table 1 Antibacterial effects of different concentrations of dimethyl sulfoxide solutions

[0056]

[0057] Table 2 Antibacterial effects of different concentrations of dimethyl sulfoxide solutions

[0058]

[0059] Example 2: Determination of Maximum Solubility of Compounds

[0060] In 1 mL of 5% and 10% dimethyl sulfoxide solutions, 10 mg of the compound was added successively until the solute reached equilibrium, which was the maximum solubility.

[0061] Example 3: Compound sensitivity test

[0062] Under a sterile environment, 7 μL of saturated compound solution was taken with a pipette and dropped on a sterile filter paper with a diameter of 6 mm. The two compounds were attached to a plate, and the blank control used dimethyl sulfoxide solutions of different concentrations (10% dimethyl sulfoxide solution was used for Escherichia coli and Pseudomonas aeruginosa, and 5% dimethyl sulfoxide solution was used for Shewanella putrefaciens). The positive control used 0.7 mg / mL norfloxacin solution. The plates were placed in a constant temperature incubator at 37°C and cultured for 24 h. According to the above steps, 3 parallel experiments were repeated for each indicator bacteria to observe whether an inhibition zone was produced, and the average diameter of the inhibition zone was used to describe the antibacterial effect.

[0063] Drug activity screening results:

[0064] As shown in Table 3, 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine were screened out from 33 compounds, and they all had obvious antibacterial effects on Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens.

[0065] Table 3 Screening results of antibacterial activity of different drugs

[0066]

[0067] Example 4: Determination of minimum inhibitory concentration

[0068] S501, prepare two groups of 10 / 9 sterilized small test tubes respectively, mark them as No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10 (Group 2 to No. 9), use a pipette to transfer 4 mL of broth into test tube No. 1, and then use a pipette to transfer 2 mL of broth into test tubes No. 2-10 respectively;

[0069] S502, weigh 600 mg of 1-Boc-4 (piperidin-4-yl) -piperazine and dissolve it in 4 mL of 50% methanol solution to prepare a saturated concentration solution; weigh 509 mg of 1-cyclopropylmethylpiperazine and dissolve it in test tube No. 1 (test tube No. 1 contains 4 mL of LB nutrient broth) to prepare a concentration of 1, and shake well;

[0070] S503, draw 2 mL of solution from test tube No. 1 and add it to test tube No. 2, mix well, draw 2 mL of solution from test tube No. 2 and add it to test tube No. 3, and so on, dilute by multiples;

[0071] S504, prepare two groups of 36 / 33 25mL sterilized conical bottles, respectively, and mark them as 1a, 1b, 1c, 2a, 2b, 2c, and so on, until 10a, 10b, 10c (the second group to 9a, 9b, 9c), blank a, blank b, blank c, control a, control b, control c, and add 10mL broth to each of them;

[0072] S505, after sterilization, aspirate 600 μL of broth (except blank and control) and add 100 μL OD 600 The value is 0.5 bacterial solution, and 500 μL of the solution in the test tubes marked 1 to 10 (groups 2 to 9) are pipetted in sequence and added to the corresponding conical flasks;

[0073] S506, control group, aspirate 100 μL broth and add 100 μL bacterial solution. Place in a light shaking incubator, set the temperature to 37°C, the speed to 180 r / min, take out after a certain interval, and measure the absorbance at a wavelength of 600 nm with a UV-visible spectrophotometer. Escherichia coli and Pseudomonas aeruginosa were cultured for 4 hours, and Shewanella putrefaciens was cultured for 5 hours.

[0074] The minimum inhibitory concentration determination data are shown in the following table:

[0075] Table 4 Minimum inhibitory concentration sample addition table

[0076]

[0077]

[0078] According to the above method, the OD values ​​of each concentration of 1-Boc-4 (piperidin-4-yl)-piperazine and the control group in different indicator bacteria were measured. 600 Value. Figure 1 , Figure 2 , Figure 3 It can be seen that the greater the concentration of 1-Boc-4 (piperidin-4-yl)-piperazine, the better the inhibitory effect on the three spoilage bacteria. In this set of concentration gradients, the minimum inhibitory concentration of 1-Boc-4 (piperidin-4-yl)-piperazine on the three spoilage bacteria is 3.75 mg / mL; analysis shows that different concentrations of 1-cyclopropylmethylpiperazine have different antibacterial activities on the three spoilage bacteria, and the absorbance values ​​at 600 nm are measured. Figure 4 , 5 , 6 It can be seen that the higher the concentration of 1-cyclopropylmethylpiperazine, the better the antibacterial activity against the three spoilage bacteria. The minimum inhibitory concentration of 1-cyclopropylmethylpiperazine against Escherichia coli, Shewanella putrefaciens and Pseudomonas aeruginosa is 6.363 mg / mL.

[0079] Example 5: In vitro cytotoxicity test

[0080] HepG2 cells, A549 cells, HeLa cells and HL-7702 cells (normal human liver cells) in the logarithmic growth phase were seeded in 96-well plates, with 8×10 3After culturing in an incubator for 24 h, 1-Boc-4(piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine at concentrations of 20, 50, 100, 200, and 500 μg / mL were added to the cells, and 50 μL of MTT was added to each well. The cells were incubated for 48 h, and then 100 μL of DMSO was added to each well to dissolve the crystals. The absorbance was measured at 570 nm with a microplate reader. Cell inhibition rate = (average OD value of the negative group - average OD value of the experimental group)) / (average OD value of the negative group - average OD value of the blank group) × 100%.

[0081] Conclusion: In order to study the antitumor effect and biosafety of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine, we incubated HepG2 cells, A549 cells, HeLa cells and HL-7702 cells with different concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine for 48 h. Figure 7 , we found that 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine had certain anti-tumor effects on HepG2 cells, A549 cells and HeLa cells, among which the anti-tumor effect on HepG2 cells was the most obvious, and there was almost no toxicity to HL-7702 cells. These results show that 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine have certain anti-tumor effects while inhibiting bacteria, and have high biological safety.

[0082] Example 6: Hemolysis Experiment

[0083] Take mouse blood from the eyeball and put it into a tube with sodium heparin. Centrifuge the whole blood of the mouse at 2000rpm for 5min, wash with physiological saline until the supernatant is no longer red, and remove the supernatant. Prepare the lower layer of red blood cells into a 4% red blood cell suspension according to the volume ratio for later use. Take 1.5mL centrifuge tubes and number them. Add 0.5mL of 4% red blood cell suspension to each centrifuge tube. Add 0.5mL of different concentrations (25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 500ug / mL) prepared with physiological saline to the sample group; add 0.5mL of physiological saline to the negative control group; add 0.5mL of water to the positive control group. Incubate the above-numbered groups at 37℃ for 4h, centrifuge at 2000rpm for 5min, then take the supernatant and add it to a 96-well culture plate. Use an enzyme marker to measure the OD value of hemoglobin in the supernatant at 540nm.

[0084] Conclusion: In order to evaluate the biosafety of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine, the hemolysis method was used to collect eye blood from healthy BALB / c mice. Different concentrations (25, 50, 100, 200 and 500 g / mL) of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine were added to 4% red blood cell suspension and incubated for 4 hours. Figure 8 As shown, with the increase of concentration, the hemolysis rate showed a slightly upward trend, but no obvious hemolysis occurred. When the concentration was 500 g / mL, the hemolysis rate was still less than 5%, indicating that 1-Boc-4 (piperidin-4-yl) -piperazine and 1-cyclopropylmethylpiperazine have good safety.

[0085] Summary: Bacillus coagulans has the advantages of high temperature resistance and easy storage, and its application as an antibacterial agent has been very extensive. 1-Boc-4 (piperidin-4-yl)-piperazine is one of the small molecules separated from its metabolites and then modified and screened. The antibacterial zone experiment was performed by the paper diffusion method to screen out the solvent suitable for drug dissolution, the optimal solvent concentration, and the drug that has an antibacterial effect on the three spoilage bacteria; different concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine solution and 1-cyclopropylmethylpiperazine were prepared by the broth dilution method, and the minimum inhibitory concentration that can inhibit the normal growth and reproduction of the three spoilage bacteria was measured to be 3.75 mg / mL and 6.36 mg / mL. This experiment can preliminarily determine that 1-Boc-4 (piperidin-4-yl)-piperazine has a certain antibacterial effect on spoilage bacteria and has good safety. At the same time, it also provides new ideas and references for the further development and utilization of antibacterial preservatives.

[0086] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the inhibitory effect of piperazine derivatives on spoilage bacteria, characterized in that: The following steps are involved: S1. Taking the endophytic bacteria Bacillus coagulans of farmed large yellow croaker as the research object, 9 cyclic dipeptide small molecule compounds were obtained from 30L fermentation broth of the bacteria, and the ring (proline-valine) with antibacterial activity was structurally modified to screen out piperazine derivatives 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine; S2, screening of solvents, washing and activating the cultured 3 bacterial strains to obtain bacterial suspensions, and culturing and observing the bacterial suspensions; S3. Determination of the maximum solubility of the compound using a dynamic method; S4. Complete compound sensitivity experiments under sterile conditions; S5. Determination of minimum inhibitory concentration: using the broth dilution method, the minimum inhibitory concentrations of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine against three spoilage bacteria were determined respectively; S6. In vitro cytotoxicity test: the inhibition rate of 1-Boc-4 (piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine on four types of cells (including human normal liver cells HL-7702) at different concentrations was determined by MTT method; S7. Hemolysis experiment: The biosafety of 1-Boc-4(piperidin-4-yl)-piperazine and 1-cyclopropylmethylpiperazine was evaluated by collecting eye blood from healthy BALB / c mice using the hemolysis method.

2. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: The S2 step includes the following steps: The activated cultured 3 bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Shewanella putrefaciens) were washed with sterile saline to obtain a bacterial suspension, and the absorbance of the bacterial suspension was adjusted to OD 600 =0.5, dilute the bacterial suspension 10 times, take 100 μL of bacterial solution to coat the plate, use 7 μL of dimethyl sulfoxide solution with different concentration gradients, drop it on a sterile filter paper with a diameter of 6 mm, place it in a 37°C constant temperature incubator and culture it for 24 h. According to the above steps, repeat 3 groups of parallel experiments for each concentration to observe whether an inhibition zone is produced, among which normal saline is the blank control group and 0.7 mg / mL norfloxacin solution is the positive control.

3. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: In the step S3, 10 mg of the compound is added successively to 1 mL of 5% and 10% dimethyl sulfoxide solutions respectively until the solute reaches equilibrium, which is the maximum solubility.

4. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: The S4 step includes the following steps: Under a sterile environment, 7 μL of saturated compound solution was taken with a pipette and dropped on a sterile filter paper with a diameter of 6 mm. The two compounds were attached to a plate, and the blank control used dimethyl sulfoxide solutions of different concentrations (10% dimethyl sulfoxide solution was used for Escherichia coli and Pseudomonas aeruginosa, and 5% dimethyl sulfoxide solution was used for Shewanella putrefaciens). The positive control used 0.7 mg / mL norfloxacin solution. The plates were placed in a constant temperature incubator at 37°C and cultured for 24 h. According to the above steps, 3 parallel experiments were repeated for each indicator bacteria to observe whether an inhibition zone was produced, and the average diameter of the inhibition zone was used to describe the antibacterial effect.

5. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: The S5 step includes the following steps: S501, prepare two groups of 10 / 9 sterilized small test tubes respectively, mark them as No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9, No. 10 (Group 2 to No. 9), use a pipette to transfer 4 mL of broth into test tube No. 1, and then use a pipette to transfer 2 mL of broth into test tubes No. 2-10 respectively; S502, weigh 600 mg of 1-Boc-4 (piperidin-4-yl) -piperazine and dissolve it in 4 mL of 50% methanol solution to prepare a saturated concentration solution, marked as test tube No. 1; in the same way, weigh 509 mg of 1-cyclopropylmethylpiperazine and dissolve it in test tube No. 1 (test tube No. 1 contains 4 mL of LB nutrient broth), prepare a concentration of 1, and shake well; S503, draw 2 mL of solution from test tube No. 1 and add it to test tube No. 2, mix well, draw 2 mL of solution from test tube No. 2 and add it to test tube No. 3, and so on, dilute by multiples; S504, prepare two groups of 36 / 33 25mL sterilized conical bottles, respectively, and mark them as 1a, 1b, 1c, 2a, 2b, 2c, and so on, until 10a, 10b, 10c (the second group to 9a, 9b, 9c), blank a, blank b, blank c, control a, control b, control c, and add 10mL broth to each of them; S505, after sterilization, aspirate 600 μL of broth (except blank and control) and add 100 μL OD 600 The value is 0.5 bacterial solution, 500 μL of the solution in the test tubes marked 1 to 10 (groups 2 to 9) are pipetted in turn and added to the corresponding conical flasks, and 100 μL of broth is pipetted out of the control group and then 100 μL of bacterial solution is added; S506. Place the culture medium in a light-shaking incubator, set the temperature to 37°C, and the rotation speed to 180 r / min. Take it out after a certain period of time, and measure the absorbance at a wavelength of 600 nm using a UV-visible spectrophotometer. Cultivate Escherichia coli and Pseudomonas aeruginosa for 4 hours, and culture Shewanella putrefaciens for 5 hours.

6. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: The S6 step includes the following steps: inoculating HepG2 cells, A549 cells, HeLa cells and HL-7702 cells (normal human liver cells) in the logarithmic growth phase into a 96-well plate, with 8×10 3 After culturing cells in an incubator for 24 h, 1-Boc-4 (piperidin-4-yl) -piperazine and 1-cyclopropylmethylpiperazine were added at concentrations of 20, 50, 100, 200, and 500 μg / mL, respectively. After incubation with the cells for 48 h, 50 μL of MTT was added to each well and the incubation continued for 4 h. Then, 100 μL of DMSO was added to each well to dissolve the crystals, and the absorbance was measured at 570 nm with a microplate reader.

7. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 1, characterized in that: The S7 step includes the following steps: taking mouse blood from the eyeball and putting it into a tube with sodium heparin, centrifuging the whole mouse blood at 2000rpm for 5min, washing with physiological saline until the supernatant is free of red, removing the supernatant, preparing the lower layer of red blood cells into a 4% red blood cell suspension according to the volume ratio for standby use, taking 1.5mL centrifuge tubes and numbering them, adding 0.5mL of 4% red blood cell suspension to each centrifuge tube, adding 0.5mL of different concentrations (25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 500ug / mL) prepared with physiological saline to the sample group; adding 0.5mL of physiological saline to the negative control group; adding 0.5mL of water to the positive control group, incubating the above-numbered groups at 37°C for 4h, centrifuging at 2000rpm for 5min, then taking the supernatant and adding it to a 96-well culture plate, and measuring the OD value of hemoglobin in the supernatant at 540nm with an enzyme marker.

8. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 6, characterized in that: In the step S6, cell inhibition rate = (average OD value of the negative group - average OD value of the experimental group) / (average OD value of the negative group - average OD value of the blank group) × 100%.

9. The method for determining the antibacterial activity of piperazine derivatives on spoilage bacteria according to claim 7, characterized in that: In the step S7, 10. Use of piperazine derivatives in inhibiting spoilage bacteria, applied to the method for determining the inhibition of spoilage bacteria by piperazine derivatives according to any one of claims 1 to 9, characterized in that: The piperazine derivatives also have anti-tumor activity and have inhibitory effects on HepG2 cells, A549 cells and HeLa cells in in vitro experiments.

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