Phosphate cross-linked nisin aggregate and preparation and application thereof

Abstract

This invention discloses a phosphate-crosslinked Nisin aggregate, its preparation, and its application. The phosphate-crosslinked Nisin aggregate of this invention can effectively improve antibacterial effects. Compared with the same mass of free Nisin, the crosslinked aggregate shows a 50% increase in antibacterial effect against the tested Gram-negative bacterium *Escherichia coli*, and a 20% increase in antibacterial effect against the tested Gram-positive bacterium *Staphylococcus aureus*. Its improved antibacterial effect against Gram-negative bacteria has significant practical value. Phosphate is a nationally approved food additive, with a permitted dosage in food exceeding that of Nisin, but its proportion in crosslinked aggregates is less than 5.0%. The use of Nisin crosslinked aggregates according to national standards ensures food safety. Phosphate is significantly cheaper than Nisin, and its use as a crosslinking agent to improve the antibacterial effect of Nisin enhances its economic value.

Description

(I) Technical Field

[0001] This invention relates to a phosphate-crosslinked Nisin aggregate, its preparation and application. (II) Background Technology

[0002] Nisin, a highly safe food preservative, has been approved for use in many countries worldwide. Due to its superior safety and antibacterial effects compared to other food preservatives, Nisin holds great promise for future applications. To improve the antibacterial efficacy of Nisin, particularly against Gram-negative bacteria, researchers both domestically and internationally have conducted numerous studies, but effective technical methods are still lacking. Currently, Nisin products produced by domestic and international companies show significantly weaker antibacterial effects against Gram-negative bacteria, such as Escherichia coli, compared to Gram-positive bacteria, which is a major drawback in practical applications.

[0003] Therefore, it is necessary to develop a Nisin preparation technology to improve its antibacterial effect against Gram-negative bacteria and obtain Nisin products with better antibacterial effects. (III) Summary of the Invention

[0004] The purpose of this invention is to provide a phosphate-crosslinked Nisin aggregate, its preparation, and its application. This invention uses phosphate-crosslinked Nisin to prepare Nisin crosslinked aggregates, which significantly improves the antibacterial effect against Gram-negative bacteria, enhancing the practical application value of the product. Furthermore, phosphates are food additives; when used according to national standards, food safety can be ensured, maintaining the high safety advantage of Nisin as a food preservative, and thus possessing ideal application prospects.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides a phosphate-crosslinked Nisin aggregate, which is prepared by the following method: (1) Nisin is dissolved in water, the pH is adjusted to 5.5-7.0, and the solution is allowed to stand at room temperature for 0.5-2.5 h to obtain a flocculated Nisin solution; (2) A phosphate aqueous solution is added to the flocculated Nisin solution in step (1) under stirring at room temperature and 200-500 rpm. After the addition is complete, the solution is stirred at room temperature and 50-150 rpm for 0.5-1.5 h until the crosslinking is completed. The precipitate is collected by centrifugation and dried until the moisture content is less than 5.0% to obtain the phosphate-crosslinked Nisin aggregate.

[0007] Furthermore, in step (1), the concentration of Nisin dissolved in water is 4.0-15 mg / mL.

[0008] Furthermore, the phosphates in step (2) include sodium hexametaphosphate, sodium tripolyphosphate, and sodium pyrophosphate.

[0009] Furthermore, in step (2), the concentration of the phosphate aqueous solution is 0.3 mg / mL, and the amount of phosphate aqueous solution added is based on the mass of phosphate, wherein the amount of phosphate added is 0.4-1.0% based on the mass of Nisin in step (1).

[0010] Further, in step (2), the phosphate aqueous solution is added at room temperature and 300 rpm at a rate of 2.7-6.0 mL / min. After the addition is complete, cross-linking is carried out at room temperature and 100 rpm.

[0011] Furthermore, in step (2), centrifugation is performed at 10,000 rpm for 30 minutes, and drying is performed at -50℃ freeze-drying for 12-15 hours.

[0012] The present invention also provides the application of the phosphate-crosslinked Nisin aggregate in the preparation of an antibacterial agent, wherein the antibacterial agent is an antibacterial agent for Gram-negative bacteria or Gram-positive bacteria, wherein the Gram-negative bacteria include Escherichia coli; and the Gram-positive bacteria include Staphylococcus aureus.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0014] The phosphate-crosslinked Nisin aggregates of this invention effectively enhance antibacterial efficacy. Compared with the same mass of free Nisin, the crosslinked aggregates show a 50% increase in antibacterial effect against the tested Gram-negative bacterium *Escherichia coli*, and a 20% increase in antibacterial effect against the tested Gram-positive bacterium *Staphylococcus aureus*. This improved antibacterial effect against Gram-negative bacteria has significant practical value.

[0015] Phosphate is a nationally approved food additive, with a permitted usage level in food exceeding that of Nisin, but its proportion in cross-linked aggregates is less than 5.0%. Nisin cross-linked aggregates are used within the permitted limits according to Chinese national standards, ensuring food safety. Phosphate is significantly cheaper than Nisin, and its use as a cross-linking agent enhances the antibacterial effect of Nisin, thus increasing its economic value. (iv) Description of the attached drawings

[0016] Figure 1 Standard curve for determining nisin using the biuret method. (V) Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0018] The room temperature mentioned in the embodiments of the present invention refers to 25-30℃.

[0019] Example 1: Preparation and antibacterial detection of phosphate-crosslinked Nisin aggregates

[0020] 1. Preparation of phosphate-crosslinked Nisin aggregates

[0021] (1) Accurately weigh 2.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 6.5 with 0.01 mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 1.5 hours to obtain Nisin flocculent solution.

[0022] (2) Weigh 0.30 g of sodium hexametaphosphate (produced by Hubei Xingfa Chemical Group Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium hexametaphosphate aqueous solution. Take 45 mL of the sodium hexametaphosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and 300 rpm (the amount of sodium hexametaphosphate added reaches 0.75% of the mass of pure Nisin), and add it over 15 minutes. Stir at room temperature and 100 rpm for 1 hour to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. Determine the Nisin content in the supernatant and calculate the crosslinking rate. Freeze-dry the precipitate obtained by centrifugation at -50℃ for 14 hours to obtain 1.72 g of phosphate crosslinked Nisin aggregate product.

[0023] 2. Detection of crosslinking rate

[0024] Biuret reagent: Weigh 6g of NaOH in a beaker, add 25mL of deionized water to the beaker to dissolve the NaOH, and set aside. Weigh 0.75g of copper sulfate (CuSO4·5H2O) and dissolve it in 500mL of deionized water. Add 2.25g of potassium sodium tartrate (KNaC4H4O6·4H2O) and 1.5g of KI. After complete dissolution, add 25mL of 6g / L NaOH solution while stirring, and bring the volume to 250mL with deionized water. Store in a sealed plastic bottle.

[0025] Preparation of Nisin standard curve: Prepare a 30 mg / mL nisin standard solution and filter it. Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into test tubes, then add deionized water to a final volume of 1 mL. Add 4 mL of biuret reagent, mix well, and let stand at room temperature for 30 min. Measure the absorbance at 540 nm. The blank is a solution without nisin. Plot the absorbance against the concentration of the nisin solution to obtain the standard curve. Figure 1 The absorbance curve of nisin solution showed a good linear relationship between absorbance and concentration within the concentration range of 5-30 mg / mL. The standard curve equation was A = 0.0142C + 0.0033; R2 =0.9998 (n=5).

[0026] Calculation of the crosslinking rate of phosphate-crosslinked Nisin aggregates: 1.0 mL of supernatant was pipetted into a test tube, 4 mL of biuret reagent was added, and the mixture was thoroughly mixed. After standing at room temperature for 30 min, the absorbance was measured at 540 nm. Finally, the Nisin content in the solution was determined according to the regression equation of the Nisin standard curve. The crosslinking rate was found to be 81.2%.

[0027] Crosslinking rate % = (1 - W1 / W) * 100%. W1 is the amount of nisin (mg) in the supernatant obtained after centrifugation after crosslinking is completed, and W is the amount of nisin (mg) weighed when preparing phosphate Nisin crosslinked aggregates.

[0028] 3. Moisture content detection

[0029] Moisture content determination: The phosphate-crosslinked Nisin aggregates prepared in step 1 were dried at 105℃ to constant weight, cooled in a desiccator, and weighed. The moisture content of the phosphate-crosslinked Nisin aggregate product was calculated based on the mass of water lost during drying. The determined moisture content was 4.3% by mass.

[0030] Moisture content % = (1 - W2 / W3) * 100%. W2 is the mass (g) of the cross-linked Nisin aggregate product dried to constant weight, and W3 is the sample mass (g) of the cross-linked Nisin aggregate product.

[0031] 4. Antibacterial test

[0032] Staphylococcus aureus and Escherichia coli were selected as test strains to conduct antibacterial tests on phosphate-crosslinked Nisin aggregates and Nisin.

[0033] Test strains: Staphylococcus aureus and Escherichia coli. Escherichia coli and Staphylococcus aureus were cultured separately in 250mL Erlenmeyer flasks on nutrient agar medium (produced by Hangzhou Microbial Reagent Co., Ltd.) at 30℃ using a shaker, with the cell number controlled at 4.5 × 10⁻⁶. 6 The concentration of CFU / mL was approximately used as the bacterial solution for the sterilization test. The nutrient agar medium consisted of 3.0g beef extract, 5.0g sodium chloride, 10g tryptone, 1L distilled water, pH 7.0–7.3, and then 20g / L agar powder.

[0034] Bactericidal test: The phosphate-crosslinked Nisin aggregates prepared in step 1 were dissolved in deionized water to prepare a bactericidal solution with a concentration of 10 mg / mL of nisin. 0.2 mL of the bactericidal solution was mixed with 9.8 mL of bacterial solution diluted 10 times (4.5 × 10⁻⁶ mg / mL).5 Mix thoroughly (CFU / mL) to achieve a nisin concentration of 200 μg / mL. Adjust the pH to 5.5. Spread 0.1 mL of each bactericidal solution onto nutrient agar plates, making three plates for each solution. Invert the plates and incubate at 30°C for 24 hours. Observe and record the number of colonies on each plate, and take the average number of colonies.

[0035] Comparative test of the bactericidal effect of nisin: Nisin was prepared into a bactericidal solution with a concentration of 10 mg / mL. 0.2 mL of this solution was mixed thoroughly with 9.8 mL of a 10-fold diluted bacterial solution. The pH was adjusted to 5.5. 0.1 mL of this solution was spread onto nutrient agar plates, with three plates evenly distributed. The plates were incubated at 30°C for 24 hours. The number of colonies on each plate was observed and recorded, and the average colony count was taken.

[0036] Phosphate sterilization control test: A 0.3 mg / mL phosphate aqueous solution was used as the control solution. 0.2 mL of phosphate aqueous solution was added to 9.8 mL of bacterial suspension diluted 10 times, and the pH was adjusted to 5.5. 0.1 mL of this solution was then spread evenly onto nutrient agar plates and incubated upside down at 30°C for 24 hours. The number of colonies on each plate was observed and recorded, and the average colony count was taken.

[0037] Blank test: Add 0.2 mL of deionized water to 9.8 mL of bacterial suspension diluted 10 times, mix and adjust the pH to 5.5. Take 0.1 mL of this solution and spread it evenly on a nutrient agar plate. Incubate at 30°C inverted for 24 h. Observe and record the number of colonies on each plate, and take the average number of colonies on each plate.

[0038] Sterilization rate = (Number of colonies in blank test - Number of colonies in test with sterilization solution) / Number of colonies in blank test * 100%

[0039] Experimental results showed that a 0.3 mg / mL sodium hexametaphosphate aqueous solution had no antibacterial effect. Nisin showed a 45.8% bactericidal rate against Escherichia coli and a 61.2% bactericidal rate against Staphylococcus aureus, while phosphate-crosslinked Nisin aggregates showed a 69.4% bactericidal rate against Escherichia coli and a 74.8% bactericidal rate against Staphylococcus aureus. Compared to nisin, phosphate-crosslinked Nisin aggregates increased the bactericidal rate against Escherichia coli by 51.6% and the bactericidal rate against Staphylococcus aureus by 22.3%.

[0040] Example 2:

[0041] (1) Accurately weigh 4.5g of Nisin (80% purity) produced by Zhengzhou Chengwang Chemical Food Additives Co., Ltd., add it to 300mL of deionized water and stir to dissolve. Adjust the pH to 5.5 with 0.01mol / L HCl aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 2.5 hours to obtain Nisin flocculent solution.

[0042] (2) Weigh 0.30 g of sodium hexametaphosphate (produced by Hubei Xingfa Chemical Group Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium hexametaphosphate aqueous solution. Take 120 mL of the sodium hexametaphosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and stirring at 300 rpm (the amount of sodium hexametaphosphate added reaches 1.0% of the mass of pure Nisin), and add it over 20 minutes. Stir at 100 rpm for 1.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 72.5%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 15 hours to obtain 2.76 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.1%.

[0043] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.3% against Escherichia coli and by 20.8% against Staphylococcus aureus.

[0044] Example 3:

[0045] (1) Weigh 2.2g of Nisin (purity 90%) produced by Wuhan Fresh Preservation Biotechnology Co., Ltd., add it to 500mL of deionized water and stir to dissolve. Adjust the pH to 7.0 with 0.01mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 0.5 hours to obtain Nisin flocculent solution.

[0046] (2) Weigh 0.30 g of sodium hexametaphosphate (produced by Hubei Xingfa Chemical Group Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium hexametaphosphate aqueous solution. Take 27 mL of the sodium hexametaphosphate aqueous solution and add it to the Nisin solution in step (1) at room temperature and stirring at 300 rpm (the amount of sodium hexametaphosphate added reaches 0.4% of the mass of pure Nisin), and add it over 10 minutes. Stir at 100 rpm for 0.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 75.7%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 12 hours to obtain 1.59 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.2%.

[0047] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.8% against Escherichia coli and by 21.4% against Staphylococcus aureus.

[0048] Example 4:

[0049] (1) Weigh 3.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 6.0 with 0.01 mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 1.5 hours to obtain Nisin flocculent solution.

[0050] (2) Weigh 0.30 g of sodium tripolyphosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium tripolyphosphate aqueous solution. Take 63 mL of the sodium tripolyphosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium tripolyphosphate added reaches 0.7% of the mass of pure Nisin), and add it over 15 minutes. Stir at 100 rpm for 1 hour to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 79.3%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 15 hours to obtain 2.19 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.1%.

[0051] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.9% against Escherichia coli and by 21.7% against Staphylococcus aureus.

[0052] Example 5:

[0053] (1) Weigh 2.2g of Nisin (purity 90%) produced by Wuhan Fresh Preservation Biotechnology Co., Ltd., add it to 400mL of deionized water and stir to dissolve. Adjust the pH to 5.5 with 0.01mol / L HCl water. Nisin is in a flocculent state. Let it stand at room temperature for 2.5 hours to obtain Nisin flocculent solution.

[0054] (2) Weigh 0.30 g of sodium tripolyphosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium tripolyphosphate aqueous solution. Take 67 mL of the sodium tripolyphosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium tripolyphosphate added reaches 1.0% of the mass of pure Nisin), and add it over 20 minutes. Stir at 100 rpm for 1.0 hour to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 73.1%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 12 hours to obtain 1.52 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.5%.

[0055] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.6% against Escherichia coli and by 20.9% against Staphylococcus aureus.

[0056] Example 6:

[0057] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 7.0 with 0.01 mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 0.5 hours to obtain Nisin flocculent solution.

[0058] (2) Weigh 0.30 g of sodium tripolyphosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water to prepare a 0.3 mg / mL sodium tripolyphosphate aqueous solution. Take 48 mL of the sodium tripolyphosphate aqueous solution and add it to the Nisin solution in step (1) at room temperature and stirring at 300 rpm (the amount of sodium tripolyphosphate added reaches 0.4% of the mass of pure Nisin), and add it over 10 minutes. Stir at 100 rpm for 0.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 75.8%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 13 hours to obtain 2.85 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.3%.

[0059] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, it was calculated that the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.6% against Escherichia coli and by 21.3% against Staphylococcus aureus.

[0060] Example 7:

[0061] (1) Weigh 2.2g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 400mL of deionized water and stir to dissolve. Adjust the pH to 6.5 with 0.01mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 2.0 hours to obtain Nisin flocculent solution.

[0062] (2) Weigh 0.30 g of anhydrous sodium pyrophosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 ml of deionized water, and prepare a 0.3 mg / mL sodium pyrophosphate aqueous solution. Take 53 mL of the sodium pyrophosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium pyrophosphate added reaches 0.8% of the mass of pure Nisin), and add it over 15 minutes. Stir at 100 rpm for 1 hour to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 76.7%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 14 hours to obtain 1.58 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.2%.

[0063] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, it was calculated that the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 50.5% against Escherichia coli and by 20.9% against Staphylococcus aureus.

[0064] Example 8:

[0065] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 7.0 with 0.01 mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 1.0 hour to obtain Nisin flocculent solution.

[0066] (2) Weigh 0.30 g of sodium phosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium phosphate aqueous solution. Take 120 mL of the sodium phosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium phosphate added reaches 1.0% of the mass of pure Nisin), and add it over 20 minutes. Stir at 100 rpm for 1.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 43.6%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 13 hours to obtain 1.71 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.1%.

[0067] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, it was calculated that the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 22.8% against Escherichia coli and by 15.2% against Staphylococcus aureus.

[0068] Example 9:

[0069] (1) Weigh 4.0 g of Nisin (90% purity) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 7.0 with 0.01 mol / L NaOH aqueous solution. Nisin will form flocculent precipitates. Let it stand at room temperature for 1.0 hour to obtain a Nisin flocculent solution.

[0070] (2) Weigh 0.30 g of sodium monohydrogen phosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium monohydrogen phosphate aqueous solution. Take 120 mL of the sodium monohydrogen phosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium monohydrogen phosphate added reaches 1.0% of the mass of pure Nisin), and add it over 20 minutes. Stir at 100 rpm for 1.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 28.6%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 13 hours to obtain 1.10 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.0%.

[0071] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, it was calculated that the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 16.3% against Escherichia coli and by 13.7% against Staphylococcus aureus.

[0072] Example 10:

[0073] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biotechnology Co., Ltd., add it to 300 mL of deionized water and stir to dissolve. Adjust the pH to 7.0 with 0.01 mol / L NaOH aqueous solution. Nisin is in a flocculent state. Let it stand at room temperature for 1.0 hour to obtain Nisin flocculent solution.

[0074] (2) Weigh 0.30 g of sodium dihydrogen phosphate (produced by Chongqing Chuandong Chemical (Group) Co., Ltd.), add 1000 mL of deionized water, and prepare a 0.3 mg / mL sodium dihydrogen phosphate aqueous solution. Take 120 mL of the sodium dihydrogen phosphate aqueous solution and add it to the entire Nisin solution from step (1) at room temperature and with stirring at 300 rpm (the amount of sodium dihydrogen phosphate added reaches 1.0% of the mass of pure Nisin), and add it over 20 minutes. Stir at 100 rpm for 1.5 hours to complete the crosslinking. Centrifuge at 10000 rpm for 30 minutes to separate the supernatant and solids. The Nisin content of the supernatant was determined using the method in Example 1, and the crosslinking rate was calculated to be 17.3%. The precipitate obtained by centrifugation was freeze-dried at -50℃ for 13 hours to obtain 0.63 g of phosphate crosslinked Nisin aggregate product, with a moisture content of 4.2%.

[0075] The antibacterial test was conducted according to the method in Example 1. Compared with the bactericidal rate of Nisin, it was calculated that the bactericidal rate of phosphate-crosslinked Nisin aggregates was increased by 9.7% against Escherichia coli and by 6.3% against Staphylococcus aureus.

Claims

1. A phosphate-crosslinked Nisin aggregate, characterized in that, The aggregates are prepared as follows: (1) Nisin is dissolved in water, the pH is adjusted to 5.5-7.0, and the solution is allowed to stand at room temperature for 0.5-2.5 h to obtain a flocculated Nisin solution; (2) Phosphate aqueous solution is added to the flocculated Nisin solution in step (1) under stirring at room temperature and 200-500 rpm. After the addition is complete, the solution is stirred at room temperature and 50-150 rpm for 0.5-1.5 h until the cross-linking is completed. The precipitate is collected by centrifugation and dried until the moisture content is less than 5.0% to obtain phosphate cross-linked Nisin aggregates. The phosphate is sodium hexametaphosphate, sodium tripolyphosphate, sodium pyrophosphate, sodium phosphate, sodium monohydrogen phosphate or sodium dihydrogen phosphate.

2. The phosphate-crosslinked Nisin aggregate as described in claim 1, characterized in that, In step (1), the concentration of Nisin dissolved in water is 4.0-15 mg / mL.

3. The phosphate-crosslinked Nisin aggregate as described in claim 1, characterized in that, In step (2), the concentration of the phosphate aqueous solution is 0.3 mg / mL, and the amount of phosphate aqueous solution added is based on the mass of phosphate. The amount of phosphate added is 0.4%-1.0% based on the mass of Nisin in step (1).

4. The phosphate-crosslinked Nisin aggregate as described in claim 1, characterized in that, Step (2) The phosphate aqueous solution is added at room temperature and 300 rpm at a rate of 2.7-6.0 mL / min. After the addition is complete, cross-linking is carried out at room temperature and 100 rpm.

5. The phosphate-crosslinked Nisin aggregate as described in claim 1, characterized in that, Step (2) centrifugation is performed at 10,000 rpm for 30 minutes, and drying is performed at -50℃ freeze drying for 12-15 hours.

6. The application of the phosphate-crosslinked Nisin aggregate of claim 1 in the preparation of an antibacterial agent, characterized in that, The antibacterial agent is an antibacterial agent for Escherichia coli or Gram-positive bacteria.

7. The application as described in claim 6, characterized in that, The Gram-positive bacteria include Staphylococcus aureus.

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