Phosphate cross-linked Nisin aggregate as well as preparation and application thereof

Abstract

The invention discloses a phosphate cross-linked Nisin aggregate and preparation and application thereof.The phosphate cross-linked Nisin aggregate can effectively improve the antibacterial effect, compared with free Nisin with the same mass, the antibacterial effect on experimental gram-negative strain escherichia coli and cross-linked aggregate is improved by 50%, and compared with free Nisin with the same mass, the antibacterial effect on experimental gram-positive strain staphylococcus aureus is improved by 50%. And the antibacterial effect of the cross-linked aggregate is improved by 20%. The antibacterial effect on gram-negative bacteria is improved, and the method has important value in practical application. Phosphate is a food additive approved by the country, the allowable dosage of phosphate in food is larger than that of Nisin, and the proportion of phosphate in the cross-linked aggregate is smaller than 5.0%. And the Nisin cross-linked aggregate is used according to the allowable dosage of the national standard, so that the food safety is fully guaranteed. The price of phosphate is obviously lower than that of Nisin, and phosphate is used as a cross-linking agent, so that the antibacterial effect of Nisin is improved, and the economic value is increased.

Description

(I) Technical field

[0001] The invention relates to a phosphate cross-linked Nisin aggregate and the preparation and application thereof. (II) Background technology

[0002] As a highly safe food preservative, Nisin has been approved for use in many countries around the world. Compared with other food preservatives, Nisin is safer and has good antibacterial effects, so it has an ideal application prospect. In order to improve the antibacterial effect of Nisin, especially the antibacterial effect on Gram-negative bacteria, domestic and foreign scientific and technological personnel have carried out many explorations, but there is still a lack of effective technical methods. At present, the antibacterial effect of Nisin products produced by domestic and foreign companies on Gram-negative bacteria, such as Escherichia coli, is significantly weaker than that on Gram-positive bacteria, which has become the main shortcoming in practical application.

[0003] Therefore, it is necessary to develop a Nisin preparation technology that improves the antibacterial effect on Gram-negative bacteria and obtain a Nisin product with better antibacterial effect. (III) Summary of the invention

[0004] The purpose of the present invention is to provide a phosphate cross-linked Nisin aggregate and its preparation and application. The present invention uses Nisin in a phosphate cross-linked aggregate state to prepare Nisin cross-linked aggregate, which can significantly improve the antibacterial effect on Gram-negative bacteria and enhance the practical application value of the product. In addition, phosphate is a food additive. When used in an amount specified by national standards, food safety can be guaranteed, and the advantage of high safety of Nisin as a food preservative is maintained, which has an ideal application prospect.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a phosphate cross-linked Nisin aggregate, which is prepared by the following method: (1) dissolving Nisin in water, adjusting the pH to 5.5-7.0, and standing at room temperature for 0.5-2.5 hours to obtain a flocculated Nisin solution; (2) adding a phosphate aqueous solution to the flocculated Nisin solution in step (1) at room temperature and stirring at 200-500 rpm, stirring at room temperature and 50-150 rpm for 0.5-1.5 hours until cross-linking is completed, centrifuging, collecting precipitates, and drying until the water content is less than 5.0% to obtain the phosphate cross-linked Nisin aggregate.

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

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

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

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

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

[0012] The present invention also provides an application of the phosphate cross-linked 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 cross-linked Nisin aggregate of the present invention can effectively improve the antibacterial effect. Compared with the free Nisin of the same mass, the antibacterial effect of the cross-linked aggregate on the tested Gram-negative bacteria Escherichia coli is increased by 50%, and the antibacterial effect of the cross-linked aggregate on the tested Gram-positive bacteria Staphylococcus aureus is increased by 20%. The antibacterial effect of the cross-linked aggregate on Gram-negative bacteria is improved, and the practical application has important value.

[0015] Phosphate is a food additive approved by the state. The permitted amount in food is greater than that of Nisin, while the proportion in cross-linked aggregates is less than 5.0%. Nisin cross-linked aggregates are used in the permitted amount according to Chinese national standards, and food safety is fully guaranteed. The price of phosphate is significantly lower than that of Nisin. When used as a cross-linking agent, it improves the antibacterial effect of Nisin and increases its economic value. (IV) Description of the drawings

[0016] Figure 1 , the standard curve of nisin was measured by the biuret method. (V) Specific implementation methods

[0017] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0018] The room temperature described in the embodiment of the present invention refers to 25-30°C.

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

[0020] 1. Preparation of phosphate cross-linked Nisin aggregates

[0021] (1) Accurately weigh 2.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and let Nisin flocculate. Let it stand at room temperature for 1.5 hours to obtain a Nisin flocculated 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 sodium hexametaphosphate aqueous solution and add it to all the Nisin solution in step (1) at room temperature and 300 rpm (the amount of sodium hexametaphosphate added is 0.75% of the mass of pure Nisin), and add it within 15 minutes. Stir at room temperature and 100 rpm for 1.0 hour to complete cross-linking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solids. Determine the Nisin content in the supernatant and calculate the cross-linking rate. The precipitate obtained by centrifugation is freeze-dried at -50°C for 14 hours to obtain 1.72 g of phosphate-crosslinked Nisin aggregate product.

[0023] 2. Detection of cross-linking rate

[0024] Biuret reagent: weigh 6g NaOH in a beaker, add 25mL deionized water to dissolve the NaOH, and set aside. Weigh 0.75g copper sulfate (CuSO 4 ·5H 2 O) was dissolved in 500 mL of deionized water, and 2.25 g of potassium sodium tartrate (KNaC 4 H 4 O 6 ·4H 2 O), and add 1.5 g of KI. After it is completely dissolved, add 25 mL of 6 g / L NaOH solution while stirring, and make up to 250 mL with deionized water. Place it in a plastic bottle and seal it for storage.

[0025] Preparation of Nisin standard curve: Prepare 30 mg / mL nisin standard solution and filter it. Take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the standard solution in a test tube, add deionized water to 1 mL, add 4 mL of biuret reagent and mix well. After standing at room temperature for 30 min, test its absorbance at a wavelength of 540 nm. The blank is a solution without nisin. Plot the absorbance against the concentration of nisin solution to obtain a standard curve ( Figure 1The relationship curve between the concentration and absorbance of the nisin solution shows that within the concentration range of 5-30 mg / mL, the absorbance of the solution and the concentration present a good linear relationship, and the standard curve equation is A=0.0142C+0.0033; R 2 =0.9998(n=5).

[0026] Calculation of the cross-linking rate of phosphate cross-linked Nisin aggregates: 1.0 mL of supernatant was taken and placed in a test tube, 4 mL of biuret reagent was added, and the mixture was fully mixed. After standing at room temperature for 30 minutes, the absorbance was measured at 540 nm. Finally, the Nisin content in the solution was calculated according to the regression equation of the Nisin standard curve. The cross-linking rate was measured to be 81.2%.

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

[0028] 3. Detection of moisture content

[0029] Moisture determination: The phosphate cross-linked Nisin aggregate prepared in step 1 was dried at 105°C to constant weight, cooled in a dryer, and weighed. The moisture content of the phosphate cross-linked Nisin aggregate product was calculated based on the mass of water lost during drying. The moisture content was determined to be 4.3%.

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

[0031] 4. Antimicrobial testing

[0032] Staphylococcus aureus and Escherichia coli were selected as test bacteria, and antibacterial tests of phosphate-cross-linked Nisin aggregates and Nisin were carried out.

[0033] Test strains: Staphylococcus aureus and Escherichia coli. Escherichia coli and Staphylococcus aureus were cultured in 250 mL conical flasks at 30 °C using nutrient agar medium (produced by Hangzhou Microbiological Reagent Co., Ltd.) and the cell number was controlled at 4.5 × 10 6 CFU / mL, used as bactericidal test bacterial solution. Nutrient agar medium composition: 3.0g beef extract, 5.0g sodium chloride, 10g tryptone, add 1L distilled water, pH 7.0-7.3, and then add 20g / L agar powder.

[0034] Sterilization test: Dissolve the phosphate-crosslinked Nisin aggregates prepared in step 1 in deionized water to prepare a sterilization solution with a concentration of 10 mg / mL nisin. Mix 0.2 mL of the sterilization solution with 9.8 mL of bacterial solution diluted 10 times (4.5×10 5 CFU / mL) were mixed evenly, and the concentration of nisin was 200μg / mL. The pH was adjusted to 5.5, and 0.1mL was taken out to coat the nutrient agar medium plate. Three plates were coated with each bactericidal solution, and inverted in a constant temperature incubator at 30℃ for 24h. The number of colonies on each plate was observed and recorded, and the average number of colonies on the plates was taken.

[0035] Comparative test of bactericidal effect of nisin: Take nisin prepared into a bactericidal solution with a concentration of 10 mg / mL, take 0.2 mL and mix it evenly with 9.8 mL of bacterial solution diluted 10 times, adjust the pH to 5.5, take out 0.1 mL and spread it on nutrient agar medium plates, spread it on 3 plates, and incubate it inverted in an incubator at a constant temperature of 30℃ for 24 hours. Observe and record the number of colonies on each plate, and take the average number of colonies on the plates.

[0036] Phosphate sterilization control test: Use 0.3 mg / mL phosphate aqueous solution as the control test solution. Add 0.2 mL of phosphate aqueous solution to 9.8 mL of 10-fold diluted bacterial solution, adjust the pH to 5.5 after mixing, and apply 0.1 mL on the nutrient agar medium plate. After evenly applying, invert and culture at a constant temperature of 30°C in an incubator for 24 hours. Observe and record the number of colonies on each plate, and take the average number of colonies on the plate.

[0037] Blank test: Add 0.2 mL of deionized water to 9.8 mL of 10-fold diluted bacterial solution, adjust the pH to 5.5 after mixing, and apply 0.1 mL on the nutrient agar medium plate. After applying evenly, invert and culture in an incubator at a constant temperature of 30°C for 24 hours. Observe and record the number of colonies on each plate, and take the average number of colonies on the plate.

[0038] Sterilization rate = (blank test colony count - sterilization liquid test colony count) / blank test colony count * 100%

[0039] The experimental results show that 0.3 mg / mL sodium hexametaphosphate aqueous solution has no antibacterial effect. The bactericidal rate of Escherichia coli and Staphylococcus aureus of nisin is 45.8%, and the bactericidal rate of Staphylococcus aureus is 61.2%. The bactericidal rate of Escherichia coli and Staphylococcus aureus of phosphate-crosslinked Nisin aggregates is 69.4%, and the bactericidal rate of Staphylococcus aureus is 74.8%. Compared with nisin, the bactericidal rate of phosphate-crosslinked Nisin aggregates against Escherichia coli increased by 51.6%, and the bactericidal rate against Staphylococcus aureus increased by 22.3%.

[0040] Embodiment 2:

[0041] (1) Accurately weigh 4.5 g of Nisin (purity 80%) produced by Zhengzhou Chengwang Chemical Food Additive Co., Ltd., add it to 300 mL of deionized water and stir to dissolve, adjust the pH to 5.5 with 0.01 mol / L HCl aqueous solution, and let the Nisin flocculate. Let it stand at room temperature for 2.5 hours to obtain a Nisin flocculated 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 sodium hexametaphosphate aqueous solution, add it to the entire Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium hexametaphosphate added is 1.0% of the mass of pure Nisin), and add it within 20 minutes. Stir at 100 rpm for 1.5 hours to complete crosslinking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is measured for Nisin content using the method in Example 1, and the crosslinking rate is calculated to be 72.5%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 15 hours to obtain 2.76 g of phosphate-crosslinked Nisin aggregate product, and the moisture content is measured to be 4.1%.

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

[0044] Embodiment 3:

[0045] (1) Weigh 2.2 g of Nisin (purity 90%) produced by Wuhan Xianbao Biotechnology Co., Ltd., add it to 500 mL of deionized water and stir to dissolve, adjust the pH to 7.0 with 0.01 mol / L NaOH aqueous solution, and let Nisin flocculate. Let it stand at room temperature for 0.5 hour to obtain a Nisin flocculated 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 sodium hexametaphosphate aqueous solution, add it to the Nisin solution of step (1) under room temperature and stirring at 300 rpm (the amount of sodium hexametaphosphate added is 0.4% of the mass of pure Nisin), and add it within 10 minutes. Stir at 100 rpm for 0.5 hours to complete crosslinking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the calculated crosslinking rate is 75.7%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 12 hours to obtain 1.59 g of phosphate cross-linked Nisin aggregate product, and the moisture content is determined to be 4.2%.

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

[0048] Embodiment 4:

[0049] (1) Weigh 3.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and let the Nisin flocculate. Let it stand at room temperature for 1.5 hours to obtain a Nisin flocculated 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 sodium tripolyphosphate aqueous solution, add it to the entire Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium tripolyphosphate added is 0.7% of the mass of pure Nisin), and add it within 15 minutes. Stir at 100 rpm for 1.0 hour to complete crosslinking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is measured for Nisin content using the method of Example 1, and the crosslinking rate is calculated to be 79.3%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 15 hours to obtain 2.19 g of phosphate cross-linked Nisin aggregate product, and the moisture content is measured to be 4.1%.

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

[0052] Embodiment 5:

[0053] (1) Weigh 2.2 g of Nisin (purity 90%) produced by Wuhan Xianbao Biotechnology Co., Ltd., add it to 400 mL of deionized water and stir to dissolve, adjust the pH to 5.5 with 0.01 mol / L HCl, and let the Nisin flocculate. Let it stand at room temperature for 2.5 hours to obtain a Nisin flocculated 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 sodium tripolyphosphate aqueous solution, add it to the entire Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium tripolyphosphate added is 1.0% of the mass of pure Nisin), and add it within 20 minutes. Stir at 100 rpm for 1.0 hour to complete crosslinking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the calculated crosslinking rate is 73.1%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 12 hours to obtain 1.52 g of phosphate cross-linked Nisin aggregate product, and the moisture content is determined to be 4.5%.

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

[0056] Embodiment 6:

[0057] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and let the Nisin flocculate. Let it stand at room temperature for 0.5 hour to obtain a Nisin flocculated solution.

[0058] (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 48 mL of sodium tripolyphosphate aqueous solution, add it to the Nisin solution of step (1) under room temperature and stirring at 300 rpm (the amount of sodium tripolyphosphate added is 0.4% of the mass of pure Nisin), and add it within 10 minutes. Stir at 100 rpm for 0.5 hours to complete the crosslinking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the crosslinking rate is calculated to be 75.8%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 13 hours to obtain 2.85 g of phosphate cross-linked Nisin aggregate product, and the moisture content is determined to be 4.3%.

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

[0060] Embodiment 7:

[0061] (1) Weigh 2.2 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological Co., Ltd., add it to 400 mL of deionized water and stir to dissolve, adjust the pH to 6.5 with 0.01 mol / L NaOH aqueous solution, and let the Nisin flocculate. Let it stand at room temperature for 2.0 hours to obtain a Nisin flocculated 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 sodium pyrophosphate aqueous solution, add it to the entire Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium pyrophosphate added is 0.8% of the mass of pure Nisin), and add it within 15 minutes. Stir at 100 rpm for 1.0 hour to complete cross-linking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the cross-linking rate is calculated to be 76.7%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 14 hours to obtain 1.58 g of phosphate cross-linked Nisin aggregate product, and the moisture content is determined to be 4.2%.

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

[0064] Embodiment 8:

[0065] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and let the Nisin flocculate. Let it stand at room temperature for 1.0 hour to obtain a Nisin flocculated 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 sodium phosphate aqueous solution, add it to all the Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium phosphate added is 1.0% of the mass of pure Nisin), and add it within 20 minutes. Stir at 100 rpm for 1.5 hours to complete the cross-linking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the cross-linking rate is calculated to be 43.6%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 13 hours to obtain 1.71 g of phosphate-crosslinked Nisin aggregate product, and the moisture content is determined to be 4.1%.

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

[0068] Embodiment 9:

[0069] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and the Nisin will be flocculated. Let it stand at room temperature for 1.0 hour to obtain a Nisin flocculated 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 sodium monohydrogen phosphate aqueous solution, add it to all the Nisin solution in step (1) under room temperature and stirring at 300 rpm (the amount of sodium monohydrogen phosphate added is 1.0% of the mass of pure Nisin), and add it within 20 minutes. Stir at 100 rpm for 1.5 hours to complete the cross-linking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is measured for Nisin content using the method of Example 1, and the cross-linking rate is calculated to be 28.6%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 13 hours to obtain 1.10 g of phosphate cross-linked Nisin aggregate product, and the moisture content is measured to be 4.0%.

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

[0072] Embodiment 10:

[0073] (1) Weigh 4.0 g of Nisin (purity 90%) produced by Tianjin Kangyi Biological 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, and let the Nisin flocculate. Let it stand at room temperature for 1.0 hour to obtain a Nisin flocculated 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 sodium dihydrogen phosphate aqueous solution, add it to all the Nisin solution in step (1) at room temperature and 300 rpm stirring (the amount of sodium dihydrogen phosphate added is 1.0% of the mass of pure Nisin), and add it within 20 minutes. Stir at 100 rpm for 1.5 hours to complete the cross-linking. Centrifuge at 10,000 rpm for 30 minutes to separate the supernatant and solid. The supernatant is determined by the method of Example 1 to determine the Nisin content, and the cross-linking rate is calculated to be 17.3%. The precipitate obtained by centrifugation is freeze-dried at -50°C for 13 hours to obtain 0.63 g of phosphate cross-linked Nisin aggregate product, and the water content is determined to be 4.2%.

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

Claims

1. A phosphate cross-linked Nisin aggregate, characterized in that: The aggregates are prepared according to the following method: (1) dissolving Nisin in water, adjusting the pH to 5.5-7.0, and standing at room temperature for 0.5-2.5 hours to obtain a flocculated Nisin solution; (2) adding a phosphate aqueous solution to the flocculated Nisin solution in step (1) at room temperature and stirring at 200-500 rpm. After the addition is completed, stirring at room temperature and 50-150 rpm for 0.5-1.5 hours until cross-linking is completed, centrifuging, collecting the precipitate, and drying until the water content is less than 5.0% to obtain phosphate-crosslinked Nisin aggregates.

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

3. The phosphate cross-linked Nisin aggregate as claimed in claim 1, characterized in that: The phosphate in step (2) includes sodium hexametaphosphate, sodium tripolyphosphate and sodium pyrophosphate.

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

5. The phosphate cross-linked Nisin aggregate as claimed in claim 1, characterized in that: Step (2) The phosphate aqueous solution is added at room temperature and 300 rpm, with an addition rate of 2.7-6.0 mL / min. After the addition is completed, cross-linking is carried out at room temperature and 100 rpm.

6. The phosphate cross-linked Nisin aggregate according to claim 1, characterized in that: In step (2), centrifugation is performed at 10,000 rpm for 30 minutes, and drying is performed by freeze drying at -50°C for 12-15 hours.

7. Use of the phosphate cross-linked Nisin aggregates according to claim 1 in the preparation of an antibacterial agent.

8. The use according to claim 7, characterized in that The bacteriostatic agent is a bacteriostatic agent for Gram-negative bacteria or Gram-positive bacteria.

9. The use according to claim 8, characterized in that The Gram-negative bacteria include Escherichia coli; the Gram-positive bacteria include Staphylococcus aureus.

Images