Separation and purification method of nisin

Abstract

The invention discloses a separation and purification method of nisin, and relates to the technical field of nisin purification. The method provided by the invention can achieve the technical effects of high separation purity, less target substance loss and less organic solvent dosage without combining with an ultrafiltration process for impurity removal in a specific pH environment, and has the technical advantages of environmental friendliness, simple separation and purification device, simplicity and convenience in process operation and suitability for industrial production.

Description

Technical Field

[0001] This invention relates to the field of nisin purification technology, and more specifically, to a method for separating and purifying nisin. Background Technology

[0002] Peptides possess antioxidant, antibacterial, and immunomodulatory activities, making them highly sought after by producers and consumers in the food, medical, and agricultural sectors. In the biological world, some protein peptides with specific physiological functions, after appropriate enzymatic hydrolysis, have their active sites activated, enabling them to exert special physiological functions. Furthermore, their absorption rate is typically faster than that of amino acids of the same composition, making them important active substances in the human body. Therefore, they have become a key focus in the development of functional food additives and peptide drugs, possessing broad market demand and development prospects.

[0003] Nisin, also known as lactococcal peptide, is a natural polypeptide compound produced by *Lactococcus lactis*. It has 34 amino acid residues and a unique inner ring structure composed of five thioether bridges. Two natural variants of nisin exist in nature: Nisin A and Nisin Z. They are essentially identical in properties, differing only in one amino acid: Nisin A has histidine at position 27, while Nisin Z has asparagine. It exhibits strong inhibitory effects against Gram-positive bacteria and also possesses immunomodulatory and probiotic activities. Nisin is highly sensitive to proteolytic enzymes, rapidly breaking down into amino acids after ingestion. It has no toxic side effects on humans and is a globally recognized, safe, and highly effective natural food preservative, as well as a good choice for feed additives. It is widely used in various food preservation applications to meet the needs of producing healthy and green foods.

[0004] Fermentation is the main route for obtaining nisin, but the fermentation products are complex and require highly sophisticated separation and purification processes, making it difficult to obtain high-purity nisin. Current methods for separating and purifying nisin require specific pH levels, ultrafiltration, and other process conditions, resulting in complex processes, stringent purification requirements, high reagent costs, and relatively low purity.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for separating and purifying nisin to solve the problems of low purity, low yield, complex purification process, and harsh conditions associated with nisin separation.

[0007] This invention is implemented as follows: This invention provides a method for separating and purifying nisin, which includes the following steps: desalting the sample to be purified using the gel filtration chromatography mode of a protein purifier, with water as the mobile phase, to obtain desalted nisin; then lyophilizing the desalted nisin, reconstituted it, and loading it onto a C18 reversed-phase column for chromatographic separation, followed by gradient elution, and collecting the eluted product. When performing chromatographic separation using a C18 reversed-phase column, the mobile phase is set to 0.1% (v / v) to 0.2% (v / v) trifluoroacetic acid aqueous solution and acetonitrile; The gradient elution procedure includes: The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 79-81% from 0 to 5 min, and the volume percentage of acetonitrile in the mobile phase was 19-21%. The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 59-61% and the volume percentage of acetonitrile in the mobile phase was 39-41% from 5 to 27 min. The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 79-81% and the volume percentage of acetonitrile in the mobile phase was 19-21% at 27-40 min.

[0008] The present invention has the following beneficial effects: This invention provides a simple method for the separation and purification of nisin. This method utilizes gel filtration chromatography in a protein purifier to remove salt components from the sample, thus contributing to improved nisin purity. High salt content in the sample reduces nisin purity, affects antibacterial activity and stability, interferes with HPLC / MS detection, increases the difficulty of downstream purification, and limits its application in low-salt foods or pharmaceuticals. To increase the concentration of nisin in the desalted product, lyophilization and reconstitution are performed. Reconstitution meets the requirements for reversed-phase chromatography, avoiding excessively low nisin concentration during sample loading, which would result in poor reversed-phase chromatography performance.

[0009] After extensive screening, nisin was obtained with high purity using a C18 reverse chromatographic column and specific gradient elution conditions, with the chromatographic purity of the nisin product exceeding 98%.

[0010] The method provided by this invention can achieve high separation purity, low loss of target substances, and low consumption of organic solvents without the need for a specific pH environment combined with ultrafiltration process for impurity removal. It has the technical advantages of being environmentally friendly, having simple separation and purification equipment, being easy to operate, and being suitable for industrial production. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 A magnified chromatogram of the semi-preparative nisin prepared in this invention; Figure 2 The ion chromatograms of the lactobacillus peptides prepared in this invention before and after desalting are shown below. Figure 3 This is a semi-preparative chromatogram of the lactobacillus peptide prepared in this invention; Figure 4 The HPLC chromatogram of the desalted and lyophilized product of nisin prepared in this invention is shown. Figure 5 The image shows the HPLC chromatogram of the lyophilized product of the desalted and purified lactis peptide prepared in this invention. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] This invention provides a method for separating and purifying nisin, which includes the following steps: desalting the sample to be purified using the gel filtration chromatography mode of a protein purifier, with water as the mobile phase, to obtain desalted nisin; then lyophilizing the desalted nisin, reconstituted it, and loading it onto a C18 reversed-phase column for chromatographic separation, followed by gradient elution, and collecting the eluted product. When performing chromatographic separation using a C18 reversed-phase column, the mobile phase is set to 0.1% (v / v) to 0.2% (v / v) trifluoroacetic acid aqueous solution and acetonitrile; The gradient elution procedure includes: The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 79-81% from 0 to 5 min, and the volume percentage of acetonitrile in the mobile phase was 19-21%. The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 59-61% and the volume percentage of acetonitrile in the mobile phase was 39-41% from 5 to 27 min. The volume percentage of trifluoroacetic acid aqueous solution in the mobile phase was 79-81% and the volume percentage of acetonitrile in the mobile phase was 19-21% at 27-40 min.

[0015] Currently, nisin is mainly precipitated via salting out, resulting in commercially available nisin containing high levels of sodium chloride. Therefore, a protein purification instrument is first used to desalt the sample. This desalting process significantly reduces the chloride ion content in the sample; tests show that it can reduce the chloride ion content in nisin from 52.76% to 10.24%, effectively improving the organic purity of nisin. The protein purification instrument offers the technical advantage of high purification efficiency.

[0016] To increase the concentration of nisin in the desalted product, lyophilization and reconstitution were performed. Reconstitution ensures that the concentration of nisin is sufficient for reversed-phase chromatography, preventing insufficient concentration during sample loading and thus ensuring optimal reversed-phase chromatography results.

[0017] After extensive screening, nisin was found to be obtained in high purity using a C18 reverse-phase column and specific gradient elution conditions, with the chromatographic purity of the nisin product exceeding 98%. Using other chromatographic columns, such as C8, resulted in a decrease in the purity of the nisin product.

[0018] The method provided by this invention can achieve high separation purity, low loss of target substances, and low consumption of organic solvents without the need for a specific pH environment combined with ultrafiltration process for impurity removal. It has the technical advantages of being environmentally friendly, having simple separation and purification equipment, being easy to operate, and being suitable for industrial production.

[0019] In a preferred embodiment of the present invention, the flow rate of the mobile phase of the protein purification instrument is set to 1~2 mL / min.

[0020] In a preferred embodiment of the present invention, during the desalination process, the effluent is started to be collected when UV ≥ 5mAU, and the collection of effluent is stopped when the conductivity of the effluent ≥ 1.0mS / cm.

[0021] "Start collecting when UV ≥ 5 mAU" is a trigger command for automatic fraction collection of the target component during chromatographic separation. It means that when the ultraviolet (UV) signal intensity of the detector reaches or exceeds 5 milliabsorbance units (mAU), the system automatically starts the fraction collector to collect the eluent. This avoids collecting solvents or impurities with no UV absorption (signal < 5 mAU), ensuring that the collected fraction is rich in target compounds (such as peptides) and reducing contamination from non-target components.

[0022] In a preferred embodiment of the present invention, during reversed-phase chromatography, the flow rate of the gradient elution mobile phase is 2-4 mL / min.

[0023] In a preferred embodiment of the present invention, gradient elution is performed, and reception begins when the impurity peaks have been eluted and stops when the main peak has been eluted, collecting the eluent from the gradient elution within this range. Preferably, the eluent from the gradient elution within the range of 24.2 to 24.8 minutes is collected. At the above retention time, high-purity target proteins can be efficiently collected. In other embodiments, the collection time can be adaptively adjusted according to actual conditions.

[0024] In a preferred embodiment of the present invention, water is used as the solvent during reconstitution. In other embodiments, the choice of solvent can be determined according to specific practical needs. Considering the absolute purity of nisin and to prevent the introduction of other impurity ions, water is the preferred solvent in the present invention.

[0025] In a preferred embodiment of the present invention, the freeze-drying conditions include: freeze-drying at -65 to -70°C for 60 to -72 hours.

[0026] In a preferred embodiment of the present invention, the chromatographic purity of the nisin in the eluted product is ≥98%.

[0027] In a preferred embodiment of the present invention, the sample to be purified is a culture of Lactococcus lactis containing nisin or a crude isolate of nisin. The sample to be purified may also be commercially available nisin.

[0028] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0029] Example 1 This embodiment provides a method for isolating and purifying nisin, including the following steps: The nisin was reconstituted with ultrapure water to obtain the sample to be treated, with a concentration of 15.0 mg / ml. The nisin was purchased from Maclean's, catalog number N874940.

[0030] The mobile phase of the AKTA protein purification instrument in gel filtration chromatography mode was set to ultrapure water at a flow rate of 2 ml / min. Two unit column volumes were equilibrated with ultrapure water. Collection was started when UV ≥ 5 mAU and stopped when conductivity ≥ 1.0 mS / cm.

[0031] Ion chromatograms before and after desalting (refer to) Figure 2 As shown in the figure, the results indicate that desalting treatment can significantly reduce the salt ion concentration in nisin.

[0032] Low-temperature freeze-drying yielded a pale yellow powder of crude nisin. The crude nisin was then reconstituted with ultrapure water, resulting in a nisin concentration of 10 mg / ml. Separation was performed using a reverse-phase C18 semi-preparative column with a gradient elution of 0.1% (v / v) trifluoroacetic acid aqueous solution and acetonitrile. The elution program is shown in Table 1. The eluent with the nisin peak range was collected. Figure 1 and Figure 3 During the process, the receiving process begins when the impurity peaks have been eluted and stops when the main peak has been eluted. The eluent from the gradient elution within the range of 24.2 to 24.8 min is collected and freeze-dried at low temperature (at -70°C for 72 h) to obtain a white powdery nisin.

[0033] Table 1 Elution procedure for Example 1

[0034] The reconstituted sample was subjected to HPLC and eluted according to the chromatographic elution program shown in Table 2. The chromatographic column was a Zorbax SB-AQ C18 (5µm, 4.6*250mm), the injection volume was 20 μL, and the detection wavelength was 199 nm. The detection results were as follows: Figure 4 As shown in the figure, the results indicate that some impurities still exist in the freeze-dried product after desalting of nisin.

[0035] Table 2 Elution procedure for Example 1

[0036] The product obtained after low-temperature freeze-drying following reversed-phase chromatography was subjected to HPLC, eluted according to the chromatographic elution program, and the detection results were as described above. Figure 5 As shown in the figure, the chromatographic purity is greater than 98%.

[0037] In summary, the method for separating and purifying nisin provided by this invention is simple to operate and can obtain nisin with high purity. This method achieves high separation purity and low organic solvent consumption without requiring a specific pH environment combined with ultrafiltration for impurity removal. It has the advantages of being environmentally friendly, having simple equipment, being easy to operate, and being suitable for industrial production.

[0038] Comparative Example 1 The method for isolating and purifying nisin provided in this comparative example differs from that in Example 1 in that the elution procedure is different; isocratic elution is used, and the specific elution procedure is shown in Table 3. Other process conditions remain unchanged, the same as in Example 1. The results show that isocratic elution has poor purification efficiency.

[0039] Table 3 Elution procedure for Comparative Example 1

[0040] Comparative Example 2 The method for separating and purifying nisin provided in this comparative example differs from that in Example 1 in that it uses methanol as the organic phase. Other process conditions remain unchanged, the same as in Example 1. The results show that the methanol mobile phase is ineffective in separating impurities.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for isolating and purifying nisin, characterized in that, It includes the following steps: the sample to be purified is desalted using the gel filtration chromatography mode of a protein purifier, with water as the mobile phase, to obtain desalted nisin; then the desalted nisin is lyophilized, reconstituted, and loaded onto a C18 reversed-phase column for chromatographic separation, followed by gradient elution, and the eluted product is collected. When performing chromatographic separation using a C18 reversed-phase column, the mobile phase is set to 0.1% (v / v) to 0.2% (v / v) trifluoroacetic acid aqueous solution and acetonitrile; The gradient elution procedure includes: The volume percentage of the trifluoroacetic acid aqueous solution in the mobile phase is 79-81% during the 0-5 min period, and the volume percentage of the acetonitrile in the mobile phase is 19-21%. The volume percentage of the trifluoroacetic acid aqueous solution in the mobile phase is 59-61% for 5-27 min, and the volume percentage of the acetonitrile in the mobile phase is 39-41%. The volume percentage of the trifluoroacetic acid aqueous solution in the mobile phase is 79-81% for 27-40 min, and the volume percentage of the acetonitrile in the mobile phase is 19-21%.

2. The method for isolating and purifying nisin according to claim 1, characterized in that, Set the flow rate of the mobile phase in the protein purification instrument to 1-2 mL / min.

3. The method for isolating and purifying nisin according to claim 2, characterized in that, During the desalination process, the effluent is collected when the UV value is ≥5mAU, and collection is stopped when the conductivity of the effluent is ≥1.0mS / cm.

4. The method for isolating and purifying nisin according to claim 1, characterized in that, During reversed-phase chromatography, the flow rate of the gradient elution mobile phase is 2-4 mL / min.

5. The method for isolating and purifying nisin according to claim 4, characterized in that, After gradient elution, the receiving process begins when the impurity peaks have been eluted and stops when the main peaks have been eluted, collecting the eluent from the gradient elution within that interval.

6. The method for isolating and purifying nisin according to claim 5, characterized in that, Collect the eluent from gradient elution within the range of 24.2 to 24.8 min.

7. The method for isolating and purifying nisin according to claim 1, characterized in that, The solvent used in the resolution process is water.

8. The method for isolating and purifying nisin according to claim 1, characterized in that, The freeze-drying conditions include: freeze-drying at -65 to -70°C for 60 to -72 hours.

9. The method for isolating and purifying nisin according to claim 1, characterized in that, The chromatographic purity of the lactobacillus peptide in the eluted product is ≥98%.

10. The method for isolating and purifying nisin according to claim 1, characterized in that, The sample to be purified is a culture of Lactococcus lactis containing nisin or a crude isolate of nisin.

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