Polypeptide amide synthetic product purification method

Abstract

The invention belongs to the technical field of cleaning products. The invention provides a polypeptide amide synthetic product purification method, which comprises: S1, adding a polypeptide amide synthetic product and methanol into a centrifuge tube, and carrying out centrifugal treatment to obtain a centrifugal product; s2, the centrifugal product is sequentially subjected to filtering and vacuum drying treatment, and a vacuum drying product is obtained; s3, sequentially performing ultrafiltration and vacuum drying treatment on the vacuum drying product to obtain purified polypeptide amide. A series of separation and purification means are adopted to purify the polypeptide amide reaction product, unreacted substances such as diethanol amine and lauric acid in the synthetic product can be effectively removed, the content of the unreacted substances meets the requirement of a standard limit value in an application scene, meanwhile, the relative content of the effective component polypeptide amide in the reaction product is increased, and the yield of the product is increased. Therefore, the decontamination effect of the purified product is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cleaning products, and in particular relates to a method for purifying a polypeptide amide synthesis product. Background Art

[0002] Peptide amide is a protein-based nonionic surfactant. The product has strong detergency, neutral pH, and is non-toxic and non-irritating to the skin. Because its chemical structure is similar to human skin protein, it also has the characteristics of moisturizing the skin, protecting the skin, protecting the hair, and having the feeling of a bath without rinsing with water after a bath. It is widely used in the production process of cosmetics such as skin care, hair care, cleansing and shampooing. At present, the synthesis method of peptide amide mainly uses gelatin, diethanolamine and lauric acid for heating reaction. The reaction method is mature and can be mass-produced.

[0003] However, in 2017, diethanolamine was listed as a Class 2B carcinogen. According to the European Detergents and Cleaning Agents Association Regulation issued by the European Chemicals Agency, the limit standard for diethanolamine in detergents is 1%, and the US Environmental Protection Agency also stipulates that the limit standard for diethanolamine should not exceed 5%. However, the existing peptide amide synthesis and subsequent skin cleansing package product production process lacks analysis of the composition and content of peptide amide reaction products. At the same time, there is no clear and effective method for separating and purifying peptide amide synthesis products. It is impossible to ensure whether the content of various components in the application product meets the standard requirements, and it is difficult to ensure the safety of skin cleansing packages. Summary of the invention

[0004] The present invention performs component analysis on the polypeptide amide synthesis product, and adopts a series of separation and purification methods to purify the polypeptide amide reaction product, which can effectively remove unreacted substances such as diethanolamine and lauric acid in the synthesis product, so that its content meets the standard limit requirements in the application scenario, and at the same time increases the relative content of the effective ingredient polypeptide amide in the reaction product, thereby improving the decontamination effect of the purified product.

[0005] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0006] The invention provides a method for purifying a polypeptide amide synthesis product, comprising: S1, adding the polypeptide amide synthesis product and methanol into a centrifuge tube, and performing centrifugal treatment to obtain a centrifugal product; S2, filtering and vacuum drying the centrifugal product in sequence to obtain a vacuum dried product; S3, ultrafiltration and vacuum drying the vacuum dried product in sequence to obtain purified polypeptide amide.

[0007] Furthermore, the centrifugal speed is 12000r / min and the time is 30min.

[0008] Furthermore, the centrifugal product is filtered and vacuum dried in sequence to obtain a vacuum dried product, including: S21, filtering the centrifugal product using a microfiltration membrane, a Buchner funnel and a suction filtration device to obtain a filtered product; S22, drying the filtered product using a vacuum oven to obtain a vacuum dried product.

[0009] Furthermore, the pore size of the microfiltration membrane is 0.1 μm to 10 μm, and the flux range is 50 LMH to 500 LMH; the operating pressure of the filtration device is 0.3 bar to 7 bar.

[0010] Furthermore, the microfiltration membrane is made of one of polyester material, polycarbonate material and polypropylene material.

[0011] Furthermore, the drying temperature is 60° C. to 80° C., and the drying time is 5 h to 6 h.

[0012] Furthermore, the vacuum-dried product is subjected to ultrafiltration and vacuum drying treatment in sequence to obtain purified polypeptide amide, including: S31, adding the vacuum-dried product and water into a beaker, and stirring to obtain a stirred product; S32, ultrafiltration treatment of the stirred product using an ultrafiltration membrane to obtain a retentate; S33, drying the retentate using a vacuum oven to obtain the purified polypeptide amide.

[0013] Furthermore, the molecular weight cutoff of the ultrafiltration membrane is 3000-10000; the ultrafiltration time is 5-8 days, the temperature is 20° C.-30° C., and the pressure is 0.1 MPa-1 MPa.

[0014] Furthermore, the drying time is 10 hours to 48 hours, and the temperature is 40° C. to 60° C.

[0015] Furthermore, the ultrafiltration membrane is one of cellulose acetate materials, cellulose acetate ester materials and polysulfone materials.

[0016] The beneficial effect of the present invention is that, compared with the prior art, a method for purifying a polypeptide amide synthesis product is provided.

[0017] 1. The present invention analyzes the components of the polypeptide amide synthesis product to determine the contents of the reaction product, unreacted raw materials and water therein. The component analysis process adopts instrumental analysis methods, which are mature in technology and easy to reproduce.

[0018] 2. The present invention reduces the concentration of unreacted raw materials diethanolamine and lauric acid in the polypeptide amide synthesis product through the steps of centrifugal separation, filtration, vacuum drying, semipermeable membrane dialysis, vacuum drying, etc., and can meet the requirements of international standard limits. The purification methods are all widely used mature technologies and are easy to reproduce. The experimental supplies required in the purification process are all commercially available and easy to obtain.

[0019] 3. The present invention conducts detergency tests on the cleaning liquid prepared from the purified polypeptide amide synthesis product, and compares the decontamination effects of the polypeptide amide synthesis product before and after treatment by various purification methods, which proves that the separation and purification process of the polypeptide amide synthesis product designed by the present invention can effectively improve the performance of the product. The detergency test is carried out using the method specified in the national standard, and the process is mature and standardized. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other embodiments can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a process flow chart of polypeptide amide synthesis reaction;

[0022] Figure 2 It is the reaction equation diagram of peptide amide synthesis;

[0023] Figure 3 This is a comparison chart of the peptide amide synthesis product and gelatin FTIR test;

[0024] Figure 4 It is a synthetic product of peptide amide 1 HNMR test spectrum;

[0025] Figure 5 This is the TGA test data diagram of the peptide amide synthesis product;

[0026] Figure 6 This is a flow chart of Example 1 for purification of polypeptide amide synthesis products;

[0027] Figure 7 This is a flow chart of Example 2 for purification of polypeptide amide synthesis products. DETAILED DESCRIPTION

[0028] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0029] Analysis of components of peptide amide synthesis products:

[0030] use Figure 1The process flow of polypeptide amide synthesis reaction is as follows: Figure 2 The peptide amide synthesis product was subjected to component analysis through the various tests shown below, and the reaction raw material gelatin was provided for comparative testing.

[0031] FTIR: Thermo Nicolet IS5 Fourier transform infrared spectrometer was used for testing. The infrared spectrum test results of peptide amide synthesis products and raw gelatin are shown in Figure 3 , where the upper figure is the test result of polypeptide amide synthesis product, and the lower figure is the test result of gelatin. Figure 3 It can be seen that gelatin contains 1235cm -1 The characteristic peak belongs to the characteristic absorption of carbonyl group, but the information of this peak is not seen in the test results of the polypeptide amide synthesis product. It is preliminarily inferred that the polypeptide amide synthesis product does not contain raw gelatin.

[0032] 1 HNMR: The test was performed using a BRUKER 400MHz nuclear magnetic resonance spectrometer. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that 2.95 ppm and 3.75 ppm are chemical shifts of diethanolamine, and 0.91 ppm, 1.31 ppm and 1.64 ppm are chemical shifts of alkane chains.

[0033] TGA: Tested using TA Q500 thermogravimetric analyzer, the results are shown in Figure 5 The test atmosphere is N2, the test temperature is between 40℃ and 800℃, and the heating rate is 20℃ / min. Figure 5 It can be seen that 204°C is the thermal weight loss temperature of the reaction raw materials diethanolamine and lauric acid; 313°C is the thermal weight loss temperature of the amide bond rupture; and 400°C is the thermal weight loss temperature of the reaction product polypeptide amide.

[0034] LCMS: Waters SQ Detector 2 single-stage quadrupole liquid-mass spectrometer was used for testing, and the test results are shown in Table 1. Among them: the sampling volume is 0.1384 g, and methanol is used to dissolve after sampling, and it is completely dissolved after vortexing for 5 minutes, the fixed volume is 50 ml, and the dilution multiple after fixed volume is 100 times. As shown in Table 1, the content of unreacted raw material diethanolamine in the sample is about 13.25%, and the content of lauric acid is about 15.25%.

[0035] Table 1 LCMS test data of peptide amide synthesis products

[0036] Object to be tested content(%) Diethanolamine 13.25 Lauric acid 15.25

[0037] Moisture analysis: The test was conducted using a METTLER TOLEDO Compact C20SX Karl Fischer titrator, and the test results are shown in Table 2. The test vials were vacuum baked at 110°C for 6 hours, then placed in a low humidity environment for 3 hours, the sample size was 0.09 g, and the drift equilibrium time was 1 hour. As shown in Table 2, the moisture content in the sample was approximately 2.70%.

[0038] Table 2 Water content analysis test data of peptide amide synthesis products

[0039] Object to be tested content(%) water 2.70

[0040] The FTIR comparison of the synthetic peptide amide products and gelatin revealed that the 1235 cm -1 The characteristic peaks of peptide amide synthesis reaction products disappeared, indicating that gelatin substances reacted and there was no obvious gelatin information in the synthesis reaction products; 1 HNMR found that the chemical shift was mainly due to polyamide substances. In addition, chemical shifts of diethanolamine were also found at 2.95ppm and 3.75ppm. TGA of the products of polypeptide amide synthesis reactions revealed that there was thermal gravimetric loss information of small molecules at around 204°C. LCMS external standard quantitative analysis of the products of polypeptide amide synthesis reactions showed that the content of diethanolamine was approximately 13.25%, and the content of lauric acid was approximately 15.25%. Finally, it was concluded that the sample contained information on polyamide, diethanolamine, and lauric acid, and no obvious information on gelatin substances.

[0041] In summary, the components and contents of the sample polypeptide amide synthesis products are shown in Table 3.

[0042] Table 3 Components and contents in the synthetic products of sample peptide amide

[0043] Component No. Component name content(%) 1 Peptide amides 68.80 2 Diethanolamine 13.25 3 Lauric acid 15.25 4 water 2.70

[0044] In actual application, the effective content of polypeptide amide in the product is 10% to 40%. If the polypeptide amide synthesis product used in this test is directly used for product production, the content of diethanolamine in the product is about 1.3% to 5.3%, which exceeds the limit values ​​required by the aforementioned European (no more than 1%) and American (no more than 5%) standards. Therefore, in order to ensure the safety of the product, the polypeptide amide synthesis product needs to be purified.

[0045] Purification of peptide amide synthesis products

[0046] Example 1

[0047] (1) Thermal dissolution: Weigh 5 g of the polypeptide amide synthesis product sample in a 250 mL beaker, add 100 mL of deionized water, heat in an 80°C water bath, and stir with a glass rod to dissolve.

[0048] (2) Filtration: The sample in (1) is filtered through a qualitative filter paper with a pore size of 10 μm, and then passed through a Buchner funnel and a suction filtration device to remove insoluble matter and suspended matter. The operating pressure of the suction filtration device is not less than 0.3 bar.

[0049] Note: Hot solution filtration is to filter the cooled liquid and retain the filtrate.

[0050] (3) Semipermeable membrane dialysis: The purified filtrate in (2) was dialyzed using a semipermeable membrane with a molecular weight cutoff of 5K, rinsed twice with 200 mL of pure water, and the dialysis time was 5 days. The retentate was collected.

[0051] (4) Vacuum drying: The retentate in (3) is dried in a vacuum oven at 60°C and 10 Pa vacuum. The drying time is 24 hours, and the purified polypeptide amide is obtained.

[0052] (5) Analysis of the composition of purified polypeptide amide: LCMS was used to test the content of unreacted raw material diethanolamine and lauric acid in the purified polypeptide amide. A certain amount of purified sample was weighed and ultrasonically dissolved in methanol for 20 minutes. After constant volume, ultrasonication was continued for 10 minutes. Finally, methanol was continued to dilute to within the range of the mark and the test was carried out on the machine. The test method and calculation steps were consistent with the above method.

[0053] The process flow chart of the above embodiment 1 is shown in Figure 6 .

[0054] Example 2

[0055] (1) Centrifugal separation: Weigh 5 g of the polypeptide amide synthesis product sample in a 50 mL centrifuge tube, add 20 mL of methanol, and centrifuge at a high speed of 12,000 r / min for 30 min.

[0056] (2) Filtration: The sample in (1) is filtered through a qualitative filter paper with a pore size of 10 μm, and then passed through a Buchner funnel and a suction filtration device to remove insoluble matter and suspended matter. The operating pressure of the suction filtration device is not less than 0.3 bar.

[0057] (3) Vacuum drying: The insoluble matter after centrifugation was dried in a vacuum oven at 60°C and 10 Pa vacuum. The drying time was 5 h.

[0058] (4) Semipermeable membrane dialysis: Add 100 mL of water to the dried sample obtained in step (3) and dissolve it in a 250 mL beaker by stirring with a glass rod. Perform dialysis using a semipermeable membrane with a molecular weight cutoff of 5K, rinse twice with 200 mL of pure water, and perform dialysis for 5 days. Collect the retentate.

[0059] (5) Vacuum drying: The retentate in (4) is dried in a vacuum oven at 60°C and 10 Pa vacuum. The drying time is 24 hours, and the purified polypeptide amide is obtained.

[0060] (6) Analysis of the composition of purified polypeptide amide: LCMS was used to test the content of unreacted raw material diethanolamine and lauric acid in the purified polypeptide amide. A certain amount of purified sample was weighed and ultrasonically dissolved in methanol for 20 minutes. After constant volume, ultrasonication was continued for 10 minutes. Finally, methanol was continued to dilute to within the range of the mark and the test was carried out on the machine. The test method and calculation steps were consistent with the above method.

[0061] The process flow chart of the above embodiment 2 is shown in Figure 7 .

[0062] The purified polypeptide amide obtained by the above-mentioned Example 2 was compared with the unpurified polypeptide amide synthesis product, and the contents of unreacted raw material diethanolamine and lauric acid were shown in Table 4. As shown in Table 4, the content of diethanolamine in the unpurified polypeptide amide synthesis product was 13.25%, and the content of lauric acid was 15.25%; after treatment by Example 1, the content of diethanolamine in the purified polypeptide amide was 5.65%, and the content of lauric acid was 10.14%; after treatment by Example 2, the content of diethanolamine in the purified polypeptide amide was 2.96%, and the content of lauric acid was 10.65%. The above results show that the purification by Example 1 and Example 2 can effectively reduce the content of unreacted raw material diethanolamine and lauric acid in the polypeptide amide synthesis product, and the content of diethanolamine is significantly reduced. According to the calculation of 10% to 40% of the polypeptide amide concentration in the aforementioned product, the content of diethanolamine in the product prepared by the polypeptide amide purified by Example 1 is about 0.6% to 2.26%, and the content of diethanolamine in the product prepared by the polypeptide amide purified by Example 1 is about 0.3% to 1.184%, both of which can meet the limit values ​​required by the aforementioned US standard (not exceeding 5%), and can partially meet the limit values ​​required by the aforementioned European standard (not exceeding 1%).

[0063] Table 4 Comparison of component contents before and after purification of peptide amide synthesis products

[0064]

[0065] Detergency test of peptide amide synthesis products

[0066] The untreated polypeptide amide synthesis product and the polypeptide amide purified by Example 1 and Example 2 were added with water to prepare a solution with a concentration of 0.2%, and the detergency was tested according to the method and steps specified in the national standard (GB / T 13174-2021). The results are shown in Table 5. As can be seen from Table 5, the detergency of the polypeptide amide purified by Example 1 is 1.6 times that before purification, and the detergency of the polypeptide amide purified by Example 1 is 1.7 times that before purification. The above results show that the above-mentioned purification treatment can not only effectively reduce the concentration of unreacted diethanolamine in the polypeptide amide synthesis product, improve the safety of the product, but also significantly improve the detergency performance of the product.

[0067] Table 5 Detergency comparison of peptide amide synthesis products before and after purification

[0068]

[0069] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or changes based on the technical scheme and inventive concept of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for purifying a polypeptide amide synthesis product, characterized in that: include: S1, adding the polypeptide amide synthesis product and methanol into a centrifuge tube, and centrifuging to obtain a centrifuge product; S2, filtering and vacuum drying the centrifuged product in sequence to obtain a vacuum dried product; S3, sequentially subjecting the vacuum-dried product to ultrafiltration and vacuum drying to obtain purified polypeptide amide.

2. The method for purifying a polypeptide amide synthesis product according to claim 1, characterized in that: The centrifugal speed is 12000 r / min and the time is 30 min.

3. The method for purifying a polypeptide amide synthesis product according to claim 1, characterized in that: The centrifugal product is filtered and vacuum dried in sequence to obtain a vacuum dried product, comprising: S21, filtering the centrifugal product using a microfiltration membrane, a Buchner funnel and a suction filtration device to obtain a filtered product; S22, using a vacuum oven to dry the filtered product to obtain a vacuum dried product.

4. The method for purifying a polypeptide amide synthesis product according to claim 3, characterized in that: The pore size of the microfiltration membrane is 0.1 μm to 10 μm, and the flux range is 50 LMH to 500 LMH; the operating pressure of the suction filtration device is 0.3 bar to 7 bar.

5. The method for purifying the polypeptide amide synthesis product according to claim 3, characterized in that: The microfiltration membrane is made of one of polyester material, polycarbonate material and polypropylene material.

6. The method for purifying a polypeptide amide synthesis product according to claim 3, characterized in that: The drying temperature is 60° C. to 80° C., and the drying time is 5 h to 6 h.

7. The method for purifying a polypeptide amide synthesis product according to claim 1, characterized in that: The vacuum dried product is subjected to ultrafiltration and vacuum drying treatment in sequence to obtain purified polypeptide amide, comprising: S31, adding the vacuum-dried product and water into a beaker and stirring to obtain a stirred product; S32, performing ultrafiltration treatment on the stirred product using an ultrafiltration membrane to obtain a retentate; S33, using a vacuum oven to dry the retentate to obtain the purified polypeptide amide.

8. The method for purifying a polypeptide amide synthesis product according to claim 7, characterized in that: The ultrafiltration membrane has a molecular weight cutoff of 3000 to 10000; the ultrafiltration time is 5 to 8 days, the temperature is 20° C. to 30° C., and the pressure is 0.1 MPa to 1 MPa.

9. The method for purifying a polypeptide amide synthesis product according to claim 7, characterized in that: The drying time is 10 hours to 48 hours, and the temperature is 40° C. to 60° C.

10. The method for purifying a polypeptide amide synthesis product according to claim 7, characterized in that: The ultrafiltration membrane is one of cellulose acetate materials, cellulose acetate ester materials and polysulfone materials.

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