Purification method and application of bacitracin A

Abstract

The invention discloses a purification method and application of bacitracin A, and relates to the field of biology. According to the method for purifying the bacitracin A, preparation of the high-purity bacitracin A is achieved through liquid chromatographic separation, the purity of the bacitracin A can reach 95% or above, the separation and purification process of the high-purity bacitracin A is simplified to a great extent, the loss of reagents in the separation and purification stage is reduced, and the yield of the high-purity bacitracin A is increased. The method has the advantages of high separation purity, less organic solvent dosage, simple device, simple and convenient process operation, environmental protection and the like, and is suitable for process production.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to a method for purifying and applying bacitracin A. Background Technology

[0002] Antimicrobial peptides, also known as antimicrobial peptides or antibiotic peptides, are a class of small polypeptides encoded by specific genes in biological cells. Their generation and release are components of the body's inflammatory response and form an important molecular barrier for the host to defend against invading pathogenic microorganisms. Antimicrobial peptides have broad inhibitory effects on bacteria, fungi, parasites, viruses, and tumor cells, and further research on antimicrobial peptides is warranted. Examples include cephalosporin, an insect antimicrobial peptide; thionine, a plant antimicrobial peptide; and bacitracin and nisin, microbial antimicrobial peptides.

[0003] Bacitracin exhibits strong inhibitory effects against both Gram-positive and Gram-negative bacteria. Clinically, bacitracin is primarily used for penicillin-resistant Gram-positive bacterial infections and for external application to skin infections. In animal husbandry, it can be used as an antibiotic feed additive, characterized by minimal residue when added in appropriate amounts, no incompatibilities, no induction of drug resistance, and long-term usability. Therefore, bacitracin is an excellent alternative to existing antibiotics.

[0004] Bacitracin is a class of polypeptides containing a thiazoline ring, mainly produced by Bacillus licheniformis and Bacillus subtilis. It contains multiple isomers, including A, B, C, D, E, F, and G, with the activity decreasing gradually from A to G. Therefore, bacitracin A is the main active ingredient. Bacitracin A is composed of 12 amino acids (L-isoleucine-D-glutamic acid-L-lysine-D-ornithine-L-aspartic acid-L-ornithine-D-ornithine-L-glycine-L-histidine-D-aspartic acid-L-threonine-L-leucine), with the molecular formula C66H103N17O16S and a relative molecular weight of 1422.72 Da. Bacitracin A can spontaneously oxidize under alkaline conditions to form bacitracin F, which has no antibacterial activity. Therefore, bacitracin A is stable under acidic or neutral conditions.

[0005] Currently, the isolation and purification of bacitracin A has many drawbacks, such as low product bioavailability and environmental pollution caused by the large-scale use of toxic reagents.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for purifying bacitracin A and its application.

[0008] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for purifying bacitracin A, comprising the following steps: The solution containing the crude bacitracin A to be purified was subjected to a first liquid chromatography separation, and the eluent was collected. The conditions for the liquid chromatography include: a reversed-phase C18 semi-preparative column; mobile phase A being an aqueous solution of ammonium formate; mobile phase B being acetonitrile; and isocratic elution.

[0009] Secondly, embodiments of the present invention provide the application of the purification method as described in the foregoing embodiments in the preparation of medicaments for the prevention and / or treatment of infections caused by pathogens.

[0010] Thirdly, the embodiments of the present invention provide the application of the purification method as described in the foregoing embodiments in the preparation of cosmetics, food or medical devices.

[0011] The present invention has the following beneficial effects: The present invention provides a method for purifying bacitracin A. High-purity bacitracin A is prepared through liquid chromatography separation under specific conditions, achieving a purity of over 95%. This method greatly simplifies the separation and purification process of high-purity bacitracin A, reduces reagent consumption during the separation and purification stage, and offers advantages such as high separation purity, low organic solvent consumption, simple equipment, easy process operation, and environmental friendliness, making it suitable for industrialized production. Attached Figure Description

[0012] 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.

[0013] Figure 1 This is a chromatogram of the crude bacitracin A prepared in this invention after separation by a semi-preparative column. Figure 2 The HPLC chromatogram of the lyophilized product of bacitracin A prepared in this invention is shown below. Figure 3 The chromatographic separation results are shown under different column temperature conditions. Figure 4 The comparison was optimized for different loading concentrations: (a) 20 mg / L, (b) 40 mg / L, (c) 60 mg / L, (d) 80 mg / L and (e) 100 mg / L. Detailed Implementation

[0014] 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.

[0015] Definition of noun The term “treatment” includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0016] The term "prevention" refers to preventing the onset, development, or worsening of a disease or health problem before or at an early stage, through proactive interventions such as medication. Its core objective is to reduce the probability of disease occurrence, delay the onset time, and / or reduce the negative impact of disease on an individual.

[0017] The term "isocratic elution" refers to an elution method in which the composition ratio of the mobile phase remains constant throughout the entire chromatographic separation process.

[0018] Based on the problems of existing separation and purification methods (including ion exchange, adsorption, precipitation, and organic solvent extraction) such as cumbersome processes, high organic solvent consumption, and low purity, the inventors developed a simpler method for separating and purifying high-purity bacitracin A. This method uses liquid chromatography to separate and purify bacitracin A, increasing the purity of crude bacitracin A from 64% to over 95%. This method achieves high separation purity and low organic solvent consumption without complicated procedures, and boasts advantages such as simple equipment, easy operation, and environmental friendliness.

[0019] Technical solution On one hand, embodiments of the present invention provide a method for purifying bacitracin A, which includes the following steps: The solution containing the crude bacitracin A to be purified was subjected to a first liquid chromatography separation, and the eluent was collected. The conditions for the liquid chromatography include: a reversed-phase C18 semi-preparative column; mobile phase A being an aqueous solution of ammonium formate; mobile phase B being acetonitrile; and isocratic elution.

[0020] In an optional embodiment, the solution containing the crude bacitracin A to be purified is made by mixing crude bacitracin A and an aqueous solution at a ratio of 10-100 mg: 1 mL. The 10-100 mg can be any one or any two of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg.

[0021] The inventors discovered that by reconstituted crude bacitracin A with an aqueous solution, and then using ammonium formate aqueous solution and acetonitrile as the mobile phase for isocratic elution with a reversed-phase C18 semi-preparative column, a better separation degree and stable peak time can be obtained.

[0022] In an optional embodiment, the crude bacitracin A refers to a product whose bacitracin A purity does not reach 95% or higher, and the purity can be below 50%, below 52%, below 54%, below 56%, below 58%, below 60%, below 62%, below 64%, below 66%, below 68%, below 70%, below 72%, below 74%, below 76%, below 78%, below 80%, below 82%, below 84%, below 86%, below 88%, below 90%, below 92%, and below 94%, etc.

[0023] In an optional embodiment, the crude bacitracin A is obtained by microbial fermentation.

[0024] In an optional embodiment, the size of the reverse-phase C18 semi-preparative column is (1–15) mm × (200–300) mm, the particle size is 1–10 µm, the pore size is 250–350 Å, and the column temperature is 30–50 °C. Alternatively, the size can be (8–12) mm × (240–260) mm. Specifically, the particle size can be any one or any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 µm; the pore size can be any one or any two of 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, and 350 Å; and the column temperature can be any one or any two of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 °C.

[0025] In an optional embodiment, the reversed-phase C18 semi-preparation column is Waters XBridge Peptide BEH C18, 10 mm × 250 mm, 5 µm, 300 Å.

[0026] In an optional embodiment, the reversed-phase C18 semi-preparation column is an Athena C18-BIO column, 10 mm × 250 mm, 5 µm, 300 Å.

[0027] In an optional embodiment, the concentration of the ammonium formate aqueous solution is 40–60 mmol / L, and the pH is 3.8–4.2. Specifically, the concentration can be any one or any two of the following: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60 mmol / L. The pH can be adjusted with formic acid, and the pH can be any one or any two of the following: 3.8, 3.9, 4.0, 4.1, and 4.2. Under these mobile phase conditions, the elution separation is improved.

[0028] In an optional embodiment, the isocratic elution procedure includes: 0-50 min, the volume percentage of mobile phase A being 70%-80%, and the volume percentage of mobile phase B being 20%-30%.

[0029] In an optional embodiment, the volume percentage of the mobile phase A can be any one or any two of 70%, 72%, 74%, 76%, 78%, and 80%.

[0030] In an optional embodiment, the volume percentage of the mobile phase B can be any one or any two of 20%, 22%, 24%, 26%, 28% and 30%, and the flow rate is 3~4 mL / min.

[0031] In an optional embodiment, the flow rate can specifically be any one or a range between any two of 3, 3.2, 3.4, 3.6, 3.8 and 4 mL / min.

[0032] In an optional embodiment, the step of collecting the eluent during the first liquid chromatography separation includes collecting the eluent over a period of 22 to 30 minutes. Specifically, this 22 to 30 minutes can be any one or any combination of 22, 23, 24, 26, 27, 28, 29, and 30 minutes.

[0033] In an optional embodiment, the purification method further includes: performing a second liquid chromatography separation on the aqueous solution of the eluent or its lyophilized product obtained from the first liquid chromatography separation, and collecting the eluent.

[0034] In an optional embodiment, the conditions for the second liquid chromatography separation include: the chromatographic column is a reversed-phase C18 semi-preparative column as described in any of the foregoing embodiments; mobile phase A is 40-60 mmol / L ammonium formate aqueous solution; mobile phase B is acetonitrile; and gradient elution is used.

[0035] In an optional embodiment, the gradient elution procedure is as follows: From 0 to 30 minutes, the volume percentage of the mobile phase B increased from 20% to 25% to 28% to 32%. Over 30–31 minutes, the volume percentage of the mobile phase B increased from 28%–32% to 85%–95%. For 31–36 minutes, the volume percentage of the mobile phase B is maintained at 85%–95%. Over 36–37 minutes, the volume percentage of the mobile phase B decreased from 85%–95% to 20%–25%. For 37 to 40 minutes, the volume percentage of the mobile phase B is maintained at 20% to 25%.

[0036] In an optional embodiment, the loading concentration for the second liquid chromatography separation is 35–60 mg / mL; specifically, it can be any one or any two of 35, 40, 45, 50, 55, and 60 mg / mL.

[0037] In an optional embodiment, the sample loading volume for the second liquid chromatography separation is 10 to 50 μL, specifically any one or any two of 10, 15, 20, 25, 30, 35, 40, 45 and 50 μL.

[0038] In an optional embodiment, the purification method further includes: freeze-drying the eluent from the first liquid chromatography separation and / or the second liquid chromatography separation.

[0039] Bacitracin A obtained based on the embodiments of the present invention has the advantage of high purity (≥95%) and can be directly used for mass spectrometry analysis.

[0040] On the other hand, embodiments of the present invention provide the use of the purification method as described in any of the foregoing embodiments in the preparation of products that inhibit pathogens, or medicaments for the prevention and / or treatment of infections caused by pathogens.

[0041] In an optional embodiment, the pathogen includes at least one of Staphylococcus aureus and Escherichia coli.

[0042] Furthermore, embodiments of the present invention also provide the application of the purification method as described in any of the foregoing embodiments in the preparation of cosmetics, food, or medical devices.

[0043] Bacitracin A prepared by the purification method described in any of the foregoing embodiments can be used as a preservative in the preparation of cosmetics, food, or medical devices.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1 This embodiment provides a method for purifying bacitracin A, which includes the following steps: (1) Solvent extraction Obtain 5g of crude bacitracin with a purity of 64% from Beijing Solarbio Technology Co., Ltd. The crude bacitracin A was dissolved at a material-to-liquid ratio of 40 mg: 1 mL (crude bacitracin A: aqueous solution).

[0046] (2) Separation and purification The above extract was separated using a reversed-phase C18 semi-preparative column. The column used was a Waters XBridge Peptide BEH C18, 10 mm × 250 mm, 5 µm, 300 Å, with a column temperature of 40 °C. A control column was also set up: an Athena C18-BIO, 10 mm × 250 mm, 5 µm, 300 Å, with a column temperature of 40 °C. The injection volume was 100 μL. The mobile phase consisted of an aqueous phase and an organic phase. The aqueous phase was a 50 mmol / L ammonium formate aqueous solution (pH = 4), and the organic phase was acetonitrile. Isocratic elution was performed.

[0047] The elution procedure is shown in Table 1.

[0048] Table 1 Elution Procedure

[0049] Collect the eluent within the peak range of bacitracin A, such as... Figure 1 The product was retained for 22-30 min, then freeze-dried at low temperature to obtain a white powder of bacitracin A, XBridge Peptide BEH C18 with a chromatographic purity of 95%. Figure 2 .

[0050] As can be seen, under the same experimental conditions and parameters, the XBridgePeptide BEH C18 column exhibits a shorter retention time for bacitracin A and demonstrates superior separation of the main component from impurities compared to the Athena C18-BIO column. Therefore, the XBridgePeptide BEH C18 column will be used as the chromatographic column in subsequent examples.

[0051] Example 2 To verify the effect of column temperature on separation performance.

[0052] Based on the purification method provided in Example 1, column temperature was used as a single-factor variable, and three experimental groups were set up, using three different column temperatures of 27℃, 35℃, and 40℃ respectively. The chromatographic column was an Xbridge Peptide BEH C18 (the chromatographic column of Example 1), and the separation of bacitracin A was compared under the same conditions. Figure 3 .

[0053] The results showed that as the column temperature increased, the number of impurities eluted increased, the impurities contained in front of the main peak were clearly separated from the main peak, the elution time of the main peak gradually advanced, and the separation degree between the main peak and impurities was best at 40℃.

[0054] Example 3 Verify the effect of mobile phase type and ratio on separation performance.

[0055] Based on the purification method provided in Example 1, multiple experimental groups were set up with the mobile phase as a variable, as shown in the table below.

[0056] Table 1. Mobile phase of the experimental group

[0057] Experimental results show that potassium dihydrogen phosphate solution in the aqueous phase cannot separate the main component and impurities. 0.2% formic acid solution and acetic acid solution can separate the main component and impurities, but the separation effect is weaker than that of 50 mmol / L ammonium formate and 0.2% trifluoroacetic acid. The separation between the main component and impurities is poor with 0.2% trifluoroacetic acid; increasing the concentration may lead to impurities in the main peak, affecting subsequent purification results. Trifluoroacetic acid may combine with basic amino acids such as lysine and histidine in bacitracin A, introducing new impurities that cannot be removed during subsequent freeze-drying, directly affecting the purity of bacitracin A. Therefore, 50 mmol / L ammonium formate solution provides the best separation effect when used as the aqueous phase.

[0058] Example 4 To verify the effect of pH on the separation efficiency of bacitracin A.

[0059] Based on the purification method provided in Example 1, the pH of the aqueous phase was used as a variable, and three experimental groups were set up. The pH of the aqueous phase was adjusted to 4, 6 and 8 with formic acid.

[0060] The results showed that as the pH gradually increased, the elution time of the main peak gradually delayed, and the separation degree between the main component and impurities gradually decreased. At pH=8, the response value of the main peak decreased, and the amount of impurities eluted was less than that in the slightly acidic and neutral mobile phases. Therefore, pH=4 of the aqueous phase is a suitable elution condition for bacitracin A.

[0061] Example 5 To verify the effect of the elution procedure on the separation efficiency of bacitracin A.

[0062] Based on the purification method provided in Example 1, two experimental groups were set up with the elution procedure as a variable.

[0063] Table 2 Elution procedure for Group 1

[0064] Table 3 Elution procedures for Group 2

[0065] Experimental data show that: In Group 1, bacitracin A and impurities were not completely separated. The main peak eluted around 27 min, and thereafter, impurity peaks were almost non-existent. This elution program showed a sharp and symmetrical main peak shape and relatively good separation from impurities. However, when the sample concentration was increased, the impurity peaks might not be completely eluted, which would be detrimental to the subsequent collection of the purified main peak solution.

[0066] Group 2 showed better separation than other gradient elution programs, but the separation of impurity peaks before and after the main peak was not ideal. Increasing the organic phase ratio from 0-15 min was considered to allow impurity peaks to elute quickly and achieve separation between impurities and the main component. However, experimental data showed that increasing the organic phase ratio during this period would cause the main peak to elute prematurely, making impurity peak elution difficult and resulting in some impurities being included in the main component.

[0067] Example 6 To verify the effect of sample concentration on the separation efficiency of bacitracin A.

[0068] The crude bacitracin was dissolved in pure water to prepare solutions of 20, 40, 60, 80, and 100 mg / mL, respectively. Purification was performed according to the method provided in Example 1, and the experimental results are as follows: Figure 4 As shown in (a), (b), (c), (d), and (e).

[0069] Comparison of chromatograms at five different concentrations revealed that the intensity of the main peak increased with increasing concentration, while the overall elution time remained relatively unchanged. The separation of the main peak from impurities was optimal at a loading concentration of 20 mg / mL, but this low concentration resulted in low purification yield.

[0070] The comparison revealed that the separation between the main peak and impurities at the loading concentration of 100 mg / mL was weaker than that at the other four concentrations. However, the purified solution could be collected when the main peak reached its highest point to reduce the impurity content. The results showed that this method was feasible and the purification yield was higher than that of the other concentrations.

[0071] Example 7 This embodiment provides a method for purifying bacitracin A, which includes the following steps: (1) First purification The purification method provided in Example 1 is used as a first purification.

[0072] (2) Secondary purification The eluent of bacitracin A collected using the purification method of Example 1 was subjected to chromatographic elution. The chromatographic column was an XBridge Peptide BEH C18 10mm×250mm, 5µm, 300Å column. The mobile phase consisted of an aqueous phase and an organic phase. The aqueous phase was 50mmol / L ammonium formate aqueous solution, and the organic phase was acetonitrile. The column temperature was 40℃, and the injection volume was 30μl.

[0073] The elution procedure is as follows.

[0074] Table 4 Elution Procedure

[0075] The collected target analytes were determined using liquid chromatography-ultraviolet (LC-UV) method. The retention time of the target analytes was consistent with that of bacitracin A in the crude product, and the area-normalized purity was 99.3%, meeting the high purity requirements.

[0076] Example 8 To verify the effect of the mobile phase on the separation effect during secondary purification.

[0077] Based on the purification method provided in Example 7, multiple experimental groups were set up with the mobile phase in the secondary purification in step (2) as a variable, as shown in the table below.

[0078] Table 5. Mobile phase of the experimental group

[0079] The results showed that when ammonium formate solution was used as the aqueous phase and acetonitrile was used as the organic phase, the impurities contained in the sample could be eluted and separated, and the separation degree from the main component was good.

[0080] Example 9 To verify the effect of sample concentration on separation efficiency.

[0081] Based on the purification method provided in Example 7, the loading concentration in step (1) was used as a variable (the sample for secondary purification was the lyophilized powder of the first purification collection liquid, which was reconstituted with water at different concentrations). Three different loading concentrations of 35 mg / mL, 40 mg / mL and 50 mg / mL were used to verify its effect on the separation of bacitracin A.

[0082] Experimental results showed that the retention time of bacitracin A remained largely consistent with increasing concentration, with only minor differences in response levels at different concentrations. To obtain a large quantity of bacitracin A, the optimal loading concentration for secondary purification and separation was set at 50 mg / mL.

[0083] Example 10 To verify the effect of the loading amount during secondary purification on the separation effect.

[0084] Based on the purification method provided in Example 7, the loading amount in step (2) was used as a variable, and three different loading amounts of 10, 20 and 30 μL were used to verify the effect on the separation of bacitracin A.

[0085] The results showed that the elution times of the main peaks were basically consistent across the three different injection volumes, with only differences in response magnitude. Therefore, the optimal injection volume for secondary purification and separation of bacitracin can be set to 30 μL.

[0086] 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 purifying bacitracin A, characterized in that, It includes the following steps: The solution containing the crude bacitracin A to be purified was subjected to a first liquid chromatography separation, and the eluent was collected. The conditions for the first liquid chromatography included: a reversed-phase C18 semi-preparative column; mobile phase A being an aqueous solution of ammonium formate; mobile phase B being acetonitrile; and isocratic elution.

2. The purification method according to claim 1, characterized in that, The concentration of the ammonium formate aqueous solution is 40-60 mmol / L, and the pH is 3.8-4.2; Optionally, the size of the reverse-phase C18 semi-preparation column is (1-15) mm × (200-300) mm, the particle size is 1-10 µm, the pore size is 250-350 Å, and the column temperature is 30-50 °C; Optionally, the reversed-phase C18 semi-preparation column is Waters XBridge Peptide BEH C18, 10 mm × 250 mm, 5 µm, 300 Å; or Athena C18-BIO, 10 mm × 250 mm, 5 µm, 300 Å.

3. The purification method according to claim 1, characterized in that, The sample concentration for the first liquid chromatography separation is 20–100 mg / mL.

4. The purification method according to claim 1, characterized in that, The isocratic elution procedure includes: 0-50 min, maintaining the volume percentage of mobile phase B at 20%-30%, and a flow rate of 3-4 mL / min.

5. The purification method according to claim 1, characterized in that, The purification method further includes: performing a second liquid chromatography separation on the aqueous solution of the eluent or its lyophilized product obtained from the first liquid chromatography separation, and collecting the eluent; Optionally, the conditions for the second liquid chromatography separation include: a reversed-phase C18 semi-preparative column; mobile phase A being 40–60 mmol / L ammonium formate aqueous solution; mobile phase B being acetonitrile; and gradient elution. Optionally, the dimensions of the reverse-phase C18 semi-preparative column are (1–15) mm × (200–300) mm, the particle size is 1–10 µm, the pore size is 250–350 Å, and the column temperature is 30–50 °C; Optionally, the reversed-phase C18 semi-preparation column is Waters XBridge Peptide BEH C18, 10 mm × 250 mm, 5 µm, 300 Å.

6. The purification method according to claim 5, characterized in that, The gradient elution procedure is as follows: From 0 to 30 minutes, the volume percentage of the mobile phase B increased from 20% to 25% to 28% to 32%. Over 30–31 minutes, the volume percentage of the mobile phase B increased from 28%–32% to 85%–95%. For 31–36 minutes, the volume percentage of the mobile phase B is maintained at 85%–95%. Over 36–37 minutes, the volume percentage of the mobile phase B decreased from 85%–95% to 20%–25%. For 37 to 40 minutes, the volume percentage of the mobile phase B is maintained at 20% to 25%.

7. The purification method according to claim 1, characterized in that, The sample concentration for the second liquid chromatography separation is 35–60 mg / mL; Optionally, the sample loading volume for the second liquid chromatography separation is 10–50 μL.

8. The purification method according to any one of claims 5 to 7, characterized in that, The purification method further includes: freeze-drying the eluent from the first liquid chromatography separation and / or the second liquid chromatography separation.

9. The use of the purification method according to any one of claims 1 to 8 in the preparation of products that inhibit pathogens, and medicaments for the prevention and / or treatment of infections caused by pathogens; Optionally, the pathogen includes: At least one of Staphylococcus aureus and Escherichia coli.

10. The use of the purification method according to any one of claims 1 to 8 in the preparation of cosmetics, food or medical devices.

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