A lipopeptide sodium whole water phase extraction process based on coupling of molecular weight screen and pH regulation

Abstract

This invention provides a full-aqueous phase extraction process for sodium lipopeptides based on molecular weight sieving and pH control coupling. The process includes the following steps: The fermentation broth is heated to denature proteins and co-precipitate them with bacterial cells; the precipitate is removed by high-speed centrifugation; the precipitate is washed with acidic brine as an "anti-solvent" to enhance impurity removal; an optimal solution pH is established before ultrafiltration using an alkaline aqueous solution to precisely retain the target lipopeptides and remove proteins, polysaccharides, etc. from the solution; after adjusting the solution pH to neutral, deep desalination and concentration are simultaneously completed using nanofiltration constant-volume washing technology; solid sodium lipopeptides are obtained by spray drying. This invention utilizes a multi-stage membrane filtration system of "microfiltration-ultrafiltration-nanofiltration" to achieve step-by-step, targeted removal of impurities; extraction is performed in a full-aqueous phase, eliminating organic solvents and avoiding organic solvent residues throughout the production process, making it more environmentally friendly and safer; the final product is sodium lipopeptide salt, improving the product's water solubility and stability, and preserving the product's bioactive structure to the greatest extent.

Description

Technical Field

[0001] This invention relates to a method for the extraction of sodium lipopeptides in aqueous phase based on the coupling of molecular weight sieve and pH control, and particularly to an aqueous phase membrane separation process using microfiltration, ultrafiltration, and nanofiltration. Technical Background

[0002] Surfactants are compounds that can significantly reduce the surface tension of water even at very low concentrations. In 1958, the Institute of Plant Protection, Chinese Academy of Sciences, successfully developed my country's first surfactant, castor oil polyoxyethylene ether, marking the formation of my country's surfactant industry. Currently, based on their chemical structure, biosurfactants can be classified into the following major categories: lipopeptides, glycolipids, phospholipids, lipoproteins, fatty acids, particulate biosurfactants, neutral lipids, and polymeric biosurfactants.

[0003] Sodium lipopeptide is a cyclic lipopeptide compound produced by Bacillus subtilis through fermentation. Structurally, it consists of a cyclic heptapeptide and a fatty acid chain. Its unique structure endows it with surface activity, making it one of the most potent biosurfactants. It can effectively reduce the interfacial tension between water and air, water and oil, and water and solid phases, lowering the surface tension of water from 72 mN•m. -1 Reduced to 27 mN•m -1 .

[0004] Sodium lipopeptide has broad application prospects in various fields such as agriculture, food, pharmaceuticals, daily chemicals, oil extraction, and bioremediation. Especially in the daily chemicals sector, sodium lipopeptide possesses excellent bioactivity and superior penetration-enhancing effects, significantly promoting the transdermal absorption of active ingredients, making it an ideal raw material for liposome construction. Furthermore, even in trace amounts, sodium lipopeptide exhibits excellent emulsification stability and dispersibility, is extremely low in irritation (similar to water), has high biodegradability, and is environmentally safe. In addition, sodium lipopeptide demonstrates strong antibacterial properties, inhibiting the growth of pathogenic bacteria and fungi such as Propionibacterium acnes, Staphylococcus aureus, Malassezia, and Aspergillus niger, while effectively removing pathogenic biofilms.

[0005] Separation and purification is one of the main production processes for preparing sodium lipopeptides. Currently, common methods for separating and purifying sodium lipopeptides include: acid precipitation, organic solvent extraction, foam separation, chromatography, adsorption, and ultrafiltration.

[0006] When using acid precipitation or salting out for precipitation, impurities such as proteins will precipitate along with the sodium lipopeptide, and many small molecule impurities will also be introduced, resulting in a lower purity product. For example, Chen et al. treated the sterilized culture medium of B. subrili SATCC21332 with 23% (NH4)2SO4, and the purity of Surfactin was only 68%. Therefore, acid precipitation is generally only used as a pretreatment and needs to be combined with a subsequent purification process. This invention utilizes the principle of protein thermal denaturation, heating the fermentation broth before acid precipitation, and using a flocculant to remove most of the protein, providing a higher purity semi-finished product for subsequent production. Furthermore, to obtain higher purity Surfactin, most people might choose to use organic solvents for extraction. For example, the article "Highly Efficient Preparation Method and Biological Activity of Four Lipopeptide Antibiotics: Surfactin, Bacitracin L, Rocromycin and Pantothecin" published by Luo Chuping et al. in the Southwest Agricultural Journal uses methanol to repeatedly extract the fermentation broth and acid precipitation to obtain a solid, which is then filtered to obtain a crude extract of surfactant. Patent CN 117756880 introduces ethanol to extract the crude lipopeptide extract, and obtains solid lipopeptides by drying the ethanol solution containing lipopeptides. The above extraction methods not only consume a large amount of organic solvents, but may also have problems such as solvent residue and poor solubility, which are very unfavorable for the industrial production and subsequent addition of sodium lipopeptides. Moreover, some organic solvents are toxic and will cause environmental pollution.

[0007] In addition, sodium lipopeptide can also be extracted using methods such as foam separation and adsorption. While these methods avoid the problem of residual organic solvents, they result in low extraction yields and low sample purity. For example, in the paper "Bioreactor design for enhanced carrier-assisted Surfactin production with Bacillus subtilis" published in Process Biochemistry by Yeh M et al., the purity of the extracted sodium lipopeptide was only 55% using the foam separation principle, requiring further purification.

[0008] Membrane filtration is a technique that utilizes the physical sieving action of semi-permeable membranes to separate, purify, or concentrate mixtures based on differences in molecular size, shape, or charge. According to pore size and molecular weight cutoff, it can be classified into microfiltration, ultrafiltration, nanofiltration, and reverse osmosis. It can achieve purification while avoiding the introduction of organic solvents or other impurities. Sodium lipopeptides possess strong surface activity and a low critical micelle concentration (CMC). Based on its property of easily aggregating to form larger micelles at concentrations above the CMC, it can be used to remove salts, small protein molecules, free amino acids, and other substances by retaining these micelles through membranes with different size cutoffs.

[0009] In previous studies, many scholars have applied membrane filtration to the extraction and purification of sodium lipopeptide, and have indicated that it can effectively improve the purity of the finished sodium lipopeptide product. For example, in CN103059108A, Sun Wen et al. used a ceramic membrane to filter the supernatant of the fermentation broth after high-speed centrifugation, and then neutralized it with sodium hydroxide solution. After spray drying, the resulting sodium lipopeptide product had a purity of about 76%, which is significantly higher than the 68% purity sample obtained by acid precipitation. In addition, membrane filtration can also use ultrafiltration membranes. For example, Sen et al. wrote in "Characterization of concentration and purification parameters and operating conditions for the small-scale recovery of surfactin" that they used one-step ultrafiltration to separate and purify Surfactin. The purity of the obtained surfactant (70%) was significantly higher than that of the product obtained by solvent extraction and recrystallization (32%). Although it effectively improved the purity of the product, compared with the 94.2% purity Surfactin prepared by Long Xuwei et al. in patent CN 110790826 A, the purity of the product obtained by one-step ultrafiltration is still relatively low, and more purification steps are required.

[0010] To further improve the purity of sodium lipopeptide, researchers have proposed further purification methods based on ultrafiltration. For example, Chen et al., in their paper "Separation of surfactin from fermentation broths by acidprecipitation and two-stage dead-end ultrafiltration processes," used a two-step ultrafiltration method. First, they used a PES 100 membrane to retain most of the surfactant. After adding 33% ethanol to destroy the surfactant micelles, they purified the H-type surfactant again through a PES 100 membrane. The purity of the H-type surfactant reached 85%, and the total recovery rate was 87%, which was significantly higher than the purity of Surfactin obtained by Sen et al.'s one-step ultrafiltration method. However, the ethanol introduced to destroy the micelles not only increases the cost of separation and purification but also hinders the promotion and use of the product in the food, pharmaceutical, and daily chemical industries. Summary of the Invention

[0011] This invention addresses the problems of the aforementioned processes by providing a fully aqueous extraction process for sodium lipopeptide based on molecular weight sieving and pH control. It innovatively constructs an integrated purification system of "molecular morphology and aggregation state changes under pH control - precise separation via multi-stage membranes." This system not only achieves stepwise, targeted removal of impurities but also completely eliminates organic solvents in the fully aqueous extraction process, ensuring no organic solvent residue in the product, making it more environmentally friendly and safer. The final product exists in the form of sodium lipopeptide salt, greatly improving the product's water solubility and stability, and preserving its bioactive structure to the greatest extent possible. This facilitates the subsequent promotion and use of the product in the food, pharmaceutical, and daily chemical industries.

[0012] To achieve the above objectives, a process for the extraction of sodium lipopeptides in aqueous phase based on the coupling of molecular weight sieving and pH control mainly includes the following steps:

[0013] (1) After the fermentation of Bacillus subtilis is completed, the temperature of the fermentation liquid is raised to 50~60℃ and maintained for 5~15 minutes to allow some of the proteins to denature due to heat.

[0014] (2) Add flocculant to co-precipitate denatured proteins and other impurities with bacterial cells; remove protein, bacterial cells and other precipitated impurities from Bacillus subtilis fermentation broth under high-speed centrifugation to obtain a clearer supernatant;

[0015] (3) Acid precipitation of the supernatant obtained in (2);

[0016] (4) Then, the water-soluble impurities are removed by microfiltration to obtain acid-precipitated solids;

[0017] (5) Wash the acid precipitate solid obtained in (4) with the prepared acidic NaCl solution to further remove water-soluble pigments, residual amino acids, polysaccharides and other impurities, which helps to improve the purity of the product.

[0018] (6) Dissolve the acid-precipitated solid washed in (5) with an alkaline aqueous solution to obtain an alkaline solution containing sodium lipopeptide;

[0019] (7) Use a suitable ultrafiltration membrane to filter the alkaline solution containing sodium lipopeptide to further remove macromolecular impurities such as proteins and polysaccharides, thus avoiding the problem of polysaccharide co-precipitation in the traditional extraction process;

[0020] (8) Adjust the solution pH to neutral;

[0021] (9) Select a suitable nanofiltration membrane to concentrate the solution in (8) to a suitable concentration and remove excess small molecules;

[0022] (10) Finally, the nanofiltration concentrate was dried by spray drying to obtain sodium lipopeptide solid. The rapid drying method is beneficial to maintaining the activity and functionality of sodium lipopeptide.

[0023] Furthermore, the flocculant mentioned in step (1) includes chitosan, gelatin, cationic starch, etc.;

[0024] Furthermore, the high-speed centrifugation conditions in step (2) are 8000~11000 rpm for 5~20 min;

[0025] Furthermore, the acid used in step (3) is preferably an inorganic acid such as hydrochloric acid, sulfuric acid, or nitric acid, and the pH is preferably 2 to 4;

[0026] Furthermore, the pore size of the microfiltration membrane used in step (3) is preferably 0.1~0.5 μm;

[0027] Furthermore, the concentration of the NaCl solution in step (4) is 0.01-1M, and the pH is 2-4;

[0028] Furthermore, the alkali used in step (5) is preferably an inorganic alkali such as NaOH or KOH, and the pH is preferably 8 to 11;

[0029] Furthermore, the pore size of the ultrafiltration membrane used in step (6) is preferably 1-100 kDa;

[0030] Furthermore, the reagent used to adjust the pH in step (7) is preferably an inorganic acid such as hydrochloric acid, sulfuric acid, or nitric acid; the nanofiltration membrane pore size is preferably 100~900 Da; and the concentration is preferably 7~20%.

[0031] Furthermore, the preferred conditions for spray drying in step (8) are an inlet air temperature of 150~200℃, an inlet air volume of 30~50Hz, and an injection rate of 8~12mL / min.

[0032] The aqueous phase extraction method for sodium lipopeptides based on different molecular weights and membrane separation described in this invention has the following advantages compared with existing sodium lipopeptide extraction methods:

[0033] (1) This invention denatures the proteins in the fermentation broth by heating and, in conjunction with the use of flocculants, significantly improves the solid-liquid separation effect, resulting in a clearer supernatant. This lays a good foundation for subsequent purification steps and helps to further improve the purity and quality of the final product.

[0034] (2) This invention innovatively adopts an all-water process to replace organic solvents such as dichloromethane and chloroform used in traditional processes, fundamentally eliminating the risk of organic solvent residue. This process is not only safer and more environmentally friendly, but also simplifies subsequent treatment steps, which is in line with the development concept of green chemistry.

[0035] (3) The present invention uses acidic salt water to wash acid precipitation, which can more effectively remove water-soluble pigments, residual sugars, amino acids and some inorganic ions from acid precipitation than traditional pure water washing, providing intermediates with higher purity for subsequent steps.

[0036] (4) The present invention uses an alkaline aqueous solution to extract sodium lipopeptide from acid-precipitated solids, which is beneficial to obtaining samples with better water solubility and is more conducive to the subsequent promotion and use of the product in the fields of food, medicine, and daily chemicals.

[0037] (5) This invention uses multi-stage membranes for precise separation, which achieves step-by-step and targeted removal of impurities. It is simple to operate and helps to improve sample purity.

[0038] (6) The present invention utilizes spray drying to dry nanofiltration concentrate to obtain sodium lipopeptide solid. The extremely rapid drying method is beneficial to maintaining the bioactive structure and functionality of sodium lipopeptide, improving the stability of the product, and facilitating the storage and transportation of the product. Detailed Implementation

[0039] The present invention will be described below through specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0040] Example 1: Preparation of sodium lipopeptide fermentation broth

[0041] Fermentation broth culture medium: glucose (40 g / L), NaNO3 (100 mM), Na2HPO4 (30 mM), KH2PO4 (30 mM), CaCl2 (7 μM), MgSO4•7H2O (800 M), EDTA-Na (4 μM) and FeSO4•7H2O (2.0 mM).

[0042] After Bacillus subtilis was activated and cultured in LB medium for 24 hours, it was inoculated into the fermentation broth medium at a 5% inoculum rate and cultured at 37°C and 600 rpm for 48 hours to obtain a fermentation broth containing sodium lipopeptide.

[0043] Example 2

[0044] Take 500g of the sodium lipopeptide fermentation broth from Example 1, centrifuge at 10000rpm for 5min, and then divide it into 5 equal portions.

[0045] The pH of the fermentation broth was adjusted to 1, 2, 3, 4 and 5 respectively using 3M HCl, and the mass of the acid precipitated solids obtained was weighed.

[0046] The mass of acid-precipitated solids at different pH values ​​is shown in Table 1. As the pH of the acid precipitation decreases, the mass of the acid-precipitated solids increases. When the pH drops below 4, the recovery rate of the acid-precipitated solids reaches over 85%. Considering subsequent processing and costs, the optimal pH for acid precipitation is 2-4.

[0047] Table 1. Recovery rate of acid precipitation solids at different pH values

[0048]

[0049] Example 3

[0050] Take 500g of the sodium lipopeptide fermentation broth from Example 1, centrifuge at 10000rpm for 5min, and then divide it into 5 equal portions.

[0051] Add 0.02%, 0.05%, 0.1%, 0.5%, and 1.0% chitosan by weight of the fermentation broth, respectively, and centrifuge at 10,000 rpm for 5 min.

[0052] The mass of each group of fermentation broth after centrifugation is shown in Table 2 below. As the mass of chitosan increases, more impurities are removed after centrifugation. Considering subsequent processing and cost, the preferred amount of chitosan added is 0.05%~0.5%.

[0053] Table 2. Impurity removal rate of fermentation broth treated with different chitosan addition amounts

[0054]

[0055] Comparative Example 4

[0056] Centrifuge 860g of the sodium lipopeptide fermentation broth from Example 1 at 10000rpm for 5min;

[0057] The pH of the fermentation broth was adjusted to 2.05 using 9.85g of concentrated sulfuric acid, yielding 89.3g of acid-precipitated solid.

[0058] Add 258.78g of deionized water and 8.4g of 20% NaOH aqueous solution, stir for 1-2 hours and then centrifuge at 10000 rpm for 10 minutes.

[0059] The supernatant after centrifugation was spray-dried under the following conditions: inlet air temperature 180℃, inlet air volume 40Hz, and injection rate 10mL / min. The recovery rate of sodium lipopeptide powder was 85%, and the purity was 48.3%.

[0060] Example 5

[0061] 588g of the sodium lipopeptide fermentation broth from Example 1 was heated at 52°C for 15 minutes;

[0062] Then add 0.1% chitosan by weight of the fermentation broth, and centrifuge at 10,000 rpm for 5 min;

[0063] The pH of the sterile supernatant was adjusted to 2.07 with 3M HCl, and the acid precipitated solid was obtained after filtration through a 0.45μm microfiltration membrane.

[0064] The acid-precipitated solid was washed with a 0.1M NaCl solution with pH=2.3. After washing, about 3 times the mass of the solid was added to the acid-precipitated solid, i.e., 100.24g of deionized water. The pH was adjusted using 20% ​​NaOH, with a target pH of about 10. The amount of 20% NaOH aqueous solution used was 3.66g.

[0065] After stirring for 1 hour, the solution was filtered through a 10 kDa ultrafiltration membrane to obtain an aqueous solution containing sodium lipopeptide. The pH of the solution was adjusted to 6.96, and the residual small molecules were removed by 600 Da nanofiltration to bring the solution concentration to 9.45%.

[0066] Subsequently, spray drying (inlet air temperature 160℃, inlet air volume 30Hz, injection rate 10mL / min) was performed to obtain powdered sodium lipopeptide. The recovery rate of powdered sodium lipopeptide was 90.54%, and the purity was 96.10%.

[0067] Example 6

[0068] The lipopeptide sodium fermentation broth of Example 1, weighing 756.31g, was heated at 58°C for 10 min, and 0.1% of the fermentation broth mass of chitosan was added. After standing and settling, the supernatant was obtained by high-speed centrifugation.

[0069] After sterile supernatant was added, 92.97 g of 3M HCl solution was added to adjust the pH to 2.65. After filtration through a 0.45 μm microfiltration membrane, 45.33 g of acid-precipitated solid was obtained.

[0070] The acid-precipitated solid was washed with 5 times its mass of acidic NaCl solution, followed by the addition of 123.48 g of water and 4.8 g of 20% NaOH aqueous solution.

[0071] After stirring, an aqueous solution containing sodium lipopeptide was obtained by separation using a 1 kDa ultrafiltration membrane. The pH of the solution was adjusted to 7.04, and the solution concentration was concentrated to 8.06% using an 800 Da nanofiltration membrane to remove residual small molecules.

[0072] Subsequently, the spray drying conditions were set as follows: inlet air temperature 180℃, inlet air volume 40Hz, and injection speed 10mL / min. Powdered sodium lipopeptide was obtained by spray drying with a yield of 92.52% and a sample purity of 95.39%.

[0073] The technical solutions of this invention are not limited to the specific embodiments described above. Any technical modifications made according to this invention, such as using equivalent substitutions, fall within the protection scope of this invention.

Claims

1. A process for the whole-aqueous phase extraction of sodium lipopeptides based on molecular weight sieving and pH control coupling, characterized in that, Includes the following steps: (1) After the fermentation of Bacillus subtilis is completed, the temperature of the fermentation broth is raised to 50~60℃ and maintained for 5~15 minutes; (2) Add flocculant and then obtain a clearer supernatant under high-speed centrifugation conditions; (3) Acid precipitation of the supernatant obtained in (2); (4) Use microfiltration membrane to remove water-soluble impurities and obtain acid-precipitated solids; (5) Wash the acid precipitate obtained in (4) with the prepared acidic NaCl solution; (6) Dissolve the acid-precipitated solid washed in (5) with an alkaline aqueous solution to obtain an alkaline solution containing sodium lipopeptide; (7) Filter the alkaline solution containing sodium lipopeptide using a suitable ultrafiltration membrane; (8) Adjust the solution pH to neutral; (9) Select a suitable nanofiltration membrane to concentrate the solution in (8) to a suitable concentration and remove excess small molecules; (10) The nanofiltration concentrate was dried by spray drying to obtain sodium lipopeptide solid.

2. The extraction process according to claim 1, characterized in that, The flocculant mentioned in step (2) includes chitosan, gelatin, and cationic starch; the high-speed centrifugation conditions mentioned in step (2) are preferably 8000~11000 rpm for 5~20 min.

3. The extraction process according to claim 1, characterized in that, The acid precipitation in step (3) is carried out using sulfuric acid, hydrochloric acid, and nitric acid.

4. The extraction process according to claim 1, characterized in that, The pH of the acid precipitation in step (3) is 2~4.

5. The extraction process according to claim 1, characterized in that, In step (4), the pore size of the microfiltration membrane is 0.1~0.5 μm.

6. The extraction process according to claim 1, characterized in that, The concentration of the NaCl solution mentioned in step (5) is 0.01-1M and the pH is 2-4.

7. The extraction process according to claim 1, characterized in that, The alkaline aqueous solution in step (6) is sodium hydroxide or potassium hydroxide; the pH in step (6) is 8~11.

8. The extraction process according to claim 1, characterized in that, The ultrafiltration membrane in step (7) has a pore size of 1-100 kDa.

9. The extraction process according to claim 1, characterized in that, The pH adjustment in step (8) uses sulfuric acid, hydrochloric acid, or nitric acid; the nanofiltration membrane in step (9) has a pore size of 100 Da to 900 Da; the concentration range after concentration in step (9) is 7 to 20%, more preferably, the concentration range after concentration is 9 to 12%.

10. The extraction process according to claim 1, characterized in that, The spray drying conditions in step (10) are: inlet air temperature of 150~200℃, inlet air volume of 30~50Hz, and injection speed of 8~12mL / min.

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