A method for separating and purifying farnesene and squalene biosynthesized by fermentation

Abstract

The present application belongs to the technical field of microbial fermentation. More particularly, it relates to a method for separating and purifying farnesene and squalene biosynthesized by fermentation. The specific separation steps of the present application include: performing solid-liquid separation on a microbial fermentation system containing farnesene and / or squalene, collecting filtrate containing farnesene and filter cake containing squalene; performing enzymatic wall-breaking treatment on the filter cake containing squalene to release intracellular target products, and collecting the post-wall-breaking feed liquid; performing solid-liquid separation on the feed liquid to remove residual bacteria, and collecting the filtrate; mixing the filtrate containing farnesene and the filtrate containing squalene with an extraction system containing a non-ionic surfactant respectively, and then performing supercritical carbon dioxide extraction to obtain extracts rich in farnesene and squalene respectively; and performing chromatographic separation on the extracts rich in farnesene and squalene respectively to obtain farnesene and squalene respectively.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology. More specifically, it relates to a method for separating and purifying farnesene and squalene produced by biosynthetic fermentation. Background Technology

[0002] Squalene is a straight-chain unsaturated olefin formed by six isoprene units linked end-to-end, with the molecular formula C30H50. It is named squalene because it was originally derived from shark liver. Squalene is an important core ingredient in vaccine adjuvants and is widely used in the formulation of various vaccines. These adjuvants can significantly enhance the immune response to vaccines and improve vaccine efficacy.

[0003] In recent years, with the rapid development of synthetic biology and metabolic engineering technologies, the biosynthesis of squalene using microbial cell factories has become a research hotspot. By constructing engineered bacteria with efficient synthetic pathways, especially modifying eukaryotic microorganisms such as yeast, heterologous synthesis of squalene and its precursors such as farnesene can be achieved. Farnesene, as a key synthetic precursor of squalene, also has significant industrial and pharmaceutical value. However, in microbial fermentation production, squalene and farnesene often coexist in complex fermentation systems. Given their similar structures and physicochemical properties, the efficient separation and purification of high-purity squalene and farnesene from the fermentation broth remains a key technological bottleneck restricting their industrial application.

[0004] Currently, traditional separation and purification methods, such as solvent extraction, distillation, and crystallization, generally suffer from problems such as lengthy steps, significant product loss, large solvent consumption, heavy environmental pollution, and difficulty in achieving highly selective separation of squalene and farnesene. Therefore, developing an efficient, green, and highly selective method for the separation and purification of biosynthesized squalene and farnesene is of great significance for promoting their industrial production and application. Summary of the Invention

[0005] The technical problem this invention aims to solve is the difficulty in efficiently separating and purifying farnesene and squalene during existing bio-fermentation processes for their production. To address this challenge, this invention provides a method for separating and purifying farnesene and squalene produced through biosynthetic fermentation.

[0006] The purpose of this invention is to provide a method for separating and purifying farnesene and squalene produced by biosynthetic fermentation.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: A method for separating and purifying farnesene and squalene produced by biosynthetic fermentation, the specific separation steps of which include: (a) Perform solid-liquid separation on a microbial fermentation system containing farnesene and / or squalene, and collect the filtrate containing farnesene and the cell filter cake containing squalene. (b) Enzymatically break down the cell wall of the bacterial filter cake containing squalene to release the target intracellular product and collect the liquid after cell wall disruption. (c) Perform solid-liquid separation on the liquid to remove residual bacteria and collect the filtrate containing squalene; (d) The filtrate containing farnesene and the filtrate containing squalene were respectively mixed with an extraction system containing a nonionic surfactant, and then extracted with supercritical carbon dioxide to obtain extracts rich in farnesene and squalene, respectively. (e) The extracts, which are respectively rich in farnesene and squalene, are subjected to chromatographic separation to obtain farnesene and squalene, respectively.

[0008] The beneficial effects of the above technical solution are as follows: Since farnesene and squalene are both lipid-soluble and non-volatile terpenoids, traditional solvent extraction methods are inefficient and prone to solvent residue. This invention, for the first time, combines a complex enzymatic hydrolysis system, a natural nonionic surfactant, and a polar gradient auxiliaries in a supercritical CO2 extraction process, producing a significant synergistic effect: using supercritical carbon dioxide extraction for separation and purification allows for the extraction of the aforementioned lipid-soluble substances by leveraging the high diffusivity, low viscosity, and controllable solubility of carbon dioxide in the supercritical state. However, since the target substances in this technology originate from biosynthetic fermentation, cell residues are easily left in the extract. Adding nonionic surfactants allows them to insert into the phospholipid bilayer of the cell membrane or the β-glucan structure of the cell wall, causing structural loosening and disruption. Combined with the swelling effect of supercritical carbon dioxide, this accelerates the rupture of cell residues, releasing internal lipid-soluble products. Ultimately, this significantly reduces the surface tension of the carbon dioxide-water-cell residue multiphase system, accelerates mass transfer during extraction, and improves extraction efficiency.

[0009] Furthermore, the nonionic surfactant is selected from any one or a combination of several of the following: tea saponin, alkyl glycosides, sophorolipids, rhamnolipids, polyglycerol fatty acid esters, and lecithin.

[0010] Furthermore, the amount of nonionic surfactant added is 0.5-1.0% of the mass of the filtrate containing farnesene or 0.5-1.0% of the mass of the precipitate after cell wall disruption.

[0011] Furthermore, the specific separation steps also include: The filtrate containing farnesene and the filtrate containing squalene were respectively mixed with an extraction system containing a nonionic surfactant, and then extracted with supercritical carbon dioxide to obtain extracts rich in farnesene and squalene, respectively. The extraction system also includes an auxiliary agent with a carbon dioxide volume flow rate of 6-8%. The additive is a compound of ethylene glycol and glycerin in a mass ratio of 3:1 to 4:1.

[0012] The beneficial effects of the above technical solution include: During the research and development process, the inventors further discovered that, in addition to the target product, the fermentation broth also contains other impurities such as lipids, pigments, or cell debris, which affect the purification efficiency and purity. Supercritical carbon dioxide has low polarity and insufficient solubility for oxygen-containing or slightly polar components. By adding the aforementioned proportionally compounded adjuvants, a polarity gradient system is formed. Ethylene glycol has moderate polarity, which can enhance the solubility of oxygen-containing intermediates and trace polar impurities, while glycerol has high polarity and can synergistically enhance the penetration and emulsification of impurities such as cell membrane debris with nonionic surfactants. A reasonable addition range can significantly improve the extraction efficiency while avoiding excessive addition that could lead to unstable carbon dioxide system pressure or difficulties in subsequent separation.

[0013] Furthermore, the extraction conditions for the supercritical carbon dioxide extraction are: pressure 15-30 MPa, temperature 35-60℃, and extraction time 1-4h.

[0014] Furthermore, the chromatographic separation is performed using silica gel column chromatography, and during the chromatographic separation process, a mixed solution of n-hexane and ethyl acetate is used for elution; The mixed solvent of n-hexane and ethyl acetate is prepared by mixing the two in a volume ratio of 1:1.

[0015] Furthermore, the specific separation steps also include: The extracts, which are respectively rich in farnesene and squalene, were subjected to chromatographic separation to obtain farnesene and squalene, respectively. Subsequently, supercritical carbon dioxide recrystallization was used for purification. The conditions for supercritical carbon dioxide recrystallization are: pressure of 8-15 MPa and temperature of 30-40℃.

[0016] Furthermore, The enzymatic hydrolysis and cell wall disruption treatment includes: adding 0.3-0.5% of the fermentation broth's mass of biological enzyme to the fermentation broth, and performing enzymatic hydrolysis and cell wall disruption treatment for 6-8 hours at a temperature of 30-35℃ and a stirring speed of 200-220 r / min.

[0017] Furthermore, the bio-enzyme is a mixture of cellulase and β-glucanase in a mass ratio of 1:1. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0019] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0020] Example 1 We provide microbial fermentation broth containing farnesene and squalene, specifically: The key indicators of the relevant microbial fermentation broth are: cell dry weight 40 g / L, squalene content 1.5 g / L, farnesene content 3.0 g / L, and main impurities: protein 3 g / L and polysaccharide 8 g / L. First solid-liquid separation: The microbial fermentation broth containing farnesene and squalene was filtered, and the filtrate containing farnesene and the bacterial filter cake containing squalene were collected. Enzymatic hydrolysis and cell wall disruption treatment: The bacterial filter cake was dispersed in water to prepare a diluted solution with a concentration of 40 g / L; Add 0.3% (by weight) of the fermentation broth bio-enzyme to the diluted solution, and perform enzymatic hydrolysis and cell wall disruption treatment for 6 hours at a temperature of 30℃, a pH of 5.0, and a stirring speed of 200 r / min to obtain the enzymatic hydrolysate. The bio-enzyme is composed of cellulase and β-glucanase in a mass ratio of 1:1. Secondary solid-liquid separation: After centrifuging the enzymatic fermentation broth at a low speed of 2000 r / min for 10 min to remove large cell residues, it was then centrifuged at a high speed of 8000 r / min for 20 min, and the supernatant was collected. Add 0.08% (by weight) of chitosan to the supernatant, stir, and let stand for 2 hours to promote sedimentation of impurities. Remove the sedimented impurities and collect the filtrate rich in squalene. Supercritical carbon dioxide extraction: The nonionic surfactant was dissolved in deionized water to prepare a stock solution with a mass fraction of 5%. Then, the squalene-rich filtrate and the stock solution were mixed in a container at 30°C and 200 r / min for 20 min to form a homogeneous emulsion. The stock solution was added at a surfactant concentration of 0.5% of the filtrate mass. Prepare another container and dissolve the nonionic surfactant in deionized water to prepare a stock solution with a mass fraction of 5%. Then, mix the filtrate rich in farnesene and the stock solution in the container at 30°C and 200 r / min for 20 min to form a homogeneous emulsion. The stock solution is added at a surfactant concentration of 0.5% of the filtrate mass. The nonionic surfactant is selected from tea saponin; Then, an auxiliary agent is added to the emulsion at a rate of 6% of the supercritical carbon dioxide volumetric flow rate. The auxiliary agent is a compound of ethylene glycol and glycerin in a mass ratio of 3:1. After the additives were added, they were transferred to the extraction vessel, with a filling rate of 70% of the extraction vessel volume. The vessel was first heated to 35°C, and then pressurized to 15 MPa. Then, dynamic extraction was performed for 1 hour at a carbon dioxide flow rate of 8 L / h. After extraction, the carbon dioxide containing the product was passed through the separation vessel, and the product was precipitated by reducing the pressure to 5 MPa and raising the temperature to 40°C, so that extracts rich in farnesene and squalene were obtained in the two extraction vessels respectively. Chromatographic separation: Chromatography was performed using a 300-mesh silica gel column with a silica gel mass 30 times the mass of the sample extract and a column height ratio of 1:10. After sample loading, elution was performed using eluent at a flow rate of 1 mL / min, and farnesene and squalene fractions were obtained. Recrystallization: The farnesene or squalene fractions obtained above were placed in crystallization vessels, with the filling amount not exceeding 50% of the solvent in the crystallization vessel. Carbon dioxide was then introduced until the pressure inside the vessel reached 8 MPa. The temperature was then raised to 30°C and maintained for 30 minutes to allow the system to reach equilibrium. The pressure was then adjusted to 10 MPa, and the temperature was 35°C (squalene) or 30°C (farnesene). The carbon dioxide flow rate was controlled at 5 L / h, and the circulation was continued for 2 hours. After recrystallization, the pressure was slowly reduced to atmospheric pressure and the temperature was lowered to 20°C. The crystallization vessel was then opened, and the precipitated crystals or high-purity oily products were collected.

[0021] Example 2 We provide microbial fermentation broth containing farnesene and squalene, specifically: The key indicators of the relevant microbial fermentation broth are: cell dry weight 50 g / L, squalene content 2 g / L, farnesene content 3.2 g / L, and main impurities: protein 4 g / L and polysaccharide 9 g / L. First solid-liquid separation: The microbial fermentation broth containing farnesene and squalene was filtered, and the filtrate containing farnesene and the bacterial filter cake containing squalene were collected. Enzymatic hydrolysis and cell wall disruption treatment: The bacterial filter cake was dispersed in water to prepare a diluted solution with a concentration of 40 g / L; Enzymatic hydrolysis and cell wall disruption treatment: Add 0.4% (by weight) of the fermentation broth bio-enzyme to the diluted solution, and perform enzymatic hydrolysis and cell wall disruption treatment for 7 hours at a temperature of 32℃, a pH of 5.4, and a stirring speed of 210 r / min to obtain the enzymatic hydrolysate. The bio-enzyme is composed of cellulase and β-glucanase in a mass ratio of 1:1. Secondary solid-liquid separation: The enzymatic fermentation broth was centrifuged at a low speed of 3000 r / min for 11 min to remove large cell residues. Then, it was centrifuged at a high speed of 8600 r / min for 25 min, and the supernatant was collected. Add 0.09% (by weight of the supernatant) of chitosan to the supernatant, stir, and let stand for 2.3 hours to promote sedimentation of impurities. Remove the sedimented impurities and collect the filtrate rich in squalene. Supercritical carbon dioxide extraction: The nonionic surfactant was dissolved in deionized water to prepare a stock solution with a mass fraction of 8%. Then, the squalene-rich filtrate and the stock solution were mixed in a container at 35°C and 250 r / min for 25 min to form a homogeneous emulsion. The stock solution was added at a surfactant concentration of 0.8% of the filtrate mass. Prepare another container and dissolve the nonionic surfactant in deionized water to prepare a stock solution with a mass fraction of 8%. Then, mix the filtrate rich in farnesene and the stock solution in the container at 35°C and 250 r / min for 25 min to form a homogeneous emulsion. The stock solution is added at a surfactant concentration of 0.8% of the filtrate mass. The nonionic surfactant is selected from alkyl glycosides; Then, an auxiliary agent is added to the emulsion at a rate of 7% of the supercritical carbon dioxide volumetric flow rate. The auxiliary agent is a compound of ethylene glycol and glycerin in a mass ratio of 3:1. After the additives were added, they were transferred to the extraction vessel, with a filling rate of 76% of the extraction vessel volume. The vessel was first heated to 40°C, and then pressurized to 20 MPa. Then, dynamic extraction was performed for 3 hours at a carbon dioxide flow rate of 9 L / h. After extraction, the carbon dioxide containing the product was passed through the separation vessel, and the product was precipitated by reducing the pressure to 6 MPa and raising the temperature to 45°C, so that extracts rich in farnesene and squalene were obtained in the two extraction vessels respectively. Chromatographic separation: Chromatography was performed using a 300-mesh silica gel column with a silica gel mass 35 times the mass of the sample extract and a column height ratio of 1:12. After sample loading, elution was performed using eluent at a flow rate of 2 mL / min, and farnesene and squalene fractions were obtained. Recrystallization: The farnesene or squalene fractions obtained above were placed in crystallization vessels, with the filling amount not exceeding 50% of the solvent in the crystallization vessel. Carbon dioxide was then introduced until the pressure inside the vessel reached 12 MPa. The temperature was then raised to 35°C and maintained for 30 minutes to allow the system to reach equilibrium. The pressure was then adjusted to 11 MPa, and the temperature was maintained at 35°C (squalene) or 30°C (farnesene). The carbon dioxide flow rate was controlled at 8 L / h, and the circulation was continued for 3 hours. After recrystallization, the pressure was slowly reduced to atmospheric pressure and the temperature was lowered to 20°C. The crystallization vessel was then opened, and the precipitated crystals or high-purity oily products were collected.

[0022] Example 3 We provide microbial fermentation broth containing farnesene and squalene, specifically: The key indicators of the relevant microbial fermentation broth are: cell dry weight 60g / L, squalene content 2.5g / L, farnesene content 3.5g / L, and main impurities: protein 5g / L and polysaccharide 10g / L. First solid-liquid separation: The microbial fermentation broth containing farnesene and squalene was filtered, and the filtrate containing farnesene and the bacterial filter cake containing squalene were collected. Enzymatic hydrolysis and cell wall disruption treatment: The bacterial filter cake was dispersed in water to prepare a diluted solution with a concentration of 40 g / L; Enzymatic hydrolysis and cell wall disruption treatment: Add 0.5% (by weight) of the fermentation broth bio-enzyme to the diluted solution, and perform enzymatic hydrolysis and cell wall disruption treatment for 8 hours at a temperature of 35℃, a pH of 6.0, and a stirring speed of 220 r / min to obtain the enzymatic hydrolysate. The bio-enzyme is composed of cellulase and β-glucanase in a mass ratio of 1:1. Secondary solid-liquid separation: After centrifuging the enzymatic fermentation broth at a low speed of 4000 r / min for 12 min to remove large cell debris, centrifuge it at a high speed of 9000 r / min for 30 min and collect the supernatant. Add 0.1% (by weight) of chitosan to the supernatant, stir, and let stand for 3 hours to promote sedimentation of impurities. Remove the sedimented impurities and collect the filtrate rich in squalene. Supercritical carbon dioxide extraction: The nonionic surfactant was dissolved in deionized water to prepare a stock solution with a mass fraction of 10%. Then, the squalene-rich filtrate and the stock solution were mixed in a container at 40°C and 300 r / min for 30 min to form a homogeneous emulsion. The stock solution was added at a surfactant concentration of 1.0% of the filtrate mass. Prepare another container and dissolve the nonionic surfactant in deionized water to prepare a stock solution with a mass fraction of 5%. Then, mix the filtrate rich in farnesene and the stock solution in the container at 40°C and 300 r / min for 30 min to form a homogeneous emulsion. The stock solution is added at a surfactant concentration of 1.0% of the filtrate mass. The nonionic surfactant is selected from sophorolipids; Then, an auxiliary agent is added to the emulsion at an amount equal to 8% of the supercritical carbon dioxide volumetric flow rate. The auxiliary agent is a compound of ethylene glycol and glycerin in a mass ratio of 4:1. After the additives were added, they were transferred to the extraction vessel, with a filling rate of 80% of the extraction vessel volume. The vessel was first heated to 60°C, and then pressurized to 30 MPa. Subsequently, dynamic extraction was performed for 4 hours at a carbon dioxide flow rate of 10 L / h. After extraction, the carbon dioxide containing the product was passed through the separation vessel, and the product was precipitated by reducing the pressure to 8 MPa and the temperature to 50°C, so that extracts rich in farnesene and squalene were obtained in the two extraction vessels respectively. Chromatographic separation: Chromatography was performed using a 400-mesh silica gel column with a silica gel mass 40 times the mass of the sample extract and a column bed height ratio of 1:15. After sample loading, elution was performed using eluent at a flow rate of 3 mL / min, and farnesene and squalene fractions were obtained. Recrystallization: The farnesene or squalene fractions obtained above were placed in crystallization vessels, with the filling amount not exceeding 50% of the solvent in the crystallization vessel. Carbon dioxide was then introduced until the pressure inside the vessel reached 15 MPa. The temperature was then raised to 40°C and maintained for 30 minutes to allow the system to reach equilibrium. The pressure was then adjusted to 12 MPa, and the temperature was set at 35°C (squalene) or 30°C (farnesene). The carbon dioxide flow rate was controlled at 10 L / h, and the circulation was continued for 4 hours. After recrystallization, the pressure was slowly reduced to atmospheric pressure and the temperature was lowered to 20°C. The crystallization vessel was then opened, and the precipitated crystals or high-purity oily products were collected.

[0023] Example 4 The difference between this embodiment and Embodiment 1 is that no additives were added, while all other conditions remained unchanged.

[0024] Example 5 The difference between this embodiment and Embodiment 1 is that an equal volume of anhydrous ethanol is used to replace the auxiliary agent, while the other conditions remain unchanged.

[0025] Example 6 The difference between this embodiment and Embodiment 1 is that glycerol was not added, while all other conditions remained unchanged.

[0026] The purity and recovery rate of the products obtained by the separation and purification methods of the above different embodiments were evaluated. The specific evaluation methods and results are as follows: purity: The farnesene and squalene products obtained in the above examples were dissolved in acetonitrile to prepare test samples with a concentration of 1 mg / mL. An Agilent ZORBAX C18 column was used with acetonitrile-isopropanol as the mobile phase, gradient elution was performed at a flow rate of 1 mL / min and a column temperature of 30 °C. Using squalene (purity ≥99%) and farnesene (purity ≥99%) as controls, the product purity was calculated using the area normalization method. The detailed calculation results are shown in Table 1. Recovery rate: Recovery rate = (final product mass × purity / target substance mass in initial fermentation broth) × 100%, and the specific calculation results are shown in Table 1; Table 1: Results of Purity and Recovery Assessment As shown in Table 1, the separation and purification steps adopted in this invention can effectively extract squalene and farnesene from the fermentation broth, and the product has high purity and considerable recovery rate.

[0027] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for separating and purifying farnesene and squalene produced by biosynthetic fermentation, characterized in that, The specific separation steps include: (a) Solid-liquid separation was performed on a microbial fermentation system containing farnesene and squalene, and the filtrate containing farnesene and the cell filter cake containing squalene were collected. (b) Enzymatically break down the cell wall of the bacterial filter cake containing squalene to release the target intracellular product and collect the liquid after cell wall disruption. (c) Perform solid-liquid separation on the liquid to remove residual bacteria and collect the filtrate containing squalene; (d) The filtrate containing farnesene and the filtrate containing squalene were mixed with an extraction system containing a nonionic surfactant, and then extracted with supercritical carbon dioxide to obtain extracts rich in farnesene and squalene, respectively. The nonionic surfactant is selected from any one or a combination of several of the following: tea saponin, alkyl glycosides, sophorolipids, and polyglycerol fatty acid esters; (e) The extracts, which are respectively rich in farnesene and squalene, are subjected to chromatographic separation to obtain farnesene and squalene, respectively; The specific separation steps also include: The filtrate containing farnesene and the filtrate containing squalene were respectively mixed with an extraction system containing a nonionic surfactant, and then extracted with supercritical carbon dioxide to obtain extracts rich in farnesene and squalene, respectively. The extraction system also includes an auxiliary agent with a carbon dioxide volume flow rate of 6-8%. The additive is a compound of ethylene glycol and glycerin in a mass ratio of 3:1 to 4:

1.

2. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 1, characterized in that, The amount of nonionic surfactant added is 0.5-1.0% of the mass of the filtrate containing farnesene or 0.5-1.0% of the mass of the precipitate after cell wall disruption.

3. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 1, characterized in that, The extraction conditions for supercritical carbon dioxide extraction are: pressure 15-30 MPa, temperature 35-60℃, and extraction time 1-4 h.

4. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 1, characterized in that, The chromatographic separation is performed using silica gel column chromatography, and during the chromatographic separation process, a mixed solution of n-hexane and ethyl acetate is used for elution. The mixed solvent of n-hexane and ethyl acetate is prepared by mixing the two in a volume ratio of 1:

1.

5. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 1, characterized in that, The specific separation steps also include: The extracts, which are respectively rich in farnesene and squalene, were subjected to chromatographic separation to obtain farnesene and squalene, respectively. Subsequently, supercritical carbon dioxide recrystallization was used for purification. The conditions for supercritical carbon dioxide recrystallization are: pressure of 8-15 MPa and temperature of 30-40℃.

6. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 1, characterized in that, The enzymatic hydrolysis and cell wall disruption treatment includes: adding 0.3-0.5% of the fermentation broth's mass of biological enzyme to the fermentation broth, and performing enzymatic hydrolysis and cell wall disruption treatment for 6-8 hours at a temperature of 30-35℃ and a stirring speed of 200-220 r / min.

7. The method for separating and purifying farnesene and squalene produced by biosynthetic fermentation according to claim 6, characterized in that, The bio-enzyme is a mixture of cellulase and β-glucanase in a mass ratio of 1:1.

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