Continuous extraction process of phytosterol oil-based phase fermentation liquor

Abstract

The invention relates to a phytosterol oil-based phase fermentation broth continuous extraction process, which comprises: carrying out partial dehydration on a fermentation broth to obtain a dehydrated fermentation broth; adding an extracting agent into the dehydrated fermentation liquor in batches, mixing, uniformly mixing the first batch of extracting agent with the dehydrated fermentation liquor, continuously discharging into a membrane separation device, and carrying out continuous cross-flow extraction and membrane separation to obtain extract liquor; in an extraction system obtained by mixing the dehydrated fermentation liquor and the extraction agent, the mass ratio of a water base to an oil phase is (5-15): (13-16); the extraction agent comprises organic alcohol; and carrying out decoloration treatment and evaporative crystallization on the extract liquor to obtain a phytosterol fermentation product. According to the method, through continuous cross-flow extraction and separation and by controlling the mass ratio of the water base to the oil phase in the extraction system, full contact and dynamic extraction of the extraction agent and the fermentation liquid are achieved, the final fermentation product is high in extraction yield and purity, small in extraction agent dosage and low in cost, and the production safety is improved.

Description

Technical Field

[0001] This invention relates to the field of steroidal fermentation extraction, specifically to a continuous extraction process for phytosterol oil-based fermentation broth. Background Technology

[0002] In modern biopharmaceutical processes, 4-hydroxyandrostenedione (4-AD) and 9-hydroxyandrostenedione (9-AD) are novel basic active pharmaceutical ingredients (APIs). These are produced through a bio-fermentation process using phytosterols, followed by extraction from the oil phase using organic solvents and multiple refining processes. In the phytosterol fermentation process, most steps utilize vegetable oil as a carrier (oil-to-water emulsion = 16%:84%). After fermentation, the product dissolves in the oil phase. The extraction process then uses solvents such as acetone and ethyl acetate as high-concentration solvents for extraction. The extract is further refined through distillation and crystallization.

[0003] In the extraction process, the fermentation broth is first repeatedly extracted with a solvent of at least 10 BV. During extraction, after the fermentation product enters the extractant phase, the solid residue, oil-based, water-based, and solvent phases are allowed to stand for a long time to separate into layers. The separated extract, oil-based, water-based, and solid bacterial residue are then discharged separately. The resulting extract is then sent to a crystallization tank for evaporation and crystallization. The solvent is recovered to obtain a crude product. The crude product undergoes 3-4 refining processes, including decolorization / adsorption, decolorization / dehydrogenation, and solvent evaporation, to obtain a refined product. The extract residue obtained from the standing separation is then filtered to obtain a solid residue phase and an emulsified oil phase. The emulsified oil phase is treated with ethyl acetate and then allowed to stand again to separate the oil and water. The separated water is discharged after meeting standards or after further treatment. The obtained oil-based phase is either reused or discarded. This traditional extraction method has the following main drawbacks: (1) Traditional extraction processes typically require 3 to 4 extractions of a batch of fermentation products to achieve relatively complete extraction. Furthermore, the extractant requires a large volume, with the fermentation broth and extractant generally mixed at a volume ratio of 1:10. The concentration of fermentation products is high during the first and second extractions. In traditional processes, only the fermentation products at this concentration are clarified and separated before proceeding to subsequent processes. The fermentation product content is relatively low in the third and fourth extractions. After settling and separation, the extracts are temporarily stored in a storage tank and mixed with the next batch of fermentation broth for further extraction. This cyclical extraction improves extractant utilization efficiency. Besides lower returns, this extraction process easily leads to batch-to-batch variations, making it difficult to control the quality of the fermentation products (each extraction involves a mixture of two batches of fermentation products). Simultaneously, subsequent impurity removal and purification processes are time-consuming and complex, resulting in significant fermentation product loss. At least 40 times the volume of extractant is required for a single complete extraction of a batch of fermentation broth, leading to a large amount of extraction equipment, high investment costs, and high solvent recovery costs.

[0004] (2) Extraction of fermentation products requires standing and separation, which takes a long time (6-8h). After separation, the clear liquid, oil base and residue phase are not completely separated, and there are high levels of impurities. Multiple refining processes are required. In addition to the product, the remaining crude mother liquor contains oil, residue and other difficult-to-treat wastes, which reduces the yield of fermentation products and makes the mother liquor treatment difficult. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a continuous extraction process for phytosterol oil-based fermentation broth, solving the technical problems of large amount of extractant and high cost required for the extraction of phytosterol oil-based fermentation broth in the prior art.

[0006] To achieve the above-mentioned technical objectives, the technical solution provided by this invention is as follows: In a first aspect, the present invention provides a continuous extraction process for phytosterol oil-based fermentation broth, comprising the following steps: S1, the fermentation broth is partially dehydrated to obtain a dehydrated fermentation broth; S2, an extractant is added to the dehydrated fermentation broth in batches and mixed, and after the first batch of extractant is evenly mixed with the dehydrated fermentation broth, it is continuously discharged into a membrane separation device for continuous cross-flow extraction and membrane separation to obtain an extract; in the extraction system obtained by mixing the dehydrated fermentation broth and the extractant, the mass ratio of the water-based phase to the oil phase is (5-15):(13-16); the extractant includes organic alcohols; S3, the extract is decolorized and evaporated to crystallize to obtain phytosterol fermentation products.

[0007] Compared with the prior art, the beneficial effects of the present invention include: This invention relates to continuous cross-flow extraction and separation of phytosterol oil-based fermentation broth. By controlling the mass ratio of the water-based to the oil phase in the extraction system, it achieves full contact and dynamic extraction between the extractant and the fermentation broth. During extraction, the fermentation products move from the oil-based phase into the extractant phase. The positions originally occupied by the fermentation products in the oil-based phase create a "cavitation effect," which is rapidly occupied and coupled by an appropriate amount of water-based phase to form large molecular charged groups in "water-in-oil" emulsions. These groups are compatible with other impurities, resulting in larger particle sizes, while the extract molecules are smaller, allowing for rapid passage through a membrane separation device. This enables continuous and rapid separation of the extract and residual substances. The separated extract has a high content of fermentation products, simplifies post-processing, and ultimately achieves high extraction yield and high purity of the fermentation products. This invention allows for continuous extraction with low extractant usage, effectively reducing raw material costs and equipment footprint, and improving production safety. Attached Figure Description

[0008] Figure 1 This is a schematic diagram of the existing phytosterol fermentation broth extraction process; Figure 2 This is a schematic diagram of the continuous extraction process of the phytosterol oil-based fermentation broth according to the present invention. Detailed Implementation

[0009] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0010] In existing technologies, the extraction of phytosterol oil-based fermentation broth, such as Figure 1 As shown, the extraction process typically includes steps such as clarification, layering, redissolution, re-extraction, re-layering, and redistillation. These steps remove impurities (such as fermentation oil base, biological protein, bacterial residue, heterocyclic byproducts, pigments, etc.) during multiple extraction processes to obtain a high-purity product. However, traditional processes are mainly discontinuous. A single extraction typically requires 10-12 times the volume of the extractant in the fermentation broth, and sometimes even 40 times the volume. This results in a large amount of extractant required, leading to extensive equipment usage, high investment costs, and significant solvent recovery costs. Furthermore, the extraction of fermentation products requires settling and separation, which takes a long time (6-8 hours). After separation, the clear liquid, oil base, and residue phases are not completely separated, resulting in high levels of impurities and requiring multiple refining processes. The refining process is time-consuming, requires complex equipment, and involves a lengthy process route. Mother liquor is generated during refining, reducing the yield of fermentation products and making mother liquor treatment difficult. The oil base has a high impurity content, with the bacterial residue and oil base mixed, making complete separation or treatment difficult. The process has a short application cycle and high oil base costs. The degree of automation is low, the investment in process equipment is large, and quality control is difficult.

[0011] Meanwhile, in traditional extraction processes, it is generally believed that the lower the degree of oil-water emulsification in the fermentation broth, the better the extraction process will be. In other words, the lower the water content, the better the extraction process will be. Otherwise, oil-water emulsification is likely to occur during the extraction process, which will greatly affect the extraction efficiency. The "miscibility" between the oil-water phase and the solid phase is difficult to separate, resulting in a large amount of entrainment and residual mother liquor. Multiple extractions are required to improve the recovery efficiency of fermentation products. Due to the multiple extraction steps, fermentation products are easily lost. In addition, due to oil-water emulsification and charge effect, the oil-based phase has a high degree of impurity fragments, and the solid phase encapsulates and entrains fermentation products, which also easily leads to the loss of fermentation products.

[0012] The applicant unexpectedly discovered that using organic alcohols as extractants while retaining a certain amount of water in the fermentation broth, so that there is a suitable amount of water during the mixed extraction process, is beneficial for the continuous and stable extraction and separation.

[0013] Based on this, the present invention is established.

[0014] Firstly, see [the following] Figure 2 This invention provides a continuous extraction process for phytosterol oil-based fermentation broth, comprising the following steps: S1, after partial dehydration of the fermentation broth, dehydrated fermentation broth is obtained; S2, the extractant is added to the dehydrated fermentation broth in batches and mixed. After the first batch of extractant is mixed evenly with the dehydrated fermentation broth, it is continuously discharged into the membrane separation device for continuous cross-flow extraction and membrane separation to obtain the extract. In the extraction system obtained by mixing the dehydrated fermentation broth and the extractant, the mass ratio of water-based to oil phase is (5-15):(13-16). The extractant includes organic alcohols. S3, the extract was decolorized and evaporated to crystallize, yielding phytosterol fermentation products.

[0015] This invention targets continuous cross-flow extraction and separation of phytosterol oil-based fermentation broth. By controlling the mass ratio of the water-based to the oil phase in the extraction system, it achieves full contact and dynamic extraction between the extractant and the fermentation broth. Simultaneously, through a membrane separation device, the oil-based, water-based, and residue phases (biological proteins, phytosterols, fermentation bacteria (residue), macromolecular pigment groups, by-product impurities, etc.) can be continuously separated from the extractant. This achieves a continuous and synchronous process of product extraction, clarification, impurity removal, separation, and purification. The extracted product has high content, few impurities, and minimal fermentation product loss. The process can be continuously and automatically controlled. The amount of extractant used is only about one-third of that used in traditional single-extraction processes, significantly reducing extractant recovery costs. The required extraction equipment is simple, and equipment investment and plant floor space can be effectively reduced.

[0016] Preferably, in step S1, the main components of the fermentation broth include 13-16 wt% oil phase, 4-5 wt% slag phase, and the remainder is water and other impurities.

[0017] It is understandable that after phytosterols undergo fermentation, the fermentation products dissolve in the oil base, meaning that the oil phase contains fermentation products; the residue phase mainly consists of fermentation bacteria and phytosterols, etc.

[0018] Preferably, in step S1, the extractant comprises an organic alcohol with a mass concentration of 95% or higher.

[0019] Preferably, in step S1, the organic alcohol is an organic alcohol with ≤4 carbon atoms; including but not limited to one or more of methanol and ethanol.

[0020] Preferably, in step S2, the extractant is added to the dehydrated fermentation broth in four batches; the continuous cross-flow extraction and separation specifically includes: S201, the dehydrated fermentation broth is mixed with the first batch of extractant to obtain the first mixture; the first mixture is continuously discharged after membrane separation to obtain the first extract and the first residue. S202, add the second batch of extractant to the first residual liquid to obtain the second mixture, and continuously discharge the second mixture after membrane separation to obtain the second extract and the second residual liquid; S203, repeat step S202, add the third batch of extractant and the fourth batch of extractant in sequence to obtain the third extract, the fourth extract and the fourth residue; The volumes of the first to fourth batches of extractant added were 1.8 to 2.2 times, 0.8 to 1.2 times, 0.4 to 0.6 times, and 0.4 to 0.6 times the volume of the dehydrated fermentation broth, respectively; the flow rate of the extractant added was greater than the discharge flow rate.

[0021] A further preferred option is a discharge flow rate of 45–100 L / m³. 2 / h.

[0022] In a further preferred embodiment, after the dehydrated fermentation broth is mixed with the first batch of extractant, it is stirred for 10 to 15 minutes and then continuously discharged. When the volume of the resulting extractant is equal to the volume of the added extractant, the next batch of extractant is added.

[0023] This invention achieves continuous cross-flow extraction and separation by adding the extractant in batches while simultaneously discharging the material continuously. Furthermore, it controls the timing of extractant addition; for example, after the first extractant is added, in the discharging step, the volume of the first extract equals the volume of the first extractant. At this point, the second extractant is added, and discharging continues. The third extractant is added when the volume of the second extract equals the volume of the second extractant. This invention mixes the dehydrated fermentation broth and extractant according to a volume ratio and a gradient addition rate, and then performs continuous cross-flow extraction and membrane separation to obtain a pure extract and a relatively uniform residual liquid.

[0024] In a further preferred embodiment, in step S2, the fourth residual liquid mainly consists of a slag-like solid phase and an oil-based phase. After centrifugation, solid slag, oil, and water are obtained, and the oil is reused.

[0025] In this invention, the residual liquid after extraction mainly consists of a slag-like solid phase, an oil-based phase (water-in-oil), and a small amount of extractant phase. After three-phase high-speed centrifugation (preferably at a speed of 3000-8000 rpm), solid, oil, and water (extractant) are separated. Since oil and water have different specific gravities, centrifugation can dehydrate and separate the oil-based phase in the form of water-in-oil. A small amount of extractant and other impurities dissolve in water. Therefore, the separated oil phase has a high quality and is easy to recycle and reuse. The water is discharged and sent to wastewater treatment.

[0026] Preferably, in step S2, the membrane pore size in the membrane separation device is below 200 nm. The thin film material with a separation precision below 200 nm used in this invention allows for continuous and sufficient contact between the extraction process and the surface of the thin film material, enabling dynamic extraction. After extraction is complete, the thin film element simultaneously purifies and separates the extractant from the fermentation broth.

[0027] Preferably, in step S2, the membrane material of the membrane separation device includes, but is not limited to, silicon carbide, alumina / zirconia materials, metal materials and other solvent-resistant materials.

[0028] The separation membrane material of this invention is oleophobic, which makes it difficult for oil-based impurities to combine with the extractant through charge coupling. This ensures that the material intercepts oil-based impurities during operation without clogging, while the extractant can pass through quickly. This achieves a dynamic and continuous extraction process and eliminates the defects caused by the extractant being left to stand for a long time and then "miscible" with oil and water-based substances due to charge coupling, which introduces impurities and byproducts, reduces product quality, and increases the need for subsequent purification processes.

[0029] Preferably, in step S2, the extraction temperature is 40–55°C.

[0030] Preferably, in step S2, after the dehydrated fermentation broth is mixed with the extractant, it is stirred at a speed of 80-120 rpm.

[0031] The organic alcohol extractant used in this invention has a low water content. Therefore, the mass ratio of water-based to oil-based phase after the first extractant is mixed with the dehydrated fermentation broth is mainly determined by the water content in the dehydrated fermentation broth. The mass ratio of water-based to oil-based phase does not change much in the subsequent stages. Therefore, this invention mainly controls the water content in the dehydrated fermentation broth.

[0032] Preferably, in step S3, the decolorization process uses activated carbon powder. This invention yields a high-purity product after removing impurities through processes such as decolorization.

[0033] Preferably, in step S3, during the evaporation and crystallization process, the extractant vapor is recovered and reused after condensation. In this invention, the extractant vapor is returned to step S1 for continuous extraction after condensation. If the moisture content is too high, it can be directly dehydrated and reused, for example, by methanol pervaporation dehydration or mixing with a high-concentration extractant, ensuring its mass concentration is above 95%.

[0034] Preferably, in step S3, the phytosterol fermentation product includes 4-hydroxyandrostenedione (4-AD), 4-hydroxyandrostenedione (9-AD), 11-hydroxyandrostenedione (11-AD), or bis(BA).

[0035] It is understood that extraction devices, membrane separation devices, and decolorization, evaporation and crystallization devices are common devices in the field. This invention does not specifically limit the devices, as long as they can achieve the corresponding extraction, separation, decolorization and evaporation and crystallization.

[0036] Specifically, after fermentation, the fermentation broth is dehydrated and then enters the extraction section. The extractant is added in batches continuously, with the batches and volumes decreasing gradually according to the content of fermentation products in the fermentation broth during continuous extraction. Continuous discharge is carried out as needed. When the extraction process is complete, the extracted product undergoes continuous decolorization and other impurity removal processes in the subsequent stages before being evaporated and crystallized to obtain a high-purity product. The extractant is returned to the extraction section to achieve system continuity. The continuous evaporation volume is greatly reduced compared to the traditional evaporation volume, resulting in significant savings in equipment, investment, and space requirements.

[0037] This invention mainly focuses on a novel continuous extraction process developed for the post-extraction of phytosterol oil-based fermentation products. Its main mechanism of action and advantages are as follows: (1) Dynamic extraction is achieved by using organic alcohol extractants and controlling the water content: During the extraction process, the fermentation products move from the oil-based phase into the extractant phase. The positions originally occupied by the fermentation products in the oil-based phase create a "cavitation effect." With the presence of an appropriate amount of water-based phase, these products can quickly occupy and couple to form large molecular charged groups in the form of "water-in-oil." These groups are also compatible with "fragment impurities (such as biological proteins, bacterial residues, etc.)," ​​resulting in larger particle sizes (water-in-oil groups). Meanwhile, the molecules in the extract (extractant and fermentation products) are smaller, allowing them to pass through a membrane separation device quickly. This membrane separation process rapidly separates the extract from the remaining substances, resulting in a low content of fermentation product impurities and high quality in the separated extract. The entire process does not require settling or stratification, enabling continuous and stable extraction and separation.

[0038] (2) Continuous batch addition of extractant: In this invention, the extraction process involves continuously adding the extractant according to the proportion of fermentation products in the fermentation broth. Based on the principles of extraction and separation, the amount of extractant added is reduced as the fermentation products are dynamically discharged and their content gradually decreases in the fermentation broth. This improves the extraction efficiency and reduces the amount of extractant used. The advantage lies in the fact that during small-batch addition, phytosterol fermentation product molecules in the fermentation broth can quickly move from the oil-based phase to the extractant phase during contact, forming "cavities." Simultaneously, the oil-based and water-based phases rapidly occupy these cavities, forming large molecular charged groups in an "oil-in-water" emulsion.

[0039] Because alcohol-based extractants, such as methanol, have strong hydrogen bonds with the water-based phase, they are highly hydrophilic and miscible with water at any concentration. In contrast, the alkyl and hydrocarbon groups in the oil-based phase have relatively weak hydrogen bonds with the water-based phase, resulting in poor hydrophilicity. However, when oil, water, and methanol are mixed and a "water-in-oil" group has been formed, the hydrogen bonds of the extractant (such as methanol) are not easily broken by the alkyl bonds of the macromolecular charged groups in the "water-in-oil" group. The inability to disperse and destroy the "water-in-oil" group allows methanol to bind with the water-based phase, thus intercepting the "water-in-oil" group without changing its state. This facilitates the separation of the oil-water group from the extractant phase. Furthermore, solid residues and biological proteins are easier to separate from the extractant phase than the oil phase, and the resulting "water-in-oil" group has a low impurity content, which is beneficial for obtaining a high-quality oil phase and facilitating recycling.

[0040] If the alcohol content and molecular weight are large, it will disrupt the water-in-oil emulsion. The alcohol group is affinity for water, and some short alkane groups in the oil phase will combine with the alcohol group at the same time, forming a miscible state of oil, alcohol and water, i.e., an emulsion state. At this time, it is impossible to completely intercept the oil group, water group, alcohol group and the formation of short-chain hydrocarbons through membrane separation, resulting in the introduction of hydrocarbons and a small amount of small molecule oil groups into the product, thus causing the product quality to be low.

[0041] (3) The process of the present invention is based on the concentration difference of the fermentation product during the extraction process, and matches the corresponding amount of extractant. The concentration of the extracted fermentation product is proportional to the mass of the extractant added. That is, when the initial concentration of the fermentation product in the fermentation liquid is high, the amount of extractant added is relatively high (the solubility of the fermentation product in the solvent is close to saturation). The content of the discharged fermentation product is high, and the addition ratio is twice the volume of the fermentation liquid. As the concentration of the fermentation product in the subsequent fermentation liquid decreases, the proportion of extractant added shows a decreasing trend, so that the fermentation product is basically completely extracted from the fermentation liquid, and the concentration difference of the discharged fermentation product is small. This achieves the purpose of thorough extraction of each batch of products and effectively avoids the defects of traditional processes that require multiple extractions and large amounts of extractant.

[0042] (4) In addition to the simple extraction process, high extraction efficiency, and low equipment investment, this invention overcomes the emulsification factor in material liquid extraction. It can achieve extraction by using membrane separation treatment and simultaneously intercept a large amount of biological protein, by-products, bacterial residue, and oil-in-water macromolecular charged group impurities generated during fermentation by using high-precision separation pores. This allows for simultaneous extraction and purification, greatly reducing equipment investment and maintenance costs. The separated "oil-in-water" and bacterial residue solids are easier to separate completely. Under the action of a high-speed centrifuge, the oil base and water base liquid and solid phases are completely separated under different centrifugation actions due to their different specific gravities, removing the mother liquor. At the same time, the quality of the separated oil base phase is improved (low impurity content, no entrainment), and the efficiency of recycling is greatly improved.

[0043] (5) The extraction process of the present invention is short, can be extracted and taken immediately without intervals, and is continuous. The yield of fermentation products extracted by the whole process can be increased from the traditional 55-65% to more than 90%.

[0044] Therefore, this invention provides a novel extraction process that is simple, low-cost, and highly efficient to meet industry needs. This invention achieves continuous extraction, shortens the overall extraction process flow, reduces operational steps, reduces investment in extraction equipment and infrastructure, lowers production costs, reduces the use of extractant (both single and total amounts of extractant can be effectively reduced), storage costs, and solvent recovery costs. After continuous extraction, the efficiency of the fermentation product is four times that of the original process, with a relatively high degree of automation, enabling unattended operation, significantly reducing labor costs and human error, saving on process equipment and investment, improving product yield (reducing fermentation product loss) and quality, and reducing mother liquor and its disposal steps.

[0045] The present invention will be further described in detail below through specific embodiments.

[0046] Example 1 In this embodiment, 4-AD was extracted from the phytosterol oil-based fermentation broth of a certain company. The fermentation product in the broth was 4-AD, of which 13% was plant oil, 5% was residue, 80% was water, and the remainder was other impurities.

[0047] A continuous extraction process for phytosterol oil-based fermentation broth includes the following steps: S1, after partial dehydration of the fermentation broth, dehydrated fermentation broth is obtained; S2, continuous cross-flow extraction and separation: S201, 15m 3 The dehydrated fermentation broth and 30m 3 The methanol was mixed and stirred at 100 rpm for 10 min to obtain the first mixture; the first mixture was subjected to membrane separation treatment (membrane pore size of 200 nm) and continuously discharged to obtain the first extract and the first residue. S202, the volume of the first extract is 30m³. 3 At that time, add 15m to the first residual liquid. 3 The methanol is used to obtain a second mixture, which is then subjected to membrane separation and continuously discharged to obtain a second extract and a second residue. S203, repeat step S202, adding 7.5m each time. 3 The methanol was added twice to obtain a third extract, a fourth extract, and a fourth residue.

[0048] In the above continuous cross-flow extraction process, the mass ratio of water-based to oil phase in the extraction system is controlled to be 10:13.

[0049] S3, the first to fourth extracts are continuously discharged, decolorized by activated carbon, and then evaporated and crystallized to obtain phytosterol fermentation products (hereinafter referred to as products). The evaporated extractant vapor is condensed and reused.

[0050] In this embodiment, the main parameters of the continuous cross-flow extraction and separation of the fermentation broth are shown in Table 1 below. The content of 4-AD in the fermentation broth and the content in the fourth residual liquid are obtained by liquid phase analysis. The content of the extractant added in the intermediate process is calculated to more clearly show the product concentration gradient change brought about by the continuous cross-flow extraction and separation of the present invention.

[0051] Table 1. Continuous cross-flow extraction and separation parameters for Example 1

[0052] As can be seen from Table 1, during the continuous extraction process, the concentration in the fermentation broth gradually decreases as the extraction process continues. In order to maintain the extraction efficiency and concentration, the amount of extractant added in subsequent extractions is gradually reduced until the final extraction is completed.

[0053] Example 2 (Investigating the effect of moisture content) Compared with Example 1, the only difference is that the mass ratio of water-based to oil phase in the extraction system is adjusted to 3:13, 5:13, 15:13 and 20:13, respectively; the other steps and conditions are the same as in Example 1.

[0054] The yield and purity of 4AD obtained under different water-based contents are shown in Table 2 below.

[0055] Table 2. Yield and purity of 4AD obtained at different moisture contents

[0056] The results in Table 2 show that when the water content is too low, it cannot effectively fill the "cavities (or pores)" inside the oil phase molecules, resulting in oil phase molecules that are too small. During separation, they will also permeate through the membrane and mix with the extract, leading to poor separation effect and low product purity. When the water content is too high, the water-in-oil system becomes saturated, and excessive water enters the extractant, resulting in a high water content in the recovered extractant, which makes reuse difficult and easily causes emulsification, resulting in a decrease in product yield and purity. Therefore, the present invention preferably controls the mass ratio of water-based to oil phase in the extraction system to be (5-15):(13-16).

[0057] Example 3 (Investigating the effect of membrane pore size) Compared with Example 1, the only difference is that the membrane pore size is adjusted as shown in Table 3 below; other steps and conditions are the same as in Example 1.

[0058] The product content and oil-based and water-based content in the extracts obtained under different membrane pore sizes are shown in Table 3 below.

[0059] Table 3. Product content and oil-based and water-based content in extracts obtained under different membrane pore sizes

[0060] As shown in Table 3, the product content in the extract is not significantly correlated with the separation precision. However, as the separation precision increases, the quality of the product in the extract gradually improves. Meanwhile, the permeation of oil-based and water-based products decreases with increasing precision. This indicates that membrane materials with different pore sizes have different permeation rates for oil-based and water-based products, and membrane materials with smaller pore sizes have higher interception rates for oil-based and water-based products.

[0061] Example 4 Compared with Example 1, the only difference is that the fermentation broth with fermentation product 9-AD is treated, while the other steps and conditions are the same as in Example 1.

[0062] The results showed that the yield and purity of 9-AD were basically the same as in Example 1, indicating that the present invention is also suitable for processing other similar phytosterol fermentation products.

[0063] Comparative Example 1 (Examining the effect of the type of extractant) Compared with Example 1, the only difference is that the extractant is changed to ethyl acetate, and the mass ratio of water-based to oil phase in the extraction system is adjusted; other steps and conditions are the same as in Example 1.

[0064] The mass ratio of the water-based and oil-based phases in the extraction system, as well as the yield and purity of the obtained products, are shown in Table 4 below.

[0065] Table 4 shows the parameter conditions and product test results for Comparative Example 1.

[0066] As can be seen from the results in Table 4 above, due to the high solubility of ethyl acetate in vegetable oil, a small portion of the oil and water will enter the subsequent process simultaneously, resulting in a lower yield and purity of the product compared to Example 1.

[0067] Comparative Example 2 (Examining the effect of the type of extractant) Compared with Example 1, the only difference is that the extractant is changed to acetone; the other steps and conditions are the same as in Example 1.

[0068] The results showed that the yield of the product was 88.5%, the purity was 89.5%, and the product quality declined.

[0069] As can be seen from Examples 1 and Comparative Examples 1-2, changing the type of extractant may cause adverse effects such as more impurities (final product purity is less than 90%). Therefore, the present invention preferably uses organic alcohols, and the number of carbon atoms in the organic alcohols should not be too high, preferably not more than 4, and more preferably not more than 2; to avoid increasing the solubility of oil bases and the probability of passing through the membrane separation device together with water, which would affect the quality of the product.

[0070] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A continuous extraction process for phytosterol oil-based fermentation broth, characterized in that, Includes the following steps: S1, after partial dehydration of the fermentation broth, dehydrated fermentation broth is obtained; S2, the extractant is added to the dehydrated fermentation broth in batches and mixed. After the first batch of extractant is mixed evenly with the dehydrated fermentation broth, it is continuously discharged into the membrane separation device for continuous cross-flow extraction and membrane separation to obtain the extract. In the extraction system obtained by mixing the dehydrated fermentation broth with the extractant, the mass ratio of the water-based phase to the oil phase is (5-15):(13-16); the extractant includes organic alcohols. S3, the extract is decolorized and evaporated to crystallize, yielding phytosterol fermentation products.

2. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S1, the extractant comprises an organic alcohol with a mass concentration of 95% or higher.

3. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S1, the organic alcohol is an organic alcohol with ≤4 carbon atoms.

4. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S2, the extractant is added to the dehydrated fermentation broth in four batches; The continuous cross-flow extraction and separation specifically includes: S201, the dehydrated fermentation broth is mixed with the first batch of extractant to obtain a first mixture; the first mixture is continuously discharged after membrane separation treatment to obtain a first extract and a first residue. S202, add a second batch of extractant to the first residual liquid to obtain a second mixture, and continuously discharge the second mixture after membrane separation to obtain a second extract and a second residual liquid; S203, repeat step S202, add the third batch of extractant and the fourth batch of extractant in sequence to obtain the third extract, the fourth extract and the fourth residue; The volumes of the first to fourth batches of extractant added are respectively 1.8 to 2.2 times, 0.8 to 1.2 times, 0.4 to 0.6 times, and 0.4 to 0.6 times the volume of the dehydrated fermentation liquid; and the flow rate of the added extractant is greater than the discharge flow rate.

5. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 4, characterized in that, The discharge flow rate is 45–100 L / m³. 2 / h; After the dehydrated fermentation broth is mixed with the first batch of extractant, it is stirred for 10 to 15 minutes and then continuously discharged. When the volume of the resulting extractant is equal to the volume of the added extractant, the next batch of extractant is added.

6. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 4, characterized in that, The fourth residual liquid mainly consists of a slag-like solid phase and an oil-based phase. The fourth residual liquid is centrifuged to obtain solid slag, oil, and water, and the oil is reused.

7. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S2, the membrane separation device has a membrane pore size of less than 200 nm; the membrane material includes silicon carbide, alumina, or zirconium oxide.

8. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S2, the extraction temperature is 40–55°C; The dehydrated fermentation broth is mixed with the extractant and stirred at a speed of 80-120 rpm.

9. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S3, the decolorization treatment is carried out using activated carbon powder; During the evaporation and crystallization process, the extractant vapor is recovered and reused after condensation.

10. The continuous extraction process of phytosterol oil-based fermentation broth according to claim 1, characterized in that, In step S3, the phytosterol fermentation product includes 4-hydroxyandrostenedione, 9-hydroxyandrostenedione, 11-hydroxyandrostenedione, or dichlorvos.

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