Method for recycling waste liquid from quercetin extraction from Sophora japonica flowers to produce spinosad

Abstract

This invention provides a method for recovering waste liquid generated from quercetin extraction from Sophora japonica flowers to produce spinosad. The method includes: preparing a liquid culture medium from the waste liquid generated from quercetin extraction from Sophora japonica flowers; inoculating the liquid culture medium with Polysporum oryzae for fermentation; and obtaining spinosad after fermentation. According to the method provided by this invention, not only can the waste liquid generated from quercetin extraction from Sophora japonica flowers be fully utilized, saving resources and reducing wastewater discharge, but spinosad can also be produced, reducing the production cost of spinosad.

Description

Technical Field

[0001] This invention relates to a method for recycling waste liquid generated from the extraction of quercetin from Sophora japonica flowers to produce spinosad, and relates to the field of microbial fermentation technology. Background Technology

[0002] Sophora japonica buds are the unopened flower buds of the Sophora japonica plant (a legume). The main component of Sophora japonica buds is rutin, with a content reaching 12-16%. Other components include quercetin, triterpenoid saponins, and sophoraecin. Quercetin, as a representative compound of flavonols, has high medicinal value. Traditional Chinese medicine preparations containing quercetin can protect against ischemia-reperfusion arrhythmias, lower blood lipids, enhance capillary lipidity, dilate coronary arteries, increase coronary blood flow, enhance myocardial contractility, relax intestinal smooth muscle, lower blood pressure, lower cholesterol, reduce platelet membrane lipid fluidity, and have immunosuppressive and anti-allergic effects, anti-cancer and cancer-preventive effects, as well as expectorant, antitussive, and antiasthmatic effects. Therefore, the extraction of quercetin from Sophora japonica buds has been widely applied.

[0003] The extraction of quercetin from Sophora japonica buds typically involves extracting rutin, then adding a dilute acid solution to the rutin for acid hydrolysis. The resulting precipitate is quercetin. The filtrate and the acidic solution generated during the washing of the precipitate are considered waste liquids. These waste liquids are directly discharged after being adjusted to neutral pH, which not only causes environmental pollution but also wastes resources. Therefore, how to recycle and reuse the waste liquid generated from the extraction of quercetin from Sophora japonica buds is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0004] This invention provides a method for recycling waste liquid generated from the extraction of quercetin from Sophora japonica flowers to produce spinosad. This method is used to recycle and utilize the waste liquid generated from the extraction of quercetin from Sophora japonica flowers, thereby solving the problems of environmental pollution and resource waste, and producing spinosad.

[0005] This invention provides a method for recovering waste liquid generated from the extraction of quercetin from Sophora japonica flowers to produce spinosad, the method comprising:

[0006] The waste liquid generated from the extraction of quercetin from Sophora japonica flowers was used to prepare a liquid culture medium.

[0007] Sporobacter spp. was inoculated into the liquid culture medium for fermentation, and spinosad was obtained after fermentation.

[0008] Sophora japonica buds are rich in rutin, quercetin, and other substances, among which quercetin has high medicinal value. Quercetin extraction typically utilizes the property that rutin molecules readily precipitate in acidic solutions. First, crude rutin is extracted from the buds. Then, rutin is refined by recrystallization, taking advantage of its high solubility in hot water and hot ethanol, but relatively low solubility in cold water and cold ethanol. Finally, a dilute acid solution is added to the rutin for acid hydrolysis to generate quercetin. The specific extraction steps can be carried out according to conventional techniques in this field. For example, place Sophora japonica flowers in a beaker, add saturated lime water to cover the flowers, heat to react, collect the filtrate while hot, adjust the pH of the filtrate to 4-5 with concentrated hydrochloric acid at 60-70℃, let it stand for a period of time, filter and collect the precipitate to obtain crude rutin. Then, dissolve the crude rutin in hot water, filter while hot and cool to refine the rutin. Next, add dilute acid solutions such as dilute sulfuric acid and dilute hydrochloric acid to the rutin, heat to acid hydrolyze for a certain time, collect the precipitate to obtain quercetin. In the process of obtaining quercetin from rutin acid hydrolysis, the remaining dilute acid solution after acid hydrolysis and the waste liquid generated during the washing of precipitated quercetin are usually adjusted to neutral pH and discharged, which not only wastes resources but also easily pollutes the environment. Studies have found that the waste liquid contains glucose, rhamnose, and various amino acids and inorganic salts. Saccharopolysporaspinosa can use the organic matter in the waste liquid for growth and metabolism and fermentation to obtain spinosad. Spinosad is a macrocyclic lactone bioactive substance that is highly effective against most lepidopteran and dipteran agricultural pests, and also has some control effect on other agricultural pests. Crucially, spinosad is very safe for humans and the environment, and is biodegradable, making it an ideal "green pesticide." Therefore, the method provided by this invention not only makes full use of the waste liquid generated after extracting quercetin from Sophora japonica flowers, saving resources and reducing wastewater discharge, but also produces spinosad, reducing its production cost.

[0009] In one specific embodiment, the method for recycling the waste liquid generated from the extraction of quercetin from Sophora japonica flowers to produce spinosad specifically includes the following steps:

[0010] Step 1: Prepare a liquid culture medium from the waste liquid generated during the extraction of quercetin from Sophora japonica flowers;

[0011] The waste liquid is mainly a dilute acid solution with a low pH of about 1. Therefore, it is necessary to first add alkaline substances to the waste liquid to adjust its pH to slightly alkaline, specifically 7-8, to reach the suitable pH range for the growth and metabolism of Polysporus spp. The alkaline substances used can be sodium hydroxide, potassium hydroxide, etc.

[0012] Subsequently, to further increase the fermentation yield of spinosad, it is necessary to add the carbon source, nitrogen source, and inorganic salts required for the growth of *Saccharomyces cerevisiae* to the waste liquid. Studies have shown that different substances have a significant impact on the fermentation yield of spinosad. For example, maltose is more effective than glucose in increasing the fermentation yield of spinosad, and soybean meal and cottonseed meal are more effective than peanut meal and fish meal in increasing the fermentation yield of spinosad. Therefore, this embodiment optimizes the carbon source, nitrogen source, and inorganic salts in the fermentation medium, specifically:

[0013] The carbon source includes maltose and soybean oil. Soybean oil not only helps increase the fermentation yield of spinosad but also acts as an antifoaming agent, reducing the generation of bubbles during fermentation. Furthermore, based on the waste liquid generated from the extraction of quercetin from the sophora japonica flowers, the added maltose is 8 g / L and the added soybean oil is 2 g / L.

[0014] The nitrogen source includes yeast powder, soybean meal powder, and cottonseed meal powder. Based on the waste liquid generated per liter of quercetin extraction from the Sophora japonica flower, the added yeast powder is 2 g / L, the soybean meal powder is 1 g / L, and the cottonseed meal powder is 3 g / L.

[0015] The inorganic salts include dipotassium hydrogen phosphate, magnesium sulfate, and calcium carbonate. Based on the waste liquid generated per liter of the extract of quercetin from the Sophora japonica flowers, the added dipotassium hydrogen phosphate is 0.1 g / L, the magnesium sulfate is 0.1 g / L, and the calcium carbonate is 0.4 g / L.

[0016] Finally, the waste liquid is stirred evenly with a mixed solution of carbon source, nitrogen source and inorganic salt, and then sterilized at high temperature. Specifically, the sterilization temperature is 121-123℃ and the time is 20-30 minutes. After sterilization, the liquid culture medium is obtained and cooled to room temperature for later use.

[0017] Step 2: Inoculate Polysporus spp. into the liquid culture medium for fermentation. After fermentation, spinosad is obtained.

[0018] The *Saccharopolyspora* strain used in this invention is a strain capable of producing spinosad, specifically the *Saccharopolyspora* disclosed in Chinese invention patent publication number CN111849807A, which was deposited on October 14, 2019, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postcode 100101), and classified as *Saccharopolyspora spinosa*, with accession number CGMCC. NO.18681; The seed culture medium includes 10 g / L glucose, 5 g / L yeast extract, 15 g / L peptone, 10 g / L soybean meal, 5 g / L dipotassium hydrogen phosphate and 2 g / L magnesium sulfate. Then, it is cultured on a shaker at a speed of 220-230 rpm and a temperature of 28-30℃ for 3-4 days. After the culture is completed, the seed liquid of Polysporum spinosum is obtained.

[0019] Next, the seed liquid is inoculated into the liquid culture medium at a volume fraction of 10%-12% for fermentation. Fermentation can be carried out according to conventional techniques in the art. Specifically, the fermentation is carried out on a shaker at a speed of 220-230 rpm, at a fermentation temperature of 28-30°C, and for 10-12 days. After fermentation, spinosad can be extracted from the fermentation liquid.

[0020] In summary, the method provided by the present invention not only makes full use of the waste liquid generated after extracting quercetin from Sophora japonica, saving resources and reducing wastewater discharge, but also produces spinosad, reducing the production cost of spinosad. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0023] The *Saccharomyces cerevisiae* used in the following examples was deposited on October 14, 2019, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.18681, and can be purchased from this collection center.

[0024] Example 1

[0025] This embodiment provides a method for fermenting waste liquid generated from the extraction of quercetin from Sophora japonica buds, specifically including the following steps:

[0026] Step 1: Add sodium hydroxide to the waste liquid to adjust its pH to 7-8;

[0027] Step 2: Add 8 g / L glucose, 2 g / L rapeseed oil, 1 g / L soybean meal powder, 2 g / L peptone, 3 g / L cottonseed meal powder, 0.2 g / L magnesium sulfate, and 0.4 g / L calcium carbonate to the waste liquid. Stir well and sterilize at 121℃ for 30 min to obtain liquid culture medium. Cool to room temperature for later use.

[0028] Step 3: Inoculate the mature *Saccharomyces cerevisiae* into a seed culture medium containing 10 g / L glucose, 5 g / L yeast extract, 15 g / L peptone, 10 g / L soybean meal, 5 g / L dipotassium hydrogen phosphate, and 2 g / L magnesium sulfate. Culture the culture on a shaker (220 rpm) at 28-30°C for 3 days to obtain the seed culture.

[0029] Step 4: Inoculate the seed culture into the liquid culture medium obtained in Step 2 at a volume fraction of 10%, and culture at 28-30℃ and 220 rpm for 10 days to obtain a fermentation broth containing spinosad. HPLC analysis showed that the spinosad content in the fermentation broth was 2316 μg / mL.

[0030] Example 2

[0031] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 1, the difference being the carbon source. Specifically, 8 g / L of glucose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of peptone, 3 g / L of cottonseed meal powder, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, stirred evenly, and then sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0032] Example 3

[0033] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 1, the difference being the carbon source. Specifically, 8 g / L of maltose, 2 g / L of rapeseed oil, 1 g / L of soybean meal powder, 2 g / L of peptone, 3 g / L of cottonseed meal powder, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, stirred evenly, and then sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0034] Example 4

[0035] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 1, the difference being the carbon source. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of peptone, 3 g / L of cottonseed meal powder, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, stirred evenly, and then sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0036] Table 1 shows the fermentation culture media and spinosad content provided in Examples 1-4.

[0037]

[0038] As shown in Table 1, when the carbon source is a combination of 8 g / L maltose and 2 g / L soybean oil for fermentation, i.e., in Example 4, the content of spinosad obtained by fermentation is the highest. Therefore, the carbon source is determined to be 8 g / L maltose and 2 g / L soybean oil.

[0039] Example 5

[0040] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 4, the difference being the nitrogen source. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of peanut cake powder, 2 g / L of peptone, 3 g / L of cottonseed cake powder, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, stirred evenly, and then sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0041] Example 6

[0042] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 5, the difference being the nitrogen source. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of yeast powder, 3 g / L of cottonseed meal powder, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, and after stirring evenly, it is sterilized at 121℃ for 30 min to obtain a liquid culture medium.

[0043] Example 7

[0044] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 5, the difference being the nitrogen source. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal, 2 g / L of peptone, 3 g / L of fish meal, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid, stirred evenly, and then sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0045] Table 2 shows the fermentation culture media and spinosad content provided in Examples 5-7.

[0046]

[0047] According to Table 2, when the nitrogen source is a combination of 1 g / L soybean meal powder, 2 g / L yeast powder, and 3 g / L cottonseed meal powder for fermentation, i.e., in Example 6, the content of spinosad obtained by fermentation is the highest. Therefore, the nitrogen source is determined to be 1 g / L soybean meal powder, 2 g / L yeast powder, and 3 g / L cottonseed meal powder.

[0048] Example 8

[0049] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 6, the difference being the different inorganic salts. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of yeast powder, 3 g / L of cottonseed meal powder, 0.1 g / L of dipotassium hydrogen phosphate, 0.2 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid. After stirring evenly, the mixture is sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0050] Example 9

[0051] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 8, the difference being the different inorganic salts. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of yeast powder, 3 g / L of cottonseed meal powder, 0.1 g / L of dipotassium hydrogen phosphate, 0.2 g / L of magnesium sulfate, and 0.2 g / L of calcium carbonate are added to the waste liquid. After stirring evenly, the mixture is sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0052] Example 10

[0053] The waste liquid fermentation method provided in this embodiment can be referred to in Embodiment 8, the difference being the different inorganic salts. Specifically, 8 g / L of maltose, 2 g / L of soybean oil, 1 g / L of soybean meal powder, 2 g / L of yeast powder, 3 g / L of cottonseed meal powder, 0.1 g / L of dipotassium hydrogen phosphate, 0.1 g / L of magnesium sulfate, and 0.4 g / L of calcium carbonate are added to the waste liquid. After stirring evenly, the mixture is sterilized at 121°C for 30 min to obtain a liquid culture medium.

[0054] Table 3 shows the fermentation culture media and spinosad content provided in Examples 8-10.

[0055]

[0056]

[0057] According to Table 3, when the inorganic salts are a combination of 0.1 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, and 0.4 g / L calcium carbonate, i.e. Example 10, the fermentation yields the highest content of spinosad. Therefore, the inorganic salts are determined to be 0.1 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, and 0.4 g / L calcium carbonate.

[0058] The optimal fermentation medium was determined through the optimization of the culture medium in Examples 1-10. Specifically, it includes a carbon source, a nitrogen source, and inorganic salts. The carbon source includes maltose and soybean oil, with the mass of maltose being 8 g / L and the mass of soybean oil being 2 g / L. The nitrogen source includes yeast powder, soybean meal, and cottonseed meal, with the mass of yeast powder being 2 g / L, the mass of soybean meal being 1 g / L, and the mass of cottonseed meal being 3 g / L. The inorganic salts include dipotassium hydrogen phosphate, magnesium sulfate, and calcium carbonate, with the mass of dipotassium hydrogen phosphate being 0.1 g / L, the mass of magnesium sulfate being 0.1 g / L, and the mass of calcium carbonate being 0.4 g / L.

[0059] Comparative Example 1

[0060] The waste liquid fermentation method provided in this comparative example can be referred to Example 1. The difference is that the waste liquid is adjusted to pH and sterilized and then directly used as a liquid culture medium. The seed liquid is inoculated into the waste liquid at a volume fraction of 10%, and cultured at 28-30℃ and 220rpm for 10 days to obtain a fermentation broth containing spinosad.

[0061] Using the same HPLC detection method as in Example 1, the spinosad content in the fermentation broth of Comparative Example 1 was found to be 452 μg / mL. This indicates that carbon source, nitrogen source and inorganic salts need to be added to the waste liquid as nutrients for the growth and metabolism of Polysporus spp. to increase the fermentation yield of spinosad.

[0062] Comparative Example 2

[0063] This comparative example provides a method for producing spinosad by fermentation of Polysporum spinosae, specifically including the following steps:

[0064] Step 1: Mix glucose (8 g / L), rapeseed oil (2 g / L), soybean meal powder (1 g / L), peptone (2 g / L), cottonseed meal powder (3 g / L), magnesium sulfate (0.2 g / L), calcium carbonate (0.4 g / L) with water until homogeneous, sterilize at 121°C for 30 min to obtain liquid culture medium, and cool to room temperature for later use.

[0065] Step 2: Inoculate the mature *Saccharomyces cerevisiae* into a seed culture medium. The seed culture medium and culture method are the same as in Example 1. After the culture is completed, the seed liquid is obtained and inoculated into the liquid culture medium at a volume fraction of 10%. The mixture is cultured at 28-30℃ and 220 rpm for 10 days to obtain a fermentation broth containing spinosad.

[0066] Using the same HPLC detection method as in Example 1, the spinosad content in the fermentation broth of Comparative Example 2 was 2061 μg / mL, which was significantly lower than that in Example 1. This indicates that using the waste liquid generated after extracting quercetin from Sophora japonica flowers, combined with other nutrients, for fermentation of Polysporus spp. can effectively increase the fermentation yield of spinosad and reduce the production cost of spinosad.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for recovering production of a pluronicillin from a waste liquid produced by extracting quercetin from sophora fruit, characterized in that, The method comprises: The waste liquid generated by extracting quercetin from the sophora japonica is configured to obtain a liquid medium; Inoculate saccharopolyspora spinosa in the liquid medium for fermentation, and obtain polysporin after the fermentation is completed; The waste liquid generated by extracting quercetin from the sophora japonica is configured to obtain a liquid medium, and the method comprises the following steps: The pH of the waste liquid generated by extracting quercetin from the sophora japonica is adjusted to 7-8; Add carbon source, nitrogen source and inorganic salt required for the growth of the saccharopolyspora spinosa to obtain the liquid medium; The carbon source is maltose and soybean oil, and the mass of the added maltose is 8 g / L and the mass of the added soybean oil is 2 g / L based on 1 liter of the waste liquid generated by extracting quercetin from the sophora japonica; The nitrogen source is yeast powder, soybean meal and cottonseed meal, and the mass of the added yeast powder is 2 g / L, the mass of the added soybean meal is 1 g / L and the mass of the added cottonseed meal is 3 g / L based on 1 liter of the waste liquid generated by extracting quercetin from the sophora japonica; The inorganic salt is dipotassium hydrogen phosphate, magnesium sulfate and calcium carbonate, and the mass of the added dipotassium hydrogen phosphate is 0.1 g / L, the mass of the added magnesium sulfate is 0.1 g / L and the mass of the added calcium carbonate is 0.4 g / L based on 1 liter of the waste liquid generated by extracting quercetin from the sophora japonica; The preservation number of the saccharopolyspora spinosa is CGMCC NO. 18681.

2. The method of claim 1, wherein, The liquid medium is sterilized, and the sterilization temperature is 121-123 DEG C and the sterilization time is 20-30 min.

3. The method of claim 1, wherein, The saccharopolyspora spinosa is inoculated in a seed culture medium for culture to obtain a seed liquid, and the seed liquid is inoculated in the liquid medium at a volume fraction of 10%-12% for fermentation.

4. The method of claim 3, wherein, The seed culture medium comprises 10 g / L of glucose, 5 g / L of yeast extract, 15 g / L of peptone, 10 g / L of soybean meal and 5 g / L of dipotassium hydrogen phosphate and 2 g / L of magnesium sulfate.

5. The method according to claim 1 or 3, characterized in that, The fermentation temperature is 28-30 DEG C and the fermentation time is 10-12 days.

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