Method for preparing sophorolipid with mass ratio of lactone type sophorolipid not less than 50% by using kitchen waste grease
By optimizing fermentation conditions and post-processing techniques, high-purity lactone-type sophorolipids were prepared using Candida strains, solving the problem of low utilization rate of kitchen waste oil and achieving efficient and low-cost sophorolipid production.
Patent Information
- Application Number
- CN202511283879.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies are insufficient to effectively utilize kitchen waste oil to prepare a high proportion of lactone-type sophorolipids, resulting in high production costs and severe environmental pollution.
Kitchen waste oil was fermented using Candida strains under specific conditions. By controlling dissolved oxygen, temperature, and pH, and by adding glucose and kitchen waste oil, the fermentation process was optimized to increase the proportion of lactone-type sophorolipids. High-purity lactone-type sophorolipids were obtained through post-processing.
The method significantly increased the content of lactone-type sophorolipids, reduced production costs, and simplified purification steps, resulting in high-purity, low-cost lactone-type sophorolipid products that meet the safety and quality requirements of high-end applications.
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Figure CN121109531A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of sophorolipid preparation technology, and more specifically, relates to a method for preparing sophorolipids with a mass ratio of not less than 50% using kitchen waste oil. Background Technology
[0002] Sophorolipids are a class of glycolipid surfactants obtained through microbial fermentation and transformation. They possess excellent emulsifying, surface / interfacial activity, and biodegradability, while also exhibiting antibacterial activity and physiological activities such as inducing tumor cell apoptosis and mutation. They have potential applications in food, cosmetics, environmental protection, medicine, and materials.
[0003] Sophorolipids are classified into two types: lactone and acidic. Generally, lactone-type sophorolipids have higher surface tension reduction and antibacterial properties, while acidic sophorolipids have better solubility and foaming ability. In addition, compared with acidic sophorolipids, lactone-type sophorolipids have better antibacterial, anti-inflammatory, and emulsifying abilities and are more stable under high temperature and high salt conditions.
[0004] The main existing method for preparing lactone-type sophorolipids is microbial fermentation. The most commonly used microorganisms in this method are *Candida* species, such as *Candida bombicola* / *Starmerella bombicola*, followed by *Saccharomyces* species. Suitable culture media (carbon sources such as glucose and vegetable oil; nitrogen sources such as yeast extract and urea; inorganic salts, etc.) are provided in specific fermenters for fermentation. In the middle and later stages of fermentation, hydrophobic inducing substrates (usually vegetable oils or long-chain fatty acid esters) are added. At this point, the microorganisms utilize the sugars and hydrophobic substrates to synthesize sophorolipids.
[0005] Kitchen waste oil, also known as restaurant waste oil, mainly consists of palmitic acid, stearic acid, oleic acid, and linoleic acid. Its fatty acid composition is basically the same as other animal and vegetable oils, with a carbon chain length of 14-18. It can be used as a substitute for hydrophobic inducing substrates such as vegetable oils in the fermentation production of sophorolipids, thus significantly reducing the production cost of sophorolipids. Currently, my country generates approximately 10 million tons of kitchen waste oil annually, of which about half is recycled by legitimate enterprises, while the other half may be illegally recycled or carelessly discarded, causing environmental pollution. Therefore, how to utilize kitchen waste oil for fermentation to prepare sophorolipids and improve the utilization rate of waste oil is the technical problem this invention aims to solve. Summary of the Invention
[0006] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for preparing sophoroliposide with a mass ratio of not less than 50% using kitchen waste oil. The method involves increasing the proportion of sophoroliposide in the final product by adjusting fermentation conditions and other methods to obtain sophoroliposide.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0008] A method for preparing sophoroliposide with a content of not less than 50% by mass using kitchen waste oil involves using Candida albicans as the production strain, glucose as the fermentation substrate, controlling dissolved oxygen and temperature to start fermentation, adding glucose and kitchen waste oil during fermentation until fermentation is completed, and obtaining sophoroliposide with a content of not less than 50% by post-treatment of the fermentation broth.
[0009] The method for preparing sophoroliposide lactone with a mass ratio of not less than 50% using kitchen waste oil, wherein the kitchen waste oil is derived from a Western-style fast food restaurant and is decolorized using activated clay.
[0010] The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil, wherein the Candida albicans is Candida albicans ATCC 22214.
[0011] The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil has the following fermentation culture medium composition: glucose 100g / L, yeast powder 20g / L, pH=4.0-6.0.
[0012] The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil involves adding glucose solution after the initial glucose is consumed during fermentation and maintaining the glucose concentration in the fermenter at no more than 10 g / L. During the fermentation period of 48 h to 216 h, kitchen waste oil is added at a maximum feeding rate of 5 g / (L·h).
[0013] The method for preparing sophoroliposide lactone with a mass ratio of not less than 50% using kitchen waste oil involves a fermentation temperature of 30℃ and a dissolved oxygen content of 50-60%.
[0014] The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil involves post-treatment of the fermentation broth as follows: heating the fermentation broth to 80°C and discharging it, allowing it to settle naturally to obtain sophoroliposide.
[0015] The method for preparing sophoroliposide with a mass ratio of not less than 50% using kitchen waste oil, wherein the content of lactone-type sophoroliposide in the prepared sophoroliposide accounts for 50-90% of the total sophoroliposide.
[0016] The method for preparing sophorolipid-type lactone-type sophorolipids with a mass ratio of not less than 50% using waste cooking oil includes the following steps:
[0017] 1) Inoculate Candida ATCC 22214 onto YPD slant medium and incubate at 30℃ for 2 days to obtain the original strain;
[0018] 2) Place 100 mL of seed culture medium in a 500 mL shake flask, sterilize at 121 °C for 20 min, and after cooling, scrape the bacterial cells from the slant and place them in the culture medium. Place the shake flask on a shaker at 30 °C and 220 rpm for 24 h to obtain the seed liquid.
[0019] 3) Add 25L of fermentation medium to a 50L fermenter, sterilize at 121℃ for 30min, and after cooling, inoculate the above seed liquid into the fermentation medium at an inoculation rate of 10% by volume. The fermentation temperature is 30℃, and the dissolved oxygen is 50-60%. During the fermentation process, when the initial glucose is consumed, glucose solution is added to maintain the glucose concentration in the fermenter at no more than 10g / L. During the fermentation period from 48h to 216h, kitchen waste oil is added at a maximum rate of 5g / (L·h), and the feeding rate is adjusted according to the oil consumption of the bacteria in the fermenter. Feeding is stopped when fermentation reaches 216h.
[0020] 4) Heat the fermentation liquid to 80°C and discharge it. Allow it to settle naturally to obtain sophorolipid.
[0021] In the preparation method of this application, the pH value of the fermentation process is controlled at 5.0-6.0. A higher pH value can inhibit the synthesis of acidic sophorolipids by the cells during fermentation, thereby reducing the proportion of acidic sophorolipids in the final product.
[0022] Beneficial effects: Compared with the prior art, the main technical advantages of this application are as follows:
[0023] 1) This application optimizes the fermentation strategy (such as specific inducers, temperature and pH control) to enable microorganisms to synthesize target lactone-type sophorolipids directly, efficiently and selectively during the fermentation process, significantly reducing or even eliminating the synthesis of acidic products, improving product specificity, and making the lactone-type sophorolipid content account for up to 85% of the total sophorolipids.
[0024] 2) Since this application directly yields a high proportion or a single lactone-type product after fermentation, it can completely eliminate the chemical lactone formation step, and also eliminates the need for forced in-situ acidification and conversion operations. This directly solves the disadvantages of chemical lactone formation / in-situ acidification, including high energy consumption, high reagent toxicity, numerous side reactions, high degradation risk, and complex steps.
[0025] 3) Since the fermentation product of this application is already a high-purity lactone-type sophorolipid (or with extremely low acid content), the separation difficulty is greatly reduced. Therefore, this application can be complemented by the development of simpler, more economical, and more environmentally friendly post-processing technologies (such as gentle crystallization, selective adsorption, and high-efficiency chromatography media), and even simple extraction, concentration, and drying can achieve the required purity. This effectively solves the problems of "difficult separation and purification, high cost, and large losses".
[0026] 4) The overall effect of this application in avoiding conversion loss, simplifying purification steps, reducing solvent / reagent consumption, and improving unit production efficiency is to significantly increase the total yield of the final lactone-type sophorolipid and greatly reduce production costs (including raw materials, energy, environmental protection, and labor).
[0027] 5) This application avoids side reactions and impurities introduced by the chemical conversion step, resulting in a lactone-type sophorolipid product with a lighter color, higher purity, and more complete structure. The production process is cleaner and more controllable, and the product better meets the safety and quality requirements of high-end applications (such as pharmaceuticals, food, and cosmetics). Attached Figure Description
[0028] Figure 1 This is a diagram showing the fermentation process results of Example 3. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0030] The kitchen waste oil used in the following examples is grease that has been used in the kitchen and has been treated by sedimentation to remove the supernatant and solid impurities. The fermentation strain used is commercially available Candida bombicola ATCC 22214, abbreviated as Candida bombicola ATCC 22214.
[0031] The seed culture medium used in the following examples consisted of 30 g / L glucose, 5 g / L yeast extract, and 10 g / L peptone.
[0032] The fermentation medium used in the following examples consisted of 100 g / L glucose, 20 g / L yeast extract, and pH = 4.0-6.0 (pH was adjusted with phosphate and ammonia).
[0033] In the following examples, the method for determining the sophorolipid content was as follows: 500 μL of fermentation broth was taken, 1 mL of ethanol was added, and the mixture was shaken and stirred at 12000 r·min.-1 Centrifuge for 10 min. Take 20 μL of the supernatant and add it to an 8 mL EP tube, then add 980 μL of distilled water (diluted 50 times). Determine the total sugar content using the anthrone-sulfuric acid method. The total sophorolipid content is calculated by removing residual glucose from the fermentation broth from the total sugar content. The sophorolipid content is determined based on the ratio between the molecular weights of sophorolipids and glucose, i.e., 1.91 g of sophorolipids is equivalent to 1 g of glucose.
[0034] Lactone-type sophorolipid content: The glucose content of lactone-type sophorolipids in the ethyl acetate supernatant was determined by extraction with ethyl acetate, and the content was then calculated. The specific steps are as follows: Take 500 μL of fermentation broth, add 1 mL of ethyl acetate, and vortex to extract thoroughly at 12000 r·min. -1 Centrifuge for 10 min. Take 20 μL of the supernatant and add it to an 8 mL EP tube, then add 380 μL of distilled water (diluted 20 times). Take the supernatant and determine the OD value of the sophorolipid sample at 620 nm using the anthrone-sulfuric acid method. Calculate the glucose content from the glucose standard curve, and obtain the lactone-type sophorolipid content at a ratio of 1:1.91.
[0035] Example 1
[0036] A method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using waste cooking oil includes the following steps:
[0037] 1) Inoculate Candida ATCC 22214 onto YPD slant medium and incubate at 30℃ for 2 days to obtain the original strain.
[0038] 2) Preparation of seed culture: Place 100 mL of seed culture medium in a 500 mL shake flask, sterilize at 121 °C for 20 min, and after cooling, scrape the bacterial cells from the slant and place them in the culture medium. Place the shake flask on a shaker at 30 °C and 220 rpm for 24 h to obtain the seed culture.
[0039] 4) Treatment of kitchen waste oil: Add a certain amount of activated clay to the kitchen waste oil collected from Western-style fast food restaurants, heat it, and then filter it for preliminary decolorization treatment.
[0040] 5) Fermentation: Add 25L of fermentation medium to a 50L fermenter, sterilize at 121℃ for 30min, and after cooling, inoculate the above secondary seed liquid into the fermentation medium at an inoculation rate of 3% by volume. The fermentation temperature is 30℃, and the dissolved oxygen is 50-60%. During the fermentation process, when the initial glucose is consumed, glucose solution is added to maintain the glucose concentration in the fermenter at no more than 10g / L. From 48h to 216h of fermentation, the treated kitchen waste oil is added at a maximum rate of 5g / (L·h), and the feeding rate is adjusted according to the oil consumption of the bacteria in the fermenter. When fermentation reaches 216h, glucose and oil feeding is stopped.
[0041] The fermentation medium consisted of 100 g / L glucose, 20 g / L yeast extract, and pH 4.0.
[0042] 6) Heat the fermentation liquid to 80℃ and discharge it. Allow it to settle naturally to obtain 14.9 kg of sophorolipid product.
[0043] The content of lactone-type sophorolipids in the finished sophorolipid product was determined by acid hydrolysis, and the content of lactone-type sophorolipids accounted for 50.3% of the total sophorolipids.
[0044] Example 2
[0045] A method for preparing lactone-type sophorolipids using kitchen waste was described, similar to Example 1, except that the pH of the fermentation medium was 5.0. 15.1 kg of sophorolipids were obtained. The lactone-type sophorolipid content was determined to be 71.5% of the total glycolipids by acid hydrolysis.
[0046] Example 3
[0047] The method for preparing lactone-type sophorolipids using kitchen waste is the same as in Example 1, except that the pH of the fermentation medium is 6.0. 15.2 kg of sophorolipids were obtained. The fermentation process is shown in the figure below. Figure 1 As shown, the content of lactone-type sophorolipids accounted for 85.3% of the total glycolipids, as determined by acid hydrolysis.
[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil, characterized in that, Using Candida albicans as the production strain and glucose as the fermentation substrate, fermentation was initiated by controlling dissolved oxygen and temperature. During the fermentation process, glucose and waste cooking oil were continuously added until the fermentation was completed. Sophorolipids with a lactone-type sophorolipid content of not less than 50% were obtained by post-processing the fermentation broth.
2. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, The Candida albicans mentioned is Candida albicans ATCC 22214.
3. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, Fermentation medium composition: glucose 100g / L, yeast extract 20g / L, pH=4.0-6.
0.
4. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, During the fermentation process, after the initial glucose is depleted, glucose solution is added to maintain the glucose concentration in the fermenter at no more than 10 g / L. During the fermentation period of 48 h to 216 h, kitchen waste oil is added at a maximum feeding rate of 5 g / (L·h).
5. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, Fermentation temperature: 30℃, dissolved oxygen: 50-60%.
6. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, The post-treatment fermentation broth was prepared by heating the fermentation broth to 80°C, discharging it, and allowing it to settle naturally to obtain sophorolipids.
7. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, In the prepared sophorolipids, lactone-type sophorolipids account for 50-90% of the total sophorolipids.
8. The method for preparing sophoroliposide of lactone type with a mass ratio of not less than 50% using kitchen waste oil according to claim 1, characterized in that, Includes the following steps: 1) Inoculate Candida ATCC 22214 onto YPD slant medium and incubate at 30℃ for 2 days to obtain the original strain; 2) Place 100 mL of seed culture medium in a 500 mL shake flask, sterilize at 121 °C for 30 min, and after cooling, scrape the bacterial cells from the slant and place them in the culture medium. Place the shake flask on a shaker at 30 °C and 220 rpm for 24 h to obtain the seed liquid. 3) Add a certain amount of activated clay to the kitchen waste oil collected from Western-style fast food restaurants, heat it, and then filter it for preliminary decolorization treatment. 4) Add 25L of fermentation medium to a 50L fermenter, sterilize at 121℃ for 30min, and after cooling, inoculate the above seed liquid into the fermentation medium at an inoculation rate of 10% by volume. The fermentation temperature is 30℃, and the dissolved oxygen is 50-60%. After 48h of fermentation, start continuously adding glucose solution and treated kitchen waste oil while maintaining the glucose concentration in the fermenter at no more than 10g / L. During the fermentation period from 48h to 216h, add kitchen waste oil at a maximum feeding rate of 5g / (L·h), and adjust the feeding rate according to the oil consumption of the bacteria in the fermenter. Stop feeding when fermentation reaches 216h. 5) Heat the fermentation liquid to 80°C and discharge it. After natural sedimentation, sophorolipids are obtained.