A method for producing sophorolipids by fermentation using palmitic acid oil as substrate

Abstract

The present invention belongs to the technical field of microbial fermentation, and provides a method for fermenting and producing sophorolipids using palmitic acid oil as a substrate. The present invention uses Candida albicans of Bombus as a base strain, and ferments and cultures after activation; the present invention uses palmitic acid oil as the main carbon source of the fermentation medium, and on the basis of reducing the raw material cost by 70%, the yield of sophorolipids is also significantly improved, and the palmitic acid oil not only does not affect the growth of the strain, but also makes the shake flask yield of sophorolipids reach 120 g / L (lactone-type sophorolipids account for 37.5%, acid-type sophorolipids account for 62.5%), and the yield of sophorolipids reaches 300 g / L under the feeding control of the 5 L bioreactor.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a culture medium and a fermentation method for producing sophorolipids by fermenting palmitic acid oil as a substrate. Background Art

[0002] Glycolipid biosurfactants are 100% bio-sourced, 100% biosynthesized and 100% biodegradable, and have been a hot topic in the industry in recent years. However, compared with petroleum-based surfactants such as SDS, bio-based surfactants such as alkyl glucoside, and amino acid surfactants, the high price of glycolipid surfactants makes them face major difficulties in commercial promotion.

[0003] Sophorolipids are the most widely studied biosurfactants with the highest known yield. They are fermented by bumblebee Candida albicans, and their main carbon sources are glucose and vegetable oil. At present, the high market price and addition amount of glucose and rapeseed oil are the main reasons for the high price of sophorolipids. Therefore, finding low-cost alternative carbon sources can effectively reduce the production cost of sophorolipids.

[0004] Palmitic acid oil is an oil obtained by acid treatment of saponins produced by palm oil refining. It is a by-product obtained after palm oil refining, in which the free fatty acid mass content is about 25~70%, and the total oil mass content is about 80~95%. The price of palmitic acid oil is only half of that of vegetable oil. Therefore, palmitic acid oil is a good substitute for vegetable oil. Summary of the invention

[0005] In order to solve the problem of high production cost of sophorolipid, the present invention provides a culture medium and a fermentation method for producing sophorolipid by fermenting with palmitic acid oil as a substrate.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for producing sophorolipids by fermentation using palmitic acid oil as a substrate, wherein the seed liquid of a sophorolipid-producing strain is inoculated into a fermentation medium for fermentation culture; the fermentation medium comprises a hydrophilic carbon source, a hydrophobic carbon source, a nitrogen source and an inorganic salt; the hydrophobic carbon source is palmitic acid oil. Preferably, the palmitic acid oil has a free fatty acid content of 25-75% by mass, a main component of palmitic acid, a moisture content and an impurity content of 1-2% by mass, an iodine value of 40 g I2 / 100 g, a total fat content of 80-99% by mass, a brown-red appearance, an insoluble impurity content of 0.1-0.5% by mass, and an unsaponifiable matter content of 0.1-0.5% by mass.

[0008] Wherein, the sophorolipid-producing strain is Candida bombus Starmerella bombicola .

[0009] Preferably, the sophorolipid-producing strain is Candida bombus PL0120, which is classified as Starmerella bombicola The deposit number is CGMCC No. 29188, the deposit date is November 30, 2023, and it is deposited in the General Microbiology Center of China Microorganism Collection Administration, referred to as CGMCC, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Wherein, the sophorolipid-producing strain seed liquid is prepared according to the following method: inoculate the sophorolipid-producing strain into a growth medium and culture it at 20-30°C for 12-48 hours. Wherein, the growth medium is a YPD liquid medium. Preferably, culture at 25-30°C for 24-36 hours, with a rotation speed of 100-300 rpm, and more preferably, culture at 30°C for 24 hours. Preferably, the sophorolipid-producing strain is activated and then inoculated into the growth medium, and the activation is to streak the glycerol bacteria stored at -80°C, and culture it in a growth incubator at 20-30°C until a single colony grows.

[0011] Wherein, the inoculation amount is 1-10% v / v, the fermentation culture temperature is 20-30°C, and the culture time is 96-168 h. Preferably, the inoculation amount is 4% v / v, the culture temperature is 25-30°C, the fermentation time is 96-144h, and the rotation speed is 100-300 rpm.

[0012] The hydrophilic carbon source is any one or more combinations of glucose, fructose, molasses and lactose; the nitrogen source is any one or more combinations of peptone, yeast extract, corn flour, trypsin and fish meal; and the inorganic salt is any one or more combinations of disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate.

[0013] Among them, in the fermentation medium, the hydrophilic carbon source is glucose, and the nitrogen source is a combination of yeast extract and trypsin, and the addition mass ratio is 1-3:1-3, preferably 1:1, 1:2, 2:3, 3:2 or 2:1, and more preferably 1:1; the inorganic salt is a combination of disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, and the addition mass ratio is 1-3:1-3:1-3, preferably 2:2:1, 1:1:1, 1:1:2, 3:3:1 or 1:1:3, and more preferably 2:2:1.

[0014] Wherein, in the fermentation medium, the concentration of the hydrophilic carbon source is 10-200 g / L, preferably 50-150 g / L; the concentration of the hydrophobic carbon source is 10-200 g / L, preferably 10-100 g / L, more preferably 10-80 g / L; the concentration of the nitrogen source is 1-20 g / L, preferably 1-10 g / L; the concentration of the inorganic salt is 1-20 g / L, preferably 1-10 g / L.

[0015] Preferably, the sophorolipid shake flask yield obtained by the method reaches 120 g / L, and the cost is reduced by 70%.

[0016] Beneficial effects:

[0017] The present invention adopts a palmitic acid oil as a substrate component for fermenting and producing sophorolipids, and designs a fermentation medium and a fermentation method. The medium and the fermentation method have a good effect on reducing the cost load and by-product treatment brought by large-scale production while preparing an equivalent sophorolipid product, which is conducive to determining its potential applicability to future industrial processes based on this. The yield of sophorolipids in a shake flask fermented with the palmitic acid oil reaches 120 g / L, which is higher than other vegetable oils. The yield reaches 300 g / L under the feeding control of a 5 L bioreactor, which is about 8% higher than the yield of rapeseed oil fermentation. Therefore, compared with using conventional vegetable oils as substrate sources, the application of the palmitic acid oil has a higher sophorolipid yield and lower raw material cost.

[0018] The present invention provides a culture medium and a fermentation method for synthesizing sophorolipids using palmitic acid oil as a substrate. In the present invention, a fermentation medium for sophorolipids is prepared by using a palmitic acid oil as a substrate source for the synthesis of sophorolipids, and a fermentation method is established using Candida albicans of Bombus as a base strain. The fermentation results show that the shake flask yield of sophorolipids fermented with the palmitic acid oil can reach 120 g / L, and the high concentration of palmitic acid oil does not affect the growth of the strain, wherein the lactone type sophorolipids account for 37.5% and the acid type sophorolipids account for 62.5%. Compared with using other vegetable oils such as rapeseed oil, soybean oil, and peanut oil as substrate sources, the raw material cost is significantly reduced, the raw material utilization rate is significantly improved, and the sophorolipid yield is significantly increased (and the proportion of acid type sophorolipids is significantly increased). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0020] Figure 1 To compare the fermentation results of palmitic acid oil and other vegetable oils as substrates for the synthesis of sophorolipids, the CK group refers to the fermentation medium with only glucose added as the carbon source without adding vegetable oil.

[0021] Figure 2 It is the state of fermentation liquid after palmitic acid oil is fermented as substrate.

[0022] Figure 3 Fermentation results of palmitic acid oil as substrate in a 5 L bioreactor. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below in conjunction with the examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Based on the examples in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0024] In the following examples, the anthrone sulfuric acid method was used to detect the sophorolipid production.

[0025] In the following examples, the palmitic acid oil was provided by Suzhou Corning Polyol Co., Ltd.

[0026] In the following examples, the bumblebee Candida PL0120 is classified and named Starmerella bombicola The deposit number is CGMCC No. 29188, the deposit date is November 30, 2023, and it is deposited in the General Microbiology Center of China Microorganism Collection Administration, referred to as CGMCC, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0027] Example 1 Synthesis of Sophorolipids Using Palmitic Acid Oil and Other Vegetable Oils as Fermentation Substrates

[0028] In this example, Candida bombus PL0120 was used as the base strain. After the strain was activated, it was inoculated into YPD liquid culture medium for culture. After culturing at 30° C. in a shaking incubator for 24 h, it was inoculated into the fermentation medium at a transfer rate of 4% v / v.

[0029] The fermentation medium is mainly composed of hydrophilic carbon source, hydrophobic carbon source, nitrogen source and inorganic salt. The specific formula includes: a palmitic acid oil or other vegetable oil (rapeseed oil, sunflower oil, soybean oil, peanut oil or palm oil), added at 80 g / L; glucose, added at 100 g / L; yeast extract and trypsin added at 2 g / L each; disodium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate added at 1 g / L, 1 g / L, and 0.5 g / L, respectively. In addition, the experiment also added only glucose (100 g / L) as the carbon source in the fermentation medium composition without adding any other vegetable oil as the CK group control.

[0030] The shake flask culture conditions were as follows: shaker culture temperature was 28 °C, shaker speed was 220 rpm, and fermentation time was 120 h. Figure 1 , Figure 2 As shown. Figure 1 As shown in the figure, all fermentation data of other experimental groups were higher than that of CK group. The fermentation yield of palmitic acid oil reached about 120 g / L. High concentration of palmitic acid oil did not affect the growth of strains. Lactone-type sophorolipids accounted for 37.5% and acid-type sophorolipids accounted for 62.5%. Compared with other vegetable oils, the palmitic acid oil is a better substrate raw material with lower cost, and the strain has the highest utilization rate of palmitic acid oil, and its residual oil content is much lower than other vegetable oils. Based on this, the addition amount of palmitic acid oil was optimized. The fermentation process was the same as above. The only difference was that the addition amount of palmitic acid oil was set to 40, 60, 80, and 100 g / L, respectively. The sophorolipid yield, cell dry weight, residual oil content, and the respective yields of lactone-type sophorolipids and acid-type sophorolipids were detected. The results showed that the optimal addition amount of palmitic acid oil was 80 g / L.

[0031] According to the GC-MS analysis of the changes in fatty acid composition and content before and after fermentation, the fatty acid composition and content of the palmitic acid oil before and after fermentation were determined. Take 2 mL of palmitic acid oil and add it to a 10 mL centrifuge tube, add 3 mL of anhydrous methanol, heat it in a water bath to completely dissolve it, add 0.2 mL of concentrated sulfuric acid, mix it thoroughly for 10 min, take it out and add 5 mL of deionized water and 0.5 mL of petroleum ether, shake it thoroughly for 3 min, let it stand for 5 min, and after stratification, take the ether phase for the next step of determination.

[0032] The fatty acid composition and content of the ether phase were determined by gas chromatography-mass spectrometry. Gas chromatography conditions: a 30 m×0.25 mm×0.25 μm column was used; the column temperature was programmed to start at 50°C, maintained for 3 min, increased to 230°C at 15°C / min, and then increased to 300°C at 5°C / min, and maintained for 5 min; the carrier gas was He, the flow rate was 1 mL / min, and the split ratio was 20:1. Mass spectrometry conditions: EI ion source, source temperature 200°C, interface temperature 250°C, solvent delay 3 min, mass scan range 45-450 amu, scan interval 0.3 s, full scan mode.

[0033] The results of the changes in the contents of related components are listed in Table 1. Palmitic acid oil contains fatty acids such as palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, and arachidenic acid, with a total fat content of 98%.

[0034] Table 1 Changes in the content of various components in palmitic acid oil before and after fermentation

[0035]

[0036] Example 2 Comparison of the fermentation of palmitic acid oil and rapeseed oil in a 5 L bioreactor

[0037] In this example, Candida bombus PL0120 was used as the base strain. After the strain was activated, it was cultured in YPD liquid medium and cultured in a shaking incubator at 30°C for 24 h. The transfer volume of 4% v / v was transferred to two 5 L bioreactors containing 3.5 L fermentation medium.

[0038] The fermentation medium mainly consists of carbon source, nitrogen source and inorganic salt, and its specific formula is as follows: a palmitic acid oil or rapeseed oil, added in an amount of 80 g / L, added 24 hours after inoculation; glucose, added in an amount of 100 g / L; yeast extract and trypsin, added in an amount of 2 g / L each; disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate, added in amounts of 1 g / L, 1 g / L and 0.5 g / L respectively.

[0039] The dissolved oxygen was controlled at 25% throughout the whole process, and the dissolved oxygen was linked to stirring, the ventilation volume was 1 vvm, the fermentation temperature was maintained at 28°C, and the glucose concentration was reduced to 40 g / L and then controlled at 25-37 g / L. The volume ratio of glucose addition to palmitic acid oil or rapeseed oil was maintained at 2.1:1. The fermentation time was 196 h, and the feed amount was consistent during the fermentation of the two bioreactors. Figure 3 As shown in the figure, the yield of palmitic acid oil fermentation reached 300 g / L (and the proportion of acid-type sophorolipids increased significantly), and the final yield of rapeseed oil fermentation reached 280g / L. The laboratory purchased rapeseed oil at an average price of 16 yuan / liter. The average price of the purchased palmitic acid oil was 4.5 yuan / liter, the raw material cost was significantly reduced, and the yield of sophorolipids was significantly improved.

[0040] The present invention provides a method and idea for producing sophorolipids by fermentation using palmitic acidified oil as a substrate. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for producing sophorolipids by fermentation using palmitic acid oil as a substrate, characterized in that: The seed liquid of the sophorolipid-producing strain is inoculated into a fermentation medium for fermentation culture; wherein the fermentation medium comprises a hydrophilic carbon source, a hydrophobic carbon source, a nitrogen source and an inorganic salt; the hydrophobic carbon source is palmitic acid oil; the sophorolipid-producing strain is Candida bombifolia PL0120, which is classified and named Starmerella bombicola, with a preservation number of CGMCC No.29188 and a preservation date of November 30, 2023.

2. The method according to claim 1, characterized in that The sophorolipid-producing strain seed solution is prepared according to the following method: inoculating the sophorolipid-producing strain into a growth medium and culturing at 20-30° C. for 12-48 hours.

3. The method according to claim 2, characterized in that The growth medium is YPD liquid medium.

4. The method according to claim 1, characterized in that: The inoculation amount is 1-10% v / v, the fermentation culture temperature is 20-30° C., and the culture time is 96-168 hours.

5. The method according to claim 1, characterized in that The hydrophilic carbon source is any one or more combinations of glucose, fructose, molasses and lactose; The nitrogen source is any one or more of yeast extract, corn meal, tryptone and fish meal; The inorganic salt is any one or more combinations of disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate.

6. The method according to claim 5, characterized in that The hydrophilic carbon source is glucose, the nitrogen source is a combination of yeast extract and trypsin; and the inorganic salt is a combination of disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate.

7. The method according to claim 6, characterized in that The added mass ratio of the yeast extract and trypsin is 1-3:1-3; the added mass ratio of the disodium hydrogen phosphate, potassium dihydrogen phosphate and magnesium sulfate is 1-3:1-3:1-3.

8. The method according to any one of claims 1 to 7, characterized in that: In the fermentation medium, the concentration of the hydrophilic carbon source is 10-200 g / L, the concentration of the hydrophobic carbon source is 10-200 g / L, the concentration of the nitrogen source is 1-20 g / L, and the concentration of the inorganic salt is 1-20 g / L.

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