Kitchen waste oil-based sophorolipid high-yield candida utilis and application thereof

Abstract

The invention discloses a kitchen waste oil-based sophorolipid high-yield candida utilis strain and an application thereof. Belongs to the technical field of directional domestication breeding. According to the method, wild candida bombicola is used as an original strain, and is sequentially domesticated in a culture medium containing kitchen waste oil with the concentration gradient of 2%, 4%, 6%, 8% and 10% to obtain the S.bombicola x10. The invention further discloses an application of the domesticated strain in fermentation production of sophorolipid, after the strain is subjected to shake flask fermentation, the sophorolipid yield reaches 60.19 g / L and is improved by 16.22% compared with that of a wild strain, and the acid type sophorolipid yield is improved by 32.11%. When the concentration of the lactone sophorolipid generated by fermentation is 1.25 mg / mL, the growth of the bacillus subtilis is completely inhibited. The invention provides an excellent production strain for industrial production of sophorolipid, and has good theoretical research significance and practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of directed domestication breeding, and more specifically, to a Candida parapsilosis strain with high yield of sophorolipid based on kitchen waste oil and its application. Background Art

[0002] Sophorolipids are a class of important amphiphilic biosurfactants. They have good environmental compatibility, biocompatibility, biodegradability, and low toxicity, and are an environmentally friendly surfactant. Due to these properties of sophorolipids, they can be widely used in industries such as daily chemicals, food, environmental protection, and oil extraction. Compared with chemically synthesized surfactants, sophorolipids not only have good surface activity but also have unique biological activities, mainly reflected in their antibacterial and antifungal activities, which can treat sepsis and inhibit the production of dermatophytes, and have activities against liver cancer, esophageal cancer, cervical cancer, etc. Sophorolipid molecules with different structures have different biological activities. Acidic sophorolipids have good surface activities such as wetting, emulsifying, and foaming abilities, while lactone-type sophorolipids have better biological activities such as reducing surface tension, antibacterial, anti-inflammatory, and antiviral activities.

[0003] In the prior art, sophorolipids can be prepared by fermenting Candida bombicola S.bombicola. However, the current yield of sophorolipids by Candida bombicola strains is relatively low and cannot meet the market and large-scale industrial production requirements. Therefore, there is an urgent need to develop strains with high yield of sophorolipids. Summary of the Invention

[0004] In view of this, the present invention provides a Candida parapsilosis strain with high yield of sophorolipid based on kitchen waste oil and its application.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A Candida parapsilosis strain with high yield of sophorolipid based on kitchen waste oil, named Candida parapsilosis x10, its taxonomic name is Starmerella bombicola, was deposited in the China General Microbiological Culture Collection Center on August 12, 2024, with the deposit number CGMCC No. 31660, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The above-mentioned Candida parapsilosis strain is used in the utilization of kitchen waste oil.

[0008] The above-mentioned Candida parapsilosis strain is used in the fermentation production of sophorolipids.

[0009] Further, after the strain is activated, it can be fermented through seed liquid fermentation and shake flask fermentation in sequence.

[0010] Furthermore, in the seed culture fermentation, the temperature was 30 °C, the rotation speed was 200 rpm, and the culture time was 30 h.

[0011] Furthermore, in the shake flask fermentation, the culture was carried out at 30 °C and 200 rpm for 7 days.

[0012] Furthermore, in the seed culture fermentation, the medium composition was as follows: glucose 20.0 g / L, peptone 20.0 g / L, yeast extract 10.0 g / L.

[0013] Furthermore, in the shake flask fermentation, the medium composition was as follows: glucose 80.0 g / L, yeast extract 3.0 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 1.0 g / L, MgSO4·7H2O 0.5 g / L, and kitchen waste oil 80 mL / L.

[0014] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0015] The present invention provides a domesticated bacterium for high-yield sophorolipid production using kitchen waste oil and its application. Currently, the reported methods for improving sophorolipid production mainly include screening and identifying new sophorolipid-producing bacteria, plasma or ultraviolet mutagenesis technology for breeding screening, optimization of culture media and culture conditions, etc. The present invention uses the concentration gradient domestication of kitchen waste oil to complete the directional selection of high-yield sophorolipid strains, providing a new approach for the directional breeding of sophorolipid strains, laying a foundation for the development of engineering bacteria for high-yield sophorolipid production using cheap substrates and large-scale industrial production of sophorolipids, with broad application prospects, environmental protection value and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0017] Figure 1 It is the schematic diagram of domestication of strains using the gradient of kitchen waste oil in Example 1 of the present invention;

[0018] Figure 2 It is the phylogenetic tree analysis of the domesticated strain S. bombicola x10 in Example 2 of the present invention;

[0019] Figure 3 It is the sophorolipid fermentation result of the strain in Example 4 of the present invention;

[0020] Figure 4This is the HPLC analysis result of sophorolipids in Example 4 of the present invention. Among them, a represents the result of the wt wild-type strain; B represents the result of S. bombicola x10; peak 1 represents C18:2 NASL, peak 2 represents C18:1 MASL, peak 3 represents C18:2 DLSL, and peak 4 represents C18:1 DLSL, where NASL is non-acetylated acid-type sophorolipid, MASL is mono-acetylated acid-type sophorolipid, and DLSL is di-acetylated lactone-type sophorolipid;

[0021] Figure 5 This is the growth curve of sophorolipid components inhibiting Bacillus subtilis in Example 6 of the present invention. Detailed implementation manners

[0022] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] General description:

[0024] Composition of YEPD liquid medium (1L): glucose 20.0 g, peptone 20.0 g, yeast extract 10.0 g; YEPD seed solid medium also adds agar powder 20.0 g.

[0025] Composition of LB liquid medium (1L): tryptone 10.0 g, yeast extract 5.0 g, sodium chloride 10.0 g, pH value 7.0.

[0026] Composition of gradient acclimation medium (1L): glucose 20.0 g, peptone 20.0 g, yeast extract 10.0 g, adding gradient concentrations of 2%, 4%, 6%, 8%, 10% (v / v) of kitchen waste oil (obtained by filtering the waste oil after repeated frying). It is used for the directional acclimation culture of strains.

[0027] Composition of fermentation medium (1L): glucose 80.0 g, yeast extract 3.0 g, KH2PO4 1.0 g, Na2HPO4·12H2O 1.0 g, MgSO4·7H2O 0.5 g, kitchen waste oil 80 mL.

[0028] Example 1

[0029] Tolerance acclimation of Candida guilliermondii to kitchen waste oil

[0030] As Figure 1As shown, the starting strain was cultured successively in liquid media containing kitchen waste oil with concentration gradients of 2%, 4%, 6%, 8%, and 10%. After culturing, single colonies with fast growth and large colonies were selected by streaking on the plate for domestication at the next concentration gradient. Finally, the strain domesticated in the medium containing 10% kitchen waste oil was obtained. The specific operation steps are as follows:

[0031] (1) Domestication of the strain with 2% kitchen waste oil: After the wild-type Candida bombicola (S. bombicola) was activated in YEPD liquid medium, the OD 600 was adjusted to 1, and it was transferred at 2% (v / v) to the gradient domestication medium containing 2% kitchen waste oil and cultured at 30 °C and 200 rpm for two days; 100 μL of the bacterial suspension was spread on the YEPD seed solid medium and cultured in an incubator at 30 °C for 2 - 3 days. The single colony with the fastest growth was selected, and the strain was named S. bombicola x2, which was used for the domestication of 4% kitchen waste oil.

[0032] (2) Domestication of the strain with 4% kitchen waste oil: The specific operation was the same as described in the above step (1). After S. bombicola x2 was activated in YEPD liquid medium, the OD 600 was adjusted to 1, and it was transferred at 2% (v / v) to the gradient domestication medium containing 4% kitchen waste oil and cultured at 30 °C and 200 rpm for two days; 100 μL of the bacterial suspension was spread on the YEPD seed solid medium and cultured in an incubator at 30 °C for 2 - 3 days. The single colony with the fastest growth was selected, and the strain was named S. bombicola x4, which was used for the domestication of 6% kitchen waste oil.

[0033] (3) Domestication of the strain with 6% kitchen waste oil: The specific operation was the same as described in the above step (1). After S. bombicola x4 was activated in YEPD liquid medium, the OD 600 was adjusted to 1, and it was transferred at 2% (v / v) to the gradient domestication medium containing 6% kitchen waste oil and cultured at 30 °C and 200 rpm for two days; 100 μL of the bacterial suspension was spread on the YEPD seed solid medium and cultured in an incubator at 30 °C for 2 - 3 days. The single colony with the fastest growth was selected, and the strain was named S. bombicola x6, which was used for the domestication of 8% kitchen waste oil.

[0034] (4) Domestication of the strain with 8% kitchen waste oil: The specific operation was the same as described in the above step (1). After S. bombicola x6 was activated in YEPD liquid medium, the OD 600= 1, transfer 2% (v / v) to the gradient acclimation medium containing 8% kitchen waste oil, culture at 30 °C and 200 rpm for two days; take 100 μL of the bacterial suspension and spread it on the YEPD seed solid medium, culture in an incubator at 30 °C for 2 - 3 days, select the single colony with the fastest growth and the largest colony, and name it S. bombicolax8 for the acclimation of 10% concentration kitchen waste oil.

[0035] (5) Strain acclimation of 10% concentration kitchen waste oil: The specific operation is the same as described in step (1) above. After activating S. bombicola x8 in the YEPD liquid medium, adjust OD 600 = 1, transfer 2% (v / v) to the gradient acclimation medium containing 10% kitchen waste oil, culture at 30 °C and 200 rpm for two days; take 100 μL of the bacterial suspension and spread it on the YEPD seed solid medium, culture in an incubator at 30 °C for 2 - 3 days, select the single colony with the fastest growth and the largest colony, and name it S. bombicolax10.

[0036] Example 2

[0037] Identification and preservation of strains

[0038] Amplify the 26s rDNA sequence of the acclimated strain S. bombicola x10 for sequencing, and conduct online alignment analysis in the NCBI database. It is found that the sequence identity with the S. bombicola strain sequence reaches 100%. Construct a phylogenetic tree as Figure 2 shown. Therefore, according to the molecular identification results, it is proved that the acclimated strain belongs to S. bombicola.

[0039] The above-mentioned S. bombicola x10 is named Candida parapsilosis x10, and its taxonomic name is Starmerella bombicola. It was preserved in the China General Microbiological Culture Collection Center on August 12, 2024, with the preservation number CGMCC No. 31660 and the preservation address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0040] Example 3

[0041] Sophorolipid fermentation

[0042] (1) Inoculate the wild strain of Candida parapsilosis (S. bombicola) preserved in a glycerol tube and the acclimated strain S. bombicola x10 (CGMCC No. 31660) into 5 mL of YEPD liquid medium respectively, and culture at 30 °C and 200 rpm for activation.

[0043] (2) Inoculate the activated bacterial liquid at an inoculation amount of 1%, and transfer it to a 300 mL Erlenmeyer flask containing 50 mL of YEPD liquid medium. Incubate at 30 °C and 200 rpm for 1 - 2 days until OD 600 = 1.

[0044] (3) In a sterile environment, transfer the bacterial suspensions of the wild strain and the domesticated strain obtained in step (2) to 300 mL conical flasks containing 50 mL of fermentation medium at an inoculation amount of 2% respectively. Culture at 25 - 30 °C and 180 - 200 rpm for 7 - 14 days to obtain the fermentation broth containing sophorolipids.

[0045] All fermentations have at least three biological replicates. The dosage for fermentation tank fermentation can be scaled up proportionally according to the dosage in shake flasks.

[0046] The preferred culture conditions for the above step (2): 30 °C, rotation speed of 200 rpm, culture for 30 h.

[0047] The preferred culture conditions for the above step (3): 28 °C, rotation speed of 200 rpm, culture for 7 days.

[0048] Example 4

[0049] Determination of sophorolipid yield

[0050] After the wild - type strain and the domesticated strain are fermented in the fermentation medium for 7 days, take the fermentation broth samples to measure various parameters such as the yields of lactone - type, acid - type and total sophorolipids, the components of sophorolipids, and the residual glucose amount.

[0051] (1) The residual glucose in the fermentation broth is measured using the biosensor SBA - 40C.

[0052] Take 1 mL of the fermentation broth, centrifuge it and take the supernatant, dilute it to an appropriate multiple and measure the glucose content with the biosensor SBA - 40C. The injection volume is 25 μL, and the glucose content is G = n×m / 100 (n is the instrument reading, m is the dilution multiple).

[0053] (2) The content of sophorolipids in the fermentation broth is measured by the anthrone - sulfuric acid method.

[0054] Add twice the volume of ethyl acetate to 0.5 mL of the fermentation broth, vortex - oscillate it, then let it stand at room temperature for 10 min, centrifuge at 10,000 rpm for 10 min, and take the supernatant containing lactone - type sophorolipids to measure the content of lactone - type sophorolipids by the anthrone - sulfuric acid method.

[0055] 3.5 mL of anhydrous ethanol was added to 0.5 mL of the fermentation broth. After vortex oscillation, it was left standing at room temperature for 10 min, and then centrifuged at 10,000 rpm for 10 min at room temperature. The total sugar content in the supernatant was determined by the anthrone-sulfuric acid method. The content of total sophorolipids was the total sugar content minus the residual glucose amount in the fermentation broth, and then multiplied by the conversion factor 1.91.

[0056] The content of acidic sophorolipids was the content of total sophorolipids minus the content of lactone-type sophorolipids.

[0057] (3) Component analysis of sophorolipids.

[0058] ① Preparation of crude sophorolipids.

[0059] Two volumes of chromatographically pure acetonitrile were added to one volume of the fermentation broth. After sufficient shaking, it was left standing at room temperature for 10 min, and then centrifuged at 8,000 - 10,000 rpm for 10 min at room temperature. The supernatant was collected and dried in an oven at 50 - 60 °C. The obtained crude sophorolipids were washed twice with n-hexane to remove the residual oleic acid, and then dried to a constant weight again.

[0060] ② The components of sophorolipids in the fermentation broth were analyzed by high-performance liquid chromatography (HPLC).

[0061] The crude sophorolipids were dissolved in chromatographically pure acetonitrile and then filtered through a 0.22 μm organic filter membrane. The sophorolipid samples were detected using analytical HPLC. The chromatographic column used was Venusil MP-C18 column (250 mm × 4.6 mm, Agela Technologies Inc., USA). Binary gradient elution was used, and the mobile phase was water and acetonitrile with a flow rate of 1.0 - 1.5 mL / min. The HPLC program was as follows: from 0 to 15 min, the acetonitrile concentration increased from 40% to 60%; from 15 to 30 min, the acetonitrile concentration slowly increased from 60% to 70%; from 30 to 40 min, the acetonitrile concentration increased from the original 70% to 90% and was held for 5 min. The injection volume was 20 - 25 μL, and the detection wavelength of the detector was 207 nm.

[0062] In the above step ①, the centrifugation speed was preferably 10,000 rpm.

[0063] In the above step ②, the flow rate of the HPLC was preferably 1.0 mL / min.

[0064] In the above step ②, the injection volume of the HPLC was preferably 20 μL.

[0065] The yield of sophorolipids was as Figure 3As shown in the figure, the high-yield sophorolipid-producing strain S. bombicola x10 obtained by the method of gradient domestication of kitchen waste oil in the present invention has a sophorolipid yield of 60.19 g / L after shake-flask fermentation, which is 16.22% higher than that of the wild-type strain. In particular, the yield of acid-type sophorolipids is increased by 32.11%.

[0066] The HPLC analysis of the sophorolipid components is as Figure 4 shown. In the S. bombicola x10 strain, the peak area of the lactone-type sophorolipid component is basically the same as that of the starting strain, while the peak 2 in the acid-type sophorolipid component is higher than that of the wild strain. The component of peak 2 is monoacetylated acid-type sophorolipid (C18:1MASL) with a fatty acid chain of 18 carbons and 1 double bond, and the peak area is increased from 1.38×10 6 mAU×s to 7.61×10 6 mAU×s.

[0067] Example 5

[0068] Preparation of crude sophorolipids

[0069] The fermentation broth of the domesticated strain S. bombicola x10 after 7 days of fermentation was added with twice the volume of ethyl acetate for extraction. After sufficient shaking, it was left to stand at room temperature for 1 - 2 h in a separating funnel, and the upper organic phase was collected and rotary evaporated under reduced pressure at 50°C. The solid was washed twice with n-hexane to remove residual grease, and was also rotary evaporated under reduced pressure at 50°C using a rotary evaporator. The solid was dried in an oven to obtain a yellowish-brown solid, which is the lactone-type sophorolipid sample.

[0070] The lower layer liquid after ethyl acetate extraction was added with 2 times the volume of absolute ethanol to dissolve. After sufficient shaking, it was left to stand at room temperature for 1 - 2 h in a separating funnel, and the upper layer liquid was collected and rotary evaporated under reduced pressure at 50°C. The solid was washed twice with n-hexane and dried to obtain the acid-type sophorolipid sample.

[0071] Example 6

[0072] Inhibition of the growth of Bacillus subtilis by sophorolipids

[0073] An appropriate amount of lactone-type sophorolipid was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 100 mg / mL. By calculation, the lactone-type sophorolipid stock solution was added to the LB liquid medium to make the sophorolipid concentration in the medium 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL respectively. 50 μL of a Bacillus subtilis bacterial suspension with OD600 = 1 was inoculated, and it was cultured at 37°C for 14 h. Samples were taken and the growth curve of the strain was measured to observe the growth of Bacillus subtilis in lactone-type sophorolipids at different concentrations, and to analyze the antibacterial performance of the lactone-type sophorolipid fermented and synthesized by the domesticated strain. The results are as Figure 5 shown.

[0074] When the sophorolipid concentration was 1.25 mg / mL, the growth of Bacillus subtilis was completely inhibited.

[0075] In summary, by using the method of gradient domestication of kitchen waste oil, the Candida parapsilosis strain S. bombicola x10 with significantly increased sophorolipid production was obtained, providing an excellent production strain for the industrial production of sophorolipids. Among them, the obtained lactone-type sophorolipid has good antibacterial activity and broad application prospects. The strain of the present invention has great theoretical research significance and practical application value.

[0076] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0077] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Candida guilliermondii strain with high yield of sophorolipid based on kitchen waste oil, characterized in that, Named Candida parapsilosis x10, its taxonomic name is Starmerella bombicola, and it was deposited in the China General Microbiological Culture Collection Center on August 12, 2024. The deposit number is CGMCC No. 31660, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Application of the Candida parapsilosis described in claim 1 in the utilization of kitchen waste oil.

3. Application of the Candida parapsilosis described in claim 1 in the fermentation production of sophorolipids.

4. The application according to claim 3, wherein After the strain is activated, it can be fermented through seed liquid fermentation and shake flask fermentation in sequence.

5. The application according to claim 4, characterized in that, In the seed liquid fermentation, at 30 °C, the rotation speed is 200 rpm, and the culture is carried out for 30 h.

6. The application according to claim 4, wherein In the shake flask fermentation, at 30 °C and 200 rpm, the culture is carried out for 7 days.

7. The application according to claim 4, characterized in that, In the seed liquid fermentation, the medium composition is as follows: glucose 20.0 g / L, peptone 20.0 g / L, yeast powder 10.0 g / L.

8. The application according to claim 4, characterized in that In the shake flask fermentation, the medium composition is as follows: glucose 80.0 g / L, yeast powder 3.0 g / L, KH2PO4 1.0 g / L, Na2HPO4·12H2O 1.0 g / L, MgSO4·7H2O 0.5 g / L, kitchen waste oil 80 mL / L.

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