WO2024147665A1 - Method for culturing candida bombicola using coffee waste, and cultured substance therefrom - Google Patents

Abstract

The present invention relates to: a Candida bombicola culturing medium composition using coffee waste; a Candida bombicola culturing method using same; and an eco-friendly cultured and purified substance of Candida bombicola that does not use an organic solvent. The present invention can obtain a cultured product having high added value by culturing Candida bombicola using discarded coffee waste, and can produce a cultured and purified substance by means of a method that does not affect the environment.

Description

Cultivation method of Candida bombicola using coffee grounds and cultures therefrom

This patent application claims priority to Korean Patent Application No. 10-2023-0001984 filed with the Korean Intellectual Property Office on January 5, 2023, the disclosure of which is incorporated herein by reference.

The present invention relates to a method for cultivating Candida bombicola using coffee grounds and cultures therefrom.

Recently, as the demand for coffee has increased significantly, a large amount of coffee grounds are being produced. Until now, coffee grounds have been used as air fresheners, dehumidifiers, and fertilizers, but only a small amount of coffee grounds are recycled, and most of the coffee grounds are discarded. According to statistics, approximately 120,000 tons of coffee grounds are generated annually in Korea, and the cost of disposing of them incurs a social cost of approximately 90 billion won.

The present inventors sought to develop a method for utilizing coffee grounds, which are the coffee grounds described above. Among them, we sought to develop a method of using coffee grounds as a culture medium component of Candida bombicola to produce sophorolipids with high added value.

Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

The present inventors made extensive research efforts to develop a method for utilizing the coffee grounds, which are the coffee grounds described above. As a result, it did. The present invention was completed by demonstrating that large quantities of sophorolipids can be produced when coffee grounds are used as a culture medium component for Candida bombicola.

Therefore, the purpose of the present invention is to provide a medium composition for cultivating Candida bombicola using coffee grounds.

Another object of the present invention is to provide a method for cultivating Candida bombicola using a culture medium composition for Candida bombicola containing the above-described coffee grounds.

Another object of the present invention is to provide a method for producing purified culture of Candida bombicola.

Another object of the present invention is to provide a cosmetic composition for promoting hair growth or preventing hair loss containing purified Candida bombicola culture.

Another object of the present invention is to provide a cleaning composition containing purified Candida bombicola culture.

Another object of the present invention is to provide a composition for pesticide cleaning containing purified Candida bombicola culture.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention provides a medium composition for culturing Candida bombicola using coffee grounds.

The medium composition for cultivating Candida bombicola includes vegetable oil, glucose, and coffee grounds. The Candida bombicola is known to produce sophorolipids.

Sophorolipids are surface-active molecules composed of dimeric sugars with hydroxy fatty acid residues linked by glycosidically bonds, and are synthesized in many yeast species to exist in lactonic and acidic forms. Although sophorolipids have been known for more than 40 years, they have only recently received attention as microbial surfactants due to their characteristics such as biodegradability, low ecotoxicity, and production from renewable sources (Ashby et al., 2006; Van Bogaert et al. , 2007). As described above, sophorolipids are synthesized through bioconversion of saccharides and/or vegetable oils/hydrocarbons by Candida sp. and are released extracellularly under appropriate growth conditions (Felse et al. , 2007; Sarubbo et al., 2007).

In one embodiment of the present invention, the coffee grounds are included at a concentration of 5 to 20% (w/v), but are not limited thereto.

In one embodiment of the present invention, the vegetable oil is shea butter, mango kernel oil, palm oil, palm kernel oil, coconut oil, Sarah Shorea Robusta Seed Butter, margarine, cocoa butter, or a combination thereof is maintained in a solid state at room temperature, but is not limited thereto.

In another embodiment of the present invention, the vegetable oil is olive oil, rapeseed oil, soybean oil, peanut oil, grape seed oil, sunflower seed oil, rice bran oil, safflower oil, cottonseed oil, corn oil, perilla oil, sesame oil, or a combination thereof at room temperature. It is maintained in a liquid state, but is not limited thereto.

In one embodiment of the present invention, the vegetable oil is 5 to 20% (w/v), 5 to 15% (w/v), 5 to 10% (w/v), 10 to 20% in the medium composition. (w/v), 10 to 15% (w/v), 5 (w/v), 10% (w/v), or 15 (w/v).

In one embodiment of the present invention, the medium composition contains 5 to 20% (w/v), 5 to 15% (w/v), 5 to 10% (w/v), 10 to 10% glucose in the medium composition. It may be included at a concentration of 20% (w/v), 10 to 15% (w/v), 5 (w/v), 10% (w/v), or 15 (w/v).

According to another aspect of the present invention, the present invention provides a method for cultivating Candida bombicola using a culture medium composition for Candida bombicola containing the above-described coffee grounds.

The culture method includes the steps of inoculating and culturing Candida bombicola in the medium composition of the present invention described above.

The culturing may be performed at a temperature of 25°C to 35°C.

In addition, the culture is performed at 150 to 500 rpm, 150 to 400 rpm, 150 to 350 rpm, 150 to 300 rpm, 200 to 500 rpm, 200 to 400 rpm, 200 to 350 rpm, 200 to 300 rpm, 250 to 500 rpm, Stirring at a speed of 250 to 400 rpm, 250 to 350 rpm, 250 to 300 rpm, 300 to 500 rpm, 300 to 400 rpm, 300 to 350 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, or 400 rpm. It can be done.

Additionally, the culture may be performed for 6 to 12 days, 7 to 12 days, 8 to 12 days, 9 to 12 days, or 10 to 12 days.

Additionally, the culture may be performed under a combination of the temperature, stirring speed, and culture time conditions described above.

According to another aspect of the present invention, the present invention provides a method for producing a purified culture of Candida bombicola comprising the following steps.

(a) removing the maintenance layer from the culture medium;

(b) removing the aqueous layer from the culture medium; and

(c) Obtaining a residual layer containing sophorolipids from the culture medium.

Hereinafter, the method for producing purified culture of Candida bombicola will be described step by step.

(a) removing the maintenance layer from the culture medium;

This step is to remove fat from the culture medium in which Candida bombicola was cultured.

The oil layer includes residual vegetable oil contained in the culture medium composition.

The step additionally includes adding hydrogenated oil to the culture medium before removing the maintenance layer.

In addition, the above step additionally includes the step of heating and then cooling the culture solution to which hydrogenated oil has been added.

In one embodiment of the present invention, hydrogenated oil refers to a liquid oil with a high unsaturated fatty acid content hydrogenated to obtain a solid state oil, and is also called hydrogenated oil. Examples of the hydrogenated oil include hydrogenated palm oil, hydrogenated palm oil, hydrogenated soybean oil, hydrogenated olive oil, thinning, margarine, etc., but are not limited thereto.

In one embodiment of the present invention, the heating may be performed up to 100 to 150°C.

In one embodiment of the present invention, the cooling may be performed to 15 to 30°C.

In a specific embodiment of the present invention, the heating may be performed at 121°C for 30 minutes, stirred at 90°C and mixed well, then stirring may be stopped and cooled to 25°C.

As described above, when hydrogenated oil is added to the culture medium, the remaining oil solidifies together with the hydrogenated oil as the culture medium cools, making recovery easy.

(b) removing the aqueous layer from the culture medium;

This step is a step of removing the aqueous layer, which is the middle layer, after removing the uppermost retaining layer in step (a).

As in step (a) above, when hydrogenated oil is added to the culture medium, heated, and the culture medium is cooled, the remaining oil solidifies together with the hydrogenated oil and the layers are separated. As a result, it is separated into three layers from the top: an oil layer, a water layer, and a residual layer containing sophorolipids.

At this time, if the oil layer (solid oil layer) is removed from the upper layer of the sterilized and cooled culture medium and the middle aqueous layer (water layer) is removed, the lower layer containing a lot of sophorolipids can be recovered in the next step.

(c) Obtaining a residual layer containing sophorolipids from the culture medium.

After removing the holding layer in step (a) and the aqueous layer in step (b), the remaining layer is obtained in step (c).

As shown in the comparative example of the present invention, a general method for purifying sophorolipids is to remove residual oil in the culture medium with hexane and separate layers with ethyl acetate to obtain bulky sophorolipids. . This purification method uses organic solvents, which can increase product costs, and the process of recovering and disposing the used solvent is cumbersome and unenvironmentally friendly.

In order to solve the above problem, the present inventors sought a method to produce sophorolipids without using a solvent. As a result, it was confirmed that when solid hydrogenated oil was added to the culture medium and subjected to heat treatment as described above, as the culture medium was cooled, the remaining oil was collected toward the solid hydrogenated oil and solidified, making recovery easier. By removing the solid oil layer from the upper layer of the sterilized and cooled culture medium and removing the middle water layer, the lower sophorolipid-rich layer can be recovered.

According to another aspect of the present invention, the present invention provides a cosmetic composition for promoting hair growth or preventing hair loss containing sophorolipids derived from Candida bombicola culture medium.

The sophorolipid derived from the Candida bombicola culture may be derived from a purified Candida bombicola culture prepared according to an aspect of the present invention described above.

In one embodiment of the present invention, the cosmetic composition may include the purified Candida bombicola culture.

As shown in one embodiment of the present invention, the sophorolipid derived from the Candida bombicola culture medium of the present invention or the Candida bombicola culture purified product has excellent proliferation effects on dermal papilla cells and VEGF activity, and is therefore useful as a composition for promoting hair growth or preventing hair loss. It can be used effectively.

In one embodiment of the present invention, the cosmetic composition of the present invention may include not only the active ingredient, sophorolipid derived from the Candida bombicola culture medium, or Candida bombicola culture purified product, but also components commonly used in cosmetic compositions. It may include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

As the carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitic acid or linolenic acid), oils and fats (wheat germ oil, Camellia oil, jojoba oil, olive oil, squalane, sunflower oil, macadamia peanut oil, Avogard oil, hydrogenated soybean lecithin or fatty acid glycerides) can be used, but are not limited to these. Additionally, surfactants, disinfectants, antioxidants, ultraviolet absorbers, anti-inflammatory agents, and fresheners can be added as needed.

Surfactants include polyoxyethylene, hydrogenated castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, polyoxyethylene, glyceryl monostearate, sorbitan monostearate, polyoxyethylene monooleate, sorbitan, Sucrose fatty acid ester, hexaglycerin monolauric acid, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N-acetylglutamine, and isostearyl ester can be used.

Disinfectants include hinocthiol, tricrosan, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, and zinc pyritaone.

As an antioxidant, any of butylhydroxyanisole, gallic acid, propyl gallic acid, and erythorbic acid can be used.

Ultraviolet absorbers include benzophenones such as dihydroxybenzophenone, melanin, ethyl para-aminobenzoate, 2-ethylhexyl para-dimethylaminobenzoic acid, synoxite, para-methoxycinnamic acid 2-ethylhexyl ester, 2-(2- Hydroxy-5-methylphenyl) benzotriazole, urochanic acid, and metal oxide fine particles can be used.

As an anti-inflammatory agent, dipotassium glycyrrhizinate or allantoin can be used, and as a refreshing agent, red pepper tincture or 1-menthol can be used.

In another embodiment of the present invention, the cosmetic composition of the present invention may be used as a solution, external ointment, gel, cream, foam, nutritional lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, ampoule, hair ampoule, dufitreet. hair treatment, hair tonic, hair conditioner, hair treatment, hair lotion, hair shampoo, hair rinse, conditioner shampoo, nourishing hair lotion, hair gel, hair wax, hair spray, hair dye, soap, liquid detergent, bath salt, sunscreen Prepared in a formulation selected from the group consisting of creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays. It may be possible, but it is not limited to this.

According to another aspect of the present invention, the present invention provides a cleaning composition containing sophorolipids derived from Candida bombicola culture medium.

The sophorolipid derived from the Candida bombicola culture may be derived from a purified Candida bombicola culture prepared according to an aspect of the present invention.

In one embodiment of the present invention, the cleaning composition may include the purified Candida bombicola culture.

In one embodiment of the present invention, the cleaning composition may be a washer fluid composition.

The cleaning composition containing the sophorolipid derived from the Candida bombicola culture of the present invention, or the purified product of the Candida bombicola culture, has initial water repellency, wet water repellency durability, and dry water repellency durability, as shown in one embodiment of the present invention. Since it has excellent cleaning properties, phase stability, and freezing performance, it can be usefully used as a cleaning composition, especially a washer fluid composition.

In one embodiment of the present invention, the cleaning composition containing the sophorolipid derived from the Candida bombicola culture of the present invention, or the purified product of the Candida bombicola culture, has initial water repellency, wet, as shown in one embodiment of the present invention. It has excellent water repellency durability, dry water repellency durability, cleaning properties, phase stability, and freezing performance, so it can be usefully used as a cleaning composition.

According to another aspect of the present invention, the present invention provides a composition for pesticide cleaning containing a sophorolipid derived from a Candida bombicola culture medium.

The sophorolipid derived from the Candida bombicola culture may be derived from a purified Candida bombicola culture prepared according to an aspect of the present invention.

In one embodiment of the present invention, the composition for pesticide cleaning may include the purified Candida bombicola culture.

As shown in one embodiment of the present invention, the composition containing the sophorolipid derived from the Candida bombicola culture of the present invention, or the Candida bombicola culture purified product, has excellent pesticide cleaning effect and can be usefully used as a pesticide cleaning composition. You can.

The present invention provides a culture medium composition for Candida bombicola using coffee grounds, a method for cultivating Candida bombicola using the same, and an eco-friendly purified culture of Candida bombicola that does not use organic solvents.

In the present invention, culture products with high added value can be obtained by cultivating Candida bombicola using discarded coffee grounds, and culture purification products can be produced in a manner that does not affect the environment.

Figure 1 is a diagram showing the production of sophorolipids according to the content of coffee grounds in the medium composition.

Figure 2 is a diagram showing the production of sophorolipids according to the culture time of the present invention.

Figures 3a and 3b are schematic diagrams comparing the eco-friendly sophorolipid purification method of the present invention and the conventional sophorolipid purification method using an organic solvent.

Figure 4 is a diagram showing a comparison of the antioxidant efficacy of the coffee waste fermentation product of the present invention, general fermentation product, and ascorbic acid.

Figure 5 is a schematic diagram of applying the purified culture medium to a composition for cosmetics, washer fluid, and pesticide detergent after purification using the eco-friendly purification method of fermented coffee waste of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1: Selection of optimal coffee waste medium concentration of coffee waste sophorolipids (flask experiment)

The present inventors cultivated Candida bombicola using coffee grounds and tested the composition of the medium for mass production of sophorolipids as follows.

In a 1L flask, 10g of rapeseed oil, 10g of glucose, coffee grounds (1%(w/v), 3%(w/v), 5%(w/v), 10%(w/v), 15%(w/v) ), 20% (w/v)) were added, and 100 mL of deionized water was added. The solution was sterilized at 121°C for 30 minutes, then 5 mL of Candida bombicola seed solution was added to the solution, and cultured for 10 days at 30°C while stirring at 160 rpm. After 10 days, the pH of the culture medium was measured.

Additionally, in order to measure the amount of sophorolipids produced, 50 mL of hexane was added to 100 mL of the culture medium, and then the hexane in the upper layer was removed to remove the oil. The above process was repeated one more time. Next, 100 mL of ethyl acetate was added to the lower water layer, and the process of recovering the supernatant was repeated three times. The recovered liquid was concentrated and the amount of bulky sophorolipid was measured.

The results are shown in Figure 1 and Table 1.

Coffee waste concentration One% 3% 5% 10% 15% 20% Sophorolipid yield (g/L) 5 3 23 54 38 14 pH after incubation 5.1 4.2 3.5 2.9 2.9 3.9

As shown in Figure 1 and Table 1, when the concentration of coffee grounds was 5 to 20%, the yield of sophorolipids was excellent and the pH was maintained below 4.0 after cultivation. The pH must be maintained below 4.0 to remove Candida bombicola from Candida bombicola. Sophorolipids are produced. Since the initial pH when the concentration of coffee grounds is 1% is 5, the pH must fall below 4 for Candida bombicola bacteria to be enriched and sophorolipids to be produced.

Example 2: Coffee waste sophorolipid 30L culture experiment (135g/L)

The present inventors added 10% (w/v) of coffee grounds, 10% (w/v) of rapeseed oil, and 10% (w/v) of glucose to deionized water and cultured the mixture for 12 days in a culture medium volume of 15 L. Cultivation was performed at 30°C, 300rpm, and 0.8vvm. As culture factors, pH and glucose (using a blood sugar meter) were measured. Additionally, 10 mL of culture medium was taken, 5 mL of hexane was added, the hexane in the upper layer was removed to remove oil, and the above process was repeated one more time. Next, 10 mL of treated ethyl acetate was added to the lower water layer, and the process of recovering the supernatant was repeated three times. The recovered liquid was concentrated and the amount of bulky sophorolipid was measured.

The results are shown in Figure 2.

As shown in Figure 2, the production of sophorolipids increased over time under the above culture conditions, and the amount of sophorolipids reached its maximum value around 9-10 days after the start of culture.

Example 3: Eco-friendly purification method (compared to conventional solvent purification method)

The culture broth cultured in a 30L fermenter was purified under two conditions. The first condition (comparative example, conventional method, solvent purification method) is a method of purification with hexane and ethyl acetate, and the second condition (the present invention, eco-friendly purification method) is a method of purification using hydrogenated oil and natural layer separation without using a solvent. am.

Comparative Example - Solvent Purification Method

As a comparative example of the present invention, solvent purification was performed as follows.

First, 1L of hexane was added to 5L of the culture medium cultured in the fermenter, and the upper oil layer was removed after waiting for the layers to separate. Layer separation and oil layer removal were repeated twice. Next, 5 L of ethyl acetate was added to the 5 L of culture medium from which the remaining oil was removed, and the ethyl acetate layer was recovered by waiting for the layers to separate. After layer separation and ethyl acetate layer recovery were repeated three times, the recovered liquid was concentrated to a solid content of more than 60%.

The present invention - eco-friendly purification method

The eco-friendly purification method of the present invention was carried out as follows.

500 g of hydrogenated oil (95% palm stearin oil, 5% glycerin fatty acid ester) was added to 5 L of the culture medium, and sterilized at 121°C for 30 minutes. The sterilized solution was stirred at 90°C and mixed well, then the stirring was stopped and allowed to naturally cool to 25°C for 1 day. After 1 day, the culture medium was separated into an upper layer (solid hydrogenated oil and residual oil present), a middle layer (water and medium components), and a lower layer (water and substances containing a large amount of sophorolipids). The lower layer was carefully recovered, and the amount of bulky sophorolipid was measured. In the comparative example, the sample recovered from 5L of culture medium using the conventional solvent purification method was 750g, and the sample recovered from 5L using the eco-friendly purification method was 2,500g. As a result of analyzing the samples recovered using an eco-friendly purification method, 65% were water components, 25% were bulky sophorolipids, 7% were culture fluid water-soluble components, and 3% were culture fluid fat-soluble components. Assuming that bulky sophorolipids are the product, the purity of products using existing solvents is 100%, and the purity of samples manufactured using eco-friendly purification methods is 83%. That is, the sophorolipid production in the comparative example was about 150 g/L, and the sophorolipid production in the example was about 120 g/L.

As can be seen from the above results, the purity of sophorolipid products is lower than that of purified products using solvent purification methods, but the eco-friendly purification method does not use solvents during purification, so the cost of expensive solvents can be reduced. , environmental pollution caused by the use of solvents can be prevented.

Example 4: Antioxidant activity (excellent antioxidant activity)

Free radical scavenging ability was confirmed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. Samples of various concentrations (0.01, 0.1, 1, 10 mg/ml) were dissolved in methanol and used. 100 μL of DPPH 300 μM solution was added to 100 μL of sample, mixed, reacted at 37°C for 30 minutes, and absorbance was measured at 517 nm. Free radical scavenging ability was calculated using the formula below.

ceremony

Antioxidant percentage(%) = (1 - absorbance of test substance added group/absorbance of no group added) x 100

Ascorbic acid, which is known to have antioxidant activity, was used as a positive control sample, and fermented product produced using coffee grounds and purified using an eco-friendly refining method was used as the experimental group. Produced using an industrial medium and then purified using a conventional solvent. The general fermented product purified by the method was used as a control. The free radical scavenging ability of each sample was compared according to the above formula.

The results are shown in Figure 4.

As shown in Figure 4, the fermented coffee waste of the present invention had much better antioxidant efficacy at all concentrations than the general fermented product.

Example 5: Sensory evaluation

The odor intensity and preference of fermented products produced using the coffee waste of the present invention and general fermented products were confirmed through sensory evaluation. The 20 panelists who participated in this test received prior training on sensory evaluation, were allowed to conduct objective evaluations in an independent space, and were allowed sufficient time to evaluate samples.

The intensity of the smell was evaluated on a 9-point scale: 1 point very weak, 3 points weak, 5 points average, 7 points strong, 9 points very strong, and the smell preference was 1 point very bad, 3 points bad, and 5 points. It was evaluated on a 9-point scale, with 7 being average, 7 being good, and 9 being very good.

The results are shown in Table 2.

Sample name intensity of odor Smell Preference General fermented product 1% 5.75±0.72 3.95±1.05 General fermented product 5% 7.30±0.92 2.90±1.12 Coffee waste fermentation 1% 4.05±0.89 6.05±0.83 Coffee waste fermentation 5% 5.55±0.89 7.05±1.23

As shown in Table 2, the odor intensity of the coffee waste fermentation product was weaker than that of the general fermentation product and the preference was higher. In addition, it was confirmed that the preference for general fermented products decreased at high concentrations, while the preference for fermented coffee bean products increased at high concentrations.

Example 6: Confirmation of dermal papilla cell proliferation effect and VEGF activity

Dermal papilla cells were inoculated into DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, U.S.A.) medium containing 10% bovine serum and cultured in a 5% CO2 incubator at 37°C. MTT assay was used to check cell proliferation. was used.

Dermal papilla cells were inoculated into a 96 well plate at a concentration of 1.5x10 ⁴ cells/well and cultured for 24 hours, then replaced with serum-free medium, treated with test samples, and cultured for 3 days. After incubation, 20 μl of MTT solution (5 mg/ml) was added and reacted at 37°C for 3 hours. The supernatant was removed. Formazan crystals were dissolved in 200 μl DMSO, reacted at room temperature for 30 minutes, and absorbance was measured at 540 nm.

The test samples were as follows:

Comparative sample 1. 100 μg/ml of general fermentation using solvent extraction method

Comparative sample 2. General fermentation using eco-friendly purification method 100 μg/ml

Sample of the present invention 1. Coffee waste fermentation using solvent extraction method 100 μg/ml

Sample 2 of the present invention: 100 μg/ml of coffee waste fermentation using an eco-friendly purification method

The cell proliferation rate was calculated as a percentage by correcting the proliferation rate of the untreated control group, and Minoxidil, which is known to be effective in the proliferation of dermal papilla cells, was used as a positive control sample.

The results are shown in Table 3.

sample Cell proliferation rate (%) Control 100 Minoxidil 100μg/ml 123.2 Comparison Sample 1 118.9 Comparison Sample 2 115.6 Invention Sample 1 121.8 Invention Sample 2 124.2

As shown in Table 3, the proliferation effect of hair papilla cells was confirmed in the fermented product produced using an eco-friendly extraction method (comparative sample 2, sample 2 of the present invention), and the fermented product produced using the conventional solvent extraction method (comparative sample 1) , it appears to be effective at an equal or higher level than the present invention sample 1) or minoxidil. In addition, the fermented coffee waste (inventive sample 1, inventive sample 2) showed a higher hair papilla cell proliferation effect than the general fermented product (comparative sample 1, comparative sample 2).

Example 7: Measurement of increase in VEGF expression by coffee waste fermentation

The effect of gene expression of vascular endothelial growth factor (VEGF) was measured using RT-PCR. Dermal papilla cells were cultured in a 5% CO2 incubator at 37°C for 24 hours, then test samples (Example 2) were added at different concentrations and cultured for 18 hours. RNA was isolated by adding 1 ml of Trizol (Invitrogen, USA) to the cells, RNA was quantified at 260 nm, and RT-PCR was performed. An RT-PCR Kit (All-in-one RT-PCR Kit, SuperBio, Korea) was used, and the reaction conditions were reverse transcription at 50°C for 30 minutes, then inactivation of the reverse transcriptase enzyme at 96°C for 3 minutes, 94°C for 30 seconds, and 58°C for 30 minutes. The polymerization reaction was performed at 40 cycles for 1 minute at 72°C.

Primer sequences are shown in Table 4 and results are shown in Table 5.

item order VEGF forward ATCCAATCGAGACCCTGGTG (SEQ ID NO: 1) VEGF reverse TGGTGATGTTGGACTCCTCA (SEQ ID NO: 2)

sample mRNA expression level (%) Control 100 Coffee waste fermentation 1μg/ml 106.2 Coffee waste fermentation 10μg/ml 110.6 Coffee waste fermentation 100μg/ml 116.8

As shown in Table 5, it was confirmed that the expression of vascular endothelial cell growth factor increased as the concentration of fermented coffee grounds increased.

In Examples 8 to 10 below, as shown in FIG. 5, cosmetics, washer fluid, and pesticide detergents containing samples using the fermented coffee waste product of the present invention were prepared, and their efficacy was compared with that of general fermented products.

Example 8: Hair loss clinical effect data

Solid soap containing 1% of fermented coffee grounds was prepared and clinically confirmed to be effective in preventing hair loss and hair growth. A test was conducted for 2 months on 10 adults aged 20-54 years with alopecia. The comparison group was asked to use bar soap that did not contain fermented coffee grounds and to cleanse hair loss areas with the test product 1-2 times a day.

The clinical effect was assessed by the investigator's visual evaluation on a 7-point scale (1 point very worse, 2 points worse, 3 points slightly worse, 4 points no change, 5 points slightly improved, 6 points improved, 7 points very improved), and each panel A satisfaction survey evaluation of the hair loss improvement effect was conducted in parallel (1 point not very satisfied, 2 points not satisfied, 3 points average, 4 points satisfied, 5 points very satisfied).

The results are shown in Table 6.

division Tester's visual evaluation (7-point scale) Panel satisfaction evaluation (5-point scale) After 4 weeks of using the product After 8 weeks of using the product After 8 weeks of using the product test group 4.8±0.6 5.9±0.7 4.8±0.4 comparison group 4.0±0.5 4.1±0.7 3.0±0.7

As shown in Table 6, the hair loss improvement effect was observed in the test group compared to the comparison group after 4 and 8 weeks of sample use, and the panel satisfaction evaluation also showed that the test group was satisfied with the hair loss improvement effect.

Example 9: Washer fluid effect

The present inventors prepared washer fluid with the composition shown in Table 7 below.

division Example Comparative example Coffee waste sophorolipids 3 - Lauryl Glucoside - 3 ethanol 30 30 glycerin One One tartaric acid 0.03 0.03 acetic acid 0.15 0.15 Tolyltriazole 0.05 0.05 Purified water 65.77 65.77 Sum 100 100

The prepared washer fluid was tested for initial water repellency, wet water repellency durability, dry water repellency durability, cleansability, phase stability, and freezing performance, and the results are shown in Table 8 below.

division Example Comparative example initial water repellency ◎ ○ Wet water repellency durability (cycles) 400 375 Dry water repellent durability (cycles) 4750 3850 Cleanability ◎ ○ Phase stability ○ ○ Freezing performance (freezing point, ℃) -26 -26

As shown in Table 8 above, in the case of the washer fluid produced using the coffee waste of the present invention and using sophorolipids purified by an eco-friendly refining method, the water repellency, water repellency durability, and cleaning power were decreased after production using an industrial medium. It was equal to or better than washer fluid prepared using sophorolipids from general fermentation purified by conventional solvent purification methods.

Example 10: Effect of pesticide detergent

The present inventors prepared a pesticide detergent with the composition shown in Table 9.

division Example Comparative example Coffee waste sophorolipids 11.7 - Sodium Lauryl Ether Ethoxy Sulfate - 11.7 ethanol 7.3 7.3 D-Limonene One One Calcium oxide aqueous solution 80 80 Sum 100 100

Pesticides 0.2ppm of azoxytrobin, 3.0ppm of thiafluzide, 0.1ppm of metalaxyl, 1.0ppm of dinotefran, 0.1ppm of clothiandin, and 0.1ppm of methyl chloride were added to 1L of deionized water. 100 g of soybeans were soaked in the above solution for 3 hours at room temperature. After recovering 100 g of soybeans from the test solution, they were dried in a hot air dryer at 80°C for 3 hours.

After soaking 10 g of pesticide-containing soybeans in tap water for 30 seconds, the pesticide concentration was calculated. Comparative Examples and Examples After soaking 10 g of soybeans in a solution of the same concentration, the moisture was removed, and the residual pesticide concentration of the soybeans was calculated. A general fermented product produced using an industrial medium and purified using a conventional solvent purification method was used as a comparative example, and a fermented product produced using coffee grounds and purified using an eco-friendly refining method was used as an example.

Pesticide name Pesticide concentration in soybeans (ppm) Water-washed soybeans (30sec) pesticide removal ability (%) Comparative example (1% sample, 30sec) Pesticide removal ability (%) Example (1% sample, 30sec) (pesticide removal ability) Azoxycrobin 0.015 100 100 100 Tiafluzide 0.371 38 75 100 metalaxyl 0.071 50 100 100 dinotephran 0.393 0 83 94 Clotiandin 0.018 5 36 68 Crosim Methyl 0.017 100 100 100

As shown in Table 10 above, in the case of the sophorolipid sample (Example) produced using the coffee waste of the present invention and purified by an eco-friendly purification method, it was produced using water or an industrial medium and then subjected to a conventional solvent purification method. It was found that the pesticide removal ability was similar or higher compared to the pesticide cleaner using sophorolipids (comparative example) of general fermented products purified by .

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention.

Claims (10)

A medium composition for culturing Candida bombicola comprising vegetable oil, glucose, and coffee grounds. The medium composition for culturing Candida bombicola according to claim 1, wherein the coffee grounds are included at a concentration of 5 to 20% (w/v). The method of claim 1, wherein the vegetable oil is shea butter, mango kernel oil, palm oil, palm kernel oil, coconut oil, and Sarasu seed butter. (Shorea Robusta Seed Butter), margarine, cocoa butter, or a combination thereof, a medium composition for Candida bombicola culture, which is a solid oil at room temperature. The method of claim 1, wherein the vegetable oil is olive oil, rapeseed oil, soybean oil, peanut oil, grape seed oil, sunflower seed oil, rice bran oil, safflower oil, cottonseed oil, corn oil, perilla oil, sesame oil, or a combination thereof maintained in a liquid state at room temperature. Phosphorus, medium composition for Candida bombicola culture. The medium composition for culturing Candida bombicola according to claim 1, wherein the vegetable oil is contained at a concentration of 5 to 20%. A method of cultivating Candida bombicola, comprising culturing Candida bombicola in the medium composition of any one of claims 1 to 5. Method for preparing a culture purified product of Candida bombicola comprising the following steps: (a) removing the maintenance layer from the culture medium; (b) removing the aqueous layer from the culture medium; and (c) Obtaining a residual layer containing sophorolipids from the culture medium. A cosmetic composition for promoting hair growth or preventing hair loss comprising a sophorolipid derived from a Candida bombicola culture or a purified Candida bombicola culture. A cleaning composition comprising a sophorolipid derived from a Candida bombicola culture, or a purified Candida bombicola culture. A composition for pesticide cleaning comprising a sophorolipid derived from a Candida bombicola culture, or a purified Candida bombicola culture.

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