KR101663898B1 - Extraction method of essential oil from citrus junos using enzyme - Google Patents

Abstract

The present invention relates to a method for extracting Yuzu essential oil by using enzymes, and an air-freshener composition comprising the Yuzu essential oil produced thereby. By extracting an essential oil, which is an aromatic component of Yuzu, by using enzymes or fungus, the problem of heat-induced denaturation in the prior art can be prevented, and the essential oil of Yuzu can be obtained in high yield without using an organic solvent, and the extraction can be performed efficiently and cost-effectively by using relatively small facilities and simple extraction process. Therefore, the present invention can be advantageously used in the fields related to food materials, cosmetic materials, aromatherapeutic agents and the like, that utilize Yuzu essential oil.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for extracting citron essential oil from an enzyme,

The present invention relates to a citron essential oil extraction method using an enzyme and a perfume composition comprising citron essential oil produced by the citron essential oil extraction method.

Citron is a crop belonging to citrus fruits produced only in China, Japan and Korea globally. It grows only in south coast areas of Goheung, Geochang, Wando, Jangheung, Kangjin, Geoje and Namhae including Jeju in Korea. Yuzu has a thick perilla and can not consume flesh immediately, but it has a limitation as an edible crop, but it has been widely used for a long period of time as a popular fruit with excellent natural direction. Among the citrus flavorings, which are highly preferred in the world, citron is a very expensive fragrance material, and the specificity of the flavor is internationally recognized. Currently, citrus is used mainly as a raw material of a flavor and food, and recently, It is also used for fragrance therapy (aromatherapy).

As a result, many researchers have studied how to extract the essential oil components present in citron due to the superiority of citron essential oil as a perfume material. Methods known to date include distillate extraction, supercritical fluid extraction by CO ₂ , cold press extraction, cold-pressing and needling, and solvent extraction. have.

Solvent extraction is a simple method and has an advantage of easily extracting oil components, but it is not preferable for use as a food because of a long extraction time, denaturation by heat and residual solvent.

Distillate extraction is a method of collecting the oil component which is evaporated by applying heat to the sample. Since the heat is applied during extraction, the essential oil of citron is mainly citrus type, which is easily oxidized by heat or collects only the evaporated essential oil. The extracted fragrance melts and the strength of the fragrance is weak and it is disadvantageous in that it is different from the natural citron aroma (Korean Patent Laid-Open Publication No. 2001-0035501).

Supercritical fluid extraction (CO ₂ ) is a method of extracting carbon dioxide as a supercritical solution in the form of a liquid and gas near the critical point by varying the pressure and temperature using a solvent. This extraction method is disadvantageous in that it is expensive in terms of equipment, limits on mass production processes, and uses carbon dioxide as a solvent (Korean Biotechnology Journal, Vol. 17, No. 2, 148-152, 2002).

Coldpress extraction and cold-pressing and needling are extracted at low temperature, so they are less denatured by heat, but they have a disadvantage that they produce emulsion by bonding with pectin substances of citron, (Korean Patent Publication No. 2000-0037001).

Korean Patent Laid-Open Publication No. 2001-0035501 Korean Patent Publication No. 2000-0037001

Korea Institute of Bioscience and Biotechnology Volume 17, Issue 2 148-152, 2002

It is an object of the present invention to provide a citron essential oil extraction method using an enzyme in order to maximize essential oil extraction efficiency existing in citron.

It is another object of the present invention to provide a fragrance composition containing citron essential oil produced by the citron essential oil extraction method.

The present invention relates to a method for preparing a citrus peel or a citrus peel, which comprises adding water to the surface of citron or citron and then adjusting pH to 4.5 to 5 in a solution containing citron or citron peel; Adding and reacting at least one member selected from the group consisting of hydrolytic enzymes and fungi to the solution containing citron or citron peel after the forming step; And a step of extracting and recovering the essential oil from citron or citron husks after the step of reacting.

In one embodiment, the hydrolytic enzyme may be at least one selected from the group consisting of a cellulase, a polygalacturonase, and a xylanase.

In another embodiment, the reacting step is performed for 6 to 9 hours.

In another embodiment, the addition amount of the hydrolytic enzyme may be 0.1 to 1% (w / w) relative to the crust of citron or citron.

In another embodiment, the extracting and recovering step may be performed by using a juicer to juice the citron or citron after the reaction, and centrifuging the juice raw material to recover the upper layer.

The present invention also provides a perfume composition comprising citron essential oil produced by the citron essential oil extraction method.

The present invention can prevent denaturation due to heat, which is a problem of the prior art, by extracting an essential oil, which is a directional component of citron, with an enzyme, and can obtain a citron essential oil at a high yield without using an organic solvent, And simple extraction process, it is economical to extract citron essential oil efficiently.

Therefore, it can be usefully used in related industry fields such as food raw materials using cosmetic essential oil, cosmetic raw materials, antiseptic drugs (aromatherapy), and the like.

1 is a schematic view showing a citron essential oil extraction method according to an embodiment of the present invention.
FIG. 2 shows a mold according to an embodiment of the present invention (A, A ': yuzu 2, B, B': yuzu 2-1, C, C ': yuzu12).
Fig. 3 shows the yield of essential oil extraction (citron) according to the enzyme reaction time.
4 shows the yield of essential oil extraction (citron shell) according to the enzyme reaction time.
FIG. 5 shows GC-MS results of enzyme reactions according to an embodiment of the present invention.
Fig. 6 shows the yield of essential oil extraction (citron shell) according to the fungal reaction time.

The present invention relates to a method for extracting citron essential oil using an enzyme or a fungus and a perfume composition comprising citron essential oil produced by the citron essential oil extraction method. The present invention can prevent denaturation due to heat, which is a problem of the prior art, by extracting an essential oil, which is a directional component of citron, by using an enzyme or a mold, and can obtain a citron essential oil at a high yield without using an organic solvent, It is economical because it can efficiently extract citron essential oil with a small facility and simple extraction process.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for preparing a citrus peel or a citrus peel, which comprises adding water to the surface of citron or citron and then adjusting pH to 4.5 to 5 in a solution containing citron or citron peel; Adding the at least one member selected from the group consisting of hydrolytic enzymes and fungi to a solution containing the citron or citron peel after the forming step; And a step of extracting and recovering the essential oil from citron or citron husks after the step of reacting.

The citrus fruits include citron fruits such as citrus fruits, flesh and seeds, preferably citronelles.

When adding water to the citron or citron peel, the amount of water can be adjusted as needed. For example, the amount of citron or citron husk and water may be 1: 2 to 1:10 (w / w).

In order to adjust the pH condition in the solution containing the citron or citron peel, a compound such as sodium bicarbonate may be added to form a pH of 4 to 7. When the pH is in the range of 4 to 7, the hydrolytic enzyme ≪ / RTI >

The hydrolytic enzyme may be an enzyme that penetrates and degrades the plant cell wall of the citron. The hydrolytic enzymes can efficiently decompose the plant cell walls of the citron husks containing citron or essential oil, thereby maximizing the yield in the citron essential oil extraction process. Specifically, the hydrolytic enzymes include cellulase, polygalacturon And may be one or more selected from the group consisting of neat and xylanase.

The step of reacting may be carried out for 3 to 12 hours, preferably for 6 to 9 hours. Particularly, when citron or citron husk and hydrolase are reacted for 6 to 9 hours, the plant cell wall is more effectively decomposed and the yield of citron oil can be further maximized.

The temperature in the step of reacting is preferably the temperature at which the enzyme reaction can be optimized. For example, in the reacting step, the temperature condition may be 18 to 35 占 폚.

Further, in the reaction step, stirring may be performed to maximize the reaction, and stirring may be performed at 100 to 200 rpm, for example.

The amount of the hydrolytic enzyme added may be 0.1 to 1% (w / w) based on citron or citron peel. In case of less than 0.1% (w / w), the amount of hydrolytic enzyme is too small to efficiently decompose the cell wall, so that the yield can not be maximized in citron extract extraction. There are too many quantities to be economical.

In the extracting and recovering step, the citron or citron husks after the reaction may be squeezed using an extractor, and then the extract may be centrifuged to recover the upper layer. The juicer is not particularly limited, but a twin-gear type juicer is preferable, and the centrifugation condition may be a centrifugation at 6 to 12,000 rpm at 4 to 18 DEG C for 20 to 40 minutes as a condition for efficiently separating citron essential oil .

The present invention also provides a perfume composition comprising citron essential oil produced by the citron essential oil extraction method.

The composition may contain one or more ingredients or excipients commonly used with flavors and fragrances, such as carrier materials, thickeners, flavor enhancers and other adjuvants commonly known and used in the art.

The composition according to the invention can be used in all applications. For example, it can include all kinds of perfume products, such as palatable cosmetics, cosmetics, consumer hygiene products, or household products such as cleansers, cleansers, soaps and toothpastes. It also includes uses in flavoring applications, for example, groceries, soft drinks, pharmaceuticals, oral hygiene products and other health products using common flavorings. The composition can also be applied to aromatherapy (aromatherapy).

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited by the following examples, but may be variously modified and changed.

Example  1. Materials Preparation

1-1. Preparation of citron

Yuza was purchased from Seil Foods Co., Ltd., which produces citron juice in Goheung, Jeonnam Province, in late December, 2013 and stored at -20 ℃ freezer. The citrus fruits were separated into perilla, The average value was obtained and expressed as a percentage with respect to the total weight of the fruit.

1-2. Enzyme and medium preparation

The availability and characteristics of the enzyme used in the pretreatment are shown in Table 1 below.

Enzyme name product name origin Usage Optimum reaction conditions activation Xylanase Shearzyme®
500L Aspergillus oryzae Improved filtration during processing 25 ° C, pH 5.5 350 EGU / g Cellulase Celluclast 1.5L Tricoderma reesei Vegetable, fruit decomposition, yield improvement 5 ° C, pH 4-7 700 EGU / g Polygalacturonase Pectinex Ultra SP-L Aspergillus aculeatus Viscosity decrease 5 ° C, pH 4-5 9500 PGU / ml

In this experiment, Potato Dextrose Broth (PDB; Dfico Laboratories), Potato Dextrose Agar (PDA; Difco Laboratories) and Agar (Bio Basic INC) were purchased and used. In addition, sodium hypochlorite solution (showa) was used for the sterilization of citron peel as a reagent and LS300 Antifoaming agent (Dowcorning) was used as a defoamer in the cultivation of fungal strains. Sodium sulphate anhydrous (Yakuri pure chemicals co.) Was used for water removal before GC-MS analysis.

1-3. Fungal Isolation and Culture

Five samples were purchased from each box (20 boxes per box, 20kg) randomly selected. Each bottle was randomly selected. To reduce the loss of water, the vials were placed in a plastic bag made of air and examined for changes in the pericarp in a 22 ° C incubator (BI-600M, Jeio tech) Respectively. After 37 days of observation, red flour was found in four citrus fruits. These four citrus fruits were observed around the nipple from the 40th day of observation. However, one citron had found red powder at about 45 days, red powder was observed, and mold was first found three days later. The first red powder was considered to be an insect by the movement. It was thought that this insect affects the production of citron microorganism, since the appearance of the insect and the mold were observed at the beginning of the insect. In order to separate microorganisms from citrus fruits, the red powder and white mold spores produced in the perilla were inoculated into the PDA medium and cultured under the same conditions as above. Two days after the culture, colonies were observed in the medium inoculated with white mold spores, but no colonies were observed in the medium inoculated with red powder. 5 ml of distilled water was added to the PDA solid medium in which the colonies were observed, and the spores were collected and then filtered using sterilized agar. 1 ml of the filtered strain was plated and cultured at 22 ° C for 5 days, and the grown fungi was observed and separated. In order to isolate the fungi that were separated first, they were inoculated at one time on the PDA medium, respectively, and the different types of the fungus were separated on the plate culture supposing them to be different bacteria, and the growth rate of the fungus and the color tone of the front and back of the colonies were observed Respectively.

As a result of the experiment to isolate the different types of fungus on the plate culture media, three kinds of fungi were able to be isolated purely and named as Yuzu 2, Yuzu 2-1, Yuzu 12 respectively. The growth rate of Yuzu 12 was the fastest among the three fungi on the PDA solid medium and the color tone of the back surface of the colonies. The density of the microflora was lower and relatively looser than those of the other two strains. On the other hand, the growth rate of yuzu 2-1 was the slowest, and the shape of the microflora was dense and denser than the other two microfoliants.

The color of the fruit body part of the Yuzu 2 colonial surface showed a greenish blue color, and the part of the mycelium on the back side showed a wrinkle color close to forsythia. The surface of Yuzu 2-1 colonies showed a grayish blue color, and the color of the white color was distinctly different from that of the other two strains at the beginning of the growth of the mycelium. The color on the back was bright yellow. The surface of Yuzu 12 showed a gray color, and the color of the back side of the inoculation center was black, while the growing hypha showed an ivory color (Fig. 2).

Example  2. The surface of citron using enzyme flavedo ) And extraction of crude oil and yield

Citrus fruit or frozen fruits were added to the lemon juice at 30 ° C in an amount of 1% of the fruit washing detergent, and washed, followed by washing with running water. After washing, when the water had fallen to some extent, it was cut into quarters or eighths, and the flavedo and albedo of citron peel were separated using a knife. Separated flabeds were placed in an Erlenmeyer flask with distilled water at a ratio of 1: 3, and then treated with enzymes xylanase, cellulase, polygalactuose After the completion of the reaction at 18 ° C and 150 rpm for 1 to 12 hours using a shaker (SI-900R, JEIO TECH), the sample was added to the flour in a concentration of 0.1% (w / w) Angel green juice 7000p). The juices were collected by centrifugation (Mega 21R, hanil science industry) at 12,000 rpm at 4 ° C for 20 min. Only the clear oil layer of the upper layer was recovered and sealed and stored frozen at -20 ° C. As a result, The yields are shown in Table 2 and FIG.

division 1 hours 3 hours 6 hours 9 hours 12 hours No enzyme treatment (control group) 0.318 g / 300 g (0.11%, w / w) Cellulase 0.75 g / 300 g
(0.25%, w / w) 2.35g / 300g
(0.78%, w / w) 3.297g / 300g
(1.1%, w / w) 3.098g / 300g
(1.03%, w / w) 2.846g / 300g
(0.95%, w / w) Polygalacturonase 0.94 g / 300 g
(0.31%, w / w) 2.221g / 300g
(0.74%, w / w) 2.686g / 300g
(0.9%, w / w) 2.665g / 300g
(0.89%, w / w) 2.452 g / 300 g
(0.82%, w / w) Cellulase +
Xylanna (1: 1) 0.89g / 300g
(0.3%, w / w) 2.265g / 300g
(0.76%, w / w) 3.268g / 300g
(1.09%, w / w) 3.236 g / 300 g
(1.08%, w / w) 3.012g / 300g
(1%, w / w) Cellulase +
Polygalactotonase (1: 1) 0.96g / 300g
(0.32%, w / w) 2.541 g / 300 g
(0.85%, w / w) 3.151g / 300g
(1.05%, w / w) 3.021g / 300g
(1.01%, w / w) 2.894g / 300g
(0.97%, w / w) Cellulase +
Xylanase +
Polygalactotonase (1: 1: 1) 0.93 g / 300 g
(0.31%, w / w) 2.611g / 300g
(0.87%, w / w) 2.95g / 300g
(0.98%, w / w) 2.851g / 300g
(0.95%, w / w) 2.613g / 300g
(0.87%, w / w)

As shown in Table 2 and FIG. 3, the extraction yield of the essential oil extract showed a sharp increase within 3 hours of the enzymatic reaction and showed the maximum yield between 6 and 9 hours, and the yield was decreased in the later time period. This means that the enzyme reaction time of citron essential oil extraction is most preferably 6 to 9 hours.

Example  3. Extraction of essential oil from whole fruit using enzyme and its yield

Citron fruit or frozen fruits were added to the lemon juice at 30 ℃ for 1% of the fruit washing detergent and washed thoroughly with running water. When the water was removed to some extent, the citron fruit flask was mixed with distilled water at a ratio of 1: 3. The enzyme xylanase, cellulase, and polygalacturonase, which were mixed individually or in a ratio of 1: 1 at a pH of 4.0 to 5.0, were added to a citron sample at a concentration of 0.1% (w / w) 900R, JEIO TECH) at 18 ° C and 150 rpm for 1 to 12 hours, and then the sample was poured (7000p of Angel juice). After centrifugation (Mega 21R, hanil science industry) at 12,000 rpm at 4,000 rpm, only the clear oil layer of the upper layer was recovered and stored at -20 ° C after cryopreservation. As a result, The yields are shown in Table 3 and FIG.

division 1 hours 3 hours 6 hours 9 hours 12 hours Enzymatic treatment 0.6 g / 300 g (0.2%, w / w) Cellulase 0.75 g / 300 g
(0.25%, w / w) 0.67 g / 300 g
(0.22%, w / w) 0.86g / 300g
(0.29%, w / w) 1.23g / 300g
(0.41%, w / w) 1.01 g / 300 g
(0.34%, w / w) Polygalacturonase 0.94 g / 300 g
(0.31%, w / w) 0.98g / 300g
(0.33%, w / w) 1.33g / 300g
(0.44%, w / w) 0.88g / 300g
(0.29%, w / w) 0.43 g / 300 g
(0.14%, w / w) Cellulase +
Xylanna (1: 1) 0.27 g / 300 g
(0.09%, w / w) 0.65g / 300g
(0.22%, w / w) 1.01 g / 300 g
(0.34%, w / w) 0.91 g / 300 g
(0.3%, w / w) 0.76 g / 300 g
(0.25%, w / w) Cellulase +
Polygalactotonase (1: 1) 0.96g / 300g
(0.32%, w / w) 0.95 g / 300 g
(0.32%, w / w) 1.19g / 300g
(0.4%, w / w) 1.52 g / 300 g
(0.51%, w / w) 0.84g / 300g
(0.28%, w / w) Cellulase +
Xylanase +
Polygalactotonase (1: 1: 1) 0.93 g / 300 g
(0.31%, w / w) 1.09 g / 300 g
(0.36%, w / w) 1.43g / 300g
(0.48%, w / w) 1.59g / 300g
(0.53%, w / w) 0.72g / 300g
(0.24%, w / w)

As shown in Table 3 and FIG. 4, the group treated with the enzyme showed an oil extraction yield of 2 to 2.5 times higher than the group not treated with the enzyme. The enzyme yield was slightly different between 6 and 9 hours, and the yield was decreased after 9 hours.

Example  4. Analysis of essential oil components

The essential components extracted by the method of Example 2 were analyzed by gas chromatography-mass spectrometry (GC-MS) to determine the essential components of the essential oil component and the oriental component. Are shown in Table 4, and the results are shown in Table 5 and FIG.

Table 5 shows GC-MS results for each enzyme reaction.

Model 7890A / 5975C, Agilent Inlet 250 ° C, Spitl mode 80: 1, 1 μl ingection Oven Min → 3 ° C / min → 100 ° C, 2 min → 3 ° C / min 175 ° C, 2min → 3 ° C / min → 246 ° C, 2 min
total 73min Column DB-WAX (30x0.25x250) MSD Scan mode

division Non Cel Pec Shea Cel + Pec Cel +
Xyl +
Pec Dimethylamine 0.04 alpha-Pinene, d-alpha-Pinene, alpha-Pinene (2902) 5.45 6.59 6.42 6.44 6.74 6.49 Camphene (2229) 0.03 0.03 0.04 0.06 0.03 0.04 beta-Pinene (2903) 2.04 2.46 2.36 2.46 2.54 2.49 Hexanal (2557) 0.03 0.04 0.03 0.03 0.03 Myrcene (2762), Sabinene 12.73 12.21 12.52 11.92 12.79 12.07 3-Carene (3821) 0.01 Myrcene (2762), Sabinene, alpha-Phellandrene (2856) 1.11 0.53 0.85 0.56 0.58 1.05 D-Limonene (2633), d, 1-Limonene, 1-Limonene 55.63 53.60 54.72 53.55 55.43 53.25 Gamma-Terpinene (3559) 14.06 14.97 14.08 14.55 14.73 14.44 3,7-Dimethyl-1,3,6-octatriene (3539) 0.48 0.50 0.52 0.87 0.96 0.90 O-Cymene (2356) 1.11 1.46 1.34 1.27 0.01 1.36 Terpinolene (3046) 1.23 1.48 1.38 1.43 1.50 1.43 Nonanal (2782) 0.01 0.02 0.01 0.02 0.02 0.02 p-alpha-Dimethyl styrene (3144) 0.10 0.13 0.11 0.10 0.11 alpha-Terpinene (3558), Terpinolene (3046) 0.01 0.47 0.01 Decanal (2362) 0.06 0.07 0.06 0.08 0.09 0.09 Trans, trans-2,4-Hexadienal (3429) 0.01 0.01 Cis-4-Decenal 0.01 0.01 0.01 0.01 0.01 0.01 Carvacryl methyl ether 0.02 1 (10), 4 (14), 5-Germacratriene 0.92 0.93 0.85 0.92 0.89 0.91 Carvone (2249), p-Mentha-1,4 (8) -duran-3-one (3560) 0.01 Linalool (2635) 1.38 1.52 1.37 1.70 1.85 1.71 1-Methyl-3-methoxy-4-isopropyl benzene (3436) 0.01 0.02 0.01 0.02 0.02 beta-Caryophyllene (2252) 0.84 0.76 0.77 0.73 0.74 0.70 Trans-2-Dodecan-1-al (2402) 0.01 Undecanal (3092) 0.02 0.02 0.02 0.02 Tridecanal 0.02 3,7,10-Humulatriene 0.17 0.15 0.15 0.14 0.14 0.14 beta-Farnesene 2.02 2.06 2.01 2.38 2.36 alpha-Terpineol (3045) 0.08 0.07 0.06 0.08 Farnesol (2478) 0.01 Lauric aldehyde (2615) 0.02 0.02 0.02 0.02 0.02 p-Mentha-1,8-dien-7-al (3557) 0.01 0.01 0.01 0.01 2,4-Decadienal (3135), Trans, trans-2,4-Decadien-1-al (3135) 0.01 0.01 0.01 0.01 0.01 0.01 Aromadendrene 0.02 0.02 0.01 0.02 Myristaldehyde (2763) 0.01 Viridiflorol 0.02 Elemol 0.02 0.01 0.01 0.01 0.01 0.01 beta-Bisabolene, Aromadendrene 0.02 0.01 Thymol (3066) 0.30 0.24 0.21 0.22 0.26 0.23 Thymol (3066), Carvacrol (2245) 0.01 0.01 0.01 0.01 0.01 Longifolene 0.01 Ethyl linoleate 0.01 Non: Enzyme disposal
Cel: Cellulase
Pec: Polygalacturonase
Shea: Cellulase + xylanase (1: 1)
Cel + Pec: Cellulase + polygalactotonase (1: 1)
Cel + Xyl + Pec: Cellulase + xylanase + polygalactotonase (1: 1: 1)
(Unit: peak area%)

As shown in Table 5 and FIG. 5, about 60 kinds of fragrance components of citron essential oil were isolated and 35 species were identified, and the difference in amount and amount of fragrance components with and without enzyme treatment was not significant. Specifically, limonene content was the highest at 53.25 ~ 55.43% and γ-terpinene was 14.08 ~ 14.87% at the enzyme-treated samples. , Indicating that there is almost no difference between the essential oil extracted after the enzyme treatment and the natural essential oil.

Example  5. The surface of citron using mold flavedo ) And extraction of crude oil and yield

5 ml of distilled water was added to the solid medium of the PDA colonies in which three kinds of fungus colonies were formed, and spores were collected and then filtered using sterilized agar. The filtered bacteria were inoculated into 100 ml of PDB liquid medium and cultured for 3 days at 22 ° C and 150 rpm using a shaker (SI-900R, JEIO TECH). To measure the dry weight of the cultivated fungus, filter 100 ml of the culture with filter paper (whatman No. 2). After drying at 105 ° C for 1 hour using a dry oven (vision scientific co., LTD), the average value measured by repeating the measurement three times was measured by dry weight. Then, 0.2 g to 0.4 g of 10% dry weight was inoculated into a liquid medium of 1 L of PDB, and the cells were cultured for 3 days under the same conditions as above.

400 g of citrus fruit was dipped in 1% hypochlorite (NaOCl) solution for 3 minutes, sterilized and washed three times with sterilized distilled water to directly immerse the spore suspension in the spore suspension. The citrus peel inoculated with the spores was placed in a sealed container sterilized in 70% ethanol, and sterilized distilled water was separately added thereto to maintain a humidity of 80% to 90%. Using an incubator (BI-600M, Jeio tech) And cultured for 10 to 20 days, respectively. Essential oil was extracted from the cultured samples by coldpress extraction method, and the extraction yield was shown in FIG.

As shown in FIG. 6, the extraction yields of the fungi were slightly higher than those of the non-fungus samples obtained by the cold-pressing method inoculation of each fungus. These results indicate that the bacterial or fungal plant It is believed that this is a result of showing the correlation with the existing studies that the plant cell wall is degraded by the enzymes that secrete in the process of penetrating the cell wall, and that it is known as the enzyme which decomposes the typical polygalacturonase such as pectin. In addition, the 10-day culture showed higher extraction yield than the 20-day cultured sample, indicating that the 20-day sample is over-digested, which is not easy to extract.

Example  6. Analysis of essential oils

In order to investigate the changes of aroma components due to fungi cultivation, essential oils were extracted from three species isolated from citrus fruits by culturing them in citron peel. The flavor components of the essential oil extract were analyzed and tabulated. The analytical conditions of the GC-MS were the same as those of the above experiment, and the results are shown in Table 6.

division A B C D E F G H I Methanamine 0.590 0.67 0.57 1.42 0.92 0.79 0.57 0.76 1.59 alpha-pinene 1.980 1.65 1.35 1.83 1.68 1.65 2.36 1.73 1.58 alpha-thujene 0.760 1.01 1.21 0.96 0.91 0.89 1.31 1.1 1.34 beta-pinene 1.010 0.93 0.89 0.97 1.08 0.91 1.28 1.01 0.94 beta-myrcene 2.950 1.2 2.56 3.16 3.02 2.55 3.78 2.51 . d-limonene 70.85 66.39 66.54 71.33 68.79 68.58 67.28 68.49 68.61 sabinene 3.480 3.1 2.65 2.66 3.18 0.34 3.31 0.78 3.43 gamma-terpinene 10.27 9.8 10.9 10.16 10.81 11.34 9.07 13.58 12.32 p-cymene 0.750 0.79 1.03 0.85 0.8 0.89 0.81 0.95 0.89 alpha-terpinene 0.850 1.4 0.64 0.6 0.62 0.56 0.47 0.56 0.66 6-Octen-2-one 0.01 0.01 0.02 0.01 0.01 0.01 0.01 alpha-dimethylstyrene 0.060 0.08 0.08 0.06 0.06 0.05 0.06 0.06 alpha-cubebene 0.020 0.04 0.04 0.05 0.05 0.09 0.02 0.09 0.06 Decanal . 0.06 0.02 0.01 p-mentha-1,3-diene 0.040 0.11 0.01 0.07 alpha-copaene 0.060 0.02 0.16 0.1 0.1 0.12 0.1 0.1 0.1 beta-linalool 0.790 0.92 0.8 0.55 0.62 0.68 0.51 0.61 0.46 alpha-phellandrene 0.020 0.03 0.04 0.02 0.03 0.02 0.02 0.03 0.05 beta-elemene 0.020 0.02 0.08 0.06 0.04 0.12 0.05 0.1 0.02 caryophyllene 0.350 0.51 0.62 0.33 0.36 0.41 0.42 0.35 . terpinen-4-ol 0.030 0.1 0.14 0.07 0.06 0.08 0.1 0.1 0.1 beta-sesquiphellandrene 0.040 0.06 0.06 0.04 0.14 0.05 0.05 0.16 beta-farnesene 1.070 1.59 1.9 1.06 1.21 1.3 1.57 1.34 1.22 alpha-humulene 0.060 0.1 0.12 0.06 0.07 0.07 0.09 0.08 0.07 alpha-terpineol 0.040 0.07 0.08 0.03 0.07 0.4 0.05 0.04 0.05 1 (10), 4 (14), 5-germacratriene 0.700 0.95 1.81 0.54 1.49 0.86 0.84 0.69 0.74 alpha-muurolene 0.040 0.09 0.1 0.05 0.06 0.07 0.08 0.05 0.08 bicyclogermacrene 1.520 2.24 2.45 1.53 1.7 1.89 2.27 1.69 1.77 delta-cadinene 0.140 0.02 0.26 0.12 0.01 0.12 0.2 0.19 0.21 camphene 0.040 0.02 0.04 0.01 0.01 0.03 0.01 0.01 beta-sesquiphellandrene 0.140 0.13 0.23 0.08 0.17 0.13 0.13 butanoic acid 0.010 0.01 0.02 0.01 0.03 0.02 alpha-amorphene 0.020 0.01 0.02 0.01 p-mentha-1,8-dien-7-ol 0.010 0.01 0.01 0.01 0.01 0.02 0.01 L-linalool 0.02 0.02 0.02 0.03 0.03 0.02 nerolidol 0.030 0.03 0.04 0.02 0.03 0.03 0.03 0.04 0.02 endo-1-bourbonol 0.350 0.11 0.08 0.03 0.04 0.12 0.02 Caprylic acid 0.01 0.01 0.02 0.01 cedrene 0.1 0.22 viridiflorol 0.010 0.01 0.03 0.01 0.02 0.02 0.02 0.02 0.02 triacetin 0.010 0.01 0.01 spathulenol 0.010 0.02 0.04 0.01 0.02 0.02 0.02 0.03 0.02 gamma-cadinene 0.020 0.02 0.03 0.02 0.01 0.02 0.02 thymol 0.170 0.18 0.22 0.15 0.19 0.17 0.2 0.17 isospathulenol 0.01 0.02 0.01 0.01 alpha.-cadinol 0.17 0.22 0.12 0.25 0.16 0.18 0.18 0.17 4-bromo 0.01 0.1 0.02 0.01 beta-eudesmol 0.090 0.01 0.01 0.01 0.01 0.01 Carvacrol 0.01 0.01 diazene 0.010 0.01 0.01 dodecanoic acid 0.010 0.03 0.03 0.01 0.01 0.02 0.02 0.02 cyclohexene 0.010 0.01 0.01 0.01 0.02 0.01 0.01 capric acid 0.010 0.01 0.09 0.02 0.01 0.01 2,3-dimethoxy
phenylacetonitrile 0.02 0.01 0.01 0.01 beta-selinen 0.01 0.02 Stearic acid 0.06 0.06 0.06 0.07 0.05 0.06 0.05 (Z) 6, (Z) 9-Pentadecadien-1-ol 0.02 0.03 0.01 oleic acid 0.020 0.14 0.11 0.02 0.04 0.02 0.1 0.09 0.12 myristic acid 0.050 0.1 0.11 0.07 0.04 0.1 0.06 0.07 0.8 linoleic acid 1.31 0.02 0.39 0.71 0.71 0.85 0.87 0.39 Palmitinic acid 0.360 0.55 0.73 0.3 0.53 0.48 0.55 0.54 0.01 linolenic acid 0.54 1.22 0.08 0.29 0.45 0.33 0.33 0.73 Heptadecanoic acid 0.050 0.07 0.06 0.07 0.07 A: Standard
B: Non inoculation after 10 days
C: Non inoculation after 20 days
D: Inoculation face 2 after 10 days
E: Inoculation face 2 after 20 days
F: Inoculation face 2-1 after 10 days
G: Inoculation face 2-1 after 20 days
H: Inoculation face 12 after 10 days
I: Inoculation face 12 after 20 day

As a result, about 63 fragrance components were detected for each mold type. All of the molds showed similar composition ratios and there was no significant difference from the reference samples. Limonene content was the highest in all samples, followed by gamma-terpinene, sabinene, myrcene, beta-pinene and alpha-pinene. The content of limonene tended to decrease by 2% when compared with the reference sample. However, the decrease of limonene content in the samples left for 10 to 20 days without culturing the fungi was larger than that of the fungal cultured samples. This result shows that some of the components of limonene due to the fungi are preserved. These results suggest that the oxidation of limonene is lower than that of non - cultured fungi by blocking the contact with oxygen while the epidermis of the sample is surrounded by the colony during the growth of the fungus. After sensory evaluation, the specimens were found to be fungus - specific and the extracts were almost in the same color as the reference samples.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be obvious that it is not. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

Adding water to the surface of the citrus peel to form a pH condition in the solution containing the citrus peel to 4.5 to 5;
Adding at least one member selected from the group consisting of a cellulase, a polygalacturonase and a xylanase to a solution containing the citron shell and reacting at 18 DEG C after the step of forming; And
And extracting and recovering the essential oil from the citron peel after the step of reacting. delete The method according to claim 1,
Wherein the step of reacting is carried out for 6 to 9 hours. The method according to claim 1,
Wherein the amount of at least one selected from the group consisting of cellulase, polygalacturonase, and xylanase is 0.1 to 1% (w / w) based on the crust of citrus peel. The method according to claim 1,
Wherein the step of extracting and recovering the juice is carried out by using a juicer to juice the citron peel after the reaction, and then centrifuging the juice raw material to recover the upper layer. A perfume composition comprising citron essential oil prepared by the citron essential oil extraction method of claim 1.

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