KR101080076B1 - Fermented Oil and Preparation method thereof - Google Patents

Abstract

The present invention comprises the steps of immersing the oil seed in water and germinating for 24 hours at 30 ℃, or at 120 ℃ steam steaming the oil seed for 30 minutes and cooling the oil seed to 30 ~ 35 ℃; Inoculating the germinated seed or the cooled seed with a strain and solid fermenting at 35 ° C. for 15 hours; Drying the fermented product and grinding to extract lipids; And purifying the extracted lipids; It provides a method for producing fermented oil comprising a.

Description

Fermented oil and preparation method thereof

The present invention relates to fermented oil obtained by steaming or germinating oilseed soybeans, sunflower seeds, grape seeds, safflower seeds, sesame seeds, etc. as a main raw material, and then solid fermenting with a Bacillus or Aspergillus strain and extracting them. Compared to the immune activity and antioxidant activity is a strong effect to help people's health while eating easily in everyday life.

Conventionally, the oilseed itself has not been fermented, but various raw materials (apples, plums, ginseng, herbal ingredients, etc.) have been used in the fermentation process by mixing functional substances produced by fermentation with oil or immersing them in oil. . Representative prior arts include garlic fermented cooking oil (Korean Patent Application No. 2005-0101015), ginseng fermented cooking oil (Korean Patent Application No. 2004-0007416), red pepper fermented cooking oil (Korean Patent Application No. 2003-0056356), plum fermented cooking oil ( Korean Patent Application No. 2003-0045567), apple fermented cooking oil (Korean Patent Application No. 2003-0058944), herbal fermented cooking oil (Korean Patent Application No. 2003-0062881), etc. Most of the manufacturing process of these patents are stripped and washed After drying, adjust the moisture content to 25 ~ 50% and mix with edible oils and fats. The mixture is aged at 5-38 ° C. for 15 days with stirring at 45-55 rpm. The matured mixture is stripped again, washed with water, compressed and then secondary fermented under the same conditions as the first fermentation. After completion of the secondary fermentation, separation and filtration produce fermented oil.

Simple fermented fermentation is characterized by simple manufacturing process, low manufacturing cost, and reduced flavor, taste, etc. of raw materials, but simple fermented cooking oil is not the fermented oil, but the taste of garlic and sour plum. The purpose is to enhance the peculiar taste of raw materials, and does not expect physiological functions such as antioxidant activity and immune activity. Therefore, fermented oil has little effect on physiological and antioxidant activity of fermented products. Since the raw material is fermented and then mixed with edible oils and fats, the dilution effect of the functional substance is large, and fermentation oil has the disadvantage that there is no fermentation-related treatment for the highest content oil.

Thus, the present inventors fermented by two methods using soybean, sunflower seed, grape seed which is an oilseed seed (oilseed) as a raw material for oil extraction.

Soybean, sunflower seeds, grape seeds are only examples and will be described as a method applicable to all oilseed (oilseed) made from cooking oil.

(a) Solid fermentation by soaking oilseed seeds and adding Aspergillus or Bacillus strains

(b) Method of solid fermentation by adding Aspergillus or Bacillus strain after steaming oilseed seeds

In order to optimize the activation of nutrients and glycosides of oilseed seeds, two methods of solid fermentation are performed for a certain period of time to maximize antioxidant activity, immune activity, and physiological activity than extracting oilseed seeds themselves. Completed. This is considered to be a very useful invention in that the oil-based solid fermented cooking oil of the present invention has increased antioxidant activity and immune activity, compared to the conventional fermented oil reported only results of taste and flavor improvement.

Accordingly, an object of the present invention is a fermented oil having increased antioxidative activity and immune activity by solid fermentation with Aspergillus or Bacillus strain after immersing, germinating, or steaming oilseed seeds (soybean, sunflower seed, grape seed, sesame, etc.). The purpose is to provide a solution.

The present invention is to solve the above-mentioned problems, the present invention is immersed in a thermostat (30 ℃, 60% RH) for 24 hours after immersion at 30 ℃ temperature for 24 hours without steaming oilseed seeds to Aspergillus or Bacillus After fermentation of solids at 35 ° C for 15 hours, it was pulverized to produce fermented oil. Also, the oil seeds were steamed at 100 ° C for a short time and then fermented at 30 ° C with Aspergillus or Bacillus for 24 hours and then pulverized to produce fermented oil. It became.

An object of the present invention is to produce a type of edible oil that can be expected to function of antioxidant activity and immune activity while being widely used as a food material by fermenting oilseed seed and processing into powder.

An object of the present invention is to prepare fermented oil as a main ingredient for each oilseed seed, and to ferment the oilseed seeds with each other in order to fully utilize the functionality of each oilseed seed.

The present invention comprises the steps of soaking and germinating soybean, sunflower seeds or grape seeds in water at 30 ℃ for 24 hours; Inoculating the germinated soybean, sunflower seed or grape seed into a strain and solid fermenting at 35 ° C. for 15 hours; Drying the solid fermented soybean, sunflower seed or grape seed and then grinding to extract lipids; And purifying the extracted lipids; It provides a method for producing fermented oil comprising a.

In addition, the present invention comprises the steps of steaming soybean, sunflower seeds or grape seeds for 30 minutes at 120 ℃ and cooling the steamed soybean, sunflower seeds or grape seeds to 30 ~ 35 ℃; Inoculating the strain with chilled soybean, sunflower seed or grape seed and solid fermenting at 35 ° C. for 15 hours; Drying the solid fermented soybean, sunflower seed or grape seed and then grinding to extract lipids; And purifying the extracted lipids; It provides a method for producing fermented oil comprising a.

Preferably, the strain is Bacillus firmus (Bacillus firmus), Bacillus subtilis (Bacillus subtilis), Aspergillus duckjae (Aspergillus oryzae), Aspergillus flavus, Aspergillus japonicas ( Aspergillus japonicus) and Aspergillus awamori (Aspergillus awamori) is selected from the group consisting of one or more, the strain provides a method for producing fermented oil inoculated 0.5% by weight of the soybean, sunflower seeds or grape seeds.

In addition, the present invention provides a fermentation oil having an antioxidant activity as a fermentation oil prepared by the above production method.

In addition, the present invention provides a fermentation oil having an immune activity as a fermentation oil prepared by the above production method.

In addition, the present invention provides a fermentation oil having anticancer activity as a fermentation oil prepared by the above production method.

The present invention provides a fermented oil having increased antioxidant activity and immune activity by solid fermentation with Aspergillus or Bacillus strains after immersing, germinating or steaming oilseed seeds (soybeans, sunflower seeds, grape seeds, safflower seeds).

Hereinafter, the present invention will be described in more detail.

(1) pre-fermentation stage

At 30 ° C., the oilseed seeds were soaked in water for 24 hours. The soaked oilseed seeds were germinated for 48 hours in a thermostat (30 ° C., 60% RH). The germination rate of germinated oilseed seeds was more than 95%. The reason for soaking oilseed seeds is to promote germination of oilseed seeds.

Or, unlike the above step, at 120 ° C., the oilseed seeds were steamed for 30 minutes and the product temperature of the oilseed seeds was cooled to 30 to 35 ° C. The reason for steaming the oilseed seeds is to sterilize the oilseed seeds and to easily infiltrate the fermentation strain into oilseed seeds to promote fermentation.

The oilseed seeds include, but are not limited to, soybean, sunflower seeds, grape seeds, green tea seeds, safflower seeds, sesame seeds, sesame seeds, rapeseed, peanuts, and all oil seeds that can be prepared with edible oil.

(2) strain inoculation and fermentation step

The strain was inoculated with 0.5% of the weight of the oilseed seed in the oilseed seed pretreated in the step (1).

The strain is Bacillus firmus (Bacillus firmus), Bacillus subtilis, Aspergillus duck (Aspergillus oryzae), Aspergillus flavus, Aspergillus japonicus (Aspergillus japonicus) And Aspergillus awamori.

Bacillus firmus JAP used in the experiment in the present invention was isolated from the Japanese Cheonggukjang (natto), Bacillus subtilis (KU-A) is isolated from the Cheonggukjang.

The detailed separation method of the strain is first 1g of each sample is added to 9ml of sterile distilled water, suspended well and left for 5 minutes, and then the supernatant is diluted to 10 ⁴ ~ 10 ⁷ each on a prepared MRS agar plate Smear to separate the strain to produce a large amount of viscous by incubating for 24 hours at 37 ℃. Then, the isolated strain is plated on an MRS agar plate and separated purely. Finally, the isolated strains were incubated for 24 hours on an MRS agar plate and identified using the Analytical Profile Index (API) kit and 16s rDNA sequencing.

In addition, the microorganisms used in the present invention are Aspergillus oryzae, Aspergillus flavus, Aspergillus japonicus, Aspergillus awamori and Nuruk It is separated from.

Inoculated with the microorganisms in oilseed seed and solid fermentation at 30 ℃ for 24 hours.

(3) purification step

The fermented product fermented in the step (3) was extracted and purified.

Extraction may be performed using an organic solvent such as nucleic acid, or may be extracted using a compactor.

In the case of extraction using an organic solvent, the extract was dried after pulverizing at 110 ° C. for 30 minutes and pulverized. In the case of extraction using a compactor, the fermented product was dried at 110 ° C. for 30 minutes to extract lipids.

The extracted lipids were added water at 250 ° C. to make water vapor and blow off odors present in the extracted lipids. In addition, 0.5% of acidic clay was added and reacted at 110 ° C for 10 minutes, followed by filtration to decolorize.

According to the present invention, excellent anti-oxidant activity was observed in both the oils extracted by fermentation and solid fermentation after immersion germination and the oils extracted by fermentation after steaming, and there was no cytotoxicity and no addition control in growth of immune B cells and T cells. It showed high activity in comparison with. The cytokine secretion of immune cells showed similar results as in the growth rate measurement, which was also effective in promoting NK cell growth. Fermentation oil is a very useful invention in the food industry because it shows high activity in the growth inhibition activity of human lung cancer cells A549 and breast cancer cells MCF-7.

Hereinafter, the configuration of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

(Example 1)

The antioxidant activity of fermented fats and oils was measured to select the optimal strains by oilseed seeds. After oily seed was solid fermented, oil was extracted with nucleic acid. As a method of evaluating the induction period of extracted oil, Rancimat (Rancimati 743, Metrohm Ltd. Herisau, switzerland) was used to determine the time required for oxidation (induction period). Experimental conditions were carried out by setting the heating temperature to 120 ℃, oxygen flow rate 20ml / hr, the correction temperature (delta T) to 1.6 ℃.

In the present invention, the antioxidant activity was determined by electron donating ability using DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical. After dissolving 56 uL of sample in isooctane, 200 uL was added to a 25 ml test tube and 250 uM DPPH isooctane ( 3 ml of isooctane) solution was added and left at room temperature for 30 minutes to measure absorbance at 517 nm. Absorbance of the control group without the sample was measured and calculated as follows.

R% = (A- / B / A) × 100

A: control

B: sample group

Oxidation stability analysis results measured by the above method are shown in Table 1 below.

TABLE 1

Strain Oilseed seeds Pretreatment AOM (120 ℃) Electron donating ability Pretreatment AOM (120 ℃) Electron donating ability Untreated
(Control group) Big head none 8.24 9.1 - - - Sunflower seeds 6.34 10.4 - - - Grape seed 7.32 13.2 - - - Bacillus Pilmus Big head




Immersion
germination
after
Fermentation








20.34 67.4




Steaming
after
Fermentation








18.54 59.4 Sunflower seeds 18.47 60.1 16.45 55.5 Grape seed 19.22 59.4 17.21 51.3 Bacillus subtilis Big head 20.34 77.9 19.65 57.2 Sunflower seeds 18.21 54.7 17.11 43.2 Grape seed 18.54 60.1 16.49 47.0 Aspergillus Duck Big head 16.54 54.2 14.23 48.9 Sunflower seeds 17.11 60.3 15.85 51.1 Grape seed 17.54 61.4 13.23 43.2 Aspergillus Flavers Big head 15.35 48.3 14.67 49.2 Sunflower seeds 16.36 51.1 17.23 50.2 Grape seed 17.43 53.2 15.87 45.3 Aspergillus japonicacus Big head 17.45 49.0 16.89 46.2 Sunflower seeds 26.42 84.2 25.21 77.5 Grape seed 22.49 79.4 21.87 73.7 Aspergillus Awamori Big head 18.43 58.4 17.77 55.3 Sunflower seeds 21.75 73.1 20.53 69.8 Grape seed 28.45 86.2 26.37 81.4

As shown in Table 1, all fermented experimental groups showed better results in induction period and electron donating ability (antioxidant activity) than oils extracted from unfermented raw materials. The efficacy was excellent in the post fermented sample.

Soybeans were found to be more effective in Bacillus subtilis, sunflower seeds in Aspergillus japonicus, and grape seeds in Aspergillus awamori. Efficacy was excellent in the period of induction and the electron donating ability of fats and oils in fermented fats and oils after immersion and germination. From the following examples, samples were prepared by immersion, germination, fermentation, and culturing into strains optimized for each oilseed.

Example 2 Cell Culture

The immune cell lines used in the experiment were human immune T cells (Jurkat, American Type Culture collection (ATCC), Manassas, VA, USA) and B cells (Raji, ATCC) for the experiment, and NK-92MI cells for measuring NK cell activity. (NK-92MI, ATCC) was used. The cancer cell line used human lung cancer cell A549 (lung carcinoma, human, ATCC) and human breast cancer cell MCF-7 (breast adenocarcinoma, human, ATCC). HEK293 (humnan embryonic kidney, ATCC) was used.

Reagents necessary for cell culture were RPMI 1640 (Invitrogen Corp., Carlsbad, CA, USA) and DMEM-F12 (Invitrogen Corp.) DMEM (Invitrogen Corp.), respectively. , UT, USA). In addition, hepes buffer (Sigma chemical Co), gentamycin sulfate (Sigma), and trysin-EDTA (Sigma Chemical Co) were used as reagents for cell culture.

Example 3 Immune Cell Growth Enhancement Effect and Cytokine Secretion Measurement

Immune function enhancement effect was verified using T-cellized B cells, which are immune cells. The growth of the cells was incubated at 5% CO ₂ , 37 ℃ in RPMI 1640 medium containing 10% FBS, the immune function enhancing effect was adjusted to a concentration of 1.0 × 10 ⁴ cells / ㎖ in a 24 well plate sample Dosing was performed by incubating for 8 days and counting the number of cells in each well using a hemacytometer every day to determine growth growth.

Cytokine was quantified using the cytokine assay kit (United Chemi-Con, Rosemont, IL, USA) for the amount of IL-6 and TNF-α secreted. 900uL each was added to a 24 well plate adjusted to a concentration of 1ⅹ10 ⁴ cells / ml, followed by incubation for 24 hours (37 ℃, 5% CO ₂ ), and the final concentration of the sample was added to 100mg in 0.5mg / ml. And cultured again (37 ° C., 5% CO ₂ ). The supernatant was collected by centrifugation of the culture medium, and the absorbance was measured using a microplate reader at 450 nm, and the amount of cytokine was measured by comparing the obtained OD value with a standard curve prepared using a standard material.

As a result, the immune-promoting effect was confirmed by measuring the growth promoting effect of B cells and T cells, immune cells that play an important role in the human immune system for the exploration of the immune activity of fermented oil prepared by oilseeds. In all fermented oils prepared by oilseed seeds, the growth of B cells was increased and concentration-dependently increased (FIG. 2).

The growth of T cells showed similar results to the growth of B cells, showing the highest value at the concentration of 0.8 mg / ml, but the growth of T cells was also increased at the other concentrations (FIG. 3).

Table 2 below is a data that can support the growth of human immune cells, and the amount of cytokines (IL-6 and TNF-α) secreted by immune cells plays an important role in the human immune system. , And the results measured in T cells. As shown in Table 2, all fermented oil added groups showed an increase in cytokine secretion.

TABLE 2

Sample

Hours
Specific secretion (10 ^-4 pg / ml) B cell T cell IL-6 TNF-α IL-6 TNF-α Control
(Normal
Soybean oil) 2 1.1 1.0 2.4 3.2 4 2.4 3.2 3.1 5.3 6 7.1 6.3 8.6 7.6 Fermented Soybean Oil
(Bacillus subtilis) 2 2.4 2.1 2.1 3.3 4 4.7 4.9 5.7 6.9 6 7.6 7.7 9.1 8.7 Fermented sunflower seed oil (Aspergillus japonicaus) 2 2.5 1.9 2.8 3.2 4 5.1 4.7 6.3 7.1 6 8.2 8.1 9.6 8.9 Fermented Grape Seed Oil
Aspergillus Awamori 2 2.2 2.0 2.4 3.1 4 4.9 5.5 5.9 7.7 6 7.9 9.2 8.9 8.6

Example 4 Immunostimulating Effect of NK Cells

NK-92MI cells were treated with 2 mM L-glutamine, 0.2 mM myoinositol, 20 mM folic acid, 2-mercaptoethanol, 12.5% FBS and 12.5% horses in α-MEM medium. Horse serum (Myelocult, StemCell Technologies Inc., Vancouver, British, Columbia, Canada) was used after diluting to a concentration of 2 × 10 ⁷ cells / ml.

Human T cells and B cells were cultured in a T-25 flask, followed by administering a sample, followed by three to four passages while observing the degree of proliferation. Dispense NK-92MI cells into 4 wells at 5 to 10 ⁴ cells / mL in 24 well plates at 900uL. After 24 hours, supernatant of T cells and B cells are administered to each plate at 100uL for 48 hours. Cell activity (nucleocounter, Chemometec, Denmark) using the cell activity was measured by measuring the viable cell number to determine the activity of NK-92MI cells.

As a result of the experiment, the activity of NK cells was measured by comparing the change in viability shown by adding the culture medium of immune B cells to which the sample was added to NK cells. Figure 4 shows the activity of the NK cells appearing when each sample is added to the B cells and cultured when the culture solution is added to the NK cells. The growth rate was observed for 6 days and showed high growth rate during the measurement period, and there was no significant difference between the samples, but the growth enhancement became apparent over time (FIG. 4).

Example 5 Measurement of Cytotoxicity and Anticancer Activity

Cytotoxicity was measured using HEK293, a human kidney cell, by means of a sulfohodamine B (SRB) assay, and anticancer activity was similarly SRB using A549, a human lung cancer cell, and MCF-7, a human breast cancer cell. It was measured by the method. Add 100uL of each cell to 96 well plate at 4 ~ 5 cells10 ⁴ cells / ml and incubate for 24 hours (37 ℃, 5% CO ₂ ). , 0.6, 0.8, 10 mg / ㎖ was prepared and incubated for 48 hours by adding 100uL. After incubation, remove the supernatant, add 100 uL of cold 10% (w / v) trichloroacetic acid (TCA), leave at 4 ° C for 1 hour, wash 4-5 times with distilled water to remove TCA, and dry the plate at room temperature. Each well was then added with 100uL of 0.4% (w / v) SRB solution dissolved in 1% (v / v) acetic acid and stained at room temperature for 30 minutes. The unbound SRB dye was washed 4-5 times with 1% acetic acid solution, dried at room temperature, and then dissolved in the dye solution by adding 100 μL of 10 mM tris buffer to the microplate reader (Thermo max, Molecular Devices, Sunnyvale, CA) at 540 nm. , USA) was used to measure the absorbance.

Selectivity was measured as the ratio of the cancer cell growth inhibitory activity to the cytotoxicity at each concentration after measuring cytotoxicity against normal cells (HEK 293) and growth inhibitory activity against each cancer cell line at each sample concentration using the SRB method. It was.

The cytotoxicity against normal cells was examined by adjusting the concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml of fermented oils used in the experiment, respectively. Cytotoxicity against HEK293, a human kidney cell, was not significantly different between samples and did not show significant cytotoxicity compared to the control group (Fig. 5).

The anticancer activity was adjusted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / ml of fermented oil samples used in the experiment, and the effects of growth inhibition on cancer cells were examined. Figure 6 shows the growth inhibitory activity for liver lung cancer cells A549 showed a tendency to increase in a concentration-dependent manner and showed a higher activity than the control (Fig. 6). FIG. 7 shows the growth inhibitory activity of MCF-7, a breast cancer cell. As in the measurement of A549, the inhibitory activity was concentration dependent on all samples and showed high anticancer activity compared to the control group (FIG. 7).

On the other hand, while the above has been described with reference to the preferred embodiments of the present invention, those skilled in the art will be variously modified within the scope of the present invention without departing from the spirit and scope of the present invention described in the claims below. It will be appreciated that modifications and variations can be made.

1 is a flowchart illustrating a process for preparing fermented oil according to the present invention.

Figure 2 is a graph measuring the growth promoting effect of B cells to explore the immune activity of the present invention.

Figure 3 is a graph measuring the growth promoting effect of T cells in order to explore the immune activity of fermented oil according to the present invention.

Figure 4 is a graph measuring the activity of NK cells of fermented oil according to the present invention.

5 is a graph measuring whether the fermented oil according to the present invention is cytotoxic to normal cells.

Figure 6 is a graph measuring the growth inhibitory activity of the fermented oil according to the invention A549 lung cancer cells.

Figure 7 is a graph measuring the growth inhibitory activity for MCF-7 fermented oil according to the present invention breast cancer cells.

Claims (6)

At 30 ° C., soaking soybean, sunflower or grape seeds in water for 24 hours and germinating; Inoculating the germinated soybean, sunflower seed or grape seed into a strain and solid fermenting at 35 ° C. for 15 hours; Drying the solid fermented soybean, sunflower seed or grape seed and then grinding to extract lipids; And Purifying the extracted lipids; Fermentation oil production method comprising a. Steaming soybean, sunflower seed or grape seed for 30 minutes at 120 ° C. and cooling the steamed soybean, sunflower seed or grape seed to 30-35 ° C .; Inoculating the strain with chilled soybean, sunflower seed or grape seed and solid fermenting at 35 ° C. for 15 hours; Drying the solid fermented soybean, sunflower seed or grape seed and then grinding to extract lipids; And Purifying the extracted lipids; Fermentation oil production method comprising a. The method according to claim 1 or 2, The strain is Bacillus firmus, Bacillus subtilis, Bacillus subtilis, Aspergillus oryzae, Aspergillus flavus, Aspergillus japonicus And Aspergillus awamori; The strain is inoculated 0.5% by weight of the soybean, sunflower seeds or grape seed production method of fermented oil. As fermented oil prepared by claim 1 or 2, Fermented oil with antioxidant activity. As fermented oil prepared by claim 1 or 2, Fermented oil with immune activity. As fermented oil prepared by claim 1 or 2, Fermented oil with anticancer activity.

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