KR20140078851A - Method for producing chlorella extract with improved functionality using supercritical fluid extraction technique - Google Patents

Abstract

The present invention relates to a method for producing chlorella extract with improved functionalities characterized by extracting chlorella powder using supercritical carbon dioxide as a solvent, the chlorella extract produced by the method and processed food including the chlorella extracts, wherein the chlorella extract of the present invention can provide extracts with improved functionalities such as antioxidant activities, chlorophyll content and anti-cancer effects compared to chlorella extracts produced by existing methods.

Description

TECHNICAL FIELD The present invention relates to a chlorella extract having improved functionality by using a supercritical fluid extraction method,

The present invention relates to a process for producing chlorella extract having enhanced functionality, which comprises extracting chlorella powder with supercritical carbon dioxide as a solvent, a chlorella extract prepared by the process, and a processed food containing the chlorella extract.

Recently, as the quality of life has improved and the interest in health has increased due to the improvement of living standards, and the consumption of health supplement foods and interest in functional foods have increased, the demand for health supplement foods and functional foods has increased. In the living body, various reactive oxygen species (ROS) including various radicals are produced as a result of various life sustaining activities such as breathing and energy generation. These reactive oxygen species irreversibly destroy cell components such as lipids, proteins, and DNA, thereby acting as a cause of cancer, inflammation, cardiovascular disease, and the like, as well as directly or indirectly causing skin diseases or aging.

Rich and diverse biological and mineral resources are still largely undeveloped in lakes and oceans that occupy most of the surface of the earth. Therefore, research and development on these resources are actively carried out in various countries around the world. Compared with ordinary agricultural vegetables, seaweed contains not only general components such as protein, lipid, and carbohydrate, but also various kinds of essential trace elements. It also contains ingredients showing specific physiological activities according to kinds, , And health food. Among them, chlorella is a spherical unicellular green algae with a diameter of 2 ~ 10 ㎛ which grows and proliferates by photosynthesis. It is a high protein food with an essential amino acid composition. Total amino acid content is much higher than beef, and protein, lipid, dietary fiber, vitamins and minerals Is nutritionally superior to other foods and contains a large amount of chlorophyll a and b.

Extraction method using supercritical fluid sets the pressure and temperature above the critical point, which is the point of the vapor phase and liquid phase of the material, so that it has the characteristics of liquid phase dissolution power, gas phase diffusion coefficient and viscosity, Method. Since the supercritical fluid is mainly formed of carbon dioxide or a small amount of co-solvent, there is no risk of residual harmful solvent compared with the solvent extraction method. In addition, since the extraction operation can be performed at a room temperature, It is a useful method for extraction.

Currently, studies on extraction of chlorella are limited to hydrothermal, enzymatic and organic solvent extraction methods, and research on the extraction of chlorella supercritical fluid is still insufficient.

Korean Patent Registration No. 0558382 discloses a method of preparing a Ginkgo biloba extract using supercritical fluid extraction, but it is different from the method of producing chlorella extract having enhanced functionality using the supercritical fluid extraction method of the present invention.

Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to solve the problems of the conventional organic solvent extraction method, such as the human body, environmental toxicity, high cost and low selectivity, The present invention aims at establishing an extraction method for producing an extract having enhanced functional components and physiological activity of chlorella using supercritical fluid technology which is attracting attention as a technology.

In order to solve the above-described problems, the present invention provides a method for producing chlorella extract having enhanced functionality, which comprises extracting chlorella powder with supercritical carbon dioxide as a solvent.

The present invention also provides a functionalized chlorella extract prepared by the above method.

The present invention also provides a health functional food containing the chlorella extract.

The chlorella extract prepared through the supercritical fluid extraction process of the present invention has antioxidative effect and anticancer effect against gastric cancer, lung cancer and cervical cancer as compared with the conventional solvent extract and supercritical extract prepared under different conditions, It is possible to utilize the chlorella extract with improved functionality to be usefully used in foods, medicines, cosmetics industry, and the like.

In addition, the greatest advantage of the present invention is that by extracting carbon dioxide, which is harmless to the human body, as a solvent, it is more environmentally friendly than the conventional organic solvent extraction method, and the process is simpler than the conventional extraction method, There is an advantage.

Figure 1 shows the supercritical fluid extraction device used in this experiment.
FIG. 2 is a graph showing the concentration of superoxide radical scavenging ability (%) of chlorella extract by concentration.
FIG. 3 is a graph comparing the amount of nitric oxide produced in chlorella extract by concentration. FIG.
FIG. 4 is a graph comparing survival rates of cancer cells (A549: lung cancer cells, HeLa: cervical cancer cells, SNU719: stomach cancer cells) according to the concentration of chlorella extract.
50% ethanol: chlorella 50% ethanol extract, 94% ethanol: chlorella 94% ethanol extract, Supercritical fluid: chlorella supercritical extract shown in FIGS.

In order to accomplish the object of the present invention, the present invention provides a method for producing chlorella extract having enhanced functionality, which comprises extracting chlorella powder with supercritical carbon dioxide as a solvent.

In the process for preparing chlorella extract of the present invention, the extraction is preferably carried out at a temperature of 45 to 55 ° C and a pressure of 180 to 220 bar for 35 to 45 minutes, more preferably at a temperature of 50 ° C and at 200 bar pressure for 40 minutes. The extraction of chlorella under the above conditions can enhance the antioxidative and anticancer effects of the extract and enhance the chlorophyll content of chlorella in the extract, compared with the conventional extraction with an organic solvent or supercritical extraction under other conditions.

In addition, in the method for preparing chlorella extract of the present invention, an auxiliary solvent may be further used to further enhance the functionality of the extract at the time of extraction. The auxiliary solvent may be ethanol, isopropanol, ethyl acetate or tetrahydrofuran, Preferably ethanol, but is not limited thereto.

In addition, in the method for producing chlorella extract of the present invention, the functionalities may be antioxidant activity, chlorophyll content or anticancer effect, and the cancer may be gastric cancer, lung cancer or cervical cancer, but is not limited thereto.

The method for preparing chlorella extract of the present invention is characterized in that chlorella powder is prepared by supplying supercritical carbon dioxide as a solvent, supplying the solvent at a flow rate of 18 to 22 mL / min and further using ethanol as an auxiliary solvent at 45 to 55 ° C Temperature and pressures of 180 to 220 bar for 35 to 45 minutes. More specifically, chlorella powder can be prepared by supplying supercritical carbon dioxide as a solvent at a flow rate of 20 mL / min and further adding ethanol Using as an auxiliary solvent and extracting at a temperature of 50 ° C and a pressure of 200 bar for 40 minutes.

The present invention also provides a functionalized chlorella extract prepared by the above method.

The present invention also provides a processed food containing the chlorella extract. There is no particular limitation on the kind of the processed food. Examples of the food to which the chlorella extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all processed foods in a conventional sense.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

1. Experimental material

Chlorella sp. Powder was used for this experiment. The sample was stored in a cow below -20 ℃ and used as a sample for extraction.

2. Sample extraction method

(1) Solvent extraction

50% ethanol and 94% ethanol were added to each sample at a ratio of 1:20, and the mixture was refluxed for 4 hours at 50 ° C (CA-1112, Eyela Co., Japan). After extraction, it was filtered using a Whatman No.2 filter paper (Whatman International Ltd, England) to remove impurities. The filtered solution was concentrated under reduced pressure using a vacuum concentrator (Model N-1N, Eyela Co., Tokyo, Japan), and lyophilized (FreeZone 2.5, Labconco Co, USA) Was used as a sample.

(2) supercritical extraction

As shown in FIG. 1, the supercritical fluid extraction apparatus used in this experiment was an HPLC pump (pu-980, JASCO Co., Japan) equipped with a cooling head and another HPLC pump for supplying an auxiliary solvent, An air-driven oven (CO-965 column oven, JASCO Co., Japan) equipped with an extraction vessel, an electronic back pressure regulator (880-01, JASCO Co., Co., Japan). The pressure and temperature of each extraction process are adjusted by a back-pressure regulator and an air-driven oven.

5 g of the sample was filled in an extraction vessel and extracted at a pressure of 200 bar and a temperature of 50 ° C for 40 minutes at a CO ₂ flow rate of 20 mL / min. Extraction was performed by adding ethanol as a co-solvent. The extracted extract was concentrated under reduced pressure with a rotary vacuum concentrator (Model N-1N, Eyela Co., Tokyo, Japan) twice by water (H ₂ O) And stored in a dark room at -70 ° C or lower.

3. Experimental Method

(1) Electron donating ability measurement

The electron donating ability of the extract was measured by the reducing power of 1,1-diphenyl-2-pycrylhydrazyl (DPPH). That is, 12 mg of DPPH reagent was dissolved in absolute ethanol (100 mL), and 100 mL of water was added to adjust the absorbance of the DPPH solution to about 1.6 at 517 nm. Then, 0.5 mL of the extract was mixed with 5.0 mL of the DPPH solution After incubation at room temperature for 15 minutes, absorbance was measured with a spectrophotometer (Ultraspec 2100pro, Amersham Co., Sweden) and calculated as follows.

Electron donating ability (%) = (1 - absorbance of sample addition group / absorbance of no addition group) x 100

(2) Measurement of ORAC (Oxygen Radical Absorbency Capacity)

Antioxidant activity was measured by ORAC (Oxygen Radical Absorbance Capacity) method, which was used by Talcott ST and Lee JH to measure antioxidant activity. In this experiment, Neutral Phosphate Buffer (61.6: 38.9 v / v, 0.75 MK ₂ HPO4 and 0.75 M NaH ₂ PO ₄ ) was used for the dilution of the test solution and standard solution and preparation of the experimental sample. In order to prepare a calibration curve, a water soluble analogue of vitamin E (6-hydroxy-2,5,7,8-tetramethlychroman-2-carboxylic acid, Aldrich Chem, Inc., USA) (Sigma, St. Louis, Mo., USA) was dissolved in phosphate buffer (50 mL), and the concentration of the fluorescent stock (Sigma, St. Louis, Mo., USA) Using a fluorescent microplate reader (Infinite M200 PRO, Tecan Co., Austria), the electrons were excited at 485 nm and emitted at 538 nm.

(3) Ferric-reducing antioxidant potential (FRAP) measurement

The antioxidant activity by FRAP method was measured by using a 300 mM acetate buffer (pH 3.6), a 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution dissolved in 40 mM HCl and a 20 mM FeCl ₃ .6H ₂ O Were mixed at a ratio of 10: 1: 1 (v / v / v), and the mixture was heated in a 37 ° C water bath. 25 μl of the sample solution and 175 μl of the FRAP substrate were mixed in a 96-well plate, and reacted at 37 ° C. for 4 minutes. The absorbance at 590 nm was measured, and FeSO ₄ · 7H ₂ O was used as a standard Calculated from the standard calibration curve obtained.

(4) Total chlorophyll content measurement

Total chlorophyll content was measured by dissolving 0.05 g of dry powder in 10 mL of distilled water and sonication for 30 minutes. 2 mL of the suspension was placed in a shaded centrifuge tube, 5 mL of an alkaline pyridine solution was added, and the mixture was ultrasonicated for 15 minutes in a water bath at 60 ° C. After centrifugation at 3000 rpm at 4 ° C for 3 minutes, the supernatant was transferred to a 10 mL brown m-flask, and the remaining residue was repeatedly treated to a final volume of 10 mL. Absorbance was measured at a wavelength of 419 nm and a wavelength of 454 nm using a solution of alkaline pyridine as a reference solution.

Total chlorophyll (mg / 100 g) = C / S x 100

C: chlorophyll (mg / L) = 8.970 × (7.19 E419 nm + 3.33 E454 nm)

S: Number of samples collected / 5

E419 nm: Absorbance at 419 nm

E454 nm: absorbance at 454 nm

(5) Superoxide radical scavenging activity measurement

Superoxide radical scavenging activity was determined by mixing 800 μL of a 20 mM Tris solution (pH 8.0) containing 62 μM NBT (nitro blue tetrazolium) and 98 μM β-nicotinamide adenine dinucleotide (NADH) in 20 μL of the diluted sample Then, 80 μl of a 20 mM Tris solution and 100 μl of 33 μM phenazine methosulfate (PMS) were added, respectively. In other words, the superoxide radicals induced by PMS / NADH nonenzymatically were measured by measuring the absorbance of the reactants at 560 nm for 10 minutes to measure formazan and the following equation.

Superoxide radical scavenging ability = [(AB) / A] x 100

A: absorbance without addition of sample

B: absorbance of sample added

(6) Cell culture

Human gastric cancer cells (SNU719), human lung cancer cells (A549), human cervical cancer cells (HeLa) and mouse macrophages Raw 264.7 cells were used for the experiment. SNU719, A549, HeLa cell line and Raw 264.7 cell line were supplemented with 10% FBS (Fetal bovine serum) and penicillin-streptomycin using DMEM medium. Cells are both 37 ℃, 5% CO ₂ And incubated in an incubator.

(7) Measurement of inhibitory activity of nitric oxide

The amount of nitric oxide produced from Raw 264.7 cells was measured using a Griess reagent as follows. That is, 100 μl each of 2 × 10 ⁴ cells / mL was added to each well of a 96-well plate, and incubated at 37 ° C. in 5% CO ₂ And cultured in an incubator for 24 hours. After culturing, various concentrations of extracts and LPS (1 μg / mL) dissolved in DMSO were treated and cultured for 24 hours. After incubation, 50 ㎕ of supernatant was mixed with the same amount of Griess reagent, incubated for 10 min, and absorbance was measured at 540 nm. The concentration of NO was calculated using the standard curve for the concentration of sodium nitrite.

(8) Measurement of anticancer activity

The cultured cancer cell lines, 2 × 10 ⁴ cell / mL was added to each 96-well plate by 100 ㎕ to 37 ℃, 5% CO ₂ After culturing for 24 hours in an incubator, the chlorella extract dissolved in DMSO was treated for each concentration and cultured for 48 hours. After the cultivation was dissolved in PBS buffer solution, MTT (5 mg / mL) solution for each well (well) 10 ㎕ added and again four hours by the CO ₂ MTT was reduced by incubation in an incubator. Formazan crystals formed after the completion of incubation were completely removed so that the culture solution was completely removed. 100 μl of DMSO was added to each of the removed wells to react for 10 minutes to dissolve the formazan crystals, and the ELISA reader And the absorbance at 540 nm was measured. Cells not treated with the extract were expressed as relative cell viability as a control.

(9) Statistical processing

Experimental results were statistically analyzed with SPSS 12.0 package and expressed as mean ± standard deviation for each sample. The significance test for each sample was analyzed by Duncan's multiple test at p <0.05 after analysis of variance.

Example  1: Extraction yield of chlorella extract

Extraction yields of supercritical fluid extraction, 94% ethanol and 50% ethanol were 5.2%, 3.4% and 8.1%, respectively, and 50% ethanol was the best. These results suggest that soluble solids were extracted in addition to the desired chlorophyll content due to the nature of the polarity.

Extraction yield of chlorella extract Extract Type Yield ^{1 )} (%, w / w) Chlorella supercritical extract 5.2 Chlorella 94% Ethanol Extract 3.4 Chlorella 50% Ethanol Extract 8.1

¹⁾ Yield (%) = solid content (g) of extract / raw material (dry weight) x 100

Example  2: of the chlorella extract Electron donating ability  And ORAC

Electron donating ability is used to donate electrons to active radicals and to inhibit lipid oxidation in foods. In the human body, it is used to inhibit aging by active radicals. Radical scavenging action prevents human diseases and aging It plays a very important role. The electron donating ability of the extract according to the extraction method of chlorella is shown in Table 2. The electron donating ability of chlorella extract was the highest at 30.15% under the supercritical extraction condition and the lowest at 50% ethanol extract was 7.95%.

The ORAC method used in this study was based on the free radicals generated by AAPH (2,2'-azobis-2-methyl-propanimidamide, dihydrochloride) in association with hydrogen electron transfer, which is the most critical step of the radical chain reaction (Ie, radical chain breaking antioxidant capacity) by measuring the antioxidant's ability to remove antioxidants, ie, hydrophobic and hydrophilic components, Have. As a result of ORAC measurement of the extracts according to the extraction method of chlorella, supercritical extract and 94% ethanol extract were 134.18 μmoles TE / g and 120.90 μmoles TE / g respectively, and 50% ethanol extract was 115.74 μmoles TE / g, Was the most excellent.

Electron donating ability of chlorella extract and ORAC Extract Type Electron donating ability (%) ORAC (μmoles TE / g) Chlorella supercritical extract 30.15 + 0.64 ^a 134.18 ± 4.97 ^a Chlorella 94% Ethanol Extract 12.21 + - 0.94 ^b 120.90 ± 0.28 ^b Chlorella 50% Ethanol Extract 7.95 + - 0.90 ^c 115.74 ± 6.32 ^b

The data represent the mean ± standard deviation from three replicate experiments

The other superscripts in each column of ^ac mean that there is a significant difference ( p <0.05)

Example  3: Total chlorophyll content of chlorella extract

The total chlorophyll content of the extract according to the extraction method is shown in Table 3. The total chlorophyll content of the supercritical fluid extract was 8,064.41 ㎎ / 100 g and the 94% and 50% ethanol extracts were 5,362.11 ㎎ / 100 g and 549.30 ㎎ / 100 g, respectively. Therefore, it was confirmed that the supercritical fluid extraction process was effective for chlorophyll extraction, which is a main functional ingredient of chlorella. The chlorophyll content of the syrup type chlorella product was reported to be 30.8 ㎎ / 100 g and the chlorophyll content of the chewable type chlorella product was 123.7 mg / 100 g. The chlorophyll content of the various food and cosmetic industries It is confirmed that the supercritical fluid extraction method can be utilized.

Total chlorophyll content of chlorella extract Extract Type Total chlorophyll content (mg / 100 g) Chlorella supercritical extract 8,064.41 ± 94.40 ^a Chlorella 94% Ethanol Extract 5,362.11 ± 140.41 ^b Chlorella 50% Ethanol Extract 549.30 ± 22.57 ^c

The data represent the mean ± standard deviation from three replicate experiments

The other superscripts in each column of ^ac mean that there is a significant difference ( p <0.05)

Example  4: Chlorella extract FRAP Antioxidant activity by

The antioxidative activity of ferric reducing antioxidant power (FRAP) is based on the principle that ferric tripyridyltriazine (Fe ^{3 +} -TPTZ) complex is reduced to ferrous tripyridyltriazine (Fe ^{2 +} -TPTZ) Most of the antioxidants are designed with a focus on the fact that they have a reducing power. The FRAP results of chlorella extract according to the extraction method are shown in Table 4.

The activity was increased in a concentration - dependent manner at all extraction conditions. However, no significant difference was observed between 47 and 46.70 μM at 50 ~ 500 ㎍ / mL concentration of 50% ethanol extract. At the concentration of 1,000 ㎍ / mL, FRAP antioxidant activity was statistically the highest at 397.17 μM in supercritical extract and 220.53 μM and 80.30 μM in 94% ethanol extract and 50% ethanol extract, respectively. This is because the ascorbic acid and the antioxidative activity of FRAP method used as a positive control means the effect of reducing the metal ion acting as a catalyst of the oxidation reaction. The reducing power of the sample is also related to the scavenging activity of the radical through electron donation The results of this study are similar to those of electron donating ability.

Antioxidant activity of FRAP of chlorella extract (FeSO ₄ eq μM) Extract Type Concentration (/ / mL) 100 250 500 1000 Supercritical extract 71.90 + - 6.84 ^a 123.06 + - 8.02 ^b 217.23 ± 7.97 ^c 397.17 + - 4.41 ^d 94% ethanol extract 78.77 ± 2.45 ^a 81.06 ± 8.41 ^a 131.46 ± 10.36 ^b 220.53 + - 7.72 ^c 50% ethanol extract 17.67 ± 1.52 ^a 47.00 ± 1.73 ^b 46.70 ± 1.92 ^b 80.30 ± 2.89 ^c ascorbic acid 993.23 ± 9.67 ^a 1710.44 + 15.01 ^b 1767.20 ± 10.01 ^c 1774.58 + - 23.41 ^c

^ad The other superscripts in each column indicate a significant difference ( p <0.05)

Example  5: Chlorella extract Superoxide  Radical ( Non - enzymatic system : NADH-PMS) scavenging activity

In the case of the superoxide radical generation system induced by the nonenzymatic PMS / NADH, NBT is reduced to purple formazan. In the presence of the superoxide radical scavenging activity in the sample, the addition of the sample inhibits the formation of formazan and decreases the absorbance. The superoxide radical scavenging activity of each concentration of chlorella extract according to the extraction method is shown in FIG. As the concentration increased, the scavenging activity was increased but the scavenging activity was not significantly different. The supernatant fluid extract showed 66.76% scavenging activity at 1,000 ㎍ / mL. The 94% ethanol extract and the 50% ethanol extract showed no significant difference in the scavenging activity between the extracts.

Example  6: Nitric oxide of chlorella extract ( NO ) Production amount

The amount of NO produced is determined by measuring the content of NO in the culture medium of RAW 264.7 cells, which is produced by the action of NO synthetase on L-arginine, and NO produced is known to inhibit the proliferation of cancer cells have. The results of investigating the effect of chlorella extract on the production of NO after treatment of 1,000 ㎍ / mL concentration, which does not affect cell survival rate, are shown in FIG. In the LPS-treated group, the NO production was 27.08 μM, and the supercritical extract increased the concentration-dependent production from 250 μg / mL. The NO production was increased from 1,000 μg / mL to 26.87 μM, which was 99.24% Enhancing activity. 94% ethanol extract and 50% ethanol extract showed 11.97 μM and 12.31 μM nitric oxide production at 1,000 ㎍ / mL, respectively.

Example  7: Inhibitory Effect of Chlorella Extract on the Growth of Cancer Cells MTT Assay )

The results of examining the cell viability by treating chlorella extract according to the extraction method with three types of cancer cells such as A549, HeLa and SNU719 are shown in FIG. The inhibitory effect of A549 cells on supercritical fluid extract was not significantly increased at 100 ㎍ / mL concentration, but showed a high inhibitory effect on cancer cell growth of 49.32 ~ 56.04% at 250 ~ 1,000 ㎍ / mL. The 94% ethanol extract showed a mild inhibitory effect of 13.79% at 1,000 ㎍ / mL and almost no effect at other concentrations. The inhibitory effect of 50% ethanol extract on the growth of cancer cells was observed at 250 ~ 1,000 ㎍ / mL at 20.83 ~ 23.2%, and supercritical fluid extract showed the highest inhibitory effect on cancer cell growth.

In the case of HeLa cells, the growth inhibition effect of cancer cells was 55.68 ~ 57.05% at 100 ~ 1,000 ㎍ / mL concentration of supercritical fluid extract, which was the highest in the case of 94% ethanol extract and 50% ethanol extract. The 94% ethanol extract showed no inhibitory effect on the growth of cancer cells and showed a 41.04% inhibitory effect on cancer cell growth at 1,000 ㎍ / mL of 50% ethanol extract.

In SNU719 cells, the supercritical fluid extract and the 50% ethanol extract at the concentration of 1,000 ㎍ / mL were 52.95 and 30.32%, respectively. The inhibitory effect of the ethanol extract on the growth of cancer cells was dose dependent.

Claims (7)

Wherein the chlorella powder is prepared by extracting the chlorella powder with supercritical carbon dioxide as a solvent at a temperature of 45 to 55 ° C and a pressure of 180 to 220 bar for 35 to 45 minutes. The method according to claim 1, wherein the auxiliary solvent is further used in the extraction, and the auxiliary solvent is ethanol, isopropanol, ethyl acetate or tetrahydrofuran. The method according to claim 1, wherein the functionalities are antioxidant activity, chlorophyll content, or anticancer effect. [Claim 5] The method according to claim 3, wherein the cancer is gastric cancer, lung cancer or cervical cancer. The method of claim 1, wherein the chlorella powder is prepared by using supercritical carbon dioxide as a solvent, supplying the solvent at a flow rate of 18 to 22 mL / min and further using ethanol as an auxiliary solvent at a temperature of 45 to 55 DEG C and a pressure of 180 to 220 bar At a pressure of 50-45 &lt; 0 &gt; C for 35-45 minutes. A functional enhanced chlorella extract prepared by the method of any one of claims 1 to 5. A processed food containing the chlorella extract of claim 6.

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