KR102421434B1 - Fermented vegetable oil with improved oxidation stability and emulsificatin and preparation thereof - Google Patents

Abstract

The present invention uses microorganisms to offset the hydrophobic interaction of oil to improve the affinity of oil and highly polar substances, thereby reducing surface tension, and improving emulsification power, oxidation stability, skin absorption, and formulation stability. have.
In addition, it is possible to provide a vegetable fermented oil with improved oxidative stability and acid odor by refining free fatty acids and removing impurities with an affinity chromatography column filled with magnesium silicate in the recovery process of the fermented vegetable oil.

Description

Fermented vegetable oil with improved oxidative stability and emulsification ability and manufacturing method thereof

The present invention relates to a vegetable fermented oil having improved oxidative stability and emulsification ability and a method for producing the same, and to an oil capable of improving emulsification power, oxidative stability, skin absorption, and formulation stability, and a method for preparing the same.

Developing various technologies capable of inducing changes in function by fermenting oil is in response to industrial demands to develop various products.

In the conventional cosmetic composition containing oil, additives such as emulsifiers and surfactants are generally used for mixing with other substances.

These additives promote the destruction of the skin barrier and allow other chemical additives contained in the cosmetic composition to penetrate into the skin, which causes skin toxicity and can dry the skin and accelerate aging. have.

Korean Patent Publication No. 10-2013-0023162 discloses a fermented vegetable oil and a composition comprising the same. In the above technique, a method for manufacturing fermented vegetable oil having an effect of improving emulsion stability, feeling of use and flavor by increasing free fatty acids and acid values was presented. However, unsaturated fatty acids have disadvantages that can cause unpleasant odors and skin irritation by reducing oxidation stability due to the influence of peroxides and free radicals formed by easily rancidity of oil due to its unstable chemical properties.

Accordingly, research is required to facilitate the emulsification of oil while minimizing the use of additives. Therefore, the present invention is a result of a study on a method to remove free fatty acids and impurities, which can be a disadvantage of vegetable fermented oil, and to improve the emulsification ability. As a result, the fermentation method and affinity of oil using Candida bombicola UA-06 The purification process of vegetable fermented oil was devised using a chromatography column.

Prior art literature

(Patent Document 1) Republic of Korea Patent Publication No. 10-2013-0023162, March 07, 2013

An object of the present invention is to solve the disadvantages of the conventional vegetable fermented oil by improving the fermentation and refining process of vegetable oil using microorganisms. That is, the main object of the present invention is to provide a vegetable fermented oil with improved oxidative stability and emulsification activity, emulsion stability, skin absorption, fermented acid odor, and the like of the vegetable fermented oil.

In order to solve the above problems, the present invention

To provide a vegetable fermented oil fermented by Candida bombicola UA-06 (Accession No.: KCTC18592P) microorganisms.

According to one embodiment, the vegetable fermented oil may have a surface tension of 1.2 to 3 times lower than before fermentation.

In addition, the vegetable fermented oil may have 1.5 to 20 times higher emulsification activity than before fermentation.

In addition, the fermented vegetable oil may have a micelle size 5 to 20 times smaller than before fermentation.

In addition, the free fatty acid content of the vegetable fermented oil may be 1 to 1.2 times the free fatty acid content of the oil before fermentation.

According to another embodiment of the present invention,

Candida bombicola UA-06 (Accession No.: KCTC18592P) First seed fermentation step of fermenting microorganisms and vegetable oil in a medium;

A secondary seed fermentation step of sub-culturing the first seed fermented product, and

Comprising the main fermentation step of fermenting the secondary seed fermented product by passage,

The medium used in the first seed fermentation step includes yeast extract, malt extract, peptone, glucose, urea and vegetable oil,

The medium used in the secondary seed fermentation step contains glycerin,

The medium used in this fermentation step includes glycerin, K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄ and yeast extract,

The first and second seed fermentation step is to ferment for 10 to 30 hours under aerobic conditions of 15 to 25 ℃,

It is possible to provide a method for producing a vegetable fermented oil in which the main fermentation step is fermented for 90 to 100 hours under aerobic conditions of 15 to 25°C.

According to one embodiment, it is possible to improve oxidation stability and emulsification ability by removing moisture, impurities, and fatty acids through the recovery and purification steps of the vegetable fermented oil.

According to another embodiment of the present invention, there may be provided a cosmetic composition comprising the vegetable fermented oil.

The specific details of other embodiments of the invention are included in the detailed description below.

The present invention can effectively produce vegetable fermented oil by fermenting and refining vegetable oil using microorganisms, and the vegetable oil fermented by the method is functional in emulsification activity, emulsion stability, skin safety, skin absorption, formulation stability, etc. This improved vegetable fermented oil can be produced. In addition, the present invention provides oxidative stability of vegetable fermented oil, which is easily oxidized, through a 2M affinity chromatography column filled with magnesium silicate, thereby reducing skin cytotoxicity and maintaining stable for a long period of time. can be usefully used as

1 is a graph showing the results of surface tension measurement with respect to oil.
2 is a photograph showing the degree of emulsification of oil after 1 hour.
3 is a photograph showing the degree of emulsification of oil after 48 hours.
4 is an optical micrograph of oil.
5 is a graph showing the results of cell viability measurement.
6 shows a result of analyzing a black-and-white balance histogram.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, a vegetable fermented oil having improved oxidative stability and emulsification ability according to an embodiment of the present invention and a manufacturing method thereof will be described in more detail.

The vegetable fermented oil according to the present invention is fermented by Candida bombicola UA-06 (Accession No.: KCTC18592P) microorganism.

Candida bombicola is a microorganism that can produce emulsified substances, and plays an important role in improving the emulsification rate and miscibility with oil without the use of additives such as surfactants, thereby improving skin absorption and formulation stability. etc. can be improved.

According to one embodiment, the fermented vegetable oil has an emulsification activity of 1.5 to 20 times, for example 3 to 15 times, due to a decrease in surface tension of 1.2 to 3 times, for example 1.5 to 2 times, than before fermentation. . In addition, the fermented vegetable oil may improve skin absorption by reducing the size of micelles 5 to 20 times, for example 7 to 15 times, compared to before fermentation.

In addition, the fermented vegetable oil can improve the oxidative stability of the oil by reducing the peroxidation rate by showing the free fatty acid content after fermentation is similar to that before fermentation, for example, 1 to 1.2 times the free fatty acid before fermentation.

Free fatty acids include linoleic acid, oleic acid, palmitic acid, stearic acid, and the like. Free fatty acids can be divided into unsaturated fatty acids and saturated fatty acids, among which linoleic acid and oleic acid belong to unsaturated fatty acids, palmitic acid, stearic acid, etc. It belongs to saturated fatty acids.

The method for producing fermented vegetable oil according to the present invention may include a primary seed fermentation step, a secondary seed fermentation step, and the main fermentation step.

Specifically, the present invention

Candida bombicola UA-06 (Accession No.: KCTC18592P) First seed fermentation step of fermenting microorganisms and vegetable oil in a medium;

A secondary seed fermentation step of sub-culturing the first seed fermented product, and

It provides a vegetable fermented oil by a method comprising the main fermentation step of fermenting the secondary seed fermented product by passage.

According to one embodiment, the primary seed fermentation is Candida bombicola in a medium containing yeast extract, malt extract, peptone, glucose, urea and vegetable oil. UA-06 (Accession No.: KCTC18592P) strain may be inoculated and fermented for 10 to 30 hours under aerobic conditions of 15 to 25 °C.

According to one embodiment, the secondary seed fermentation is performed using a culture medium in which glucose is replaced with glycerin in the primary seed fermentation medium composition, and the primary fermentation product is subjected to aerobic conditions at 15 to 25 ° C. It may be subcultured for 10 to 30 hours.

According to one embodiment, the main fermentation is passaged for 90 to 100 hours at aerobic conditions at 15 to 25° C. using a culture medium containing glycerin, K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄ and yeast extract. It may be culturing.

According to one embodiment, it may further include a purification process step after the main fermentation step. Specifically, for purification of moisture and microorganisms, the fermented product is left standing after main fermentation to separate the oil layer located on the upper layer and the medium and impurity layer located on the lower layer to discard the lower layer, and then add MgSO ₄ and stir An oil from which residual impurities and moisture are removed can be obtained by using a centrifugal separator.

In addition, in order to purify the fatty acids and impurities in the fermented product after the main fermentation, a chromatography method may be used. Specifically, by using magnesium silicate as a filler in an affinity chromatography column, fatty acids and impurities can be adsorbed and filtered.

According to the present invention, since the surface tension can be reduced by canceling the hydrophobic interaction of the oil by using the metabolites by the microorganisms and improving the affinity of the oil and the highly polar substances, surfactants, emulsifiers, etc. It can easily improve emulsification power, absorption power, formulation stability, acid odor, etc. without additives.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Examples and Comparative Examples

Comparative Example 1 and Examples 1 to 5: Preparation of vegetable fermented oil

The oil of Comparative Example 1 was prepared with a medium composition according to Table 1 below.

primary culture

Pseudozyma antarctica (KCTC7640) strain was inoculated into 1 L of medium and cultured at 25° C., 300 rpm, aerobic conditions for 48 hours.

secondary culture

Pseudozyma antarctica (KCTC7640) strain was inoculated into 100L of the same culture medium as the primary culture conditions, and the primary culture was performed for 48 hours, then 100L of olive oil was added to the culture medium and cultured at 25℃, 500rpm, aerobic conditions for 96 hours. did.

collect

The vegetable oil after secondary fermentation is recovered.

In the present invention, Candida bombicola UA-06 (Accession No.: KCTC18592P) strain was used, the primary culture conditions were the same, and the secondary seed fermentation medium composition substituted glycerin instead of glucose, and the rest of the composition was the same. did. And, the fermentation conditions were 20 ℃, 300~500 rpm, and the production time was shortened and the efficiency was increased by performing the fermentation for 24 hours under aerobic conditions. In addition, by adding a refining process in the recovery stage of vegetable fermented oil, high free fatty acids and impurities were removed, thereby improving the marketability of the oil.

The oil of Examples was prepared with a medium composition according to Table 1 below.

1st seed fermentation

Candida bombicola UA-06 (Accession No.: KCTC18592P) strain was inoculated into 1L of medium, and primary seed fermentation was performed for 24 hours under aerobic conditions of 20°C, 300rpm, and 1NL/min (1vvm). The medium used was Yeast extract 3g/L, Malt extract 3g/L, Peptone 5g/L, Glucose 10g/L, Urea 1g/L, Oil 50g/L, Distilled water 928g/L in order to optimize the growth and metabolism of microorganisms. It was set as the composition.

Secondary seed fermentation

In the composition of the primary seed fermentation medium, except that Glucose 10g/L was replaced with Glycerin 20g/L, 10L of culture medium of the same composition was placed under aerobic conditions of 20℃, 300rpm, 10NL/min (1vvm) for 2 hours for 24 hours. Tea seed fermentation was performed.

main fermentation

The secondary seed fermentation culture medium was glycerin 20g/L, K2HPO₄4 25g/L, KH2PO4 7g/L, NH4NO3 3g/L, MgSO4 2g/L, yeast extract 10g/L, distilled water 433g/L, oil 500g/L. The main fermentation was carried out for 96 hours under aerobic conditions of 20° C., 300 rpm, and 100 NL/min (1 vvm) by subculture at 100 L.

refine

In order to remove microorganisms, etc., after the main fermentation, the fermented product was allowed to stand and separated into an upper layer (oil) and a lower layer (medium and impurities), all of the lower layers were discarded, and MgSO ₄ 20g/L was added to remove moisture. and stirred. After 2 hours of stirring, microbial cells, MgSO ₄ , impurities and moisture remaining in the oil layer were removed using a continuous centrifuge equipment to obtain an oil.

In order to remove fatty acids and impurities, magnesium silicate was used as a filler inside an affinity chromatography column with a diameter of 45 pie and a length of 2M, which was made to order, and fatty acids and impurities were adsorbed and filtered at a flow rate of 30 L/hour.

Examples 6 and 7: Formulations comprising fermented vegetable oils

Example 6: Lotion

An external lotion was prepared according to the composition of Table 2 below. Specifically, after sequentially adding raw materials 2 to 5 to raw material No. 1 in Table 2 below, stirring and heating to 60 to 70 ° C. After dissolution by heating, it was put into the raw material reaction vessel Nos. 1 to 5 and emulsified. After the completion of emulsification, the 10th raw material was added, uniformly dissolved and mixed, cooled to 40°C, and then the 11th raw material was added and stirred, cooled to 30°C, and aged.

Example 7: Cream

Creams for external use were prepared according to the composition of Table 3 below. Specifically, the raw materials 2 to 6 are sequentially added to the raw material No. 1 in Table 3 below, stirred, heated to 60 to 70 ° C. to dissolve, and the raw material No. 7 to 10 is put in a separate container, and 60 to 70 After dissolution by heating to ℃, it was put into the raw material reaction vessel Nos. 1 to 6 to emulsify. After the completion of emulsification, raw materials No. 11 and 12 were put in, uniformly dissolved and mixed, cooled to 40° C., and then the No. 13 materials were added and stirred, cooled to 30° C., and aged.

Experimental example

Experimental Example 1: Surface tension

A processor tensiometer (K14, KRUSS) was used to measure the surface tension before and after fermentation of Examples 1 to 4 and the surface tension of Comparative Example 1. Certified grade hexadecae (Acros organics, γ=26.6 dyne/cm) was used, and pure water by Millipore Mill-Q-water system was used. The surface tension was 72.4 dyne/cm, and the pH was 6.4. The surface tension measurement result is shown in FIG. 1 below.

As shown in FIG. 1 , when the oils according to Comparative Example 1 and Example 3 (olive oil) are compared, it can be confirmed that the surface tension of Example 3 is 1.5 to 2 times lower than that of Comparative Example 1. These results confirm that the fermented oil according to the present invention can improve the ability to promote emulsion dispersion by reducing the interfacial tension between two different phases.

Experimental Example 2: Emulsifying Activity

Experimental Example 2-1: Absorbance Measurement

In order to analyze the emulsification activity before and after fermentation of Examples 1 to 4, fermented oil and distilled water were mixed at a ratio of 1:9, stirred vigorously for 1 minute to emulsify, and left still for 10 minutes to collect the solution of the lower layer and measure the absorbance. . Emulsification activity was confirmed by measuring the absorbance at 660 nm using a spectrophotometer (Microplate, BIOTECK, USA), and the results are shown in Table 4 below.

As shown in Table 4, it can be confirmed that the emulsification activity of the oil after fermentation is 4 to 10 times higher than before fermentation.

Experimental Example 2-2: Visual evaluation

The oil was visually evaluated to confirm the emulsifying activity. Specifically, for each of the oils before and after fermentation according to Examples 1 to 4 and Comparative Example 1, they were mixed with distilled water 1:1 and stirred for 5 minutes, and the emulsified state after 1 hour is shown in FIG. 2 below, 48 The emulsified state after standing for time is shown in FIG. 3 below.

As shown in FIGS. 2 and 3 , when the emulsified state with distilled water was observed for each time in Comparative Example 1, before and after fermentation, the oil after fermentation was dispersed in a stable form, whereas In Comparative Example 1 and the oil before fermentation, the separation between distilled water and oil was evident over time.

Experimental Example 2-3: Microscopic observation

For the composition according to Table 5 below, the micelle shape, particle size, and number were measured using an optical microscope (Mitoc AE2000), and the distribution was analyzed using Mitoic Images Plus 3.0 (x64). Optical micrographs and analysis results are shown in FIGS. 4 and 6 below.

As shown in Table 6, since uniform and small emulsified particles can be observed in Example 3 (olive oil) according to the present invention, it can be confirmed that the emulsion stability is improved compared to Comparative Example 1.

Experimental Example 3: Oxidative stability

Experimental Example 3-1: Acid value measurement

Acid value was measured using the Acid Value Test Method of the Food Code. After taking 5 g of oil in an Erlenmeyer flask, add 100 ml of an ethanol-ether mixture (1:2, v/v) to dissolve the oil, add 100 μl of 1% phenolphthalein solution, mix, and 0.1N ethanolic hydroxide until it turns pale red It was titrated with potassium solution. A blank test was performed using distilled water instead of oil.

The calculation formula is as follows.

Acid value = (5.611×a×f)/s

s = amount of sample taken

a=0.1N Consumption of KOH solution (ml)

f=0.1N KOH solution titer

The results are shown in Table 7 below.

Comparative Example 1 had an acid value of 12.34 mg KOH/g, which was more than 17 times higher than that of Example 3 (olive oil) before fermentation, and the fermented oils of Examples 1 to 4 according to the present invention were 1 to 1 compared to the oil before fermentation. It can be seen that there is a double increase.

Experimental Example 3-2: Peroxide value measurement

Peroxide value was measured using the peroxide value test method of the Food Industry Association. After taking 1 g of oil in an Erlenmeyer flask, add 25 ml of a mixed solution of acetic acid:chloroform=3;2 (v/v), cover with a lid, shake lightly, and store in a cool and dark place for 10 minutes. Then, 30 ml of distilled water was added, shaken vigorously, 1 ml of starch solution was added, and then titrated with 0.01 N sodium thiosulfate solution until colorless. A blank test was performed using distilled water instead of oil. The results are shown in Table 8 below.

As shown in Table 8, in Comparative Example 1, the peroxide value of the oil before fermentation was 289 meq/Kg, which was 36 times higher than in Example 3 (olive oil) before fermentation of the present invention, and the fermented oil of the present invention was fermented. The peroxide value increased by 1.3 to 2.5 times compared to before. This result is due to the difference in the fatty acid composition of Comparative Example 1 and the fermented oil according to the present invention, and it is determined that the automatic oxidation rate is increased because the sample of Comparative Example 1 has a high free fatty acid content, and the measurement of the free fatty acid content is performed in the following experiment proceeded in the example.

Experimental Example 4: Measurement of free fatty acids

To measure the oil and free fatty acids of Comparative Example 1 before and after fermentation of Example 3 (olive oil), HPLC analysis was performed, and Waters Alliance2690 liquid chromatograph system (Waters, USA) and Waters 2420 evaporative light scattering detector were used. . As a column, PLRP-S 100 Å 5 μm, 250×4.6 mm (P/N PL1512-5800) was used. Analysis conditions are shown in Table 9 below.

The free fatty acid analysis results are shown in Table 10 below.

As shown in Table 10, in Comparative Example 1, the total free fatty acid content was 165 times higher than before fermentation, but the olive oil after fermentation according to Example 3 of the present invention showed the same level of free fatty acid content as before fermentation.

Experimental Example 5: Cell proliferation and toxicity

In order to verify the human fibroblast proliferation effect and toxicity of the fermented oil according to Examples 1 to 3, an MTT test was performed. Human fibroblasts, Hs68 cells, were cultured in a 96-well culture plate at a concentration of 1x10 ⁴ cells/well at 37°C and 5% CO ₂ conditions for 24 hours. After 2 hours of serum starvation, the cells were treated with vegetable fermented oil at different concentrations (0-1%) and cultured for 48 hours. To measure the viability of cells, all of the culture medium was removed and treated with MTT reagent and cultured for 2 hours. The formed formazan was dissolved in DMSO and absorbance was measured at 570 nm with a microplate reader. The MTT test method is a test method that counts live cells by reducing the mitochondrial reductase soluble MTT reagent to water-insoluble formazan. It is one of the test methods for measuring cell viability, cytotoxicity, and cell proliferation rate. The results are shown in FIG. 5, and when Examples 1 to 3 were treated by concentration, the highest cell proliferation rate of 0.0625% to 115 to 125% was exhibited, and the cell viability up to 0.5% was more than 80%.

Experimental Example 6: Skin Absorption

The skin absorbency of the fermented oil applied to the skin was measured using oil paper. As the fermented oil, Examples 2 (grape seed oil) and 5 (argan oil) before fermentation and Examples 2 and 5 fermented by the method according to the present invention were used.

For each oil, drop one drop at a time with a dropper to absorb it on the skin for 30 seconds, and then absorb the remaining oil with oil paper. Photo the black and white balance of the oil paper? The comparison was made with the histogram analysis function, and the results are shown in FIG. 6 below.

6, the more oil is absorbed in the oil paper, the darker the black color, and when the oil is not absorbed, the original color of the oil paper is light drinking. When the oil paper is represented as a black-and-white distribution diagram, the leftmost part of the graph shows the distribution of the darkest black part of the oil paper, and the rightmost part shows the distribution of the whitest part of the oily paper. The more the graph is skewed to the left, the darker the black part and the larger the area, the more oil is absorbed by the oil paper, which means that the amount of oil absorbed into the skin is small and there is a lot of oil remaining on the skin. As a result, it can be confirmed that the skin absorption is higher in Examples 2 and 5 fermented by the method according to the present invention than in Example 2 (grape seed oil) before fermentation and Example 5 (argan oil) before fermentation.

Experimental Example 7: Sensory Test

For the sensory test of Comparative Examples 1 and 3, 20 women in their 20s to 50s were selected as subjects. After using each of Comparative Examples and Examples, a questionnaire was conducted on the subjects for 4 items of absorbency, stickiness, spreadability, and odor, and the results are shown in Table 11 below.

As shown in Table 12, Example 3 showed superior results compared to Comparative Example 1 in all items of absorbency, stickiness, spreadability, and odor. In Comparative Example 1, it can be seen that the number of subjects who selected very bad in the odor item was high as 7 people.

As can be seen from the above results, according to the present invention, metabolites of vegetable oil cancel the hydrophobic interaction of oil, thereby improving affinity with other highly polar substances to reduce surface tension, emulsifying power, skin absorption, and It can be confirmed that formulation stability can be improved.

The above description is merely illustrative of the technical idea of the present invention, and various modifications and variations are possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. In addition, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the equivalent range should be construed as being included in the scope of the present invention.

Claims (8)

Candida bombicola UA-06 (Accession No.: KCTC18592P) First seed fermentation step of fermenting microorganisms and vegetable oil in a medium;
A secondary seed fermentation step of sub-culturing the first seed fermented product, and
Comprising the main fermentation step of fermenting the secondary seed fermented product by passage,
The medium used in the first seed fermentation step includes yeast extract, malt extract, peptone, glucose, urea and vegetable oil,
The medium used in the secondary seed fermentation step contains glycerin,
The medium used in this fermentation step includes glycerin, K₂HPO₄, KH₂PO₄, NH₄NO₃, MgSO₄ and yeast extract,
The first and the second seed fermentation step is to ferment for 10 to 30 hours under aerobic conditions of 15 to 25 ℃,
The method for producing fermented vegetable oil, wherein the main fermentation step is fermented for 90 to 100 hours under aerobic conditions of 15 to 25°C. A vegetable fermented oil prepared by the method according to claim 1 . 3. The method of claim 2,
The surface tension of the vegetable fermented oil is 1.2 to 3 times lower than before fermentation, vegetable fermented oil. 3. The method of claim 2,
The emulsification activity of the vegetable fermented oil is 1.5 to 20 times higher than before fermentation, the vegetable fermented oil. 3. The method of claim 2,
The fermented vegetable oil is that the micelle size is 5 to 20 times smaller than before fermentation, vegetable fermented oil. 3. The method of claim 2,
The free fatty acid content of the vegetable fermented oil is 1 to 1.2 times the free fatty acid content of the oil before fermentation, the vegetable fermented oil. The method of claim 1,
After the main fermentation step, the fermentation product is left standing to separate the upper layer and the lower layer, the lower layer is discarded, and MgSO ₄ is added thereto, followed by centrifugation to remove microorganisms and moisture;
A method for producing fermented vegetable oil comprising the step of recovering the oil from which fatty acids and impurities have been removed by using magnesium silicate as a filler in an affinity chromatography column. A cosmetic composition containing the vegetable fermented oil according to any one of claims 2 to 6.

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