KR101761006B1

KR101761006B1 - Osmotic Enzyme Fermentation

Info

Publication number: KR101761006B1
Application number: KR1020150107015A
Authority: KR
Inventors: 옥민; 윤경훈; 박진철; 이성은; 성혜영; 이진아; 홍우종; 박성수; 강민정; 박노환
Original assignee: (주)엠앤씨생명과학; 옥민
Priority date: 2015-07-29
Filing date: 2015-07-29
Publication date: 2017-07-26
Also published as: KR20170014150A

Abstract

The present invention relates to a method for producing a microorganism which comprises washing a substrate with (i) a sugar, (ii) adding Saccharomyces cerevisiae as a yeast and Lactobacillus fermentum as a lactic acid bacterium, in Primary fermentation step, Filtering the solid content after the first fermentation and then fermenting the fermentation product at a temperature of 20 to 50 degrees Celsius; filtering the solid content after the second fermentation; storing the fermentation product at 0 to 10 degrees Celsius and aging; to provide.

Description

Osmotic Enzyme Fermentation < RTI ID = 0.0 >

The present invention relates to a method of fermenting an osmotic enzyme using a microorganism and a fermented product produced by the method.

Conventionally, in order to obtain useful components from a substrate such as a natural raw material, a method of adding a solvent such as water to the substrate and extracting it by applying heat has been used. This conventional extraction method has disadvantages in that some nutrients such as functional proteins, enzymes and vitamins which are vulnerable to heat are destroyed, and browning generally occurs.

In order to overcome such drawbacks, there has been used a method (direct method) of eluting water and an active ingredient in a substrate by adding a high concentration of sugar to a substrate and using an osmotic pressure phenomenon. Through this, the loss of active ingredient by heat could be reduced. However, in this method, there is no process of inoculating microorganisms, so that the fermentation process is slow and it is difficult to obtain a uniform extract repeatedly depending on the state and kind of microorganisms present in the substrate.

It is also known how to proceed with fermentation after proceeding with the current method. Nevertheless, there is a continuing need for a fermentation method capable of quickly obtaining homogeneous results while simultaneously proceeding with the fermentation of the strain and the strain.

Abdel-Rahman et al., 2011; John et al., 2009

It is an object of the present invention to provide a new fermentation method which is capable of obtaining a fast and uniform result, as well as an excellent fermentation effect and a fermented product produced by the method.

In one embodiment of the present invention with sugar (i) After washing the substrate, (ii) a yeast MY process three Levy Jia in Saccharomyces (Saccharomyces cerevisiae ) and / or Lactobacillus fermentum as a lactic acid bacterium at 20 to 50 degrees Celsius Primary fermentation step,

Filtering the solid matter after the first fermentation, and then performing secondary fermentation at 20 to 50 degrees Celsius,

And filtering the solid content after the second fermentation, and then storing the fermented product at 0 to 10 degrees Celsius for aging.

Another embodiment of the present invention is a method of treating a substrate, comprising washing the substrate (i) with a saccharide (ii) saccharomyces cerevisiae cerevisiae ) and / or Lactobacillus fermentum as a lactic acid bacterium. Primary fermentation step,

Sterilization is carried out at 100 to 140 degrees Celsius after filtering out the solid content after the first fermentation,

(Ii) addition of Saccharomyces cerevisiae as a yeast and / or Lactobacillus fermentum as a lactic acid bacterium after sterilization,

A second fermentation step at 20 to 50 degrees Celsius,

Yet another embodiment of the present invention provides a fermented product obtained by the above method, a cosmetic composition comprising the fermented product as an active ingredient, and a food composition comprising the fermented product as an active ingredient.

According to the method of the present invention, it is possible to obtain a fermented product which is fast and uniformly free from loss of nutrients due to heat, and has an excellent effect of inhibiting the formation of NO. Further, the fermented product obtained by the method of the present invention is excellent in the effect of inhibiting the formation of NO, and can be used for cosmetic compositions, food compositions and the like.

1 shows a fermentation process according to a first embodiment of the present invention.
2 shows a fermentation method according to a second embodiment of the present invention.
Fig. 3 shows changes in the number of viable cells of yeast according to the use of the fermented product according to Examples 1 to 3 and Comparative Example 1. Fig.
Fig. 4 shows changes in the number of viable cells of the lactic acid bacteria according to the use of the fermented product according to Examples 1 to 3 and Comparative Example 1. Fig.
5 shows the invert activities (reduction equivalents) of fermentations of Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 (yeast, lactic acid bacteria inoculation) and Comparative Example 1 (non-inoculation, immediate fermentation) It shows the change graph.
Fig. 6 shows the inhibition of NO production of fermentation products of Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 (yeast, lactic acid bacteria inoculation) and Comparative Example 1 (non-inoculation, general immediate fermentation) Represents an effect graph.
7 is a graph showing the cytotoxicity (toxicity) change of fermentations of Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 (yeast, lactic acid bacteria inoculation) and Comparative Example 1 .
Fig. 8 shows the results of NO production (Fig. 8) when fermented according to Example 3 (5% inoculation), Example 4 (1% inoculation), Example 5 (3% inoculation) and Example 6 The graph shows the inhibition effect.
FIG. 9 is a graph showing a change in invert activity (reducing equivalent) of the fermented product of Example 3 (using yellow sugar), Example 7 (using white sugar) and Example 8 (using raw sugar).
Fig. 10 shows graphs of inhibition of NO production of fermentations of Example 3 (using yellow sugar), Example 7 (using white sugar), and Example 8 (using raw sugar).
11 and 12 are graphs showing the ratio of aloe vera to raw sugar in the ratio of 1: 0 (Comparative Example 2), 1: 0.5 (Example 9), 1: 1 (Example 8), 1: (Fig. 11) and a graph of the change in the number of lactic acid bacteria living cells (Fig. 12), and Fig. 13 shows a graph of the invert activity change of the fermented product.
FIG. 14 is a graph showing the interbase activity change of a fermented product using an incubator (Example 11) at 20 degrees Celsius, an incubator (Example 12) at 30 degrees Celsius, an incubator (Example 13) at 37 degrees Celsius and an incubator at 45 degrees Celsius Fig. 15 is a graph showing the effect of inhibiting the NO production of such a fermented product, and Fig. 16 is a graph showing a change in cell stability of the fermented product.
FIG. 17 shows a graph of invert activity change of the fermented product of Example 8 and Comparative Example 3 (the strain used in Example 8 is different), and FIG. 18 shows the effect of inhibiting the formation of NO of such fermented product.
19 shows the NO production inhibitory effect of the fermented product of Example 15 and Comparative Examples 4 to 6. FIG.
Fig. 20 shows the NO production inhibitory effect of the fermented product depending on the kind of the substrate.
Fig. 21 is a graph showing the change in protein measurement of the osmotic enzyme fermented product of Example 8 and the hydrothermal fermentation product of Comparative Example 6, and Fig. 22 shows the effect of inhibiting the formation of NO of such fermented product.
FIG. 23 is a graph showing the change in protein measurement of the osmotic enzyme fermented product of Example 8 and the fermented hot water product of Comparative Example 8, FIG. 24 is a graph showing the invert activity effect of such fermented product, and FIG. Effect.

Hereinafter, a fermentation method according to the present invention will be described.

[First embodiment]

The fermentation method according to the first embodiment of the present invention is shown in Fig. This will be described in detail below.

1. Washing and drying the substrate

As a substrate, there are aloe, kale, rhododendron, red cola bee, aloe vera, aloe avoresense, lucerne, greenschist, garlic, radish, onion, omija, red paprika, mushroom (mushroom, Apple, seokjangpo, fern, cucumber, mushroom and the like can be used. Two or more of these substrates may be used together.

Wash the substrate with water, remove the water completely, and cut to a uniform size in both width, height and height.

2. Addition of sugars (mixing of substrate and sugar)

Washed with water and the dried substrate is mixed at a weight ratio of 1: 0.5 to 2 with sugar. Sugars such as yellow sugar, white sugar, and raw sugar can be used. It is preferable to use a yellow sugar or a raw sugar, and it is more preferable to use a raw sugar.

3. Inoculation with microorganisms

Immediately after mixing the substrate and the sugar, the yeast and / or lactic acid bacteria are inoculated into the mixture of the substrate and the saccharide by an amount of 1 to 10% based on the total weight of the substrate and the sugar, respectively.

As yeast, Saccharomyces cerevisiae is used. For example, Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), Saccharomyces cerevisiae (KCTC 7904) can be used. As yeast, Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP) is preferably used.

As lactic acid bacteria, Lactobacillus fermentum is used. For example, Lactobacillus fermentum Miev L1106 (KCTC 12082BP), Lactobacillus fermentum (KCTC 3112) can be used. As the lactic acid bacteria, Lactobacillus fermentum Miev L1106 (KCTC 12082BP) is preferably used.

Only one of the yeast and the lactic acid bacteria may be used, but it is more preferable to use the yeast and the lactic acid bacteria together in terms of inhibiting the formation of NO. When yeast and lactic acid bacteria are used together, they are used in a weight ratio of 1: 0.5 ~ 2.

4. Primary Fermentation (Dissolution Period of Active Ingredient)

The nonwoven fabric is fixed to the inlet of the fermentation vessel so that only the oxygen passes through it, so that the anaerobic condition does not occur. The nonwoven fabric is fixed using rubber band or the like, and then stored in an Incubator at 20 to 50 캜, preferably at 25 to 45 캜, Ferment.

Here, the reason why the anaerobic condition does not occur is that, under anaerobic conditions, alcohol is generated when the sugar is decomposed by the yeast, and the activity of the enzyme is inhibited.

The pH is measured for each day to check for contamination, and the bubbles accumulated in the upper layer of the liquid phase are removed. Determine the amount of enzyme produced per day by the protein assay method (BCA assay) and determine the reference value in the range of 400 to 1000 μg / ml (for example, 800 μg / ml). There is no restriction on the primary fermentation time, but it is preferable to ferment for 5 days to 9 days. The active ingredient is eluted from the substrate through the primary fermentation.

5. Second fermentation (Intensive fermentation period by various enzymes)

After the first fermentation is completed, the solid content is filtered using a mesh (300 mesh), and only the liquid is put into a new glass fermentation container having been completely disinfected. After that, it is fermented at 20 ~ 50 ℃ incubator and the pH is measured by date to check whether there is contamination and remove the bubbles accumulated in the upper layer of the liquid phase. The invertase activity, which is a digestive enzyme, is checked for each day, and the fermentation is carried out until the reduced sugar amount falls within the range of 3.0 to 10.0 mg / ml and the standard value (for example, 4.0 mg / ml) Go ahead. There is no restriction on the secondary fermentation time, but fermentation is preferable for 5 to 18 days. Intensive fermentation takes place through secondary fermentation.

6. Third fermentation (fermentation)

After the secondary fermentation is completed, the solid content is filtered using a mesh (300 mesh), transferred to a new fermentation vessel having been completely disinfected, and stored in the refrigerator for aging. The storage temperature is preferably 0 to 10 degrees Celsius, and the aging time is not limited, but is preferably 24 to 60 hours.

[Second embodiment]

The fermentation method according to the second embodiment of the present invention is shown in Fig. This will be described in detail below.

Washing and drying of substrate, addition of sugar, inoculation of microorganism, primary fermentation and removal of solids proceed in the same manner as in the first embodiment of the present invention.

However, after the removal of the solid content, sterilization is carried out at 100 to 140 degrees Celsius for 5 to 30 minutes,

After the sterilization, the microorganisms are inoculated in the same manner as the first inoculation (second inoculation), and then the second fermentation and the third fermentation proceed as in the first embodiment.

According to the second embodiment of the present invention, the production of the final enzyme can be remarkably increased by conducting the microbial inoculation twice.

The osmotic enzyme fermentation method according to one embodiment of the present invention focuses on the progress of fermentation by enzymes, so that the time point at which the enzyme is sufficiently generated is the ending point of the first fermentation and the end of the second fermentation The fermentation is proceeded by setting as a time point.

The fermented product or the composition containing the fermented product produced by the fermentation method according to the present invention can be used for foods, medicines, cosmetics and the like. Specifically, the fermented product produced by the fermentation method according to the present invention or a composition containing the fermented product may be effective for improving inflammation, anti-diabetic, joint health, etc.

The composition may be used as a cosmetic composition, such as an essence, lotion, conditioner, foam, gel, hair stick, blow, zymo coat, spray, mousse, aerosol, pomade, powder, soap, pack, ointment or cream. Specifically, it can be used as a cosmetic composition for hair for use such as a shampoo, a rinse, a tonic, a hair conditioner, a hair essence, etc., or as a cosmetic composition for a face or body for use as a body shower, body lotion, foundation, and soap.

The composition may also be used as a food composition. For example, it can be used in a variety of foods such as beverages, carbonated water, mineral water, fruit drinks, vegetable beverages, beverage syrups, alcoholic beverages, fruit juices, vegetable juices, edible yeast extract, edible lactobacillus extract, , Paste enzyme, edible vegetable extract, edible fruit extract, edible glucose substitute edible saccharide, natural sweeteners, seasoning sauce, vinegar, salad sauce, salad dressing, incense vinegar, fruit tea, vegetable tea, mushroom tea, / Fruit / mushroom juice, it can be used as general processed food, health functional food, and the like.

[Example]

1. Preparation of substrate

As a substrate, aloe produced in Geoje city, Gyeongsangnam-do was prepared. The aloe vera leaves were washed with running water, and then the water was completely removed and then cut to a size of 2 cm in both width, height and height.

2. Preparation of microorganisms

Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP) was used as a yeast, and Lactobacillus fermentum Miev L1106 (KCTC 12082BP) was used as a lactic acid bacterium.

[Examples 1 to 4, Comparative Example 1, Experimental Examples 1 to 4]

Example 1

1. Addition of yellow sugar (a mixture of aloe vera and yellow sugar)

Aloe vera washed with water and dried was mixed with the sulfur sugar at a weight ratio of 1: 1, mixed well, and placed in a fermentation vessel.

2. Inoculation with microorganisms

Immediately after mixing aloe vera and sulfur sugar, saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), a yeast strain, was mixed with aloe vera and sulfur sugar to produce aloe vera And yellow sugar.

3. Primary fermentation (Dissolution period of active ingredient)

Nonwoven fabric was used to prevent the anaerobic condition by blocking the entrance of the fermentation vessel to only pass oxygen, and the fermentation was kept at 30 ℃ Incubator for primary fermentation.

The pH was measured on each day to confirm whether there was contamination, and the bubbles accumulated in the upper layer of the liquid phase were removed.

The amount of enzyme produced per day by the protein assay method (BCA assay) was measured to confirm that it exceeded 800 μg / ml. Immediately after inoculation of the strains, the samples were regarded as 0-day samples and sampled at regular intervals. It exceeded 800 ㎍ / ml around 7 days.

4. Second fermentation (intensive fermentation period by various enzymes)

After the first fermentation was completed, the solids were filtered using a mesh (300 mesh), and only the liquid was added to a new glass fermentation vessel having been completely disinfected. After that, fermentation was carried out at 30 ° C Incubator, and the pH was measured on each day to check for contamination, and the bubbles accumulated on the upper layer of the liquid phase were removed. In addition, fermentation was carried out until the reducing sugar amount was 4.0 mg / ml or less by checking the enzyme activity of invertase (invertase) for each day.

5. Third fermentation (fermentation)

After the secondary fermentation was completed, the solid content was filtered using a mesh (300 mesh), transferred to a new fermentation vessel having been completely disinfected, stored in a refrigerator at 4 ° C and aged.

Example 2

Lactobacillus fermentum Miev L1106 (KCTC 12082BP), a lactic acid bacterium instead of yeast, was inoculated with a mixture of aloe vera and yellow sugar by an amount of 5% of the total weight of aloe vera and yellow sugar mixed with yeast The fermentation was carried out in the same manner as in Example 1.

Example 3

Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), which is a yeast strain, and Lactobacillus fermentum Miev L1106 (KCTC 12082BP), a lactic acid bacterium, were mixed with aloe vera and sulfur sugar The fermentation was carried out in the same manner as in Example 1, except that the mixture of aloe vera and the yellow sugar was inoculated in an amount of 5% by weight.

Comparative Example 1

Fermentation was carried out in the same manner as in Example 1, except that the microorganisms were not inoculated (non-inoculated). That is, the fermentation proceeded immediately.

Experimental Example 1 - Variation of viable cell counts after fermentation

9 milliliters of sterilized water was added to 1 milliliter of each of the fermentations of Example 1, Example 2, Example 3, and Comparative Example 1, diluted in steps of 10 ⁵ times, and plated on a BPB agar medium at a constant temperature of 37 ° C After culturing for 48 h in the incubator, the cells were expressed as colony forming unit (CFU) / ml. Changes in the number of viable cells in yeast and the change in the number of viable cells in lactic acid bacteria are shown in Figs. 3 and 4, respectively.

As shown in FIG. 3 and FIG. 4, no bacteria were found in yeast and lactic acid bacteria when the microorganism was not inoculated (as indicated by "Vera (sterile) sulfur" in FIGS. When yeast was inoculated only as in Example 1 (yeast in 'Vera (yeast)' in FIGS. 3 and 4), only yeast was grown, and when lactic acid bacteria were inoculated as in Example 2 ("Vera (lactic acid bacteria) Sulfur ') showed only lactic acid bacteria. When yeast and lactic acid bacteria were inoculated together as shown in Example 3 ("Vera (yeast + lactic acid bacteria) sulfur" in FIGS. 3 and 4), yeast and lactic acid bacteria were all grown.

Experimental Example 2 - Interbase activity (reduction equivalent) change due to fermentation

Since the fermented sugar used in fermentation is a non-reducing sugar, in order to produce microorganisms, the invertase must convert the non-reducing sugar, sulfur sugar, to the reducing sugar, monosaccharide, by using a digesting enzyme. As the microorganism grows, the reducing sugar is consumed and the reducing sugar gradually decreases. Therefore, the microbial activity on the progress of the fermentation can be indirectly confirmed by comparing the difference in the degree of reduction of the reducing sugar.

2 ml of the DNS solution (3,5-dinitrosalicylic acid) was added to 1 ml of fermented aloe vera solution, which was reacted at 100 ° C for 10 minutes and immediately cooled in ice water. UV spectrophotometer was used to determine the quantification relative to the standard curve at 550 nm. The standard curve was prepared by mixing 1 ml of this solution with 2 ml of the DNS reagent, reacting at 100 ° C for 10 minutes, cooling immediately in ice water and UV spectrophotometer at 550 nm Invert activity was measured.

Invert activity (reduction equivalent) change in the fermented product of Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 (yeast, lactic acid bacteria inoculation) and Comparative Example 1 (non-inoculation, immediate fermentation) The graph is as shown in Fig.

As shown in FIG. 5, there was almost no change in invert activity with time in Comparative Example 1 (non-inoculated bacteria), whereas in Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 Yeast, and lactobacillus), the amount of reducing sugars decreased significantly over time.

Experimental Example 3 - Inhibition of NO formation by fermentation

RAW 264.7 cells, a murine macrophage cell line, were purchased from the American Type Culture Collection (ATCC) and cultured in high glucose DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) , USA) at 37 ° C and 5% CO2.

1 × 10 ⁴ cells / well of RAW 264.7 cells were cultured on a 96-well plate, and the cells were incubated for 24 hours. The fermented extracts were treated for 1 hour at 37 ° C, 5% CO 2, and treated with 1 μg / mL of LPS for 18 hours. 50 μL of 1% sulfanilamide, 5% phosphoric acid, and 0.1% naphthylethylenediamine were added to 50 μL of the cell culture, followed by incubation for 10 min with 50 μL of Griess reagent (Sigma Chemical Co., St. Louis, Mo., USA) The absorbance was measured at 540 nm using a plate reader (TECAN / infinite M200, Switzerland). The average absorbance of each sample group was calculated, and the degree of NO production was expressed as a percentage in comparison with the absorbance value of the control group. The results are shown in Fig.

As shown in FIG. 6, when the inhibitory effect on the NO production of LPS was 100, the NO production inhibitory effect of Comparative Example 1 (non-inoculation, immediate fermentation) was 95.6, while Example 1 (yeast inoculation) In case 2 (lactic acid bacteria inoculated), 82.4 and 78.9, respectively, showed excellent NO production inhibitory effect. In particular, in Example 3 (yeast and lactic acid bacteria were inoculated together), 66.4% showed very excellent NO production inhibitory effect.

EXPERIMENTAL EXAMPLE 4 - CELL SAFETY (TOXICITY) CHANGES DURING FERMENTATION

RAW 264.7 cells (1 × 10 ⁵ cells / ml) were pre-cultured for 18 hours and then cultivated in the same manner as in Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation), Example 3 (yeast, lactic acid bacteria inoculation) (MTT, Sigma) of 1 mg / ml was incubated for 48 hours, and the cells were treated with LPS (1 μg / ml) mL < / RTI > for 4 hours. Dimethylsulfoxide (DMSO), Sigma) or 100 μl of isopropanol was added to dissolve the formazan precipitate produced by the reduction of MTT and the absorbance was measured at 570 nm using a microplate reader. The average absorbance value of each sample group was obtained and compared with the absorbance value of the control group, the cell survival rate was expressed as a percentage. The results are shown in Fig.

As a result of the experiment, it was found that all the fermentations of Example 1 (yeast inoculation), Example 2 (lactic acid bacteria inoculation) and Example 3 (yeast and lactic acid bacteria inoculation) were safely contained in the range of 0.1 to 2.5% by weight based on 100% It was found to be applicable.

[Examples 4 to 6 and Experimental Example 5]

Example 4

Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), which is a yeast strain, and Lactobacillus fermentum Miev L1106 (KCTC 12082BP), a lactic acid bacterium, were mixed with aloe vera and sulfur sugar Fermentation was carried out in the same manner as in Example 3, except that the mixture of aloe vera and yellow sugar was inoculated by an amount of 1% by weight.

Example 5

Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), which is a yeast strain, and Lactobacillus fermentum Miev L1106 (KCTC 12082BP), a lactic acid bacterium, were mixed with aloe vera and sulfur sugar The fermentation was carried out in the same manner as in Example 3, except that the mixture of aloe vera and the yellow sugar was inoculated in an amount of 3% by weight.

Example 6

Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP), which is a yeast strain, and Lactobacillus fermentum Miev L1106 (KCTC 12082BP), a lactic acid bacterium, were mixed with aloe vera and sulfur sugar The fermentation was carried out in the same manner as in Example 3, except that a mixture of aloe vera and yellow sugar was inoculated by an amount of 10% by weight.

Experimental Example 5 - Inhibition of NO formation by fermentation

The inhibitory effect of NO production was measured in the same manner as in Experimental Example 3 with respect to Examples 3 to 6, and the results are shown in Fig.

As shown in FIG. 8, when the inhibitory effect on the NO production of LPS was 100, the inhibitory effect on NO production was 90.85 in Example 4 (1% inoculation), while the inhibitory effect on NO production in Example 5 (3% 74.58, indicating excellent NO production inhibitory effect. The inhibition of NO production was very good in Example 3 (5% inoculation) and in Example 6 (10% inoculation), 67.80 and 67.38, respectively. Therefore, the inhibitory effect of NO production is increased in proportion to the inoculation concentration of microorganisms, and it can be estimated that the inoculation concentration does not increase greatly when the concentration exceeds 10%.

[Examples 7 to 8, Experimental Examples 6 to 7]

Example 7

Fermentation was carried out in the same manner as in Example 3, except that white sugar was used as a sugar.

Example 8

Fermentation was carried out in the same manner as in Example 3 except that a raw sugar was used as a sugar.

Experimental Example 6 - Interbase activity (reduction equivalent) change due to fermentation

The changes in invert activity (reduction equivalent) were measured for Examples 3, 7 and 8 in the same manner as in Experimental Example 2, and the results are shown in FIG.

As shown in Fig. 9, in the case of Example 3 (yellow sugar), Example 7 (white sugar) and Example 8 (raw sugar), significant activity of the invertase was exhibited over time. However, in Example 7 (white sugar), the activity of invertase was not significantly maintained during the secondary fermentation after 7 days of the primary fermentation, and the activity was relatively low. Example 4 (yellow sugar), Example 8 (Raw sugar) also showed significant activity during the second fermentation.

Experimental Example 7 - Inhibition of NO formation by fermentation

The inhibitory effects of NO production were measured in the same manner as in Experimental Example 3 for Examples 3, 7 and 8, and the results are shown in FIG.

As shown in FIG. 10, the inhibitory effect of NO formation was found to be larger in the order of Example 7 (white sugar) <Example 3 (yellow sugar) and Example 8 (raw sugar).

[Examples 9 to 10, Comparative Example 2, Experiments 8 to 9]

Example 9

Fermentation was carried out in the same manner as in Example 3 except that raw sugar was used as a saccharide and aloe vera and raw sugar were mixed at a ratio of 1: 0.5.

Example 10

Fermentation was carried out in the same manner as in Example 9, except that aloe vera and raw sugar were mixed at a ratio of 1: 2.

Comparative Example 2

Fermentation was carried out in the same manner as in Example 9, except that the raw sugar was not used (ratio of 1: 0).

Experimental Example 8 - Number of viable cells after fermentation

The changes in the number of viable cells following fermentation were measured for Examples 8 to 10 and Comparative Example 2 in the same manner as in Experimental Example 1, as shown in FIG. 11 (number of yeast live cells) and FIG. 12 (number of lactic acid bacteria live cells).

As a result of measuring the change in the number of viable cells per day up to the 14th day, no yeast or lactic acid bacteria were found in Comparative Example 1 (ratio of 1: 0 not using sugar) as shown in Fig. 11 and Fig. When the ratio of aloe vera to sugar was adjusted to 1: 0.5 (Example 9), 1: 1 (Example 8), and 1: 2 (Example 10), yeast was in the early stage of fermentation (Example 9) in which the ratio of aloe vera to sugar was 1: 0.5, but grew better in the high sugar condition (Example 10) in which the ratio was 1: 2 toward the latter half of fermentation. The lactic acid bacteria were found to grow the most in the low mortality condition (Example 9). Lactic acid bacteria did not grow well due to sugar inhibition in the early stage due to inhibition of sugar,

Experimental Example 9 - Interbase activity (reduction equivalent) change due to fermentation

The change in invert activity (reduction equivalent) was measured for Examples 8 to 10 and Comparative Example 2 in the same manner as in Experimental Example 2, and the results are shown in FIG.

(1: 0.5), Example 8 (1: 1), and Example 10 (1: 2), except for the case of Comparative Example 2 in which sugar was not added, And showed significant inversion activity with the passage of time. In the case of Comparative Example 2 in which sugar was not added, invertase activities were not exhibited in both primary and secondary fermentations.

[Examples 11 to 14, Examples 10 to 12]

Example 11

Fermentation was carried out in the same manner as in Example 3 except that raw sugar was used as a saccharide and aloe vera and raw sugar were added in the same amount of 1: 1, and incubator was used at 20 ° C.

Example 12

Fermentation was carried out in the same manner as in Example 11, except that an incubator at 30 ° C. was used.

Example 13

Fermentation was carried out in the same manner as in Example 11, except that an incubator at 37 ° C was used.

Example 14

Fermentation was carried out in the same manner as in Example 11, except that an incubator at 45 ° C was used.

Experimental Example 10 - Interbase activity (reduction equivalent) change due to fermentation

The change in invert activity (reduction equivalent) was measured for Examples 11 to 14 in the same manner as in Experimental Example 2, and the results are shown in Fig.

In the case of Examples 11 to 14, which were performed at different temperatures, the fermentation activity was significantly changed over time up to the first fermentation day 7. As the time elapsed, in the case of Example 13 (37 ° C experimental group) and Example 14 (45 ° C experimental group), there was a change in the property according to the evaporation in a relatively high temperature environment, and thus the amount of reducing sugar was also relatively increased . The activity was similar in Example 11 (20 ° C experimental group) and Example 12 (30 ° C experimental group), but in Example 12, invert activity was relatively high.

Experimental Example 11 - Inhibitory effect of NO on fermentation

The inhibitory effect of NO production was measured in the same manner as in Experimental Example 3 with respect to Examples 11 to 14, and the results are shown in Fig.

As shown in FIG. 15, the higher the fermentation temperature, the higher the inhibitory effect of NO production.

EXPERIMENTAL EXAMPLE 12 - CELL SAFETY (TOXICITY) CHANGES DURING FERMENTATION

The changes in cell stability (toxicity) following fermentation were measured for Examples 11 to 14 in the same manner as in Experimental Example 4, and the results are shown in FIG.

As shown in FIG. 16, the safety was somewhat low in Example 14 (45 ° C experimental group) where the fermentation temperature was high, but the safety was also improved as the fermentation temperature was lowered to 37 ° C and 30 ° C (respectively, Examples 13, Example 12). In particular, the safety was relatively high in Example 12 (30 ° C experimental group).

[Comparative Example 3, Experimental Examples 13 to 14]

Comparative Example 3

Saccharomyces cerevisiae is used as a saccharide, and other strains of the same genus and the same species as the strain, i.e., cerevisiae KCTC 7904 and Lactobacillus fermentum KCTC 3112 as a lactic acid bacterium were each inoculated in an amount of 5% of the combined weight of aloe vera and raw sugar, respectively.

Experimental Example 13 - Interbase activity (reduction equivalent) change due to fermentation

The fermentation was carried out in the same manner as in Experimental Example 2 with respect to Example 8 and Comparative Example 3, and the change in invert activity (reducing equivalent) was measured using aloe vera as a substrate by applying raw sugar, and the results are shown in FIG. .

The strain group of Example 8 fermented by the osmotic enzyme fermentation method showed relatively higher invertase activity over the first fermentation and the second fermentation as compared with the other strain group of Comparative Example 3. In the case of Comparative Example 1, the fermentation was carried out in a natural fermentation state without microbial inoculation, and the fermentation progressed relatively slowly.

Experimental Example 14 - Inhibitory effect of NO production upon fermentation

The inhibitory effect of NO production was measured for Example 8 and Comparative Example 3 in the same manner as in Experimental Example 3, and the results are shown in FIG.

As shown in FIG. 18, when the strain according to the present invention of Example 8 was used, the NO production inhibitory effect was 54.7%, which was superior to that of Comparative Example 3 by 61.8%.

[Examples 15, 16, Comparative Examples 4 to 5, Experimental Examples 15 to 16]

Example 15

Except that the solid content was removed after the first fermentation and sterilization was carried out at 121 ° C for 15 minutes, and the microorganism was inoculated in the same manner as the first inoculation after the sterilization (second inoculation). The same fermentation was carried out.

Example 16

( Saccharomyces cerevisiae KCTC 7904) and Lactobacillus fermentum KCTC 3112 (lactic acid bacteria: Lactobacillus fermentum KCTC 3112) were mixed at a concentration of 5% of the total weight of aloe vera and raw sugar, respectively, using 1: 1, the fermentation was carried out under the same conditions as in Example 15.

Comparative Example 4

Fermentation was carried out in the same manner as in Example 15, except that the microorganisms were not inoculated.

Comparative Example 5

Fermentation was carried out in the same manner as in Example 15, except that the fermentation group was hydrothermally extracted.

Experimental Example 15 - Inhibition of NO formation by fermentation

The inhibitory effects of NO production were measured in the same manner as in Experimental Example 3 for Examples 15 and 16 and Comparative Examples 4 and 5, and the results are shown in FIG.

As shown in FIG. 19, the strains of Examples 15 and 16 showed excellent NO production inhibitory effect, and the strain of Example 15 showed particularly excellent NO production inhibitory effect.

Experimental Example 16 - Inhibitory effect of NO on the substrate

The inhibitory effect of NO production was measured in the same manner as in Experimental Example 3, except that the kinds of substrates were different. The results are shown in Fig.

[Comparative Example 6, Experimental Examples 17 and 18]

Comparative Example 6

Fermentation was carried out under the same conditions as in Example 8, except that the fermentation was carried out at 121 ° C for 15 minutes without using sugar.

Experimental Example 17

Bovine serum albumin (BSA) standard (Sigma) and BCA working solution were prepared. BCA working solution was prepared by mixing Bicinchoninic Acis Solution (Sigma) and Copper (II) Sulfate Pentahydrate 4% Solution (Sigma) at a ratio of 50: 1. Each of the standard and unknown samples was pipetted into each 96-well plate. After adding 80 ul of BCA working solution to each 96-well plate, 96 well plates were shaken for 30 seconds and cover plates were incubated at 37 ° C for 30 minutes. After incubation, the absorbance at 562 nm was measured with an ELISA reader (TECAN). The average absorbance and ug / ul concentration of each BSA standard were plotted and a standard curve was drawn. These standard curves were used to determine the protein concentration of each unknown sample. The results are shown in Fig.

As a result of measuring osmotic enzyme fermentation of Example 8 and the protein change of hydrothermal fermentation of Comparative Example 6, the amount of protein increased with time, but was not increased after 8 days. The osmotic enzyme fermentation of Example 8 was twice as much as that of the hydrothermal fermentation of Comparative Example 6.

Experimental Example 18 - Inhibitory Effect of NO on Formation of Substrate

The osmotic enzyme fermentation of Example 8 and hydrothermal fermentation of Comparative Example 6 were measured in the same manner as in Experimental Example 3, and the NO production inhibitory effect was measured. The results are shown in Fig.

[Comparative Example 7, Experimental Example 19]

Comparative Example 7

Aloe vera and raw sugar were mixed at a ratio of 1: 1 and saccharified for 7 days. Then, lactic acid bacteria were inoculated into the saccharified solution, which had been removed from the solid content using a mesh (300 mesh), and fermented for 7 days.

Experimental Example 19

The amount of protein accumulation was measured in the same manner as in Experimental Example 18 and shown in a graph. The results are shown in Fig.

As shown in FIG. 23, it can be seen that the osmotic enzyme fermentation group of Example 8 produced a larger amount of protein from the fermentation period to the fermentation termination period than the post-glycation fermentation group of Comparative Example 7.

Experimental Example 20 - Interbase activity (reduction equivalent) change due to fermentation

The change in invert activity (reduction equivalent) was measured in the same manner as in Experimental Example 2, and the results are shown in Fig.

As shown in FIG. 24, the strain fermented by the osmotic enzyme fermentation method of Example 8 showed relatively higher invertase activity than that of the fermentation method after fermentation after the saccharification in Comparative Example 7.

Experimental Example 21 - Inhibitory effect of NO production upon fermentation

The inhibitory effect of NO production was measured for Example 8 and Comparative Example 7 in the same manner as in Experimental Example 3, and the results are shown in FIG.

As shown in FIG. 25, the strain of Example 8 according to the osmotic enzyme fermentation showed an excellent NO production inhibitory effect as compared with the strain of Comparative Example 7 due to saccharification fermentation.

Claims

The substrate,
(i), (ii) Saccharomyces cerevisiae as a yeast, and Lactobacillus fermentum as a lactic acid bacterium.
Mixed at 20 to 50 degrees Celsius for 5 to 9 days at a time Primary fermentation step,
Filtering the solid matter after the first fermentation, and then performing secondary fermentation at 20 to 50 degrees Celsius for 5 to 18 days,
Filtering the solid content after the second fermentation, and storing the fermented product at 0 to 10 degrees Celsius for aging. According to the osmotic fermentation method,
Wherein the yeast and lactic acid bacteria are used at a weight ratio of 1: 0.5 to 2 during the primary fermentation.

The substrate,
(ii) Saccharomyces cerevisiae as a yeast and Lactobacillus fermentum as a lactic acid bacterium are mixed at a temperature of 20 to 50 degrees Celsius for 5 to 9 days at a time Fermenting step,
Sterilization is carried out at 100 to 140 degrees Celsius after filtering out the solid content after the first fermentation,
(Ii) Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP) as yeast or Lactobacillus fermentum Miev L1106 (KCTC 12082BP) as lactic acid bacteria after sterilization, , Or by further mixing both the yeast and the lactic acid bacteria and inoculating them,
Secondary fermentation at 20 to 50 degrees Celsius for 5 to 18 days,
Filtering the solid content after the second fermentation, and storing the fermented product at 0 to 10 degrees Celsius for aging. According to the osmotic fermentation method,
Wherein the yeast and lactic acid bacteria are used at a weight ratio of 1: 0.5 to 2 during the primary fermentation.

3. The method according to claim 1 or 2,
Saccharomyces cerevisiae as yeast ( Saccharomyces < RTI ID = 0.0 > cerevisiae MAB Y1 (KCTC 11386BP) is used.

3. The method according to claim 1 or 2,
Lactobacillus momentum spread (Lactobacillus fermentum) Miev L1106 (KCTC 12082BP) osmotic pressure enzymatic fermentation method is used as lactic acid bacteria.

3. The method according to claim 1 or 2,
The substrate is selected from the group consisting of aloe, kale, rhododendron, red cola bean, aloe vera, aloe avocence, yujiao, garlic, garlic, radish, onion, omija, red paprika, mushroom, apple, Wherein the osmotic enzyme fermentation method comprises the steps of:

6. The method of claim 5,
The osmotic enzyme fermentation method wherein the substrate is aloe.

3. The method according to claim 1 or 2,
A method for fermenting an osmotic enzyme by mixing a substrate and a sugar at a weight ratio of 1: 0.5 to 2.

3. The method according to claim 1 or 2,
Wherein the saccharide is at least one selected from the group consisting of white sugar, sulfur sugar and raw sugar.

3. The method according to claim 1 or 2,
An osmotic enzyme fermentation method wherein the sugar is a sugar.

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3. The method according to claim 1 or 2,
Yeast and lactic acid bacterium in an amount of 1 to 10% by weight based on the total weight, respectively.

3. The method according to claim 1 or 2,
The yeast and lactic acid bacteria are inoculated, and then the entrance of the fermentation vessel is blocked so that the anaerobic condition does not occur.

A fermentation product obtained by the osmotic enzyme fermentation method according to any one of claims 1 and 2.

A cosmetic composition comprising the fermented product of claim 13 as an active ingredient.

14. A food composition comprising the fermented product of claim 13 as an active ingredient.

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