KR20030036187A - Process for producing antioxidant - Google Patents

Abstract

(1) a seed culture step of obtaining a seed culture of photosynthetic bacteria,

(2) a preculture step of preculturing the seed culture in the presence of an antioxidant and under anaerobic light conditions to obtain a preculture;

(3) The culture was carried out until the cells were inactivated under aerobic cancer conditions in the presence of antioxidants, with or without adding a substrate to the preculture, and the culture solution was treated with a pH of 6.4 to 6.6 to give an aqueous solution of antioxidants. Get

Description

Production method of antioxidant substance {PROCESS FOR PRODUCING ANTIOXIDANT}

At present, there is a need for an antioxidant substance for suppressing oxidation of sebum by UV-A or UV-B, aging of the skin and damage to DNA by free radicals generated in the process.

Here, conventional antioxidants such as butylhydroxyanisole (BHA) and butylhydroxytoluene (BHT) have been used in food, cosmetics, and pharmaceuticals. However, they are problematic in terms of safety, and there has been a demand for antioxidants that have improved these points from consumers.

In addition, there is a need for a safe natural antioxidant that can be stored for a long time in the production of agricultural and livestock products.

The present invention relates to a process for the manufacture of antioxidants.

1 is a graph showing the ultraviolet absorption spectrum curve of the antioxidant liquid according to the present invention.

Figure 2 is a redox potential measurement of the addition of iron to the antioxidant liquid and water according to the present invention.

[Initiation of invention]

The present invention has been made in view of the above circumstances, and is suitable for agricultural products, livestock products, foods, cosmetics, medicines, and concrete, and antioxidant substances by microbial reaction without using an organic solvent for extracting antioxidant substances having excellent antioxidant activity. It is an object of the present invention to provide a method for producing an antioxidant substance which is obtained.

The inventors of the present invention have conducted research and application of the useful microbial group for producing antioxidants, and as a result, have completed the production technology for producing large amounts of antioxidants.

That is, in order to achieve the above object, a method for producing an antioxidant substance according to the present invention,

(1) a seed culture step of obtaining a seed culture of photosynthetic bacteria,

(2) a preculture step of preculturing the seed culture in the presence of an antioxidant and under anaerobic light conditions to obtain a preculture;

(3) The substrate was added to the preculture, and the culture was carried out until the cells were inactivated under aerobic cancer conditions in the presence of an antioxidant substance, and the culture solution was treated at pH 6.4 to 6.6 to obtain an aqueous solution of antioxidant substance. This process is included.

Moreover, in another form, the manufacturing method of the antioxidant substance which concerns on this invention,

(1) a seed culture step of obtaining a seed culture of photosynthetic bacteria,

(2) a preculture step of preculturing the seed culture in the presence of an antioxidant and under anaerobic light conditions to obtain a preculture;

(3) The present culture is carried out in the presence of an antioxidant substance until the cells are inactivated under aerobic cancer conditions, and the culture medium is treated to pH 6.4 to 6.6 to obtain an aqueous antioxidant substance solution. That is, it can also be performed as an aspect which implements this process, without adding a substrate to the said preculture.

Best Mode for Carrying Out the Invention

EMBODIMENT OF THE INVENTION Below, embodiment of the manufacturing method of the antioxidant substance which concerns on this invention is described in detail.

Seed culture process

In the present invention, first, a species culture of photosynthetic bacteria is obtained.

A cultivation is obtained by culturing the lyophilized cells as photosynthetic bacteria in a seed medium.

Such seed cultures may be cultured separately from lyophilized bacteria of a plurality of photosynthetic bacteria to use a plurality of seed cultures.

In the present invention, at least one species of photosynthetic bacteria, which are basic bacteria, are used. In particular, the bacteria belonging to Rhodopseudomonas palustris, Rhodopseuaomonas spheroides, or Rhodopseuaomonas capsulata.

In addition, in this specification, when describing the quantity using "a species culture | cultivation", a "preculture", and a "complex culture", it is generally expressed by the quantity containing the culture medium (cultivation liquid) which cultured cells. In addition, in this invention, a "culture medium", a "preculture", and a "complex culture" are suitably prepared as a culture liquid.

Preculture Process

Next, the seed culture is precultured in the presence of an antioxidant and under anaerobic bright conditions to obtain a preculture.

That is, in this culture step, an antioxidant substance is added to the seed culture and cultured in a container under an anaerobic bright environment at a temperature of 25 ° C. to 30 ° C. under various other conditions for 30 to 40 days. Thereby, the preculture of pH 6.4-7.0 is obtained. When using a plurality of seed cultures, a plurality of such seed cultures are mixed and used. By using an antioxidant substance, excessive microorganisms are suppressed and oxidative action is suppressed.

This culture is suitably prepared as a photosynthetic bacterial culture solution containing 10 ⁸ to ^{10 10} / mL of individual photosynthetic bacteria. Incidentally, the number of bacteria is also included in the present specification, and the number of bacteria measured in accordance with the plate culture method is described in this specification.

Examples of antioxidants that can be used in this step include vitamin A, vitamin B group, and vitamin E. Among them, it is particularly preferable to use KMX of vitamin B12, a product of KORIN KOREA Co., Ltd. Such antioxidants are suitably used in the form of aqueous solutions.

It is also suitable to preculture by adding a substrate to the species culture. As such a substrate, a fish sorbable is suitable. Fish solvable is the heat treatment of fish residues. It consists of 16 kinds of amino acid composition. It is suitable to heat-treat again or use what was processed to pH 3.5 or less with lactic acid. Moreover, rice bran, fish tailings, powdered kelp, etc. can also be used or used as a substrate.

Tofu plant drainage, starch plant drainage, urine drainage, livestock manure drainage, and fish processing drainage are also available as substrates. Antioxidants can be obtained by culturing photosynthetic bacteria using these as substrates.

In addition, the said anaerobic light condition means the environment which the air | atmosphere which is interrupted | blocked from the outer space, and the air mixed at the time of airtight at the time of airtight in the airtight container exists. When culturing photosynthetic bacteria under anaerobic light conditions, the substrate may be selected to generate hydrogen sulfide. In this case, various bacteria are suppressed, and photosynthetic bacteria propagate well. In pre-culture, it is suitable to mix | blend 1-5 weight part of antioxidant liquids containing 1-3 weight part of seed cultures, 0.01-0.05 weight% antioxidant substance, and 0.5-5 weight part of a board | substrate, respectively.

This process

In this step, the substrate is added to the preculture, and in the presence of an antioxidant, the cells are cultured until the cells are inactivated under aerobic cancer conditions, and the culture solution is treated with a pH of 6.4 to 6.6 to obtain an aqueous solution of antioxidants. It is also possible to carry out without adding a substrate to the preculture.

That is, in this step, the preculture and the substrate are added to the aqueous solution of antioxidant substance, and the cells are cultured while maintaining the water temperature under the conditions of 25 ° C to 30 ° C under aerobic dark conditions. This culture is performed until the cells are self-extinguishing and inactivated. Ammonia is generated by the self-extinguishing of the cells and the culture solution is highly alkaline, but ammonia is driven out under aerobic cancer conditions, and the culture solution is treated to pH 6.4 to 6.6 as described above. In addition, the aerobic state is maintained by passing oxygen or air through the culture liquid. Thereby, the aqueous solution of antioxidant substance can be obtained. The antioxidant substance obtained by the present invention is vitamin B12. This is Lactobacillus delbrueckii subspecies. It has been confirmed by a microorganism identification method using a strain of Lactobacillus Delbrueckii subsp.lactis (ATCC 7830).

As the substrate, it is suitable to use a substrate such as fish sorbable used in the preculture process.

This substrate may not be added. At that time, the cells do not undergo a proliferation process and mainly cause self-extinguishing action. The subsequent process is the same as when the substrate is added.

Examples of antioxidants that can be used in this step include vitamin A, vitamin B group, and vitamin E. Among them, it is particularly preferable to use KMX of vitamin B12, a product of KORIN KOREA CO., LTD. Such antioxidants are suitably used in the form of aqueous solutions.

In addition, an aerobic dark condition means the environment which ventilates from outer space and air circulates in a sealed container and light does not enter.

In carrying out this culture, it is suitable to mix 1 to 30 parts by weight of the whole culture medium, 1 to 5 parts by weight of an antioxidant liquid containing 0.01 to 0.05% by weight of an antioxidant substance, and 0.5 to 5 parts by weight when a substrate is added. Do.

After completion of the incubation, the impurities in the liquid are allowed to settle naturally and passed through a filter at the bottom of the tank to obtain an aqueous antioxidant substance solution.

Below, the Example of the manufacturing method of the antioxidant substance which concerns on this invention is given.

Example 1

Preparation of Speculum A of Photosynthetic Bacteria

As a photosynthetic bacterium in probiotics, freeze dried JCM 2524 was selected from the JCM microbial catalog published by the Institute of Microbiology and Preservation, published by the Institute of Microbiological Sciences, Yeast Extract, 2 g, sodium L-malate. 2 g, sodium glutamate 2 g, potassium hydrogen phosphate 1 g, sodium hydrogen carbonate 0.5 g, magnesium sulfate heptahydrate 0.2 g, potassium chloride dihydrate 0.2 g, manganese sulfate hydrate 2 mg, iron sulfate heptahydrate 0.5 mg, cobalt chloride hexahydrate 0.5 mg, thiamine-HCL 1 mg, nicotinic acid 1 mg, biotin 0.01 mg, distilled water 1 L, pH 7 medium at 30 ℃, irradiated with an illuminance 2000 Lux fluorescent lamp to anaerobic culture, 10 ⁹ / mL Species Culture A was obtained.

Example 2

Preparation of Speculum B of Photosynthetic Bacteria

Among the probiotics, another type of photosynthetic bacterium was selected from the ATCC Bacteria and Bacteriophages catalog published by the America Type Culture Collection, using freeze dried ATCC 17023, 2.5 g of malic acid, 1 g of yeast extract, and 1.25 ammonium sulfate. g, magnesium sulfate dihydrate 0.2 g, potassium chloride dihydrate 0.07 g, ferric citrate 0.01 g, ethylenediamine tetraacetic acid 0.02 g, dipotassium hydrogen phosphate 0.6 g, potassium dihydrogen phosphate 0.9 g, trace elements 1 ml (citric acid) Ferric citrate 0.3 g, MnSO ₄ 0.002 g, H ₃ BO ₃ 0.0001 g, (NH ₄ ) 6 Mo ₇ O ₂₄ 4H ₂ O 0.002 g, EDTA 0.05 g, CaCL ₂ · 2H ₂ O 0.02 g, Prepared from 100 ml of distilled water), 7.5 ml of vitamin (0.2 g of nicotinic acid, 0.2 g of nicotinamide, 0.4 g of thiamine HCL, 0.008 g of biotin, prepared from 1 L of distilled water), 1 L of distilled water, and a medium having a pH of 6.9 Anaerobic incubation with irradiation of Illumina 2000 Lux fluorescent lamp at 30 ° C. and cultured B of 10 ⁹ / mL Got.

Example 3

Preparation of Preculture

5 parts by weight of the antioxidant liquid containing 3 parts by weight of the seed cultures A, B and 0.01% by weight of the antioxidant substance KMX, and 3 parts by weight of the fish solving, and in a container under an anaerobic environment, 25 ° C to 30 ° C Incubated for 35 days at a temperature of ℃. This obtained the preculture which is pH 7.0. When using a plurality of seed cultures, a plurality of such seed cultures are mixed and used.

This culture is prepared as a photosynthetic bacterial culture containing 10 ⁸ / mL of individual photosynthetic bacteria.

Example 4

Main culture A

To an aqueous solution of 5 parts by weight of KMX liquid (product of KORIN KOREA Co., Ltd.) containing 0.01% by weight of KMX as an antioxidant, 5 parts by weight of the preculture and 0.5 parts by weight of fish sorbent were added. The cells were cultured while maintaining under the condition of -30 ° C until the cells were inactivated, and the culture solution was finally prepared at pH 6.5. Thereafter, impurities were naturally precipitated and passed through a filter at the bottom of the tank to obtain an aqueous solution of an antioxidant substance. Antioxidant substances in aqueous solution are Lactobacillus delbruecchia subspecies. It was confirmed that it is vitamin B12 by the microorganism identification method using the strain of Lactis (ATCC 7830).

Example 5

Main culture B

To the aqueous solution of 5 parts by weight of KMX liquid (product of KORIN KOREA Co., Ltd.) containing 0.01% by weight of KMX as an antioxidant, 30 parts by weight of the preculture was added, and the water temperature was maintained under the conditions of 25 ° C to 30 ° C under aerobic dark conditions. While culturing the cells and carrying out the main culture until the cells became inactivated, the culture solution was finally prepared at pH 6.5. Thereafter, impurities were naturally precipitated and passed through a filter at the bottom of the tank to obtain an aqueous solution of antioxidant substance. Antioxidant substances in aqueous solution are Lactobacillus delbruecchia subspecies. It was confirmed that it is vitamin B12 by the microorganism identification method using the strain of Lactis (ATCC 7830).

Example 6

Single-dose toxicity test using mice of natural antioxidants of the present invention

20 g / Kg of the test substance (an antioxidant substance obtained in Example 4) was orally administered to the mouse, and the change in general state and the change in weight were observed for 7 days, and then dissected and each organ was visually observed. As a result, in each group, there was no death from immediately after administration to the day after the end of observation, and no abnormalities in hair growth, diarrhea, sweating, deep breathing, and behavior were observed after the test period. Observation of the necropsy was not confirmed abnormally. This proved that there was no toxicity as a single-dose 20 g / Kg test substance (Table 1, Table 2).

group Disclosure mouse (number of animals) Mouse average weight (g) Dosage (ml) Observation period (days) Abnormal and dead mice (number of animals) Test substance 10 28.8 0.57 7 0 contrast 10 28.5 0.57 7 0

Mouse weight change (g) Specimen Mouse number Measurement days 0 days 2 days 4 days 7 days Test substance 1.2.3.4.5.6.7.8.9.10. 28.1829.8728.6429.0027.8827.7727.4829.8729.8629.28 30.2131.7830.9831.4930.2130.1729.6933.8832.4433.06 30.7732.6032.2731.9231.8631.0931.0635.5333.6734.33 31.4633.6533.5233.5432.8432.1432.6137.6434.0535.05 Mean SD 28.780.93 31.391.40 32.511.55 33.651.73 contrast 1.2.3.4.5.6.7.8.9.10. 28.9928.7627.9928.3927.8429.0629.5028.2428.2728.00 31.0930.4829.7930.3328.9130.6131.2430.8630.5130.30 33.1031.8230.7730.9629.7832.4932.5131.3232.4230.71 33.4731.1231.5629.9730.2832.3033.4431.8631.4831.41 Mean SD 28.500.55 30.410.67 31.561.05 31.691.16

Example 7

UV absorption spectrum of antioxidant liquid according to the present invention

Among the ultraviolet rays of the sun, a part of UV-B having a short wavelength and UV-C are absorbed by the ozone layer, and UV-A (400 nm to 320 nm) and UV-B (320 nm to 290 nm) reach the surface, and the UV -A causes suntan, and after long-term exposure, it accelerates the aging of the skin and enhances the reaction of UV-B, and UV-B has the property of causing sunburn on the epidermis. In addition, DNA absorbed ultraviolet rays having a peak of 260 nm, and the antioxidant liquid solution obtained in Example 4 was tested in the ultraviolet absorption spectrum (FIG. 1, Table 3).

Wavelength (nm) 200 202 204 206 208 210 Absorbance 1.024 1.013 0.989 0.951 0.899 0.832 Wavelength (nm) 212 214 216 218 220 222 Absorbance 0.751 0.661 0.564 0.469 0.383 0.309 Wavelength (nm) 224 226 228 230 232 234 Absorbance 0.250 0.199 0.159 0.128 0.105 0.086 Wavelength (nm) 236 238 240 242 244 246 Absorbance 0.072 0.063 0.057 0.054 0.051 0.048 Wavelength (nm) 248 250 255 260 265 270 Absorbance 0.047 0.045 0.044 0.042 0.040 0.038 Wavelength (nm) 275 280 285 290 295 300 Absorbance 0.036 0.035 0.033 0.032 0.032 0.030 Wavelength (nm) 310 320 330 340 350 360 Absorbance 0.028 0.025 0.023 0.021 0.019 0.018 Wavelength (nm) 370 380 390 400 Absorbance 0.017 0.015 0.015 0.013

The absorbance of Table 3 shows the absorbance of 1.24 when irradiated with UV light at 200 nm, and then the absorbance decreases rapidly as the wavelength is increased.The absorbance decreases rapidly at 0.03 at 234 nm, 0.042 at 260 nm, 0.030 at 300 nm. At 400 nm, the absorbance was 0.013. From this test, the absorbances of UV-A (400 nm to 320 nm) and UV-B (320 nm to 290 nm) wavelengths of ultraviolet rays were very low to protect sebum. In this test, a 100-fold dilution of the liquid of the antioxidant substance of Example 4 was used. Moreover, it turned out that the absorbance falls with the wavelength of an ultraviolet-ray from this test.

Example 8

Antioxidant test

20 g of iron (purity 55.85) was added to each of the antioxidant liquid of Example 4 and 1 L of water, respectively, and the change was tested by using a redox potentiometer (ORP). The results are shown in FIG.

The redox potential of the water to which iron was added showed -27 mv on the third day, the iron turned gray, and the iron browned on the fifth day. However, iron in the natural antioxidant liquid was maintained at the time of addition even after 9 days of testing. This test proved the antioxidant activity.

As is apparent from the foregoing, according to the present invention, an antioxidant substance suitable for agricultural products, livestock products, food products, cosmetics, medicines, and concrete, and having an excellent antioxidant activity is obtained by the microbial reaction without using an organic solvent extraction method. It is possible to provide a method for producing an antioxidant substance to obtain That is, the antioxidant substance obtained by this invention suppresses deterioration, discoloration, discoloration, discoloration, and the damage by an ultraviolet-ray, and therefore the field of use is wide.

Claims (2)

(1) a seed culture step of obtaining a seed culture of photosynthetic bacteria, (2) a preculture step of preculturing the seed culture in an anaerobic condition in the presence of an antioxidant substance to obtain a preculture; (3) adding the substrate to the preculture, carrying out the main culture until the cells become inactivated under aerobic cancer conditions in the presence of an antioxidant substance, and treating the culture solution at pH 6.4 to 6.6 to obtain an aqueous antioxidant substance solution. Method for producing an antioxidant substance. (1) a seed culture step of obtaining a seed culture of photosynthetic bacteria, (2) a preculture step of preculturing the seed culture in an anaerobic condition in the presence of an antioxidant substance to obtain a preculture; (3) carrying out the main culture of the whole culture in the presence of an antioxidant substance until the cells are inactivated under aerobic cancer conditions, and treating the culture medium at pH 6.4 to 6.6 to obtain an aqueous solution of antioxidant substance. Manufacturing method.