WO2001090394A1 - Process for producing antioxidant - Google Patents

Abstract

An aqueous solution of an antioxidant is obtained by the following steps: (1) the seed culture step for obtaining a seed culture medium of a photosynthetic bacterium; (2) the preculture step for obtaining a preculture medium by pre-culturing the above seed culture medium in the presence of an antioxidant under anaerobic and bright conditions; and (3) the main culture step of effecting the main-culture of the above preculture medium, to which a substrate has been optionally added, in the presence of an antioxidant under aerobic and dark conditions until the cells are inactivated and then adjusting the liquid culture medium to pH 6.4 to 6.6.

Description

 Specification

 Method for producing antioxidants

 Technical field

 The present invention relates to a method for producing an antioxidant.

 Background art

 At present, antioxidants are required to suppress sebum oxidation by UV-A and UV-B, and to prevent skin aging and damage to DNA by active oxygen generated in the process.

 Here, conventionally, synthetic antioxidants such as butylhydroxydisole (BHA) and butylhydroxyltoluene (BHT) have been used in foods, cosmetics, pharmaceuticals, and the like. However, these have many problems in terms of safety, and consumers have been demanding antioxidant substances that have improved such points.

 There is also a strong need for safe natural antioxidants that can be used for long-term storage of agricultural and livestock products.

 Disclosure of the invention

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

 The inventors of the present invention have conducted research on applied technology and experiments on useful microorganisms that produce antioxidants, and as a result, have completed a production technology for producing antioxidants in large quantities.

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

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

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

(3) A substrate is added to the preculture, main culture is carried out in the presence of antioxidants under aerobic dark conditions until the cells are inactivated, and the culture solution is adjusted to pH 6.4 to 6.4. .6 to obtain an antioxidant aqueous solution. In another aspect, the method for producing an antioxidant according to the present invention comprises:

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

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

 (3) Main culture of the preculture under aerobic dark conditions in the presence of antioxidants until the cells are inactivated, and treatment of the culture solution to ρΉ6.4-6.6. This step of obtaining an antioxidant aqueous solution. That is, the present step can be carried out without adding a substrate to the preculture.

 BRIEF DESCRIPTION OF THE FIGURES

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

 FIG. 2 is a diagram showing an oxidation-reduction potential measurement in which iron powder is added to an antioxidant solution and water according to the present invention.

 BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of a method for producing an antioxidant according to the present invention will be described in detail. In the present invention, first, a seed culture of a photosynthetic bacterium is obtained.

 Seed cultures are obtained by culturing the freeze-dried cells as photosynthetic bacteria in a seed medium.

 For such a seed culture, a plurality of freeze-dried bacteria of photosynthetic bacteria can be separately seed-cultured, and a plurality of seed cultures can be used.

 In the present invention, one or more species of photosynthetic bacteria that are anaerobic bacteria are used. In particular, it is a bacterium belonging to Rhodopseuumonas palust ris, Rhodopseumonas spheroides, Rhodopseudomonas sphero ides, and Rhodops euaomo n "ascapsu 1 ata belonging to Rhodops euaomo n" ascapsu 1 ata. Is preferred.

In this specification, the terms “seed culture”, “pre-culture”, and “complex culture” are used to describe the amount. Culture medium) Expressed as the amount included. In the present invention, the “seed culture”, “preculture”, and “complex culture” are preferably prepared as a culture solution.

 Preculture step

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

 That is, in this pre-culture step, an antioxidant is added to the seed culture, and in a container under an anaerobic environment at a temperature of 25 ° C to 30 ° C, depending on other conditions, 30 to 4 Incubate for 0 days. This gives a preculture of pH 6.4-7.0. When a plurality of seed cultures are used, the plurality of seed cultures are mixed and used. The use of antioxidants suppresses excessive germs and suppresses oxidative effects.

The pre-culture, it is preferable that the individual photosynthetic bacteria prepared as ^{1 0 8 ~1 0 1 (1} / m L photosynthetic bacteria cultures contained. It should be noted, including the number of bacteria, hereby In this document, the number of bacteria is described as the number of bacteria measured according to the plate culture method.The 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 KORINK REA Co., Ltd. Such antioxidants are preferably used in the form of an aqueous solution.

 It is also preferable to preculture the seed culture by adding a substrate. As such a substrate, fish solution pull is preferable. Fish Solupur is heat-treated fish residue. It consists of 16 amino acid compositions. It is preferable to use a material that has been subjected to heat treatment again or treated with lactic acid to a pH of 3.5 or less. In addition, as a substrate, rice bran, fish cake, powdered kelp, and the like can be used or used in combination.

 In addition, wastewater from tofu plants, wastewater from starch plants, human wastewater, livestock manure wastewater, and fishery processing wastewater can also be used as substrates. By culturing photosynthetic bacteria using these as substrates, antioxidants can be obtained.

The above anaerobic condition refers to an environment in which air is shut off from the outside world, and in a sealed container, air mixed in at the time of sealing exists but light enters. Disgust When culturing photosynthetic bacteria under light conditions, the substrate can be selected so that hydrogen sulfide is generated. In this case, various bacteria are suppressed, and photosynthetic bacteria grow well. When performing pre-culture, the seed culture is 1 to 3 parts by weight, the antioxidant solution containing 0.01 to 0.05% by weight of antioxidant is 1 to 5 parts by weight, and the substrate is 0.5 to 0.5 parts by weight. It is preferable to mix it by 5 to 5 parts by weight.

 This process

 In this step, the substrate is added to the preculture, the main culture is performed under aerobic conditions under the presence of antioxidants until the cells are inactivated, and the culture solution is adjusted to pH 6.4 to 6. 6 to obtain an antioxidant aqueous solution. The preculture can be performed without adding a substrate.

 That is, in this step, the preculturer and the substrate are added to the antioxidant aqueous solution, and the cells are cultured under aerobic and dark conditions while maintaining the water temperature at 25 ° C to 30 ° C. This culture is performed until the cells are self-digested and inactivated. Ammonia is generated by autolysis of the cells, and the culture solution becomes highly alkaline.However, under aerobic conditions, ammonia is expelled, and the culture solution has a pH of 6.4 to 6.6 as described above. Will be processed. The aerobic state is maintained by passing oxygen or air through the culture solution. Thereby, an aqueous solution of an antioxidant can be obtained. The antioxidant obtained according to the present invention is a vitamin; B12. This has been confirmed by a microorganism identification method using a strain of Lact obac i 11 us De lbruecki i subsp. Lact is (ATCC 7830).

 As the above-mentioned substrate, it is preferable to use a substrate such as fish zollible used in the preculture step.

 This substrate may not be added. At that time, the cells mainly cause autolysis without going through the growth process. The subsequent process is the same as when the substrate is added.

Antioxidants that can be used in this process include vitamin A, vitamin B group, and vitamin E. Among them, especially, KMX of vitamin B12, a product of KOR IN K 0 REA Co., Ltd. It is preferable to use Such antioxidants are preferably used in the form of an aqueous solution. The aerobic and dark conditions refer to an environment in which air is circulated from the outside world and air circulates in a closed container so that light does not enter.

 When performing the main culture, 1 to 30 parts by weight of the preculture, 1 to 5 parts by weight of an antioxidant solution containing 0.01 to 0.05% by weight of an antioxidant, and a substrate are added. Is preferably blended in an amount of 0.5 to 5 parts by weight.

 After completion of the main culture, the liquid impurities are allowed to settle naturally, and an antioxidant aqueous solution is obtained through a filter from the lower part of the tank.

 Example

 Examples of the method for producing the antioxidant substance according to the present invention will be described below.

 Example 1

 Preparation of seed culture A of photosynthetic bacteria

As a photosynthetic bacterium among the viable bacterial agents, a freeze-dried bacterium (freezedried) JCM2524 was selected from the JCM microbial strain published by the RIKEN Microorganism Preservation Facility and used. Yeast extract 2 g, L-sodium maleate (Sodi urn L—malate) 2 g, sodium glutamate 2 g, potassium hydrogen phosphate lg, 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 7 hydrate 0.5 m :, cobalt chloride hexahydrate 0.5 mg, thiamine mono-HCL lmg, nicotinic acid lmg, biotin 0.01 mg, distillation A culture medium consisting of 1 L of water and having a pH of ^{7 was} anaerobically cultured at 30 ° C. by irradiating a 200 OLux fluorescent lamp with illuminance to obtain 109 ZmL of a seed culture A.

 Example 2

 Preparation of photosynthetic bacteria seed culture B

As a further photosynthetic bacterium, a freeze-dried ATCC 17023 is selected from the ATCCB acteria and B acteriophages log of America Type Copper Culture Co 11ection. , Malic acid 2.5 _g , Yeast extract lg, Ammonium sulfate 1.25 g, Magnesium sulfate heptahydrate 0.2 g, potassium chloride dihydrate 0.07 g, ferric citrate 0.01 g, ethylenediaminetetraacetic acid 0.02 g, dipotassium hydrogen phosphate 0.6 g, diphosphate potassium hydrogen 0. 9 g, trace elements 1 ml (Beautique down iron (f erri cc it rat e) 0. 3g, MnSO 4 0. 002 g, H3BO3 0. 001 g, (NH 4) 6Μο 7 0 24 · 4Η 2 0 0. 002 g, EDTAO. 05 g, CaCL 2 · 2 H20 0. 02 g, distilled water 100 m 1 or et those prepared), vitamins 7. 5 ml (nicotinic acid 0. 2 g, Nikochina Mi de 0 2 g, thiamine HCL 0.4 g, biotin 0.008 g, prepared from distilled water 1 L), distilled water 1 L, pH 6.9 medium at 30 ° C with 2000 Lux fluorescent lamp. irradiated anaerobically cultured to obtain 10 ⁹ / mL of the seed culture B. Example 3

 Preparation of preculture

 3 parts by weight of each of seed cultures A and B, 5 parts by weight of an antioxidant solution containing 0.01% by weight of antioxidant KMX, and 3 parts by weight of fish soluble, mixed in a container under anaerobic environment The cells were cultured at a temperature of 25 ° C to 3.0 ° C for a period of 35 days. Thus, a preculture of PH 7.0 was obtained. When a plurality of seed cultures are used, the plurality of seed cultures are mixed and used.

The preculture was prepared as photosynthetic bacteria culture individual photosynthetic bacteria contain 10 ⁸ ZML. '

 Example 4

 Main culture A

To an aqueous solution of 5 parts by weight of KMX solution (product of KORIN KORE A) containing 0.01% by weight of KMX as an antioxidant, 5 parts by weight of the above preculture and 0.5 parts by weight of fish solution were added. In addition, under aerobic and dark conditions, maintain the water temperature at 25 ° C to 30 ° C, and perform the main culture until the cells are inactivated and the cells are completely inactivated. PH 6.5. After that, the impurities were allowed to settle naturally and passed through a filter from the lower part of the tank to obtain an aqueous solution of an antioxidant. In aqueous solution The antioxidant was identified as vitamin B12 by a microorganism identification method using a strain of Lact obacil lus De lbrecki is ubsp. Lact is (ATCC 7830).

 Example 5

 Main culture B

To an aqueous solution of 5 parts by weight of KMX solution (product of KORIN KOREA Co., Ltd.) containing 0.01% by weight of KMX as an antioxidant, 30 parts by weight of the above preculture was added. While maintaining the temperature at 30 ° C, the culture was continued until the cells were inactivated, and the culture was adjusted to pH 6.5. After that, the impurities were allowed to settle naturally and passed through a filter from the lower part of the tank to obtain an aqueous solution of an antioxidant. Antioxidants in the aqueous solution by Lact obac ll lus De lbrue cki isubsp . Lact is (ATCC 7830) microorganism identification method using a strain of, were confirmed as a vitamin B _12.

 Example 6

 Single dose toxicity test using natural antioxidant substances of the present invention in mice

 Test substance (antioxidant obtained in Example 4 above) 20 g ZKg was orally administered to mice, and changes in general condition and body weight were observed for 7 days, and then dissected, and each organ was visually observed. As a result, there were no deaths in each group from immediately after administration to the day after the end of observation, and no piloerection, diarrhea, sweating, deep breathing or abnormal behavior was observed throughout the test period. No abnormalities were observed by gross observation at necropsy. This proved that there was no toxicity with a single dose of the test substance of 20 g / Kg (Tables 1 and 2).

) o group Test mice Average of mice Observation period Abnormality and death

 (Number of animals) Body weight (g) (ml) (Remarks) Mouse (animal) Test 10 28. 8 0. 57 7 0 Substance

Reference 10 28.5 0.57 7 0 Table 2 Changes in mouse weight (g)

 Days of sample mass measurement

 Ν 0 n

 U hi Δ d 4 d 7 Q

1 28. 18 30. 21 30. 77 31. 46

2 29. 87 31. 78 32. 60 33. 65

3 28. 64 30.q «Q 9 o 7 · 52 Subject 4 2 9 · 0 0 3 1.A qoqo QQ · 5 4 Test 5 27.88 30.21 31.86 32.84 Object 6 27.77 30 17 31 · 09 32. 14 Quality 7 27. 48 29. 69 31 · 06 32. 61

 8 29 87 33. 88 35. 53 37. 64

9 29.86 32.44 33.67 34.05

10 29.28 33.06 34.33 35 05 ean o o

 1 o 丄. 39 32.51 33 Ό O

U. Ω Q 丄, 40 1.55 1.

1 28. 99 31. 09 33. 10 33. 47

2 28. 76 30. 48 31. 82 31. 12

3 27.99 29. ((o 丄 56 to 4 28.39 30

 o o o U 9 b Δ y. 97 5 27 84 28.Q 1? Q 7 ί 28

 6 29. 06 30. 61 32. 49 32

7 29. 50 31. 24 32. 51 33. 44

8 28. 24 30. 86 31. 32 31. 86

9 28. 27 30. 51 32. 42 31. 48

10 28.00 30.30 30.71 31.1.

Mean 28. 50 30. 1 31. 56 31. 69

SD 0.55 0.67 1.05 1.16 Example 7

 Ultraviolet absorption spectrum of antioxidant liquid according to the present invention

Of the ultraviolet rays of the sun, a part of short wavelength UV-B and UV-C are absorbed by the ozone layer, and UV-A (400 ηπ! ~ 320 nm) and UV-B (320 nn! ~ 29 Onm) ) Reaches the ground surface, UV-A causes suntan, has a property that accelerates aging of skin and prolongs UV-B anti-aging property when exposed for a long time, and UV-B has a property that causes sunburn on the epidermis. is there. In addition, DNA has an ultraviolet peak at 26 Onm. Here, the antioxidant solution obtained in Example 4 was tested with an ultraviolet absorption spectrum (FIG. 1, Table 3).

 Table 3

 Looking at the absorbance in Table 3, when irradiated with ultraviolet light at 20 Onm, it shows an absorbance of 1.24, and then the absorbance sharply decreases as the wavelength becomes longer. At 234ηm, the absorbance is 0.086, 26 Onm At 0.042 and 30 Onm, the absorbance was 0.030 and at 40 Onm, the absorbance was 0.013. From this test, the absorbance of UV-A (400 nm to 320 nm) and UV-B (320 nm to 290 nm) ultraviolet rays was extremely low, and it was possible to protect sebum. In this test, a 100-fold diluted solution of the antioxidant substance of Example 4 was used. From this test, it was found that the absorbance was reduced by the wavelength of the ultraviolet light.

 Example 8

 Antioxidant test

20 g of iron powder (purity 55.85) for each 1 L of the antioxidant solution and water of Example 4 )

And the change was tested using an oxidation-reduction potentiometer (ORP). The results are shown in Fig. 2. The oxidation-reduction potential of the water into which the iron powder has been introduced shows a state of 127 mv from the third day, the iron powder turns gray, and on the fifth day the iron powder turns brown. However, the iron powder in the natural antioxidant liquid maintained its state at the time of injection even after the 9-day test. This test demonstrated an antioxidant effect.

 Industrial applicability

 As is clear from the above description, according to the present invention, a method for extracting an antioxidant which is suitable for agricultural products, livestock products, food, cosmetics, pharmaceuticals, and concrete and has excellent antioxidant activity with an organic solvent is provided. It is possible to provide a method for producing an antioxidant in which an antioxidant is obtained by a microbial reaction without using the antioxidant. That is, the antioxidant obtained by the present invention suppresses deterioration, deterioration, discoloration, discoloration and harm caused by ultraviolet rays, and therefore has a wide field of application.

Claims

The scope of the claims  1. (1) a seed culture step of obtaining a seed culture of a photosynthetic bacterium,  (2) a preculture step of preculturing the seed culture under anaerobic conditions in the presence of an antioxidant to obtain a preculture,  (3) Add a substrate to the preculture, perform main culture under aerobic dark conditions until the cells are inactivated in the presence of antioxidants, and adjust the culture solution to pH 6.4 to 6.6. A step of obtaining an aqueous solution of an antioxidant by treating the solution.  2. (1) a seed culture step of obtaining a seed culture of photosynthetic bacteria,  (2) a preculture step of preculturing the seed culture under anaerobic conditions in the presence of an antioxidant to obtain a preculture,  (3) Main culture is performed on the preculture under aerobic dark conditions until the cells are inactivated in the presence of antioxidants, and the culture is treated to pH 6.4 to 6.6. A step of obtaining an aqueous solution of an antioxidant by the above method.