JP5503631B2 - Method for producing antioxidant - Google Patents

Description

  The present invention relates to a method for producing an antioxidant by fermentation of soybean residue using yeast, and an antioxidant exhibiting high SOD activity obtained by the production method.

  In recent years, active oxygen is considered to cause aging such as skin wrinkles and reduction of tension, and a method of removing active oxygen is known as one of methods for preventing aging. Superoxide dismutase (SOD, also called antioxidant enzyme) and the like have been used as active oxygen scavengers. In addition to the effect of suppressing the generation of active oxygen by SOD, it is said that rough skin, spots and wrinkles caused by active oxygen can also be prevented (see Non-Patent Document 1).

  In recent years, therefore, research has been conducted on antioxidant substances exhibiting SOD activity. For example, an SOD activator having an active ingredient such as a birch extract composition (see Patent Document 1) or an extract of horse chestnut seed (see Patent Document 2) has been proposed. However, these SOD activators are all extracted from plant raw materials, and this extraction operation takes a long time, and the availability of natural resources is limited, so that the production cost is high.

  On the other hand, in addition to plant-derived antioxidants, fermented products such as lactic acid bacteria have also been found to have an antioxidant effect. For example, Patent Document 3 proposes a culture derived from lactic acid bacteria that exhibits degradation characteristics for a wide range of peroxides. However, the culture is a cell suspension containing cells recovered from a culture obtained by isolating and culturing lactic acid bacteria from a source such as soybean cake, and is free from peroxides such as hydrogen peroxide. On the other hand, it only shows decomposition characteristics. The decomposition of peroxide may not be decomposition by SOD because it may be decomposition by a reducing substance. Non-patent documents 2 to 5 disclose that fermented products obtained from fermented natto or lactic acid bacteria fermented have an antioxidant effect. However, these researches are focused on the antioxidant action, hydroxy radical scavenging ability, hydrogen peroxide scavenging activity, etc. in the linoleic acid system of okara fermented products. It is not known until it has SOD activity.

  Further, Patent Documents 4 and 5 propose an antioxidant composition derived from natto-fermented guava leaves. However, according to the examples, since the SOD activity is low in any case where only guava extract (sample 1) or rice bran, only soybean fermented product (sample 2) is used, its active oxygen scavenging effect is sufficient. It can not be said. Similarly, this method is practically difficult because the availability of guava leaves is limited depending on the season.

As described above, various substances having active oxygen scavenging ability are known, but among them, a high SOD activity is not seen and few are put into practical use.
In addition, microorganisms belonging to the genus Pichia are known to have the following uses. For example, methanol can be used to produce useful substances such as antibiotics and biological proteins, or it can be used as a genetically engineered bacterium.
Microorganisms belonging to the genus Candida are known to have the following uses. For example, Candida valida is used for lipase production. Candida metapsilosis, Candida utilis, etc. can also be used in the production of brews, glycerin, edible yeast, organic acids and enzyme agents, and the feed industry.
However, there is no production example regarding an antioxidant by soybean residue fermentation using a microorganism belonging to the genus Pichia or Candida.

JP 2008-88109 A JP 2007-291069 A JP 2009-191276 A JP 2004-24004 A JP 2004-43505 A

Food Functional Materials II, issued April 27, 2001, CMC Corporation, pp. 44-63 Takaki Tamura et al., “The Journal of Japan Society for Food Science and Technology”, 1999, 46, 9, 561-569 Watanabe Yasunari et al., “Saitama Industrial Technology Center Research Report”, 2005, Volume 3 B. Moktan et al., “Food Research International”, 2008, 41, 586-593 Y.P. Zhu et al., `` Food Control '', 2008, Vol. 19, pp. 654-661

  The subject of this invention is providing the antioxidant obtained by the manufacturing method of the antioxidant by the fermentation method using a safe microorganism and a safe food material, and a fermentation method.

  From the above, as a result of intensive studies to solve the above problems, the present inventors have cultivated a specific yeast in a medium containing soybean residue, and in particular, Pichia anomala, Pichia angui A microorganism belonging to the genus Pichia such as Pichia guilliermondii, or Candida such as Candida zeylanoides, Candida parapsilosis and Candida metapsilosis ) It is possible to increase the antioxidant ability (SOD activity) of the culture supernatant obtained by culturing microorganisms belonging to the genus and centrifuging the culture solution, and to obtain an antioxidant having high SOD activity at a low cost and efficiently. As a result, the present invention has been completed.

That is, this invention provides the manufacturing method of an antioxidant which culture | cultivates yeast in the culture medium containing a soybean residue, and extract | collects an antioxidant from a culture supernatant.
Moreover, this invention provides the antioxidant which shows the high SOD activity obtained by the manufacturing method of the said antioxidant.
Moreover, this invention provides use of the fermented extract by yeast of soybean residue as an antioxidant.
The present invention also provides the use of a fermented extract of soybean residue yeast for the production of antioxidants.
Furthermore, this invention provides the foodstuffs, cosmetics, and pharmaceutical which mix | blended the said antioxidant.

  According to the method for producing an antioxidant according to the present invention, an antioxidant can be obtained inexpensively and efficiently by using a specific microorganism and using soybean residue, which is a by-product of the food processing industry, as a main raw material of the culture medium. Can do. Since the antioxidant has an active oxygen scavenging effect, particularly high SOD activity, is excellent in safety, and is easy to handle, it can be used in fields such as foods, cosmetics, and pharmaceuticals.

The yeast used in the present invention is preferably a microorganism belonging to the genus Pichia or Candida.
As microorganisms belonging to the genus Pichia (Pichia anomala), Pichia guilliermondii (Pichia guilliermondii), Pichia burtonii (Pichia burtonii), Pichia membranaefaciens (Pichia membranaefaciens), or Examples include Pichia Kluyveri. The microorganisms belonging to the genus Candida include Candida zeylanoides, Candida parapsilosis, Candida metapsilosis, Candida albicans, and Candida. -Candida holmii or Candida valida is exemplified. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included. The microorganisms are cultivated in a medium containing soybean residue, thereby producing an antioxidant that exhibits high SOD activity from the culture supernatant. Among them, Pichia anomala or Pichia guilliermondii belonging to the genus Pichia, or Candida zeylanoides belonging to the genus Candida, Use of Candida parapsilosis or Candida metapsilosis is preferable because an antioxidant having a higher active oxygen scavenging effect can be obtained.
All of the yeasts according to the present invention include the Chinese Common Microorganisms Storage Center (CGMCC) and the Chinese Typical Culture Storage Center (CCTCC), the Chinese High School Industrial Microorganism Resources and Information Center (CICIM). Universities) and other commercial routes.

  The method for producing an antioxidant of the present invention is to obtain an antioxidant from a culture supernatant by culturing the above microorganisms in a medium containing soybean residue. Details of the method are described below.

<Medium>
In the present invention, the main component of the liquid medium is soybean residue. Other components include saccharides and water. Moreover, you may add a peptone and yeast powder suitably to a culture medium. Since soybean residue is a readily available raw material, the culture medium of the present invention has an advantage that it is cheaper and superior than the conventional one.
The soybean residue used in the present invention is obtained by immersing soybean in water, absorbing water, grinding and filtering, and removing water. As described above, it is possible to use soybean residue, okara, etc., which is a residual residue obtained by squeezing soy milk in the food industry and the like.
As the saccharide, for example, sucrose, maltose, sucrose and the like can be used alone or in combination, and sucrose can be preferably used from the viewpoint of enhancing fermentation productivity and availability.

In the present invention, in order to increase the SOD activity, the medium is preferably composed of soybean residue, sucrose and water. The soy residue is preferably contained in the medium at 5 to 15 w / v%, more preferably 8 to 12 w / v%, still more preferably 10 w / v%. When the content of soybean residue falls within that range, high SOD activity is obtained. From the same point, sucrose is preferably contained in an amount of 0.2 to 2 w / v%, more preferably 0.8 to 1.2 w / v%.
Hereinafter, the medium used for producing the antioxidant as described above is referred to as “antioxidant production medium”.

<Fermentation culture conditions>
In the fermentation culture process, one or two or more microorganisms described above can be used. When two or more microorganisms are used, fermentation is performed by adding them to the antioxidant production medium sequentially or simultaneously.
Fermentation culture conditions for producing antioxidants are 20 to 40 ° C., more preferably 27 to 35 ° C. from the viewpoint of optimizing the temperature and time necessary for yeast growth and enhancing SOD activity. The culture is preferably performed at a temperature of 1 to 7 days, more preferably 48 to 96 hours, and even more preferably 68 to 76 hours.

<Recovery and purification of antioxidants>
The antioxidant of the present invention can be used as an antioxidant by removing the cells from the culture solution by centrifugation or the like, recovering the culture supernatant (hereinafter sometimes referred to as a fermented extract of soybean residue). it can. Further, it may be further purified by appropriately performing known separation / purification techniques, for example, ultrafiltration, ammonium sulfate precipitation and the like.

  In the present invention, the antioxidant effect of an antioxidant (ie, active oxygen scavenger), ie, the active oxygen scavenging effect, is confirmed by measuring the SOD activity value. The larger the SOD activity value, the higher the SOD activity, the higher the active oxygen scavenging ability, and the suppression of aging due to skin oxidation. The “high SOD activity” in the present invention means that the SOD activity value measured and calculated by the method of Examples described later is 75% or more. Here, when the SOD activity of the antioxidant (active oxygen scavenger) of the present invention is 75 to less than 100%, it can be determined that the SOD activity is moderate, and when it is 100 to 120%, the SOD activity is even higher. It can be judged as active.

It was confirmed that the fermented extract of soybean residue obtained by culturing the yeast of the present invention has high SOD activity, as shown in Examples below. It was found that by using this fermented extract, high active oxygen scavenging effects such as antioxidant and anti-aging can be obtained.
The fermented extract of soybean residue obtained by culturing the yeast of the present invention can be used for therapeutic or non-therapeutic purposes as an antioxidant (active oxygen scavenger). Non-therapeutic uses include cosmetic use and use for maintaining health. In addition, the therapeutic use and the non-therapeutic use as the above antioxidant (active oxygen scavenger) can be performed separately.

  Moreover, the fermented extract of soybean residue obtained by culturing the yeast of the present invention can be used for producing an antioxidant (active oxygen scavenger).

  The antioxidant (active oxygen scavenger) of the present invention has a very high active oxygen scavenging effect and extremely high safety, and is caused by an increase in the amount of active oxygen in the living body, hypertension, arteriosclerosis, diabetes and cancer. It is useful for the improvement and prevention of various diseases such as illness and cosmetic disorders. The active enzyme scavenger of the present invention can be formulated into ordinary foods, cosmetics and pharmaceuticals to impart an active oxygen scavenging effect and increase its commercial value.

  Moreover, as a food dosage form, normal food forms, such as a confectionery, chocolate, gum, a candy, a drink, are employable, for example. Examples of cosmetic dosage forms include skin external preparations applied to the skin, such as lotions, emulsions, gels, essences, packs, cleaning agents, bath preparations, foundations, lipsticks, ointments and the like. Examples of pharmaceutical dosage forms include powders, granules, tablets, capsules, suspensions, solutions, emulsions, injection solutions, and the like. In these foods, cosmetics and pharmaceuticals, arbitrary components can be used according to the purpose of use.

  The blending amount of the antioxidant (active oxygen scavenger) of the present invention may be appropriately changed depending on its form, but is 0.001 to 10% by mass in terms of solid matter with respect to the total amount of food, cosmetics, pharmaceuticals and the like. Preferably 0.01-1 mass% can be mix | blended. If it is less than 0.001% by mass, a sufficient active oxygen scavenging effect is hardly expected. On the other hand, when the content exceeds 10% by mass, the improvement of the effect is hardly recognized and it is uneconomical. In addition, the addition method may be added in advance or during the production, and may be appropriately selected in consideration of workability.

Hereinafter, the present invention will be described with reference to examples. However, the present invention should not be limited to these examples.
(Example 1 to Example 8)
(Production of antioxidants (active oxygen scavengers))
As microorganisms for producing an antioxidant (active oxygen scavenger) from soybean residues, microorganisms belonging to the genus Pichia in Table 1 below were used.

A 300 mL Erlenmeyer flask containing 100 mL of an antioxidant production medium (10 w / v% soybean residue, 1 w / v% sucrose) was obtained by culturing the above strain obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium). And inoculated at 30 ° C. for 72 hours. Here, the soybean residue was prepared by putting soybean in water and impregnating it for 12 hours, allowing the soybean to absorb water sufficiently, grinding and grinding the soybean that absorbed water, filtering and drying.
Next, the microbial cells were removed from the obtained culture solution by centrifugation at 3000 rpm for 10 mins, and the culture supernatant was collected. Each was designated as samples 1-8.
Further, as a control, only the medium (not inoculated with microorganisms) is cultured and treated in the same manner as in samples 1 to 8, and the supernatant is collected and used for the next measurement of SOD activity.

(Measurement of SOD activity)
The SOD activity value is measured with a 96-well plate using SOD Assay Kit-WST (manufactured by Dojindo Laboratories) for the samples 1 to 8 and the control obtained as described above. Use the reagents that come with the kit.
Using the samples 1 to 8 and the control as samples, 20 μl is transferred to the “sample” & “Blank 2” hole of the 96-well plate, and 20 μl of sterile water is added to the “Blank 1” & “Blank 3” hole. Thereafter, 200 μl of [WST working solution] is added to each hole and well stirred with a plate mixer. Add 20 μl of [Dilution buffer] to the “Blank2” and “Blank3” holes. Add 20 μl of [Enzyme working solution] to each of the “sample” & “Blank 1” holes and mix well. After heating and incubating at 37 ° C. for 20 minutes, the plate is placed in a Bioreader and the absorbance value at 450 nm is measured.

  Next, the SOD activity value is calculated by the following formula. Tables 2 and 3 show 64.11 to less than 75% as Δ (having general SOD activity) and 75 to less than 100% as ○ (having high SOD activity). In addition, it shows that antioxidant activity is so high that the value of SOD activity is high.

(Equation 1)
SOD activity (%) = [(A _B1 −A _B3 ) − (A _S −A _B2 )] / (A _B1 −A _B3 ) × 100

A _S : Absorbance when the sample is added to the sample and the reaction is performed A _B1 : Absorbance when the reaction is performed without adding the sample to the “Blank1” hole A _B2 : In the “Blank2” hole Absorbance of sample itself A _B3 : Absorbance of plate itself and [Dilution buffer] in “Blank3” hole

<Result>
The results are shown in Tables 2 and 3.

  From the above results, high SOD activity was observed in any of the samples obtained in Examples 1 to 8.

(Example 9 to Example 17)
(Production of antioxidants (active oxygen scavengers))
As microorganisms for producing an antioxidant (active oxygen scavenger) from soybean residues, microorganisms belonging to the genus Candida in Table 4 below were used.

The above strain obtained by culturing at 30 ° C. in YPD medium (Yeast Extract Peptone Dextrose Medium) was added to a 300 mL Erlenmeyer flask containing 100 mL of antioxidant production medium (10 w / v% soybean residue, 1 w / v% sucrose). Inoculated and incubated at 30 ° C. for 72 hours. Here, the soybean residue was prepared by putting soybean in water and impregnating it for 12 hours, allowing the soybean to absorb water sufficiently, grinding and grinding the soybean that absorbed water, filtering and drying.
Next, the microbial cells were removed from the obtained culture solution by centrifugation at 3000 rpm for 10 mins, and the culture supernatant was collected. Each was designated as Samples 9-17.
Further, as a control, only the medium (not inoculated with microorganisms) is cultured and treated in the same manner as in Samples 9 to 17, and the supernatant is collected and used for the next measurement of SOD activity.

(Measurement of SOD activity)
The SOD activity value is measured with a 96-well plate using SOD Assay Kit-WST (manufactured by Dojindo Laboratories) with respect to the samples 9 to 17 and the control obtained as described above. Use the reagents that come with the kit.
Using the samples 9 to 17 and the control as samples, 20 μl is transferred to the “sample” & “Blank 2” holes of the 96-well plate, and 20 μl of sterile water is added to the “Blank 1” & “Blank 3” holes. Thereafter, 200 μl of [WST working solution] is added to each hole and well stirred with a plate mixer. Add 20 μl of [Dilution buffer] to the “Blank2” and “Blank3” holes. Add 20 μl of [Enzyme working solution] to each of the “sample” & “Blank 1” holes and mix well. After heating and incubating at 37 ° C. for 20 minutes, the plate is placed in a Bioreader and the absorbance value at 450 nm is measured.

  Next, the SOD activity value is calculated by the following formula. 64.11 to less than 75% is Δ (having general SOD activity), 75 to less than 100% is ○ (having moderately high SOD activity), and 100 to 120% is ◎ (having higher SOD activity) Tables 5 and 6 show. In addition, it shows that antioxidant activity is so high that the value of SOD activity is high.

(Equation 2)
SOD activity (%) = [(A _B1 −A _B3 ) − (A _S −A _B2 )] / (A _B1 −A _B3 ) × 100

A _S : Absorbance when the sample is added to the sample and the reaction is performed A _B1 : Absorbance when the reaction is performed without adding the sample to the “Blank1” hole A _B2 : In the “Blank2” hole Absorbance of sample itself A _B3 : Absorbance of plate itself and [Dilution buffer] in “Blank3” hole

<Result>
The results are shown in Tables 5 and 6.

  From the above results, high SOD activity was observed in any of the samples obtained in Examples 9-17. Among them, samples 15 to 17 obtained by using Candida metapsilosis in Examples 15 to 17 were found to have the highest SOD activity.

  Therefore, it was confirmed that the fermented extract of soybean residue of the present invention exhibits high SOD activity, has an excellent active oxygen scavenging effect, and can be used as an antioxidant (active oxygen scavenger).

  From the above, the antioxidant by fermenting soybean residue using yeast has an excellent active oxygen scavenging effect and can be used for cosmetics, foods, pharmaceuticals and the like.

Claims (15)

Cultivating and culturing yeast selected from Pichia, Candida zeylanoides, Candida parapsilosis and Candida metapsilosis in a medium containing soybean residues A method for producing an antioxidant, wherein an antioxidant is collected from the supernatant. The method for producing an antioxidant according to claim 1 , wherein the microorganism belonging to the genus Pichia is Pichia anomala or Pichia guilliermondii. The manufacturing method of the antioxidant of Claim 1 or 2 which contains 5-15 w / v% of the said soybean residue in the said culture medium. The manufacturing method of the antioxidant as described in any one of Claims 1-3 which contains the said soybean residue in the said culture medium 8-12 w / v%. The manufacturing method of the antioxidant as described in any one of Claims 1-4 which contains the said soybean residue in the said culture medium 10 w / v%. The method for producing an antioxidant according to any one of claims 1 to 5 , wherein the culture temperature is 27 to 35 ° C. The method for producing an antioxidant according to any one of claims 1 to 6 , wherein the culture time is 1 to 7 days. By centrifugation of the culture solution obtained by the culture, and collecting the culture supernatant as an antioxidant, a manufacturing method of an antioxidant according to any one of claims 1-7. The antioxidant which shows the high SOD activity obtained by the manufacturing method as described in any one of Claims 1-8 . The antioxidant of Claim 9 whose SOD activity of the said antioxidant is 75 to 120%. The antioxidant of claim 9 , wherein the antioxidant has an SOD activity of less than 75-100%. The antioxidant of Claim 9 whose SOD activity of the said antioxidant is 100 to 120%. A food comprising the antioxidant according to any one of claims 9 to 12 . A cosmetic comprising the antioxidant according to any one of claims 9 to 12 . A pharmaceutical comprising the antioxidant according to any one of claims 9 to 12 .