Aspergillus oryzae fermented product with high vitamin B content

Abstract

The objective is to obtain a fermented product of Aspergillus oryzae with a high concentration of vitamin B. [Solution] The present invention relates to a fermented product of Aspergillus oryzae derived from grain raw materials, having an arabinan concentration of 5 mg / g or less. The present invention also relates to a method for producing a fermented product of Aspergillus oryzae derived from grain raw materials, comprising treating the grain raw materials with arabinase and cellulase, and fermenting the treated raw materials using Aspergillus oryzae.

Description

[Technical Field]

[0001] The present invention relates to a fermented product of Aspergillus oryzae and a method for producing the same. [Background technology]

[0002] Vitamins are organic compounds necessary for maintaining normal bodily functions, but they are hardly synthesized in the body and must be obtained from food. Among the vitamins, vitamin B is a collective term for eight nutrients: vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folic acid, and biotin.

[0003] Regarding folic acid, a type of vitamin B, the folic acid contained in food is the natural polyglutamate form, while the folic acid used as a food additive is the synthetic monoglutamate form. Concerns have been reported that if pregnant women continue to take supplements containing the synthetic monoglutamate form of folic acid, the incidence of asthma in their children may increase (Non-Patent Literature 1).

[0004] Meat is a nutritionally important food source, rich in nutrients such as vitamins, proteins, and minerals. However, meat also contains a lot of fat, especially saturated fatty acids, and is high in energy compared to other foods. Therefore, excessive consumption of meat can be a cause of various chronic diseases, including metabolic syndrome.

[0005] Therefore, in order to curb excessive meat consumption, the use of meat-like foods, which are processed meat products, is attracting attention. Meat-like foods are foods that use ingredients that contain less saturated fatty acids and fewer calories than meat, while containing about the same amount of protein. Replacing meat products with meat-like foods may lead to the prevention and improvement of lifestyle-related diseases such as metabolic syndrome.

[0006] As a representative example of meat-like foods, foods made from grains such as soybeans are processed for various purposes and used in food compositions and foods.

[0007] Patent Document 1 discloses a method for producing koji and miso using Aspergillus oryzae, which has high productivity of vitamin B2 compounds. Furthermore, as a method for producing novel biological materials derived from bean pulp, Patent Document 2 discloses a method of treating okara (soybean pulp) with cellulase to promote liquefaction or gelation of bean pulp. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent No. 5974254 [Patent Document 2] Japanese Patent No. 3888539 [Non-patent literature]

[0009] [Non-Patent Document 1] Melissa J. Whitrow et al., “Effect of Supplemental Folic Acid in Pregnancy on Childhood Asthma: A Prospective Birth Cohort Study”, American Journal of Epidemiology, 2009, Vol. 170, No. 12, pages 1486-1493. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] As mentioned above, vitamins are nutrients that must be obtained from food, and it is desirable to obtain natural vitamins. Therefore, meat-like foods that are rich in vitamins are in demand.

[0011] It has been reported that vitamin B, such as folic acid, can be increased through koji mold fermentation. However, in order to obtain a fermented product with high vitamin B content and high nutritional value, it was necessary to use koji mold that had been mutated to produce high vitamin B productivity (Patent Document 1). In addition, it is known that okara (soy pulp) can be treated with cellulase (Patent Document 2), but the effect of cellulase treatment on the amount of vitamin B was unknown.

[0012] Therefore, the present invention aims to obtain a fermented product of Aspergillus oryzae with a high vitamin B concentration. [Means for solving the problem]

[0013] As a result of diligent research to solve the above problems, the inventors of the present invention have found that the vitamin B concentration in Aspergillus oryzae fermented products obtained by treating grain-derived raw materials with arabinase and cellulase before fermenting with Aspergillus oryzae increases compared to Aspergillus oryzae fermented products obtained by treating grain-derived raw materials with arabinase and cellulase without treatment.

[0014] The present invention is as follows: [1] A fermented product of Aspergillus oryzae derived from grain raw materials, with an arabinan concentration of 5 mg / g or less. [2] The koji mold fermented product described in [1] above, having a vitamin B concentration of 40 μg / 100g or more. [3] The koji mold fermented product described in [2] above, wherein the vitamin B is folic acid. [4] The koji mold fermented product according to any one of [1] to [3] above, wherein the grain-derived raw material is derived from legume residue. [5] The koji mold fermented product described in [4] above, wherein the raw material derived from the soybean pulp is okara. [6] The koji mold fermented product according to any one of [1] to [3] above, wherein the koji mold is a koji mold of the genus Aspergillus. [7] A method for producing a fermented product of grain-derived raw materials by koji mold, comprising treating grain-derived raw materials with arabinase and cellulase, and fermenting the treated raw materials with koji mold. [8] The method for producing the product according to [7] above, wherein the arabinan concentration in the koji mold fermented product is 5 mg / g or less. [9] The production method according to [7] or [8] above, wherein the ratio of arabinase to cellulase is 1:3 to 9:1 based on units.

[10] The production method according to [7] or [8] above, wherein the amount of arabinan contained in the treated raw material is reduced by 70% by mass or more from the amount of arabinan in the cereal-derived raw material before the treatment.

[11] A koji fermentation product of a cereal-derived raw material, obtained by treating the cereal-derived raw material with arabinase and cellulase and fermenting the treated raw material with Aspergillus oryzae.

[12] The koji fermentation product according to

[11] above, having an arabinan concentration of 5 mg / g or less.

[13] The koji fermentation product according to

[11] or

[12] above, having an increased vitamin B concentration as compared with the vitamin B concentration in the cereal-derived raw material before treatment with arabinase and cellulase. [Effect of the Invention]

[0015] According to the production method which is one aspect of the present invention, that is, a production method of a koji fermentation product of a cereal-derived raw material, which comprises treating the cereal-derived raw material with arabinase and cellulase and fermenting the treated raw material with Aspergillus oryzae, by fermenting the cereal-derived raw material treated with arabinase and cellulase with Aspergillus oryzae, a koji fermentation product having an increased vitamin B concentration can be obtained as compared with the koji fermentation product obtained without treating the cereal-derived raw material with arabinase and cellulase. [Brief Description of the Drawings]

[0016] [Figure 1A] FIG. 1A is a graph showing the amount of N-acetylglucosamine in the koji fermentation product treated with each carbohydrase. [Figure 1B] FIG. 1B is a graph showing the amount of folic acid in the koji fermentation product treated with each carbohydrase. [Figure 2A] FIG. 2A is a graph showing the amount of N-acetylglucosamine in the koji fermentation product fermented using Aspergillus oryzae strain. [Figure 2B]Figure 2B is a graph showing the amount of folic acid in the koji mold fermentation product fermented using Aspergillus oryzae strain. [Figure 3A] Figure 3A is a graph showing the amount of N-acetylglucosamine in koji mold fermented using Aspergillus awamori, Aspergillus inui, Aspergillus usami, or Aspergillus cytoi. [Figure 3B] Figure 3B is a graph showing the amount of folic acid in koji mold fermented using Aspergillus awamori, Aspergillus inui, Aspergillus usami, or Aspergillus cytoi. [Figure 4A] Figure 4A is a graph showing the amount of N-acetylglucosamine in koji mold fermented products using okara, wheat bran, or corn flour as grain-derived raw materials. [Figure 4B] Figure 4B is a graph showing the amount of folic acid in koji mold fermented products using okara, wheat bran, or corn flour as grain-derived raw materials. [Figure 5A] Figure 5A is a graph showing the relative levels of folic acid in Aspergillus oryzae fermented products treated under different conditions with varying ratios of arabinase and cellulase. [Figure 5B] Figure 5B is a graph showing the relative levels of folic acid in Aspergillus oryzae fermented products treated under different conditions with varying ratios of arabinase and cellulase. [Figure 6] Figure 6 is a graph showing the glucose content of the enzyme-treated grain-derived raw material solution. [Figure 7] Figure 7 is a graph showing the viscosity of koji mold fermented products made from grain-derived raw materials. [Modes for carrying out the invention]

[0017] The structure and preferred embodiments of this invention will be described in more detail below. In this specification, "A to B" indicates a range and means "A or greater and B or less." Also, in this specification, "weight" and "mass," and "weight%" and "mass%" are treated as synonyms.

[0018] 1. Method for producing fermented products from grain-derived raw materials using Aspergillus oryzae. One method for producing a fermented product of grain-derived raw materials according to this embodiment is to treat the grain-derived raw materials with arabinase and cellulase, and then ferment the treated raw materials using Aspergillus oryzae.

[0019] <Grain-derived ingredients> Grain-derived raw materials include, for example, those containing at least one of carbon and nitrogen. Since Aspergillus oryzae can efficiently utilize arabinose, a component of arabinan, it is preferable that the grain-derived raw material contains arabinan.

[0020] Grain-derived raw materials include, for example, protein-derived raw materials from whole soybeans, defatted soybeans, soy protein, wheat gluten, peas, broad beans, adzuki beans, etc., and starch-derived raw materials from wheat, barley, rye, wheat bran, rice, rice bran, corn, starch residue, etc. Grain-derived raw materials may also be legume residue, wheat bran, beer husk, or corn flour. Wheat bran is the outer layer of the wheat grain and is also called wheat bran. Beer husk is the residue left over when making beer; it is the solid material that remains after crushed malt is mixed with hot water, stirred, and filtered to remove the wort. These can be used individually or in combination.

[0021] From the standpoint of containing high levels of carbon and nitrogen sources essential for the growth of Aspergillus oryzae, the grain-derived raw material is preferably bean pulp, and even more preferably okara. Okara is the pulp left over from soybeans.

[0022] <Enzyme treatment> In the manufacturing method of this embodiment, grain-derived raw materials are treated with arabinase and cellulase. By treating grain-derived raw materials with arabinase and cellulase, the vitamin B content increases. The reason for this is not clear, but it is speculated that factors such as a change in the balance of sugars that can be assimilated by Aspergillus oryzae, leading to high expression of genes involved in vitamin B metabolism, or an increase in the amount of primary metabolites that serve as substrates for vitamin B may be involved.

[0023] Examples of the aforementioned cellulase include hemicellulase, glucanase, and glucosidase, and examples of the aforementioned hemicellulase include xylanase.

[0024] The grain-derived raw materials may be subjected to heating and pressurizing treatment before processing with arabinase and cellulase. Heating and pressurizing treatment is preferably carried out using an autoclave, extruder, or high-pressure heated tube reactor. Such heating and pressurizing treatment allows the enzymes to act efficiently on the grain-derived raw materials.

[0025] In the manufacturing method of this embodiment, the ratio of arabinase to cellulase used to process grain-derived raw materials is preferably 1:3 to 9:1 on an enzyme unit basis (hereinafter also referred to as "unit" or "U"), and more preferably 1:3 to 3:1. A ratio of arabinase to cellulase of (1 or more):3 on an enzyme unit basis has the advantage of further increasing the amount of vitamin B.

[0026] In the manufacturing method of this embodiment, it is preferable to treat 1 g of grain-derived raw material with 4 U or more of arabinase and 1.6 U or more of cellulase. It is even more preferable that the concentrations of arabinase and cellulase per 1 g of grain-derived raw material are 4 to 15 U and 10 to 12.5 U, respectively. The advantage of having concentrations of arabinase and cellulase of 4 U or more and 1.6 U or more, respectively, is that the raw material is sufficiently decomposed and the amount of vitamin B increases, while the advantage of having concentrations of 15 U or less and 12.5 U or less, respectively, is that the cost used during manufacturing can be reduced. Furthermore, in the culture medium substrate containing grain-derived raw materials, the concentrations of arabinase and cellulase are preferably 250-900 mU / ml and 100-750 mU / ml, respectively.

[0027] The temperature, pH, and processing time in the treatment of grain-derived raw materials with the above-mentioned arabinase and cellulase can be appropriately determined considering the concentrations of arabinase and cellulase. The temperature is preferably 0 to 80°C, more preferably 20 to 70°C, and even more preferably 30 to 60°C. A temperature within this range has the advantage of allowing the enzyme to exhibit high activity and efficiently decompose the raw materials. The pH is preferably 2 to 8, more preferably 3 to 7, and even more preferably 4 to 6. A pH within this range has the advantage of allowing the enzyme to exhibit high activity and efficiently decompose the raw materials. The processing time is preferably 1 to 72 hours, more preferably 3 to 24 hours, and even more preferably 5 to 20 hours. A processing time of 72 hours or less has the advantage of shortening the manufacturing period. Furthermore, a processing time of 1 hour or more has the advantage of allowing the raw materials to be sufficiently decomposed. When treating grain-derived raw materials with the above-mentioned arabinase and cellulase, stirring is preferable because, for example, increasing the contact surface between the raw materials and the enzymes promotes decomposition.

[0028] When treating grain-derived raw materials with arabinase and cellulase, the order of treatment does not matter. Grain-derived raw materials treated with arabinase may be treated with cellulase, or grain-derived raw materials treated with cellulase may be treated with arabinase. Alternatively, both arabinase and cellulase may be added to the grain-derived raw materials and the enzymatic treatment may be performed simultaneously.

[0029] It is preferable that the amount of arabinan contained in the grain-derived raw material treated with arabinase and cellulase decreases by 80% by mass or more compared to the amount of arabinan in the grain-derived raw material before treatment with arabinase and cellulase, and more preferably by 90% by mass or more. A decrease of 80% by mass or more in the amount of arabinan contained in the grain-derived raw material treated with arabinase and cellulase compared to the amount of arabinan in the grain-derived raw material before treatment with arabinase and cellulase has the advantage of increasing the amount of arabinose in the culture medium, which changes the balance of sugars that can be assimilated by Aspergillus oryzae, and thereby increases the amount of vitamin B. Genes and transcription factors involved in sugar metabolism in Aspergillus oryzae are activated by various sugars (Kagaku to Seibutsu, 2019, 57(9), p532-540). Since B vitamins are required as coenzymes in glycolysis (Chemico-Biological Interactions, 2006, 163(1-2), 94-112), it is thought that the production of B vitamins necessary for activated sugar metabolism is promoted, leading to an increase in B vitamin content.

[0030] <Fermentation using Aspergillus oryzae> In the manufacturing method of this embodiment, a grain-derived raw material treated with enzymes is fermented using Aspergillus oryzae to produce an Aspergillus oryzae fermented product.

[0031] Before fermentation by Aspergillus oryzae, other sources of carbon (C) and nitrogen (N), metal ions, or defoaming agents to eliminate foam during cultivation may be added to the enzyme-treated grain-derived raw materials to create conditions conducive to vigorous growth of Aspergillus oryzae. The pH may also be adjusted to 3-7.

[0032] While there are no particular restrictions on the type of koji mold used, koji mold of the genus Aspergillus is preferred from the perspective of being a fungus used in food production. Among these, Aspergillus oryzae, Aspergillus awamori, Aspergillus inuii, Aspergillus usamii, and Aspergillus saitoi are particularly preferred.

[0033] For Aspergillus oryzae, strains ATCC1011, ATCC22788, NISL1365, NISL2074, and NISL1018 are preferred, with ATCC1011 and ATCC22788 being more preferred. For Aspergillus awamori, strain JCM22312 is preferred. For Aspergillus inuii, strain NISL1608 is preferred. For Aspergillus usamii, strain ATCC11364 is preferred. For Aspergillus saitoi, strain NISL1541 is preferred.

[0034] Strains ATCC1011, ATCC22788, and ATCC11364 are available from The Global Bioresource Center. Strain JCM22312 is available from the RIKEN (National Research and Development Institute).

[0035] Aspergillus spores are present in 1 x 10⁶ units per gram of enzyme-treated grain-derived raw material. 4 One or more, preferably 1 x 10 6 ~1 × 10 8 It is preferable to add them in such a way that they form individual particles.

[0036] The enzyme-treated grain-derived raw material may be placed in a container that can suppress contamination by harmful microorganisms, and fermentation by Aspergillus oryzae may be carried out in this container. Here, a container that can suppress contamination by harmful microorganisms can be any container that has a structure that can seal the inside of the container from the outside air. Experimentally, sterile wide-mouthed polypropylene bottles or glass media bottles can be used, while industrially, jar fermenters or pressurized fermentation tanks that have the function of supplying sterilized air into the container can be used. In addition, for air sterilization, a filter that can collect 99.97% or more of dust particles of 0.3 μm or larger, such as a HEPA filter, can be used. Furthermore, stirring is preferable when carrying out fermentation by Aspergillus oryzae.

[0037] Fermentation with Aspergillus oryzae may be carried out at 10-40°C, preferably 25-37°C, for 1-5 days, preferably 2-3 days. A fermentation temperature within this range has the advantage of promoting vigorous growth of Aspergillus oryzae. Furthermore, a fermentation time of 5 days or less has the advantage of reducing fermentation costs and environmental impact. Additionally, a fermentation time of 1 day or more has the advantage of allowing sufficient growth of Aspergillus oryzae.

[0038] Preferably, the amount of vitamin B contained in the koji mold fermented product is 150% or more by mass compared to the amount of vitamin B in the grain-derived raw material before arabinase and cellulase treatment, and more preferably 200% or more.

[0039] Furthermore, the amount of vitamin B contained in the koji mold fermented product treated with arabinase and cellulase per unit of dried koji mold fermented product is preferably 110% or more by mass, more preferably 120% or more, and particularly preferably 150% or more, compared to the amount of vitamin B contained in the koji mold fermented product not treated with enzymes such as arabinase and cellulase per unit of dried koji mold fermented product.

[0040] In the manufacturing method of this embodiment, it is preferable not to add vitamin B from an external source. This is because food additives are generally avoided by consumers. Furthermore, in practical terms, there are challenges such as an increase in the number of work steps required for vitamin B preparation and an increase in manufacturing costs due to the addition of vitamin B.

[0041] In the manufacturing method of this embodiment, post-treatments such as grinding, sterilization, disinfection, concentration, membrane separation, and drying may be performed. Examples of drying methods include freeze-drying, vacuum drying, and heat drying.

[0042] The aforementioned post-treatment may be carried out, for example, at either the enzymatic treatment or the fermentation treatment stage. Furthermore, these post-treatments may be carried out individually or in combination.

[0043] 2. Fermented products of Aspergillus oryzae derived from grains. The koji mold fermented product of this embodiment is a koji mold fermented product made from grain-derived raw materials, having an arabinan concentration of 5 mg / g or less. A koji mold fermented product refers to a product that has been fermented by koji mold.

[0044] In this embodiment, the arabinan in the koji mold fermented product may have an arabinose polymer as its main chain, and may also have other constituent sugars in its side chains or main chain.

[0045] The arabinan concentration in the koji mold fermented product of this embodiment is 5 mg / g or less. This concentration of 5 mg / g or less has the advantage of increasing the amount of vitamin B. The vitamin B content increases when grain-derived raw materials are treated with arabinase and cellulase. While the exact reason is unclear, it is presumed that the enzyme treatment alters the balance of sugars that koji mold can utilize, such as arabinose and glucose, thereby changing the metabolism of vitamin B in the koji mold. Furthermore, the increase in vitamin B, a coenzyme necessary for glycolysis, is thought to promote sugar metabolism and increase cell growth. The arabinan concentration can be measured using, for example, an arabinan measurement kit (manufactured by Megazyme).

[0046] In this embodiment, it is preferable that the amount of arabinan in the koji mold fermented product is 5 mg or less per gram of dried fermented product.

[0047] The vitamin B in the koji mold fermented product of this embodiment may be any of vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folic acid, or biotin, and these may be used individually or in combination. Of these, folic acid is particularly preferred as the vitamin B in the koji mold fermented product of this embodiment.

[0048] The vitamin B concentration in the koji mold fermented product of this embodiment is preferably 40 μg / 100g or more, more preferably 100 μg / 100g or more, and particularly preferably 120 μg / 100g or more. Here, it is preferable that the vitamin B in the koji mold fermented product of this embodiment does not contain vitamin B derived from additives. Folic acid derived from additives is of the monoglutamate type, which is different from polyglutamate type, which is naturally derived folic acid.

[0049] In this embodiment, the amount of vitamin B per 100g of dried fermented product is preferably 40μg or more, more preferably 100μg or more, and particularly preferably 120μg or more.

[0050] When the koji mold fermented product of this embodiment is a koji mold fermented product of grain-derived raw materials obtained by treating grain-derived raw materials with arabinase and cellulase, and then fermenting the treated raw materials with koji mold, it is preferable that the amount of vitamin B contained in the koji mold fermented product increases by 150% or more by mass from the amount of vitamin B in the grain-derived raw materials before treatment with arabinase and cellulase, and more preferably by 200% or more.

[0051] The vitamin B content can be measured using, for example, a folic acid testing kit (manufactured by VitaFast).

[0052] N-acetylglucosamine is formed by chitin, a component of the cell wall of Aspergillus oryzae. Since the amount of N-acetylglucosamine produced over a certain period of time is proportional to the concentration of the substrate cells, the amount of Aspergillus oryzae in the Aspergillus oryzae fermented product of this embodiment can be estimated by measuring N-acetylglucosamine.

[0053] In the koji mold fermented product of this embodiment, the ratio of vitamin B to N-acetylglucosamine is 1:1 × 10⁻¹⁶ by mass. -5 ~1:1 × 10 -3 Preferably, 1:1 × 10 -5 ~1:5×10 -4 It is more preferable that the ratio is 1:1 × 10 -5~1:3×10 -4 is more preferably. 1:(1×10 -3 or less), there is an advantage that a fermented product with a high content of vitamin B can be obtained.

[0054] The content of N-acetylglucosamine can be measured using, for example, the hydrochloric acid decomposition method, the method using Aspergillus enzyme (also referred to as "Aspergillus decomposition enzyme"), near-infrared spectroscopic analysis method, etc. In particular, the fermented product is decomposed with Aspergillus enzyme, and the released N-acetylglucosamine is measured by the method of JL Reissing et al. (Jose L. Reissing et al., “A MODIFIED COLORIMETRIC METHOD FOR THE ESTIMATION OF N-ACETYLAMINO SUGARS”, Journal of Biological Chemistry, 1955, Volume 217, Issue 2, Pages 959-966). This method is common.

[0055] The Aspergillus fermented product of the present embodiment may contain salt, umami components, etc.

[0056] The Aspergillus fermented product of the present embodiment can be used as a substitute for meat. Further, the Aspergillus fermented product of the present embodiment may be subjected to cutting processing such as chopping or dicing in advance. Further, the Aspergillus fermented product of the present embodiment is added to and kneaded with a mixture of cut processed plant foods, vegetables, and other foods, and then the kneaded product is subjected to heat cooking to obtain heat-cooked products in the form of tempura, shigure stew, hamburger, meatball, meatloaf, mentchikatsu, gyoza, shumai, wantan, spring roll, meat bun, etc. that exhibit a meaty flavor. At this time, meat may be added as other ingredients. The heat-cooked products are not limited to those described above, and examples include heat-cooked products that use soy sauce as a normal seasoning ingredient. Further, the Aspergillus fermented product of the present embodiment preferably does not contain meat.

[0057] As described above, the following matters are disclosed in this specification. In other words, the gist of the present invention is as follows: <1> A fermented product of Aspergillus oryzae derived from grain raw materials, with an arabinan concentration of 5 mg / g or less. <2> The above, where the vitamin B concentration is 40 μg / 100g or higher. <1> The koji mold fermented product described above. <3> The above vitamin B is folic acid. <2> The koji mold fermented product described above. <4> The above grain-derived raw material is a raw material derived from legume residue. <1> ~ <3> A koji mold fermented product listed in any one of the following documents. <5> The raw material derived from the bean pulp is okara. <4> The koji mold fermented product described above. <6> The koji mold mentioned above is a koji mold of the genus Aspergillus. <1> ~ <5> A koji mold fermented product listed in any one of the following documents. <7> A method for producing a fermented product of grain-derived raw materials using Aspergillus oryzae, comprising treating grain-derived raw materials with arabinase and cellulase, and then fermenting the treated raw materials using Aspergillus oryzae. <8> The arabinan concentration in the koji mold fermented product is 5 mg / g or less. <7> The manufacturing method described above. <9> The ratio of arabinase to cellulase is 1:3 to 9:1 on a unit basis, as described above. <7> or <8> The manufacturing method described above. <10> The amount of arabinan contained in the processed raw material is reduced by 70% by mass or more compared to the amount of arabinan in the grain-derived raw material before processing. <7> ~ <9> A manufacturing method described in any one of the following. <11> A fermented product of grain-derived raw materials obtained by treating grain-derived raw materials with arabinase and cellulase, and then fermenting the treated raw materials with Aspergillus oryzae. <12> The above, where the arabinan concentration is 5 mg / g or less. <11> The koji mold fermented product described above. <13> Compared to the vitamin B concentration in grain-derived raw materials before treatment with arabinase and cellulase, the vitamin B concentration increased as described above. <11> or <12> The koji mold fermented product described above. [Examples]

[0058] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0059] First, prior to the examples, the following materials were prepared.

[0060] (Grain-derived ingredients) The okara (soy pulp) used was a by-product of tofu or soy milk production. The wheat bran used was a by-product of wheat flour production. The corn flour used was POLENTA (manufactured by Nuvitality).

[0061] (enzyme) For arabinase, we used "PECLYVE FILTRATION" (manufactured by Soufflet Biotechnologies), and for cellulase, we used "CELLULYVE 50L" (manufactured by Soufflet Biotechnologies). For pectinase, we used "PECTINASE ULTRA SP-L" (manufactured by Novozyme). For mannanase, we used "Mannanase BGM "Amano" 10" (manufactured by Amano Enzyme Co., Ltd.). (others) Commercially available, top-grade reagents were used for all types of reagents.

[0062] [Preparation of culture medium substrate] In Examples 2 to 8 described later, a culture medium suitable for the growth of Aspergillus oryzae was prepared by enzymatically treating grain-derived raw materials using the following procedure.

[0063] 6 g of dried grain-derived raw material was added to 100 mL of distilled water and autoclaved at 121°C for 30 minutes. The enzyme solution, filtered through a 0.22 μm filter, was added to the grain-derived raw material solution to a final concentration of 1 U / ml, and the mixture was stirred overnight at 55°C. After the reaction, the mixture was sterilized at 121°C for 30 minutes to obtain the culture medium substrate. The types of grain-derived raw materials and enzymes used are as described in each example.

[0064] [Preparation of fermented products] In Examples 2-5, 7, and 8 described later, the fermented product was prepared by the following procedure.

[0065] Maltodextrin agar (Sigma) was inoculated with one of the Aspergillus oryzae shown in Table 1 to obtain spores. The spores were suspended in a surfactant solution (Tween20 0.01%) to a final concentration of 5.0 × 10⁶. 5 The culture medium substrate was inoculated to a concentration of cells / ml, and cultured with shaking (30°C, 180 rpm) for 2 days. After culturing, the fermented product was heat-sterilized (80°C, 20 minutes). Subsequently, the fermented product was homogenized using a homogenizer (VIOLAMO) and freeze-dried. The types of Aspergillus oryzae used are as described in each example.

[0066] [Table 1]

[0067] [Measurement of N-acetylglucosamine content in fermented products] In Examples 2 to 4 described later, the amount of N-acetylglucosamine was measured by the following procedure.

[0068] 40 mg of the dried ferment was suspended in 5 ml of 20 mM phosphate buffer (pH 7.0), and the supernatant was removed by centrifugation (3500 rpm, 10 minutes). This washing procedure was repeated three times, and then the mixture was suspended in 2 mg / ml Yatalase (Takara Bio Inc.) solution and reacted at 37°C for 3 hours. After the reaction, the supernatant was collected by centrifugation (3500 rpm, 10 minutes) and used to measure the amount of N-acetylglucosamine.

[0069] N-acetylglucosamine was measured according to the method of JL Reissing et al. Standard solutions of N-acetylglucosamine were prepared at concentrations of 10, 50, 100, and 250 μg / ml. 20 μl of 0.8 M boric acid solution (pH 9.1) was added to 200 μl of each standard solution, mixed, and reacted in a boiling bath for 3 minutes. After cooling the samples, 1.2 ml of 100 mg / ml para-dimethylaminobenzaldehyde (hydrochloric acid:acetic acid = 1:7) solution was added and mixed, and the mixture was reacted at 37°C for 20 minutes. After the reaction, the absorbance at 585 nm was measured to create a calibration curve. The samples from each test example obtained in the following examples were similarly colored, and the amount of N-acetylglucosamine was determined based on the calibration curve.

[0070] [Measurement of folic acid content in grain-derived raw materials and fermented products] In Examples 1 to 5 described later, the amount of folic acid was measured using the following procedure. Folic acid content was measured by microbiological quantification using 1 g of dried grain-derived raw material or fermented product. Preparation was carried out according to the method of the folic acid measurement kit (manufactured by VitaFast), and after reaction at 37°C for 2 days, the absorbance at 340 nm was measured.

[0071] <Example 1> [Evaluation of folic acid content in raw materials derived from various grains] Folic acid content was measured in okara (soy pulp), wheat bran, and corn flour, which were grain-derived raw materials that had not undergone enzymatic reaction or koji mold fermentation, as shown in Test Examples 1-3 in Table 2. Among the evaluated grain-derived raw materials, it was found that okara contained the most folic acid.

[0072] [Table 2]

[0073] <Example 2> [Evaluation of growth rate and folic acid content in okara culture medium treated with various glycosphagnum enzymes] Using a culture medium prepared with okara (soy pulp) as the grain-derived raw material, test examples 5-9 were treated with the respective glycosphagocytes shown in Table 3. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. Subsequently, the NISL1018 strain was inoculated and cultured.

[0074] [Table 3]

[0075] The N-acetylglucosamine and folic acid content of the obtained Aspergillus oryzae fermentation product was evaluated. The results are shown in Figures 1A and 1B. As a result, an increase in N-acetylglucosamine levels was confirmed in Test Example 7, which was treated with arabinase, and in Test Example 8, which was treated with cellulase. Furthermore, a further increase in N-acetylglucosamine levels was confirmed in Test Example 9, which was treated with both arabinase and cellulase. In addition, a significant increase in folic acid levels was observed in Test Example 9.

[0076] This indicates that treating grain-derived raw materials with arabinase and cellulase increases the amount of microbial cells and folic acid in the fermented product.

[0077] <Example 3> [Evaluation of growth rate and folic acid content in each Aspergillus species] Using a culture medium prepared with okara (soy pulp) as a grain-derived raw material, test examples 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, and 36 were treated with the respective sugar-degrading enzymes shown in Tables 4 and 5. The enzyme concentrations are the final concentrations after adding the enzyme solution to the grain-derived raw material solution. Subsequently, the Aspergillus oryzae shown in Tables 4 and 5 were inoculated and cultured.

[0078] [Table 4]

[0079] [Table 5]

[0080] The results of evaluating the N-acetylglucosamine and folic acid content in koji mold fermented using Aspergillus oryzae, as listed in Table 4, are shown in Figures 2A and 2B, respectively. It was confirmed that treatment with arabinase and cellulase in each strain increased the amount of N-acetylglucosamine and folic acid per unit of dried koji mold ferment compared to the untreated condition.

[0081] Furthermore, Figures 3A and 3B show the results of evaluating the amount of N-acetylglucosamine and folic acid in koji mold fermented using Aspergillus awamori, Aspergillus inui, Aspergillus usami, or Aspergillus cytoi, as listed in Table 5. It was confirmed that treatment with arabinase and cellulase increased the amount of N-acetylglucosamine and folic acid per unit of dried koji mold ferment compared to the untreated condition.

[0082] <Example 4> [Evaluation of Aspergillus oryzae growth and folic acid content in raw materials derived from various grains] As shown in Table 6, culture media prepared using okara, wheat bran, or corn flour as grain-derived raw materials were used as substrates, and test examples 38, 40, and 42 were treated with arabinase and cellulase, respectively. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. Subsequently, culture was performed using the ATCC1011 strain.

[0083] [Table 6]

[0084] The results of evaluating the N-acetylglucosamine and folic acid content in Aspergillus oryzae fermented products are shown in Figures 4A and 4B, respectively. In all Aspergillus oryzae fermented products using grain-derived raw materials, treatment with arabinase and cellulase increased the amount of N-acetylglucosamine per unit of dried Aspergillus oryzae fermented product compared to the untreated condition. Furthermore, in all Aspergillus oryzae fermented products using grain-derived raw materials, treatment with arabinase and cellulase increased the amount of folic acid compared to the untreated condition.

[0085] This indicates that when grain-derived raw materials containing arabinan are used as culture medium substrates, treatment with arabinase and cellulase increases the amount of folic acid in the Aspergillus oryzae fermented product compared to when these enzymes are not used.

[0086] <Example 5> [Effect of enzyme ratio on folic acid levels] To confirm the effects of different arabinase and cellulase ratios on the amount of Aspergillus oryzae and folic acid in the fermented product, culture media prepared using okara (soy pulp) as the grain-derived raw material were used as substrates. Tests 44-48 and 50-54 were treated with the enzymes shown in Table 7. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. Subsequently, culture was performed using either strain ATCC1011 or strain ATCC22788.

[0087] [Table 7]

[0088] Figure 5A shows the results of evaluating the amount of folic acid in Aspergillus oryzae fermented with strain ATCC1011, and Figure 5B shows the results of evaluating the amount of folic acid in Aspergillus oryzae fermented with strain ATCC22788. The folic acid value in Aspergillus oryzae fermented with arabinase alone is set to 1, and the relative values ​​for the other test examples are shown. In all test examples with unit-based ratios of arabinase to cellulase of 1:3, 3:1, and 9:1, the amount of folic acid increased compared to the test examples treated with arabinase alone or cellulase alone. From this, it was confirmed that treatment with enzymes in a unit-based ratio of arabinase to cellulase of 1:3 to 9:1 increases the amount of folic acid in Aspergillus oryzae fermented products.

[0089] <Example 6> [Glucose level measurement] Since cellulase treatment breaks down cellulose in the substrate into glucose, the glucose concentration before culturing was measured to observe the effect of the glucose level before culturing on the folic acid level.

[0090] As a substrate, a culture medium prepared using okara (soy pulp) as a grain-derived raw material was used, and test examples 56-60 were treated with the enzymes shown in Table 8. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. 0.2 ml of the enzyme-treated solution was taken and centrifuged at 14,000 rpm for 10 minutes, and the supernatant was collected. The glucose concentration of the obtained supernatant was measured using a YSI Biochemistry Analyzer (YSI Life Sciences).

[0091] [Table 8]

[0092] Figure 6 shows the glucose concentrations for each test example. It was confirmed that the glucose concentration increased when enzymatic treatment was performed with both arabinase and cellulase compared to when only arabinase treatment was performed. This indicates that arabinase and cellulase treatment increases the glucose concentration in the culture medium. Glucose is one of the sugars that Aspergillus oryzae can assimilate, and it is presumed that a change in the balance of assimilated sugars alters the metabolism of vitamin B in Aspergillus oryzae, leading to an increase in vitamin B in the Aspergillus oryzae fermented product.

[0093] <Example 7> [Arabinane content measurement] As a grain-derived raw material, a culture medium prepared using okara (soy pulp) was used as the substrate, and test examples 62-67 were treated with the enzymes shown in Table 9. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. Subsequently, test examples 65-67 were cultured using the ATCC22788 strain.

[0094] The amount of arabinane in test examples 61-67 was measured. An arabinane testing kit (manufactured by Megazyme) was used to measure the amount of arabinane. In Test Examples 61-64, the supernatant of the culture medium was collected as a non-fermented sample, and the filtrate containing arabinan, obtained by removing free arabinose by passing it through the attached PD-10 column, was used as the sample. In Test Examples 65-67, the lyophilized fermentation product was suspended in distilled water and similarly passed through the PD-10 column for use.

[0095] The kit contains endoarabinase, α-L-arabinofuranosidase, and galactose mutarotase. These enzymes convert arabinan to β-L-arabinose. NAD +In the presence of β-galactose dehydrogenase, β-L-arabinose is converted to L-arabinonic acid and NADH. The amount of free NADH was measured by measuring the absorbance at 340 nm, and the arabinan concentration was quantified. The measured arabinan concentrations are shown in Table 9. In Table 9, the arabinan concentrations for Test Examples 61-64 are shown as the weight of arabinan per unit weight of dried raw material, and for Test Examples 65-67, the weight of arabinan per unit weight of dried Aspergillus oryzae fermented product.

[0096] [Table 9]

[0097] In Test Example 61, where neither arabinase nor cellulase enzymatic reactions were performed and no fermentation by Aspergillus oryzae was carried out, the arabinan concentration was 38.0 mg / g per unit weight of the raw material. On the other hand, in Test Example 67, where the material was treated with 1 U / ml of arabinase and fermented with Aspergillus oryzae, the amount of arabinan was 3.7 mg / g per unit weight of the dried Aspergillus oryzae fermented product. Furthermore, in Test Example 65, where the arabinase to cellulase ratio was 1:3 on a unit basis and fermented with Aspergillus oryzae, the amount of arabinan was 5.1 mg / g per unit weight of the dried Aspergillus oryzae fermented product. In Test Example 66, where the arabinase to cellulase ratio was 9:1 on a unit basis and fermented with Aspergillus oryzae, the amount of arabinan was 3.1 mg / g per unit weight of the dried Aspergillus oryzae fermented product. From this, it was confirmed that the amount of arabinan per unit weight of the dried Aspergillus oryzae fermented product using enzyme-treated okara (soy pulp) is between 3.1 mg / g and 5.1 mg / g.

[0098] <Example 8> [Measurement of culture viscosity] A culture medium prepared using okara (soy pulp) as a grain-derived raw material was used as a substrate and treated with the enzymes shown in Table 10. The enzyme concentration is the final concentration after adding the enzyme solution to the grain-derived raw material solution. Subsequently, culture was performed using the ATCC22788 strain.

[0099] [Table 10]

[0100] The viscosity of the cultured sample was measured. For viscosity measurement, 50 ml of the culture collected from a 100 ml flask was used, and a viscometer (TVB-10M, manufactured by Toki Sangyo Co., Ltd.) was used. A rotor of type M3 was used, and the stirring speed was 60 rpm. The results are shown in Figure 7.

[0101] Compared to test example 68, which did not undergo enzyme treatment, the viscosity increased in test example 70, which was treated with cellulase, but it was confirmed that the viscosity did not increase significantly in test example 71, which was treated with both arabinase and cellulase. The fact that the viscosity does not increase is an advantage in terms of reducing the yield and ease of handling when recovering the final product of the fermentation. [Industrial applicability]

[0102] The koji mold fermented product of this embodiment can be used as a meat substitute or other food ingredient.

[0103] Although various embodiments have been described above, it goes without saying that the present invention is not limited to these examples. It is clear to those skilled in the art that various modifications or alterations can be conceived within the scope of the claims, and these will naturally also fall within the technical scope of the present invention. Furthermore, the components in the above embodiments may be combined in any way without departing from the spirit of the invention.

[0104] This application is based on the Japanese Patent Application No. 2022-085521 filed on May 25, 2022, and its contents are incorporated herein by reference.

Claims

1. A koji mold fermented product made from grain-derived raw materials, wherein the vitamin B concentration is 40 μg / 100g or more, the grain-derived raw material is derived from legume pulp, and the koji mold is Aspergillus oryzae.

2. The koji mold fermented product according to claim 1, wherein the vitamin B is folic acid.

3. The koji mold fermented product according to claim 1, wherein the arabinan concentration is 5 mg / g or less.

4. The koji mold fermented product according to claim 1, wherein the raw material derived from the bean pulp is okara.

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