Fermented seasonings

Abstract

To provide a natural matter-derived fermented seasoning which can be added to a food and drink, causing the food and drink to have a better flavor.SOLUTION: It has been found that a liquid raw material for fermentation including at least one of leucine or valine can be fermented with Bacillus subtilis to produce a Bacillus subtilis fermented seasoning including at least one of isovaleric acid or isobutyric acid.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a Bacillus subtilis fermented seasoning, a method for producing the same, a food or drink added with the fermented seasoning, and a method for producing the same, etc.

Background Art

[0002] A fermented seasoning using a spore-forming ability-deficient strain of Bacillus subtilis is disclosed in Patent Document 1. Further, in Patent Document 2, saccharides are added to soy milk, natto bacteria or completed natto is added thereto, and dehydration treatment is performed at the stage when the soy milk has finished aggregating, and if necessary, the water content is further reduced to produce a plant-based dairy product using soy milk as a basic raw material, which is made from natto or a combination of natto bacteria and soy milk. A method for making it is disclosed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention provides a fermented seasoning derived from natural products that can be used to produce a food or drink with a better flavor by adding it to the food or drink.

Means for Solving the Problems

[0005] The inventors have found that a Bacillus subtilis fermented seasoning containing at least one of isovaleric acid or isobutyric acid can be produced by fermenting a liquid raw material for fermentation containing at least one of leucine or valine with Bacillus subtilis, and have completed the present invention.

[0006] That is, the present invention relates to the following aspects [1] to [8]. [1] A Bacillus subtilis fermented seasoning comprising a Bacillus subtilis fermented product obtained by fermenting a liquid raw material for fermentation containing one or more of leucine or valine with Bacillus subtilis, A Bacillus subtilis fermented seasoning containing either isovaleric acid or isobutyric acid, with the total amount of isovaleric acid and isobutyric acid being 0.15% by weight or more based on the solid content of the Bacillus subtilis fermented seasoning. [2] The Bacillus subtilis fermented seasoning according to [1], characterized by using a liquid raw material for fermentation containing a total of 0.01% by weight or more of leucine and valine. [3] The Bacillus subtilis fermented seasoning according to [1] or [2], which is a Bacillus subtilis fermented product obtained by fermenting with Bacillus subtilis and then enzymatically treating it with an enzyme having lipase activity. [4] A method for producing a Bacillus subtilis fermented seasoning, comprising the step of fermenting a liquid raw material for fermentation containing one or more of leucine or valine with Bacillus subtilis to obtain a Bacillus subtilis ferment product, A method for producing a Bacillus subtilis fermented seasoning, comprising one or more isovaleric acid or isobutyric acid, wherein the total amount of isovaleric acid and isobutyric acid is 0.15% by weight or more based on the solid content of the Bacillus subtilis fermented seasoning. [5] A method for producing a Bacillus subtilis fermented seasoning according to [4], characterized by using a fermentation liquid raw material containing 0.01% by weight or more of leucine and valine in total. [6] A method for producing a Bacillus subtilis fermented seasoning according to [4] or [5], comprising the step of further treating a Bacillus subtilis fermented product with an enzyme having lipase activity. Food and beverages containing isovaleric acid and isobutyric acid in total at a rate of 0.0001% by weight or more per solid content of the food and beverage, with the addition of a Bacillus subtilis fermented seasoning as described in any of [7][1] to [3]. A method for producing food and beverages, comprising adding a Bacillus subtilis fermented seasoning described in any of [8][1] to [3] to food and beverages such that the total amount of isovaleric acid and isobutyric acid is 0.0001% by weight or more per unit of solid content of the food and beverage. [Effects of the Invention]

[0007] The present invention provides a naturally derived Bacillus subtilis fermented seasoning that can impart a fermented flavor and reduce unpleasant odors in food and beverages. By adding this seasoning, food and beverages can be made to have a better flavor and be more delicious. [Modes for carrying out the invention]

[0008] The Bacillus subtilis fermented seasoning of the present invention can be produced by a manufacturing method that includes the step of fermenting a fermentation liquid raw material containing one or more of leucine or valine with Bacillus subtilis to obtain a Bacillus subtilis ferment product, and may further include a step of enzymatic treatment using an enzyme having lipase activity, and the obtained Bacillus subtilis ferment product contains one or more of isovaleric acid or isobutyric acid and can be used as a Bacillus subtilis fermented seasoning.

[0009] The Bacillus subtilis described in this invention is not particularly limited as long as it is Bacillus subtilis, but natto bacteria such as Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC3335, and Bacillus subtilis NBRC13169 are preferred, and can be obtained from the National Institute of Technology and Evaluation, etc., and spore-forming-deficient strains are preferred.

[0010] The aforementioned spore-forming ability-deficient strain can be obtained by mutation, such as by genetic recombination or spontaneous mutation, but spontaneous mutation is preferred. The method for obtaining a spore-forming ability-deficient strain by spontaneous mutation is not particularly limited, and examples include high-temperature culture, a random method for distinguishing wild-type and deficient strain colonies by the coloration of melanin pigment, and a method utilizing a catabolite repression-like phenomenon (JFMichel, B.Cami, P.Schaeffer: Ann.Inst.Pasteur, 114, 11; 21 (1968)), but the method utilizing a catabolite repression-like phenomenon is preferred (see Japanese Patent Publication No. 6019528). Spore-forming ability-deficient strains obtained by the method utilizing a catabolite repression-like phenomenon lack spore-forming ability, lysozyme activity, and transformation ability, and can be subcultivated without problems due to lysis, thus maintaining the trait of spore-forming ability deficiency. Using a spore-forming-deficient strain allows for sterilization under mild conditions below 100°C, thus preventing contamination of manufacturing equipment due to insufficient sterilization, a problem with spore-forming strains, and facilitating addition to various foods and formulations.

[0011] The fermentation liquid raw material used in this invention may be any liquid containing components that allow Bacillus subtilis to grow, and may be a liquid culture medium in which a carbon source, nitrogen source, inorganic substances, etc. are dissolved. As a carbon source, organic acids such as citric acid, sugars such as glucose, sucrose, dextrin, and starch, sugar alcohols such as glycerin and mannitol, molasses, etc., can be used, as well as plant extracts containing sugars, sugar alcohols, or citric acid, such as brown algae extracts such as kelp extract, mushroom extract, fruit juices of aromatic citrus fruits such as lemon, lime, shikwasa, yuzu, sudachi, kabosu, and bitter orange, fruit juices of fruits belonging to the Prunus subgenus such as plum, apricot, and Japanese apricot, etc., and fruit juices containing citric acid can be used, and one or more of these may be used. As a nitrogen source, inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, and ammonium phosphate, nitrogen-containing natural products such as peptone, meat extract, yeast extract, malt extract, and corn steep liquor can be used. Examples of inorganic substances include monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and ferric chloride, and components found in natural products can be utilized.

[0012] The present invention provides a fermented seasoning that can be obtained by using a fermentation liquid raw material containing one or more of leucine or valine. A fermentation liquid raw material to which one or more of leucine or valine has been added, or a culture medium component containing one or more of leucine or valine in a relatively large amount, such as an amino acid mixture, a protein hydrolysate, or a culture medium combining these, can be used. The content of leucine or valine in the fermentation liquid raw material is not particularly limited as long as the fermented seasoning of the present invention can be obtained. However, when the fermentation liquid raw material containing one or more of leucine or valine is considered to be 100% by weight, it is preferable that the total amount of leucine and valine be 0.01% by weight or more, more preferably 0.02 to 10% by weight, even more preferably 0.04 to 8% by weight, particularly preferably 0.08 to 5% by weight, and most preferably 0.1 to 2% by weight. Leucine or valine may be in L-form, D-form or racemic form, but the L-form is preferred.

[0013] The fermentation conditions in this invention can be set as appropriate, but aerobic liquid fermentation by aeration, shaking, stirring, etc. is preferred. The fermentation temperature can be exemplified as 10 to 50°C, preferably 20 to 45°C. The fermentation time can be exemplified as 2 to 72 hours, preferably 4 to 48 hours, and more preferably 6 to 36 hours. The pH can be exemplified as 4.0 to 9.0, and preferably 5.0 to 8.5. The pH may be adjusted during fermentation within the above range, preferably in the range of 5.5 to 8.0, and more preferably in the range of 6.0 to 7.5. For pH adjustment, organic acids such as citric acid, sodium hydroxide, ammonia, etc., can be used.

[0014] The manufacturing method of the present invention may include a step of further enzymatic treatment of a Bacillus subtilis fermentation product fermented with Bacillus subtilis using an enzyme having lipase activity, and the enzymatic treatment can be carried out in parallel with or after the fermentation, and an enzyme-treated Bacillus subtilis fermentation product can be obtained. The enzymatic treatment can be carried out by adding an enzyme having lipase activity during or after fermentation, but it is preferable to add ethanol and carry out the enzymatic treatment in the presence of ethanol. The ethanol concentration should be such that the enzyme acts appropriately, but if the ethanol concentration is too high, the enzyme activity tends to decrease, so it is preferable to contain 0.01 to 60% by weight, more preferably 0.1 to 50% by weight, even more preferably 0.3 to 40% by weight, and particularly preferably 0.5 to 30% by weight, when the total liquid during enzymatic treatment is considered as 100% by weight.

[0015] In the present invention, the enzyme having lipase activity is not particularly limited as long as it is an enzyme having lipase activity that can be used in food processing, and may contain the enzyme, or an enzyme preparation may be used. Examples of enzyme preparations include the lipase preparations Lipase OF (manufactured by Meito Sangyo Co., Ltd.), Lipase A "Amano" 6 (manufactured by Amano Enzyme Co., Ltd.), and Lilipase® A-10D (manufactured by Nagase ChemteX Corporation). The amount of enzyme added can be set as appropriate, but when the total liquid before enzyme addition is considered to be 100% by weight, examples of enzyme preparation addition amounts can be 0.0005 to 2.0% by weight, preferably 0.001 to 1.0% by weight, and more preferably 0.002 to 0.5% by weight.

[0016] The enzyme treatment conditions in this invention are not particularly limited as long as the conditions allow the enzymatic reaction to proceed. For example, the treatment temperature can be 10 to 70°C, preferably 15 to 60°C, more preferably 20 to 50°C. The treatment time can be 10 minutes to 24 hours, preferably 20 minutes to 12 hours, more preferably 30 minutes to 6 hours. The pH conditions can be set appropriately considering the optimal pH of the enzyme, pH stability, etc. For example, pH 3.0 to 9.0 can be exemplified, with pH 3.5 to 8.0 being preferred. Before enzyme treatment, the pH can be adjusted to this range using organic acids such as citric acid, sodium hydroxide, ammonia, etc. Neutralization may also be performed after enzyme treatment.

[0017] In the present invention, heating is preferable after fermentation, or after fermentation and enzyme treatment, as heating can sterilize and / or deactivate the enzymes. The heating conditions are not particularly limited as long as they are general sterilization or enzyme deactivation methods, but heating at 60-120°C for 1-30 minutes or 80-100°C for 5-20 minutes is preferable, for example. Furthermore, the product may be powdered by spray drying, vacuum and / or freeze-drying, and general excipients can be used.

[0018] The Bacillus subtilis fermented seasoning of the present invention is a Bacillus subtilis fermented seasoning comprising a Bacillus subtilis fermented product obtained by fermenting a fermentation liquid raw material containing one or more leucine or valine with Bacillus subtilis, and may also be a Bacillus subtilis fermented seasoning comprising a Bacillus subtilis fermented product obtained by further enzymatic treatment of the Bacillus subtilis fermented product with an enzyme having lipase activity, and contains one or more isovaleric acid or isobutyric acid, and the total amount of isovaleric acid and isobutyric acid is 0.15% by weight or more, preferably 0.2 to 50% by weight, more preferably 0.5 to 40% by weight, even more preferably 1.0 to 30% by weight, and particularly preferably 2.0 to 20% by weight, based on the solid content of the Bacillus subtilis fermented seasoning.

[0019] The Bacillus subtilis fermented seasoning of the present invention can impart a fermented flavor when added to various food and beverage products, and food and beverage products of the present invention with better flavor can be obtained. Further, it has the effect of enhancing the flavor of food and beverage products, is useful for enhancing the flavor of food and beverage products, and in particular, can be used for enhancing the fermented flavor of fermented food and beverage products. In addition, since it can reduce the unpleasant taste of food and beverage products, it is also useful for masking, and in particular, can be used for masking the smell of vegetable protein. The food and beverage products to be added are not particularly limited, and examples include seasonings such as miso, sauce, dressing, and tare, beverages such as non-alcoholic beverages and alcoholic beverages, liquid foods such as soup, functional foods, etc. Also, protein supplements, protein bars, protein jelly, etc. for protein supplementation using vegetable protein, beverages such as soy milk, soups containing vegetable protein, meat-like foods, plant protein-containing food and beverage products such as imitation dairy products, etc. can be exemplified. While suppressing the unpleasant taste of food and beverage products, a fermented flavor can be imparted, so the flavor of food and beverage products can be enhanced, and more delicious food and beverage products can be provided. Further, the addition to fermented food and beverage products can be used for enhancing yogurt flavor, cheese flavor, flavor enhancement of pickles such as kimchi and pickled Chinese cabbage, flavor enhancement of fermented seasonings such as miso, flavor enhancement of natto flavor, and flavor enhancement of brewing liquors such as the aroma of ginjo sake of Japanese sake. The content of the Bacillus subtilis fermented seasoning in the food and beverage products added with the Bacillus subtilis fermented seasoning of the present invention is not particularly limited as long as more delicious food and beverage products can be obtained, but it is preferably contained at 0.01 to 40% by weight in the food and beverage products, more preferably 0.05 to 20% by weight, and even more preferably 0.1 to 10% by weight.

[0020] The food and beverage products of the present invention added with the Bacillus subtilis fermented seasoning of the present invention contain any one or more of isovaleric acid or isobutyric acid, and the total of isovaleric acid and isobutyric acid is 0.0001% by weight or more per solid content of the food and beverage products, preferably 0.0005 to 20% by weight, more preferably 0.001 to 10% by weight, and even more preferably 0.005 to 5% by weight.

Examples

[0021] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited by the following examples. In the present invention, % is all weight % unless otherwise specified.

Example

[0022] From Bacillus subtilis NBRC3335, a kind of Bacillus subtilis, by natural mutation utilizing the phenomenon of catabolite repression, Bacillus subtilis lacking the ability to form spores: Bacillus subtili TIFF0007899999000001.tif6170 was obtained by the method described in the examples of Japanese Patent No. 6019528, and it was confirmed that the strain lacked the ability to form spores by the method described in the publication.

[0023] 24 g (2.0%) of 5-fold concentrated transparent lemon juice (manufactured by Nippon Fruit Processing Co., Ltd.), 12 g (1.0%) of yeast extract (Mist (registered trademark) P1G, manufactured by Asahi Group Foods Co., Ltd.), 6 g (0.5%) of L-leucine (food additive), and 1,158 g of tap water were mixed, adjusted to pH 6.0 with a 20% aqueous sodium hydroxide solution, heat-sterilized at 121 °C for 15 minutes, and then cooled. The above-mentioned Bacillus subtilis was inoculated into the liquid fermentation raw material, and aerated fermentation was carried out at 35 °C and pH 7.5 for 20 hours to obtain 1,200 g of Bacillus subtilis fermented product (Product 1-1: solid content 2.18%).

[0024] 24 g (2.0%) of kelp stock solution CIE-8828 (manufactured by Ikeda Sugar Industry Co., Ltd.), 12 g (1.0%) of yeast extract (Mist (registered trademark) P1G), 6 g (0.5%) of L-valine (food additive), and 882 g of tap water were mixed, adjusted to pH 6.0 with a 20% aqueous sodium hydroxide solution, heat-sterilized at 121 °C for 15 minutes, and then cooled. The above-mentioned Bacillus subtilis was inoculated into the liquid fermentation raw material, and aerated fermentation was carried out at 35 °C and pH 6.0 for 20 hours to obtain 1,200 g of Bacillus subtilis fermented product (Product  1-2: solid content 1.59%).

[0025] Mix 24 g (2.0%) of 5-fold concentrated transparent lemon juice, 12 g (1.0%) of yeast extract (Mist (registered trademark) P1G), 2.4 g (0.2%) of L-leucine (food additive), 2.4 g (0.2%) of L-valine (food additive) and 1,159.2 g of tap water. After adjusting the pH to 6.0 with 20% sodium hydroxide aqueous solution, heat and sterilize at 121 °C for 15 minutes and then cool. Inoculate the obtained liquid raw material for fermentation with the said Bacillus subtilis and perform aeration fermentation at 30 °C and pH 7.0 for 24 hours to obtain 1,200 g of Bacillus subtilis fermented product (Product 1-3: solid content 1.60%).

[0026] Filter the Bacillus subtilis fermented products of Products 1-1 to 1-3 with a cellulose acetate filter (pore size 0.45 μm). For the samples fluorescently labeled with a fluorescent reagent for fatty acid analysis (ADAM, manufactured by Funakoshi Corporation), measure the concentrations of isobutyric acid and isovaleric acid under the following HPLC conditions and show them in Table 1.

[0027] <HPLC measurement conditions> · Detector: Fluorescence detector (excitation wavelength: 365 nm, fluorescence wavelength 412 nm) · Column: InertSustain C18 (inner diameter 4.6 mm, length 250 mm) · Mobile phase: 80% by volume acetonitrile aqueous solution · Flow rate: 1.0 ml / min · Column temperature: 40 °C · Standard: Dissolve isobutyric acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) and isovaleric acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) in methanol to prepare a calibration curve.

[0028]

Table 1

[0029] [Evaluation test] Each of the Bacillus subtilis fermented products (1-1 to 1-3) or the comparative product was added to soy milk at a concentration of 5%, and a sensory evaluation was conducted by a panel of three trained individuals. The results are shown in Table 2. The sensory evaluation assessed the presence or absence of fermented flavor or soybean odor. If 3 people judged the presence of fermented flavor to be "present," it was marked as "○," 2 as "△," and 0-1 as "×." If 3 people judged the absence of soybean odor to be "absent," it was marked as "○," 2 as "△," and 0-1 as "×." The results are shown in Table 2. In addition, as an overall evaluation of the soy milk after the addition of each sample, those that were more delicious were marked as "○," and those that were not more delicious were marked as "×," as shown in Table 2.

[0030] Furthermore, comparative product 1-1 was prepared by diluting the reagent isobutyric acid and isovaleric acid with the fermentation liquid raw material of Example 1-1 to achieve the isobutyric acid and isovaleric acid concentrations of product 1-1. Comparative product 1-2 was prepared by diluting the reagent isobutyric acid and isovaleric acid with the fermentation liquid raw material of Example 1-2 to achieve the isobutyric acid and isovaleric acid concentrations of product 1-2. Comparative product 1-3 was prepared by diluting the reagent isovaleric acid and isovaleric acid with the fermentation liquid raw material of Example 1-3 to achieve the isobutyric acid and isovaleric acid concentrations of product 1-3.

[0031] [Table 2]

[0032] As shown in Table 2, all of the Bacillus subtilis fermented products 1-1 to 1-3, when added to soy milk, were able to suppress the unpleasant soy odor and impart a fermented flavor. However, when aqueous solutions of comparative products 1-1 to 1-3, which contained similar levels of isobutyric acid or isovaleric acid, were added, they were unable to impart a fermented flavor or suppress the unpleasant soy odor.

[0033] Therefore, simply adding isobutyric acid or isovaleric acid to soy products did not result in a more delicious soy milk compared to the original soy milk, as it did not produce a fermented flavor and retained an unpleasant odor derived from the soy products. However, by adding the Bacillus subtilis fermented product of the present invention, which contains isobutyric acid and / or isovaleric acid, it was found that the soy milk could be made more delicious than the original soy milk, demonstrating the usefulness of the Bacillus subtilis fermented product as a Bacillus subtilis fermented seasoning. [Examples]

[0034] The Bacillus subtilis fermented product of test 1-1 was adjusted to pH 4.0 with a 50% citric acid aqueous solution, then 0.05% of the lipase preparation Lipase OF (manufactured by Meito Sangyo Co., Ltd.) was added (Example 2-1), or 0.05% of lipase and 20% 95% ethanol were added (Example 2-2). After enzymatic treatment at 40°C for 1 hour, the product was heat-treated at 90°C for 10 minutes to obtain the enzymatically treated Bacillus subtilis fermented products of test 2-1 and 2-2.

[0035] [Evaluation Test] Enzyme-treated Bacillus subtilis fermented products 2-1 and 2-2, or comparative product 1-1, were each added to soy milk at a concentration of 5%, and sensory evaluations were conducted by a trained panel of three people. The results are shown in Table 3. The sensory evaluation assessed the presence or absence of a fruity fermented flavor or soybean odor. The number of people who judged the fruity fermented flavor to be "present" was rated as "○", 2 as "△", and 0-1 as "×". The number of people who judged the soybean odor to be "absent" was rated as "○", 2 as "△", and 0-1 as "×". The results are shown in Table 2.

[0036] [Table 3]

[0037] As shown in Table 3, both of the enzyme-treated Bacillus subtilis fermented products of test 2-1 and 2-2, when added to soy milk, were able to suppress the soy odor and impart a fermented flavor with a fruity aroma.

[0038] Therefore, it was found that by further enzymatic treatment of the Bacillus subtilis fermented product with an enzyme having lipase activity, either in the presence or absence of ethanol, it is possible to create a fermented flavor that has a masking effect on the soybean odor and a fruity aroma. Enzymatic treatment gives the Bacillus subtilis fermented product an even more desirable flavor, making it a Bacillus subtilis fermented seasoning that can be imparted to the product.

Claims

1. A method for producing a Bacillus subtilis fermented seasoning, comprising the steps of preparing a liquid raw material for fermentation by adding one or more of leucine or valine to a liquid culture medium in which a carbon source, a nitrogen source, and an inorganic substance are dissolved, and obtaining a Bacillus subtilis fermented product by inoculating the liquid raw material for fermentation with Bacillus subtilis and performing aerobic liquid fermentation for 6 to 36 hours, A method for producing a Bacillus subtilis fermented seasoning, comprising one or more isovaleric acid or isobutyric acid, wherein the total amount of isovaleric acid and isobutyric acid is 0.15% by weight or more based on the solid content of the Bacillus subtilis fermented seasoning.

2. The method for producing a Bacillus subtilis fermented seasoning according to Claim 1, characterized in that, when the liquid raw material for fermentation is 100% by weight, leucine and valine are added in a total amount of 0.01% by weight or more.

3. A method for producing a Bacillus subtilis fermented seasoning according to claim 1 or 2, comprising the step of further enzymatically treating a Bacillus subtilis fermented product obtained by fermenting with Bacillus subtilis using an enzyme having lipase activity.

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