Antifungal agent for grain processed foods

Abstract

To provide an antifungal agent containing an enzyme, which shows antifungal properties while improving the flavor of processed grain food.SOLUTION: An antifungal agent for processed grain food contains exomaltotetraohydrolase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antifungal agent for processed cereal foods, a method for inhibiting mold growth in processed cereal foods using the same, a method for producing processed cereal foods, and processed cereal foods. The present invention also relates to processed cereal foods in which the amount of nonenzymatic antifungal agent used is reduced, and a method for reducing the amount of nonenzymatic antifungal agent used in processed cereal foods. [Background technology]

[0002] In order to extend the shelf life of foods, it is necessary to suppress the growth of microorganisms, especially mold. It is known that chemical substances such as calcium propionate have antifungal properties, but they can deteriorate the flavor of foods, and there is strong consumer resistance to adding chemical substances.

[0003] It is also known to use enzymes as naturally occurring antifungal agents. Patent Document 1 discloses an antifungal agent for food containing chitinase.

[0004] On the other hand, exomaltotetrahydrolase (G4 synthase) is an exo-type amylase that cleaves oligosaccharides in maltotetraose units from the non-reducing end of starch, and is used as an enzyme to produce maltooligosaccharides in the starch saccharification industry. Patent Document 2 discloses that exomaltotetrahydrolase has the effect of preventing staling of bread. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2014-195444 A [Patent Document 2] Special Publication No. 2007-526752 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an enzyme-containing antifungal agent that exhibits antifungal properties while improving the flavor of processed grain foods. [Means for solving the problem]

[0007] The present inventors have studied the effects of various enzymes in preventing the growth of mold in processed cereal foods and have found that exomaltotetraohydrolase is useful as an antifungal agent, which has led to the completion of the present invention.

[0008] That is, the present invention relates to an antifungal agent for processed grain foods, which contains exomaltotetrahydrolase.

[0009] Preferably, the exomaltotetraohydrolase is derived from Pseudomonas stutzeri.

[0010] It is preferred that the exomaltotetrahydrolase is a non-recombinant enzyme.

[0011] The processed cereal food is preferably bread.

[0012] The present invention also relates to a method for preventing mold growth in processed grain foods, which comprises the step of blending the above-mentioned antifungal agent with a processed grain food material or a processed grain food.

[0013] The present invention also relates to a processed grain food containing the above-mentioned antifungal agent and using a reduced amount of a non-enzymatic antifungal agent.The processed grain food preferably does not contain a non-enzymatic antifungal agent.

[0014] The present invention also relates to a method for producing a processed grain food, which comprises a step of blending the antifungal agent with a processed grain food material or a processed grain food.

[0015] The present invention also relates to a method for producing a processed grain food, which comprises a step of blending the above-mentioned antifungal agent and a non-enzymatic antifungal agent with a processed grain food material or a processed grain food.

[0016] The present invention also relates to a method for reducing the amount of a non-enzymatic anti-fungal agent used in a processed grain food, which comprises a step of blending the anti-fungal agent with a processed grain food material or a processed grain food. Effect of the Invention

[0017] The fungicide of the present invention is highly effective against molds that are forcibly inoculated into cereal processed foods. Furthermore, since it contains exomaltotetrahydrolase as an active ingredient, it can simultaneously achieve the fungicidal effect and the flavor improving effect of cereal processed foods. [Brief description of the drawings]

[0018] [Figure 1A] 1 shows the growth scores of mold-separated bread in bread produced by the straight method. [Figure 1B] 1 shows the growth scores of mold-separated bread in bread produced using the sponge method. [Figure 2A] 4 shows the growth scores of Penicillium expansum in bread produced by the straight method. [Figure 2B] 1 shows the growth scores of Penicillium expansum in bread produced using the sponge method. [Figure 3A] 4 shows the growth scores of Aspergillus niger in bread produced by the straight method. [Figure 3B] 4 shows the growth scores of Aspergillus niger in bread produced using the sponge method. [Figure 4A] The growth scores of Penicillium chrysogenum in bread produced by the straight method are shown. [Figure 4B] The growth scores of Penicillium chrysogenum in bread produced using the sponge method are shown. [Diagram 5]1 shows the growth scores of mold-separated bread for two loaves of bread containing super barley produced by the straight method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] (1) Exomaltotetrahydrolase The antifungal agent for processed grain foods of the present invention contains exomaltotetraohydrolase, which is an enzyme that hydrolyzes starch in an exo form and produces maltotetraose consisting of four glucose molecules.

[0020] The origin of exomaltotetrahydrolase is not particularly limited, and may be derived from microorganisms, animals, or plants. However, from the viewpoint of availability, exomaltotetrahydrolase derived from microorganisms is preferred. Examples of microorganisms include Pseudomonas and Bacillus. Examples of microorganisms of the Pseudomonas genus include Pseudomonas stutzeri, Pseudomonas saccharophilia, and Pseudomonas sp. Examples of microorganisms of the Bacillus genus include Bacillus circulans and Bacillus sp. Among these, it is more preferred to be derived from the Pseudomonas genus, and even more preferred to be derived from Pseudomonas stutzeri. In addition, the exomaltotetrahydrolase may be extracted from the original microorganism, animal, or plant, or may be mass-produced in microbial cells. Furthermore, recombinant exomaltotetrahydrolase can also be used, but non-genetically modified (Non-GMO) products are preferred.

[0021] The exomaltotetrahydrolase is preferably a polypeptide of the following (A), (B), or (C): (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1; (B) a polypeptide that exhibits 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and has the activity of exohydrolyzing starch to produce maltotetraose; (C) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and / or added in the amino acid sequence shown in SEQ ID NO:1, and having the activity of exo-hydrolyzing starch to produce maltotetraose.

[0022] The sequence identity between exomaltotetrahydrolase and the amino acid sequence shown in SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more, and most preferably 100%. The sequence identity of the amino acid sequence is expressed by comparing the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence to be evaluated, dividing the number of positions where amino acids match in both sequences by the total number of amino acids, and multiplying the result by 100.

[0023] The number of deleted, inserted, substituted and / or added amino acids is preferably 82 or less, more preferably 54 or less, even more preferably 27 or less, even more preferably 10 or less, and particularly preferably 5 or less.

[0024] In addition, the exomaltotetrahydrolase is preferably a polypeptide encoded by the DNA of the following (a), (b), or (c): (a) DNA comprising the nucleotide sequence shown in SEQ ID NO:2; (b) a DNA which shows 85% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 and encodes a polypeptide having an activity of exohydrolyzing starch to produce maltotetraose; (c) DNA consisting of a base sequence in which one or more bases are deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO: 2, and encoding a polypeptide having the activity of exohydrolyzing starch to produce maltotetraose.

[0025] The sequence identity between the DNA encoding exomaltotetrahydrolase and the base sequence shown in SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, particularly preferably 99% or more, and most preferably 100%. The sequence identity of the base sequence is expressed by comparing the base sequence shown in SEQ ID NO: 2 with the base sequence to be evaluated, dividing the number of positions where bases match in both sequences by the total number of bases compared, and further multiplying the result by 100.

[0026] DNA consisting of a base sequence in which one or more bases have been deleted, inserted, substituted and / or added in the base sequence shown in SEQ ID NO:2 and encoding a polypeptide having the activity of exo-hydrolyzing starch to produce maltotetraose can be prepared in accordance with known gene modification methods.

[0027] The number of deleted, inserted, substituted and / or added bases is preferably 246 or less, more preferably 164 or less, even more preferably 82 or less, even more preferably 32 or less, and particularly preferably 16 or less.

[0028] The amino acid sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 2 are the amino acid sequence of exomaltotetrahydrolase possessed by Pseudomonas stutzeri strain MO-19 and the base sequence of the gene thereof, respectively.

[0029] The content of exomaltotetrahydrolase in the fungicide is not limited, but it is preferable that the activity is 6000U or more per 1g of the total weight of the fungicide, and more preferably 6500U or more. The upper limit is preferably as high as possible, and is not particularly limited, but is generally 100000U or less or 50000U or less per 1g of the total weight of the fungicide. Here, the activity of exomaltotetrahydrolase can be measured by acting the enzyme on the substrate starch and quantifying the reducing power of the resulting reducing sugar by the modified Somogyi-Nelson method. The unit of enzyme activity is the amount of enzyme that generates reducing power equivalent to 1μmol of glucose per minute, which is defined as 1U.

[0030] (2)Optional components The fungicide of the present invention may contain, in addition to exomaltotetrahydrolase, ingredients acceptable for food. Examples of such ingredients include various sugars such as reduced lactose, trehalose, xylitol, sorbitol, and hemicellulose hydroxide, and mixtures of emulsifiers and polymerized phosphates. The fungicide of the present invention may also contain excipients such as dextrin, starch, modified starches, and lactose.

[0031] The form of the antifungal agent is not particularly limited, and examples thereof include powder, granule, liquid, paste, and solid. In the case of powder, exomaltotetrahydrolase may be dissolved in a solvent such as water or sugar liquid, and then an excipient such as dextrin may be added as necessary, followed by drying to obtain a powder.

[0032] (3) Anti-mold effect In the present invention, antifungal refers to inhibiting the growth of mold. The growth of mold can be measured by the method described in the Examples.

[0033] Exomaltotetrahydrolase produces maltotetraose from starch. When amylase is contained in a cereal processed food, the amylase decomposes maltotetraose to produce maltose, and carbohydrates containing maltose as the main component accumulate in the cereal processed food. Since sugars such as maltose improve the flavor of cereal processed foods, the antifungal agent of the present invention can improve the flavor of the food as well as the antifungal effect.

[0034] In the present invention, the fungi that are the subject of the antifungal effect are not particularly limited, but include Penicillium genus, Aspergillus genus, Cladosporium genus, and Fusarium genus. Examples of Penicillium genus include Penicillium expansum, Penicillium chrysogenum, Penicillium citrinum, and Penicillium islandicum. Examples of Aspergillus genus include Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, and Aspergillus versicolor. Among these, it is preferable that the antifungal agent of the present invention has an antifungal effect against Penicillium expansum, Penicillium chrysogenum, and Aspergillus niger.

[0035] Since the fungicide of the present invention contains exomalttetrahydrolase as an active ingredient, the amount of nonenzymatic fungicide used can be reduced in the processed grain food containing the fungicide of the present invention. The processed grain food containing the fungicide of the present invention preferably does not contain a nonenzymatic fungicide. Examples of the nonenzymatic fungicide include propionic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, citric acid, glycine, polylysine, or a salt thereof. Examples of the salt include calcium salt, sodium salt, and potassium salt. Examples of the nonenzymatic fungicide include orthophenylphenol, biphenyl, thiabendazole, and imazalil. In the processed grain food containing the fungicide of the present invention, the propionic acid content is preferably 0.25% by weight or less, and the benzoic acid content is preferably 0.25% by weight or less.

[0036] (4) Processed grain foods The cereal processed food to which the antifungal agent is applied is not particularly limited as long as it is a food obtained by processing cereals. Examples of cereals include wheat, rice, red beans, corn, tapioca, kudzu, rye, barley, super barley, buckwheat, sweet potato, potato, yam, taro, lily root, lotus root, lentil, chickpea, kidney bean, pea, broad bean, peanut, white kidney bean, soybean, and green pea. Among these, wheat and rice are preferred. Examples of wheat processed foods include bread, cake, cookies, castella, udon, pasta, and wheat gluten. Examples of rice processed foods include mochi, manju, dumplings, rice balls, okowa, porridge, and arare. Examples of red bean processed foods include red bean paste, red rice, red bean porridge, green pea bean snacks, yokan, sweet red bean soup, zenzai, and ice candy. Examples of corn processed foods include corn cream, corn cream croquette, and corn cream stew. Examples of corn processed foods also include sweets such as milk pudding using cornstarch. Examples of tapioca processed foods include warabi mochi, tapioca pearls, tapioca drinks, kudzu mochi, and pão de queijo. Examples of kudzu processed foods include kudzu mochi and sesame tofu. Among these, wheat processed foods are preferred, and bread, cake, and castella are more preferred, in that they are easy to achieve the flavor improvement effect and mold prevention effect due to exomaltotetrahydrolase.

[0037] The method of applying the antifungal agent to the cereal processed food is not particularly limited, and the antifungal agent may be added to the ingredients during the production of the cereal processed food, or may be added after the production of the cereal processed food. An example of a method of adding the antifungal agent after the production of the cereal processed food is a method of spraying a liquid containing the antifungal agent from the outside of the cereal processed food. Therefore, another aspect of the present invention is a method for preventing mold growth in a cereal processed food, which includes a step of adding the antifungal agent of the present invention to a cereal processed food material or a cereal processed food.

[0038] When the fungicide is mixed with the grain processed food material during the production of the grain processed food, if the production method of the grain processed food includes a heating step, the fungicide may be added before or after the heating step. When the fungicide is added before the heating step, the fungicide acts before the heating step. When the fungicide is added after the heating step, the fungicide maintains its antifungal effect for a long period after the production of the grain processed food. On the other hand, the enzyme protein exomaltotetrahydrolase is thermally denatured and loses its activity by treatment at pH 7.0 and 55°C or higher for 60 minutes or at pH 7.0 and 60°C or higher for 15 minutes. When the fungicide is mixed into the grain processed food and heated at a high temperature, the thermally denatured exomaltotetrahydrolase is digested and absorbed in the body like other proteins contained in the food.

[0039] The amount of exomaltotetrahydrolase to be added varies depending on the type of processed grain food, but is preferably 20 to 500 U per 100 g of food, more preferably 50 to 300 U, and even more preferably 80 to 120 U. The activity of exomaltotetrahydrolase is defined as above.

[0040] (5) Bread The method for producing bread as a processed grain food is described below. The anti-mold agent may be applied to the bread after it has been produced, but it is preferable to add it to the dough material, as this makes it easier to achieve both flavor improvement and anti-mold effects.

[0041] The main ingredients of bread dough include wheat flour (weak flour, medium flour, strong flour, whole wheat flour, graham flour, etc.), barley flour, super barley flour, oat flour, rye flour, soy flour, rice flour, etc. Other ingredients include yeast (e.g., fresh yeast, dry yeast, instant dry yeast, etc.), sugar (e.g., white sugar, granulated sugar, brown sugar, brown sugar, isomerized sugar, powdered syrup, starch syrup, sugar alcohol, oligosaccharides, trehalose, etc.), salt, dairy ingredients (e.g., milk, cream, whole milk powder, skim milk powder, milk protein, concentrated milk, etc.), fats and oils (e.g., shortening, margarine, butter, liquid oil, emulsified fats and oils, etc.), water, eggs (whole eggs, egg yolks, egg whites, dried eggs, frozen eggs, etc.), baking powder, etc.

[0042] When the mold inhibitor is added to the dough material, it is preferable to mix the dough material with the mold inhibitor and knead it. "Kneading" refers to mixing the dough material and kneading it. Kneading can be performed under conditions used in ordinary bread making.

[0043] The dough can be produced and baked by a conventional method, including the sponge method, the straight dough method, the refrigeration method, the freezing method, the liquid dough method, the sourdough method, the sake dough method, the hop dough method, the soaking method, the Chorleywood method, and the continuous bread making method.

[0044] In the sponge method, part or all of the wheat flour is fermented first to produce a sponge, and then the remaining wheat flour and ingredients are added and kneaded. In the sponge method, mold inhibitors are effective whether they are added to the sponge or the kneading ingredients, but it is preferable to add them to the sponge.

[0045] In the straight method, all ingredients are mixed from the beginning and fermented to produce dough. In the case of the straight method, it is preferable to mix the mold inhibitor with other dough ingredients from the beginning.

[0046] The content of the antifungal agent in the bread dough can be appropriately selected depending on the conditions used in the bread making method. For example, 50 to 400 ppm (350 to 2800 U / kg strong flour) is preferable, 100 to 300 ppm (700 to 2100 U / kg strong flour) is more preferable, and 150 to 250 ppm (1050 to 1750 U / kg strong flour) is even more preferable. The definition of exomaltotetrahydrolase activity is as described above.

[0047] Methods for heating bread dough include baking and steaming. The heating temperature can be appropriately selected depending on the type of bread, but in the case of baking, it is preferably 170 to 250°C, more preferably 190 to 220°C. In the case of steaming, it is preferably 100 to 140°C, more preferably 115 to 125°C. The heating time of the bread dough is preferably 10 to 70 minutes, more preferably 15 to 60 minutes, even more preferably 20 to 50 minutes, and even more preferably 20 to 40 minutes.

[0048] Examples of breads to which the antifungal agent of the present invention can be applied include white bread, bread made by putting two dough balls into a case and fermenting and baking them (hereinafter referred to as two-ball bread), healthy bread, sweet bread (sweet bean bread, jam bread, cream bread, etc.), roll bread, French bread, steamed bread, cooked bread, buns, fruit bread, corn bread, butter roll, buns, sandwiches, croissants, Danish pastry, hardtack, bagels, and pretzels. Among these, sweet breads such as sweet bean bread, jam bread, and cream bread, and butter rolls are collectively called variety breads. EXAMPLES

[0049] (1) Manufacture of anti-mold agents The enzyme powder (exomaltotetrahydrolase) was mixed with a component (dextrin) that is acceptable for use as an excipient in food products so that the content was approximately 3% exomaltotetrahydrolase and approximately 97% dextrin, and then the mixture was ground to produce a powdered antifungal agent.

[0050] (2) Production of bread containing antifungal agent (Example) (2-1) Bread production using the straight method A mold inhibitor was added and bread was made using the straight method. The amounts of ingredients are shown in Table 1. In Table 1, the content of each ingredient other than the mold inhibitor is shown in parts by weight when the wheat flour contained in the final bread starter is taken as 100 parts by weight.

[0051] [Table 1]

[0052] The ingredients in Table 1 were mixed and baked into square loaf bread according to the process in Table 2. One day after baking, the baked bread was sliced ​​to a thickness of 1.5 cm.

[0053] [Table 2]

[0054] (2-2) Manufacturing bread using sponge method A mold inhibitor was added and bread was made using the sponge method. The amounts of ingredients are shown in Table 3. In Table 3, the content of each ingredient other than the mold inhibitor is shown in parts by weight when the wheat flour contained in the final dough after mixing with the kneading ingredients is taken as 100 parts by weight.

[0055] [Table 3]

[0056] The sponge ingredients in Table 3 were mixed, and sponge was produced according to steps 1 and 2 in Table 4. The sponge thus produced was mixed with the main kneading ingredients in Table 3, and square bread was baked according to steps 3 to 10 in Table 4. The baked square bread was sliced ​​to a thickness of 1.5 cm one day after baking.

[0057] [Table 4]

[0058] (2-3) Manufacturing two-loaf bread using the straight method A mold inhibitor was added and two-loaf bread containing super barley was produced using the straight method. The amounts of ingredients are shown in Table 5. In Table 5, the contents of each ingredient other than the mold inhibitor and vitamin C are shown in parts by weight when the wheat flour contained in the final bread starter is taken as 100 parts by weight.

[0059] [Table 5]

[0060] The ingredients in Table 5 were mixed and two-loaf bread containing super barley was baked according to the process in Table 6. The baked bread was sliced ​​to a thickness of 1.5 cm one day after baking.

[0061] [Table 6]

[0062] (3) Bread production (Comparative example) Except for not adding any mold inhibitor, we baked square loaves of bread using the straight and sponge method, and two-loaf bread with super barley using the straight method, following the same procedure as above. The baked bread was sliced ​​to a thickness of 1.5 cm one day after baking.

[0063] (4) Antifungal properties test (4-1) Bread made by the straight method and sponge method Spores of the following microorganisms were suspended in physiological saline and adjusted to a concentration of 5 spores (low concentration seeding) or 10 spores (high concentration seeding) per drop (approximately 10 μl) to prepare a spore suspension. (i) Bread-isolated mold (isolated from mold naturally occurring on bread) (ii) Penicillium expansum NRBC 6096 (iii) Aspergillus niger NRBC 33023 (iv)Penicillium chrysogenum NRBC 32030

[0064] As described above, bread was baked using the straight or sponge method, and the sliced ​​surface was seeded with mold by dropping spore suspension onto 9 spots. Two pieces of bread were used for each test. After seeding, the bread was left to stand in a plastic bag at room temperature of about 25°C, and the growth of mold hyphae and spores on the sliced ​​surface was checked with the naked eye over time. For each spot, 0.5 points were given if hyphae were observed near the mold seeding area, and 1 point was given if spores were observed. The total points for all spots were used to determine the mold growth score. The relationship between the standing time and the mold growth score is shown in Figures 1A to 4B.

[0065] As shown in Figures 1 to 4, the bread of the Example was able to retard the growth of all molds compared to the bread of the Comparative Example. In addition, as long as the bread contained an antifungal agent, the growth of mold could be retarded regardless of the bread manufacturing method. The antifungal agent not only retarded the growth of Penicillium expansum, Penicillium chrysogenum, and Aspergillus niger, but also reduced the maximum mold growth score.

[0066] (4-2) Two-bread containing super barley produced by the straight method Spores of the following microorganisms were suspended in physiological saline to prepare a spore suspension containing 3 spores per drop (approximately 10 μl). (i) Bread-isolated mold (isolated from mold naturally occurring on bread)

[0067] As described above, two loaves of bread containing super barley baked by the straight method were sliced, and the sliced ​​surfaces were seeded with mold by dropping 18 spots of the spore suspension. Two pieces of bread were tested for each test. After seeding, the bread was left to stand in a plastic bag at room temperature of about 25°C, and the growth of mold hyphae and spores on the sliced ​​surfaces was checked with the naked eye over time. For each spot, a score of 0.5 was given if hyphae were observed near the mold seeding area, and a score of 1 was given if spores were observed. The total score for all spots was used to determine the mold growth score. The relationship between the standing time and the mold growth score is shown in Figure 5.

[0068] As shown in FIG. 5, mold growth was delayed in the bread of the embodiment compared to the bread of the comparative example.

Claims

1. A mold inhibitor for processed grain foods, containing exomaltotetrahydrolase.

2. 2. The antifungal agent according to claim 1, wherein the exomaltotetrahydrolase is derived from Pseudomonas stutzeri.

3. The antifungal agent according to claim 1 or 2, wherein exomaltotetrahydrolase is a non-genetically modified enzyme.

4. The antifungal agent according to any one of claims 1 to 3, wherein the processed grain food is bread.

5. A method for preventing mold growth in processed cereal foods, comprising the step of blending the antifungal agent according to any one of claims 1 to 4 with a processed cereal food material or a processed cereal food.

6. A method for producing a processed grain food, comprising a step of blending the antifungal agent according to any one of claims 1 to 4 with a processed grain food material or a processed grain food.

7. A method for producing a processed grain food, comprising a step of blending the antifungal agent according to any one of claims 1 to 4 and a non-enzymatic antifungal agent with a processed grain food material or a processed grain food.

8. A method for reducing the amount of a non-enzymatic anti-fungal agent used in a processed cereal food, comprising the step of blending the anti-fungal agent according to any one of claims 1 to 4 with a processed cereal food material or the processed cereal food.

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