Beta-amylase compositions, methods of stabilizing beta-amylases and methods of preserving

Abstract

The present application provides a beta-amylase composition, a beta-amylase stabilization method and a preservation method, the beta-amylase composition as a food use, which does not use chemicals that can affect the human body, and can stably store amylase for a long time. The present application relates to an amylase composition containing: a saccharide containing glucose as a structural unit, a polyol, and an amylase.

Description

[0001] This application is a divisional application, which targets the Chinese national application number 202080000776.7, the international application number PCT / JP2020 / 010428, the application date of which was March 11, 2020, the entry date into China of which was May 21, 2020, and the invention title of which is "Amylase Composition". Technical Field

[0002] This invention relates to amylase compositions. Background Technology

[0003] β-amylase is used in the manufacture of maltose and syrup, and also as an anti-aging agent for rice cakes, pastries, and desserts. β-amylase exists in plant-derived forms such as soybeans, barley, wheat, and sweet potatoes, as well as microbial forms. Compared to liquid β-amylase preparations from barley, soybean-derived β-amylase exhibits higher heat resistance, remaining active even at temperatures around 60°C, thus promising wider applicability under more complex conditions. Furthermore, soybean-derived β-amylase produces equivalent saccharification effects at half the dosage of barley-derived β-amylase, suggesting the potential for reduced enzyme usage and lower costs.

[0004] Stabilizers are typically added to liquid enzyme preparations to maintain enzyme activity or for preservation. To achieve high preservative effects, chemicals such as benzoic acid, para-hydroxybenzoic acid, and sorbic acid are sometimes added. However, these chemicals are equivalent to restricted additives for food use, and due to increased concerns about food safety, they tend to be avoided in the market.

[0005] To improve the shelf-life stability of products, salts such as sodium chloride can be added to enzyme preparations to reduce water activity or increase osmotic pressure, thereby inhibiting the growth of microorganisms in the enzyme preparation. However, when using β-amylase preparations containing salts in the manufacture of maltose, the salts may have adverse effects during the purification process using ion exchange resins, especially in the decolorization and desalting processes.

[0006] Patent document 1 discloses the addition of polyols and sugars to prevent self-decomposition caused by proteases and to prevent precipitation in aqueous solvents. However, methods to improve the long-term storage stability of amylases are still unknown.

[0007] Existing technical documents

[0008] Patent documents

[0009] [Patent Document 1] Japanese Patent Application Publication No. 2017-029129 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] The objective of this invention is to provide an amylase composition for food use that does not contain any chemicals that may have an effect on the human body and can stably preserve the amylase for a long period of time.

[0012] Methods for solving problems

[0013] Through in-depth research, the inventors discovered that by combining polyols and sugars containing glucose as structural units, long-term preservation stability of amylase was achieved, thus completing this invention.

[0014] That is, the present invention relates to an amylase composition comprising: a sugar containing glucose as a structural unit, a polyol and an amylase.

[0015] The total amount of sugars containing glucose as a structural unit and polyols is preferably 70% by weight or less.

[0016] The polyols are preferably selected from the group consisting of glycerol, sorbitol and propylene glycol.

[0017] The sugars are preferably selected from one or more of the group consisting of sucrose, trehalose, dextrin, maltose, and maltitol.

[0018] The preferred amylase is β-amylase.

[0019] In addition, the present invention relates to food additives containing the above-mentioned amylase composition.

[0020] In addition, the present invention relates to food containing the above-mentioned food additives.

[0021] In addition, the present invention relates to a method for stabilizing amylase, which includes a step of mixing amylase, polyol and sugar containing glucose units.

[0022] Invention Effects

[0023] For food use, the amylase composition of the present invention does not contain chemicals that may affect human health and improves the long-term storage stability of the amylase. Furthermore, an essential element of the amylase composition of the present invention is that it is salt-free, thus not placing a burden on the maltose manufacturing process. Attached Figure Description

[0024] Figure 1 The relative activity (residual potency) of the amylase compositions in Comparative Examples 1-4 is indicated.

[0025] Figure 2 The relative activity (potency residual rate) of the amylase compositions in Examples 1-3 and Comparative Examples 5-6 is indicated.

[0026] Figure 3 The relative activity (potency residual rate) of the amylase compositions in Examples 4-7 and Comparative Example 7 is indicated.

[0027] Figure 4 The relative activity (potency residual rate) of the amylase compositions in Examples 8-12 and Comparative Examples 7-8 is indicated.

[0028] Figure 5 The relative activity (potency residual rate) of the amylase compositions in Examples 13 and Comparative Examples 8-13 is indicated. Detailed Implementation

[0029] <<Amylase Composition>>

[0030] The amylase composition of the present invention comprises: a sugar containing glucose as a structural unit, a polyol, and an amylase.

[0031] <Amylase>

[0032] Amylase is an enzyme that cleaves the α-1,4-bonds of starch or glycogen to produce maltose. Amylases are further classified into α-amylase, β-amylase, glucosylamylase, isoamylase, maltose-amylase, etc. The amylase used in this invention is not particularly limited, but β-amylase capable of preparing formulations with high heat resistance is preferred.

[0033] The source of the amylase in this invention is not particularly limited, and examples include plants, animals, and microorganisms. Among these, amylases derived from soybeans, barley, wheat, sweet potatoes, and microorganisms such as those produced by *Aspergillus*, *Bacillus*, and *Streptomyces* are preferred due to prior culinary experience and ease of use in food. Soybean-derived β-amylases are even more preferred. Soybean-derived β-amylases exhibit excellent heat resistance and reactivity, and therefore, compared to β-amylases from sources other than soybeans, such as barley-derived β-amylases, they are expected to be useful in a wide range of applications.

[0034] Amylase can be any type of amylase extracted from plants, animals, or microorganisms, or amylase produced in large quantities using transgenic technology. Additionally, wild-type amylase or mutant amylase can also be used.

[0035] As a method for obtaining amylase, amylase accumulates within the cells of the source organism, and the tissues and cells are broken down, yielding a cell-free extract through centrifugation or similar means. This extract can then be used as amylase. Alternatively, the cell-free extract can be used as a starting material and purified using general protein purification methods such as salting out, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. When amylase is produced by microorganisms and secreted extracellularly, it can be purified from the culture medium. The amylase used in this invention is not limited to pure products but can also be in the form of crude purified products such as plant extracts like soybean whey or cell-free extracts of microorganisms.

[0036] To maintain a high amylase potency, the amylase content in the amylase composition is preferably 30% by weight or more, more preferably 50% by weight or more, even more preferably 70% by weight or more, even more preferably 80% by weight or more, and particularly preferably 90% by weight or more.

[0037] <Polyols>

[0038] Polyols are any alcohols having two or more hydroxyl groups that can reduce the reactivity of water; there are no particular limitations. Specific examples of polyols include glycerol, sorbitol, propylene glycol, polyvinyl alcohol, pentaerythritol, ethylene glycol, diethylene glycol, triethylene glycol, and polyethylene glycol. Among these, glycerol is preferred.

[0039] In the amylase composition, the polyol content is preferably 30-60% by weight, more preferably 30-55% by weight. When it is below 30% by weight, there is a tendency that the amylase cannot be sufficiently stabilized, and when it exceeds 60% by weight, the amylase potency in the composition is excessively diluted.

[0040] <Carbohydrates that contain glucose as a structural unit>

[0041] Sugars can be any sugar containing glucose, and can be any type of monosaccharide, disaccharide, trisaccharide, or polysaccharide; there are no particular limitations. Disaccharides or polysaccharides are substances formed by the polymerization of monosaccharide molecules through glycosidic bonds; any sugar containing glucose as one of the monosaccharide molecules is acceptable, without particular limitations. In the case of disaccharides or polysaccharides, glucose is preferably α-1,4-bonded. Specific examples of sugars containing glucose as a structural unit include dextrin, maltose, maltitol, sucrose, lactose, trehalose, and cellobiose. Among these, dextrin, sucrose, trehalose, and maltitol, which do not contain reducing groups that would reduce product quality due to browning during storage, or have a low proportion of reducing groups, are preferred.

[0042] In the amylase composition, the content of sugars containing glucose as a structural unit is preferably 1 to 20% by weight, more preferably 2 to 15% by weight. When it is less than 1% by weight, there is a tendency that the amylase cannot be sufficiently stabilized, and when it exceeds 20% by weight, the amylase potency in the composition cannot be improved.

[0043] Furthermore, in the amylase composition, the total amount of sugars containing glucose as a structural unit and polyols is preferably 70% by weight or less, more preferably 60% by weight or less, even more preferably 50% by weight or less, and even more preferably 45% by weight or less. When it exceeds 70% by weight, the concentration of amylase in the amylase composition decreases. To increase the amylase concentration in the amylase composition, it is preferable to have a small total amount of sugars containing glucose as a structural unit and polyols, but the lower limit is generally 30% by weight.

[0044] The form of the amylase composition is not particularly limited and can be either liquid or solid. Examples of liquid forms include aqueous solutions, suspensions, and slurries. Examples of solid forms include powders, granules, and tablets. From the viewpoint of cost and operation, a liquid form is preferred. Conventionally, liquid forms have been difficult to maintain the enzymatic activity of amylase and inhibit microbial contamination, but the amylase composition of the present invention can stably maintain the activity of amylase.

[0045] When the amylase composition is in liquid form, the pH of the amylase composition is preferably 4 to 9, more preferably 4.5 to 7, and even more preferably 5 to 6. Below pH 4, there is a tendency for precipitates to form. Above pH 9, the activity of the amylase tends to be impaired. The pH of the amylase composition can be adjusted using acids such as hydrochloric acid and sulfuric acid, and bases such as sodium hydroxide and potassium hydroxide.

[0046] The enzyme potency of the amylase composition is not particularly limited, but is generally preferred to be 1,000 to 1,000,000 units / g or less. Here, one unit of enzyme potency is defined as the amount of enzyme that produces 1 mg of maltose in 1 hour under conditions of pH 5.5 / 60°C.

[0047] The amylase composition of the present invention contains polyols and sugars comprising glucose as structural units, thus enabling it to maintain amylase activity stably over a long period. After storage at 40°C for 3 months, the amylase composition preferably maintains more than 70% of its activity compared to before storage, and more preferably more than 80%. The activity can be evaluated using the enzyme titer described above.

[0048] The amylase composition of the present invention can prevent the proliferation of live bacteria in the composition. Preferably, even after storage at 40°C for 3 months, the number of live bacteria does not increase to more than 10,000.

[0049] (Method for manufacturing the composition)

[0050] Amylase compositions can be manufactured by mixing the components in any order. Alternatively, the components can be mixed and then filtered or sterilized by contacting a porous material or passing through a filter.

[0051] In addition to sugars, polyols, and amylases containing glucose as a structural unit, the amylase composition may also contain any other ingredients.

[0052] <<Food Additives>>

[0053] The food additive of the present invention is characterized by containing the above-described amylase composition. In addition to the amylase composition, the food additive may also contain other food-acceptable ingredients. Examples of such other ingredients include excipients, pH adjusters, enzymes, thickening polysaccharides, emulsifiers, mixtures of emulsifiers and polyphosphates, dairy products, extracts, sweeteners, fermentation flavorings, eggs, inorganic salts, preservatives, organic acids, metals, and filter aids. The content of these ingredients is not particularly limited and can be selected in any amount by those skilled in the art.

[0054] Examples of pH adjusters include ascorbic acid, acetic acid, dehydroacetic acid, lactic acid, citric acid, gluconic acid, succinic acid, tartaric acid, fumaric acid, malic acid, and adipic acid, as well as their sodium (Na), calcium (Ca), and potassium (K) salts; carbonic acid, phosphoric acid, and pyrophosphate, as well as their Na and K salts.

[0055] Examples of enzymes include α-amylase, glucosylamylase, amylopectinase, isoamylase, maltotriase, cyclodextrin glucosyltransferase, transglucosidase, glucanase, glucose isomerase, cellulase, xylanase, hemicellulase, mannanase, pectinase, pectin methyl esterase, invertase, lactase, inulinase, α-galactosidase, chitinase, chitosanase, alginate lyase, and other sugar-related enzymes; proteases, peptidases, collagenases, glutaminase, and other protein / amino acid-related enzymes; lipases, phospholipases, esterases, and other lipid-related enzymes; and other catalases, glucose oxidases, ureases, tannic acidases, deaminases, etc.

[0056] Examples of thickening polysaccharides include processed starch, gums, alginic acid, alginic acid derivatives, pectin, carrageenan, curd, amylopectin, gelatin, cellulose derivatives, agar, tamarind polysaccharide, psyllium polysaccharide, and glucomannan.

[0057] Examples of emulsifiers include glycerol fatty acid esters, polyglycerol fatty acid esters, sucrose fatty acid esters, propylene glycol fatty acid esters, sorbitol fatty acid esters, lecithin, enzymatically hydrolyzed lecithin, and saponins.

[0058] Examples of dairy products include milk, skim milk powder, whole milk powder, whey powder, casein, cheese, yogurt, condensed milk, fermented milk, and cream.

[0059] Examples of extracts include yeast extract and malt extract.

[0060] Examples of sweeteners include stevia, aspartame, glycyrrhizin, acesulfame potassium, sucralose, neotame, etc.

[0061] Examples of inorganic salts include table salt, ammonium sulfate, sodium sulfate, calcium chloride, and polyphosphates.

[0062] Examples of preservatives include propionic acid, propionate, sulfites, benzoates, sorbic acid, sorbates, protamine, polylysine, glycine, and acetate. Examples of salts include sodium (Na) salts, calcium (Ca) salts, and potassium (K) salts.

[0063] <<Food>>

[0064] The food product of the present invention is characterized by containing the aforementioned food additives. The food product of the present invention is preferably a grain-processed food. Examples of grains include rice, red beans, rye, barley, buckwheat, wheat, sweet potato, potato, cassava, kudzu, corn, yam, taro, lily root, lotus root, lentils, chickpeas, kidney beans, peas, broad beans, peanuts, white kidney beans, soybeans, and cooked sweet peas. Processed rice and wheat products are preferred. Examples of rice-processed foods include cooked rice, glutinous rice with red beans, zongzi (sticky rice dumplings), rice balls, sushi, fried rice, and rice cakes. Examples of wheat-processed foods include bread, cakes, pastries, and noodles.

[0065] Due to starch aging, processed grain foods tend to harden and lose palatability over long-term storage. Starch aging is caused by the partial β-conversion of α-starch. Amylases cleave maltose, which is a glucose dimer, at the ends of sugar chains, shortening the sugar chains and preventing partial β-conversion, thereby preventing starch aging. The food products of this invention contain an amylase composition, thus inhibiting starch aging.

[0066] When manufacturing the food products of the present invention, there is no particular limitation on the timing of adding the food additives of the present invention. The food products can be manufactured after adding and / or mixing the food additives into the raw materials, or the food additives can be added during the manufacturing process of the food products to enable the amylase to function.

[0067] In food manufacturing methods that include a heating step, the food additive of the present invention can be added either before or after the heating step. When added before the heating step, the amylase functions before the heating step and maintains its anti-aging effect for a long period after heating. When added after the heating step, the anti-aging effect is maintained for a long period after food manufacturing.

[0068] The preferred temperature for adding the food additive of the present invention is 4–70°C, more preferably 25–65°C, and even more preferably 50–60°C. When using β-amylase from soybeans, its high heat resistance allows it to function at high temperatures, such as above 60°C. On the other hand, to reduce the amount of amylase added while achieving the effect of preventing aging, it is preferable to add the food additive of the present invention after the food has reached a low temperature.

[0069] The food products of this invention can also be preserved after being mixed with the food additives of this invention. The preferred storage temperature is -80 to 30°C. Specifically, the preferred storage temperature during refrigeration is -20 to 0°C, more preferably -10 to -4°C. The preferred storage temperature during refrigeration is 0 to 10°C, more preferably 0 to 4°C. The preferred storage temperature for room temperature storage is 15 to 25°C. Through the action of amylase, starch aging can be prevented, and the texture can be maintained even after low-temperature storage.

[0070] <<Methods for Stabilizing Amylase>>

[0071] The amylase stabilization method of the present invention includes a step of mixing amylase, polyol, and sugars containing glucose units. The amylase, polyol, and sugars containing glucose units are as described above. The mixing order of the amylase, polyol, and sugars containing glucose units is not particularly limited.

[0072] [Example]

[0073] The present invention will now be described with reference to specific embodiments, but the present invention is not limited to these embodiments. In the following text, unless otherwise stated, “parts” or “%” means “parts by weight” or “% by weight”, respectively.

[0074] (1) Materials used

[0075] Soy whey (Showa Sangyo Co., Ltd.)

[0076] Glycerin (Sakamoto Pharmaceutical Co., Ltd.: Food Additive Glycerin RG)

[0077] Dextrin (Matsutani Chemical Industry Co., Ltd.: Max1000)

[0078] Sorbitol (Ueno Pharmaceutical Co., Ltd.: Sorbitol Ueno 20M)

[0079] Maltose (Habayashihara Co., Ltd.: SUNMALT S)

[0080] Maltitol (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0081] Filter aid: TOPCO Perlite #54 (TOKO PERLITE INDUSTRY Co., Ltd.) KC FLOCK W-100 (Naiwai Flour Milling Co., Ltd.) (2) Preparation of amylase composition TOPCO perlite #54 and KC FLOCK W-100 were added to soybean whey at a weight ratio of 0.3%, and the mixture was clarified and filtered through a filter press. The filtered sample was then concentrated using a UF membrane (DAICEN MEMBRANE FS10-FS-FUY03A1) to obtain a liquid soybean whey concentrate.

[0082] Soy whey concentrate, polyol, and sugars containing glucose units were mixed in the proportions described in Tables 1-5. Sodium hydroxide solution was added to adjust the pH to 5.2, and the mixture was stirred for 1 hour. The stirred sample was then clarified and filtered using TOPCO perlite and KC FLOCK W-100. The filtered sample was sterilized using a 0.2 μm filter (Toyo Filter Paper Co., Ltd.: C020A047A) to obtain the amylase composition.

[0083] (3) Determination of β-amylase activity

[0084] For freshly manufactured amylase compositions and amylase compositions stored at 40°C for 0.5 months, 1 month, 2 months, and 3 months, the activity was determined using the following method, and the relative activity at which the freshly manufactured activity was 100% was calculated. The results are as follows: Figures 1-5 As shown.

[0085] β-amylase activity was determined using a quantitative method based on 3,5-dinitrosalicylic acid (DNS method) to measure reducing sugars. As a test method, 1 ml of amylase composition was added to 9 ml of a 1.1% glucose substrate solution at pH 5.5 containing phosphate buffer, and the mixture was allowed to react at 60°C for 30 minutes. 30 minutes after the start of the reaction, 1 ml of this reaction solution was added to 3 ml of DNS solution, and the mixture was boiled for 15 minutes. After boiling and cooling to room temperature, distilled water was added to bring the volume to 25 ml, and the absorbance was measured at 550 nm. The concentration of glucose reduced was calculated from the absorbance using a standard curve. One unit was defined as the amount of enzyme required to generate 1 mg of maltose in 1 hour under pH 5.5 / 60°C conditions.

[0086] Table 1

[0087] Table 2

[0088] Table 3

[0089] Table 4

[0090] Table 5

[0091] exist Figure 1 In the comparative examples, when the glycerol concentration was 35-50% (35-50%), the enzyme stability at 40°C was low, and the relative activity was significantly lost after storage at 40°C for 3 months. When the glycerol concentration was 60-70% (3-4), the relative activity could be maintained due to the reduced moisture content, but the amylase concentration could not be increased to a high level in the composition.

[0092] exist Figure 2 In this study, the stability of amylase was significantly improved by combining 10% of sugars containing glucose as a structural unit in 50% glycerol (Examples 1-3). The amylase storage stability was lower in the test areas containing only 60% glycerol (Comparative Example 5), 50% glycerol, and 10% sorbitol (Comparative Example 6) compared to the test areas containing sugars with glucose as a structural unit.

[0093] exist Figure 3 In this study, the stability of amylase can be greatly improved by adding 2.5% to 10% of glucose as a constituent sugar to 50% glycerol (Examples 4-7).

[0094] exist Figure 4 In this study, by combining 2.5% to 12.5% ​​of glucose as a constituent sugar in 40% glycerol, the amylase in the composition can be at a high concentration, and the stability of the amylase can also be improved (Examples 8 to 12).

[0095] exist Figure 5 In this study, the stability of amylase was improved when 10% of a sugar containing glucose as a structural unit was added to 40% glycerol (Example 13). The stability of amylase was low when glycerol or a sugar containing glucose as a structural unit was used alone (Comparative Examples 8-13).

[0096] (4) The number of viable bacteria in the amylase composition

[0097] For freshly manufactured amylase compositions and amylase compositions stored at 40°C for 0.5 months, 1 month, 2 months, and 3 months, the viable bacterial count in the compositions was determined using the following method. The results are listed in Table 6.

[0098] Weigh 25 ml of the amylase composition, add 225 ml of physiological saline to dilute it 10 times, and mix thoroughly. Prepare 10 [units of solution] from the 10-fold diluted amylase composition using phosphate buffer. 3 ~10 6 A series of dilutions were prepared. 1 ml of each diluted solution was placed in a 90 mm diameter plate, and 15–20 ml of Tryptone Glucose Yeast Extract medium (46℃±1℃) was added and mixed to prepare the plate. For each dilution, the viable count was determined using n=2. The plates were incubated at 30℃±1℃ for 72 hours, and the number of colonies appearing on the plates was counted.

[0099] Additionally, when the colony count exceeds 300 CFU on the plate with the highest dilution, only plates at that dilution are counted. When the colony count is below 30 CFU, only plates with the lowest dilution are counted. For the range of 30–300 CFU, the average value of the two dilutions is calculated.

[0100] Table 6

[0101] In Example 13, no bacterial proliferation was observed after 3 months. Bacterial proliferation occurred in Comparative Examples 9-13. In Comparative Example 8, as... Figure 5 As shown, the amylase has low stability, but no bacterial proliferation was observed.

Claims

1. A β-amylase composition comprising one or more sugars selected from the group consisting of sucrose, dextrin, and maltitol, glycerol, and β-amylase. The amount of sugars mixed is 2.5% to 12% by weight. The amount of glycerol mixed is 40% to 55% by weight.

2. The β-amylase composition according to claim 1, wherein, The total amount of sugars and glycerol in the mixture is less than 60% by weight.

3. The β-amylase composition according to claim 1 or 2, wherein, The sugar is selected from one or more of the group consisting of dextrin and maltitol.

4. The β-amylase composition according to claim 1 or 2, wherein, The amount of glycerol in the mixture is 40% to 50% by weight.

5. A method for stabilizing β-amylase, comprising the step of mixing β-amylase, glycerol, and one or more sugars selected from the group consisting of sucrose, dextrin, and maltitol to obtain a β-amylase composition. The amount of glycerol mixed in is 40% to 55% by weight relative to the total weight of the β-amylase composition. The amount of sugars mixed in is 2.5% to 12% by weight relative to the total weight of the β-amylase composition.

6. A method for preserving β-amylase, comprising the steps of mixing β-amylase, glycerol, and one or more sugars selected from the group consisting of sucrose, dextrin, and maltitol to obtain a β-amylase composition; and the step of preserving the composition for more than 3 months. The amount of glycerol mixed in is 40% to 50% by weight relative to the total weight of the β-amylase composition. The amount of sugars mixed in is 2.5% to 12% by weight relative to the total weight of the β-amylase composition. After the mixing process, the bacteria were stored at 40°C for 3 months. Compared with before storage, the β-amylase activity was more than 70%, and the number of live bacteria did not increase to more than 10,000.

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