Methods for producing plant-based protein foods.

Abstract

This invention addresses the problem of providing a novel method for the production of plant-based protein foods. This involves treating plant protein substrates with both lipase and protein deamides (for example, (Glutamidase protein) is added to that to improve the flavor of plant-based protein foods;

Description

Method for producing plant protein food

[0001] The present invention relates to a novel method for producing a plant-based protein food using an enzyme. For example, the present invention is useful for producing a fermented food (particularly a plant-based yogurt).

[0002] Due to factors such as allergy issues, an increase in vegetarians, and religious reasons, plant-based proteins such as soybeans, grains, and nuts have become popular as alternative ingredients to food and beverages that use animal-derived dairy protein sources, such as milk.

[0003] When dairy protein ingredients are replaced with plant-derived protein ingredients, the types and functionality of proteins and fats contained therein, or the components that constitute aroma and flavor, are different, and therefore there is a demand for improvements in texture, taste, aroma, etc. For example, a method is known in which odorless and smooth protein is obtained by treating soy protein with lipase and then removing the resulting hydrolyzate (Patent Document 1).

[0004] Meanwhile, in recent years, interest in health has grown, leading to an increased demand for fermented foods such as yogurt. Various findings have been made regarding the use of enzymes in fermented foods, including a method for enhancing cheese flavor using lactase and lipase, a method for smoothing the texture of yogurt using transglutaminase, a method for improving the texture and flavor of yogurt using protein deamidating enzymes, and a method for improving the flavor of yogurt using lipase (see, for example, Patent Documents 2, 3, 4, and 5). However, these documents make no mention of improving the flavor or properties of plant protein foods such as soy milk and soy milk yogurt. Furthermore, a method for improving the flavor of soy milk yogurt using fumaric acid is known (see, for example, Patent Document 6).

[0005] Japanese Patent Laid-Open No. 6-30710, Special Publication No. 2011-525356, Japanese Patent Laid-Open No. 6-197688, International Publication No. 2006 / 075772, Pamphlet No. 2003-250482, Japanese Patent Laid-Open No. 7-31371

[0006] An object of the present invention is to provide a method for producing a vegetable protein food product that is at least excellent in flavor, and preferably has excellent properties in addition to flavor.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that, when producing a vegetable protein food, treating a protein material with lipase and protein deamidase can improve flavor (for example, impart a distinct fermented odor to fermented foods) and, in some cases, can improve properties (for example, improve smoothness) in addition to the flavor improvement effect, thereby completing the present invention described below. [1] A method for producing a vegetable protein food, characterized by treating a vegetable protein material containing protein and fat with lipase and protein deamidase. [2] A method for producing a vegetable protein food according to [1], wherein the vegetable protein material is a soybean-derived material, an oat-derived material, an almond-derived material, or a coconut-derived material. [3] A method for producing a vegetable protein food according to [1] or [2], comprising the following steps: (1) preparing a vegetable protein material containing protein and fat, and (2) treating the prepared vegetable protein material with lipase and protein deamidase. [4] A method for producing a plant protein food according to [3], wherein the plant protein food is a fermented food, and the method comprises the following step after step (2): (3) a step of fermenting by a microorganism. [5] A method for producing a plant protein food according to [4], wherein the fermented food is a lactic acid fermented food, and the microorganism in step (3) is a lactic acid bacterium. [6] A method for producing a plant protein food according to any one of [1] to [5], wherein the lipase is a lipase derived from Candida cylindraceae. [7] A method for producing a plant protein food according to any one of [1] to [6], wherein the protein deamidase is an enzyme that acts on glutamine residues in proteins. [8] A method for producing a plant protein food according to [7], wherein the protein deamidase is protein glutaminase. [9] A plant protein improving agent containing lipase and protein deamidase.

[10] The plant protein improving agent according to [9], wherein the lipase is a lipase derived from Candida cylindraceae.

[11] The vegetable protein improving agent according to [9] or

[10] , wherein the protein deamidase is an enzyme that acts on glutamine residues in proteins.

[12] The vegetable protein improving agent according to

[11] , wherein the protein deamidase is protein glutaminase.

[13] The vegetable protein improving agent according to any of [9] to

[11] , which is used as a fermentation odor enhancer for vegetable fermented foods.

[14] The vegetable protein improving agent according to any of [9] to

[12] , which is used as a smoothness improver for vegetable protein foods.

[0008] 1. Method for Producing a Vegetable Protein Food The method for producing a vegetable protein food of the present invention is characterized by allowing lipase and protein deamidating enzyme to act on a vegetable protein raw material containing protein and fats and oils.

[0009] 1-1. Vegetable Protein Foods The vegetable protein foods obtainable by the production method of the present invention are not particularly limited. Examples of such vegetable protein foods include fermented vegetable foods, which are imparted with at least an improved flavor (particularly a distinct fermented odor) by the production method of the present invention, and preferably those to which improved properties (particularly smoothness) are imparted in addition to the improved flavor. Specific examples of vegetable fermented foods include lactic acid-fermented vegetable foods, more specifically, fermented dairy substitute foods, and even more specifically, yogurt substitutes (also known as vegetable yogurt; having a solid content other than fats and oils of 8.0% or more by weight), dairy product lactic acid bacteria beverage substitutes (having a solid content other than fats and oils of 3.0% or more but less than 8.0% by weight), lactic acid bacteria beverage substitutes (having a solid content other than fats and oils of less than 3.0% by weight), and cheese substitutes (also known as vegetable cheese; a coagulated form of fermented dairy substitute foods). Examples of such plant protein foods include plant protein beverages (e.g., milk substitutes (also called plant-based milk)), tofu, meat substitutes prepared from plant materials, and dairy substitute products prepared from plant materials (e.g., processed plant-based milk products such as fermented milk substitute foods), which have been imparted with at least an improved flavor (particularly a distinct fermented odor) by the production method of the present invention, and preferably have been imparted with improved properties (particularly smoothness) in addition to the improved flavor. Examples of fermented milk substitute foods are as described above. Of the above examples, preferred plant protein foods for the production method of the present invention are lactic acid-fermented plant foods, and among lactic acid-fermented plant foods, fermented milk substitute foods are preferred, and yogurt substitutes (plant-based yogurts) are particularly preferred.

[0010] 1-2. Vegetable protein ingredients There are no particular restrictions on the origin or properties of the vegetable protein ingredients used to make vegetable protein foods.

[0011] For example, the origin of the protein contained in the plant protein raw material is not particularly limited as long as it is a plant, and specific examples include beans such as soybeans, green peas, lentils, chickpeas, black beans, broad beans, mung beans, lupin beans, and kidney beans; grains such as wheat, barley, oats, rice, rye, buckwheat, barnyard millet, foxtail millet, and teff; nuts such as almonds, coconuts, peanuts, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, and pine nuts; and seeds such as hemp seeds, chia seeds, quinoa, amaranth, canary seeds, and flaxseed. Proteins contained in the vegetable protein raw material may also be those obtained by chemical partial hydrolysis of the above proteins using acid, alkali, etc., enzymatic partial hydrolysis using proteases, etc., proteins chemically modified with various reagents, or synthetic peptides. In the present invention, these proteins may be derived from a single plant or may be derived from two or more plants in combination. Among these proteins, preferred are proteins derived from beans, grains, and nuts, and more preferred are proteins derived from soybeans, almonds, oats, and coconuts.

[0012] Furthermore, vegetable protein raw materials contain fats and oils in addition to proteins. The origin of the fats and oils is not particularly limited as long as it is a plant. Specific examples of plants from which the fats and oils are derived can be selected from the specific examples of plants listed as the origin of proteins. Furthermore, the plant from which the fats and oils are derived may be the same as or different from the plant from which the protein is derived, but is preferably the same as the plant from which the protein is derived.

[0013] The form of the vegetable protein raw material to be subjected to the enzyme treatment is not particularly limited, but preferably includes a liquid, a slurry, or a paste, and more preferably includes a liquid, a slurry, or a paste formed as an emulsion with fats or oils.

[0014] A preferred example of the vegetable protein raw material to be subjected to enzyme treatment in the present invention is vegetable milk, more preferred examples are vegetable milk made from beans, vegetable milk made from grains, and vegetable milk made from nuts, and even more preferred examples are soy milk, almond milk, oat milk, and coconut milk.

[0015] The method for preparing the vegetable protein material is not particularly limited. For example, the vegetable protein material can be prepared by the following preparation method. As the preparation method, a person skilled in the art can appropriately select from known methods a method capable of preparing a mixture containing the above protein and fat / oil, preferably an emulsion in which the above fat / oil is dispersed in water containing the above protein.

[0016] Specific examples of methods for preparing vegetable protein materials include (i) dispersing dried powder of the plant from which the protein and oil are derived in water; (ii) crushing or grinding the plant from which the protein and oil are derived in water, dispersing the crushed or ground plant, and filtering it if necessary; (iii) a mixture obtained by removing at least a portion of the components other than the plant-derived protein and oil from the liquid obtained by method (i) or (ii), thereby increasing the content of the plant-derived protein and oil; (iv) a liquid obtained by further diluting the liquid obtained by method (i) or (ii) with water; and (v) a liquid obtained by dissolving and / or dispersing a solid, such as a powder, prepared by drying any of the liquids (i) to (iv) in water. When in liquid form, the vegetable protein materials prepared by methods (i) to (iv) can be used as vegetable milk.

[0017] Another specific example of a method for preparing a vegetable protein raw material is a method in which a protein purified from any of the above plants is mixed with an oil or fat purified from any of the above plants, for example, in water.

[0018] In addition to the above, particularly when using plant milk as a plant protein raw material, commercially available plant milk can be used as is, diluted with water, or concentrated by removing water.

[0019] The protein content and fat / oil content, as well as their ratio, in the vegetable protein material are not particularly limited and can be appropriately determined depending on the desired properties and state of the vegetable protein food, the type of plant from which it is derived, and other factors. For example, the protein content in the vegetable protein material may be, for example, 0.1 to 30% by weight, preferably 0.3 to 20% by weight, 0.5 to 15% by weight, and more preferably 0.9 to 11% by weight. The fat / oil content in the vegetable protein material may be, for example, 0.5 to 30% by weight, preferably 1 to 25% by weight, 2 to 20% by weight, and more preferably 2.5 to 15% by weight. Furthermore, the protein to fat content ratio in the vegetable protein material may be, for example, 0.05 to 50 parts by weight, 0.1 to 30 parts by weight, preferably 0.3 to 20 parts by weight, 0.5 to 10 parts by weight, and more preferably 0.7 to 7 parts by weight, in terms of the fat / oil content per part by weight of protein.

[0020] Furthermore, the vegetable protein raw material may contain salts, sugars, proteins other than vegetable proteins, flavorings, moisturizers, coloring agents, etc., as needed.

[0021] 1-3. Lipase and Protein Amide Enzyme The lipase referred to in the present invention is an enzyme that liberates fatty acids from fats and oils by hydrolysis. The type and origin of the lipase that can be used in the present invention are not particularly limited. The origin may be, for example, animal, plant, or microbial. For example, lipases derived from the genus Rhizopus, Penicillium, Burkholderia, Aspergillus, Candida, Pichia, Chromobacterium, Pseudomonas, Mucor, Thermomyces, or Geotrichum can be used. Preferably, lipase derived from the genus Candida is used. An example of a lipase derived from the genus Candida is a lipase produced by Candida cylindracea (specifically, lipase AY (Amano Enzyme Co., Ltd.)).

[0022] The protein deamidase referred to in the present invention is an enzyme that deamidates the amide groups of glutamine residues and asparagine residues in proteins. The type and origin of the protein deamidase that can be used in the present invention are not particularly limited. The origin may be, for example, animal-derived, plant-derived, or microbial-derived. Well-known examples of enzymes that deamidate glutamine residues in proteins include, but are not limited to, protein glutaminase from Chryseobacterium proteolyticum (Eur J Biochem, 268 (5), 1410, 2001, Protein-glutaminase From Chryseobacterium Proteolyticum, an Enzyme That Deamidates Glutaminyl Residues in Proteins. Purification, Characterization and Gene Cloning, S Yamaguchi 1 , DJ Jeenes, DB Archer or Front Microbiol , 9, 1975, 2018, Complete Genome Sequence and Characterization of a Protein-Glutaminase Producing Strain, Chryseobacterium proteolyticum QSH1265, Ruidan Qu, Xiaoyu Zhu, Min Tian, ​​Yingjie Liu, Wenjuan Yan, Jian Ye, Hongliang Gao, Jing Huang). Enzymes that deamidate asparagine residues in proteins are disclosed, for example, in WO2015 / 133590, but are not limited thereto. Protein deamidating enzymes as used herein also include enzymes that deiminate arginine residues. Known examples of enzymes that deiminate arginine residues include arginine deiminase derived from Fusarium graminearum.

[0023] Deamidation of glutamine and asparagine residues in proteins, generally resulting in the generation of carboxyl groups, increases the protein's negative charge, lowering its isoelectric point and increasing its hydration potential. Furthermore, increased electrostatic repulsion leads to decreased protein-protein interactions, i.e., decreased association. These changes significantly increase the protein's solubility and water dispersibility. Furthermore, the increased negative charge of proteins unfolds the protein, altering its conformation and exposing hydrophobic regions previously buried in the interior of the molecule to the molecular surface. Therefore, deamidated proteins possess amphiphilic properties, making them ideal surfactants, significantly improving their emulsifying power, emulsion stability, foaming ability, and foam stability. Thus, protein deamidation improves various functional properties of proteins, dramatically expanding their applications (e.g., Molecular Approaches to Improving Food Quality and Safety, D. Chatnagar and T.E. Cleveland, eds., Van Nostrand Reinhold, New York, 1992, p. 37). Deimination of arginine residues in a protein also increases the hydrophobicity of the protein, changing the higher-order structure of the protein.

[0024] The method for obtaining the lipase and protein deamidase usable in the present invention is not particularly limited. For example, when the lipase and / or protein deamidase is an enzyme derived from a microorganism, it may be accumulated either intracellularly or extracellularly. Furthermore, not only naturally occurring enzymes but also enzymes produced by genetic engineering or cell engineering techniques may be used. Furthermore, enzyme proteins modified by protein engineering techniques may also be used. Furthermore, it is desirable to use lipase and protein deamidase (e.g., protein glutaminase) that have been purified to a high purity, but the purity is not important as long as the desired reaction is possible. Furthermore, enzyme preparations may be used as the lipase and protein deamidase, and in such cases, various salts, sugars, proteins, lipids, surfactants, etc. may be added to the enzyme preparation as an enzyme stabilizer.

[0025] The amounts of the enzymes used are not particularly limited. Usually, the amounts of the enzymes are 0.01 to 2000 U, preferably 0.1 to 1000 U, more preferably 1 to 500 U, and even more preferably 10 to 400 U, of lipase and 0.1 to 10000 U, preferably 1 to 1000 U, more preferably 2 to 100 U, even more preferably 3 to 50 U, and even more preferably 4 to 20 U, of protein deamidase, relative to 1 g of protein, as measured by a kit method.

[0026] Furthermore, when the vegetable protein raw material is made from beans (preferably soybeans), more preferably soy milk, the amount of lipase used per 1 g of protein is, for example, 1 to 500 U, preferably 5 to 300 U, more preferably 10 to 230 U, in terms of activity value measured by a kit method, and the amount of protein amide enzyme used per 1 g of protein is, for example, 1 to 40 U, ​​preferably 2 to 20 U, more preferably 4 to 16 U.

[0027] When the plant raw material is a cereal (preferably oats), more preferably oat milk, the amount of lipase to be used per 1 g of protein, in terms of activity value measured by a kit method, is, for example, 50 to 600 U, preferably 100 to 500 U, more preferably 200 to 400 U, and even more preferably 250 to 350 U, and the amount of protein deamidating enzyme to be used per 1 g of protein is, for example, 0.5 to 15 U, preferably 2 to 10 U, and more preferably 4 to 7 U.

[0028] When the plant raw material is a nut (preferably almond), more preferably almond milk, the amount of lipase to be used per 1 g of protein, in terms of activity value measured by a kit method, is, for example, 100 to 700 U, preferably 200 to 600 U, more preferably 300 to 500 U, and even more preferably 350 to 400 U, and the amount of protein deamidating enzyme to be used per 1 g of protein is, for example, 0.5 to 15 U, preferably 2 to 10 U, and more preferably 4 to 7 U.

[0029] When the plant raw material is a nut (preferably coconut) raw material, more preferably coconut milk, the amount of lipase to be used per 1 g of protein, in terms of activity value measured by a kit method, is, for example, 1 to 100 U, preferably 5 to 50 U, more preferably 10 to 25 U, and even more preferably 15 to 20 U, and the amount of protein deamidating enzyme to be used per 1 g of protein is, for example, 5 to 40 U, ​​preferably 10 to 20 U, and more preferably 12 to 17 U.

[0030] 1-4. Operating Procedures, etc. The reaction time, temperature, and pH of the reaction solution for reacting lipase and protein deamidase with a vegetable protein material are not particularly limited. The reaction temperature is, for example, 5 to 80°C, preferably 20 to 70°C, and more preferably 30 to 60°C. The pH of the reaction solution is, for example, 2 to 10, preferably 4 to 8. The reaction time is, for example, 10 seconds to 48 hours, preferably 10 minutes to 24 hours. The above reaction conditions change the physical properties of the vegetable protein material, improving at least the flavor, and in some cases improving the properties in addition to the flavor. These reaction conditions are appropriately selected depending on the desired vegetable protein food. The optimal reaction conditions can be determined through preliminary experiments.

[0031] In the method for producing a vegetable protein food of the present invention, lipase and protein deamidase are allowed to act on a vegetable protein material. In other words, the vegetable protein material is treated with lipase and protein deamidase. The order in which the enzymes are acted on (i.e., the order of lipase treatment and protein deamidase treatment) is not particularly limited, but simultaneous treatment is preferred for the purpose of improving work efficiency, etc. It has been confirmed that simultaneous treatment with lipase and protein deamidase produces excellent effects (see Examples below).

[0032] By using the production method of the present invention, it is possible to produce a food product with at least an improved flavor, and preferably a food product with improved properties in addition to the flavor. One embodiment of the production method of the present invention for a vegetable protein food product comprises the following steps (1) and (2). Note that an enzyme deactivation step may be added after step (2): (1) a step of preparing a vegetable protein material containing protein and fats and oils; and (2) a step of treating the prepared vegetable protein material with lipase and protein deamidating enzyme.

[0033] When the vegetable protein food to be produced is a fermented food, the above step (2) is followed by the following fermentation step (3): (3) Step of fermenting with microorganisms

[0034] Various microorganisms (mold, yeast, bacteria) are used in the fermentation process. Microorganisms that are suitable for the plant protein food to be produced are used. For example, when producing a lactic acid fermented plant protein food, lactic acid bacteria can be used. In other words, in this example, a "step of fermentation using lactic acid bacteria" is carried out.

[0035] 2. Vegetable Protein Foods The present invention also provides a vegetable protein food obtained by the above-described production method. The vegetable protein of the present invention is not particularly limited as described above, and examples thereof include fermented vegetable foods, which are imparted with at least an improved flavor (particularly a distinct fermented odor) by the production method of the present invention, and preferably are imparted with improved properties (particularly smoothness) in addition to the improved flavor. Specific examples of fermented vegetable foods include lactic acid-fermented vegetable foods, more specifically, fermented dairy substitute foods, and even more specifically, yogurt substitutes (also known as vegetable yogurt; having a solid content other than fats and oils of 8.0% or more by weight), dairy product lactic acid bacteria beverages (having a solid content other than fats and oils of 3.0% or more but less than 8.0% by weight), lactic acid bacteria beverage substitutes (having a solid content other than fats and oils of less than 3.0% by weight), and cheese substitutes (also known as vegetable cheeses; a coagulated form of fermented dairy substitute foods). Examples of the plant protein foods of the present invention include plant protein beverages (e.g., milk substitutes (also called plant-based milk)), tofu, meat substitutes prepared from plant materials, and dairy substitute products prepared from plant materials (e.g., processed plant-based milk products such as fermented milk substitute foods), which have been imparted with at least an improved flavor by the production method of the present invention, and preferably have been imparted with improved properties (particularly smoothness) in addition to the improved flavor. Examples of fermented milk substitute foods are as described above. Of the above examples, preferred plant protein foods of the present invention are lactic acid-fermented plant foods, and among lactic acid-fermented plant foods, fermented milk substitute foods are preferred, and yogurt substitutes (plant-based yogurts) are particularly preferred.

[0036] 3. Protein Improving Agent The present invention also provides a protein improving agent that can be used to improve vegetable proteins. The protein improving agent of the present invention is typically used in the production method of the present invention. The protein improving agent of the present invention contains lipase and protein deamidating enzyme as active ingredients. Details of the lipase and protein deamidating enzyme are as described above (section 1. Production method of vegetable protein food), and therefore further explanation will be omitted.

[0037] The protein improving agent of the present invention can impart a distinct fermented odor to the resulting plant-based fermented food, particularly when producing such a food. This makes it possible to impart a flavor closer to that of an animal-based fermented food (preferably a lactic-fermented animal-based fermented food, particularly yogurt) when producing a substitute for the animal-based fermented food (i.e., a plant-based fermented food, preferably a lactic-fermented plant-based fermented food, particularly plant-based yogurt). Therefore, the protein improving agent of the present invention is particularly useful when used as a fermentation odor enhancer for a plant-based fermented food.

[0038] The protein improving agent of the present invention can, in some cases, impart smoothness to the resulting vegetable protein food. Specific examples of how smoothness is imparted include enhancing softness, homogeneity, and / or viscosity. A preferred example of a case in which the protein improving agent of the present invention imparts the above-mentioned smoothness is when the protein contained in the vegetable protein raw material on which the protein improving agent of the present invention is applied is derived from the beans and nuts listed in 1-2 above, more preferably soybeans and coconuts. Another preferred example of a case in which the protein improving agent of the present invention imparts the above-mentioned smoothness is when the vegetable protein food obtained by using the protein improving agent of the present invention is a vegetable fermented food, more preferably a lactic acid fermented vegetable fermented food, particularly a vegetable yogurt. Therefore, the protein improving agent of the present invention is also useful when used as a smoothness improver for vegetable protein foods.

[0039] The present invention will now be further described with reference to examples.

[0040] In the following test examples, lipase hydrolysis activity was measured by the following method. <Activity measurement method (kit method)> Lipase activity measurement (kit method) was performed using Lipase Kit S (manufactured by SB Biosciences) according to the manual attached to the kit. However, the buffer used was the included buffer adjusted to pH 7, and acetone was used as the reaction stop solution. Activity values ​​were calculated using a calibration curve prepared using Lipase AY "Amano" 30SD (30,000 u / g).

[0041] Meanwhile, the enzyme activity of protein glutaminase was measured using Z-Gln-Gly as a substrate according to the method described below. Activity Measurement Method: 10 μl of enzyme solution was added to 100 μl of 176 mmol / L phosphate buffer (pH 6.5) containing 10 mmol / L Z-Gln-Gly. The mixture was incubated at 37°C for 60 minutes, and then 100 μl of 12% trichloroacetic acid solution was added to terminate the reaction. After centrifugation (15,000 rpm, 4°C, 5 minutes), the supernatant was measured using F-kit ammonia (Boehringer Mannheim) as follows, yielding a measurement value (A1). Separately, a similar measurement was performed using water instead of the enzyme solution, yielding a measurement value (A2). 10 μl of the supernatant and 190 μl of water were added to 100 μl of F-kit ammonia Reagent 2, and the mixture was left at room temperature for 5 minutes. The absorbance at 340 nm (E1) was measured using 100 μl of the mixture. To the remaining 200 μl, 1.0 μl of Reagent 3 (glutamate dehydrogenase) was added, and the mixture was left at room temperature for an additional 20 minutes. The absorbance (E2) of the remaining 200 μl was measured at 340 nm. The amount of enzyme required to liberate 1 μmol of ammonia per minute under the above conditions was defined as 1 unit (1 u), and calculated according to the following formula: u / ml = 1.76 × [A1(E1-E2)-A2(E1-E2)]

[0042] In the following test examples, the flavor was evaluated based on the following criteria: In addition to the flavor, the properties were also evaluated based on the following criteria.

[0043] <Evaluation of the effect of improving flavor 1 (aroma)> ---: No fermentation odor, strong raw material odor ---: No fermentation odor, weak raw material odor ---: No fermentation odor -: Weak fermentation odor +: Slightly strong fermentation odor ++: Strong fermentation odor

[0044] <Evaluation of the effect of improving flavor 2 (aroma)> -: Weak sourness +: Slightly strong sourness ++: Strong sourness

[0045] <Evaluation of the improvement effect on property 1 (smoothness)> ×: Not smooth (hard and inhomogeneous) △: Smooth (soft and homogeneous) ○: Remarkably smooth (remarkably soft and homogeneous) <Evaluation of the improvement effect on property 2 (smoothness)> ×: Not smooth (low viscosity) △: Slightly smooth (slightly viscous) ○: Smooth (sufficiently viscous)

[0046] Test Example 1: 200 g of soybeans were soaked in 400 mL of water, drained, and peeled. 500 mL of water was added to the peeled soybeans, which were then ground in a blender for 1 minute and filtered to obtain soy milk. 75 μg of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (5 μg per gram of raw protein) and 3400 μg (kit method) of lipase (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (300 μg per gram of raw oil) were added to 150 mL of the resulting soy milk (protein concentration 10 wt. %, oil concentration 7.5 wt. ), and the mixture was treated at 50°C for 1 hour. The enzymes were inactivated by heat treatment at 72°C for 15 minutes, followed by cooling to 40°C. To 100 mL of the obtained enzyme-treated soy milk, 3 g of sugar and 15 g of commercially available soy milk yogurt (Rivon Soygurt, manufactured by Thai-Dairy Co., Ltd.) as a starter were added, and the mixture was fermented at 42°C for 10 hours, followed by cooling to 4°C to obtain soy milk yogurt (Example 1). For comparison, soy milk yogurt was obtained in the same manner as in Example 1, except that only protein glutaminase was used for the enzyme treatment (Comparative Example 2). As a control, soy milk yogurt was obtained in the same manner as in Example 1, except that the enzyme treatment was omitted (Comparative Example 1).

[0047] The soy milk yogurt thus obtained was subjected to a sensory test for the above-mentioned flavor 1 and property 1. The results of the sensory test are shown in Table 1.

[0048]

[0049] As shown in Table 1, the soy milk yogurt without enzyme treatment (Comparative Example 1) had a strong soybean smell and was hard and inhomogeneous, resulting in a lack of smoothness. Furthermore, although PG treatment reduced the soybean smell and provided a smooth texture (Comparative Example 2), the improvement in these properties was insufficient. Surprisingly, adding lipase treatment to PG treatment (i.e., the combined use of PG and lipase) resulted in a strong fermented smell similar to that of regular yogurt, and further improved smoothness (Example 1).

[0050] Test Example 2: 200 g of soybeans were soaked in 400 mL of water. After draining, the soybeans (with skins) were added to 500 mL of water and ground in a blender for 1 minute. The mixture was then filtered to obtain soy milk. To 150 mL of soy milk (protein concentration 10 wt%, oil concentration 7.5 wt%), 75 μl or 225 μl of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (5 μl or 15 μl per gram of raw protein) and 170 μl (kit method) of lipase AY (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (15 μl per gram of raw oil) were added and the mixture was treated at 50°C for 1 hour. The enzymes were inactivated by heat treatment at 72°C for 15 minutes, followed by cooling to 40°C. To 100 mL of the obtained enzyme-treated soy milk, 3 g of sugar and 15 g of commercially available soy milk yogurt (Rivon Soygurt, manufactured by Thai-Dairy Co., Ltd.) as a starter were added, and the mixture was fermented at 42°C for 10 hours, followed by cooling to 4°C to obtain soy milk yogurt (Example 2). For comparison, soy milk yogurt was obtained in the same manner as in Example 2, except that only protein glutaminase was used for the enzyme treatment (Comparative Examples 3 and 4). As a control, soy milk yogurt was obtained in the same manner as in Example 2, except that the enzyme treatment was omitted (Comparative Example 5).

[0051] The soy milk yogurt thus obtained was subjected to a sensory test for the above-mentioned flavor 1 and property 1. The results of the sensory test are shown in Table 2.

[0052] As shown in Table 2, even when whole soybeans (soybeans with skin) were used, the combined use of PG and lipase was able to impart a strong fermented odor favorable for yogurt, replacing the soybean odor, and resulting in a smooth texture (Example 2).On the other hand, treatment with PG alone only slightly reduced the soybean odor, and although the texture became soft and smooth, the improvement was insufficient (Comparative Examples 3 and 4).

[0053] Test Example 3: 200 g of almonds were soaked in 400 mL of water. 500 mL of water was added to the drained almonds, which were then ground in a blender for 1 minute. The mixture was then filtered to obtain almond milk. 45 μg of protein glutaminase (Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.) (5 μg per gram of raw protein) and 3400 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, manufactured by Amano Enzyme Inc.) (160 μg per gram of raw oil) were added to 150 mL of almond milk (protein concentration 6% by weight, oil concentration 14% by weight) and treated at 50°C for 1 hour. The enzymes were inactivated by heat treatment at 72°C for 15 minutes, followed by cooling to 40°C. To 100 mL of the resulting enzyme-treated almond milk, 3 g of sugar and 15 g of commercially available almond yogurt (Hooray almond yogurt, manufactured by Crossmax Retail Co. Ltd.) were added as a starter, and the mixture was fermented at 42°C for 10 hours and then cooled to 4°C to obtain almond yogurt (Example 3). For comparison, almond yogurt was obtained in the same manner as in Example 3, except that only protein glutaminase was used for the enzyme treatment (Comparative Example 6). Furthermore, almond yogurt was obtained in the same manner as in Example 3, except that the enzyme treatment was omitted (Comparative Example 7).

[0054] The obtained almond yogurt was subjected to a sensory test for the above-mentioned flavor 1 and flavor 2. The results of the sensory test are shown in Table 3.

[0055] As shown in Table 3, in the case of almond yogurt, although PG treatment alone showed some improvement in aroma, the improvement was insufficient (Comparative Example 6), but it was found that the fermented odor could be strengthened by further adding lipase treatment (Example 3).Furthermore, in terms of taste, although the effect of enhancing sourness by PG treatment alone was not confirmed (Comparative Example 6), it was found that the strong sourness characteristic of yogurt could be imparted by further adding lipase treatment (Example 3).

[0056] Test Example 4: 200 g of oats were soaked in 400 mL of water. 500 mL of water was added to the drained oats, which were then ground in a blender for 1 minute. The mixture was then filtered to obtain oat milk. 23 μg of protein glutaminase (Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.) (5 μg per gram of raw protein) and 1300 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, manufactured by Amano Enzyme Inc.) (300 μg per gram of raw oil) were added to 150 mL of oat milk (protein concentration 3 wt.%, oil concentration 2.9 wt.), and the mixture was treated at 50°C for 1 hour. The enzymes were inactivated by heat treatment at 72°C for 15 minutes, followed by cooling to 40°C. To 100 mL of the resulting enzyme-treated oat milk, 3 g of sugar and 15 g of commercially available almond yogurt (Hooray almond yogurt, manufactured by Crossmax Retail Co. Ltd.) were added as a starter, and the mixture was fermented at 42°C for 10 hours and then cooled to 4°C to obtain oat yogurt (Example 4). For comparison, oat yogurt was obtained in the same manner as in Example 4, except that only protein glutaminase was used for the enzyme treatment (Comparative Example 8). Also, oat yogurt was obtained in the same manner as in Example 4, except that the enzyme treatment was omitted (Comparative Example 9).

[0057] The resulting oat yogurt was subjected to a sensory test for the above-mentioned flavor 1. The results of the sensory test are shown in Table 4.

[0058] As shown in Table 4, PG treatment alone also improved the flavor of oat yogurt, but the degree of improvement was insufficient (Comparative Example 8). However, it was found that by further combining lipase treatment, a strong fermented odor that is desirable for yogurt could be imparted (Example 4).

[0059] Test Example 5: 50 g of coconut milk was added to 100 mL of water to obtain diluted coconut milk (liquid). 22 μg of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (15 μg per gram of raw protein) and 27 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (3 μg per gram of raw oil) were added to 150 mL of the diluted coconut milk (protein concentration 1 wt%, fat concentration 6 wt%) and treated at 50°C for 1 hour. 3 g of modified starch and 4.5 g of sugar were added, and the mixture was heated at 85°C for 30 minutes to inactivate the enzymes, followed by cooling to 40°C. To 100 mL of the resulting enzyme-treated coconut milk, 15 g of commercially available coconut yogurt (Agrilife Cocogurt, Earth Born Co. Ltd.) was added, and the mixture was fermented at 42°C for 10 hours, followed by cooling to 4°C to obtain coconut yogurt (Example 5). For comparison, coconut yogurt was obtained in the same manner as in Example 5 except that only protein glutaminase was used for the enzyme treatment (Comparative Example 10), and coconut yogurt was also obtained in the same manner as in Example 5 except that the enzyme treatment was omitted (Comparative Example 11).

[0060] The obtained coconut yogurt was subjected to a sensory test for the above-mentioned flavor 1 and property 2. The results of the sensory test are shown in Table 5.

[0061] As shown in Table 5, it was found that the combined use of PG and lipase in coconut yogurt not only altered the odor to a strong fermented odor that is favorable for yogurt, but also imparted viscosity and a smooth texture (Example 5).On the other hand, treatment with PG alone provided a smooth texture, but did not change the coconut odor and did not impart any fermented odor at all (Comparative Example 10).

[0062] According to the production method of the present invention, a plant protein food with excellent flavor can be obtained. Examples of plant protein foods produced by the present invention include fermented plant foods (e.g., lactic acid fermented plant foods, more specifically, milk substitute fermented foods, even more specifically, yogurt substitutes (plant-based yogurts), dairy substitute lactic acid bacteria drinks, lactic acid bacteria drinks, and cheese substitutes (plant-based cheeses)); plant protein drinks (e.g., milk substitutes (plant-based milk)), tofu, meat substitutes prepared from plant materials, and dairy substitutes prepared from plant materials (e.g., plant-based milk processed products such as milk substitute fermented foods).

[0063] The present invention is not limited to the above-described embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, and other publications explicitly stated in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for the production of plant protein foods, which includes lipase and protein deamides to act on plant protein raw materials containing protein, fat, and oil.

2. A method for the production of plant protein foods under claim 1, where the plant protein raw materials are soybean derivatives, oat derivatives, almond derivatives, or coconut derivatives.

3. A method for the production of plant protein foods under claim 1 or 2, where the method includes the following steps: (1) the preparation step of the plant protein raw materials containing protein, fat, and oil; and (2) the treatment step of the prepared plant protein raw materials with lipase and protein deamides.

4. A method for the production of plant protein foods under claim 3, where the plant protein food is a fermented food, and the method includes the following steps after step (2): (3) the microbial fermentation step.

5. A method for the production of plant protein foods under claim 4, where the fermented food is a lactic acid fermented food, and the microbial fermentation step (3) is Lactobacillus. 6.A method for the production of plant protein foods according to one of the claims 1 through 5, where lipase is a lipase derivative of Candidacylindracea.

7. A method for the production of plant protein foods according to one of the claims 1 through 6, where protein deamicase is an enzyme acting on glutamine residue in protein.

8. A method for the production of plant protein foods according to claim 7, where protein deamicase is a protein glutaminese.

9. A plant protein modifier comprising lipase and protein deamicase.

10. A modifier. Plant protein modifiers under claim 9, where lipase is a lipase derived from Candidacylindracea11. Plant protein modifiers under claim 9 or 10, where protein amidase is an enzyme acting on glutamine residue in proteins12. Plant protein modifiers under claim 1I, where protein amidase is a protein glutaminase13. Plant protein modifiers under one of claims 9 through 11, where the plant protein modifier is used as a fermentation flavor enhancer for plant-fermented foods14.Plant protein improvers under any of the claims 9 through 12, whereby the plant protein improvers are used as smoothing enhancers for plant-fermented foods;