Method for producing plant protein food

Abstract

The present invention addresses the problem of providing a novel production method of a vegetable protein food. This method comprises treating a starting vegetable protein material with both of a lipase and a protein deamidase (for example, protein glutaminase) to thereby improve the flavor of a vegetable protein food.

Description

[Technical Field]

[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). [Background technology]

[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, as well as the components that make up their aroma and flavor, are different, and therefore improvements in texture, taste, aroma, etc. For example, a method is known in which odorless, smooth protein is obtained by treating soy protein with lipase and then removing the resulting hydrolyzate (Patent Document 1).

[0004] Meanwhile, with growing interest in health in recent years, there has been an increase in 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 using fumaric acid is known as a method for improving the flavor of soy milk yogurt (see, for example, Patent Document 6). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 6-30710 [Patent Document 2] Special Publication No. 2011-525356 [Patent Document 3] Japanese Patent Application Publication No. 6-197688 [Patent Document 4] International Publication No. 2006 / 075772 Pamphlet [Patent Document 5] Japanese Patent Application Laid-Open No. 2003-250482 [Patent Document 6] Japanese Patent Application Publication No. 7-31371 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that, when producing plant protein foods, allowing lipase and protein deamidase to act on a protein raw material can provide a flavor-improving effect (e.g., the effect of imparting a distinct fermented odor to fermented foods), and in some cases, can provide a property-improving effect (e.g., an effect of improving smoothness) in addition to the flavor-improving effect, and have thus completed the present invention described below. [1] A method for producing a vegetable protein food, characterized by reacting lipase and protein deamidating enzyme with a vegetable protein raw material containing protein and fats and oils. [2] A method for producing a vegetable protein food according to [1], wherein the vegetable protein raw material is a soybean-derived raw material, an oat-derived raw material, an almond-derived raw material, or a coconut-derived raw material. [3] A method for producing the plant protein food according to [1] or [2], comprising the following steps: (1) preparing a vegetable protein raw material containing protein and fat; (2) A process in which the prepared vegetable protein raw material is treated with lipase and protein deamidating enzyme. [4] The 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) The process of fermentation by microorganisms. [5] The 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 described in any one of [1] to [5], wherein the lipase is a lipase derived from Candida cylindracea. [7] The 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] The method for producing a plant protein food according to [7], wherein the protein deamidase is protein glutaminase. [9] A vegetable protein improver containing lipase and protein deamidating enzyme.

[10] The vegetable protein improver according to [9], wherein the lipase is a lipase derived from Candida cylindracea.

[11] The vegetable protein improver according to [9] or

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

[12] The vegetable protein improver according to

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

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

[12] , which is used as a smoothness improver for vegetable protein foods. DETAILED DESCRIPTION OF THE INVENTION

[0008] 1. Manufacturing method of plant 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. Plant-based protein foods The plant protein foods obtainable by the production method of the present invention are not particularly limited. Examples of such plant protein foods include plant fermented 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 plant fermented foods include lactic acid fermented plant foods, more specifically, dairy fermented foods, and even more specifically, yogurt substitutes (also known as plant-based yogurt; solids other than fats and oils: 8.0% by weight or more), dairy lactic acid bacteria beverage substitutes (solids other than fats and oils: 3.0% by weight or more but less than 8.0% by weight), dairy lactic acid bacteria beverage substitutes (solids other than fats and oils: less than 3.0% by weight), and cheese substitutes (also known as plant-based cheese; a coagulated form of dairy fermented foods). Examples of such plant protein foods include plant protein drinks (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. Plant-based protein ingredients There are no particular restrictions on the origin, properties, etc. of the vegetable protein raw materials that are 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 vegetable protein raw materials may also be those obtained by chemical partial hydrolysis of the above proteins using acids, alkalis, 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 used in combination with two or more plant proteins. Of 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 a 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 a 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 a 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 a 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 enzymes 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. Lipases derived from the genus Candida are preferred. 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 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, it may be not only naturally occurring but also one produced by genetic engineering or cell engineering techniques. It may also be an enzyme protein modified by protein engineering techniques. 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, an enzyme preparation may be used as the lipase and protein deamidase, and in this case, the enzyme preparation may contain various salts, sugars, proteins, lipids, surfactants, etc. 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 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, in terms of activity value measured by a kit method, 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. Operation procedures, etc. The reaction time, temperature, and pH of the reaction solution for reacting lipase and protein deamidating enzyme 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. 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 the Examples below).

[0032] By using the production method of the present invention, it is possible to produce foods that have at least improved flavor, and preferably foods that have improved properties in addition to improved flavor. One embodiment of the production method of the plant protein food of the present invention comprises the following steps (1) and (2). Note that an enzyme inactivation step may be added after step (2). (1) A process for preparing a vegetable protein raw material containing protein and fat. (2) A process of treating the prepared vegetable protein raw 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) The process of fermentation by 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. Plant-based protein foods The present invention also provides a plant protein food obtained by the above-mentioned production method. The plant protein of the present invention is not particularly limited as described above, and examples include plant-based fermented 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 plant-based fermented foods include lactic acid-fermented plant foods, more specifically, dairy fermented 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 lactic acid bacteria beverage substitutes (3.0% or more by weight but less than 8.0% by weight of solids other than fats and oils), dairy 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 dairy fermented foods). Further, 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 improvers 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 foods), and therefore further explanation is 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 such an 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 can be imparted include enhancing softness, homogeneity, and / or viscosity. A preferred example of how the protein improving agent of the present invention imparts 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 how the protein improving agent of the present invention imparts 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 as a smoothness improver for vegetable protein foods.

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

[0040] In the following test examples, the hydrolytic activity of lipase 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 solution used was 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] On the other hand, the enzyme activity of protein glutaminase was measured by the method described below using Z-Gln-Gly as a substrate. <Activity measurement method> Ten microliters of enzyme solution was added to 100 μl of 176 mmol / L phosphate buffer (pH 6.5) containing 10 mmol / L Z-Gln-Gly, and the mixture was incubated at 37°C for 60 minutes. The reaction was then stopped by adding 100 μl of 12% trichloroacetic acid solution. 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 at 340 nm was measured. 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 the absorbance was 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 fermented smell, strong raw material smell ---: No fermentation smell, weak raw material smell --: No fermented smell -: Weak fermented odor +: Slightly strong fermented odor ++: Strong fermented odor

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

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

[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, ground in a blender for 1 minute, and then filtered to obtain soy milk. To 150 mL of the resulting soy milk (protein concentration 10 wt%, oil concentration 7.5 wt%), 75 μg of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (5 μg per 1 g of raw protein) and 3400 μg (kit method) of lipase (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (300 μg per 1 g of raw oil) were added and the mixture was heated at 50°C for 1 hour. The enzymes were inactivated by heat treatment at 72°C for 15 minutes, and then cooled 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] [Table 1]

[0049] As shown in Table 1, 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, PG treatment reduced the soybean smell and provided a smooth texture (Comparative Example 2), but the improvement in these properties was insufficient. Surprisingly, adding lipase treatment to PG treatment (i.e., using PG and lipase in combination) 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 husks) 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. [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. After draining, the almonds were mixed with 500 mL of water and 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, Amano Enzyme Inc.) (5 μg per gram of raw protein) and 3400 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (160 μg per gram of raw oil) were added to 150 mL of almond milk (protein concentration 6 wt%, oil concentration 14 wt%) 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 obtained 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, followed by cooling 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. [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 by further adding lipase treatment, the fermented odor could be strengthened (Example 3).Furthermore, with regard to taste, although the effect of enhancing sourness by PG treatment alone was not confirmed (Comparative Example 6), it was found that by further adding lipase treatment, it was possible to impart a strong sourness characteristic of yogurt (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 and filtered to obtain oat milk. 23 μg of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (5 μg per gram of raw protein) and 1300 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (300 μg per gram of raw oil) were added to 150 mL of oat milk (protein concentration 3 wt%, fat concentration 2.9 wt%) 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 obtained 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, followed by cooling 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). Furthermore, 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. [Table 4]

[0058] As shown in Table 4, PG treatment alone improved the flavor of oat yogurt, but the improvement was insufficient (Comparative Example 8). However, it was found that by further adding 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). To 150 mL of the diluted coconut milk (protein concentration 1 wt%, fat concentration 6 wt%), 22 μg of protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) (15 μg per g of raw protein) and 27 μg (kit method) of lipase AY (Lipase AY "Amano" 30SD, Amano Enzyme Inc.) (3 μg per g of raw fat) were added and incubated 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 obtained enzyme-treated coconut milk, 15 g of commercially available coconut yogurt (Agrilife Cocogurt, manufactured by 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. [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 resulted in a smooth texture, but did not change the coconut odor and did not impart any fermented odor at all (Comparative Example 10). [Industrial Applicability]

[0062] According to the production method of the present invention, vegetable protein foods with excellent flavor can be obtained. Examples of vegetable protein foods produced by the present invention include fermented vegetable foods (e.g., lactic acid fermented vegetable foods, more specifically, fermented dairy substitute foods, even more specifically, yogurt substitutes (vegetable yogurts), dairy substitute lactic acid bacteria drinks, lactic acid bacteria substitute drinks, and cheese substitutes (vegetable cheeses) as well as vegetable protein drinks (e.g., milk substitutes (vegetable milk)), tofu, meat substitutes prepared from plant materials, and dairy substitutes prepared from plant materials (e.g., processed vegetable milk products such as fermented dairy substitute 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 producing plant-based yogurt, characterized by allowing lipase and protein glutaminase to act on a plant-based protein raw material containing protein and fats and oils.

2. The method for producing plant-based yogurt according to claim 1, wherein the plant protein raw material is a soybean-derived raw material, an oat-derived raw material, an almond-derived raw material, or a coconut-derived raw material.

3. A method for producing a plant-based yogurt according to claim 1 or 2, comprising the following steps: (1) preparing a vegetable protein raw material containing protein and fat; (2) treating the prepared vegetable protein raw material with lipase and protein glutaminase; and (3) Fermentation by lactic acid bacteria.

4. The method for producing plant-based yogurt according to any one of claims 1 to 3, wherein the lipase is a lipase derived from Candida cylindracea.

5. The vegetable protein improver contains lipase and protein glutaminase and is used as a fermentation odor enhancer for vegetable yogurt.

6. 6. The vegetable protein improving agent according to claim 5, wherein the lipase is a lipase derived from Candida cylindracea.

7. A vegetable protein improver as described in claim 5 or 6, used as a smoothness enhancer for vegetable yogurt.

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