Agent for inhibiting change in taste of vegetable-protein-containing liquid composition

Abstract

The purpose of the present invention is to provide a processing technique with which it is possible to suppress any change in taste while improving the solubility of a vegetable-protein-containing liquid composition (i.e., further increasing the amount of protein dissolved in water over that when the liquid composition is solubilized only by a protein deamidase). A vegetable-protein-containing liquid composition is treated with a protein deamidase and a bacteria-derived protease, whereby any change in taste resulting from solubilization of the liquid composition can be suppressed.

Description

Agent for suppressing taste change in liquid composition containing vegetable protein

[0001] The present invention relates to a processing technique for a vegetable protein-containing liquid composition, which can suppress changes in taste caused by solubilization treatment of the liquid composition.

[0002] Due to a variety of factors, including the recent health boom, addressing allergy issues, religious reasons, and increased opportunities to refrain from going out due to the spread of infectious diseases, plant-based protein drinks (plant-based milk), which are nutritious and have a long shelf life, are becoming increasingly popular as an alternative to animal milk.

[0003] On the other hand, plant proteins are generally inferior in properties such as solubility compared to proteins contained in animal milk, and therefore their uses are inevitably limited. For this reason, plant milk has not yet been able to fully replace animal milk, and its use or application has not been fully pursued.

[0004] Deamidation, i.e., hydrolysis of the amide group in the side chain of glutamine or asparagine residues in the protein, is known to be an effective method for improving the solubility of vegetable proteins. For example, Patent Document 1 shows that treating vegetable milk with protein deamidating enzymes can suppress aggregation when added to hot liquid foods and beverages.

[0005] International Publication No. 2020 / 171106

[0006] Further improvements in processing technology are needed to broaden and deepen the penetration of plant-based protein beverages into the general market. Furthermore, protein solubility remains an important processing characteristic. While the use of protein deamidating enzymes is known as a means of improving the solubility of plant-based proteins, its solubilizing effect is limited. Combining proteases with proteases is considered as an additional solubilizing method. However, because proteases have a different mode of action than protein deamidating enzymes, they can undesirably alter the component composition of plant-based protein beverages, resulting in undesirable changes in taste.

[0007] Therefore, an object of the present invention is to provide a processing technology that can improve the solubility of a vegetable protein-containing liquid composition treated with protein deamidase (i.e., increase the amount of protein that dissolves in water compared to when the vegetable protein is solubilized only by protein deamidase) while suppressing changes in taste.

[0008] The present inventors have found that treating a vegetable protein-containing liquid composition with a bacterial protease, such as a Bacillus licheniformis-derived protease or a Bacillus amyloliquefaciens-derived protease, in combination with protein deamidase can improve solubility compared to solubilization with protein deamidase alone, while suppressing changes in taste compared to solubilization with protein deamidase alone. The present invention was completed based on these findings and through further investigations.

[0009] That is, the present invention provides the following aspects of the invention. Item 1. A method for suppressing a change in taste while improving the solubility of a vegetable protein-containing liquid composition treated with protein deamidase, comprising a step of treating the vegetable protein-containing liquid composition with the protein deamidase and a bacterial protease. Item 2. The method for suppressing a change in taste according to Item 1, wherein the bacterial protease is selected from the group consisting of Bacillus licheniformis, Bacillus amyloliquefaciens, and proteases derived from these Geobacillus species. Item 3. The method for suppressing a change in taste according to Item 1 or 2, wherein the vegetable protein is selected from the group consisting of pea protein, soybean protein, and almond protein. Item 4. Item 5. The method for suppressing a change in taste according to any one of Items 1 to 3, wherein the amount of the bacterial protease used per 1 g of the vegetable protein is 13 to 250 U. Item 6. The method for suppressing a change in taste according to any one of Items 1 to 4, wherein the amount of the protein deamidase used per 1 g of the vegetable protein is 4 U or more. Item 7. The method for suppressing a change in taste according to any one of Items 1 to 5, wherein the amount of the bacterial protease used per 1 U of the protein deamidase is 1 to 45 U. Item 7. A taste change inhibitor comprising a bacterial protease and used to improve the solubility and suppress a change in taste of a vegetable protein-containing liquid composition treated with protein deamidase. Item 8. Item 7. The taste change inhibitor according to Item 7, wherein the bacterial protease is selected from the group consisting of Bacillus licheniformis, Bacillus amyloliquefaciens, and proteases derived from these bacteria belonging to the Geobacillus genus. Item 9. The taste change inhibitor according to Item 7 or 8, wherein the vegetable protein is selected from the group consisting of pea protein, soybean protein, and almond protein. Item 10. The taste change inhibitor according to any one of Items 7 to 9, which is further used to improve the foaming properties of a vegetable protein-containing liquid composition that is treated with protein deamidase.Item 11. The taste change suppressor according to any one of Items 7 to 10, which is further used to improve the dispersion stability of a vegetable protein-containing liquid composition that is treated with protein deamidating enzyme.

[0010] According to the present invention, there is provided a processing technique that can improve the solubility of a vegetable protein-containing liquid composition treated with protein deamidase while suppressing changes in taste.

[0011] 1. Method for Suppressing a Change in Taste The method for suppressing a change in taste of the present invention is a method for improving the solubility of a vegetable protein-containing liquid composition treated with protein deamidase (i.e., increasing the amount of protein soluble in water compared to when the vegetable protein is solubilized by protein deamidase alone), while suppressing a change in taste compared to when the vegetable protein is solubilized by protein deamidase alone. Specifically, the method for suppressing a change in taste of the present invention is a method for improving the solubility of a vegetable protein-containing liquid composition treated with protein deamidase and suppressing a change in taste, and is characterized by comprising a step of treating the vegetable protein-containing liquid composition with the protein deamidase and a bacterial protease. Preferably, the method for suppressing a change in taste of the present invention further improves the foaming property and / or dispersion stability of the vegetable protein-containing liquid composition treated with protein deamidase. The method for suppressing a change in taste of the present invention is described in detail below.

[0012] 1-1. Vegetable Protein-Containing Liquid Composition The vegetable protein-containing liquid composition used in the present invention is not particularly limited as long as it is a liquid in which vegetable protein is dissolved and / or dispersed in water. Specific examples of vegetable protein-containing liquid compositions include: (i) a liquid obtained by dispersing in water a dry powder (a form of vegetable protein material) of a plant (typically a food material) from which the vegetable protein is derived; (ii) a liquid obtained by dispersing in water a dry powder (a form of vegetable protein material) of a plant (typically a food material) from which the vegetable protein is derived, which has been increased by removing at least one component other than the protein; (iii) a liquid obtained by crushing and dispersing in water a plant (typically a food material, a form of vegetable protein material) from which the vegetable protein is derived, and optionally removing insoluble matter derived from the skins of these plants by any means such as centrifugation, filtration, a filter bag, or a sieve; (iv) a liquid obtained by removing at least one component other than the vegetable protein from any of the liquids (i) to (iii) above to increase the protein concentration; and (v) a liquid obtained by mixing with water a dry powder (a form of vegetable protein material) prepared from any of the liquids (i) to (iv) above. A preferred example of the vegetable protein-containing liquid composition is vegetable milk.

[0013] Examples of vegetable proteins include pea protein, soy protein, and almond protein. These vegetable proteins may be used alone or in combination.

[0014] Among the above-mentioned plants from which the vegetable protein is derived, pea and soybean are preferred, from the viewpoint of further enhancing the effect of improving the solubility of the vegetable protein-containing liquid composition to be treated with protein deamidating enzyme.

[0015] Among the above-mentioned plants from which the vegetable protein is derived, almond is preferred from the viewpoint of further enhancing the foaming property-improving effect of the vegetable protein-containing liquid composition treated with protein deamidating enzyme.

[0016] Among the above-mentioned plants from which the vegetable protein is derived, pea and soybean are preferred, from the viewpoint of further enhancing the effect of improving the dispersion stability of the vegetable protein-containing liquid composition treated with protein deamidating enzyme.

[0017] The vegetable protein content of the vegetable protein material is not particularly limited, but may be, for example, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, or 1% by weight or more, preferably 5% by weight or more, 10% by weight or more, or 20% by weight or more, more preferably 30% by weight or more, or 35% by weight or more. The upper limit of this content range is not particularly limited, but may be, for example, 95% by weight or less, preferably 90% by weight or less, and more preferably 82% by weight or less. Specific vegetable protein content ranges of the vegetable protein material include, for example, 0.01 to 95% by weight, 0.05 to 95% by weight, 0.1 to 95% by weight, or 1 to 90% by weight, preferably 5 to 90% by weight, 10 to 90% by weight, or 20 to 82% by weight, more preferably 30 to 82% by weight, or 35 to 82% by weight. The lower limit of this specific range may be 40% by weight, 50% by weight, 60% by weight, 70% by weight, or 75% by weight, and the upper limit of this specific range may be 60% by weight, 50% by weight, 45% by weight, or 40% by weight. More specific examples of the vegetable protein content contained in the vegetable protein material include the following. The pea protein content contained in the pea protein material is preferably 20% by weight or more, more preferably 40% by weight or more, even more preferably 60% by weight or more, still more preferably 70% by weight or more, or 75% by weight or more. It is preferably 95% by weight or less, more preferably 90% by weight or less, and even more preferably 82% by weight or less. Specifically, it is preferably 20 to 95% by weight, more preferably 40 to 90% by weight, even more preferably 60 to 90% by weight, and still more preferably 70 to 82% by weight, or 75 to 82% by weight. The soy protein content of the soy protein material is preferably 10% by weight or more, more preferably 20% by weight or more, even more preferably 30% by weight or more, and even more preferably 35% by weight or more. Preferably, it is 60% by weight or less, more preferably 50% by weight or less, even more preferably 45% by weight or less, or 40% by weight or less. Specifically, it is preferably 10 to 60% by weight, more preferably 20 to 50% by weight, even more preferably 30 to 45% by weight, and even more preferably 35 to 40% by weight.The almond protein content of the almond protein material is preferably 10% by weight or more, more preferably 20% by weight or more, even more preferably 30% by weight or more, or 35% by weight or more, and even more preferably 40% by weight or more. Preferably, it is 60% by weight or less, more preferably 50% by weight or less, and even more preferably 45% by weight or less. Specifically, it is preferably 10 to 60% by weight, more preferably 20 to 50% by weight, even more preferably 30 to 50% by weight, or 35 to 45% by weight, and even more preferably 40 to 45% by weight.

[0018] The content (in terms of dry weight) of the vegetable protein material in the vegetable protein-containing liquid composition is not particularly limited, and may be, for example, 0.1 to 50 wt%, 0.5 to 40 wt%, or 1 to 30 wt%, preferably 1.5 to 25 wt%, 1.5 to 15 wt%, or 2 to 10 wt%, more preferably 2.5 to 7 wt%, 3 to 6.5 wt%, 3 to 5 wt%, 3 to 4 wt%, 3 to 7 wt%, 4 to 7 wt%, 5 to 7 wt%, or 5.5 to 7 wt%. More specific examples of the content (in terms of dry weight) of the vegetable protein material in the vegetable protein-containing liquid composition include the following: - Content (in terms of dry weight) of pea protein material: preferably 1.5 to 25 wt%, more preferably 2 to 7 wt%, even more preferably 2.5 to 6.5 wt%, and even more preferably 3 to 5 wt% or 3 to 4 wt%. - Content of soy protein material (dry weight equivalent): preferably 1.5 to 25% by weight, more preferably 2 to 7% by weight, even more preferably 2.5 to 6.5% by weight, even more preferably 3 to 5% by weight, or 3 to 4% by weight. - Content of almond protein material (dry weight equivalent): preferably 3 to 25% by weight, more preferably 4 to 15% by weight, even more preferably 5 to 10% by weight, even more preferably 5.5 to 7% by weight.

[0019] The vegetable protein content in the vegetable protein-containing liquid composition is not particularly limited, and examples thereof include 0.001 to 50 wt%, 0.005 to 40 wt%, 0.01 to 20 wt%, 0.05 to 15 wt%, 0.1 to 10 wt%, or 0.3 to 7 wt%, preferably 0.6 to 4 wt%, more preferably 0.8 to 3 wt%, even more preferably 1 to 2.7 wt%, 1 to 2 wt%, 1 to 1.5 wt%, 1.5 to 3 wt%, or 2 to 3 wt%. Pea protein content: preferably 0.6 to 4 wt%, more preferably 1 to 3 wt%, even more preferably 1.5 to 3 wt%, and even more preferably 2 to 3 wt%. Soybean protein content: preferably 0.6 to 4 wt%, more preferably 0.8 to 3 wt%, even more preferably 1 to 2.7 wt%, even more preferably 1 to 2 wt%, or 1 to 1.5 wt%. Almond protein content: preferably 0.6 to 4% by weight, more preferably 1 to 3% by weight, even more preferably 1.5 to 3% by weight, and even more preferably 2 to 3% by weight.

[0020] 1-2. Protein deamidating enzyme The protein deamidating enzyme used to treat the vegetable protein-containing liquid composition is an enzyme that exhibits an action of decomposing an amide group-containing side chain of a protein without cleaving a peptide bond or crosslinking a protein, and the type, origin, etc. of the enzyme are not particularly limited.

[0021] Examples of protein deamidating enzymes include enzymes that deamidate glutamine residues in proteins and convert them to glutamic acid (e.g., protein glutaminase), and enzymes that deamidate asparagine residues in proteins and convert them to aspartic acid (e.g., protein asparaginase).

[0022] More specific examples of protein deamidases include protein deamidases derived from the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, Myroides, Luteimicrobium, Agromyces, Microbacterium, or Leifsonia. These protein deamidases are known, and reference can be made to, for example, JP2000-50887A, JP2001-218590A, WO2006 / 075772A1, WO2015 / 133590, etc. These protein deamidases may be used alone or in combination of two or more.

[0023] Among these protein deamidating enzymes, from the viewpoint of the solubility-enhancing effect and / or the taste change-inhibiting effect, or, in addition to the aforementioned effects, further enhancing the foaming property and / or dispersion stability, preferred are protein deamidating enzymes derived from the genus Chryseobacterium, more preferred are protein glutaminases derived from the genus Chryseobacterium, even more preferred are protein glutaminases derived from the species Chryseobacterium proteolyticum, and even more preferred are protein glutaminases derived from the Chryseobacterium proteolyticum strain 9670.

[0024] Protein deamidase can be prepared from a culture solution of a microorganism from which the above-mentioned protein deamidase is derived. Specific preparation methods include methods of recovering protein deamidase from the culture solution or cells of the above-mentioned microorganism. For example, when a protein deamidase-secreting microorganism is used, the cells can be recovered from the culture solution in advance by filtration, centrifugation, or the like, as necessary, and the enzyme can then be separated and / or purified. Furthermore, when a protein deamidase-nonsecreting microorganism is used, the cells can be recovered from the culture solution in advance by pressure treatment, ultrasonic treatment, or the like, as necessary, and the enzyme can then be separated and / or purified. The enzyme separation and / or purification method can be any known protein separation and / or purification method, without any particular limitation, and examples thereof include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc. The separated and / or purified enzyme can be powdered by a drying method such as freeze-drying or vacuum drying, and can also be powdered using an appropriate excipient and / or drying aid in the drying method. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration.

[0025] The amount of protein deamidase used is not particularly limited, and may be, for example, 4 U or more per 1 g of vegetable protein. From the viewpoint of achieving the solubility-improving effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects, the amount of protein deamidase used per 1 g of vegetable protein is preferably 4.5 U or more, more preferably 4.8 U or more. The upper limit of the range of the amount of protein deamidase used per 1 g of vegetable protein is not particularly limited, and may be, for example, 20 U or less, 15 U or less, 11 U or less, 10 U or less, or 5.5 U or less. Specific ranges of the amount of protein deamidase used per 1 g of vegetable protein include, for example, 4 to 20 U, preferably 4.5 to 15 U, 4.5 to 11 U, or 4.5 to 10 U, and more preferably 4.8 to 5.5 U.

[0026] The amount of protein deamidase used per 1 g of vegetable protein material is, for example, 0.01 U or more. From the viewpoint of achieving the solubility-improving effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects, the amount of protein deamidase used per 1 g of vegetable protein material is preferably 1 U or more, more preferably 1.5 U or more, and even more preferably 1.8 U or more. The upper limit of the amount of protein deamidase used per 1 g of vegetable protein material is not particularly limited, and examples include 15 U or less, 8 U or less, or 5 U or less. Specific ranges of the amount of protein deamidase used per 1 g of vegetable protein material are, for example, 0.01 to 15 U, preferably 1 to 15 U, more preferably 1.5 to 8 U, and even more preferably 1.8 to 5 U.

[0027] Regarding the activity of protein deamidase, one unit (1 U) is defined as the amount of enzyme that liberates 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate.

[0028] 1-3. Bacterial Protease The bacterial protease is not particularly limited as long as it is an enzyme derived from bacteria that hydrolyzes peptide bonds in proteins. Bacterial proteases can further enhance the solubility-improving effect of protein deamidase while suppressing changes in taste.

[0029] Examples of bacterial proteases include proteases derived from Bacillus licheniformis, Bacillus amyloliquefaciens, and those of the genus Geobacillus (i.e., Geobacillus licheniformis and Geobacillus amyloliquefaciens). Hereinafter, the genera Bacillus and Geobacillus will be collectively referred to as the "genus (geo)bacillus."

[0030] As the bacterial protease, one of these proteases may be used alone, or two or more of them may be used in combination. Among these bacterial proteases, from the viewpoint of improving solubility and / or suppressing a change in taste, or further enhancing foaming property and / or dispersion stability in addition to these effects, proteases derived from (Ge)bacillus amyloliquefaciens are preferred, and proteases derived from Bacillus amyloliquefaciens are more preferred.

[0031] Bacterial proteases can be prepared by known methods. For example, proteases derived from the genus (Geo)bacillus can be easily prepared by culturing bacteria of the genus (Geo)bacillus and isolating the protease using known means, or by using genetic engineering techniques. Commercially available bacterial proteases may also be used.

[0032] The amount of bacterial protease used is not particularly limited, but may be, for example, 13 to 250 U per gram of vegetable protein.

[0033] More specifically, the amount of (Geo)bacillus licheniformis-derived protease used per 1 g of vegetable protein is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: Amount of (Geo)bacillus licheniformis-derived protease used per 1 g of pea protein: preferably 15 to 200 U, more preferably 30 to 200 U, even more preferably 45 to 200 U, 45 to 150 U, 45 to 120 U, 48 to 110 U, 48 to 80 U, 48 to 60 U, or 48 to 55 U. Amount of (Geo)bacillus licheniformis-derived protease used per 1 g of soybean protein: preferably 15 to 250 U, more preferably 30 to 250 U, even more preferably 90 to 250 U, or 100 to 250 U, still more preferably 160 to 250 U, 180 to 250 U, 190 to 250 U, or 190 to 230 U. Amount of (Geo)bacillus licheniformis-derived protease used per 1 g of almond protein: preferably 13 to 150 U, more preferably 13 to 120 U, even more preferably 15 to 110 U (the lower limit of this range may be 30 U, 50 U, 70 U, or 90 U, and the upper limit of this range may be 100 U, 80 U, 60 U, 40 U, ​​or 20 U).

[0034] More specifically, the amount of (Geo)Bacillus amyloliquefaciens-derived protease to be used per 1 g of vegetable protein is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: Amount of (Geo)Bacillus amyloliquefaciens-derived protease to be used per 1 g of pea protein: preferably 13 to 250 U, or 15 to 250 U, more preferably 35 to 230 U, or 35 to 200 U, even more preferably 45 to 140 U, ​​48 to 140 U, ​​or 50 to 140 U, ​​and even more preferably 80 to 140 U, ​​or 90 to 120 U. Amount of (Geo)bacillus amyloliquefaciens-derived protease used per 1 g of soybean protein: preferably 15 to 250 U, more preferably 30 to 250 U, or 50 to 250 U, even more preferably 70 to 230 U, or 80 to 200 U, and even more preferably 90 to 150 U. Amount of (Geo)bacillus amyloliquefaciens-derived protease used per 1 g of almond protein: preferably 13 to 150 U, more preferably 13 to 120 U, and even more preferably 15 to 110 U (the lower limit of this range may be 30 U, 50 U, 70 U, or 90 U, and the upper limit of this range may be 100 U, 80 U, 60 U, 40 U, ​​or 20 U).

[0035] The amount of bacterial protease used per 1 g of vegetable protein material is, for example, 5 to 200 U.

[0036] More specifically, the amount of (Geo)bacillus licheniformis-derived protease to be used per 1 g of vegetable protein material is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: Amount of (Geo)bacillus licheniformis-derived protease to be used per 1 g of pea protein material: preferably 12 to 160 U, more preferably 24 to 160 U, even more preferably 36 to 160 U, 36 to 120 U, 36 to 96 U, 38 to 88 U, 38 to 64 U, 38 to 48 U, or 38 to 44 U. - Amount of (Geo)bacillus licheniformis-derived protease used per 1 g of soy protein material: preferably 5 to 100 U, more preferably 10 to 100 U, even more preferably 35 to 100 U or 38 to 100 U, still more preferably 60 to 100 U, 70 to 100 U, 75 to 100 U, or 75 to 90 U. - Amount of (Geo)bacillus licheniformis-derived protease used per 1 g of almond protein material: preferably 5 to 60 U, more preferably 5 to 48 U, even more preferably 6 to 45 U (the lower limit of the range may be 12 U, 20 U, 30 U, or 35 U, and the upper limit of the range may be 40 U, ​​32 U, 24 U, 16 U, or 8 U).

[0037] More specifically, the amount of (Geo)Bacillus amyloliquefaciens-derived protease to be used per 1 g of vegetable protein material is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste-change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: Amount of (Geo)Bacillus amyloliquefaciens-derived protease to be used per 1 g of pea protein material: preferably 10 to 200 U, or 12 to 200 U, more preferably 28 to 185 U, or 28 to 160 U, even more preferably 36 to 110 U, 38 to 110 U, or 40 to 110 U, and even more preferably 64 to 110 U, or 72 to 95 U. - Amount of (Geo)bacillus amyloliquefaciens-derived protease used per 1 g of soy protein material: preferably 5 to 100 U, more preferably 10 to 100 U, or 20 to 100 U, even more preferably 27 to 90 U, or 30 to 80 U, and even more preferably 35 to 60 U. - Amount of (Geo)bacillus amyloliquefaciens-derived protease used per 1 g of almond protein material: preferably 5 to 60 U, more preferably 5 to 48 U, and even more preferably 6 to 45 U (the lower limit of this range may be 12 U, 20 U, 28 U, or 36 U, and the upper limit of this range may be 40 U, ​​32 U, 24 U, 16 U, or 8 U).

[0038] Protease activity is measured by the Folin method using casein as a substrate. That is, protease activity is measured by a standard enzyme reaction using casein as a substrate, and one unit (1 U) is the amount of enzyme that increases the amount of Folin test solution color substance equivalent to 1 μg of tyrosine per minute.

[0039] The amount of bacterial protease to be used per 1 U of protein deamidase is, for example, 1 to 45 U.

[0040] More specifically, the amount of the (Geo)bacillus licheniformis-derived protease to be used per 1 U of protein deamidase is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: When pea protein is used as the vegetable protein, the amount of the (Geo)bacillus licheniformis-derived protease to be used per 1 U of protein deamidase: preferably 3 to 40 U, ​​more preferably 6 to 40 U, ​​even more preferably 9 to 40 U, ​​9 to 30 U, 9 to 25 U, 9.5 to 22 U, 9.5 to 16 U, 9.5 to 12 U, or 9.5 to 11 U. When soybean protein is used as the vegetable protein, the amount of the (Geo)bacillus licheniformis-derived protease used per 1 U of protein deamidase is preferably 1.5 to 25 U, more preferably 2.8 to 25 U, even more preferably 9 to 25 U, or 9.5 to 25 U, and even more preferably 16 to 25 U, 18 to 25 U, 19 to 25 U, or 19 to 23 U. When almond protein is used as the vegetable protein, the amount of the (Geo)bacillus licheniformis-derived protease used per 1 U of protein deamidase is preferably 2.5 to 30 U, more preferably 2.5 to 24 U, and even more preferably 3 to 22 U (the lower limit of the range may be 6 U, 10 U, 14 U, or 18 U, and the upper limit of the range may be 20 U, 16 U, 12 U, 8 U, or 4 U).

[0041] More specifically, the amount of the protease derived from (Geo)bacillus amyloliquefaciens to be used per 1 U of protein deamidase is preferably as follows, from the viewpoint of achieving the solubility-enhancing effect and / or the taste change-suppressing effect, or further enhancing the foaming property and / or dispersion stability in addition to these effects: When pea protein is used as the vegetable protein, the amount of the protease derived from (Geo)bacillus amyloliquefaciens to be used per 1 U of protein deamidase: preferably 2.5 to 45 U, or 5 to 45 U, more preferably 7 to 45 U, or 7 to 40 U, ​​even more preferably 9 to 28 U, 9.5 to 28 U, or 10 to 28 U, and even more preferably 16 to 28 U, or 18 to 24 U. When soybean protein is used as the vegetable protein, the amount of the (Geo)bacillus amyloliquefaciens-derived protease used per 1 U of protein deamidase is preferably 1.5 to 25 U, more preferably 2.8 to 25 U, or 5 to 25 U, even more preferably 7 to 23 U, or 8 to 20 U, and even more preferably 9 to 15 U. When almond protein is used as the vegetable protein, the amount of the (Geo)bacillus amyloliquefaciens-derived protease used per 1 U of protein deamidase is preferably 2.5 to 30 U, more preferably 2.5 to 24 U, and even more preferably 3 to 22 U (the lower limit of the range may be 6 U, 10 U, 14 U, or 18 U, and the upper limit of the range may be 20 U, 16 U, 12 U, 8 U, or 4 U).

[0042] 1-4. Reaction Conditions, etc. In the step of treating the vegetable protein-containing liquid composition with the protein deamidase and the bacterial protease, the order in which the bacterial protease and the protein deamidase are allowed to act is not particularly limited, and the enzymes may be allowed to act sequentially in any order, or both enzymes may be allowed to act simultaneously.

[0043] The temperature for the treatment with the bacterial protease and protein deamidase is not particularly limited and can be appropriately determined by a person skilled in the art depending on the optimum temperature of the enzyme used and / or the thermal properties of the vegetable protein-containing liquid composition, and examples thereof include 40 to 70°C, preferably 48 to 62°C.

[0044] The enzyme treatment time for the vegetable protein-containing liquid composition is not particularly limited and may be determined appropriately depending on the preparation scale of the composition, the timing of enzyme addition, etc., but may be, for example, 30 minutes or more, preferably 50 minutes or more. The upper limit of the range of the enzyme treatment time is not particularly limited, but may be, for example, 80 minutes or less, 70 minutes or less, or 60 minutes or less. Specific ranges for the enzyme treatment time include, for example, 30 to 80 minutes, or 30 to 70 minutes, preferably 50 to 60 minutes.

[0045] The vegetable protein-containing liquid composition after the enzyme treatment is subjected to an enzyme inactivation treatment, cooled, and optionally subjected to post-treatment such as filtration to obtain a processed vegetable protein-containing liquid composition.

[0046] The obtained processed vegetable protein-containing liquid composition has improved solubility compared to a vegetable protein-containing liquid composition treated only with protein deamidase, while undergoing processing that suppresses changes in taste. Furthermore, in a preferred embodiment of the obtained processed vegetable protein-containing liquid composition, the foaming property and / or dispersion stability have also been improved compared to a vegetable protein-containing liquid composition treated only with protein deamidase.

[0047] The resulting processed vegetable protein-containing liquid composition can be dried and then redispersed in water to form a solid vegetable protein composition that exhibits improved protein solubility in water while suppressing changes in taste, and preferably also exhibits improved foaming properties and / or dispersion stability when redispersed in water. The drying method is not particularly limited, and examples include freeze-drying, vacuum drying, and spray drying. The solid vegetable protein composition can be in the form of a powder, fine particles, granules, or the like.

[0048] 2. Agent for suppressing taste change in a vegetable protein-containing liquid composition As described above, a bacterial protease can suppress a change in taste of a vegetable protein-containing liquid composition treated with protein deamidase while improving the solubility of the protein in water. Therefore, the present invention also provides an agent for suppressing taste change, which contains a bacterial protease and is used to improve the solubility of a vegetable protein-containing liquid composition treated with protein deamidase and suppress a change in taste.

[0049] Specific modes of use of the taste change inhibitor of the present invention include a mode in which a vegetable protein-containing liquid composition is treated using the taste change inhibitor and protein deamidase simultaneously, a mode in which a vegetable protein-containing liquid composition is treated with protein deamidase and then with the taste change inhibitor, and a mode in which a vegetable protein-containing liquid composition is treated with a taste change inhibitor and then with protein deamidase.

[0050] In a preferred embodiment of the taste change inhibitor of the present invention, in addition to being used to improve the solubility and suppress taste change of a vegetable protein-containing liquid composition treated with protein deamidase, it is also used to improve the foaming properties and / or dispersion stability of the vegetable protein-containing liquid composition treated with protein deamidase.

[0051] In the above-mentioned taste change inhibitor, the type and amount of bacterial protease to be used, the target vegetable protein-containing liquid composition, and the protein deamidating enzyme to be used in combination and the amount to be used, etc. are as shown in the section "1. Method for inhibiting taste change."

[0052] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0053] [Materials and enzymes used]

[0054] [Method for measuring enzyme activity] (1) Method for measuring protease activity: 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was heated at 37°C for 10 minutes, after which 1 mL of a sample solution containing protease was added and immediately shaken. This solution was then allowed to stand at 37°C for 10 minutes, followed by the addition of 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid), shaking, and again at 37°C for 30 minutes, followed by filtration. The first 3 mL of filtrate was discarded, and 2 mL of the next filtrate was measured. 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin's test solution (1→3) were added, shaken well, and allowed to stand at 37°C for 30 minutes. The absorbance AT of this solution (enzyme reaction solution) at a wavelength of 660 nm was measured, using water as a control.

[0055] Separately, 1 mL of a sample solution containing protease was measured, 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) was added, and the mixture was shaken. 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was then added, the mixture was immediately shaken, and the mixture was allowed to stand at 37°C for 30 minutes. This mixture (blank) was treated in the same manner as the enzyme reaction solution described above, except that the absorbance AB was measured.

[0056] The amount of enzyme that causes an increase in the colored substance of Folin's test solution equivalent to 1 μg of tyrosine per minute was defined as 1 unit (1 U).

[0057] 1 mL, 2 mL, 3 mL, and 4 mL of a 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid) were measured, and 0.2 mol / L hydrochloric acid test solution was added to each to make a 100 mL solution. 2 mL of each solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The mixture was immediately shaken and left at 37°C for 30 minutes. For each of these solutions, 2 mL of 0.2 mol / L hydrochloric acid test solution was measured and the resulting solution was used as a control. Absorbances A1, A2, A3, and A4 at a wavelength of 660 nm were measured. A calibration curve was created, plotting absorbances A1, A2, A3, and A4 on the vertical axis and the amount of tyrosine (μg) in 2 mL of each solution on the horizontal axis, and the amount of tyrosine (μg) per 2 mL of each solution was calculated.

[0058]

[0059] (2) Protein deamidase activity measurement method: 0.1 mL of a sample solution containing protein deamidase was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture was allowed to stand at 37°C for 10 minutes. Then, 1 mL of 0.4 M TCA (trichloroacetic acid) solution was added to stop the reaction. As a blank, 1 mL of 0.4 M TCA solution was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of the sample solution containing protein deamidase was further added, followed by standing at 37°C for 10 minutes.

[0060] The amount of ammonia generated in the reaction solution was measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.). The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0061] The activity of protein deamidase was calculated using the following formula, where 1 unit (1 U) is the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0062]

[0063] Test Examples (1) Production of a liquid composition containing processed vegetable protein (1-1) Production of a liquid composition containing processed pea protein 1.565 g of pea protein material was suspended in 50 mL of water and pre-incubated at 50°C for 10 minutes with stirring. Protein glutaminase (protein deamidating enzyme) and various proteases were added in the amounts shown in Table 2A, and the mixture was treated at 50°C for 1 hour. The enzyme activity was then inactivated by treating at 85°C for 15 minutes. After the enzyme inactivation, the mixture was cooled to room temperature to obtain a liquid composition containing processed pea protein.

[0064] (1-2) Production of liquid composition containing processed soy protein 1.565 g of soy protein material was added to 50 mL of water to form a suspension, and the suspension was pre-incubated at 50°C for 10 minutes with stirring. Protein glutaminase and various proteases were added in the amounts shown in Table 3A, and the suspension was treated at 50°C for 1 hour. The enzyme activity was then inactivated by treating at 85°C for 15 minutes. After the enzyme inactivation, the suspension was cooled to room temperature to obtain a liquid composition containing processed soy protein.

[0065] (1-3) Production of liquid composition containing processed almond protein 3.05 g of almond protein material was added to 50 mL of water to form a suspension, and the suspension was pre-incubated at 50°C for 10 minutes with stirring. Protein glutaminase and various proteases were added in the amounts shown in Table 4A, and the suspension was treated at 50°C for 1 hour. The enzyme activity was then inactivated by treating at 85°C for 15 minutes. After the enzyme inactivation, the suspension was cooled to room temperature to obtain a liquid composition containing processed almond protein.

[0066] (2) Evaluation of Solubility Improvement Effect The processed vegetable protein-containing liquid composition obtained in (1) above was suspended using a vortex mixer and then centrifuged at 3,000 rpm for 20 minutes. The supernatant was collected without removing the cloudy upper layer, and the soluble protein was analyzed by an external organization. The analysis was performed using a LECO FP828 Series Combustion (Dumas method). The amount of soluble protein was calculated using the following formula. In the formula below, 6.25 is the nitrogen-protein conversion coefficient.

[0067]

[0068] In addition, the relative value of the amount of soluble protein in the processed plant protein-containing liquid composition of each Example and Comparative Example was calculated, assuming that the amount of soluble protein in a processed plant protein-containing liquid composition obtained using protein deamidating enzyme alone without using protease was set to 1. The results are shown in Tables 2A to 4A.

[0069] (3) Evaluation of Inhibitory Effect on Taste Change The taste of the processed plant protein-containing liquid composition obtained in (1) above was evaluated. Evaluation was carried out by four panelists, and the taste (specifically, bitterness) of the processed plant protein-containing liquid composition obtained using protein deamidase alone without using protease was used as the standard to compare whether or not there was a change in the taste of the processed plant protein-containing liquid compositions of each Example and Comparative Example (specifically, whether or not the bitterness became stronger). Specifically, if there was a change in taste, it was evaluated as "X", and if there was no change in taste, it was evaluated as "O". The results are shown in Tables 2A to 4A.

[0070] (4) Emulsifying Property Evaluation 300 mL of the processed vegetable protein-containing liquid composition obtained in (1) above was placed in a 400 mL beaker and set in a Silverson homogenizer. The homogenizer timer was set for 5 minutes, and the stirring speed was set to 5,000 RPM, and the processed vegetable protein-containing liquid composition was homogenized. After homogenization, the beaker containing the processed vegetable protein-containing liquid composition was removed, and a portion was taken for emulsifying property evaluation, and the absorbance at a wavelength of 600 nm was measured. The results are shown in Tables 2B to 4B.

[0071] (5) Evaluation of Improvement in Dispersion Stability The processed vegetable protein-containing liquid composition prepared in (4) above was refrigerated for one week, and the supernatant was collected. The turbidity was measured using a Portable Microprocessor Turbidity Meter (manufactured by HANNA). The results are shown in Tables 2B to 4B.

[0072] (6) Evaluation of Improvement in Foaming Ability The processed vegetable protein-containing liquid composition obtained in (1) above was thoroughly stirred until homogeneous. After stirring, 70 mL was measured using a measuring cylinder and poured into a HadinEEon Milk Frother, followed by whipping for 1 minute. After whipping, the processed vegetable protein-containing liquid composition was poured into the measuring cylinder, and the foam volume was measured. The results are shown in Tables 2B to 4B.

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] As is clear from Tables 2A, 3A, and 4A, treating a pea protein-containing liquid composition, a soy protein-containing liquid composition, or an almond protein-containing liquid composition with protein deamidase and a Bacillus licheniformis-derived protease (Protease 1) or a Bacillus amyloliquefaciens-derived protease (Protease 2) increased the solubility of the protein while suppressing changes in taste compared to using protein deamidase alone (Examples 1 to 17). Furthermore, as is clear from Tables 2B, 3B, and 4B, treating a pea protein-containing liquid composition, a soy protein-containing liquid composition, or an almond protein-containing liquid composition with protein deamidase and a Bacillus amyloliquefaciens-derived protease (Protease 2) showed almost no change in emulsifiability itself, but improved dispersion stability and foamability (Examples 5, 12, and 17).

Claims

1. A method for improving the solubility of a plant protein-containing liquid composition treated with a protein deamidase and suppressing taste changes, the method comprising the step of treating the plant protein-containing liquid composition with the protein deamidase and a bacterium-derived protease, the taste change suppressing method.

2. The taste change suppressing method according to claim 1, wherein the bacterium-derived protease is selected from the group consisting of proteases derived from Bacillus licheniformis, Bacillus amyloliquefaciens, and Geobacillus spp.

3. The taste change suppressing method according to claim 1, wherein the plant protein is selected from the group consisting of pea protein, soybean protein, and almond protein.

4. The taste change suppressing method according to claim 1, wherein the amount of the bacterium-derived protease used per 1 g of the plant protein is 13 to 250 U.

5. The taste change suppressing method according to claim 1, wherein the amount of the protein deamidase used per 1 g of the plant protein is 4 U or more.

6. The taste change suppressing method according to claim 1, wherein the amount of the bacterium-derived protease used per 1 U of the protein deamidase is 1 to 45 U.

7. A taste change inhibitor used for improving the solubility of a plant protein-containing liquid composition treated with a protein deamidase and suppressing taste changes, the taste change inhibitor containing a bacterium-derived protease.

8. The taste change inhibitor according to claim 7, wherein the bacterium-derived protease is selected from the group consisting of proteases derived from Bacillus licheniformis, Bacillus amyloliquefaciens, and Geobacillus spp.

9. The taste change inhibitor according to claim 7, wherein the plant protein is selected from the group consisting of pea protein, soybean protein, and almond protein.

10. The taste change inhibitor according to claim 7, further used for improving the foaming property of a plant protein-containing liquid composition treated with a protein deamidase.

11. The taste change inhibitor according to claim 7, which is used to improve the dispersion stability of a plant protein-containing liquid composition treated with a protein deamidase.