METHOD FOR PRODUCING PROCESSED PLANT-BASED PROTEIN-CONTAINING LIQUID COMPOSITION

Abstract

The purpose of the present invention is to provide a processing technology that exhibits an excellent solubilization effect on a plant-based protein-containing liquid composition. This method for producing a processed plant-based protein-containing liquid composition comprises a step for processing a plant-based protein-containing liquid composition by using a protein deamidase and a protease. The processed plant-based protein-containing liquid composition obtained from said production method has increased solubility.

Claims

1. A method for producing a processed plant protein-containing liquid composition, the method comprising a step of treating a plant protein-containing liquid composition with a protease and a protein deamidase.

2. The method according to claim 1, wherein the plant protein-containing liquid composition is treated with the protease and then treated with the protein deamidase.

3. The method according to claim 1, wherein the protease is a protease derived from a filamentous fungus.

4. The method according to claim 1, wherein the protease is derived from Aspergillus oryzae.

5. The method according to claim 1, wherein the plant protein contains a protein of a plant selected from the group consisting of oats, peas, chickpeas, rice, and almonds.

6. The method according to claim 1, wherein the plant protein-containing liquid composition is a plant milk.

7. A solubilizer for a plant protein-containing liquid composition, the solubilizer comprising a protease and a protein deamidase.

8. The method according to claim 1, wherein the protease is a neutral protease and wherein a change in taste of the plant protein-containing liquid composition is suppressed.

9. The method of claim 3, wherein a change in taste of the plant protein-containing liquid composition is suppressed.

Description

EXAMPLES

[0081] Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not to be construed as being limited to the following Examples.

Used Enzyme

[0082] KSSD-8 (Kleistase SD8): -Amylase derived from Bacillus amyloliquefaciens [0083] PR-ASD (Protease A Amano SD): Neutral protease derived from Aspergillus oryzae [0084] TH-PC10F (THERMOASE PC10F): Metal protease derived from Geobacillus stearothermophilus [0085] PR-UFSD (Acid Protease UF Amano SD): Acidic protease derived from Aspergillus niger [0086] PR-HF150SD (Protease HF Amano 150SD): Acidic protease derived from Aspergillus oryzae [0087] PG-500 (Protein-glutaminaseAmano500): Protein-glutaminase (protein deamidase) derived from Chryseobacterium proteolyticum

Method of Measuring Enzyme Activity

(1) Method of Measuring Protease Activity

[0088] After heating 5 mL of a 0.6% (v/w) casein solution (0.05 mol/L sodium hydrogen phosphate, pH 8.0 [in the case of TH-PC10F] or pH 6.0 [in the case of PR-ASD]) or a 0.6% (v/w) casein solution (0.7% (v/w) lactic acid, pH 3.0 [in the case of PR-UFSD or PR-HF150SD]) at 37 C. for 10 minutes, 1 mL of a sample solution containing a protease was added, and the mixture was immediately shaken. After this liquid was allowed to stand at 37 C. for 10 minutes, 5 mL of a trichloroacetic acid reagent (trichloroacetic acid containing 1.8% trichloroacetic acid, 1.8% sodium acetate and 0.33 mol/L acetic acid [in the case of TH-PC10F], or 0.44 mol/L trichloroacetic acid [in the case of PR-ASD, PR-UFSD, or PR-HF150SD]) was added, and the mixture was shaken, allowed to stand at 37 C. for 30 minutes again, and filtered. The first filtrate (3 mL) was removed, and the next filtrate (2 mL) was measured out, 5 mL of a 0.55 mol/L sodium carbonate reagent and 1 mL of a folin reagent (1.fwdarw.3) were added, and the mixture was well shaken and allowed to stand at 37 C. for 30 minutes. This liquid (enzyme reaction liquid) was measured using water as a control to determine the absorbance AT at a wavelength of 660 nm.

[0089] Separately, a liquid (blank) was obtained by an operation similar to that performed to obtain the above-described enzyme reaction liquid, except the following procedure. A 1 mL of a sample solution containing a protease was measured out, 5 mL of a trichloroacetic acid reagent (trichloroacetic acid containing 1.8% trichloroacetic acid, 1.8% sodium acetate and 0.33 mol/L acetic acid [in the case of TH-PC10F], or 0.44 mol/L trichloroacetic acid [in the case of PR-ASD, PR-UFSD, or PR-HF150SD]) was added, the mixture was shaken, then 5 mL of a casein solution having a measured pH set for each sample was added, and the mixture was immediately shaken and allowed to stand at 37 C. for 30 minutes. The obtained liquid (blank) was measured to determine the absorbance AB.

[0090] The amount of the enzyme that increased a folin reagent-coloring substance by an amount equivalent to 1 g of tyrosine per minute was defined as 1 unit (1 U).

[0091] A 1 mg/mL tyrosine standard stock solution (0.2 mol/L hydrochloric acid) was measured out in amounts of 1 mL, 2 mL, 3 mL, and 4 mL, and to each measured out solutions, a 0.2 mol/L hydrochloric acid reagent was added so that the resulting liquid had an amount of 100 mL. Each liquid was measured out in an amount of 2 mL, 5 mL of a 0.55 mol/L sodium carbonate reagent and 1 mL of a folin reagent (1.fwdarw.3) were added, and the mixture was immediately shaken and allowed to stand at 37 C. for 30 minutes. These liquids were each measured using, as a control, a liquid obtained by measuring out 2 mL of a 0.2 mol/L hydrochloric acid reagent and performing an operation similar to the above-described operation to determine the absorbances A1, A2, A3, and A4 at a wavelength of 660 nm. A calibration curve was prepared by plotting the absorbances A1, A2, A3, and A4 on the vertical axis and plotting the amount of tyrosine (g) in 2 mL of each liquid on the horizontal axis, and thus the amount of tyrosine (g) with respect to the absorbance difference 1 was determined.

Protease activity (U/g, U/mL)=(ATAB)F11/21/101/M [Math. 1]

AT: Absorbance of enzyme reaction liquid
AB: Absorbance of blank
F: Amount (g) of tyrosine when absorbance difference is 1, determined from tyrosine calibration curve
11/2: Conversion factor to total liquid amount after stop of reaction
1/10: Conversion factor to value per minute of reaction time
M: Amount (g or mL) of sample in 1 mL of sample solution

(2) Method of Measuring Protein Deamidase Activity

[0092] To 1 mL of a 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, 0.1 mL of a sample solution containing a protein deamidase was added, the mixture was allowed to stand at 37 C. for 10 minutes, and then 1 mL of a 0.4 M TCA solution was added to stop the reaction. To 1 mL of a 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, 1 mL of a 0.4 M TCA reagent was added, 0.1 mL of a sample solution containing a protein deamidase was further added, and the mixture was allowed to stand at 37 C. for 10 minutes as a blank.

[0093] The solution obtained above was measured using Ammonia Test Wako (FUJIFILM Wako Pure Chemical Corporation) to determine the amount of ammonia generated in the reaction liquid. A calibration curve representing the relationship between the ammonia concentration and the absorbance (630 nm) was prepared using an ammonia standard solution (ammonium chloride), and from the calibration curve, the ammonia concentration in the reaction liquid was determined.

[0094] The amount of the enzyme that produces 1 mol of ammonia per minute was defined as 1 unit (1 U), and the activity of the protein deamidase was calculated from the following formula. In the formula, the reaction liquid amount is 2.1, the enzyme solution amount is 0.1, and Df is a dilution rate of the enzyme solution. Furthermore, 17.03 is a molecular weight of ammonia.

Protein deamidase activity (U/mL)=ammonia concentration in reaction liquid (mg/L)(1/17.03)(reaction liquid amount/enzyme solution amount)(1/10)Df [Math 2]

(3) Method of Measuring -Amylase

[0095] After heating 10 mL of a 1% potato starch substrate solution (0.1 mol/L acetic acid (pH 5.0)) at 37 C. for 10 minutes, 1 mL of a sample solution containing -amylase was added, and the mixture was immediately shaken. After this solution was allowed to stand at 37 C. for 10 minutes, 1 mL of this solution was added to 10 mL of a 0.1 mol/L hydrochloric acid reagent, and the mixture was immediately shaken. Next, 0.5 mL of the resulting liquid was measured out, 10 mL of a 0.0002 mol/L iodine reagent (Japanese Pharmacopoeia) was added, and the mixture was shaken and then measured using water as a control to determine the absorbance (AT) at a wavelength of 660 nm. Separately, a similar operation was performed except that 1 mL of water was added instead of the sample solution, and the absorbance (AB) was measured. The amount of the enzyme that reduces coloring of potato starch due to iodine by 10% per minute was defined as 1 unit (1 U).

-Amylase activity (U/g, U/mL)=(ABAT)/AB1/W [Math 3]

AT: Absorbance of reaction liquid
AB: Absorbance of blank liquid
W: Amount (g or mL) of sample in 1 mL of sample solution

Test Example 1

(1) Method

[0096] To 90 mL of water, 10 g of oat flour (equivalent to 10 g of an oat ingredient (whole grains) and having a protein content of 1.4 g) and 50 mg (40 U/1 g of oat flour) of -amylase KSSD-8 were added and suspended, protease PR-ASD was added in an amount shown in Table 1, the mixture was stirred for 5 minutes and then treated at 60 C. for 45 minutes, then protein-glutaminase PG-500 was added in an amount shown in Table 1, and the resulting mixture was treated at 50 C. for 1 hour, boiled for 10 minutes, and cooled to room temperature. Thus, a processed oat milk was obtained.

(2) Evaluation of Solubilization

[0097] The obtained processed oat milk was centrifuged at 15000 rpm for 15 minutes, then the supernatant was collected 2 times so as not to take the cloudy upper layer, and the supernatant was measured with the Bradford method to determine the protein concentration (mg/mL). The protein concentration obtained in Comparative Example 1 in which a protein deamidase was used singly was regarded as 1, and thus the relative concentration of the protein concentration obtained in each Example was calculated. Table 1 shows the results. A processed oat milk obtained by a similar treatment using none of a protease and a protein deamidase had a protein concentration determined as described above of almost 0 mg/L, but in Comparative Example 1, the protein concentration was improved to more than about 2 mg/mL.

(3) Evaluation of Property of Suppressing Change in Taste

[0098] From the viewpoint of creamy feeling and milk feeling, the tastes of the processed oat milks in Examples were compared using, as a reference, the taste (perceived as creamy feeling and milk feeling) of the processed oat milk in Comparative Example 1 in which a protein deamidase was used singly, and all of the scores corresponding to the following five items were added for each processed oat milk to obtain an index of the property of suppressing a change in taste. The highest score of this index is +1, and the lower the score indicates lower property of suppressing a change in taste. The term creamy feeling refers to a taste that is perceived when a processed oat milk is put into the mouth and perceived to be rich due to feeling such that the processed oat milk remains on the tongue by combination of the fineness and the viscosity of the processed oat milk. The term milk feeling refers to a milky flavor. Table 1 shows the results. Regarding the taste of the processed oat milk obtained by a similar treatment using none of a protease and a protein deamidase, no creamy feeling was perceived with the rough texture.

[0099] No change occurred in taste +1 point

[0100] Creaminess was slightly reduced and slight lightness was generated 1 point

[0101] Creaminess was reduced and lightness was generated 2 points

[0102] Milk feeling was slightly reduced 1 point

[0103] Milk feeling was reduced 2 points

TABLE-US-00001 TABLE 1 Protein Property of concentration suppressing PR-ASD PG-500 (relative change in taste (U/1 g of oat) (U/1 g of protein) (U/1 g of oat) (U/1 g of protein) concentration) (point) Comparative 0 0 0.4 3 1 (Reference) Example 1 Example 1 0.0084 0.064 0.4 3 1.28 +1 Example 2 0.084 0.64 0.4 3 1.40 +1 Example 3 0.84 6.4 0.4 3 1.48 +1 Example 4 8.4 64 0.4 3 1.20 +1

[0104] As is apparent from Table 1, treating an oat milk with a protease and a protein deamidase further improved the solubility. In the case of using PR-ASD, the taste was not changed while the solubility was improved, and thus an excellent property of suppressing a change in taste was also recognized.

Test Example 2

[0105] A processed oat milk was prepared in the same manner as in Test Example 1 except that the enzyme shown in Table 2 was used as the protease in an amount indicated, and the solubilization and the property of suppressing a change in taste were evaluated. Table 2 shows the results.

TABLE-US-00002 TABLE 2 Protein Property of concentration suppressing TH-PC10F PG-500 (relative change in taste (U/1 g of oat) (U/1 g of protein) (U/1 g of oat) (U/1 g of protein) concentration) (point) Comparative 0 0 0.4 3 1 (Reference) Example 1 Example 5 0.014 0.11 0.4 3 1.47 3 Example 6 0.14 1.1 0.4 3 1.35 3 Example 7 1.4 11 0.4 3 1.59 4

[0106] As is apparent from Table 2, treating an oat milk with a protease and a protein deamidase further improved the solubility.

Test Example 3

[0107] A processed oat milk was prepared in the same manner as in Test Example 1 except that the enzyme shown in Table 3 was used as the protease in an amount indicated, and the solubilization and the property of suppressing a change in taste were evaluated. Table 3 shows the results.

TABLE-US-00003 TABLE 3 Protein Property of concentration suppressing PR-UFSD PG-500 (relative change in taste (U/1 g of oat) : (U/1 g of protein) (U/1 g of oat) (U/1 g of protein) concentration) (point) Comparative 0 0 0.4 3 1 (Reference) Example 1 Example 8 0.028 0.21 0.4 3 1.13 3 Example 9 0.28 2.1 0.4 3 1.07 3

[0108] As is apparent from Table 3, treating an oat milk with a protease and a protein deamidase further improved the solubility.

Test Example 4

(1) Production of Processed Plant Milk

(1-1) Production of Processed Rice Milk

[0109] In 50 g of water, 15 g of an unpolished rice powder (protein content: 7.1 wt %) was dispersed, 50 mg of an amylase and a protease of the type and the amount shown in Table 4 were added, the mixture was treated at 60 C. for 1 hour, then a protein deamidase of the amount shown in Table 4 was added, and the resulting mixture was treated at 50 C. for 1 hour. The treated rice milk composition was boiled for 10 minutes, allowed to dissipate heat on ice, and cooled to obtain a processed rice milk.

(1-2) Production of Processed Almond Milk

[0110] In 60 g of water, 10 g of an almond powder (protein content: 19.6 wt %) was dispersed to prepare an almond milk, a protease of the type and the amount shown in Table 4 was added, the mixture was treated at 60 C. for 80 minutes, then a protein deamidase of the amount shown in Table 4 was added, and the resulting mixture was treated at 50 C. for 1 hour. The treated almond milk composition was boiled for 10 minutes, allowed to dissipate heat on ice, and cooled to obtain a processed almond milk.

(1-3) Production of Processed Chickpea Milk

[0111] In water, 300 g of a chickpea (protein content: 20 wt %) was immersed overnight, and the resulting product was ground with a mixer. The total amount was adjusted to 2,700 mL with water to obtain a chickpea milk. The chickpea milk was divided into portions of 100 mL, a protease of the type and the amount shown in Table 5 was added, the mixture was treated at 60 C. for 1 hour, then a protein deamidase of the amount shown in Table 5 was added, and the resulting mixture was treated at 50 C. for 1 hour. The treated chickpea milk composition was boiled for 10 minutes, allowed to dissipate heat on ice, and cooled to obtain a processed chickpea milk.

(1-4) Production of Processed Pea Milk

[0112] To 10 g of a pea protein ingredient (protein content: 79 wt %), 3.6 g of a sunflower oil was added, water was further added to adjust the total amount to 240 mL, and then the mixture was homogenized at 14,000 rpm for 3 minutes to prepare a pea milk. To the pea milk, a protease and a protein deamidase of the types and the amounts shown in Table 6 were added, and the resulting mixture was treated at 50 C. for 2 hours. The treated pea milk composition was boiled for 15 minutes, allowed to dissipate heat on ice, and cooled to obtain a processed pea milk.

(2) Evaluation of Effect of Improving Solubility

[0113] The obtained processed plant milk was centrifuged at 15000 rpm for 15 minutes, then the supernatant was collected 2 times so as not to take the cloudy upper layer, and the supernatant was measured with the Bradford method to determine the protein (soluble in water) concentration (mg/mL). The protein concentrations obtained in Comparative Examples 2, 3, 4, and 5 in which a protein deamidase was used singly were each regarded as 1, and thus the relative concentration of the protein concentration obtained in each Example was calculated. Tables 4 to 6 show the results. The relative protein concentration was classified in accordance with the following criteria, and the degree of the effect of improving the solubility was evaluated. Tables 4 to 6 show the results. [0114] ++++ Relative protein concentration is 1.15 or more [0115] +++ Relative protein concentration is 1.1 or more and less than 1.15 [0116] ++ Relative protein concentration is 1.05 or more and less than 1.1 [0117] + Relative protein concentration is 1 or more and less than 1.05 [0118] Relative protein concentration is less than 1

(3) Evaluation of Effect of Suppressing Change in Taste

[0119] The tastes of the processed plant milks in Examples were compared using, as a reference, the tastes of the processed plant milks in Comparative Examples 2, 3, 4, and 5 in which a protein deamidase was used singly, and a case where no change occurred in the taste was evaluated as O, and a case where a change occurred in the taste was evaluated as x. Tables 4 to 6 show the results.

TABLE-US-00004 TABLE 4 Comparative Comparative Example 2 Example 10 Example 11 Example 3 Example 12 Food Rice Contained 1.64% 1.64% 1.64% ingredient Almond protein (*1) 2.80% 2.80% Chickpea Pea Protease PR-ASD (*1) 0.00164 ppm 0.0164 ppm 0.028 ppm derived (*2) 0.005 U 0.05 U 0.05 U from filamentous (*3) 0.00036 U 0.0036 U 0.0098 U fungus Protein (*1) 82 ppm 82 ppm 82 ppm 140 ppm 140 ppm deamidase PG-500 (*2) 2.5 U 2.5 U 2.5 U 2.5 U 2.5 U (*3) 0.18 U 0.18 U 0.18 U 0.49 U 0.49 U Effect of improving Relative protein 1 1.16 1.19 1 1.16 concentration solubility Evaluation Reference ++++ ++++ Reference ++++ Effect of suppressing Evaluation Reference Reference change in taste (*1) Weight concentration (in plant milk composition) (*2) Activity value with respect to 1 g of protein (*3) Activity value with respect to 1 g of food ingredient

TABLE-US-00005 TABLE 5 Comparative Example 4 Example 13 Example 14 Example 15 Example 16 Food Rice Contained ingredient Almond protein (*1) Chickpea 2.22% 2.22% 2.22% 2.22% 2.22% Pea Protease PR-ASD (*1) 0.00222 ppm 0.0222 ppm 2.22 ppm derived (*2) 0.005 U 0.05 U 5 U from (*3) 0.001 U 0.01 U 1 U filamentous PR-HF (*1) 0.0222 ppm fungus 150SD (*2) 0.15 U (*3) 0.03 U Protein PG-500 (*1) 111 ppm 111 ppm 111 ppm 111 ppm 111 ppm deamidase (*2) 2.5 U 2.5 U 2.5 U 2.5 U 2.5 U (*3) 0.5 U 0.5 U 0.5 U 0.5 U 0.5 U Effect of improving Relative protein 1 1.04 1.12 1.13 1.07 solubility concentration Evaluation Reference + +++ +++ ++ Effect of suppressing Evaluation Reference change in taste (*1) Weight concentration (in plant milk composition) (*2) Activity value with respect to 1 g of protein (*3) Activity value with respect to 1 g of food ingredient

TABLE-US-00006 TABLE 6 Comparative Example Example Example Example Example Example Example 5 17 18 19 20 21 22 Food Rice Contained ingredient Almond protein (*1) Chickpea Pea 3.29% 3.29% 3.29% 3.29% 3.29% 3.29% 3.29% Protease PR-ASD (*1) 3.29 ppm derived (*2) 5 U from (*3) 3.95 U filamentous PR-HF (*1) 0.0329 0.329 3.29 fungus 150SD ppm ppm ppm (*2) 0.15 U 1.5 U 15 U (*3) 0.12 U 1.19 U 11.9 U Protease TH- (*1) 0.0329 ppm 0.329 ppm derived PC10F (*2) 0.09 U 0.9 U from (*3) 0.071 U 0.71 U bacterium Protein PG-500 (*1) 164.5 ppm 164.5 ppm 164.5 ppm 164.5 ppm 164.5 ppm 164.5 ppm 164.5 ppm deamidase (*2) 2.5 U 2.5 U 2.5 U 2.5 U 2.5 U 2.5 U 2.5 U (*3) 1.98 U 1.98 U 1.98 U 1.98 U 1.98 U 1.98 U 1.98 U Effect of improving Relative 1 1.05 1.06 1.06 1.01 1.02 1.03 protein solubility concentration Evaluation Reference ++ ++ ++ + + + Effect of suppressing Evaluation Reference X X change in taste (*1) Weight concentration (in plant milk composition) (*2) Activity value with respect to 1 g of protein (*3) Activity value with respect to 1 g of food ingredient

[0120] As is apparent from the comparison between Comparative Example 2 and Examples 10 and 11, the comparison between Comparative Example 3 and Example 12, the comparison between Comparative Example 4 and Examples 13 to 16, and the comparison between Comparative Example 5 and Examples 17 to 22, treating a plant milk with a protease and a protein deamidase enhanced the solubility of the protein of the plant milk (Examples 10 to 22). Furthermore, as is apparent from the comparison between Examples 10 to 20 and Examples 21 to 22, treatment with a protease derived from a filamentous fungus and a protein deamidase enhanced the solubility of the protein without a change in taste of the plant milk (Examples 10 to 20).