Environment-friendly preparation method of distiller's grains-based lyocell fiber pulp

Abstract

The present application relates to the technical field of textile raw material preparation, and particularly relates to an environment-friendly preparation method of distiller's grains-based lyocell fiber pulp. The present application discloses an environment-friendly preparation method of distiller's grains-based lyocell fiber pulp, which comprises the following steps: distiller's grains are sequentially subjected to drying, grinding into powder, and sieving to obtain distiller's grains powder; the distiller's grains powder is sequentially subjected to high-temperature and high-pressure cooking treatment, caustic soda / sodium sulfite treatment, amylase treatment, and protease treatment to obtain the distiller's grains-based lyocell fiber pulp. The present application adopts the treatment process of high-temperature and high-pressure cooking-caustic soda / sodium sulfite-biological enzyme to treat the distiller's grains, and the obtained distiller's grains pulp has a cellulose purity of more than 90% and a cellulose molecular weight of more than 30000, and can be used for preparing lyocell fiber.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of textile raw material preparation, and particularly relates to an environment-friendly preparation method of distiller's grains-based lyocell fiber pulp. BACKGROUND

[0002] China is one of the world's famous wine-producing countries, and the annual output of wine is huge. For example, in 2020, the output of Chinese yellow wine was still as high as 185.71 million liters. According to the approximate proportion of 6 kg of distiller's grains generated per 1 kg of yellow wine, China's yellow wine industry produces more than 10 million tons of distiller's grains every year. At present, most distilleries still use traditional methods such as landfill or incineration to dispose of distiller's grains, which not only pollutes the environment but also causes great waste of resources.

[0003] In view of the fact that distiller's grains contain abundant cellulose, which is a polymer that can be used for wet spinning, and lysine, methionine, vitamin B, E and other skin-friendly amino acids and vitamins, some scholars have used distiller's grains to produce and prepare regenerated cellulose fibers with excellent biodegradability. For example, the patent application with the application publication number CN114808173A discloses a method for preparing green regenerated cellulose fibers using distiller's grains as raw materials. The method includes the following steps: immersing the distiller's grains after wine brewing in a dilute alkali solution, filtering, washing with water, drying, separating, crushing the obtained distiller's grains lignocellulose, alkalinizing with an alkali solution, and then removing lignin. The obtained distiller's grains cellulose is dissolved in N-methyl morpholine-N-oxide (NMMO) aqueous solution, and the regenerated cellulose fibers are obtained by spinning. The fibers are used in the field of textiles.

[0004] The patent application with the application publication number CN115368474A discloses a preparation method of distiller's grains-based lyocell fiber pulp. The method involves the following steps: 1) the distiller's grains raw material is dried, ground into powder, and sieved to obtain distiller's grains powder; 2) dilute sulfuric acid is used to remove starch; 3) sodium hydroxide / sodium sulfite is used to remove impurities such as protein, pectin, fatty acids (esters), part of lignin, and ash; 4) p-toluenesulfonic acid aqueous solution is used to remove hemicellulose and lignin; and 5) the powder is dried and crushed to obtain distiller's grains-based lyocell fiber pulp. The distiller's grains-based pulp cellulose produced by this process has a purity of more than 90%, and can be used to prepare lyocell fibers.

[0005] The distiller's grains contain starch, protein, pectin, fat, ash, lignin, hemicellulose and other types of impurities, which makes it difficult to separate cellulose with high purity. Therefore, the existing methods use high concentrations of caustic soda, p-toluenesulfonic acid, sulfuric acid and other chemicals when removing impurities, which inevitably causes secondary pollution when preparing lyocell pulp and increases production costs. Therefore, it is necessary to develop an environmentally friendly and low-cost method to remove various impurities in the distiller's grains, so as to obtain pulp with high purity and high quality cellulose, so that it can be used to form spinning solution for the preparation of lyocell fiber. SUMMARY

[0006] The present application provides an environmentally friendly preparation method of distiller's grain-based lyocell fiber pulp, which greatly reduces the amount of caustic soda and sodium sulfite required for purifying cellulose in the distiller's grains by using high-temperature and high-pressure cooking and biological enzyme treatment, and omits the process step of removing hemicellulose and lignin in the distiller's grains with p-toluenesulfonic acid. The purity of the cellulose obtained by the preparation is still higher than 90%, which can be used for preparing lyocell fiber.

[0007] The specific technical solutions are as follows:

[0008] An environmentally friendly preparation method of distiller's grain-based lyocell fiber pulp, comprising the following steps: high-temperature and high-pressure cooking treatment, caustic soda / sodium sulfite treatment, alpha-amylase treatment and protease treatment;

[0009] The step of high-temperature and high-pressure cooking treatment is that the distiller's grains are sequentially dried, ground into powder, and sieved to obtain distiller's grain powder; the distiller's grain powder is mixed into a suspension I with water as a reaction medium; the suspension I is cooked at a pressure of 0.1-0.7 MPa and a temperature of 120-160℃ for 1-4h; after cooking, the solid residue is obtained by filtration, washed with water and dried;

[0010] The step of alpha-amylase treatment is that the distiller's grain powder is treated with 0.1-5g / L of alpha-amylase at a pH of 6.0-8.0; after the alpha-amylase treatment, the treated distiller's grain powder is obtained by sequentially performing solid-liquid separation, washing and drying; the treatment temperature is 70-80℃, and the treatment time is 0.5-2h;

[0011] The step of protease treatment is that the distiller's grain powder is treated with 0.1-5g / L of protease at a pH of 5.0-10.0; after the protease treatment, the distiller's grain-based lyocell fiber pulp is obtained by sequentially performing solid-liquid separation, washing and drying; the treatment temperature is 40-50℃, and the treatment time is 1-4h.

[0012] The particle size of the distiller's grain powder is between 50-300 mesh. The particle size and mass fraction of the distiller's grain powder help to improve the reaction efficiency of sulfuric acid treatment.

[0013] The mass percentage of the distiller's grains in the suspension I is 5-10%.

[0014] The specific steps of the caustic soda / sodium sulfite treatment are as follows: the distiller's grains after the high-temperature and high-pressure cooking treatment are prepared into a suspension II in a water reaction medium, a certain mass of caustic soda and sodium sulfite are sequentially added into the suspension II under stirring, the temperature is raised to 95-100 DEG C, and after stirring, the suspension is filtered, washed with water until neutral, and dried to obtain the distiller's grains after the caustic soda / sodium sulfite treatment.

[0015] The mass percentage of the distiller's grains in the suspension II is 3-6%.

[0016] The mass ratio of the caustic soda to the distiller's grains after the high-temperature and high-pressure cooking treatment is 1:4-1:8, and the mass ratio of the sodium sulfite to the distiller's grains after the high-temperature and high-pressure cooking treatment is 1:15-1:20.

[0017] The stirring time is 1-4h.

[0018] The mass percentage of the distiller's grains after the alpha-amylase treatment or the distiller's grains after the protease treatment is 2%-10%.

[0019] The preferred step sequence is as follows: (1) high-temperature and high-pressure cooking treatment, (2) caustic soda / sodium sulfite treatment, (3) alpha-amylase treatment, and (4) protease treatment.

[0020] The present application first uses the high-temperature and high-pressure cooking to pretreat the distiller's grains, melts and degrades and removes the hemicellulose, part of the lignin and starch in the distiller's grains, and washes with distilled water until neutral; then, a low-concentration caustic soda / sodium sulfite mixed aqueous solution is selected to treat the distiller's grains after the high-temperature and high-pressure cooking treatment at a high temperature of 95-100 DEG C, to remove the pectin, fatty acid (ester), ash, part of the protein, residual lignin and the like in the distiller's grains, and wash with distilled water until neutral; then, the alpha-amylase and the protease are used to remove the residual starch and protein in the distiller's grains after the caustic soda / sodium sulfite treatment, and the distiller's grains are filtered, washed and dried to obtain the distiller's grains pulp. The distiller's grains pulp prepared by the method of the present application has a high cellulose purity (≥90%), has good solubility in N-methyl morpholine-N-oxide solvent, and can be used to prepare lyocell fibers through a conventional wet spinning method.

[0021] The cellulose in the coarse fiber (i.e. the combination of cellulose, hemicellulose and lignin) in the distiller's grains is an effective component for spinning lyocell fibers, and the step (1) of high-temperature and high-pressure cooking treatment for removing hemicellulose, part of lignin, starch and the step (2) of caustic soda / sodium sulfite treatment for removing pectin, fatty acid (ester), ash, part of protein, residual lignin and the like are all for removing impurities, if the conditions of steps (1) and (2) are changed, the impurities cannot be removed completely, the burden of removing starch and protein by biological enzymes is increased, and the dissolution of pulp in NMMO and spinning are not conducive. The pulp helps to make the range of processing biological-based fibers more extensive.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] (1) The present application adopts a treatment process of high-temperature and high-pressure cooking-caustic soda / sodium sulfite-biological enzyme to treat the distiller's grains, and the obtained distiller's grains pulp has a fiber purity of more than 90%.

[0024] (2) The lyocell fiber pulp of the present application needs to be finally applied to fabrics, so the present application needs to ensure that the molecular weight of the obtained cellulose is more than 30000.

[0025] (3) The present application can complete the removal of starch in the distiller's grains, the separation of cellulose and hemicellulose and lignin without consuming strong acids such as sulfuric acid and p-toluenesulfonic acid, thereby reducing environmental pollution and reducing production costs.

[0026] (4) The present application greatly reduces the amount of caustic soda and sodium sulfite used in the process of removing protein and lignin, reduces the pollution caused by alkali discharge to the environment, and reduces production costs. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The present application is a process diagram of the environmentally friendly preparation method of the distiller's grains-based lyocell fiber pulp. DETAILED DESCRIPTION

[0028] The present application will be further described below in combination with specific embodiments, and the following examples are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto.

[0029] The detection methods and specific contents involved in the following examples are as follows: crude starch content detection method in raw materials: GB / T 5009.9-2008 Determination of starch in food; crude protein content detection method in raw materials: GB / T 5009.5-2010 Determination of protein in food; crude fiber content detection method in raw materials: GB / T 5009.88-2003 Determination of insoluble dietary fiber in food; crude fat content detection method in raw materials: GB / T 5009.6-2010 Determination of fat in food; ash content detection method in raw materials: GB / T 5009.4-2010 Determination of ash in food; lignin, hemicellulose and cellulose content detection method in crude fiber: NY / T 3494-2019 Determination of cellulose, hemicellulose and lignin in agricultural biomass raw materials.

[0030] Example 1

[0031] In this example, the distiller's grains come from a Zhejiang yellow wine production factory with glutinous rice as the main raw material; the specific preparation method of the distiller's grains-based lyocell fiber pulp is as follows:

[0032] 1) The distiller's grains raw material was finely ground in a mortar and passed through a 70-mesh sieve, and then dried to absolute dryness in a vacuum drying oven. According to the component analysis, the mass proportions of the main components of the dried distiller's grains raw material, i.e. crude starch, crude protein, crude fiber, crude fat and ash, were 28.20%, 35.52%, 18.81%, 9.25% and 3.11%, respectively.

[0033] The ground distiller's grains powder with a dry weight of 100 g was dispersed in 1900 g of distilled water to prepare a distiller's grains suspension with a mass concentration of 5%, and then transferred into a digester. The suspension was cooked at a steam pressure of 0.2 MPa and a cooking temperature of 120°C for 1 h. The cooked distiller's grains were washed with distilled water until neutral, and then dried to obtain high-temperature and high-pressure cooked distiller's grains powder.

[0034] According to the component analysis, the mass proportions of the main components of the high-temperature and high-pressure cooked distiller's grains powder, i.e. crude starch, crude protein, crude fiber, crude fat and ash, were 21.03%, 45.00%, 18.40%, 11.95% and 1.52%, respectively.

[0035] 2) The high-temperature and high-pressure cooked distiller's grains powder with a dry weight of 50 g was dispersed in 950 g of distilled water to prepare a distiller's grains suspension with a mass concentration of 5%, and then transferred into a 2000 mL three-necked flask equipped with a mechanical stirrer, a thermometer and a dropping funnel. Under the condition of mechanical stirring, 12.5 g of sodium hydroxide and 3.3 g of sodium sulfite were added to the suspension in sequence, and the temperature was raised to 100°C. The suspension was stirred at this temperature for 2 h. The treated distiller's grains were washed with distilled water until neutral, and then dried to obtain sodium hydroxide / sodium sulfite treated distiller's grains powder.

[0036] According to the component analysis, the main components of the caustic soda / sodium sulfite treated distiller's grains powder are only cellulose, crude starch and crude protein, and the mass percentages are 22.57%, 34.08% and 41.35%, respectively.

[0037] 3) 0.3 g of α-amylase and 10 g of the above dry caustic soda / sodium sulfite treated distiller's grains powder were poured into 150 mL of pH 6 sodium phosphate dibasic-sodium phosphate monobasic buffer, and enzymolysis was carried out at 70°C for 1 h. After solid-liquid separation, washing and drying, the α-amylase treated distiller's grains powder was prepared.

[0038] According to the component analysis, the main components of the α-amylase treated distiller's grains powder are only cellulose and crude protein, and the mass percentages are 34.18% and 62.72%, respectively.

[0039] 4) 0.3 g of protease and 10 g of the above dry α-amylase treated distiller's grains powder were poured into 150 mL of pH 8 sodium phosphate dibasic-sodium phosphate monobasic buffer, and enzymolysis was carried out at 40°C for 2 h. After solid-liquid separation, washing and drying, the distiller's lyocell pulp was prepared. According to the component analysis, it was found that the purity of cellulose in the distiller's lyocell pulp was 91.22%, and the molecular weight of cellulose was 37260.

[0040] Example 2

[0041] In this embodiment, the distiller's grains came from a liquor production factory in Zhejiang which used corn as the main raw material. The specific preparation method of the distiller's lyocell pulp was as follows:

[0042] 1) The distiller's grains raw material was finely ground in a mortar and passed through a 60 mesh sieve, and then was dried to absolute dryness in a vacuum drying oven.

[0043] According to the component analysis, the mass percentages of the main components of the dried distiller's grains raw material, such as crude starch, crude protein, crude fiber, crude fat and ash, were 29.05%, 26.12%, 25.62%, 10.08% and 2.15%, respectively.

[0044] The ground distiller's grains powder with a dry weight of 140 g was dispersed in 1860 g of distilled water to prepare a distiller's grains suspension with a mass concentration of 7%, and then was moved into a digester. The distiller's grains were cooked at a steam pressure of 0.4 MPa and a cooking temperature of 130°C for 1.5 h. After filtration, the cooked distiller's grains were washed with distilled water until neutral, and then were dried to prepare the high-temperature high-pressure cooked distiller's grains powder.

[0045] According to the component analysis, the mass percentages of the main components of the dilute sulfuric acid treated distiller's grains powder, such as crude starch, crude protein, crude fiber, crude fat and ash, were 20.75%, 35.78%, 26.88%, 14.16% and 1.07%, respectively.

[0046] 2) Disperse the above-mentioned high-temperature and high-pressure cooked lees powder with a dry weight of 30 g in 970 g of distilled water to prepare a lees suspension with a mass concentration of 3%. Then transfer it into a 2000 mL three-necked flask equipped with a mechanical stirrer, thermometer and dropping funnel. Under mechanical stirring, add 5 g of caustic soda and 1.8 g of sodium sulfite to the suspension in sequence, raise the temperature to 95℃, stir at this temperature for 1 h, filter, wash thoroughly with distilled water until neutral, and dry to obtain caustic soda / sodium sulfite treated lees powder.

[0047] According to the composition analysis, the main components of the caustic soda / sodium sulfite treated distiller's grains powder are only cellulose, crude starch and crude protein, with a mass ratio of 23.94%, 46.50% and 27.26%, respectively.

[0048] 3) Take 0.4 g of α-amylase and the above-mentioned 10 g dry weight of caustic soda / sodium sulfite treated disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 8, and enzymatically hydrolyze at 75℃ for 2 h. After solid-liquid separation, washing and drying, α-amylase treated disodium phosphate powder is obtained.

[0049] According to the composition analysis, the main components of the α-amylase-treated distiller's grains powder are only cellulose and crude protein, with a mass ratio of 35.86% and 61.34%, respectively.

[0050] 4) Take 0.4 g of protease and 10 g of α-amylase-treated distiller's grains powder (dry weight) and pour them into 200 mL of pH 8 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. Incubate at 45°C for 1 h. After solid-liquid separation, washing, and drying, distiller's grains-based lyocell fiber pulp is obtained. Component analysis revealed that the purity of cellulose in this distiller's grains-based lyocell fiber pulp was 92.58%, and the molecular weight of cellulose was 36450.

[0051] Example 3

[0052] In this embodiment, the brewer's grains come from a beer manufacturer in Zhejiang Province that uses barley as the main raw material; the specific preparation method of the brewer's grains-based lyocell fiber pulp is as follows:

[0053] 1) Grind the raw materials of distiller's grains finely in a mortar, pass them through a 50-mesh sieve, and dry them in a vacuum drying oven until completely dry.

[0054] According to the composition analysis, the main components of the dried distiller's grains raw material, crude starch, crude protein, crude fiber, crude fat and ash, accounted for 23.42%, 24.50%, 29.35%, 9.61% and 3.25% by mass, respectively.

[0055] The dry weight of 200 g of the ground distiller's grains was dispersed in 1800 g of distilled water to prepare a distiller's grains suspension with a mass concentration of 10%, and then was moved into a digester to be cooked at a steam pressure of 0.7 MPa and a cooking temperature of 150°C for 2 h. The cooked distiller's grains were filtered and washed with distilled water until neutral, and then were dried to obtain high-temperature and high-pressure cooked distiller's grains powder.

[0056] According to the component analysis, the mass proportions of the main components of the distiller's grains powder treated with dilute sulfuric acid were 17.31% for crude starch, 33.20% for crude protein, 31.24% for crude fiber, 14.03% for crude fat, and 1.64% for ash.

[0057] 2) The dry weight of 60 g of the high-temperature and high-pressure cooked distiller's grains powder was dispersed in 940 g of distilled water to prepare a distiller's grains suspension with a mass concentration of 6%, and then was moved into a 2000 mL three-necked flask equipped with a mechanical stirrer, a thermometer, and a dropping funnel. Under the condition of mechanical stirring, 12 g of caustic soda and 3.2 g of sodium sulfite were sequentially added to the suspension, and the temperature was raised to 98°C. The suspension was stirred at this temperature for 3 h. The treated distiller's grains were filtered and washed with distilled water until neutral, and then were dried to obtain caustic soda / sodium sulfite treated distiller's grains powder.

[0058] According to the component analysis, the main components of the caustic soda / sodium sulfite treated distiller's grains powder were only cellulose, crude starch, and crude protein, with mass proportions of 19.54%, 45.58%, and 32.67%, respectively.

[0059] 3) 0.2 g of α-amylase and 10 g of the caustic soda / sodium sulfite treated distiller's grains powder were poured into 100 mL of a sodium phosphate dibasic-sodium phosphate monobasic buffer with a pH of 6, and were enzymatically hydrolyzed at 80°C for 1.5 h. After solid-liquid separation, washing, and drying, α-amylase treated distiller's grains powder was obtained.

[0060] According to the component analysis, the main components of the α-amylase treated distiller's grains powder were only cellulose and crude protein, with mass proportions of 40.56% and 56.35%, respectively.

[0061] 4) 0.2 g of protease and 10 g of the α-amylase treated distiller's grains powder were poured into 100 mL of a sodium phosphate dibasic-sodium phosphate monobasic buffer with a pH of 8, and were enzymatically hydrolyzed at 50°C for 2.5 h. After solid-liquid separation, washing, and drying, distiller's based lyocell pulp was obtained. According to the component analysis, the purity of cellulose in the distiller's based lyocell pulp was 92.75%, and the molecular weight of cellulose was 38070.

[0062] Examples 4-8 (all the same as Example 1, except for the enzyme concentration of α-amylase and protease, the treatment temperature, time, and pH, with specific parameters as follows)

[0063] Alpha-amylase treatment temperature Alpha-amylase treatment time Alpha-amylase pH Alpha-amylase enzyme concentration Protease treatment temperature Protease treatment time Protease pH Protease enzyme concentration Molecular weight of cellulose Example 4 70℃ 0.5h 6.0 1 g / L 40℃ 1.5h 5.0 1 g / L 35640 Example 5 75℃ 1.0h 7.0 2 g / L 45℃ 2.0h 6.0 2 g / L 34830 Example 6 80℃ 2.0h 8.0 3 g / L 50℃ 3.0h 7.0 3 g / L 33210 Example 7 90℃ 3.0h 6.0 6 g / L 40℃ 3.0h 7.0 6 g / L 25110 Example 8 95℃ 2.0h 7.0 7 g / L 50℃ 3.0h 8.0 7 g / L 22680

[0064] Example 9-13 (all the same as Example 1, except that the treatment pressure, temperature and time during the high-temperature high-pressure cooking treatment of the distiller's grain powder are different, and the specific parameters are as follows)

[0065] Treatment pressure Treatment temperature Treatment time Molecular weight of cellulose Example 9 0.1 MPa 130℃ 1.0h 38880 Example 10 0.3 MPa 140℃ 1.5h 34020 Example 11 0.5 MPa 150℃ 2.0h 32400 Example 12 0.7 MPa 170℃ 5.0h 29160 Example 13 0.8 MPa 150℃ 6.0h 25920

[0066] The present application has been described in detail with reference to specific embodiments and illustrative examples, but these are not to be construed as limiting the present application. Those skilled in the art will understand that various equivalent substitutions, modifications and improvements can be made to the technical solutions and embodiments of the present application without departing from the spirit and scope of the present application, and these all fall within the scope of the present application. The scope of protection of the present application is subject to the appended claims.

Claims

1. An environmentally friendly method for preparing a distiller's grain-based lyocell fiber pulp, characterized by, It comprises the following steps: high-temperature and high-pressure cooking treatment, caustic soda / sodium sulfite treatment, α-amylase treatment and protease treatment; The high-temperature and high-pressure cooking treatment is as follows: after drying, grinding into powder and sieving, the distiller's grains are obtained; the distiller's grains are mixed into a suspension I with water as the reaction medium; the suspension I is cooked at a pressure of 0.1-0.7 MPa and a temperature of 120-160℃ for 1-4h; after cooking, the solid residue is obtained by filtration, washed with water and dried; The α-amylase treatment is as follows: the distiller's grains are treated with 0.1-5g / L of α-amylase at a pH of 6.0-8.0; after treatment, the solid-liquid separation, washing and drying are sequentially performed to obtain the α-amylase-treated distiller's grains; the treatment temperature is 70-80℃ and the treatment time is 0.5-2h; The protease treatment is as follows: the distiller's grains are treated with 0.1-5g / L of protease at a pH of 5.0-10.0; after treatment, the solid-liquid separation, washing and drying are sequentially performed to obtain the distiller's grains-based lyocell pulp; the treatment temperature is 40-50℃ and the treatment time is 1-4h; The caustic soda / sodium sulfite treatment is as follows: the suspension II is prepared with the high-temperature and high-pressure cooked distiller's grains and water as the reaction medium; under stirring, a certain amount of caustic soda and sodium sulfite are sequentially added to the suspension II; the temperature is raised to 95-100℃; after stirring, the suspension is filtered, washed with water until neutral and dried to obtain the caustic soda / sodium sulfite-treated distiller's grains; The mass percentage of the distiller's grains in the suspension II is 3-6%. The mass ratio of caustic soda to the high-temperature and high-pressure cooked distiller's grains is 1:4-1:8; the mass ratio of sodium sulfite to the high-temperature and high-pressure cooked distiller's grains is 1:15-1:

20. The stirring time is 1-4h.

2. An environmentally friendly preparation method of a distiller's grain-based lyocell fiber pulp according to claim 1, characterized by: The particle size of the distiller's grains is 50-300 mesh.

3. An environment-friendly preparation method of a distiller's grain-based lyocell fiber pulp according to claim 1, characterized in that: The mass percentage of the distiller's grains in the suspension I is 5-10%.

4. An environmentally friendly preparation method of a distiller's grain-based lyocell fiber pulp according to claim 1, characterized by: The mass percentage of the α-amylase-treated or protease-treated distiller's grains is 2%-10%.

5. The environmentally friendly preparation method of the distiller's grain-based lyocell fiber pulp of claim 1, characterized in that, The steps are as follows: (1) high-temperature and high-pressure cooking treatment, (2) caustic soda / sodium sulfite treatment, (3) α-amylase treatment and (4) protease treatment.

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