Corn protein hydrolysate and preparation method thereof

Abstract

The present invention provides a composition and method for preparing a corn protein hydrolysate, the method comprising obtaining a corn protein composition having a corn protein concentration of at least about 75% by weight, adding an enzyme to a corn protein suspension containing the corn protein composition at a ratio of about 1:100 to about 1:20 by weight of enzyme to corn protein, controlling the pH and temperature of the corn protein suspension to hydrolyze the corn protein, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate, the corn protein hydrolysate having a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4. The corn protein hydrolysate can be used in a variety of food, feed, beverage, and other applications.

Description

[0001] Priority declaration

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 755,175, filed on November 2, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present patent application relates to the field of corn protein used in food and beverage products. More specifically, the present patent application relates to a corn protein hydrolysate with high solubility and a preparation method thereof. Background Art

[0004] Whether protein is inherent in food or added to food, it can have a significant impact on processing and eating experience. Protein is an important nutritional component of the diet, and manufacturers and consumers often look for protein ingredients to provide additional benefits. The beneficial effects may be sensory or economic. Recently, people have been paying attention to replacing relatively expensive animal-derived proteins with cheaper plant-derived proteins. However, the functional property combination of plant-derived protein ingredients may not match the functional property combination of animal-derived proteins. This requires the behavior of plant proteins to be modified to better match the animal proteins being replaced.

[0005] Methods for modifying ingredients include purification, physical processing and enzyme modification. Modification of plant proteins with enzymes is well known in the art, but it is not simple to apply enzymes to proteins to achieve specific behaviors. Different enzymes show different specific ranges and strengths. This means that when applied to the same protein, two enzymes may lead to very different results. Reaction conditions (such as pH, temperature, time and substrate-enzyme ratio) can change the effect of a single enzyme on a single protein substrate. The way to handle proteins (exposure to different combinations and sequences of hydration, shearing, temperature, pH, etc.) can affect the ability of enzymes to react with proteins, thereby leading to different results. Summary of the invention

[0006] Corn protein has received little attention as a potential food ingredient in the food industry. For example, there are currently no commercial corn protein concentrates or isolates suitable for food use and having a protein content greater than 65% by weight on the market. Corn protein does not contain allergens, which makes it suitable for widespread use and reduces the cleaning costs associated with mixed allergen / non-allergen production. However, corn protein has poor solubility, which limits its use in some food applications. There is still a need in the food industry to provide soluble corn protein for a range of product categories. Specifically, corn protein hydrolysates as described herein have improved solubility for use in various food and beverage applications.

[0007] In addition, it has been found that by selecting corn protein raw materials and controlling the degree of hydrolysis of protein, the obtained corn protein hydrolysate shows excellent flavor characteristics on the one hand. On the one hand, the obtained corn protein hydrolysate shows a low degree of perceptible bitter flavor, as evaluated by the test panel analysis. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value less than 0.25g / L. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value less than 0.23g / L. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value less than 0.21g / L. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.25g / L. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.23g / L. On the one hand, the obtained corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.21g / L. In one aspect, the resulting corn protein hydrolysate has a caffeine equivalent value of 0.15 g / L to 0.25 g / L. In one aspect, the resulting corn protein hydrolysate has a caffeine equivalent value of 0.15 g / L to 0.23 g / L. In one aspect, the resulting corn protein hydrolysate has a caffeine equivalent value of 0.15 g / L to 0.21 g / L.

[0008] In one aspect, a corn protein hydrolysate having a corn protein concentration of at least about 75% by weight and a solubility of about 7% to about 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4 is provided. In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 20% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 15% to about 28% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 24% to about 35% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4.

[0009] In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 17% (based on total protein). In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 7%. In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 5%. In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 8% to about 14%. In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 10% to about 17%. In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 3% to 8%. In one aspect, the corn protein hydrolysate has a degree of hydrolysis of about 4% to about 6%.

[0010] In one aspect, the method for preparing corn protein hydrolysate comprises obtaining a corn protein composition (i.e., raw material), adding an enzyme to a corn protein suspension containing the corn protein composition at a ratio of about 1:100 to about 1:20 by weight of enzyme to corn protein, controlling the pH and temperature of the corn protein suspension to hydrolyze the corn protein, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate, the corn protein hydrolysate having a hydrolysis degree of about 1% to about 17% and a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the corn protein hydrolysate thus provided is subsequently dried.

[0011] In one aspect, the method for preparing a corn protein hydrolysate comprises obtaining a corn protein composition (i.e., a raw material) having a corn protein concentration of at least about 75% by weight, adding the corn protein composition to water at a temperature of about 40° C. to about 55° C. to obtain a 5% (w / v) corn protein suspension, adjusting the pH of the corn protein suspension to a pH level of about 5.0 to about 6.0, adding an enzyme to the corn protein composition at a ratio of about 1:100 to about 1:20 by weight of enzyme to corn protein, and hydrolyzing the corn protein suspension while maintaining the temperature and pH, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate having a hydrolysis degree of about 1% to about 17% and a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4. In one aspect, the corn protein hydrolysate thus provided is subsequently dried.

[0012] The enhanced solubility of corn protein hydrolysate as described herein can be beneficial to the use of corn protein hydrolysate in various product categories such as food, beverage and feed. In one aspect, corn protein hydrolysate can be included in various non-liquid food products as a protein source additive. Corn protein hydrolysate of the present invention can advantageously increase the protein content of food products without introducing offensive flavors. In addition, corn protein hydrolysate as described herein shows excellent texture characteristics, and provides the protein content of improvement to food without adversely affecting organoleptic properties such as mouthfeel. For example, unhydrolyzed corn protein can have a granular or sandy texture, and corn protein hydrolysate of the present invention can show excellent, smoother mouthfeel. Enhanced solubility can increase other functions that are important in food, and protein modification can change those functions without changing solubility.

[0013] Corn protein is a valuable source of protein nutrition. Nutritional benefits can be described in many ways, and protein consumption has a well-described impact on physiological function. Leucine is one of the amino acids present in corn protein, and corn protein is one of the more abundant sources of leucine in protein. Leucine is particularly important for stimulating muscle protein synthesis. This is of concern to consumers of all ages, especially for middle-aged and elderly people. Young consumers interested in increasing muscle mass usually consume proteins containing sufficient leucine. Some of these proteins are expensive or only available from animal sources. Corn protein is cheaper than most animal proteins and has plant-based sustainable benefits. One of the common ways for people seeking muscle protein synthesis to consume protein is in the form of beverages. Unmodified corn protein has poor solubility and dispersibility in water, but the product of the present invention is more suitable for preparing beverages. With improved solubility and low bitterness, corn protein can be formulated alone or in combination with other proteins to produce nutritional beverages with desired organoleptic properties. Modified protein may also be more suitable for use in other applications.

[0014] This summary is intended to provide an overview of the subject matter of this patent application. It is not intended to provide an exclusive or exhaustive explanation of the invention. The detailed description is included to provide additional information about this patent application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In the drawings, which are not necessarily drawn to scale, like numbers may describe similar parts in different views. Like numbers with different letter suffixes may represent different instances of similar parts. The drawings generally illustrate various embodiments discussed in this document by way of example and not by way of limitation.

[0016] Figure 1 is a graph showing the relationship between protein solubility at pH 3.4 and pH 7.0.

[0017] Figure 2 It is a graph showing the relationship between protein solubility and degree of hydrolysis.

[0018] Figure 3 is a graph showing the solubility of corn protein hydrolysates produced under various hydrolysis conditions.

[0019] Figure 4 is a graph of the effect of pH and temperature during hydrolysis on the solubility of corn protein hydrolysate at pH 3.4.

[0020] Figure 5 is a graph of the effect of pH and temperature during hydrolysis on the solubility of corn protein hydrolysate at pH 7.0.

[0021] Figure 6 A is a graph of the effect of pH on the solubility of unheat treated corn protein hydrolysate.

[0022] Figure 6 B is a graph of the effect of pH on the solubility of heat-treated corn protein hydrolysate.

[0023] Figure 7 A is a graph of the effect of protein concentration during hydrolysis on solubility at pH 3.4.

[0024] Figure 7 B is a graph of the effect of protein concentration on solubility at pH 7 during hydrolysis.

[0025] Figure 8 is a diagram showing the protein secondary structures of CP1, DH5, DH10, DH16, and DH30**.

[0026] Fig. 9 is a graph showing the surface hydrophobicity of corn protein before and after enzyme treatment. DETAILED DESCRIPTION

[0027] "Corn protein composition" refers to a composition comprising corn protein that has not undergone hydrolysis; in other words, it is the raw material used in the hydrolysis reaction. The corn protein content in such a composition may be less than 100% protein. The corn protein content in such a composition is in the range of at least 75% by weight and at least 79% by weight protein. The protein content is determined by AACCI 46-30.01 (crude protein-combustion method) using a nitrogen analyzer (LECO TruSpecNTM, St. Joseph, Michigan USA) and a conversion factor of 6.25.

[0028] "Corn protein hydrolysate" or "hydrolysate" refers to a corn protein composition that has undergone limited hydrolysis under controlled conditions.

[0029] "Degree of hydrolysis (DH)" refers to the proportion of cleaved peptide bonds in the hydrolyzate. DH is determined by the o-phthalaldehyde (OPA) method.

[0030] "Protein solubility" refers to the concentration of protein present in the liquid phase relative to the amount of protein present in the liquid and solid phases at equilibrium. Protein solubility can be reported as a percentage and is determined by measuring the amount of protein in the supernatant after applying centrifugal force to a solution prepared at a specific protein content, pH, and salt concentration relative to the total protein in the solution before centrifugation.

[0031] Corn protein hydrolysate

[0032] A corn protein hydrolysate having a corn protein concentration of at least about 75% by weight and a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4 is provided. In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 37% at any pH from 3.4 to 7, or the corn protein hydrolysate has a solubility of about 7% to about 37% at any pH from 3.4 to 9.

[0033] In one aspect, the corn protein concentration is at least about 79% by weight. In one aspect, the corn protein concentration is at least about 82% by weight. In one aspect, the corn protein concentration is at least about 85% by weight. In one aspect, the corn protein concentration is at least about 89% by weight. In one aspect, the corn protein concentration is at least about 92% by weight. In one aspect, the corn protein concentration is at least about 98% by weight.

[0034] In one aspect, the corn protein concentration is from about 79% to about 89% by weight. In one aspect, the corn protein concentration is from about 82% to about 89% by weight. In one aspect, the corn protein concentration is from about 85% to about 89% by weight. In one aspect, the corn protein concentration is from about 85% to about 98% by weight. In one aspect, the corn protein concentration is from about 89% to about 98% by weight. In one aspect, the corn protein concentration is from about 92% to about 98% by weight. In one aspect, the corn protein concentration is from about 95% to about 100% by weight. In one aspect, the corn protein concentration is from about 98% to about 100% by weight.

[0035] In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 37% at pH 7.0. In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 20% at pH 7.0. In one aspect, the corn protein hydrolysate has a solubility of about 15% to about 28% at pH 7.0. In one aspect, the corn protein hydrolysate has a solubility of about 24% to about 35% at pH 7.0; or wherein the solubility is about 16% to about 24% at pH 7.0.

[0036] In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 37% at pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 20% at pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 15% to about 28% at pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 24% to about 35% at pH 3.4; or wherein the solubility at pH 3.4 is about 16% to about 24%.

[0037] It has been found that the corn protein hydrolysate as described herein can advantageously exhibit desired solubility characteristics without being sensitive to pH. In other words, when measuring solubility, a given corn protein hydrolysate composition will have substantially the same solubility characteristics regardless of whether the pH of the composition is at any pH from 3.4 to 9. For convenience, testing the solubility of the corn protein hydrolysate composition at pH 3.4, 5, and 7 is an indication of the sensitivity of the solubility characteristics of the composition at the relevant pH value. The composition as described herein exhibits excellent solubility characteristics at pH values ​​between 3.4 and 7, for example, at pH 5.

[0038] In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 37% at a pH of both pH 7.0 and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 7% to about 20% at a pH of both pH 7.0 and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 15% to about 28% at a pH of both pH 7.0 and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 24% to about 35% at a pH of both pH 7.0 and pH 3.4. In one aspect, the corn protein hydrolysate has a solubility of about 30% to about 37% at a pH of both pH 7.0 and pH 3.4.

[0039] Generally speaking, the desired properties of corn protein hydrolysate are achieved when the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 17%. Certain ranges of degree of hydrolysis can provide specific beneficial effects for certain food products. In one aspect, the corn protein hydrolysate can be in the range of about 1% to about 7%. In one aspect, the degree of hydrolysis can be in the range of about 8% to about 14%. In one aspect, the degree of hydrolysis can be in the range of about 10% to about 17%. In one aspect, the degree of hydrolysis can be about 3% to 8%. In one aspect, the degree of hydrolysis can be about 4% to about 6%.

[0040] In one aspect, the corn protein hydrolysate can be provided in the form of a solution or slurry. In one aspect, the corn protein hydrolysate can be provided in the form of a concentrated solution, a paste or a slurry, for example, with a solid content of about 40% by weight to about 80% by weight solids, or with a solid content of about 40% by weight to about 60% by weight solids. Providing corn protein hydrolysate provides a processing advantage, which is convenient for adding and mixing the hydrolysate into a liquid, and avoids the challenge of handling powders. In one aspect, the corn protein hydrolysate is provided in the form of a solution, a paste or a slurry in an aseptic package. In one aspect, the corn protein hydrolysate can be obtained in powder form. The powder composition of the corn protein hydrolysate can include less than 100% corn protein. In one aspect, the powder composition of the corn protein hydrolysate can include at least about 70% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate can include at least about 75% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate can include at least about 80% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate may comprise about 79% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate may comprise about 80% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate has a moisture content of less than 10%.

[0041] Method for preparing hydrolysate

[0042] In the method of the present invention, controlled and limited hydrolysis of a corn protein composition is performed to provide a corn protein hydrolysate having an indicated solubility property. In one aspect, the method comprises obtaining a corn protein composition, adding an enzyme to the corn protein composition, and hydrolyzing the corn protein composition under controlled conditions to form a corn protein hydrolysate having a desired degree of hydrolysis.

[0043] As a first step, a corn protein composition having a corn protein concentration of at least about 75% by weight is obtained. In one aspect, the corn protein composition is a concentrate or isolate suitable for food use. In one aspect, the corn protein composition is a substantially destarched corn gluten material that has been washed with a solvent comprising water and a water-miscible organic solvent to obtain a corn protein composition with a desired protein weight % on a dry weight basis. "Destarched" refers to a starting corn gluten material having residual insoluble starch solids in the range of about 0.1% by weight to 3.0% by weight (ds) as measured by Ewers polarimetry ISO 10520:1997. In at least some aspects, the residual starch solids in such corn gluten raw materials can be in the range of about 0.1% by weight to 2.0% by weight (ds), about 0.1% by weight to 1.0% by weight (ds), or about 0.1% by weight to 0.75% by weight (ds). Methods are described, for example, in WO 2016 / 154441 and WO 2018 / 058150 assigned to Cargill Incorporated, which are incorporated herein by reference. In one aspect, washing a corn protein composition source to provide a corn protein composition raw material can remove many undesirable non-protein components (pigments, mycotoxins, carbohydrates (such as sugars), organic acids, oils, etc.) from the corn raw material.

[0044] In one aspect, the corn protein concentration is at least about 79% by weight. In one aspect, the corn protein concentration is at least about 82% by weight. In one aspect, the corn protein concentration is at least about 85% by weight. In one aspect, the corn protein concentration is at least about 89% by weight. In one aspect, the corn protein concentration is at least about 92% by weight. In one aspect, the corn protein concentration is at least about 98% by weight.

[0045] In one aspect, the corn protein concentration is from about 79% to about 89% by weight. In one aspect, the corn protein concentration is from about 82% to about 89% by weight. In one aspect, the corn protein concentration is from about 85% to about 89% by weight. In one aspect, the corn protein concentration is from about 85% to about 98% by weight. In one aspect, the corn protein concentration is from about 89% to about 98% by weight. In one aspect, the corn protein concentration is from about 92% to about 98% by weight. In one aspect, the corn protein concentration is from about 95% to about 100% by weight. In one aspect, the corn protein concentration is from about 98% to about 100% by weight.

[0046] In one aspect, the corn protein composition is a substantially de-starched corn gluten material that has been treated with an oxidant to provide a corn protein composition raw material having a free sulfite concentration of less than 150 ppm as is. In one aspect, the oxidant may be, for example, but not limited to, hydrogen peroxide, ozone gas, air, sodium hypochlorite, a combination of potassium bromate and ethanol, catalase, peroxidase, or a combination thereof. In a preferred aspect, the oxidant is hydrogen peroxide. Methods for such treatment are described in WO 2017 / 165748 assigned to Cargill Incorporated, which is incorporated herein by reference.

[0047] At a temperature and amount suitable for providing a corn protein suspension, the corn protein composition is added to a solvent system comprising water. In one aspect, the solvent system is water. In one aspect, the solvent system comprises water and a suitable food-grade co-solvent. In one aspect, the corn protein composition is added to the solvent system at a temperature of about 40°C to about 70°C. In one aspect, the corn protein composition is added to the solvent system at a temperature of about 45°C to about 55°C. In one aspect, the corn protein composition is added to the solvent system in an amount to obtain a corn protein suspension having a solid content of about 1% (w / v) to about 25% (w / v). In one aspect, the corn protein composition is added to the solvent system in an amount to obtain a corn protein suspension of about 1% to about 15% (w / v). In one aspect, the corn protein composition is added to the solvent system in an amount to obtain a corn protein suspension of about 3% to about 8% (w / v). In one aspect, the corn protein composition is added to the solvent system in an amount to obtain a corn protein suspension of about 5% (w / v).

[0048] The enzyme is added to a zein suspension comprising a zein composition at a ratio of about 1:100 to about 1:20 by weight of enzyme to zein. In one aspect, the pH of the zein suspension is adjusted and / or maintained at a desired level prior to the addition of the enzyme. In one aspect, the pH of the zein suspension is adjusted and / or maintained at about 5.0 to about 6.0 prior to the addition of the enzyme.

[0049] In one aspect, the enzyme can be added in a ratio of about 1:55 to about 1:20 by weight of the enzyme and corn protein. In one aspect, the enzyme can be added in a ratio of about 1:55 to about 1:45 by weight of the enzyme and corn protein. In one aspect, the enzyme can be added in a ratio of about 1:30 to about 1:20 by weight of the enzyme and corn protein. In one aspect, the enzyme can be added in a ratio of about 1:50 to about 1:25 by weight of the enzyme and corn protein. In one aspect, the ratio of the enzyme to corn protein is about 1:50. In one aspect, the ratio of the enzyme to corn protein is about 1:37.5. In one aspect, the ratio of the enzyme to corn protein is about 1:25. In one aspect, the ratio of the enzyme to corn protein is about 1:100.

[0050] The term "enzyme" refers to a composition having an active enzyme product. One skilled in the art will appreciate that the enzyme activity and inclusion level in the enzyme product can vary. In one aspect, the enzyme is a protease. In one aspect, the protease is derived from a fungus. In one aspect, the protease is derived from the fungus Aspergillus oryzae. In one example, the fungal enzyme can be Protease M "Amano" SD from Amano Enzyme Inc. Without being bound by theory, it is believed that fungal enzymes, especially when used in a hydrolysis method as described herein, target specific sites on the protein, thereby resulting in the release of hydrophilic peptides that are not perceived as bitter, and when used under conditions as described herein, the fungal enzyme can minimize protein off-flavors.

[0051] In one aspect, the pH and temperature of the corn protein suspension comprising the enzyme are controlled to be sufficient to hydrolyze the corn protein to the desired degree of hydrolysis. In one aspect, the pH of the corn protein suspension during hydrolysis is about 5.0 to about 6.0. In one aspect, the pH of the corn protein suspension during hydrolysis is about 5.5. In one aspect, the temperature of the corn protein suspension during hydrolysis is about 40°C to about 70°C. In one aspect, the temperature of the corn protein suspension during hydrolysis is about 45°C to about 55°C. In one aspect, the temperature of the corn protein suspension during hydrolysis is about 50°C. In one aspect, the hydrolysis of the corn protein suspension is carried out for a time of about 15 minutes to about 180 minutes. In one aspect, the hydrolysis of the corn protein suspension is carried out for a time of about 30 minutes to about 120 minutes. In one aspect, the hydrolysis of the corn protein suspension is carried out for a time of about 45 minutes to about 90 minutes.

[0052] In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 17%. In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 7%. In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 8% to about 14%. In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 10% to about 17%. In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 3% to 8%. In one aspect, the hydrolysis of the corn protein suspension is terminated when the corn protein hydrolysate has a degree of hydrolysis of about 4% to about 6%.

[0053] In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 7% to about 20% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 15% to about 28% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 24% to about 35% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 16% to about 24% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension containing the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 24% to about 35% at a pH selected from pH 7.0, pH 3.4, pH 5, and all pHs of pH 7.0, pH 5, and pH 3.4. In one aspect, the pH and temperature of the corn protein suspension comprising the enzyme are controlled for a time sufficient to hydrolyze the corn protein so that the corn protein hydrolysate has a solubility of about 30% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4.

[0054] In one aspect of the method of the present invention, the corn protein hydrolysate is dried after the desired hydrolysis is completed. In one aspect, the drying is performed by freeze drying or spray drying. In one aspect, the powder composition of the corn protein hydrolysate has a moisture content of less than 10%.

[0055] In one aspect, the corn protein hydrolysate is provided in powder form. In one aspect, the powder composition of the corn protein hydrolysate comprises at least about 75% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate comprises at least about 79% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate comprises at least about 80% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate comprises at least about 85% by weight of corn protein. In one aspect, the powder composition of the corn protein hydrolysate comprises at least about 90% by weight of corn protein.

[0056] In one aspect, a method for preparing a corn protein hydrolysate may include obtaining a corn protein composition having a corn protein concentration of at least about 75% by weight, mixing with water at a temperature of about 45° C. to about 55° C. to obtain a 5% (w / v) corn protein suspension, adjusting the pH of the corn protein suspension to about 5.0 to about 6.0, adding an enzyme to the corn protein suspension at a ratio of about 1:100 to about 1:25 by weight of enzyme to corn protein, and hydrolyzing the corn protein suspension while maintaining the temperature and pH, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate having a solubility of about 7% to about 37% at a pH selected from pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4. In one aspect, the step of hydrolyzing the corn protein suspension is performed for a time of about 30 minutes to about 120 minutes while maintaining the temperature and pH to obtain the corn protein hydrolysate.

[0057] In one aspect, the hydrolysis of the corn protein composition can be carried out at a temperature between about 50° C. and about 60° C., preferably 50° C. In one aspect, the hydrolysis of the corn protein composition can be carried out at a pH range between about 5.0 and about 6.0, preferably a pH of 5.5. In one aspect, the time for hydrolyzing the corn protein composition is between about 30 minutes and about 120 minutes, preferably 60 minutes.

[0058] In one aspect, a method for preparing a corn protein hydrolysate may include obtaining a corn protein composition, mixing with water at a temperature of about 50° C. to obtain a 5% (w / v) corn protein suspension, adding an enzyme to the corn protein suspension at a ratio of about 1:50 by weight of enzyme to corn protein, adjusting the pH of the corn protein suspension to about 5.5, and hydrolyzing the corn protein suspension for about 60 minutes while maintaining the temperature and pH to obtain a corn protein hydrolysate. The corn protein hydrolysate produced under controlled and limited hydrolysis as described herein may have a total protein hydrolysis degree of about 1.5% to about 3% or about 2.4% and a solubility of at least about 16% to about 20% or about 17% at pH 7.0.

[0059] In one aspect, a method for preparing a corn protein hydrolysate may include obtaining a corn protein composition, mixing with water at a temperature of about 48° C. to obtain a 5% (w / v) corn protein suspension, adding an enzyme to the corn protein suspension at a ratio of about 1:50 by weight of enzyme to corn protein, adjusting the pH of the corn protein suspension to about 5.5, and hydrolyzing the corn protein suspension for about 120 minutes while maintaining the temperature and pH to obtain a corn protein hydrolysate. The corn protein hydrolysate produced under controlled and limited hydrolysis as described herein may have a total protein hydrolysis degree of about 4% to about 6% or about 5% and a solubility of at least about 21% to about 24% or about 21% at pH 7.0.

[0060] In one aspect, a method for preparing a corn protein hydrolysate may include obtaining a corn protein composition, mixing with water at a temperature of about 50° C. to obtain a 5% (w / v) corn protein suspension, adding an enzyme to the corn protein suspension at a ratio of about 1:25 by weight of enzyme to corn protein, adjusting the pH of the corn protein suspension to about 5.5, and hydrolyzing the corn protein suspension for about 90 minutes while maintaining the temperature and pH to obtain a corn protein hydrolysate. The corn protein hydrolysate produced under controlled and limited hydrolysis as described herein may have a hydrolysis degree of about 6% to about 10% or about 8% to about 9% and a solubility of at least about 15% to about 25% at pH 7.

[0061] Generally speaking, hydrolyzed proteins usually have a bitter or astringent taste. Surprisingly, the corn protein hydrolyzate of the present patent application has no bitter taste.

[0062] In one aspect, a method for preparing a corn protein hydrolysate comprises:

[0063] obtaining a corn protein composition;

[0064] adding the corn protein composition to water at a temperature of about 45°C to about 55°C to obtain a 5% (w / v) corn protein suspension;

[0065] adjusting the pH of the corn protein suspension to about 5.0 to about 6.0;

[0066] adding the enzyme to the corn protein suspension at a ratio of about 1:100 to about 1:25 enzyme to corn protein by weight;

[0067] hydrolyzing the corn protein suspension for about 30 minutes to about 120 minutes while maintaining the temperature and pH to obtain a corn protein hydrolysate, wherein the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 32% and a solubility of at least about 7% to about 47% at pH 7.0; and

[0068] Dried corn protein hydrolysate.

[0069] In one aspect, a method for preparing a corn protein hydrolysate comprises:

[0070] obtaining a corn protein composition;

[0071] adding the enzyme to the corn protein composition at a ratio of about 1:100 to about 1:25 by weight of enzyme to corn protein; and

[0072] The corn protein composition is hydrolyzed within a pH range of about 5.0 to about 6.0 and within a time range of about 30 minutes to about 120 minutes to obtain a corn protein hydrolysate, wherein the corn protein hydrolysate has a degree of hydrolysis of about 1% to about 20% and a solubility of at least about 7% to about 36% at pH 7.0.

[0073] In one aspect, corn protein hydrolysate is provided as an ingredient in a food product such as a beverage or a non-liquid food. In one aspect, corn protein hydrolysate is present in about 1% by weight to about 10% by weight of a beverage. In one aspect, corn protein hydrolysate is present in about 2% by weight to about 10% by weight of a beverage. In one aspect, corn protein hydrolysate is present in about 3% by weight to about 10% by weight of a beverage. In one aspect, corn protein hydrolysate is present in about 2% by weight to about 8% by weight of a beverage. In one aspect, corn protein hydrolysate is present in about 2% by weight to about 6% by weight of a beverage. In one aspect, corn protein hydrolysate is present in about 2% by weight to about 5% by weight of a beverage. In one aspect, a beverage comprising the corn protein hydrolysate described in this paragraph shows a low degree of perceptible bitter flavor, as evaluated by a test panel analysis. In one aspect, a beverage comprising corn protein hydrolysate has a caffeine equivalent value less than 0.25g / L. In one aspect, a beverage comprising corn protein hydrolysate has a caffeine equivalent value less than 0.23g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of less than 0.21g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.25g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.23g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.1g / L to 0.21g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.15g / L to 0.25g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.15g / L to 0.23g / L. In one aspect, the beverage comprising corn protein hydrolysate has a caffeine equivalent value of 0.15g / L to 0.21g / L.

[0074] Example

[0075] The present patent application will be further described in the following examples, which do not limit the scope of the invention in the claims.

[0076] Example 1

[0077] Two samples of corn protein compositions were purified according to WO 2016 / 154441, WO 2017 / 165748, and WO 2018 / 058150 assigned to Cargill Incorporated, which are incorporated herein by reference. The protein concentration of each sample is shown in Table 1. The sample CP1 composition contained 82.88% protein by weight, and the sample CP-RTE composition contained 79.44% protein by weight.

[0078] Table 1: Corn protein composition

[0079] sample protein(%) CP1 82.88 CP-RTE 79.44

[0080] Corn protein hydrolysates were prepared from sample CP1 using the fungal enzyme Protease M (Amano Enzyme Inc.) at enzyme to protein ratios (E:P) of about 1:100, 1:50, 1:37.5, and 1:25. Time (30 minutes, 60 minutes, 90 minutes, 100 minutes, and 120 minutes), temperature (40°C, 45°C, 50°C, and 55°C), and pH (5.0, 5.5, and 6.0) were monitored during hydrolysis. The DH and solubility properties of the CP1 hydrolysates prepared under different hydrolysis conditions are shown in Tables 2 and 3, respectively.

[0081] Hydrolysis of sample CP1 was performed under the conditions listed in Table 2 after mixing a 5% protein solution in water on a small scale (volume of hydrolysate was 150 ml) for 1 hour. The enzyme (Protease M) was then added and the pH was adjusted every 10 minutes (30 minutes incubation) or 15 minutes (60 minutes or longer incubation) to maintain the desired pH. After hydrolysis, neutralization to pH 7.0 was performed with 1 M NaOH and the enzyme was inactivated by heating to 75° C. for 5 minutes. After hydrolysis, the sample was freeze-dried.

[0082] Table 2: Preparation conditions and DH of CP1 hydrolysate

[0083]

[0084]

[0085] 1. Samples "DH5", "DH10", "DH16", "DH30**" are identified in the table and are reported in the subsequent examples and shown in the figures. The DH number in the sample identifier is used to facilitate identification of the sample being tested. The DH number generally corresponds to the degree of hydrolysis of the soluble fraction (i.e., supernatant) of the sample measured.

[0086] The degree of hydrolysis of the supernatant was determined using the OPA method described in Nielsen, Petersen and Dambmann, "Improved Method for Determining Food Protein Degree of Hydrolysis", Journal of Food Science, 2001, pp. 642-646. Corn protein (0.01 g) was mixed with 1 ml of 1% sodium dodecyl sulfate (SDS) and stirred overnight at room temperature. The sample was centrifuged at 13,000 rpm for 10 minutes, and 100 μL of the supernatant was diluted 10 times with double distilled water. OPA reagents, serine standards, and sample testing were performed as described in the Nielsen et al. article. The BCA assay was performed following the instructions of the Pierce BCA protein assay kit (Thermo Scientific, #23227) to determine the protein concentration of the supernatant. The total protein content of the samples was determined by AACCI 46-30.01 (crude protein-combustion method) using a nitrogen analyzer (LECO TruSpec NTM (St. Joseph, Michigan, USA)) and a conversion factor of 6.25. The solubility of untreated CP1 (CP1 treated with all conditions except enzyme addition) and CP1 hydrolysate was determined at pH 3.4 and 7 with and without heat treatment. A protein solution (10 mL, 5% protein) was prepared based on the protein content of the powder (AACCI 46-30.01) and a conversion factor of 6.25 at pH 3.4 and 7.0, with continuous stirring for 1 hour. The protein content of a 200 μL aliquot was also determined. To evaluate the thermal stability of the solubility, the samples were heated at 85°C for 30 minutes. The samples (heat treated or not) were centrifuged at 13,000 rpm for 10 minutes, and 200 μL of the supernatant was analyzed for protein content. The protein solubility percentage was calculated based on the following formula:

[0087] Solubility (%) = protein content in supernatant / protein content before centrifugation * 100

[0088] Table 3 shows the solubility results of various CP1 hydrolysates.

[0089] Table 3: Solubility of various CPI hydrolysates

[0090]

[0091]

[0092] The CP1 hydrolysate showed a strong positive correlation between the solubility measured at pH 3.4 and pH 7 for both heated and unheated samples ( Figure 1 In addition, the solubility of the hydrolysate (regardless of the pH at which it was measured) showed a positive linear correlation with DH ( Figure 2 ).

[0093] In general, solubility increases with the independent variables of time and enzyme dosage and decreases with increasing pH. Figure 3 It is a visualization of solubility at pH 7 after heating (Z-axis) for a certain time range (X-axis) and temperature range (Y-axis) at different pH (5.0, 5.5 and 6.0) and fixed enzyme dosage (1:25, 1:37.5 and 1:50). The lines on the graph represent conditions with the same solubility. For example, in the case of pH=5.0 and enzyme fixed at 1:25 (lower left block), the contour line on the far right is the 35% solubility line. Each set of time and temperature associated with this line produces 35% soluble material. These lines are not straight because there are interactions between the independent variables. Low solubility is light shaded, and high solubility is dark shaded. Solubility increases with time and temperature, and when longer time is combined with higher temperature, solubility decreases.

[0094] In one aspect, at a pH of 5.5, a temperature of 50°C, a hydrolysis time of 60 minutes, and an E:P concentration ratio of 1:50, the CP1 hydrolysate produces a DH of about 2.4%, has a higher solubility (17% to 20%) than the CP1 hydrolysate produced at pH and temperature combinations of 5.0 and 45°C, 5.5 and 40°C, 5.5 and 45°C, 5.5 and 55°C, 6.0 and 50°C, and 6.0 and 55°C, and has a similar DH of about 2.4% or less ( Figure 4 and Figure 5 ).

[0095] The particle size of the prepared hydrolyzate is determined by laser diffraction, which measures the particle size distribution determined by the angular variation of the scattered light intensity as the laser beam passes through the dispersed particle sample. The dispersed particle sample is generated as a slurry in mineral oil. These measurements are performed using a Malvern Mastersizer 3000 laser diffraction particle size analyzer. The observed particle size distribution of the selected samples is shown in Table 4 below, where D10 is the diameter when 10% of the sample mass is composed of particles with a diameter less than this value, D50 is the diameter when 50% of the sample mass is composed of particles with a diameter less than this value (i.e., median particle size), D90 is the diameter when 50% of the sample mass is composed of particles with a diameter less than this value, and D[4,3] (μm) is the average particle size.

[0096] Table 4: Selected compositions exhibit the following particle sizes :

[0097]

[0098] Example 2

[0099] Effect of Suspension pH on Final Solubility

[0100] The solubility of intact CP1 and hydrolyzed CP1 was determined at pH 3, pH 4, pH 5, pH 6, pH 7, pH 8 and pH 9 with and without heat treatment (30 minutes at 85°C). Based on the protein content of the powder (determined by the Dumas method), a protein solution (10 mL, 5% protein) was prepared at the above pH while stirring continuously for 1 hour. The protein content of 200 μL aliquots was determined by the Dumas method. In order to evaluate thermal stability, the samples were heated at 85°C for 30 minutes. The samples (heat treated or not) were centrifuged at 13,000 rpm for 10 minutes, and 200 μL of the supernatant was analyzed for protein content. The solubility percentage of the protein was calculated based on the following formula:

[0101] Protein solubility (%) = 100*(protein concentration in supernatant) / (protein concentration in suspension)

[0102] Proteins are heterogeneous polymers that may contain positive, negative, and neutral amino acid side chains. Solubility is enhanced when the protein has a net charge, and solubility can be minimized when the net charge on the protein is about zero. Performance-wise, this means that some proteins precipitate near their isoelectric points, which are typically between 4 and 6. Natural soy protein isolate (SPI) and casein show inverted bell-shaped solubility curves, with higher solubility observed at ≤3 and ≥6, and a minimum at pH 4 to 5.5.

[0103] The effect of suspension pH on the solubility of CP1, DH5, DH10, DH16 and DH30** (as identified in Table 2) with and without heat treatment (A) is shown in Figure 6 A and Figure 6 In B. Separate ANOVAs were performed for each sample, heat treated and not. Bars associated with the same letter could not be clearly distinguished (one-way ANOVA, Tukey's comparison test, p≤0.05).

[0104] The CP1 samples behaved differently. The solubility of all intact CP1 and hydrolyzed CP1 that had not been heat treated ( Figure 6A) is insensitive to changes in suspension pH, while CP1, DH5, DH10, and DH16 have slightly higher solubility after heat treatment at pH 8 and pH 9 than at ≤ pH 7. DH30** has no solubility change with pH, ​​even after heat treatment ( Figure 6 B) For example, whey protein isolate exhibits minimal pH sensitivity prior to heating, but heating at pH 4 to pH 5 causes the protein to lose solubility. This is not observed with corn protein or its hydrolyzed forms.

[0105] In many food processes where pH changes occur, solubility stability is desired under mildly acidic conditions with or without heating. Manufacturers do not need to worry that temporary deviations in pH or temperature will have serious detrimental effects on the process, equipment, and final product.

[0106] Example 3

[0107] Effect of protein content during hydrolysis on final solubility and degree of hydrolysis

[0108] Example 1 describes preparation at a low solids concentration (5%), but a scaled-up process would advantageously use less water while achieving the same results. Greater solids can enable better capital utilization and lower operating costs, less energy usage, and possible advantages in powder property management.

[0109] Under the conditions optimized for CP1_DH5 hydrolysate, CP1 hydrolysis was carried out when mixing 5%, 10%, 15% and 20% protein solution in water for 1 hour, and the optimized conditions were pH 5.5 and 50°C (the volume of hydrolysate was 150ml). Then enzyme (protease M) was added with an enzyme / substrate (E / S) ratio of 1:100, and pH was adjusted every 15 minutes in 60 minutes to keep the desired pH. After hydrolysis, neutralize with 1M NaOH and inactivate the enzyme by being heated to 75°C for 5 minutes. After hydrolysis, the sample was freeze dried. Solubility was measured at pH 3.4 and pH 7.

[0110] There were no significant differences in final solubility at various protein concentrations tested at either pH 3.4 or pH 7, with or without heat treatment. There was greater variation between replicates observed at 15% and 20% compared to 5% or 10%. This result shows that at the appropriate ratio of protease to substrate, this enzymatic hydrolysis is equally feasible over the range of 5% to 20% solids. The DH of the total hydrolysate from 10%, 15%, and 20% protein suspensions was below 4.

[0111] The effect of protein concentration during hydrolysis on solubility at pH 3.4 (A) and pH 7 (B) is shown in Figure 2 for heat-treated and non-heat-treated samples, respectively. Figure 7 A and Figure 7 In B. Separate ANOVAs were performed with and without heat treatment. Bars associated with different letters are significantly different (one-way ANOVA, Tukey's comparison test, p≤0.05).

[0112] Example 4

[0113] Analysis of protein secondary structure by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR)

[0114] CP1 and hydrolyzed corn protein samples were analyzed on a Thermo Scientific Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA) equipped with a horizontal multi-reflection diamond accessory using OMNIC 8 software. -1 Up to 1700cm -1 The secondary structure of intact and hydrolyzed samples was determined by the second derivative spectra of ). The spectral regions were assigned as follows: 1600 cm for β-sheets -1 Up to 1635cm -1 , for α-helix is ​​1636 cm -1 Up to 1649cm -1 , for random structures it is 1650 cm -1 Up to 1680cm -1 , and 1681 cm for the β-turn structure -1 Up to 1700cm -1 The second derivative area of ​​each secondary structure region was divided by the total area of ​​the amide I region. A minimum of three spectra were recorded for each sample.

[0115] For all samples (intact and hydrolyzed), the α-helices contained Figure 8 The dominant protein secondary structure shown. With increasing DH, the β-sheet content decreases with increasing random structure. This may reflect the release of peptides without secondary structure from already structured protein domains. The α-helical content remains almost unaffected by enzymatic hydrolysis up to DH16, but it is lower in DH30** compared to CP1. Our results suggest that Protease M may be more specific for amino acid regions with β-sheets. The slight reduction in the helix may mean that adjacent hydrolysis events directly attack the helix or destabilize it.

[0116] The protein secondary structures of CP1, DH5, DH10, DH16 and DH30** are shown in Figure 8 middle.

[0117] Example 5

[0118] Treatment of proteins with proteolytic enzymes generally selects specific sites for hydrolysis. Depending on the combination of enzyme activity and protein structure, the composition of the soluble fraction may differ from the initial composition in amino acid ratios. At very high DH, the soluble fraction will be closer to the starting material, but at lower DH there may be significant differences.

[0119] Table 5 shows the amino acid composition of the soluble fraction of the material after treatment with Protease M as described in 1. To prepare the samples, the treated protein was suspended in water at pH 7 for 60 minutes and then centrifuged at 9500 rpm for 15 minutes. The supernatant and pellet were freeze-dried separately and submitted for amino acid analysis (AOAC official method 988.15, chp. 45.4.04, 2006 for tryptophan; AOAC official method 982.30E (a, b, c), chp 45.3.05 for the remaining amino acids).

[0120] Expressed as a 100% protein basis, the inferred composition of the soluble fraction (if it fully reflects the raw material) can be calculated by multiplying the raw material composition by the solubility. After adjusting to a 100% protein basis again, the actual composition can be compared to the inferred composition. In this example (Table 5), the soluble fraction is relatively rich in aspartic acid, threonine, glycine, valine, lysine, histidine, arginine and tryptophan (bold). In general, there is relatively little leucine (italics) in the soluble fraction. Glutamic acid, proline and phenylalanine are relatively depleted at lower DH, but are less obvious as DH increases.

[0121] Table 5: Amino acid composition of intact and soluble fractions after protease M treatment and observed and calculated Excess / deficient. Excess is in bold, while deficiency is in italics. .

[0122]

[0123] The insoluble material can be similarly compared to the expected concentration. At lower DH, the insoluble fraction is expected to be more similar to the raw material, because relatively less material is fractionated into the soluble phase, and this is what is observed (Table 4). The insoluble fraction is not excessive, but lysine and arginine are insufficient in the entire treatment, and tryptophan becomes insufficient at higher DH.

[0124] Table 6: Amino acid composition of intact and soluble fractions after protease M treatment and observed and calculated Excess / deficient. Excess is in bold, while deficiency is in italics. .

[0125]

[0126] Example 6

[0127] Surface hydrophobicity

[0128] Changes in protein conformation can alter the exposure of polar and nonpolar amino acid side chains. The relative effectiveness of such changes can be assessed by measuring the binding of hydrophobic compounds to proteins, a metric known as surface hydrophobicity.

[0129] The fluorescence spectrometry method uses the aromatic fluorescent probe 1-aniline 8-naphthalenesulfonate (ANS), which emits detectable light when excited by light of the appropriate wavelength (Kato and Nakai, 1980; Alizadeh-Pasdar and Li-Chan, 2000). A stock solution of ANS was prepared by suspending 0.03976 g of ANS in 10 mL of 0.1 M pH 7.4 phosphate buffer and storing the stock solution in the dark (stable for 6 months). A working solution of ANS was prepared freshly each working day by diluting 133 μL of the ANS stock solution in 3734 μL of citric acid: sodium phosphate pH 7 buffer. Protein solutions (0.05% w / v) were prepared by weighing the amount of powder required to achieve 5 mg of protein each into 15 mL centrifuge tubes, adding 10 mL of 0.1 M phosphate buffer (pH 7.4) to each tube, and adjusting the pH to 7.0. Using 0.05% protein solution, concentrations of 0.025, 0.02, 0.015, 0.01 and 0.005 w / v were prepared. 200 μL of 0.005% to 0.050% protein samples were loaded into white opaque 96-well plates. The blank control contained only citric acid: sodium phosphate pH buffer. All samples and blank control samples were prepared in triplicate. The relative fluorescence index (RFI) was measured by setting the excitation wavelength and emission wavelength to 400 / 30nm (excitation wavelength / full width at half maximum) and 460 / 40nm, respectively. The gain was set to 25. 20 μL of ANS probe solution was added to each sample and blank control sample. The plate was shaken for 1 minute, then left to stand in the dark for 15 minutes, and then the RFI was measured again.

[0130] Calculate the net RFI: Average the blank controls (wells containing either citric acid:sodium phosphate buffer alone or citric acid:sodium phosphate buffer with ANS added) in each plate separately. For each plate, subtract the appropriate blank control mean from each sample. Calculate the net RFI by subtracting the RFI of the sample without the ANS probe added from the RFI of the corresponding sample with ANS. Plot the net RFI versus protein concentration (%) as a linear regression trend line. The initial slope of the linear regression is the protein surface hydrophobicity.

[0131] Upon hydrolysis of CP1, the hydrophobic region becomes more accessible, resulting in increased surface hydrophobicity in DH5, DH10, and DH16 (Figure B1); DH30** is indistinguishable from CP1.

[0132] Fig. 9 The surface hydrophobicity of zein before and after enzyme treatment is shown. In the case of α=0.05, bars with the same letter cannot be distinguished.

[0133] Example 7

[0134] Sensory evaluation

[0135] method

[0136] Samples for sensory evaluation were prepared by dispersing corn protein (5% w / v) and bitter reference standard caffeine (#1 = 0.107 g / L, #2 = 0.153 g / L, #3 = 0.2 g / L, #4 = 0.246 g / L and #5 = 0.293 g / L) in deionized water. A total of 21 people familiarized themselves with the caffeine reference solution series before evaluating the protein samples. To taste the samples, the assessors dispensed approximately 2 mL of each sample into their mouths via a pipette and dispersed by moving their tongues. Panelists tasted each protein solution and assigned a bitterness score in comparison to their perception of the caffeine reference solution. Between samples, panelists had free access to water and rice crackers for mouth cleansing.

[0137] Results Table 7

[0138]

[0139] Summary

[0140] All samples showed a bitterness score equal to or less than 3.67, which corresponds to a caffeine equivalent equal to or less than 0.23 (g / L).

[0141] Hydrolysis under the given conditions did not introduce significant differences in bitterness compared to intact CP1.

[0142] DH5, which had the lowest degree of hydrolysis, had the lowest bitterness score of 2.42 with a caffeine equivalent of 0.17 (g / L), while DH30**, which had the highest degree of hydrolysis, received a score of 2.97 with a caffeine equivalent of 0.20 (g / L).

[0143] Example 8

[0144] Enzyme characterization

[0145] Commercial proteases are typically mixtures of catalytic abilities derived from multi-specific single enzymes and mixtures of enzymes with different specificities. The presence and absence of function both affect the result of enzyme modification. In order to understand the impact of the enzyme modification of proteins, it is useful to understand the mixing and intensity of specific hydrolytic activity in the enzyme product. There may be a variety of ways to characterize the enzyme product, so a limited set of activities provides a practical description, and will not be too time-consuming to perform. The following method is used to assess the activity in the method of the invention. As used in the present embodiment, any reference to "enzyme" is a reference to an "enzyme product" comprising additional non-enzyme components.

[0146] a) General hydrolysis of azocasein at pH 7

[0147] Azocasein (Sigma A2765) is used as a substrate to detect non-specific protease activity. A 2 wt % solution of azocasein is prepared in 50mM KH2PO4-NaOH. The reaction mixture is formed by adding 0.5mL of the same buffer to a 2mL centrifuge tube. The tube is set for the time points of 0 minute, 10 minutes, 20 minutes and 30 minutes. After adding 50 μL of 100 wt % trichloroacetic acid aqueous solution, the time zero tube is immediately placed on ice. 50 μL aliquots of the diluted enzyme are added to each tube, and the tube is heated in a 50°C water bath. At the time interval, 0.4mL azocasein solution is added and timing is started. A blank control sample is prepared, but the enzyme is replaced by 50 μL buffer. At the specified time point, 50 μL of 100 wt % TCA is added to stop the reaction and the stopped reactant is immediately transferred to an ice bath.

[0148] The enzyme was diluted (or dissolved and diluted) into the above reaction buffer. A series of dilutions were tested to confirm the assay linearity for enzyme concentration. Only dilutions in which the pigment release rate was linear were included in subsequent analysis.

[0149] When all samples have stopped, centrifuge the samples at 16,000 x g for 7.5 min to precipitate unreacted proteins.

[0150] 100 μL aliquots of the supernatant were placed in the wells of a 96-position microplate reader. 100 μL of buffer and 100 μL of 1 M NaOH were added to each well. Plates were read at 440 nm with a BioTek Synergy HT using Gen 5.1.11 software. A blank control without enzyme was used to generate an average blank control value, which was subtracted from all "active" units; any negative values ​​were set to zero for time point 0. The slope of the absorbance change was calculated.

[0151] One activity unit is defined as ΔA 440 / minute represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 440 / min / g enzyme product. Individual enzyme assays were checked for linearity before being included in the analysis.

[0152] b) General hydrolysis of BSA at pH 7

[0153] Bovine serum albumin BSA (Sigma A2153-50G) is used as substrate to measure non-specific protease activity. A solution of 10 mg / mL BSA in 25 mM sodium phosphate buffer is prepared at pH 7. Enzyme is prepared by serial dilution of 25 mM sodium phosphate buffer at pH 7. Microcentrifuge tubes are marked and arranged by enzyme concentration and time point (0 minute, 10 minutes, 20 minutes and 30 minutes). Using an automatic pipette, 950 μL BSA solution is added to each tube. The time zero tube is immediately placed in an ice bath, and all other tubes are placed in a water bath set to 50 ° C for balance. In order to inhibit enzyme activity and precipitate protein, 50 μL 100 wt % trichloroacetic acid (TCA) in water is added to the time zero tube. 50 μL of appropriate enzyme solution is added to each time zero tube. Once the time point tubes of 10 minutes, 20 minutes and 30 minutes reach equilibrium, 50 μL of appropriate enzyme solution is added to the corresponding tube at the time interval. Start the timer when the enzyme is added for the first time. A blank control was prepared by replacing the enzyme solution with 50 μL of buffer. At the appropriate time point, 50 μL of 100 wt % TCA was added to terminate the reaction, and the tube was removed from the water bath, shaken, and placed in an ice bath for at least 30 minutes. The sample was then centrifuged at 9100 × g for 10 minutes.

[0154] To obtain an accurate reading at 280 nm, the pH of the supernatant was adjusted to approximately 9.0. Sodium hydroxide (120 μL 0.5 M) was added to each well of a UV-transparent 96-well microplate. 190 μL of supernatant was added to the wells and the plate was gently agitated to mix. The plate was read at 280 nm using a microplate reader with Gen5 software.

[0155] The free amino acids of the supernatant were also analyzed using TNBS (2,4,6-trinitrobenzenesulfonic acid). Prior to analysis, a 0.5% solution of TNBS in ultrapure water was prepared and stored in a refrigerator until use. Using a micropipette, 50 μL of each standard solution (0 mM to 6 mM leucine in 0.01 N HCl) was transferred in duplicate to a 96-deep well plate. A 40 μL aliquot of each sample (supernatant) was transferred to a deep well plate along with 10 μL of 2.5% borate reagent at pH 9.5. Using a multichannel pipette, 1 mL of 2.5% borate reagent was added to each standard well and sample well. To start the reaction, 20 μL of 0.5% TNBS was added to each well, and a silicone cover was placed on the plate to seal each well. The plate was shaken to mix, and placed in a dark place at room temperature for the reaction to occur. After 30 minutes, remove the cover and add 500 μL of freshly prepared 1M sodium dihydrogen phosphate to each well to stop the reaction. Shake the plate again with the silicone cover and transfer 300 μL aliquots of each standard solution and sample solution to a 96-well microplate. Measure the absorbance at 420nm and calculate the leucine equivalent concentration using the average standard curve. One activity unit is defined as the release of 1 μmol leucine equivalent / minute. In order to derive the unit / g of commercial enzyme product, the activity of each dilution is plotted against the enzyme mass in the assay and the regression line of activity relative to the enzyme is calculated. The slope represents the activity / g enzyme product.

[0156] c) General hydrolysis of hemoglobin

[0157] Two different metrics were used to measure the enzymatic decomposition of hemoglobin. A 20 mg / mL bovine hemoglobin (Sigma H2625) solution was prepared in 25 mM HEPES-HCl (pH 6.5). The enzyme was prepared by serial dilution in ice-cold 25 mM HEPES buffer. The buffer (0.45 mL 25 mM HEPES-HCl (pH 6.5)) was pipetted into a 2 mL microcentrifuge tube. For the time zero sample, 50 μL 100% TCA was added. Enzyme dilution (50 μL) was added to each tube and the tube was placed in a 50°C water bath for balance. The reaction was initiated by adding 500 μL hemoglobin solution. At 10 minutes, 20 minutes, and 30 minutes, 50 μL 100% TCA was added and the tube was transferred to an ice bath. The sample was centrifuged at 16,000 g for 7.5 minutes. The change in A280 was calculated as described above.

[0158] The release of alpha amino groups was measured using TNBS as described above.

[0159] For measurement of peptide release as indicated by absorbance at 280 nm, 120 μL of 0.5 M NaOH was added to a flat bottom 96 well UV compatible plate. A 190 μL aliquot of supernatant sample was added to each plate and gently mixed. The absorbance at 280 nm was recorded.

[0160] d) Glycine-specific hydrolysis

[0161] The cleavage at the glycine place is tested with a synthetic chromogenic substrate to have specific enzymatic activity. Prepare the 1mM solution of glycine 4-nitroaniline-HCl (Sigma G4254) in the following manner: 5mg is dissolved in 0.25mL DMSO, then diluted to 25mL with 25mM Na-HEPES-HCl (pH6.75). Prepare the enzyme by serial dilution in ice-cold 25mM HEPES buffer. 100 μL enzyme aliquots are added to the wells of 96 microtiter plates. Initiate the reaction by adding 100 μL substrate solutions. Plate is placed in 50 ℃ (preheating) BioTek Synergy HT devices running Gen 5.1.11 software. Measure the absorbance at 405nm at 0 minute, 10 minutes and 20 minutes after shaking.

[0162] One activity unit is defined as ΔA 405 / minute represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 405 / min / g enzyme product. Individual enzyme assays were checked for linearity before being included in the analysis.

[0163] e) Leucine-specific hydrolysis

[0164] The cleavage at the leucine position was tested using a synthetic chromogenic substrate with specific enzyme activity. A 1 mM solution of L-leucine-p-nitroaniline (Sigma L2158) was prepared in the following manner: 6.3 mg was dissolved in 0.25 mL DMSO and then diluted to 25 g with 25 mM Na-HEPES-HCl (pH 6.75). The enzyme was prepared by serial dilution in 25 mM HEPES buffer. 100 μL enzyme aliquots were added to the wells of a 96-position microtiter plate. The reaction was initiated by adding 100 μL substrate solution. The plate was placed in a 50 °C (preheated) BioTek Synergy HT device running Gen 5.1.11 software. The absorbance at 405 nm was measured at intervals of 0 to 30 minutes after shaking.

[0165] One activity unit is defined as ΔA 405 / minute represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 405 / min / g enzyme product. Individual enzyme assays were checked for linearity before being included in the analysis.

[0166] f) Methionine-specific hydrolysis

[0167] The enzyme activity specific for the cleavage at the methionine position was tested using a synthetic chromogenic substrate. A 10 mM solution of L-methionine p-nitroaniline (Sigma M3529) was prepared in the following manner: 4.8 mg was dissolved in 1.78 mL of acetone. The enzyme was prepared by serial dilution in ice-cold 25 mM MOPS buffer (pH 7.5). 50 μL enzyme aliquots were added to the wells of a 96-position microtiter plate together with 130 μL buffer at room temperature. The reaction was initiated by adding 20 μL substrate solution. The plate was placed in a 50°C (preheated) BioTek Synergy HT device running Gen 5.1.11 software. The absorbance at 405 nm was measured at intervals of 0 to 45 minutes after intermittent shaking.

[0168] One activity unit is defined as ΔA 405 / minute represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 405 / min / g enzyme product. Individual enzyme assays were checked for linearity before being included in the analysis.

[0169] g) Specific hydrolysis of arginine

[0170] The enzyme activity specific to the cleavage at arginine was tested using a synthetic chromogenic substrate. A 10 mM solution of Nα-benzoyl-L-arginine 4-nitroaniline hydrochloride (Sigma B4875) was prepared in the following manner: approximately 10 mg of substrate was dissolved in 0.25 mL of DMSO and then diluted to 25 g with 25 mM Na-HEPES (pH 6.75). The enzyme was prepared by serial dilution in ice-cold 25 mM Na-HEPES (pH 6.75). 100 μL enzyme aliquots were added to the wells of a 96-position microtiter plate. The reaction was initiated by adding 100 μL substrate solution. The plate was placed in a 50 ° C (preheated) BioTek Synergy HT device running Gen 5.1.11 software. The absorbance at 405 nm was measured at intervals of 0 to 15 minutes after intermittent shaking.

[0171] One activity unit is defined as ΔA 405 / minute represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 405 / min / g enzyme product. Individual enzyme assays were checked for linearity before being included in the analysis.

[0172] h) Tyrosine-specific hydrolysis

[0173] The enzyme activity specific to the cleavage at tyrosine was tested using a synthetic chromogenic substrate. A 10 mM solution of Nα-benzoyl-L-arginine 4-nitroaniline hydrochloride (Sigma B4875) was prepared in the following manner: 11.6 mg of substrate was dissolved in 2.32 mL of acetone. The enzyme was prepared by serial dilution in ice-cold 25 mM MOPS-HCl (pH 7.5). An aliquot of 350 μL of room temperature buffer and 100 μL of diluted enzyme were added to a microcentrifuge tube. The reaction was initiated by adding 50 μL of substrate. The tube was mixed and moved to a 50°C water bath. The reaction was stopped at 1 hour, 3.5 hours, and 23 hours by adding 50 μL of 100% w / w TCA, and the tube was cooled until all samples were collected. For time zero, TCA was added before adding substrate. When completed, the sample was centrifuged at 16,000 g for 7.5 minutes to precipitate the protein and unreacted substrate. Samples (200 μL) were loaded onto 96-well microtiter plates and read on a BioTek Synergy HT device running Gen5.1.11 software. The absorbance at 405 nm was recorded. This reaction was very slow, so the only data point used for further analysis was the one at 23 hours. As a check, pancreatin was also tested against the substrate in the same buffer system and showed a ΔA greater than 0.63 within the first hour. 405 .

[0174] One activity unit is defined as ΔA 405 / 23 hours represents one unit change. To derive units / g of commercial enzyme product, the activity of each dilution was plotted against the mass of enzyme in the assay and a regression line of activity versus enzyme was calculated. The slope represents activity / mg enzyme product, which is converted to ΔA 405 / 23 hours / g enzyme product.

[0175] The activity of Protease M in this set of assays is shown in Table 8 below. .

[0176] Substrate unit Activity (SE) Azocasein <![CDATA[ΔA 440 / min / g]]> 200(17) BSA <![CDATA[ΔA 280 / min / g]]> 53.4(2.8) BSA μmol leucine equivalent / min / g 17.3(0.5) Bovine hemoglobin <![CDATA[ΔA 440 / min / g]]> 21.9(1.0) Bovine hemoglobin <![CDATA[ΔA 280 / min / g]]> 61.9(3.2) Glycine 4-nitroaniline-HCl <![CDATA[ΔA 405 / min / g]]> 0.535(0.023) L-Leucine-p-nitroaniline <![CDATA[ΔA 405 / min / g]]> 10,399(714) L-Lysine p-nitroaniline dihydrobromide <![CDATA[ΔA 405 / min / g]]> 1283(44.9) L-Methionine 4-nitroaniline <![CDATA[ΔA 405 / min / g]]> 90.8(4.2) Nα-Benzoyl-L-arginine 4-nitroaniline hydrochloride <![CDATA[ΔA 405 / min / g]]> 2.28(0.12) Nα-Benzoyl-L-tyrosine 4-nitroaniline hydrochloride <![CDATA[ΔA 405 / 23 hours / g]]> 35.9(5.0)

[0177] In the example where 1 g of Protease M is applied to 100 g of protein substrate, the activity applied can also be described as approximately: 200 azocasein degradation units, 53 casein-derived A 280 The enzyme activity characteristics of the present invention are as follows: 1,000 glycine nitroaniline hydrolyzing units, 17 casein-derived alpha amine-releasing units, 0.54 glycine nitroaniline hydrolyzing units, 10,400 leucine nitroaniline hydrolyzing units, 1280 lysine nitroaniline hydrolyzing units, 91 methionine nitroaniline hydrolyzing units, 2.3 benzoylarginine nitroaniline hydrolyzing units, and 36 benzoyltyrosine nitroaniline hydrolyzing units. Those skilled in the art will recognize that other measures of enzyme general and specific activity can be used to further specify the enzyme activity characteristics useful in achieving the desired results of the methods of the present invention.

[0178] The above-mentioned detailed description includes reference to the accompanying drawings, which form a part of the detailed description. The accompanying drawings show specific aspects in which the present invention can be practiced by way of example. These aspects are also referred to as "embodiments" in this article. Such embodiments may include elements other than those shown or described. However, the inventors also contemplate embodiments in which only those elements shown or described are provided. In addition, the inventors also contemplate embodiments using any combination or arrangement of those elements shown or described (or one or more aspects thereof) with respect to a specific embodiment (or one or more aspects thereof) or with respect to other embodiments (or one or more aspects thereof) shown or described herein.

Claims

1. A corn protein hydrolysate having a corn protein concentration of at least 75% by weight and a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, in, The corn protein hydrolysate is obtained by hydrolyzing corn protein with a protease obtained from Aspergillus oryzae, and the protease has an activity that can be described as: 200 azocasein degradation units, 53.4 bovine serum albumin-derived A280-releasing units, 17.3 bovine serum albumin-derived leucine-releasing units, 21.9 bovine hemoglobin degradation units, 61.9 bovine hemoglobin-derived A280-releasing units, 0.535 glycine 4-nitroaniline-HCl hydrolysis units, 10,399 L-leucine p-nitroaniline hydrolysis units, 1283 L-lysine p-nitroaniline dihydrobromide hydrolysis units, 90.8 L-methionine 4-nitroaniline hydrolysis units, 2.28 Nα-benzoyl-L-arginine 4-nitroaniline hydrochloride hydrolysis units and 35.9 Nα-benzoyl-L-tyrosine 4-nitroaniline hydrochloride hydrolysis units, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

2. A corn protein hydrolysate having a corn protein concentration of at least 75% by weight and a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, in, The corn protein hydrolysate is obtained by hydrolyzing corn protein with a protease obtained from Aspergillus oryzae, wherein the protease includes protease M, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

3. The corn protein hydrolysate according to claim 1 or 2, wherein the solubility is 7% to 20% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.

4.

4. The corn protein hydrolysate according to claim 1 or 2, wherein the solubility is 15% to 28% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.

4.

5. The corn protein hydrolysate according to claim 1 or 2, wherein the solubility is 24% to 35% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.

4. The corn protein hydrolyzate according to claim 1 or 2, wherein the solubility is 16% to 24% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.

4.

7. The corn protein hydrolysate according to claim 1 or 2, wherein the solubility is 30% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.

4.

8. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein concentration is at least 79% by weight.

9. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein concentration is 79 wt% to 89 wt%.

10. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein concentration is 85 wt% to 98 wt%.

11. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein concentration is 95 wt% to 100 wt%.

12. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein hydrolysate has a hydrolysis degree of 1% to 7%.

13. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein hydrolysate has a degree of hydrolysis of 8% to 14%.

14. The corn protein hydrolysate according to claim 1 or 2, wherein the corn protein hydrolysate has a degree of hydrolysis of 10% to 17%.

15. A method for preparing a corn protein hydrolysate, the method comprising: a) obtaining a corn protein composition having a corn protein concentration of at least 75% by weight; b) adding an enzyme to a corn protein suspension comprising the corn protein composition at a ratio of 1:100 to 1:20 by weight of enzyme to corn protein; c) controlling the pH and temperature of the corn protein suspension to hydrolyze the corn protein; as well as d) terminating said hydrolysis of said corn protein to provide a corn protein hydrolysate having a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, wherein the enzyme is a protease obtained from Aspergillus oryzae, and the protease has an activity that can be described as: 200 azocasein degradation units, 53.4 bovine serum albumin-derived A280-releasing units, 17.3 bovine serum albumin-derived leucine-releasing units, 21.9 bovine hemoglobin degradation units, 61.9 bovine hemoglobin-derived A280-releasing units, 0.535 glycine 4-nitroaniline-HCl hydrolysis units, 10,399 L-leucine p-nitroaniline hydrolysis units, 1283 L-lysine p-nitroaniline dihydrobromide hydrolysis units, 90.8 L-methionine 4-nitroaniline hydrolysis units, 2.28 Nα-benzoyl-L-arginine 4-nitroaniline hydrochloride hydrolysis units and 35.9 Nα-benzoyl-L-tyrosine 4-nitroaniline hydrochloride hydrolysis units, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

16. A method for preparing a corn protein hydrolysate, the method comprising: a) obtaining a corn protein composition having a corn protein concentration of at least 75% by weight; b) adding an enzyme to a corn protein suspension comprising the corn protein composition at a ratio of 1:100 to 1:20 by weight of enzyme to corn protein; c) controlling the pH and temperature of the corn protein suspension to hydrolyze the corn protein; as well as d) terminating said hydrolysis of said corn protein to provide a corn protein hydrolysate having a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, Wherein, the enzyme is a protease obtained from Aspergillus oryzae, and the protease includes protease M, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

17. A method for preparing a corn protein hydrolysate, the method comprising: a) obtaining a corn protein composition having a corn protein concentration of at least 75% by weight; b) adding the corn protein composition to water at a temperature of 45° C. to 55° C. to obtain a 5% (w / v) corn protein suspension, the corn protein suspension having a corn protein content of 5% (w / v) to 25% (w / v); c) adjusting the pH of the corn protein suspension to a pH level of 5.0 to 6.0; d) adding enzyme to the corn protein suspension at a ratio of 1:100 to 1:20 by weight of enzyme to corn protein; as well as e) hydrolyzing the corn protein suspension while maintaining the temperature and the pH, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate having a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, wherein the enzyme is a protease obtained from Aspergillus oryzae, and the protease has an activity that can be described as: 200 azocasein degradation units, 53.4 bovine serum albumin-derived A280-releasing units, 17.3 bovine serum albumin-derived leucine-releasing units, 21.9 bovine hemoglobin degradation units, 61.9 bovine hemoglobin-derived A280-releasing units, 0.535 glycine 4-nitroaniline-HCl hydrolysis units, 10,399 L-leucine p-nitroaniline hydrolysis units, 1283 L-lysine p-nitroaniline dihydrobromide hydrolysis units, 90.8 L-methionine 4-nitroaniline hydrolysis units, 2.28 Nα-benzoyl-L-arginine 4-nitroaniline hydrochloride hydrolysis units and 35.9 Nα-benzoyl-L-tyrosine 4-nitroaniline hydrochloride hydrolysis units, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

18. A method for preparing a corn protein hydrolysate, the method comprising: a) obtaining a corn protein composition having a corn protein concentration of at least 75% by weight; b) adding the corn protein composition to water at a temperature of 45° C. to 55° C. to obtain a 5% (w / v) corn protein suspension, the corn protein suspension having a corn protein content of 5% (w / v) to 25% (w / v); c) adjusting the pH of the corn protein suspension to a pH level of 5.0 to 6.0; d) adding enzyme to the corn protein suspension at a ratio of 1:100 to 1:20 by weight of enzyme to corn protein; as well as e) hydrolyzing the corn protein suspension while maintaining the temperature and the pH, and terminating the hydrolysis of the corn protein to provide a corn protein hydrolysate having a solubility of 7% to 37% at a pH selected from the group consisting of pH 7.0, pH 3.4, pH 5, and all of pH 7.0, pH 5, and pH 3.4, Wherein, the enzyme is a protease obtained from Aspergillus oryzae, and the protease includes protease M, The corn protein hydrolysate has a hydrolysis degree of 1% to 17%.

19. The method of any one of claims 15-18, wherein the corn protein concentration is at least 79% by weight.

20. The method of any one of claims 15-18, wherein the corn protein concentration is 79% to 89% by weight.

21. The method of any one of claims 15-18, wherein the corn protein concentration is 85% to 89% by weight.

22. The method of any one of claims 15-18, wherein the corn protein concentration is 95% to 100% by weight.

23. The method of any one of claims 15-18, wherein the corn protein hydrolysate has a degree of hydrolysis of 1% to 7%.

24. The method of any one of claims 15-18, wherein the corn protein hydrolysate has a degree of hydrolysis of 8% to 14%.

25. The method of any one of claims 15-18, wherein the corn protein hydrolysate has a degree of hydrolysis of 16% to 18%.

26. The method of any one of claims 15-18, wherein the corn protein hydrolysate has a solubility of 7% to 20% at pH 7.

0.

27. The method of any one of claims 15-18, wherein the corn protein hydrolysate has a solubility of 7% to 20% at pH 3.

4.

28. The method of any one of claims 15-18, wherein the temperature of the corn protein suspension during hydrolysis is between 40°C and 60°C.

29. The method of any one of claims 15-18, wherein the pH of the corn protein suspension during hydrolysis is between 5.0 and 6.

0.

30. The method of any one of claims 15-18, wherein the hydrolysis of the corn protein suspension is performed at a pH range of 5.0 to 6.0 and a temperature range of 40°C to 60°C for 30 minutes to 120 minutes.

31. The method of any one of claims 15-18, further comprising mixing the corn protein suspension for at least 30 minutes to 60 minutes prior to adding the enzyme.

32. The method of any one of claims 15-18, wherein the ratio of the enzyme to the corn protein suspension is 1 :40 to 1 :25 by weight of enzyme to corn protein.

33. The method of any one of claims 15-18, wherein the hydrolysis of the corn protein is terminated by neutralizing the corn protein suspension to pH 7.0 and inactivating the enzyme by heating to a temperature of at least 75°C for 5 minutes.

34. The method of any one of claims 15-18, further comprising the step of drying the corn protein hydrolysate.

35. A beverage comprising the corn protein hydrolysate of any one of claims 1 to 34.

36. The beverage of claim 35, wherein the corn protein hydrolysate is present in an amount of 1% to 10% by weight of the beverage.

37. A beverage according to any one of claims 35 to 36, wherein the beverage has a caffeine equivalent value of less than 0.25 g / L.

38. The corn protein hydrolysate according to any one of claims 1 to 2, wherein the corn protein hydrolysate has a caffeine equivalent value of less than 0.25 g / L.

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