Enzymatic process for producing plant protein extracts

Abstract

Disclosed is a method of producing a plant protein extract comprising treating a plant protein raw material under alkaline conditions, recovering the protein, and spray-drying the recovered protein wherein a protein deamidase is added before, during, or after, alkali treatment. The obtained plant protein extract has improved solubility and can be used in food or beverages.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to an enzymatic method for producing a plant protein extract. In particular, the present invention relates to a method for producing a plant protein concentrate or a plant protein isolate having high solubility. Background Art

[0004] The growing world population needs protein-rich foods that can be produced sustainably. Leguminosae have long been considered as a valuable and low-cost source of high-quality protein products (such as concentrates and isolates). Industrial-scale production typically uses soybeans as a protein source, and the most promising alternative to soy protein products is pea protein isolate. Vegetable protein has a high potential value due to its properties (especially suitable for health-oriented applications), so its use (scope of application) is also expected to increase, such as in food, beverages, etc. However, vegetable protein generally has lower solubility and poorer functional properties. It is known that deamidation can improve the solubility of vegetable protein, and therefore improves functional properties (including foaming activity, foaming stability, emulsifying activity and emulsifying stability). This has been observed in several vegetable protein substrates (including cereal proteins such as oats, wheat, zein, and leguminous proteins such as soybeans and pea proteins, coconut protein, etc.). Protein deamidase can be applied to almost all types of proteins (plant proteins, animal proteins, fermented proteins, etc.), where the enzyme will lower the isoelectric point of the protein and, when the protein is applied at a pH above the isoelectric point, will improve solubility, improve electrostatic repulsion, improve different types of functions (such as foaming, emulsification, water binding, etc.).

[0005] Market acceptance of the use of plant-based proteins in food or beverages has been low, and thus there remains a need for the efficient production of plant protein extracts with improved physical properties, particularly solubility, to increase the usefulness of plant proteins. Summary of the invention

[0006] The inventors of the present invention have unexpectedly discovered that in the process of preparing plant protein extracts from plant protein raw materials, some processing processes and / or conditions directly and significantly affect the functional properties of the prepared plant protein extracts (for example, the solubility properties of the obtained protein extracts), which makes the obtained protein extracts more suitable for different applications (such as food and beverages) to achieve the purpose of improving nutritional value.

[0007] Therefore, in one aspect, the present invention relates to a method for producing a plant protein extract, the method comprising treating a plant protein raw material under alkaline conditions, recovering the protein and spray drying the recovered protein, wherein a protein deamidase is added before, during or after the alkaline treatment. Preferably, the plant protein extract is a plant protein concentrate or a plant protein isolate.

[0008] In other aspects, the present invention also relates to the obtained plant protein extract and food or beverage containing the plant protein extract. DETAILED DESCRIPTION

[0009] The present invention relates to a method for producing a plant protein extract, which comprises treating a plant protein raw material under alkaline conditions, recovering the protein and spray drying the recovered protein, wherein a protein deamidase is added before, during or after the alkaline treatment. Preferably, the plant protein composition is a plant protein concentrate or a plant protein isolate.

[0010] Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] The term "about" indicates and encompasses the indicated value and ranges above and below that value. In certain embodiments, the term "about" indicates that the specified value is plus or minus 10%, plus or minus 5%, or plus or minus 1%. In certain embodiments, the term "about" indicates that the specified value is plus or minus one standard deviation of that value.

[0013] In some embodiments of the present invention, the plant protein raw material is treated with a protein deamidase to significantly increase the solubility of the resulting plant protein extract.

[0014] Plant proteins are usually applied to food ingredients as products extracted from a pre-processed state (i.e., gluten flour, seeds or pulp). Plant protein products with higher protein content are typically more popular with customers. Therefore, manufacturers often require the use of plant protein extracts with high protein content in industrial processes. In some embodiments of the present invention, the plant protein composition is a plant protein concentrate or a plant protein isolate. The term "plant protein concentrate" refers to a composition in which the protein content is increased compared to the pre-processed state (i.e., the plant protein raw material) due to the extraction or concentration of protein. Typically, such a concentrate refers to a composition in which the protein content is 40% to 80% (w / w), for example, some commercial soy protein concentrates and pea protein concentrates contain 60%-80% (w / w) protein content, while commercially available peanut protein concentrates, potato protein concentrates and rice protein concentrates have a protein content of about 50%-70% (w / w). The term "plant protein isolate" refers to a composition in which the protein content is increased compared to the pre-processed state (i.e., the plant protein raw material) due to the extraction or concentration of protein. Typically, such an isolate refers to a composition wherein the protein content is 80% (w / w) or higher (e.g., in the range of 80%-99% (w / w)). The protein content of some commercial soy protein isolates or pea protein isolates is about 85% (w / w) or higher, and the protein content of some purified soy protein isolates or pea protein isolates can reach 90% (w / w) or higher.

[0015] In the present invention, plant protein raw material refers to the plant material containing protein. The plant protein raw material subjected to enzyme treatment is not particularly limited. Usually, the raw material for plant protein extraction can be untreated plant raw material or the raw material extracted by other components (i.e. oil, glucan or starch). Some of these raw materials contain higher oil content, which needs to be removed by squeezing, organic extraction or supercritical extraction of carbon dioxide to become the raw material for plant protein extraction, such as soybean, peanut, walnut, perilla, sesame, cashew, hazelnut, hemp seed, etc. Other plant raw materials are rich in materials such as starch and fiber, and can be used for plant protein extraction after often washing, enzymatic degradation, etc., and these raw materials are such as corn, rice, wheat, oats, rye, quinoa, peas, mung beans, broad beans (broad bean), potatoes, etc. Preferably, specific examples of the vegetable protein raw material include peas, chickpeas, soy beans, fava beans, lentils, oats, rye, barley, corn, amaranth, sesame, almonds, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, coconuts, chestnuts, pine nuts, hemp seeds, quinoa, rice and chia seeds. Preferably, the vegetable protein raw material is in the form of powder or flour.

[0016] In some embodiments of the invention, the protein deamidase is a protein glutaminase. Protein glutaminase is referred to as protein-glutamine glutaminase (also referred to as glutaminyl peptide glutaminase) activity, as described in EC3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or substituted at both the α-amino and carboxyl positions, such as L-glutaminyl glycine and L-phenylalanyl-L-glutaminyl glycine. Therefore, deamidase can deamidate the glutamine residues in proteins to glutamic acid residues, and deamidase is also referred to as protein glutamine deamidase. Deamidase enzymes include a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al., "Crystal structures of protein glutaminase and its proforms converted into enzyme-substrate complex," Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and are included in InterPro entry IPR041325.

[0017] Protein glutaminase activity is measured by using a fluorescent substrate containing a glutamine residue and a fluorescence quencher group. The glutamine residue is converted to a glutamic acid residue by deamidase activity, and then the substrate is cleaved by glutamyl endopeptidase to remove the fluorescence quencher group.

[0018] In some embodiments of the present invention, the protein deamidase is a protein asparaginase. Protein asparaginase can directly act on the amide group of the side chain of the asparagine residue contained in the protein to release ammonia, thereby converting the asparagine residue into an aspartic acid residue. In some embodiments of the present invention, as the protein deamidase, any one of protein glutaminase and protein asparaginase can be used, or the two can be used in combination.

[0019] The type or source of the protein deamidase used in the present invention is not particularly limited. The example of protein deamidase includes protein deamidase derived from Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium or Myroides, and commercially available protein glutaminase derived from Chryseobacterium. Protein glutaminase derived from Chryseobacterium proteolyticum can be commercially available as protein-glutaminase "Amano" 500 manufactured by Amano Enzyme Inc., for example, and this commercially available product can be used. Preferred examples include protein deamidase derived from Chryseobacterium, and preferred examples also include protein deamidase derived from Chryseobacterium proteolyticum. In some embodiments of the invention, examples of protein deamidase include protein deamidase derived from Chryseobacterium species 62563, Chryseobacterium gambrini, Chryseobacterium culicis, or Chryseobacterium defluvii or Chryseobacterium gleum. In some embodiments of the invention, protein deamidase can be derived from Chryseobacterium species 62563 and is disclosed as SEQ ID NO: 1 in this article.

[0020] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) to determine the output of sequence identity between two amino acid sequences as " longest identity ", this algorithm is as EMBOSS software package (EMBOSS:TheEuropean Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics trend] 16:276-277) (preferred 6.6.0 version or updated version) Ni Deer program is implemented.The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.In order to make Ni Deer program report the longest identity, must specify non-simplified option in command line.The output calculation of " longest identity " of Ni Deer mark is as follows:

[0021] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0022] In one embodiment, the protein deamidase is selected from a polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1.

[0023] In the context of the present invention, the term "variant" means a polypeptide having endopeptidase activity comprising an alteration (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following the amino acid occupying a position.

[0024] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0025] Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0026] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are changed. For example, these amino acid changes can affect the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0027] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for endopeptidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0028] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0029] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0030] The crushed material or the ground material (powder) of the vegetable protein raw material is usually subjected to enzyme treatment to allow the enzymatic reaction to proceed efficiently. The protein deamidase can be added before the alkali treatment step, during the alkali treatment step, or after the alkali treatment. In particular, the protein deamidase is added to a suspension or solution of the crushed material or the ground material of the vegetable protein raw material to cause the reaction.

[0031] In some embodiments of the present invention, the amount of protein deamidase added can be any amount of 0.001-100 mg enzyme protein / gram of plant protein raw material. Preferably, a ratio of 0.01-10 mg enzyme protein / gram of plant protein raw material can be used, and more preferably, a ratio of 0.1-1 mg enzyme protein / gram of plant protein raw material can be used.

[0032] The vegetable protein raw material can be preferably incubated with the enzyme at a temperature of 5°C-80°C, preferably 10°C-65°C, for example 15°C-55°C. It should be understood that the temperature can be adjusted during incubation. The reaction time can be controlled in the range of, for example, 5 minutes to 1 day, preferably in the range of 10 minutes to 480 minutes, more preferably in the range of 20 minutes to 120 minutes.

[0033] As long as the treatment with protein deamidase is effective for the effect of the present invention (i.e., increasing and / or improving the physical properties of the obtained plant protein extract), the conditions of the treatment are not particularly limited. In preliminary experiments, the plant protein raw material concentration, reaction temperature, reaction pH, reaction time, and the amount of enzyme added (enzyme concentration) can be adjusted, and the optimal reaction conditions can be established according to the enzyme used.

[0034] Plant protein extracts (e.g., plant protein concentrates or plant protein isolates) can be extracted and / or concentrated by alkali treatment and isoelectric precipitation methods. When such methods are used, the following steps are typically performed: (1) after protein deamidase treatment, a step of adjusting the pH of the enzyme reaction solution and performing an alkali treatment to separate soluble components, (2) a step of recovering protein from the separated soluble components by isoelectric precipitation methods, and (3) a step of subjecting the recovered protein to a neutralization process.

[0035] In some embodiments of the present invention, the alkali treatment method and the membrane separation method are used in combination. In addition, as described above, the protein isolate is produced by the protein concentrate through the steps of membrane concentration, etc. In addition, according to the production method of the present invention, a plant protein extract with a protein content in the range of 40% to 99% can be obtained.

[0036] In some embodiments of the present invention, during the alkali treatment (preferably alkali extraction) step, preferably, the treated or untreated plant protein raw material can be dissolved at a raw material powder: water ratio (w / w) in the range of 1:2 to 1:20 (corresponding to a dry matter concentration of 5%-50%), and any suitable alkali (e.g., sodium hydroxide, potassium hydroxide, and calcium hydroxide) can be used for such pH adjustment. For example, a 50% food-grade sodium hydroxide solution (w / w) can be used to adjust the pH of the slurry to achieve the pH required for alkali extraction, thereby dissolving the plant protein into the aqueous solution. In some embodiments, the extraction is carried out at a pH between about 7.0 and about 11.5, for example, between about 7.5 and about 11.0, preferably, the extraction is carried out at a pH between about 7.5 and about 10, or between about 8 and about 10, or between about 7.5 and about 9.0. In some embodiments, in some embodiments, the alkali treatment is treated at 10°C to 55°C for a specified time (e.g., 5 minutes to 24 hours, preferably 10 minutes to 2 hours).

[0037] Protein is recovered from the separated soluble components by an isoelectric precipitation method. For example, when the soluble components are recovered as supernatant after centrifugation, the pH of the supernatant (which may be diluted or concentrated before pH adjustment) is adjusted to about 3 to 6 to precipitate the protein, and the precipitate is then collected by centrifugation. As used herein, the term "isoelectric point" refers to the pH at which the protein has a minimum net ionic charge. The pH of the isoelectric point of proteins and protein compositions can be determined by techniques known in the art. In a preferred embodiment, the pH of the solution containing the protein is adjusted in the range of 3.0 to 6.0, preferably 4.5 to 5.5, preferably 4.5 to 5.0, and the pH can be adjusted by adding an acid (e.g., sulfuric acid or hydrochloric acid, citric acid or malic acid). At the isoelectric point, most proteins precipitate or aggregate.

[0038] After isoelectric precipitation, the precipitated components can be separated by centrifugation, membrane separation, etc. During centrifugation, a desktop centrifuge, a disc centrifuge, a horizontal spiral centrifuge, or a tubular centrifuge can be used to recover the protein by separating and collecting the precipitate. In some embodiments, a desktop centrifuge is used for centrifugation at a speed of 4000 rpm for 10 minutes.

[0039] In some embodiments of the present invention, it is necessary to sterilize the recovered protein, typically UHT, but some techniques such as pasteurization and / or flash sterilization may also be used. In some preferred embodiments, the sample is pasteurized and heated in a water bath at 70°C-90°C for about 10-30 minutes.

[0040] Typically, the collected precipitate is suspended in a suitable solvent (e.g., using water), and then a base (e.g., NaOH or sodium carbonate) is added to neutralize the suspension. After the neutralization process, the plant protein extract is obtained by concentration (e.g., membrane concentration, vacuum evaporation, etc.), drying method (e.g., spray drying, freeze drying, etc.).

[0041] In some embodiments of the present invention, during the spray drying step, dry hot air is typically used to evaporate the water in the uniformly atomized suspension containing the plant protein extract to achieve the purpose of separating the water and dry matter in the suspension. Usually, the suspension containing the plant protein extract is sprayed and atomized with compressed air, and the water and dry matter are separated under a dry hot air environment. The dry matter is collected in the form of powder or particles under the dual action of the induced draft fan and gravity. In some preferred embodiments, the atomized suspension with a dry matter concentration of 5%-20% (w / w) is mixed with dry hot air with an inlet temperature of 130°C-170°C, and the temperature is gradually reduced to 80°C-100°C during the evaporation of water until it reaches the outlet and is then collected. Usually, the water content of the collected plant protein extract is not higher than 15% (w / w), preferably not higher than 10% (w / w).

[0042] Typically, during the freeze drying step, the suspension containing the plant protein extract needs to be pre-frozen, and then the container containing the pre-frozen feed liquid is placed in the closed environment of a freeze dryer with a pressure of 0-200Pa, and gradually thawed and dried. After sublimation, the water in the liquid is condensed in the cold trap of the freeze dryer and separated from the liquid to achieve the purpose of drying. In some preferred embodiments, the concentration of the pre-frozen feed liquid is 5%-20% (w / w), the pre-freezing temperature is -20°C, the cold trap temperature is -50°C, and the ambient pressure is 50Pa. Usually, the feed liquid is freeze-dried under the above conditions and reaches a collectable state. Usually, the water content of the collected plant protein extract is not higher than 10% (w / w).

[0043] Uses of plant protein extracts

[0044] The plant protein extract prepared by the present invention shows greatly improved solubility and other functional properties compared with known materials, and can be added to a variety of food materials to form high-quality protein fortified foods. For example, the plant protein extract can be directly used as food, and has certain commercial value. In addition, the plant protein extract can also be used as food raw materials in food, beverage, fermented beverage, alcoholic beverage, condiment, baked food, dairy products, health products, dietary supplements and other products. The plant protein extract can also be applied to animal feed and pet food. The plant protein extract of the present invention can be used in various forms, including but not limited to powder, granule, rod, baked biscuit or other products, tablet candy, soft capsule or hard capsule, soft candy or hard candy, chewing gum or gel, paste, ice cream, jam, sauce, dried meat, meat substitute (alternative meat) or vegetables, dairy products or fermented dairy products.

[0045] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0046] Examples

[0047] strain

[0048] Chryseobacterium sp. strain 62563 was isolated from a soil sample collected in September 2013 in Sibhult, Sweden.

[0049] Enzymes

[0050] The protein glutaminase is derived from Chryseobacterium sp. 62563 and disclosed as SEQ ID NO:1.

[0051] Example 1 Production of plant protein isolate using protein glutaminase

[0052] Determination

[0053] Pea protein content determination:

[0054] The protein content of pea flour and pea protein isolate was determined by LECO FP-528 nitrogen analyzer. This method is based on the combustion of the sample to determine the nitrogen content, in which CO 2 , H 2 O, NOx, N 2 Pass through different kinds of "filters" to exclude everything except nitrogen. Nitrogen in helium carrier is measured by thermal conductivity cell. EDTA with 9.57% nitrogen is used as calibration standard. 6.25 is used as nitrogen-protein index.

[0055] Determination of enzyme protein (EP) content:

[0056] The enzyme protein (protein glutaminase) content was determined by Pierce BCA protein assay kit (commercially available from Thermo Scientific).

[0057] Production of Pea Protein Isolate

[0058] Pea flour from yellow peas was produced by coarse grinding of shelled pea seeds followed by further grinding with a grinder to obtain a finer ground meal which was sieved through a 0.5 μm sieve with a protein content of 25.7% (w / w).

[0059] 6g of pea flour was suspended in water in a shake flask at a mass ratio of 1:6 (pea flour: water). The pH was then adjusted to 6.5 (for water soaking) and 9.0 (for alkali soaking) with 6mol / L NaOH, respectively, followed by the addition of protein glutaminase at a concentration of 0.4 (mg EP) / (g pea protein). The flask containing the pea flour was then transferred to an orbital shaker and incubated at 50°C / 200rpm for 1 hour. After incubation, the slurry was centrifuged at 4,000rpm for 10min. The supernatant was then collected. Subsequently, the pH of the obtained supernatant was adjusted to 4.5 with 6mol / L HCl. After another round of centrifugation at 4,000rpm / 10min, the precipitate was collected and resuspended in water. After the pH was adjusted to 6.8 with 6mol / L NaOH, the slurry was dried in a freeze dryer or a spray dryer to obtain a pea protein isolate. A control was obtained in the same manner as described above, except that protein glutaminase was not added during the soaking process.

[0060] Drying conditions

[0061] Freeze drying was performed in a freeze dryer (LGJ-10C, Goring Technology Development (Beijing) Co., Ltd.). The freeze drying conditions were as follows: a sample with a dry matter content of 10% (w / v) was dried in a freeze dryer at an air pressure of less than 50 Pa until the water content of the sample was less than 10% (w / w).

[0062] In the spray dryer (Mobile Minor TM The spray drying was carried out in 2000, GEA (GEA). The spray drying conditions were set as follows: a sample with a dry matter content of 8% (w / v) was fed at a rate of 2.4 L / h by a constant flow pump. The inlet temperature of the drying air was 170°C, the pressure was 0.2 MPa, and the outlet temperature of the vegetable protein powder and air was 73°C.

[0063] Solubility determination:

[0064] The obtained 50 mg samples of pea protein isolate from different batches were resuspended in 0.95 ml of 0.01 mol / L phosphate buffer. The phosphate buffer was prepared according to the following steps:

[0065] a) Solution 1: Prepare 0.2 mol / L NaHPO 4 ·H2O solution, 35.01g of NaHPO 4 ·H 2 O was dissolved in distilled water and water was added until the volume of the solution was 1 L.

[0066] b) Solution 2: Prepare 0.1 mol / LC 4 H 2 O 7 ·H2O solution. 21.01 g of C 4 H 2 O 7 ·H 2 O was dissolved in distilled water and water was added until the volume of the solution was 1 L.

[0067] c) The pH of Solution 1 was adjusted to the final desired pH of 7.0 by adding Solution 2 dropwise to Solution 1.

[0068] Such samples were vortexed and shaken at 50°C / 1,200 rpm for 45 min. The mixture was then centrifuged at 12,500 rpm for 15 min at room temperature. TM The kit measures the soluble protein concentration in the supernatant. Bovine serum albumin was used to construct a standard curve. Protein solubility was determined by dividing the soluble protein content of the supernatant by the total protein content in the sample (×100%).

[0069] Relative protein solubility determination:

[0070] The relative protein solubility of the samples was calculated by dividing the protein solubility of the control sample from the water soaking process by the protein solubility of the samples from the other processes (for both freeze drying conditions and spray drying conditions). Therefore, the relative protein solubility of the control sample from the water soaking process was set to 100%.

[0071] According to Tables 1 and 2, the addition of protein glutaminase in the water soaking process and the alkaline soaking process slightly improved the relative protein solubility under freeze-drying conditions compared to the control without the addition of protein glutaminase. However, when spray drying was used without protein glutaminase treatment, the batch associated with the alkaline soaking process showed a lower relative protein solubility, while when we combined the alkaline soaking process with spray drying and protein glutaminase treatment, the relative protein solubility was the highest, reaching 142%. These results indicate that in the process of preparing plant protein extracts from plant protein raw materials, such treatments can significantly affect the functional properties of the prepared plant protein extracts, for example, the solubility of the obtained pea protein extracts, which makes the resulting pea protein extracts more suitable for different applications (e.g., food and beverages).

[0072] Table 1. Relative solubility of pea protein isolate obtained by freeze drying

[0073]

[0074] Table 2. Relative solubility of spray-dried pea protein isolates obtained by spray drying

[0075]

[0076]

Claims

1. A method for producing a plant protein extract, the method comprising treating a plant protein raw material under alkaline conditions, recovering the protein and spray drying the recovered protein, wherein a protein deamidase is added before, during or after the alkaline treatment.

2. The method of claim 1, wherein the protein deamidase is protein glutaminase.

3. The method according to claim 1, wherein the plant protein raw material is one or two or more beans, grains or seeds selected from the group consisting of peas, chickpeas, soybeans, beans, lentils, oats, rye, rice, barley, corn, amaranth, sesame, almonds, peanuts, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, Brazil nuts, coconuts, chestnuts, pine nuts, hemp seeds, quinoa and chia seeds.

4. The method according to claim 1, wherein the protein deamidase is an enzyme derived from a microorganism of the genus Chryseobacterium, preferably derived from Chryseobacterium species 62563.

5. The method of claim 1, wherein the plant protein extract is a plant protein isolate or a plant protein concentrate. The method according to claim 1 , wherein the alkali treatment is performed at a pH of 7.5 to 11.

7. The method according to claim 1, wherein the step of recovering the protein is performed by recovering the protein from the separated soluble component by an isoelectric precipitation method.

8. The method of claim 1, wherein the plant protein extract has improved solubility.

9. A plant protein extract obtained by the method according to any one of claims 1 to 8.

10. A food or beverage containing a plant protein extract obtained by the method according to any one of claims 1 to 8.

Images