Use of peptidylarginine deiminase to obtain improved food products - Patent Application 20070122997

Abstract

The present invention describes a process for modifying the sweetness, licorice, astringency, powdery / mealiness, body, thickness, and / or digestibility and / or protease inhibitor activity of protein-containing foods. [Selection diagram] None

Description

Detailed Description of the Invention

[0001] [Field] The present invention relates to the field of food.

[0002] [background] In a world with a growing population, the demand for protein is increasing. To meet this growing demand for protein, it is necessary to focus on a wider range of uses for protein. In addition, since plants are considered to be a more sustainable source of protein than animals, it is desirable to find plant proteins as an alternative to animal proteins. The use of plant proteins in food is still limited, in part due to their poor digestibility, flavor, and nutritional value.

[0003] The poor digestibility of proteins in diets containing less refined cereals and legumes is due to the presence of poorly digestible protein fractions, high levels of insoluble fiber, and / or high concentrations of antinutritional factors present or formed during processing. Dietary antinutritional factors (ANFs) are known to adversely affect protein digestibility, amino acid bioavailability, and the protein quality of foods (G. Sarwar Gilani et al., British Journal of Nutrition (2012), 108, S315-S332).

[0004] The presence of high levels of trypsin inhibitors in soybeans and other legumes has been shown to cause a significant reduction (up to 50%) in protein digestibility in rats and pigs (G. Sarwar Gilani et al., British Journal of Nutrition (2012), 108, S315-S332). Based on the residue at their inhibitory site, these inhibitors are classified as either lysine-type or arginine-type inhibitors. Soybeans are the main source of trypsin inhibitors, accounting for approximately 6% of the protein in defatted soybeans. There are two main types of trypsin inhibitors: Kunitz soybean trypsin inhibitor (KSTI), which has arginine at the reactive site, and Bowman-Birk inhibitor (BBI), which has lysine at the inhibitory site.

[0005] Moist heat treatment of soybeans (100°C for 20 minutes) deactivates some of the inhibitors, but significant original trypsin activity remains. The prolonged heating required to completely destroy the inhibitory activity will significantly reduce the digestibility and quality of the protein in soy products (G. Sarwar Gilani et al., British Journal of Nutrition (2012), 108, S315-S332). Some commercially available soy beverages have been reported to contain approximately 70% or less of the total inhibitory activity. Similarly, soy-based infant formulas have been found to contain 28%. Trypsin inhibitors in raw soybeans cause growth retardation and pancreatic dilation in susceptible animals.

[0006] Another important factor in creating plant-based protein drinks is their flavor and mouthfeel.Even at low protein content, plant proteins have the disadvantage of losing flavor, such as bitterness, and increasing mouthfeel.In addition, many plant protein drinks contain very little protein, making them less nutritious and less tasty foods.

[0007] WO 2008 / 000714 discloses the protein arginine deiminase and the use of this enzyme in the preparation of foodstuffs with increased amounts of citrulline.

[0008] WO 2017 / 009100 discloses a process for improving the solubility of plant proteins, such as pea, soy, and rice proteins, in which the plant proteins are incubated with peptidylarginine deiminase. The foam volume of plant proteins, such as pea proteins, was reduced after incubation with peptidylarginine deiminase.

[0009] There is a need in the art to improve the properties of foods containing (plant) proteins. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the change in absorbance over time (BAEE continuous assay described in Example 1), which directly relates to trypsin activity during the assay. The sample "min PAD" represents trypsin activity in the presence of inhibitory protein, whereas "plus PAD" represents trypsin activity measured in the presence of inhibitory protein pretreated with PAD enzyme. The arrows indicate the degree of reduction in trypsin inhibitory activity (TIA) due to the action of the enzyme PAD. [Figure 2] Figure 2 shows the NH2 released from RPI over time in the SYMPHYD digestion model without (open circles) and with (filled circles) PAD enzyme treatment. Arrows indicate additional time points: 5 minutes for pepsin and 80 minutes for pancreatin, respectively. [Figure 3]3 shows a sensory panel evaluation of soy milk with and without PAD enzyme treatment as described in Example 4. The black bars represent the sensory profile of the soy drink, and the patterned bars represent the sensory profile of the soy drink treated with PAD enzyme. The attribute "powdery-chalk-mf" represents powdery / chalky mouthfeel, "fulness-mf" represents body mouthfeel, and "thick-mf" represents thickness in the mouth. [Figure 4] 4 shows the sensory attributes of 2% solutions of rapeseed protein isolate with and without PAD treatment. Squares: sensory attributes of rapeseed protein isolate solutions not treated with PAD; Diamonds: sensory attributes of rapeseed protein isolate solutions treated with 2U PAD per liter; Triangles: sensory attributes of rapeseed protein isolate solutions treated with 20U PAD per liter; Circles: sensory attributes of rapeseed protein isolate solutions treated with 60U PAD per liter. The sensory attributes are mouthfeel (I), taste intensity (II), sweet taste (III), bitter taste (IV), licorice taste (V), bitter aftertaste (VI), aftertaste length (VII), and astringent aftertaste (VIII). [Figure 5] Figure 5 shows an isoelectric focusing (IEF) gel of various plant-based drinks incubated with PAD enzyme: Lane 1 - pea drink (Bolthouse Farm unsweetened); Lane 2 - pea drink incubated with PAD (Bolthouse Farm unsweetened); Lane 3 - soy drink (Provamel unsweetened); Lane 4 - soy drink incubated with PAD (Provamel unsweetened); Lane 5 - pea drink (Ripple unsweetened); Lane 6 - pea drink incubated with PAD (Ripple unsweetened), and Lane 7 pl marker. [Figure 6]Figure 6 shows the relative stability of PAD enzymes at various pHs and temperatures. Enzyme activity was set to 100% for incubation at pH 7 and 4°C before activity measurement. Triangles indicate enzyme stability at 37°C; circles indicate enzyme stability at 4°C as described in Example 7.

[0011] [Sequence table] SEQ ID NO: 1 Peptidylarginine deiminase from Fusarium graminearum

[0012] [overview] The present invention relates to a process for modifying the sweetness, licorice, astringency, powdery / mealiness, body, thickness, and / or digestibility and / or protease inhibitor activity of protein-containing foods.

[0013] The present invention also relates to the use of peptidylarginine deiminase (PAD) to modify sweetness, licorice, astringency, powdery / cheekiness, body, thickness, and / or digestibility and / or protease inhibitory activity.

[0014] The present invention also relates to dietary supplements containing peptidylarginine deiminase (PAD).

[0015] [Detailed explanation] The poor digestibility of vegetable proteins is also due to high levels of antinutritional factors (ANFs), such as trypsin inhibitors. The mouthfeel of drinks prepared from vegetable proteins is usually described as unacceptable. Until the present invention, no acceptable solution was available to overcome these shortcomings of vegetable protein drinks. This is generally true for protein-containing foods as well.

[0016] PAD deiminates arginine residues in proteins to citrulline. For trypsin inhibitors where arginine occupies the reactive site, the action of PAD will render the inhibitor inactive against trypsin. Chymotrypsin protease inhibitors and α-amylase inhibitors, like trypsin inhibitors, also contribute to the reduced digestibility of foods. Some of these inhibitors have been shown to have critical arginine residues that are important for their inhibitory function (Gideon M. Polya, Atta-ur-Rahman Ed., Studies in Natural Products Chemistry, vol. 29, p. 567-641).

[0017] Approximately 10% of all protease inhibitors contain an arginine (R) residue at the reactive P1 site (309 of the 3,300 sequences found in the MEROPS database for protease inhibitors). Trypsin inhibitors with an "R" at the P1 site can be found in all classes of plant proteins, such as legumes, cereals, and seeds (soybean, rapeseed, nuts, wheat, corn, barley, potato, rice, oats, tomatoes, etc.), although not all inhibitory activities are found to be distributed equally among different species. For example, approximately 40% of the proteins in potatoes are protease inhibitors, and there are many different classes of protease inhibitors (more than 10 different protease inhibitors have been identified), with the distribution of these inhibitors depending on the potato variety (Pouvreau L. et al., J. Agric. Food Chem., 2001, vol. 49, pp. 2864-2874).

[0018] The deactivation of Kunits soybean trypsin inhibitor was previously demonstrated using mammalian PAD enzymes (H. Takahara et al., 1985, The Journal of Biological Chemistry, Vol. 260, No. 14, pages 8378-8383). However, deactivation of TIA (trypsin inhibitor activity) in complex protein matrices, such as soy flour, was not demonstrated. PAD activity in complex food matrices is essential for the usefulness of food products. In addition, mammalian PAD has a narrow selectivity for arginine-containing protein sources, and not all proteins containing arginine residues can be modified using mammalian PAD2 enzymes. It has been shown that chicken egg white ovomucoid, which contains an arginine residue at one of the trypsin inhibitor reactive sites, could not be modified by mammalian PAD enzymes. In contrast, the inventors herein demonstrate that microbial PAD can reduce trypsin inhibitory activity in chicken egg white ovomucoid, making this enzyme suitable for use in a wide variety of protein foods containing protease inhibitory activity. Furthermore, previous studies have shown that the trypsin-chymotrypsin inhibitor Bill in peanuts can be modified by mammalian PAD enzymes, although not all arginine residues involved in protease inhibitor activity can be modified (Tomofumi Kurokawa et al., J. Biochem, 1987, vol. 101, pp. 1361-1367). This study, however, focused on the effect of the enzyme on purified peanut Bill protease inhibitor from peanuts. In the present invention, we demonstrate that microbial PAD can reduce not only Bill trypsin inhibitory activity but also the trypsin inhibitory activity of whole peanuts. The effects of the microbial enzyme PAD in the present invention can also be seen in more complex foods, such as processed peanut butter.

[0019] Surprisingly, the inventors of the present invention show that various aspects such as organoleptic and / or mouthfeel and / or digestibility and / or protease inhibitory activity of protein-containing foods can be improved using PAD.

[0020] In one aspect, the present invention provides a process for modifying the sweetness, licorice, astringency, powdery / cheekiness, body, thickness, and / or digestibility and / or protease inhibitor activity of a protein-containing food product, the process comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. The present invention relates to a process including:

[0021] Preferably, the process of the present invention modifies at least one of the listed properties, i.e., the process of the present invention provides a method for modifying (preferably reducing) the sweetness of a protein-containing food. Alternatively, the present invention provides a process for modifying (preferably reducing) the licorice of a protein-containing food. The present invention also provides a process for modifying (preferably reducing) the astringency of a protein-containing food. The present invention further provides a process for modifying (preferably reducing) the powdery / mealiness of a protein-containing food. Alternatively, the present invention provides a process for modifying (preferably reducing) the kokumi (body character) of a protein-containing food. The present invention also provides a process for modifying (preferably reducing) the thickness of a protein-containing food. The present invention further provides a process for modifying (preferably increasing) the digestibility of a protein-containing food. The present invention also provides a process for modifying (preferably reducing) the protease inhibitor activity of a protein-containing food. In another preferred embodiment, the process of the present invention modifies at least two, three, or four of the listed properties, such as a process for modifying the powdery / mealiness, body, and thickness of a protein-containing food product.

[0022] As used herein, the term "sweetness" refers to the basic flavor most commonly perceived when eating foods rich in sugar.

[0023] As used herein, the term "licorice" means ,vinegar Present in the sweet root of Glycyrrhiza glabra or stevia plant species taste / Refers to the constituent components.

[0024] The term "astringency" as used herein refers to a dry, unflattering mouthfeel similar to that caused by tannins found in many fruits, for example, or by proteins, especially in an acidic environment.

[0025] As used herein, the term "powdery / chalky" refers to the degree to which a product feels powdery or granular in the mouth.

[0026] The term "kokumi" as used herein refers to a rich, mellow feeling in the mouth.

[0027] The term "thickness" as used herein refers to the consistency in the mouth and corresponds to the force required to force the product between the tongue and the roof of the mouth.

[0028] The term "digestibility" as used herein refers to the tendency to be digested and relates to the amount of food retained by the body relative to the amount ingested. The digestibility of a protein is directly related to the degree of hydrolysis carried out by digestive proteases, such as pepsin and pancreatic peptidase, upon incubation with the protein under physiologically relevant conditions.

[0029] As used herein, the term "protease inhibitor activity" refers to an activity (preferably a protein) that inhibits the function of a protease (an enzyme that helps break down proteins).

[0030] The term "modification" refers to a decrease or an increase, depending on the desired goal. For example, the present invention provides a process for increasing the digestibility of a protein-containing food product, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. The present invention provides a process including:

[0031] The present invention also provides a process for reducing sweetness, licorice, astringency, powdery / cheekiness, body, and / or thickness in a protein-containing food product, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. Also provided is a process comprising:

[0032] The present invention also provides a process for reducing the protease inhibitor activity of a protein-containing food product, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. Also provided is a process comprising:

[0033] Whether any of the listed properties increase or decrease is determined by comparison with an otherwise identical prepared food product in which the protein solution is not incubated with PAD.

[0034] The term "protein-containing food" refers to a food that contains protein as an integral component or as an additional component, i.e., a food that contains protein. Protein-containing foods preferably contain at least 0.1% protein. Any suitable protein may be used in the process of the present invention. Advantageously, the protein contains at least 1 mol%, 2, 3, 4, 5, or at least 6 mol% of arginine bound to the protein.

[0035] A protein solution (or: food protein ingredient) is a liquid protein composition containing protein. A protein solution contains protease inhibitor activity if the digestibility of a food containing the protein is modified (preferably increased). A protein solution contains protease inhibitor activity if the protease inhibitor activity is modified (preferably decreased). As used herein, the term "protease inhibitor activity" refers to a peptide that, when added to a protease, will reduce the protease activity. The term "protein solution" also includes solutions in which not all components are dissolved, i.e., the term "protein solution" also includes protein suspensions.

[0036] The step of "incubating the protein solution with peptidyl arginine deiminase (PAD)" can be carried out at any suitable enzyme concentration, for any suitable time, and at any suitable pH. Those skilled in the art can easily determine the appropriate enzyme amount, incubation temperature, incubation pH, or incubation time. For example, the protein can be incubated with peptidyl arginine deiminase at a pH between 4 and 9, such as between 5 and 8.5, such as between 5.5 and 8, such as between 6 and 7, or between 6.2 and 6.8, e.g., about 6.5. Suitable temperatures for incubating the protein with PAD can be between 20 and 60 degrees Celsius, between 30 and 50 degrees Celsius, or between 35 and 45 degrees Celsius.

[0037] The PAD-treated protein solution can be a final product, such as, but not limited to, a protein drink, or alternatively, a food ingredient that can be subsequently further processed into a protein-containing food product.

[0038] As described above, any suitable protein can be used in the process of the present invention. In a preferred embodiment, the protein in the process of the present invention is a plant protein or vegetable protein (these terms are used interchangeably herein). Preferably, the protein solution used in the process of the present invention is a solution containing a plant protein such as soybean protein, rapeseed protein, wheat protein, buckwheat protein, corn protein, barley protein, potato protein, rice protein, oat protein, pea protein, (pea) nut protein, lupin protein, almond protein, etc., or the protein is egg protein, milk protein, gelatin protein, or microbial protein.

[0039] The process of the present invention includes at least one step of incubating a protein solution with peptidylarginine deiminase (PAD). As described above, and depending on whether the protein solution is a food ingredient or a final protein-containing food product, the process of the present invention optionally includes a step of processing the PAD-treated protein solution into a protein-containing food product. Depending on the starting material, further options may be included in the process of the present invention. If the starting material is a flour, such as, but not limited to, a plant protein flour, the process of the present invention will include a step of dissolving the flour to obtain a protein solution. Dissolving the flour typically involves adding a liquid (e.g., water or a buffer) to the flour and allowing the flour to at least partially dissolve in the liquid. Depending on the characteristics of the flour, mixing the liquid and the flour for a certain period of time, optionally with some heating to improve / speed up dissolution, may be required. Alternatively, a suspension is prepared in water or a suitable buffer. That is, an optional additional step of the method of the present invention includes dissolving the flour to obtain a protein solution or preparing a suspension from the flour.

[0040] The terms protein arginine deiminase and peptidylarginine deiminase (PAD) are used interchangeably herein. Protein or peptidylarginine deiminase belongs to a family of enzymes (EC 3.5.3.15) that convert peptide- or protein-bound arginine to peptide- or protein-bound citrulline. This process is called deamination or citrullination. In the arginine-to-citrulline reaction, one of the nitrogen atoms at the end of the arginine side chain is replaced with oxygen. The reaction uses one molecule of water and produces ammonia as a byproduct (http: / / en.wikipedia.org / wiki / Citrullination). Arginine is positively charged at neutral pH, while citrulline is uncharged. Surprisingly, it was found that proteins in which at least a portion of the arginine was converted to citrulline, thereby resulting in a less charged protein, exhibited modified sweetness, licorice, astringency, powdery / mealiness, body, thickness, and / or digestibility.

[0041] Peptidylarginine deiminase (PAD) may be derived from any suitable source, for example, mammalian or microbial. The PAD used in the present invention is advantageously derived from a microbial source, i.e., the PAD used in the process of the present invention is a microbial PAD. For example, the PAD may be derived from a fungal source, such as from Fusarium species, such as Fusarium graminearum, Chaetomium globosum, or Phaesphaeria nodorum, or from a bacterial source, such as the bacterium Streptomyces, e.g., Streptomyces scabies or Streptomyces clavuligeres. The phrases "derived from" or "may be derived from" with respect to the origin of a polypeptide disclosed herein mean that when a BLAST search is performed with a polypeptide disclosed herein, the polypeptide may be derived from a natural source, such as a microbial cell, and the endogenous polypeptide will show the highest percentage of homology or identity with the polypeptide disclosed herein.

[0042] Peptidylarginine deiminases are known, for example, from WO 2008 / 000714, which discloses a process for enzymatically treating proteins with protein arginine deiminases, in which at least 30% of the arginine is converted to citrulline.

[0043] The peptidylarginine deiminase may be a pure or purified peptidylarginine deiminase. A pure, purified peptidylarginine deiminase is an enzyme that is at least 50% pure, e.g., at least 60% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 80% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, or 99.9% pure, as determined, for example, by SDS-PAGE or any other analytical method suitable for this purpose and known to those of skill in the art.

[0044] Preferably, the peptidylarginine deiminase used is Ca 2+ More preferably, the peptidylarginine deiminase used is a microbial PAD, and is Ca-independent. 2+ It is independent.

[0045] Advantageously, the peptidylarginine deiminase used in the process of the invention is a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:1 or to the mature amino acid sequence of SEQ ID NO:1, wherein the polypeptide has peptidylarginine deiminase activity.

[0046] For purposes of the present invention, to determine the percentage of sequence identity between two amino acid sequences, it is described herein that the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced into either of the two sequences being compared. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments can be performed over a shorter length, e.g., about 20, about 50, about 100, or more amino acids. Sequence identity refers to the percentage of exact matches between the two sequences over the reported alignment region. The percent sequence identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid and nucleotide sequences can be aligned algorithmically. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as the substitution matrix. Optional parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5. While all of these different parameters will yield slightly different results, those skilled in the art will appreciate that the overall percentage identity of two sequences will not change significantly when using different algorithms.

[0047] A "mature polypeptide" is defined herein as a polypeptide in its final form, obtained after translation of mRNA into a polypeptide and post-translational modification of said polypeptide, including N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and cleavage to remove leader sequences, such as signal peptides, propeptides, and / or prepropeptides.

[0048] The mature polypeptide sequence of SEQ ID NO: 1 may comprise or contain amino acids 19, 20, 21, 22, 23, 24 to 640 of the amino acid sequence of SEQ ID NO: 1, and conveniently the mature polypeptide sequence of SEQ ID NO: 1 comprises or contains amino acids 22 to 640 of SEQ ID NO: 1, with the methionine at position 1 in SEQ ID NO: 1 being counted as number 1.

[0049] The term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. As used herein, the term "protein" is synonymous with the term "polypeptide" and may refer to two or more polypeptides. Thus, the terms "protein" and "polypeptide" can be used interchangeably. Polypeptides may optionally be modified to add functionality (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, and the like). Polypeptides that exhibit activity in the presence of specific substrates under certain conditions may be called enzymes.

[0050] Peptidylarginine deiminase or a polypeptide having peptidylarginine deiminase activity may be produced in any suitable host organism by methods known in the art, such as the fungi Aspergillus, e.g., Aspergillus niger or Aspergillus oryzae, Trichoderma, or the yeasts Saccharomyces and Kluyveromyces, or bacteria of the genera Streptomyces or Bacillus. Suitable methods for expressing a polypeptide having peptidylarginine deiminase activity in Aspergillus niger are disclosed, for example, in Examples 3 and 4 of WO 2008 / 000714, which is incorporated herein by reference.

[0051] As used herein, the term "protein-containing food" refers to any type of food, as long as the food contains at least protein. The term food refers to solid meals and drinks.

[0052] In one embodiment of the present invention, the invention provides a process for modifying the sweetness, licorice, astringency, powdery / cheekiness, body, thickness, and / or digestibility and / or protease inhibitor activity of a protein-containing food product, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product; The protein-containing food product provides a process for producing a protein-containing drink, i.e., a liquid intended for ingestion.

[0053] The protein-containing drink can be any type of drink, but in a preferred embodiment, the protein-containing drink is a plant protein drink or a fermented plant protein product. Examples of suitable plant protein drinks include soybean drinks, pea drinks, peanut drinks, barley drinks, rice drinks, oat drinks, quinoa drinks, almond drinks, cashew drinks, coconut drinks, hazelnut drinks, hemp drinks, sesame seed drinks, wheat drinks, potato drinks, and sunflower seed drinks. Examples of suitable fermented plant protein products include fermented soybean products, fermented pea products, fermented peanut products, fermented barley products, fermented rice products, fermented oat products, fermented quinoa products, fermented almond products, fermented cashew products, fermented coconut products, fermented hazelnut products, fermented hemp products, fermented sesame seed drinks, fermented wheat products, fermented potato products, and fermented sunflower seed products.

[0054] Soybean proteins are commonly used to prepare baby, follow-on, or infant drinks. Properties such as sweetness, liquorice, astringency, powdery / chatteriness, body, thickness, and / or digestibility can be improved by using the process of the present invention. The present invention therefore provides a process for modifying the sweetness, liquorice, astringency, powdery / chatteriness, body, thickness, and / or digestibility and / or protease inhibitor activity of a protein-containing food product, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. wherein the protein-containing food product is a protein-containing drink, the drink being an infant, follow-on, or toddler drink, and the protein is soy protein.

[0055] The protein-containing drinks produced can be drinks produced without or with added protein. Protein-enriched (plant) protein drinks offer benefits such as improved nutritional value, but are typically considered difficult to consume due to undesirable properties such as powdery / chalky, body, and thickness. Protein-enriched (plant) protein drinks prepared by the process of the present invention have improved (reduced) powdery / chalky, body, and thickness, and thus the present invention provides a process for modifying the sweetness, licorice, astringency, powdery / chalky, body, thickness, and / or digestibility and / or protease inhibitor activity of a protein-containing food, comprising: Incubating the protein solution with peptidylarginine deiminase (PAD) and Optionally, processing the PAD-treated protein solution into a protein-containing food product. and the protein-containing food product is a protein-enriched (plant) protein drink. An example of a protein-enriched drink is a protein-enriched drink for the elderly or people recovering (after surgery) to increase calorie intake. Alternatively, such a protein-enriched drink may be a medicinal or medical drink or a sports drink.

[0056] Protein-containing drinks prepared by the process of the present invention can have a neutral or acidic pH.

[0057] The present invention further provides the use of peptidylarginine deiminase (PAD) for modifying sweetness, licorice, astringency, powdery / cheekiness, richness, thickness, and / or digestibility and / or protease inhibitory activity. Preferably, the present invention provides the use of peptidylarginine deiminase (PAD) for modifying licorice, astringency, powdery / cheekiness, richness, thickness, and / or digestibility and / or protease inhibitory activity. Preferably, the present invention provides the use of peptidylarginine deiminase (PAD) for modifying licorice, astringency, powdery / cheekiness, richness, thickness, and / or digestibility and / or protease inhibitory activity. Preferably, the present invention provides the use of peptidylarginine deiminase (PAD) for modifying powdery / cheekiness, richness, thickness, and / or digestibility and / or protease inhibitory activity. The features described above for the process of the invention apply equally to the part of use.

[0058] Products obtained directly from any of the processes described herein are also within the scope of the present invention.

[0059] In another aspect, the present invention provides a nutraceutical composition comprising a peptidylarginine deiminase (PAD), preferably a microbial peptidylarginine deiminase (PAD). More preferably, the microbial peptidylarginine deiminase (PAD) has at least 80% identity to SEQ ID NO:1 or has at least 80% identity to the mature amino acid sequence of SEQ ID NO:1.

[0060] The term nutraceutical as used herein indicates usefulness in both nutritional and pharmaceutical applications.Thus, the novel nutraceutical compositions can find use as supplements to foods and beverages, as well as pharmaceutical preparations or medicaments for enteral or parenteral application, which may be in solid formulations such as capsules or tablets, or in liquid formulations such as solutions or suspensions.As is clear from the above, the term nutraceutical composition also includes food and drink and supplement compositions, such as dietary supplements, obtained by any of the methods described herein.

[0061] The term dietary supplement, as used herein, refers to a product taken by mouth containing "dietary ingredients" intended to supplement the diet. The "dietary ingredients" in these products may include substances such as vitamins, minerals, herbs or other botanicals, amino acids, and enzymes, as well as organ tissues, glands, and metabolites. Dietary supplements can also be extracts or concentrates and may be found in many forms, such as tablets, capsules, softgels, gelcaps, liquids, or powders. They can also take other forms, such as bars, although if so, the information on a dietary supplement label would generally not describe the product as a conventional food or a standalone item of diet or drink.

[0062] The present invention will be explained in more detail in the following examples, which do not limit the invention.

[0063] [Experimental section] [material] [Cloning and expression of peptidylarginine deiminase] Cloning and expression of a polypeptide with peptidylarginine deiminase activity according to SEQ ID NO: 1 was carried out as disclosed in Examples 3 and 4 of WO 2008 / 000714.

[0064] [Peptidylarginine deiminase (PAD) activity] Peptidylarginine deiminase activity was determined by measuring the formation of citrulline residues in α-N-benzoyl-L-arginine ethyl ester (BAEE). The incubation mixture contained 100 mM Tris-HCl buffer (pH 7.5), 5 mM CaCl, 10 mM DTT, and 10 mM BAEE in a final volume of 700 μl. Incubation was carried out at 55°C for 30 min, and the reaction was stopped by adding 100 μl of 8 N HClO. Citrulline was determined colorimetrically by the method of Guthöhrlein and Knappe (1968) Anal. Biochem. 26, 188.

[0065] One unit of peptidylarginine deiminase is expressed as 1 μmol citrulline formed / min / mg protein.

[0066] [Example 1] [Reduction of trypsin inhibitor activity (TIA) in proteins treated with PAD enzymes] Various proteins containing TIA were incubated with PAD and the reduction of TIA was measured by two different assays. The preparation of proteins with and without PAD incubation and the assays used to measure TIA are described below.

[0067] Soy drink sample preparation: 400 μl of commercial soy drink (Provamel unsweetened) from a local store was diluted with 600 μl of tap water. Subsequently, 0.17 U / ml PAD was added, and the solution was incubated at 40°C (600 rpm thermomixer) for 2.5 hours. A control sample was incubated with the same volume of water instead of PAD.

[0068] Soy flour sample preparation: 1 g of soybeans was mixed with 49 g of 0.01 N NaOH. The pH of the suspension was adjusted to 10 with 4 N HCl. The suspension was thoroughly mixed with magnetic stirring for 3 hours at ambient temperature. The sample was then heated to 3200 * The supernatant (without the lipid-containing upper layer) was centrifuged again at 20817 g for 10 min. To the resulting 150 μl of supernatant, 0.04 U of PAD was added, and the solution was incubated at 45°C for 30 min (thermomixer 600 rpm). A control sample was incubated with the same volume of water instead of PAD.

[0069] Chicken egg white ovomucoid sample preparation: Ovomucoid (Sigma) was solubilized (0.25 mg / ml) in 100 mM HEPES, 5 mM CaCl, 5 mM DTT, pH 7.2. 0.17 U / ml PAD was added and incubated at 37°C. The reaction was stopped with 25 μl 0.2 M EDTA.

[0070] Rapeseed meal sample preparation: 100 mg of rapeseed meal (obtained from cold-pressed rapeseed oil seed meal) was supplemented with 900 μl 2% NaCl and 0.17 units of PAD. The rapeseed was solubilized at 55°C in a thermomixer at 800 rpm for 30 minutes. After this, the sample was centrifuged at 20817 g for 10 minutes. A control rapeseed meal sample was solubilized in the absence of PAD.

[0071] Rapeseed Protein Isolate (RPI) Sample Preparation: RPI was prepared as described in patent WO 2018 / 007492, starting from cold-pressed rapeseed oil cake. A 2% (w / v) solution of RPI was prepared from this sample. Subsequently, 0.17 U / ml of PAD was added, and the solution was incubated at 40°C for 2.5 hours (thermomixer 600 rpm). A control sample was incubated with the same volume of water instead of PAD.

[0072] Preparation of peanut and peanut butter samples: Peanuts (with shells) were purchased from a local store. The shells were removed and 1 part peanuts was mixed with 4 parts water (w / w). Peanut butter was purchased from a local store (Terra Sana brand) and mixed with 3 parts water (w / w). To 500 μl of this suspension, 0.4 U of PAD or water (control) was added and incubated at 40°C for 2 hours. Subsequently, 1 part was diluted with 2 parts 10 mM acetic acid. 125 μl was analyzed as described below (TIA measurement).

[0073] [Barley sample preparation] One gram of barley was mixed in 10 ml of water (magnetic stirring). 0.4 U of PAD was added to 500 μl of this suspension and incubated at 45° C. for 90 minutes (thermomixer 600 rpm). As a control, water was added instead of PAD.

[0074] [Preparation of buckwheat flour and buckwheat drink samples:] A 10% solution of buckwheat flour purchased from a local store was prepared in water by mixing. Biological buckwheat drink (brand name Isola) purchased from a local store was used as is. To 500 μl of this suspension, 0.4 U of PAD was added and incubated at 45°C for 100 min (thermomixer 600 rpm). For control incubations, water was added instead of PAD. Trypsin inhibitory activity was measured using the AzoCasein assay as described below.

[0075] Preparation of lupin samples Lupin seeds were obtained from a local producer and further processed into a 10% flour suspension in water. To 500 μl of this suspension, 0.4 U of PAD was added and incubated at 45°C for 100 min (thermomixer 600 rpm). For control incubations, water was added instead of PAD. Trypsin inhibitory activity was measured using the AzoCasein assay as described below.

[0076] [TIA measurement] [Measurement of TIA by BAEE assay] The assay used herein was essentially as described in the Sigma protocol "assay method for trypsin inhibitor activity" (https: / / www.sigmaaldrich.com / technical-documents / protocols / biology / enzymatic-assay-of-trypsin-inhibitor.html). 25 μl of 1 mg / ml trypsin (Sigma; bovine pancreatic trypsin; 15267 units / mg solids) in 1 mM HCl was added to 1.875 ml of 1 mM HCl. Subsequently, 100 μl of protein sample (with or without incubation with enzyme) prepared as described above was added to the reaction mixture. As a control, 100 μl of 1 mM HCl was added instead of the inhibitor in the trypsin activity measurement. These solutions were incubated at ambient temperature for 5 minutes.

[0077] To 2.7 ml of 67 mM sodium phosphate, pH 7.6 (25 °C), 300 μl of BAEE (Sigma; Na-benzoyl-L-arginine ethyl ester hydrochloride, 0.86 mg / ml in the same buffer) and 100 μl of 1 mM HCl were added. Trypsin activity was determined after the addition of 100 μl of trypsin solution, and the reaction was monitored at 253 nm. The spectrophotometer was calibrated with a blank containing 100 μl of 1 mM HCl instead of trypsin solution. The reaction was continued for 20 min, with three data points taken every minute (Figure 1).

[0078] An increase in absorbance at 253 nm (increasing slope of the line) indicates cleavage of BAEE by trypsin. When trypsin is incubated with a protein sample before the addition of the substrate BAEE, the absorbance at 253 nm decreases within a certain time period, indicating inhibition of trypsin enzyme activity. When the protein sample is incubated with PAD before addition to trypsin, the absorbance at 253 nm returns (to some extent), indicating less trypsin inhibition by the protein sample. From the slopes of the samples with and without PAD treatment, the reduction in TIA levels by PAD can be calculated. Trypsin inhibition (%) was calculated by: 100 - (sample - sample background) / (trypsin - blank).

[0079] Measurement of TIA using the Azo Casein Assay The level of TIA in protein materials was assessed using the assay described in "Quantitative Determination of Trypsin Inhibitory Activity in Complex Matrices," Robin E. J. Spelbrink et al.; The Open Food Science Journal, 2011, 5, 42-46. Briefly, 125 μl of protein sample (or 125 μl of 10 mM acetic acid for the blank) was mixed with 25 μl of 0.35 mg / ml trypsin in 1 mM hydrochloric acid. The reaction was initiated by the addition of 30 mg / ml azocasein in 100 mM Tris, 5 mM calcium chloride, pH 8.5. For background signals, trypsin was replaced with 1 mM HCl. After 30 min at 37°C, the samples were quenched with 150 μl of 15% TCA. Unhydrolyzed protein material and other insolubles were analyzed by 15000 μl HCl at 4°C. * The supernatant was removed by centrifugation at 1000 x g for 10 minutes. 100 μl was transferred to a 96-well microtiter plate and mixed with 100 μl of 1.5 M NaOH. The absorbance at 450 nm was measured. Trypsin inhibition (%) was calculated as follows: 100 - (sample - sample background) / (trypsin - blank).

[0080] Table 1 shows the reduction in TIA measured by the BAEE assay or the Azocasein assay for the proteins listed above.

[0081] [Table 1]

[0082] [Example 2] Quantification of TIA in soy drinks and soy flour using international standard analytical methods. The international standard analytical method for measuring trypsin inhibitory activity was used at Eurofin labco (EN-ISO 14902:2001) to measure the trypsin inhibitory activity of soy flour and soy drinks before and after enzymatic treatment with PAD.

[0083] [Enzyme treatment of soy drinks and soy flour:] 300 g of soy drink (Provamel unsweetened) purchased from a local store was incubated with 0.04 U PAD / g soy drink in a water bath at 45°C for 90 minutes (magnetic stirring). As a control, the soy drink was incubated without any additions. The samples were then stored at -20°C overnight. After this, the samples were freeze-dried.

[0084] Soy flour: A 10% suspension (w / w) of raw soy flour was prepared in a solution of 0.01 M NaOH. The pH was adjusted to pH 10. The suspension was then mixed (magnetic stirring) at ambient temperature for 3 hours. After this, the suspension was divided into two equal parts (200 g) and transferred to a 45°C water bath. After 15 minutes, 0.05 U PAD / g soy flour suspension was added to one part and incubated for 30 minutes. The sample was then stored overnight at -20°C. After this, the sample was freeze-dried.

[0085] The results of the TIA measurements are shown in Table 2 below.

[0086] [Table 2]

[0087] [Example 3] Improving rapeseed protein digestibility using an in vitro model. Processing of rapeseed protein samples with PAD: Rapeseed protein isolate (RPI) was prepared as described in patent WO 2018 / 007492. 43 U PAD was added to 500 ml of 10% RPI in water. The suspension was incubated at 40-45°C for 3 hours. The samples were then frozen and lyophilized.

[0088] In vitro digestibility was measured at the NIZO Food Research Center using the SYMPHYD platform as described in He, Tao & Giuseppin, Marco. (2013). Slow and fast dietary proteins differentially modulate postprandial metabolism. International journal of food sciences and nutrition. 65.10.3109 / 09637486.2013.866639.

[0089] The results of the in vitro model for proteolytic digestion of RPI and RPI treated with PAD are shown in FIG.

[0090] The increase in released ammonia (measured by the OPA method) indicates a higher digestibility of the protein sample. Compared to the untreated sample, an increase in digestibility of more than 20% is observed for the RPI treated with PAD at the end of digestion.

[0091] [Example 4] [Improvement of mouthfeel of soy drinks treated with PAD] [Incubation of soybean drinks with PAD] Soybean drink Provamel unsweetened: 1000ml (4 * Samples (250 ml) were incubated with 0.27 U / ml U PAD for 4 hours at 45°C in a water bath (shaking at 40 rpm). As a control, soy drink was incubated without any additions. The samples were then heated to 65°C in a microwave oven and held at that temperature for 5 minutes to inactivate the PAD enzyme. After this, the samples were cooled in ice water. A sensory panel evaluation was then performed on these samples. The samples were evaluated by a panel (n=12) for product-relevant attributes in the test using quantitative descriptive analysis (QDA) as described in Meilgaard M., Civil GV., Carr BT. 2007. Sensory Evaluation Techniques. 4th ed. Boca Raton, FL. CRC Press. During the test, samples were presented according to an optimized design to ensure unbiased results and scored in duplicate on an unstructured line scale from 0 to 100 using EyeQuestion. Data were analyzed by ANOVA to find significant differences between individual samples. Differences at p<0.05 were considered significant. The results of the sensory panel evaluation are shown in Figure 3. A significant decrease in mouthfeel was observed for the enzyme-treated drinks in terms of powdery, rich, and thick attributes in the mouth.

[0092] [Example 5] [Sensory evaluation of rapeseed protein treated with PAD enzyme] Per sample, 1000 ml of 2% protein flour suspension was made in tap water (plant protein flour was adjusted to the protein content) and the pH was adjusted to pH-6.5 with 4 M H2S04.

[0093] The suspensions were incubated at 45°C for 2 hours with and without various amounts of PAD enzyme. The enzyme was inactivated by heating the materials to 65°C, followed by a 5-minute hold time. Samples were assessed by a sensory panel (n = 13) using descriptive analysis (QDA) for relevant product attributes. During testing, samples were presented according to an optimized design to ensure unbiased results and scored in duplicate on an unstructured line scale from 0 to 100 using the EyeQuestion. Data were analyzed by ANOVA to identify significant differences between individual samples. Differences at p < 0.05 were considered significant (Figure 4). Treatment of RPI with PAD enzyme reduced all sensory attributes tested, including sweetness, licorice, astringency, bitterness, and aftertaste duration (as described in the figure legends), with increasing amounts of added PAD.

[0094] [Example 6] [Reduction of IEF in pea and soy protein drinks treated with PAD] Samples were diluted 4-fold with H2O. Subsequently, samples were diluted 1:1 with IEF sample buffer. 10 μl of sample was added to a Novex™ pH 3-7 IEF Protein Gel. Total run time was 2.5 hours (per Invitrogen protocol). The decrease in pl for soy and pea drinks after PAD treatment is shown in Figure 5. The decrease in pl for these proteins is consistent with PAD enzyme activity on these proteins, reducing the number of positive charges on the proteins. A similar decrease in the isoelectric point of almond proteins was measured in almond flour treated with PAD (data not shown). The shift in pl for these proteins to more acidic values ​​allows for better solubility of these proteins in neutral beverage formulations.

[0095] [Example 7] [Stability of PAD enzyme at acidic pH] The stability of the PAD enzyme was determined after 40 minutes of incubation at two temperatures (on ice at 4°C and in a water bath at 37°C) over a pH range of 3 to 7. For pHs 3 to 5, PAD samples were diluted 6-fold in 100 mM citrate buffer, and the pH was increased by adding 1N sodium hydroxide. For pHs above 5, the enzyme was diluted in 50 mM potassium phosphate buffer. Enzyme activity was measured as described in WO 2017 / 009100 A1. Figure 6 shows the results of this experiment. The PAD enzyme had maximal activity at pH 7 in this experiment, but at pH 4, approximately 50% of the activity remained after 40 minutes of enzyme incubation at 37°C, indicating that the enzyme can be active in conditions similar to those found in the mammalian stomach.

Claims

1. A process for modifying the sweetness, licorice taste, powdery / cheekiness, richness, and / or thickness of a protein-containing food, the process comprising incubating a protein solution with peptidylarginine deiminase (PAD) and optionally processing the PAD-treated protein solution into a protein-containing food.

2. The process of claim 1 , wherein the protein solution is a food ingredient.

3. 3. The process according to any one of claims 1 to 2, wherein the protein is a plant protein selected from the group consisting of soy protein, rapeseed protein, wheat protein, buckwheat protein, corn protein, barley protein, potato protein, rice protein, oat protein, pea protein, peanut protein, tree nut protein, lupin protein, and almond protein, or the protein is egg protein, milk protein, gelatin protein, or microbial protein.

4. 4. The process of any one of claims 1 to 3, further comprising dissolving the flour to obtain a protein solution.

5. The process according to any one of claims 1 to 4, wherein the PAD is a microbial PAD.

6. 6. The process of any one of claims 1 to 5, wherein the peptidylarginine deiminase (PAD) has at least 90% identity to SEQ ID NO: 1 or has at least 90% identity to the mature amino acid sequence of SEQ ID NO: 1, and wherein the mature amino acid sequence of SEQ ID NO: 1 is obtained by at least one post-translational modification of the amino acid sequence of SEQ ID NO: 1 selected from the group consisting of N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of a leader sequence.

7. The process according to any one of claims 1 to 6, wherein the protein-containing food product is a protein-containing drink.

8. 8. The process of claim 7, wherein the protein-containing drink is a plant protein milk or a fermented plant protein product.

9. 9. The process of claim 7 or 8, wherein the protein-containing drink is a soybean drink, a pea drink, a peanut drink, a barley drink, a rice drink, an oat drink, a quinoa drink, an almond drink, a cashew drink, a coconut drink, a hazelnut drink, a hemp drink, a sesame seed drink, a wheat drink, a potato drink, or a sunflower seed drink.

10. 10. The process of claim 9, wherein the drink is a baby, follow-on, or toddler drink and the protein is soy protein.

11. The process according to any one of claims 7 to 10, wherein the protein-containing drink is a protein-fortified protein drink.

12. The process according to any one of claims 7 to 11, wherein the protein-containing drink is a drink with a neutral or acidic pH.

13. The process according to any one of claims 7 to 12, wherein the protein-containing drink is a medicated drink or a sports drink.

14. Use of peptidylarginine deiminase (PAD) to modify the licorice taste, powdery / mealiness, body, and / or thickness of protein-containing foods.

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