Acid-stable soy protein or almond protein

Abstract

The present invention relates to the field of food. Disclosed is a method for preparing soy protein or almond protein containing coffee or tea, comprising adding soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

Description

Detailed Description of the Invention

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

[0002] [Background of the Invention] Recent trends among consumers are leaning toward healthy / vegetarian / vegan lifestyles, leading to growing demand for plant-based milk alternatives. The main plant sources for milk alternatives on the market are oat, soybean and almond. These ingredients are compositionally diverse in terms of nutritional properties and techno-functional properties, so they can function in many applications. In particular, barista products are manufactured to provide alternatives to dairy milk for coffee applications. One of the main drawbacks in the application of these products is the lack of acid stability in solutions containing coffee. The exact mechanism explaining the coagulation of these products remains unelucidated. To solve this problem, manufacturers rely on adding 0.5% (w / w) dipotassium phosphate (K2HPO4) to buffer the acidity in coffee. The addition of dipotassium phosphate is not desirable as it is an additive with an E number. Accordingly, it is desirable to provide a solution for achieving acceptable acid stability of plant-based dairy alternatives in coffee or tea while reducing or avoiding the use of K2HPO4.

[0003] The present invention provides such a solution.

[0004] WO 2017009100 pamphlet (DSM) describes a process for improving the physical properties of plant proteins, which comprises incubating a plant protein that is pea, soybean, rice or cereal protein with peptidylarginine deiminase.

[0005] International Publication No. 2019233920 (DSM) describes a process for modifying the sweetness, licorice flavor, astringency, powderiness / powdery, richness, viscosity and / or digestibility and / or protease inhibitory activity of protein-containing foods, the process comprising incubating a protein solution with peptidylarginine deiminase (PAD). An example in this patent application describes an improvement in the mouthfeel of a soy beverage as a result of treating soy protein with PAD.

[0006] [Overview of the prefecture] Surprisingly, the inventors have found that using enzymes such as protein arginine deiminase or proline-specific endoprotease on plant-based protein sources such as soybeans or almonds yields an enzyme-treated plant-based milk substitute that has acceptable stability in acidic beverages such as coffee or tea.

[0007] The following is disclosed herein: - A method for preparing soy protein or almond protein containing coffee or tea, comprising adding soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), - A method for improving the stability of soy protein or almond protein when used in coffee or tea, comprising incubating soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain treated soy protein or treated almond protein. - Processed soy protein or processed almond protein, which can be obtained by the methods described above. - A method for preparing soy protein or almond protein containing coffee or tea, comprising adding the processed soy protein or processed almond protein described above to coffee or tea. - Coffee or tea containing soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), and / or - Use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to improve the stability of soy protein or almond protein in coffee or tea.

[0008] [Sequence List] PAD protein sequence from Trichoderma harzianum containing Sequence ID No. 1 pro sequence

[0009] PAD protein sequence from Verticillium longisporum containing Sequence ID No. 2 pro sequence

[0010] PAD protein sequence from Bionectra ochroleuca containing sequence number 3 pro sequence

[0011] PAD protein sequence from Fusarium longipes containing sequence number 4 pro sequence

[0012] PAD protein sequence derived from Fusarium solani species complex containing sequence number 5 pro sequence

[0013] PAD protein sequence from Akanthomyces lecanii RCEF 1005 containing sequence number 6 pro sequence

[0014] PAD protein sequence derived from the Fusarium incarnatum-equiseti species complex containing sequence number 7 pro sequence

[0015] PAD protein sequence from Ophiocordyceps australis containing sequence number 8 pro sequence

[0016] PAD protein sequence from Verticillium longisporum containing sequence number 9 pro sequence

[0017] PAD protein sequence from wild-type Fusarium graminearum containing sequence number 10 pro sequence

[0018] Protein sequences derived from Fusarium graminearum mutants FgraPADmut_01, FgraPADmut_02, and FgraPADmut_03, which contain the sequence number 11 pro sequence.

[0019] Sequence numbers 1-11 do not contain signal sequences.

[0020] Sequence ID 12: Polynucleotide sequence encoding the protein of Sequence ID 1.

[0021] Sequence ID 13: Polynucleotide sequence encoding the protein of Sequence ID 2.

[0022] Sequence ID 14: Polynucleotide sequence encoding the protein of Sequence ID 3.

[0023] Sequence ID 15: Polynucleotide sequence encoding the protein of Sequence ID 4.

[0024] Sequence ID 16: Polynucleotide sequence encoding the protein of Sequence ID 5.

[0025] SEQ ID NO: 17 Polynucleotide sequence encoding the protein of SEQ ID NO: 6

[0026] SEQ ID NO: 18 Polynucleotide sequence encoding the protein of SEQ ID NO: 7

[0027] SEQ ID NO: 19 Polynucleotide sequence encoding the protein of SEQ ID NO: 8

[0028] SEQ ID NO: 20 Polynucleotide sequence encoding the protein of SEQ ID NO: 9

[0029] SEQ ID NO: 21 Polynucleotide sequence encoding the protein of SEQ ID NO: 10

[0030] SEQ ID NO: 22 Polynucleotide sequence encoding the protein of SEQ ID NO: 11

[0031] [Detailed Description of the Invention] In a first aspect, the present disclosure provides a method of preparing a low-pH beverage comprising soy protein or almond protein, the method comprising adding the soy protein or almond protein to the low-pH beverage, wherein the soy protein or almond protein is soy protein or almond protein that has been treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

[0032] "Low pH beverage containing soy protein or almond protein" is defined herein as a beverage containing soy protein or almond protein, typically coffee or tea. Soy protein or almond protein is typically part of a soy or almond beverage (also referred herein as a soy milk or almond milk substitute). Soy or almond-based beverages can be used, for example, as a dairy milk substitute to whiten coffee or tea. As used herein, the term low pH beverage refers to a beverage having a pH of less than 7, preferably 6.5 or less than 6. Preferably, low pH beverages have a pH in the range of 4 to 6.5. For example, espresso and filter coffee typically have a pH of about 5. Cold brew typically has a slightly higher pH of about 6.

[0033] The phrase "adding soy protein or almond protein to a low-pH beverage" typically includes adding a beverage containing soy protein or almond protein to a low-pH beverage.

[0034] Preferably, the present disclosure provides a method for preparing soy protein or almond protein containing coffee or tea, comprising adding soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

[0035] Suitable examples of coffee include espresso, filter coffee, or cold brew.

[0036] Suitable examples of tea include black tea, green tea, or white tea.

[0037] The phrase "add soy protein or almond protein to coffee or tea" typically includes adding a beverage containing soy protein or almond protein to coffee or tea.

[0038] The terms "protein arginine deiminase" and "peptidylarginine deiminase" (PAD) are used interchangeably herein. Protein arginine deiminase or peptidylarginine deiminase belongs to the enzyme family (EC 3.5.3.15) and converts peptides or protein-bound arginine to peptides or protein-bound citrulline. This process is called deamination or citrullination. In the reaction from arginine to citrulline, one of the nitrogen atoms at the end of the arginine side chain is replaced by oxygen. One water molecule is used in this reaction, and ammonia is obtained as a byproduct (http: / / en.wikipedia.org / wiki / Citrullination). Arginine is positively charged at neutral pH, while citrulline is uncharged.

[0039] Peptidylarginine deiminase (PAD) may be derived from any suitable source, e.g., mammalian or microbial origin. The PAD used in the present invention is advantageously derived from a microbial source; that is, the PAD used in the process of the present invention is a microbial PAD. For example, the PAD may be derived from fungal origins such as Fusarium sp., such as Fusarium graminearum, Chaetomium globosum, and Phaesphaeria nodorum, or from bacterial origins such as Streptomyces, e.g., Streptomyces scabies and Streptomyces clavuligeres. The terms "derived from" or "originating from" with respect to the origin of polypeptides such as those disclosed herein mean that when a BLAST search is performed on polypeptides such as those disclosed herein, the polypeptide may originate from a natural source such as microbial cells, but its endogenous polypeptide shows the highest percentage of homology or identity with the polypeptides disclosed herein.

[0040] Peptidylarginine deiminase is known, for example, from International Publication No. 2008 / 000714, which discloses a process of enzymatic treatment of a protein with protein arginine deiminase, in which at least 30% of the arginine is converted to citrulline.

[0041] Peptidylarginine deiminase can be pure or purified peptidylarginine deiminase. Pure or purified peptidylarginine deiminase is an enzyme that, when determined by, for example, SDS-PAGE or any other analytical method suitable for this purpose and known to those skilled in the art, may have a purity of at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 80%, at least 90%, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0042] Advantageously, the peptidylarginine deiminase is a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10 or the mature amino acid sequence of SEQ ID NO: 10 when used in a process as described herein, and the polypeptide has peptidylarginine deiminase activity.

[0043] For the purposes of this disclosure, aligning two amino acid sequences for the purpose of optimal comparison is defined herein to determine the percentage of sequence identity between the two sequences. To optimize the alignment between the two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment may be performed over the entire length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, e.g., about 20, about 50, about 100, or more amino acids. Sequence identity is the percentage of perfect match between the two sequences over the reported alignment region. The percentage of sequence identity between two amino acid sequences may be determined using the Needleman-Wunsch algorithm to align the two sequences. (Needleman, S.B. and Wunsch, CD (1970) J.Mol. Biol. 48, 443-453). This algorithm can align both amino acid sequences and nucleotide sequences. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of this invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or later, 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 was used as the substitution matrix. The optional parameters used were a gap start penalty of 10 and a gap extension penalty of 0.5. While all of these different parameters produce slightly different results, those skilled in the art will understand that the overall percentage of identity between the two sequences does not change significantly even when using different algorithms.

[0044] "Mature polypeptide" is defined herein as the polypeptide in its final form, obtained after translating mRNA into a polypeptide and post-translational modification of the polypeptide. Post-translational modification includes N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of leader sequences such as signal peptides, propeptides, and / or prepropeptides by cleavage.

[0045] The mature polypeptide sequence of SEQ ID NO: 10 may contain or include amino acids 19, 20, 21, 22, 23, 24-640 of the amino acid sequence of SEQ ID NO: 10, and more favorably, the mature polypeptide sequence of SEQ ID NO: 10 may contain or include amino acids 22-640 of SEQ ID NO: 26, and the methionine at position 1 of SEQ ID NO: 10 is counted as the number 1.

[0046] The term "polypeptide" refers to a molecule containing 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 also refer to two or more polypeptides. Thus, the terms "protein" and "polypeptide" may be used interchangeably. Polypeptides can be optionally modified to confer functional groups (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.). Polypeptides that exhibit activity under specific conditions and in the presence of a specific substrate may be called enzymes.

[0047] Peptidylarginine deiminase or polypeptides having peptidylarginine deiminase activity can be produced by methods known in the art in any suitable host organism, such as the fungi Aspergillus, e.g., Aspergillus niger or Aspergillus oryzae, Trichoderma, or the yeasts Saccharomyces and Kluyveromyces, or the bacteria Streptomyces or Bacilli. A suitable method for expressing polypeptides having peptidylarginine deiminase activity in Aspergillus niger is disclosed, for example, in Examples 3 and 4 of International Publication No. 2008 / 000714, which is included herein by reference.

[0048] Instead, use one of the following PADs, for example: (a) A first domain having at least 80% sequence identity with amino acids 1-477 of SEQ ID NO: 1, and optionally containing 1-4 amino acids from amino acids 478-481 of SEQ ID NO: 1, A second domain containing at least 80% sequence identity with amino acids 512-600 of SEQ ID NO: 1, and optionally containing 1-2 amino acids from amino acids 510-511 of SEQ ID NO: 1. Isolated and / or recombinant PAD polypeptides containing, but not containing amino acids 482-509 of SEQ ID NO: 1 (also referred to as TharPAD_01, TharPAD_03, and TharPAD_02), (b) A first domain having at least 80% sequence identity with amino acids 1-478 of SEQ ID NO: 2, and optionally containing 1-4 amino acids from amino acids 488-491 of SEQ ID NO: 2, The second domain contains at least 80% sequence identity with amino acids 524-612 of SEQ ID NO: 2. Isolated and / or recombinant PAD polypeptides containing, but not containing amino acids 492-523 of SEQ ID NO: 2 (also referred to as VlonPAD_01 and VlonPAD_02), (c) A first domain having at least 80% sequence identity with amino acids 1-467 of SEQ ID NO: 3, and optionally containing 1-4 amino acids from amino acids 468-471 of SEQ ID NO: 3, The second domain contains at least 80% sequence identity with amino acids 498-587 of SEQ ID NO: 3. Isolated and / or recombinant PAD polypeptides containing the above, which do not contain amino acids 472-497 of SEQ ID NO: 3 (also referred to as BochPAD_01, BochPAD_02, and BochPAD_03), (d) A first domain having at least 80% sequence identity with amino acids 1-483 of SEQ ID NO: 4, and optionally containing 1-4 amino acids from amino acids 484-487 of SEQ ID NO: 4, The second domain contains at least 80% sequence identity with amino acids 528-615 of SEQ ID NO: 4. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 488-527 of SEQ ID NO: 4 (also referred to as FlonPAD_01), (e) A first domain having at least 80% sequence identity with amino acids 1-489 of SEQ ID NO: 5, and optionally containing 1-4 amino acids from amino acids 490-493 of SEQ ID NO: 5, The second domain contains at least 80% sequence identity with amino acids 543-630 of SEQ ID NO: 5. Isolated and / or recombinant PAD polypeptides containing, but not containing amino acids 494-542 of SEQ ID NO: 5 (also referred to as FsolPAD_01 and FsolPAD_02), (f) A first domain having at least 80% sequence identity with amino acids 1-489 of SEQ ID NO: 6, and optionally containing 1-4 amino acids from amino acids 490-493 of SEQ ID NO: 6, The second domain contains at least 80% sequence identity with amino acids 531-618 of SEQ ID NO: 6. Isolated and / or recombinant PAD polypeptides containing, but not containing amino acids 494-530 of SEQ ID NO: 6 (also referred to as AlecPAD_01 and AlecPAD_02), (g) A first domain having at least 80% sequence identity with amino acids 1-481 of SEQ ID NO: 7, and optionally containing 1-4 amino acids from amino acids 482-485 of SEQ ID NO: 7, The second domain contains at least 80% sequence identity with amino acids 517-604 of SEQ ID NO: 7. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 486-516 of SEQ ID NO: 7 (also referred to as FincPAD_01), (h) A first domain containing at least 80% sequence identity with amino acids 1-504 of SEQ ID NO: 8, and optionally containing 1-4 amino acids from amino acids 505-508 of SEQ ID NO: 8, The second domain contains at least 80% sequence identity with amino acids 535-626 of SEQ ID NO: 8. Isolated and / or recombinant PAD polypeptides containing, but not containing amino acids 509-534 of SEQ ID NO: 8 (also referred to as OausPAD_01 and OausPAD_02), or (i) A first domain having at least 80% sequence identity with amino acids 1-489 of SEQ ID NO: 9, and optionally containing 1-4 amino acids from amino acids 490-493 of SEQ ID NO: 9, The second domain contains at least 80% sequence identity with amino acids 525-613 of SEQ ID NO: 9. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 494-524 of SEQ ID NO: 9 (also referred to as VertPAD_01) This is used.

[0049] more, The first domain contains at least 80% sequence identity with amino acids 1-477 of SEQ ID NO: 1, and optionally contains 1-4 amino acids from amino acids 478-481 of SEQ ID NO: 1. A second domain containing at least 80% sequence identity with amino acids 512-600 of SEQ ID NO: 1, and optionally containing 1-2 amino acids from amino acids 510-511 of SEQ ID NO: 1. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 482-509 of SEQ ID NO: 1 (also known as TharPAD_03), or The first domain contains at least 80% sequence identity with amino acids 1-467 of SEQ ID NO: 3, and optionally contains 1-4 amino acids from amino acids 468-471 of SEQ ID NO: 3. The second domain contains at least 80% sequence identity with amino acids 498-587 of SEQ ID NO: 3. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 472-497 of SEQ ID NO: 3 (also referred to as BochPAD_02) This is used.

[0050] Most preferably, The first domain contains at least 80% sequence identity with amino acids 1-467 of SEQ ID NO: 3, and optionally contains 1-4 amino acids from amino acids 468-471 of SEQ ID NO: 3. The second domain contains at least 80% sequence identity with amino acids 498-587 of SEQ ID NO: 3. Isolated and / or recombinant PAD polypeptide containing, but not containing amino acids 472-497 of SEQ ID NO: 3 (also referred to as BochPAD_02) This is used.

[0051] Instead, a mutant PAD polypeptide, the mutant Fusarium graminearum PAD polypeptide, is used, which, when aligned with the PAD of SEQ ID NO: 10, includes at least substitutions of amino acid residues corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251, 365, 439, 467 and 560, the positions defined with reference to SEQ ID NO: 10, the mutant has a pI of less than 9, and the mutant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NO: 10. Preferably, the mutant Fusarium graminearum PAD polypeptide includes substitutions N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251D, N365D, Q439E, Q467E, and N560D, the substitutions being defined with reference to Sequence ID No. 10.

[0052] A "proline-specific endoprotease (PSP)" is a protease that hydrolyzes a protein or peptide at a position containing a proline residue. Proline-specific endoproteases may have proline-specific endoprotease activity and / or proline-specific oligopeptidase activity (EC3.4.21.26). Preferably, a proline-specific endoprotease is an enzyme that hydrolyzes the peptide bond at the carboxyl terminus of a proline residue, producing a peptide and / or polypeptide fragment having proline at the C terminus.

[0053] Proline-specific endoproteases may be derived from Aspergillus niger or Penicillium chrysogenum, for example, as disclosed in International Publication No. 2002 / 046381 and International Publication No. 2009 / 144269, respectively.

[0054] Other proline-specific endoproteases are publicly known from International Publication No. 2012 / 174127, which discloses proline-specific proteases derived from Botryotinia fuckeliana, Aspergillus clavatus, Sclerotinia sclerotiotum, Mycosphaerelly graminicola, Neuropspora crasse, Talaromyces stipitatus, and Gibberella zeae.

[0055] Suitable proline-specific endoproteases can be obtained from dsm-firmenich under the names DelvoPlant PSP or DelvoPlant® PSP.

[0056] "Soy protein or almond protein treated with PAD or PSP" is typically obtained by incubating a solution containing soy protein or almond protein (also referred to as soy protein or almond protein in solution) with PAD or PSP, during which incubation is carried out so that PAD or PSP can exert its enzymatic activity. Enzyme incubation can be carried out at any suitable pH, for any suitable time, and at any suitable enzyme concentration. Those skilled in the art can very well establish suitable enzyme amounts or suitable incubation conditions, such as a suitable incubation temperature, a suitable incubation pH, or a suitable incubation time. In the case of PSP, a suitable incubation temperature is 30 to 60 degrees Celsius, a suitable pH is pH 2 to pH 6, and a suitable incubation time is 30 minutes to 4 hours. For PAD, preferred conditions are a pH of 4–9, for example, 5–8.5, for example, 5.5–8, for example, 6–7, or 6.2–6.8, for example, about 6.5, and a preferred temperature for PAD may be 20–60 degrees Celsius, for example, 30–50 degrees Celsius, or 35–45 degrees Celsius. Those skilled in the art will find it quite possible to adjust the amount of enzyme in relation to the amount of substrate, and / or adjust the incubation time and / or temperature.

[0057] In an alternative embodiment, the Disclosure provides a method for improving the stability of soy protein or almond protein when used in low-pH beverages, comprising incubating the soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain treated soy protein or treated almond protein.

[0058] More preferably, the present disclosure provides a method for improving the stability of soy protein or almond protein when used in coffee or tea, comprising incubating soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain treated soy protein or treated almond protein. This may also be described as a method for improving the stability of soy protein or almond protein, comprising incubating soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain treated soy protein or treated almond protein.

[0059] Whether the stability of soy protein or almond protein in coffee or tea is improved can be determined, for example, by comparing soy protein or almond protein treated with PAD or PSP with soy protein or almond protein incubated without either enzyme, and determining visual parameters such as separation, precipitation, or coagulation. The experimental section provides examples of methods for measuring the stability of soy protein or almond protein treated with PAD or PSP in low pH beverages such as coffee or tea.

[0060] The disclosure also provides a method for improving the stability of soy protein or almond protein when used in coffee or tea, and includes a method for obtaining a treated soy protein or treated almond protein by incubating the soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

[0061] The Disclosure further provides a method for preparing soy protein or almond protein containing coffee or tea, comprising adding the processed soy protein or processed almond protein to coffee or tea, wherein the processed soy protein or processed almond protein is obtained by a method for improving the stability of the soy protein or almond protein when used in coffee or tea, comprising incubating the soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain the processed soy protein or processed almond protein.

[0062] Preferably, the coffee or tea described in any of the above methods has a pH in the range of 4.8 to 6.0, more preferably 5.0 to 6.0, and most preferably 5.0 to 5.2. Preferably, the pH of the (liquid, i.e., prepared) coffee or tea is the pH of the coffee or tea itself, i.e., the pH before soy protein or almond protein is added to the coffee or tea, i.e., the pH before soy milk or almond milk substitute (containing the soy protein or almond protein) is added to the coffee or tea. Most preferably, the coffee or tea described in any of the above methods has a pH in the range of 5.0 to 5.2. The upper limit of any of the above-described pH ranges is included in this range; that is, the ranges mentioned include the upper limit.

[0063] Preferably, soy protein or almond protein is part of the soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v), thereby obtaining 1 volume of milk for 1 to 3 volumes of coffee or tea. The 1:1 (v / v) to 1:3 (v / v) milk portion refers to the milk mixed with coffee and therefore does not include any foam layer that may form. The foam layer is irrelevant to the ratio mentioned because it is not in complete contact with the coffee or tea.

[0064] The temperature of soy milk or almond milk (containing protein processed with PAD or PSP) should preferably be room temperature before adding it to coffee or tea.

[0065] Soy milk or almond milk (containing proteins treated with PAD or PSP) may be frothed or unfrothed. The experimental section of this specification describes examples where the milk was not frothed. However, examples where the soy milk or almond milk was frothed (not further described herein) showed similar results to the unfrothed examples.

[0066] In a preferred embodiment, the present invention provides a method for preparing soy protein or almond protein containing coffee or tea, comprising adding the soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), the coffee or tea has a pH in the range of 4.8 to 6.0, more preferably in the range of 5.0 to 6.0, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v). Most preferably, the present invention provides a method for preparing soy protein or almond protein containing coffee or tea, comprising adding soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), the coffee or tea has a pH in the range of 5.0 to 5.2, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v).

[0067] Coffee or tea produced by the methods described herein is unfermented coffee or tea.

[0068] Any of the methods described does not involve the addition of buffers. Buffers are typically added to plant-based dairy milk substitutes (especially plant-based milk used in coffee) to improve protein stability. Such buffers prevent a rapid change in pH when the plant-based beverage comes into contact with acidic coffee.

[0069] A preferred embodiment is, - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), (i.e., in this specification, the method of preparing coffee is most preferred), - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), and the coffee has a pH in the range of 4.8 to 6.0, - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), and the coffee has a pH in the range of 5.0 to 6.0, - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), and the coffee has a pH in the range of 5.0 to 5.2, and as described above, in this specification, a pH range of 5.0 to 5.2 is most preferred, - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), the coffee has a pH in the range of 4.8 to 6.0, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), the coffee has a pH in the range of 5.0 to 6.0, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP), the coffee has a pH in the range of 5.0 to 5.2, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD), the coffee has a pH in the range of 4.8 to 6.0, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD), the coffee has a pH in the range of 5.0 to 6.0, the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein or almond protein containing coffee, comprising adding soy protein or almond protein to coffee, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD), the coffee having a pH in the range of 5.0 to 5.2, the soy protein or almond protein being part of a soy milk or almond milk substitute, and the method further comprising adding the soy milk or almond milk substitute to coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v), (wherein herein, soy protein or almond protein treated with PAD combined with coffee having a pH in the range of 5.0 to 5.2) - A method for preparing soy protein containing coffee, comprising adding soy protein to coffee, wherein the soy is soy protein treated with proline-specific endoprotease (PSP), the coffee has a pH in the range of 4.8 to 6.0, the soy protein is part of a soy milk substitute, and the method further comprises adding the soy milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein containing coffee, comprising adding soy protein to coffee, wherein the soy protein is soy protein treated with proline-specific endoprotease (PSP), the coffee has a pH in the range of 5.0 to 6.0, the soy protein is part of a soy milk substitute, and the method further comprises adding the soy milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). - A method for preparing soy protein containing coffee, comprising adding soy protein to coffee, wherein the soy protein is soy protein treated with proline-specific endoprotease (PSP), the coffee has a pH in the range of 5.0 to 5.2, the soy protein is part of a soy milk substitute, and the method further comprises adding the soy milk substitute to the coffee in a ratio of 1:1 (v / v) to 1:3 (v / v). That is the case.

[0070] The coffee described in any of the above method claims is preferably espresso, filter coffee, or cold brew.

[0071] The tea described in any of the above method claims is preferably black tea, green tea, or white tea.

[0072] The disclosure also provides coffee or tea containing soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP). The coffee is preferably espresso, filter coffee, or cold brew. The tea is preferably black tea, green tea, or white tea.

[0073] Preferably, the coffee or tea does not contain a buffering agent, such as potassium phosphate, potassium citrate, sodium citrate, calcium phosphate, calcium carbonate, or potassium carbonate.

[0074] This disclosure also provides the use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to improve the stability of soy protein or almond protein in low pH beverages, preferably coffee or tea. Preferably, the use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to improve the stability of soy protein or almond protein in coffee is also provided. Preferred embodiments, such as those described in the method claims, include, but are not limited to, the pH range of the coffee and the ratio of soy milk or almond milk to coffee, and are equally applicable to the uses described herein.

[0075] The term "stability" refers to the stability of soy protein or almond protein in the absence of typical stabilizers, i.e., stability in the absence of buffers, such as coffee, which does not contain potassium phosphate, potassium citrate, sodium citrate, calcium phosphate, calcium carbonate, or potassium carbonate. Accordingly, this disclosure provides the use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to improve the stability of soy protein or almond protein in coffee in the absence of buffers.

[0076] The present invention will be further illustrated in the following embodiments.

[0077] [Examples] [Materials and Methods] [Soybean material] The soy-based materials were prepared as follows: a) Prepare a 12% w / w hulled soybean solution in SPA* water (approximately 5 mg / L). SPA* is a trademark of light mineral Belgian mineral water produced by Spa Monopole. b) Wet grinding at 85°C for 10 minutes. A Thermomix was used, set to 85°C, 10 minutes, and speed 8. The smaller the particles, the more efficient the enzymatic treatment. c) Cool, then centrifuge at 1000G for 10 minutes. d) The recovered supernatant is separated from the pellet and heat-treated at 95°C for 15 minutes to inactivate the endogenous enzymes. e) Cool the substrate, measure the pH, and store it at 4°C.

[0078] [Almond ingredients] A commercially available almond beverage (Zonnatura) containing only water and 5% w / w almonds was used.

[0079] Alternatively, the almond solution can be prepared by the following: a) Measure out 5% almond paste and mix it with water at 45°C. b) Stir until completely dissolved.

[0080] [PAD] The experimental section of this specification used two different pads.

[0081] The first is PAD, as described in International Publication No. 2008 / 000714. The sample used had an activity of 56.6 PAD U / g.

[0082] The second PAD used, described above as BochPAD_02, had an activity of 313 PAD U / g and 19.2 mg protein / mL.

[0083] [PSP] Characteristics of Delvo® Plant PSP: Product yields 7.5 PSP U / g. Dosage used: 1200 ppm.

[0084] [Experimental setup] Table 1 shows an overview of the protein content of the materials used and the PAD dosage used.

[0085] [Table 1]

[0086] The 0.3% w / w PAD dosage mentioned means that 0.3 grams of formulated PAD product are administered per 100g of raw material protein.

[0087] Previous experimental studies determined the protein content of the soybean base material. Nitrogen content was quantified using the Dumas method. The conversion factor used to quantify protein content was 5.7, which was the value specified for soybeans. The protein content of the almond material was based on the label information of the commercially available material used.

[0088] Table 2 shows an overview of the negative and positive controls.

[0089] [Table 2]

[0090] The negative control is a soy-based material that does not contain K2HPO4 or PAD. This sample is used to demonstrate the instability of plant protein solutions in coffee.

[0091] The positive control is a solution containing 0.5% w / w K2HPO4, which is used to provide a stable coffee solution that does not coagulate.

[0092] The PAD treatment involves adding a solution containing 0.3% w / w formulated PAD enzyme based on the protein content of the raw material (for example, 0.3 g of formulated PAD protein was added to 100 g of soy-based protein). The PAD treatment included the following steps: a) Treat each solution in a thermomixer at 55°C for 2 hours. b) Inactivate the PAD by treating it at 95°C for 15 minutes. c) In the case of soybeans, PAD treatment was performed after the grinding process. d) In the case of almonds, commercially available beverages were processed based on the protein content mentioned on the packaging.

[0093] [Stability of plant protein solutions in coffee] To measure the acid stability of the plant protein solution, a concentrated coffee solution was prepared by placing 50g of ground coffee (supermarket brand: Perla, flavor intensity number 6, Arabica and Robusta beans, medium roast) into a French press, adding 600mL of boiling water, stirring, and waiting for 5 minutes.

[0094] For analysis, a) A solution was used in which plant protein solution and coffee were mixed in a 1:3 ratio. b) First, hot coffee was added to a clear container, followed by the solution to be tested. c) After 4 minutes, the resulting mixture was visually evaluated for separation, precipitation, or coagulation. If one of the solutions exhibited any of the aforementioned behaviors, the sample was characterized as poor. A solution was classified as good if it had the appearance of clear coffee with milk.

[0095] [Tests regarding stability (or lack thereof) at various pH levels] a) Different acid solutions were prepared and their reactions to different plant protein solutions treated with PAD were analyzed. b) The pH of the coffee (as described above) was 5.2. To test the acid stability of the plant protein solution treated with PAD, the pH was adjusted to five different pH points. The five pH points were 4.5, 5.0, 5.2, 5.4, and 6.0. c) Coffee was prepared as described above, and the pH was adjusted with 0.5 M citric acid (to lower the pH) or 0.5 M dipotassium phosphate (to raise the pH). d) To check the reaction, a solution of plant protein solution and coffee in a 1:3 ratio was used in a small tube. The water bath was set to 55°C to keep the coffee warm, and the tube containing the coffee was held so that it was exposed before the enzyme-treated plant protein solution was added. e) The pH was measured and a photograph of the result (not shown in this specification) was taken.

[0096] [Example 1] [Acid stability of soy-based materials in coffee-pads]

[0097] [Table 3]

[0098] It can be concluded that adding 0.3% PAD to a soy protein base material has a positive effect on the acid stability of the soy base material in coffee.

[0099] PAD can be used as a substitute for buffer solutions such as K2HPO4.

[0100] [Example 2] [Coffee-PAD-Acid Stability of Soy-Based Substrates at Different pH Levels]

[0101] [Table 4]

[0102] The following conclusions can be drawn: - Without the addition of K2HPO4 or PAD (i.e., negative control), the soybean base is stable in coffee with pH ≥ 5.4. - In both the positive control K2HPO4 and PAD treatments, stable coffee was obtained at pH ≥ 5.0. - PAD can be used as a substitute for buffer solutions such as K2HPO4.

[0103] [Example 3] [Acid stability of almond-based material in coffee-pads] In Example 3, the almond protein substrate was processed in the same manner as described in Examples 1 and 2.

[0104] The following conclusions can be drawn: - Untreated (-control) almonds were stable at pH 6.0. - In PAD, the stability of the almond solution at pH ≥ 5.4 was improved. This indicates that the enzyme slightly improved the buffering capacity of the almond solution.

[0105] Further experiments showed that increasing the PAD dosage (from 0.3% to 0.6%) in the same coffee (50g / 600ml water) significantly improved the stability of almond milk in the coffee.

[0106] Furthermore, another experiment showed that when a 0.3% PAD dose was tested in coffee prepared using 33 grams of ground coffee and 600 ml of water, the almond beverage in the coffee solution remained stable.

[0107] [Example 4] [Acid stability of soy-based substrates in coffee-PSP]

[0108] [Table 5]

[0109] The following conclusions can be drawn: - Soybean substrate treated with 1200 ppm PSP was stable under strong coffee conditions (50g / 600mL). - 1200 ppm PSP has buffering properties for coffee applications, making it a potential alternative to adding K2HPO4 to soy beverages.

Claims

1. A method for preparing soy protein or almond protein containing coffee or tea, comprising adding soy protein or almond protein to coffee or tea, wherein the soy protein or almond protein is soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

2. A method for improving the stability of soy protein or almond protein when used in coffee or tea, comprising incubating soy protein or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain treated soy protein or treated almond protein.

3. Processed soy protein or processed almond protein that can be obtained by the method of claim 2.

4. A method for preparing soy protein or almond protein containing coffee or tea, comprising adding the processed soy protein or processed almond protein according to claim 3 to coffee or tea.

5. The method according to claim 1 or 4, wherein the coffee or tea has a pH in the range of 4.8 to 6.0, preferably in the range of 5.0 to 6.0, and most preferably in the range of 5.0 to 5.

2.

6. The method according to any one of the preceding claims, wherein the soy protein or almond protein is part of a soy milk or almond milk substitute, and the method further comprises adding the soy milk or almond milk substitute to coffee or tea in a ratio of 1:1 (v / v) to 1:3 (v / v).

7. The method according to any one of the preceding claims, wherein no buffering agent is added.

8. The method according to any one of claims 1, 2, 4, 5, 6, or 7, wherein the coffee is espresso, filter coffee, or cold brew.

9. The method according to any one of claims 1, 2, 4, 5, 6, or 7, wherein the tea is black tea, white tea, or green tea.

10. Coffee or tea containing soy protein or almond protein treated with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP).

11. The coffee or tea according to claim 11, which does not contain a buffering agent, for example, potassium phosphate, potassium citrate, sodium citrate, calcium phosphate, calcium carbonate, or potassium carbonate.

12. Use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to improve the stability of soy protein or almond protein in coffee or tea.

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