Acid stable SOY or almond protein

Abstract

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

Description

[0001] ACID STABLE SOY OR ALMOND PROTEIN

[0002] Field of the invention

[0003] The present invention relates to the field of food.

[0004] Background of the invention

[0005] The current trend of consumers leaning towards a healthy / vegetarian / vegan lifestyle has increased the demand for plant-based milk alternatives. The primary plant sources for milk replacements in the market are oat, soy, and almond. Due to their compositional diversity in terms of nutrition and techno-functional properties, these materials can serve in many applications. Among those, barista products have been manufactured to provide an alternative for dairy milk in coffee applications. One of the main drawbacks in the application of these products is the lack of acid stability in solution with coffee. The precise mechanism of which explains the curdling of these products have not yet been elucidated. To solve this issue, manufacturers rely on the application of 0.5% (w / w) dipotassium phosphate (K2HPO4) to buffer the acidity in the coffee. The addition of dipotassium phosphate is undesired because it is an additive with an E-number. Therefore, it is desired to provide solutions to reduce or to avoid the use of K2HPO4 and to still get an acceptable acid stability of plant-based dairy alternatives in coffee or tea.

[0006] The present invention provides such a solution.

[0007] WO2017009100 (DSM) describes a process to improve a physical property of a plant protein comprising incubating the plant protein with peptidyl arginine deiminase wherein the plant protein is pea, soy, rice or cereal protein.

[0008] WO2019233920 (DSM) describes a process for modifying sweetness, liquorice, astringency, powdery / chalk, fulness, thickness and / or digestibility and / or protease inhibitory activity of a protein comprising food comprising incubating a protein solution with a peptidyl arginine deiminase (PAD). Examples in this patent application describe that soy protein was treated with PAD, resulting in improved mouthfeel of a soy drink.

[0009] Summary of the invention

[0010] Surprisingly, the present inventors found that the use of an enzyme such as protein arginine deiminase or proline-specific endoprotease on a plant-based protein source such as soy or almond results in an enzyme treated plant-based milk alternative which has acceptable stability in an acidic beverage such as coffee or tea.

[0011] Herein disclosed is a method for preparing soy or almond protein comprising coffee or tea, comprising adding soy or almond protein to coffee or tea and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, a method for improving stability of soy or almond protein when used in coffee or tea comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein, treated soy protein or treated almond protein obtainable by the method as described above, a method for preparing soy or almond protein comprising coffee or tea comprising adding treated soy protein or treated almond protein as described above to coffee or tea, coffee or tea comprising protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, and / or use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) for improving stability of soy protein or almond protein in coffee or tea.

[0012] Sequence listing

[0013] SEQ ID NO: 1 PAD protein sequence from Trichoderma harzianum including the pro-sequence SEQ ID NO: 2 PAD protein sequence from Verticillium longisporum including the pro-sequence SEQ ID NO: 3 PAD protein sequence from Bionectra ochroleuca including the pro-sequence SEQ ID NO: 4 PAD protein sequence from Fusarium longipes including the pro-sequence

[0014] SEQ ID NO: 5 PAD protein sequence from Fusarium solani species complex including the prosequence

[0015] SEQ ID NO: 6 PAD protein sequence from Akanthomyces lecanii RCEF 1005 including the prosequence

[0016] SEQ ID NO: 7 PAD protein sequence from Fusarium incamatum-equiseti species complex including the pro-sequence

[0017] SEQ ID NO: 8 PAD protein sequence from Ophiocordyceps australis including the pro-sequence SEQ ID NO: 9 PAD protein sequence from Verticillium longisporum including the pro-sequence SEQ ID NO: 10 PAD protein sequence from wildtype Fusarium graminearum including the prosequence

[0018] SEQ ID NO: 11 protein sequence from Fusarium graminearum variant FgraPADmut_01 , FgraPADmut_02 and FgraPADmut_03 including the pro-sequence

[0019] SEQ ID NO: 1 -11 do not comprise a signal sequence

[0020] SEQ ID NO: 12 polynucleotide sequence encoding the protein of SEQ ID NO: 1

[0021] SEQ ID NO: 13 polynucleotide sequence encoding the protein of SEQ ID NO: 2

[0022] SEQ ID NO: 14 polynucleotide sequence encoding the protein of SEQ ID NO: 3

[0023] SEQ ID NO: 15 polynucleotide sequence encoding the protein of SEQ ID NO: 4

[0024] SEQ ID NO: 16 polynucleotide sequence encoding the protein of SEQ ID NO: 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 SEQ ID NO: 20 polynucleotide sequence encoding the protein of SEQ ID NO: 9

[0028] SEQ ID NO: 21 polynucleotide sequence encoding the protein of SEQ ID NO: 10

[0029] SEQ ID NO: 22 polynucleotide sequence encoding the protein of SEQ ID NO: 11

[0030] Detailed description of the invention

[0031] In a first aspect, the disclosure provides a method for preparing a low pH beverage comprising soy or almond protein, comprising adding soy or almond protein to a low pH beverage and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein.

[0032] A “low pH beverage comprising soy or almond protein” is defined herein as a beverage - typically coffee or tea - which comprises soy or almond protein. The soy or almond protein is typically part of a soy or almond drink (herein also referred to as a soy or almond milk alternative). Soy or almond based drinks can be used as a dairy milk alternative, for example for coffee or tea whitening. The term low pH beverage as used herein refers to a beverage which has a pH below pH 7, preferably below pH 6.5 or 6. Preferably, the low pH beverage has a pH in the range between 4 to 6.5. For example, espresso and filter coffee typically have a pH of around 5. Cold brew typically has a pH of around pH 6 and slightly higher.

[0033] The phrase “adding soy or almond protein to a low pH beverage” typically comprises adding a soy or almond protein comprising drink to a low pH beverage.

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

[0035] A suitable example of coffee is expresso, filter coffee or cold brew.

[0036] A suitable example of tea is black tea, green tea or white tea.

[0037] The phrase “adding soy or almond protein to coffee or tea” typically comprises adding a soy or almond protein comprising drink to coffee or tea.

[0038] The term “protein arginine deiminase” and “peptidyl arginine deiminase” (PAD) are used interchangeably herein. Protein or peptidyl arginine deiminases belong to a family of enzymes (EC 3.5.3.15) which convert peptide or protein bound arginine into peptide or protein bound citrulline. This process is called deamination or citrullination. In the reaction from arginine to citrulline, one of the terminal nitrogen atoms of the arginine side chain is replaced by an oxygen. The reaction uses one water molecule and yields ammonia as a side product (htp: / / en.wikiDedia.org / wiki / Citrullination). Whereas arginine is positively charged at a neutral pH, citrulline is uncharged.

[0039] Peptidyl arginine deiminase (PAD) may be derived from any suitable origin, for instance from mammalian or microbial origin. PAD’S used in the present invention are advantageously derived from a microbial source, i.e. the PAD used in a process of the invention is a microbial PAD. For instance, PAD’S may be derived from fungal origin such as from Fusarium sp. such as Fusarium graminearum, Chaetomium globosum, Phaesphaeria nodorum or from bacterial origin such as from the bacteria Streptomyces, eg Streptomyces scabies, Streptomyces clavuligeres. The wording “derived” or “derivable” from with respect to the origin of a polypeptide as disclosed herein, means that when carrying out a BLAST search with a polypeptide as disclosed herein, the polypeptide may be derivable from a natural source, such as a microbial cell, of which an endogenous polypeptide shows the highest percentage homology or identity with the polypeptide as disclosed herein.

[0040] Peptidyl arginine deiminases are for instance known from W02008 / 000714. W02008 / 000714 discloses a process for enzymatically treating a protein with a protein arginine deiminase, wherein at least 30% of the arginine is transformed into citrulline.

[0041] A peptidyl arginine deiminase may be a pure or purified peptidyl arginine deiminase. A pure of purified peptidyl arginine deiminase is an enzyme that may be 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, at least 90% pure, or at least 95% pure, 96%, 97%, 98%, 99%, 99.5%, 99.9% pure for instance as determined by SDS-PAGE or any other analytical method suitable for this purpose and known to the person skilled in the art.

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

[0043] For the purpose of this disclosure, it is defined here that in order to determine the percentage of sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out overthe full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region. The percent sequence identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443- 453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this 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 for the substitution matrix. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. A “mature polypeptide” is defined herein as a polypeptide in its final form and is obtained after translation of a mRNA into a polypeptide and post-translational modifications of said polypeptide. Post -translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation and removal of leader sequences such as signal peptides, propeptides and / or prepropeptides by cleavage.

[0044] A mature polypeptide sequence of SEQ ID NO: 10 may comprise or contain amino acids 19, 20, 21 , 22, 23, 24 to 640 of the amino acid sequence of SEQ ID NO: 10, advantageously the mature polypeptide sequence of SEQ ID NO: 10 comprises or contains amino acids 22 to 640 of SEQ ID NO: 26, wherein methionine at position 1 in SEQ ID NO: 10 is counted as number 1.

[0045] The term "polypeptide" refers to a molecule comprising amino acid residues linked by peptide bonds and containing more than five amino acid residues. The term "protein" as used herein is synonymous with the term "polypeptide" and may also refer to two or more polypeptides. Thus, the terms "protein" and "polypeptide" can be used interchangeably. Polypeptides may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, and the like) to add functionality. Polypeptides exhibiting activity in the presence of a specific substrate under certain conditions may be referred to as enzymes.

[0046] A peptidyl arginine deiminase, or polypeptide having peptidyl arginine deiminase activity may be produced in any suitable host organism by known methods in the art, for instance in fungi Aspergilli, eg Aspergillus niger or Aspergillus oryzae, Trichoderma, or the yeasts Saccharomyces, and Kluyveromyces or the bacteria of the genus Streptomyces or Bacilli. A suitable method to express a polypeptide having peptidyl arginine deiminase activity in Aspergillus niger is for instance disclosed in Examples 3 and 4 in WQ2008 / 000714, which is herein included by reference.

[0047] Alternatively, any of the PADs as described below is used such as an isolated and / or recombinant PAD polypeptide comprising

[0048] (a) a first domain comprising at least 80% sequence identity to amino acids 1 to 477 of SEQ ID NO:1 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 478 to 481 of SEQ ID NO:1 , a second domain comprising at least 80% sequence identity to amino acids 512 to 600 of SEQ ID NO: 1 and wherein said second domain optionally comprises 1 to 2 amino acids of amino acids 510 to 511 of SEQ ID NO:1 , and wherein said polypeptide does not comprise amino acids 482 to 509 of SEQ ID NO: 1 (also referred to as TharPAD_01 , TharPAD_03 and TharPAD_02),

[0049] (b) a first domain comprising at least 80% sequence identity to amino acids 1 to 487 of SEQ ID NO:2 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 488 to 491 of SEQ ID NO:2, a second domain comprising at least 80% sequence identity to amino acids 524 to 612 of SEQ ID NO: 2 and wherein said polypeptide does not comprise amino acids 492 to 523 of SEQ ID NO: 2 (also referred to as VlonPAD_01 and VlonPAD_02), (c) a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO:3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO:3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3 (also referred to as BochPAD_01 , BochPAD_02 and BochPAD_03),

[0050] (d) a first domain comprising at least 80% sequence identity to amino acids 1 to 483 of SEQ ID NO:4 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 484 to 487 of SEQ ID NO:4, a second domain comprising at least 80% sequence identity to amino acids 528 to 615 of SEQ ID NO: 4 and wherein said polypeptide does not comprise amino acids 488 to 527 of SEQ ID NO: 4 (also referred to as FlonPAD_01),

[0051] (e) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:5 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:5, a second domain comprising at least 80% sequence identity to amino acids 543 to 630 of SEQ ID NO: 5 and wherein said polypeptide does not comprise amino acids 494 to 542 of SEQ ID NO: 5 (also referred to as FsolPAD_01 and FsolPAD_02),

[0052] (f) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:6 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:6, a second domain comprising at least 80% sequence identity to amino acids 531 to 618 of SEQ ID NO: 6 and wherein said polypeptide does not comprise amino acids 494 to 530 of SEQ ID NO: 6 (also referred to as AlecPAD_01 and AlecPAD_02),

[0053] (g) a first domain comprising at least 80% sequence identity to amino acids 1 to 481 of SEQ ID NO:7 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 482 to 485 of SEQ ID NO:7, a second domain comprising at least 80% sequence identity to amino acids 517 to 604 of SEQ ID NO: 7 and wherein said polypeptide does not comprise amino acids 486 to 516 of SEQ ID NO: 7 (also referred to as FincPAD_01),

[0054] (h) a first domain comprising at least 80% sequence identity to amino acids 1 to 504 of SEQ ID NO:8 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 505 to 508 of SEQ ID NO:8, a second domain comprising at least 80% sequence identity to amino acids 535 to 626 of SEQ ID NO: 8 and wherein said polypeptide does not comprise amino acids 509 to 534 of SEQ ID NO: 8 (also referred to as OausPAD_01 and OausPAD_02), or

[0055] (i) a first domain comprising at least 80% sequence identity to amino acids 1 to 489 of SEQ ID NO:9 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 490 to 493 of SEQ ID NO:9, a second domain comprising at least 80% sequence identity to amino acids 525 to 613 of SEQ ID NO: 9 and wherein said polypeptide does not comprise amino acids 494 to 524 of SEQ ID NO: 9 (also referred to as VertPAD_01).

[0056] More preferably used are an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 477 of SEQ ID NO:1 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 478 to 481 of SEQ ID NO:1 , a second domain comprising at least 80% sequence identity to amino acids 512 to 600 of SEQ ID NO: 1 and wherein said second domain optionally comprises 1 to 2 amino acids of amino acids 510 to 511 of SEQ ID NO:1 , and wherein said polypeptide does not comprise amino acids 482 to 509 of SEQ ID NO:

[0057] 1 (also referred to as TharPAD_03), or an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO: 3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO: 3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3

[0058] (also referred to as BochPAD_02).

[0059] Most preferably used is an isolated and / or recombinant PAD polypeptide comprising a first domain comprising at least 80% sequence identity to amino acids 1 to 467 of SEQ ID NO: 3 and wherein said first domain optionally comprises 1 to 4 amino acids of amino acids 468 to 471 of SEQ ID NO: 3, a second domain comprising at least 80% sequence identity to amino acids 498 to 587 of SEQ ID NO: 3 and wherein said polypeptide does not comprise amino acids 472 to 497 of SEQ ID NO: 3 (also referred to as BochPAD_02).

[0060] Alternatively, a variant PAD polypeptide which is a variant Fusarium graminearum PAD polypeptide is used and wherein the variant Fusarium graminearum PAD polypeptide has an amino acid sequence which, when aligned with the PAD of SEQ ID NO: 10, comprises at least substitutions of amino acids residue corresponding to amino acids 29, 76, 92, 107, 153, 190, 219, 251 , 365, 439, 467 and 560, said positions being defined with reference to SEQ ID NO: 10 and wherein the variant has a pl below 9 and wherein said variant Fusarium graminearum PAD polypeptide has at least 80% sequence identity with SEQ ID NO:10. Preferably, the variant Fusarium graminearum PAD polypeptide comprises substitutions N29E, P76E, A92D, S107E, N153D, Q190E, G219D, N251 D, N365D, Q439E, Q467E and N560D, wherein said substitutions are defined with reference to SEQ ID NO: 10.

[0061] A "proline-specific endoprotease (PSP)" is a protease that hydrolyses a protein or peptide at a position where the protein or peptide contains a proline-residue. A proline-specific endoprotease may have proline-specific endopotease and / or proline-specific oligopeptidase activity (EC3.4.21 .26). A proline-specific endoprotease is preferably an enzyme that hydrolyses a peptide bond at the carboxy-terminal end of proline residues, resulting in a peptide and / or polypeptide fragment with a C-terminal proline

[0062] A proline-specific endoprotease may for instance be derived from Aspergillus niger or Penicillium chrysogenum, such as disclosed in W02002 / 046381 and W02009 / 144269 respectively.

[0063] Other proline-specific endoproteases are known from WO2012 / 174127. WO2012 / 174127 discloses proline-specific protease from Botryotinia, fuckeliana, Aspergillus clavatus, Sclerotinia sclerotiotum, Mycosphaerelly graminicola, Neuropspora crasse, Talaromyces stipitatus and Gibberella zeae

[0064] A suitable proline-specific endoprotease may be obtained from dsm-firmenich under the name Delvoplant PSP or DelvoPlant® PSP.

[0065] A “PAD or PSP treated soy or almond protein” is typically obtained by incubating a solution comprising soy or almond protein (also referred to as a soy or almond protein comprising solution) with PAD or PSP and allow incubation to take place such that PAD or PSP can perform its enzymatic activity. Enzyme incubation can be performed at any suitable pH for any suitable time and with any suitable enzyme concentration. The skilled person is very well capable of establishing a suitable enzyme amount or suitable incubation conditions such as a suitable incubation temperature or a suitable incubation pH or a suitable incubation time. For PSP a suitable incubation temperature is between 30 and 60 degrees Celsius, a suitable pH is between pH 2 and pH 6 and a suitable incubation time is between 30 min and 4 hours. For PAD, suitable conditions are: a pH of between 4 and 9, such as a pH of between 5 and 8.5, such as a pH of between 5.5 and 8, such as a pH between 6 and 7, or a pH of between 6.2 and 6.8, for instance at a pH of about 6.5 and a suitable temperature for PAD may be between 20 and 60 degrees Celsius, such as a between 30 and 50, or between 35 and 45 degrees Celsius. The skilled person is very well capable to adjust the amount of enzyme compared to the amount of substrate and / or to adjust the incubation time and / or temperature.

[0066] In an alternative aspect, the disclosure provides a method for improving stability of soy or almond protein when used in a low pH beverage comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein. More preferably, the disclosure provides a method for improving stability of soy or almond protein when used in coffee or tea comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein. This can also be described as a method for improving stability of soy or almond protein comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein.

[0067] Whether or not stability of soy or almond protein is improved in coffee or tea can for example be determined be comparing a PAD or PSP treated soy or almond protein to soy or almond protein which has been incubated without any enzyme being present and determining visual parameters such as separation, sedimentation or curdling. The experimental part provides examples on how to determine the stability of PAD or PSP treated soy or almond protein in a low pH beverage such as coffee or tea.

[0068] The disclosure also provides treated soy protein or treated almond protein obtainable by a method for improving stability of soy or almond protein when used in coffee or tea comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein.

[0069] The disclosure further provides a method for preparing soy or almond protein comprising coffee or tea comprising adding treated soy protein or treated almond protein to coffee or tea, wherein said treated soy protein or treated almond protein is obtainable by a method for improving stability of soy or almond protein when used in coffee or tea comprising incubating soy or almond protein with protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) to obtain a treated soy protein or a treated almond protein.

[0070] Preferably, the coffee or tea as described in any of the above methods has a pH in the range of 4.8 to 6.0, more preferably in the range of 5.0 to 6.0, most preferably in the range of 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 as such, i.e. before the soy or almond protein is added to the coffee or tea, i.e. before the soy or almond milk alternative (comprising said soy or almond protein) is added to the coffee or tea. Most preferably, the coffee or tea as described in any of the above methods has a pH in the range of 5.0 to 5.2. The upper limit in any of the above-described pH ranges is included in the range, i.e. the mentioned ranges include the upper limit.

[0071] Preferably, the soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v) resulting in one volume milk versus one to three volumes of coffee or tea. The milk part of the 1 :1 (v / v) to 1 :3 (v / v) refers to milk which is mixed with coffee and hence it does not include a possible foam layer. The foam layer is not relevant for the mentioned ratios because it is not fully in contact with the coffee or tea.

[0072] The temperature of the soy or almond milk (with PAD or PSP treated protein) is preferably room temperature before it is being added to coffee or tea. The soy or almond milk (with PAD or PSP treated protein) may be non-frothed or frothed. The experimental part herein describes examples with non-frothed milk. However, examples (not further described herein) with frothed soy or almond milk showed similar results as the non-frothed examples.

[0073] In a preferred embodiment, the invention provides a method for preparing soy or almond protein comprising coffee or tea, comprising adding soy or almond protein to coffee or tea and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein the coffee or tea has a pH in the range of 4.8 to 6.0, more preferably in the range of 5.0 and 6.0 and wherein the soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v). Most preferred, the invention provides a method for preparing soy or almond protein comprising coffee or tea, comprising adding soy or almond protein to coffee or tea and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein the coffee or tea has a pH in the range of 5.0 to 5.2 and wherein the soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v).

[0074] The coffee or tea produced in a method as described herein is non-fermented coffee or tea.

[0075] Any of the described methods is a method wherein a buffer is not added. A buffer is typically added to plant based dairy milk alternatives (specifically for plant-based milks which are used in coffee) for increasing the stability of proteins. Such a buffer prevents a rapid change in pH when the plant drink comes into contact with the acidic coffee.

[0076] Preferred embodiments are a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein (i.e. a method for preparing coffee is most preferred herein); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 4.8 to 6.0; a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 6.0; a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 5.2; as described earlier a pH range of 5.0 to 5.2 is most preferred herein a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 4.8 to 6.0 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 6.0 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 5.2 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) treated soy or almond protein, wherein said coffee has a pH in the range of 4.8 to 6.0 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 6.0 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy or almond protein comprising coffee, comprising adding soy or almond protein to coffee and wherein said soy or almond protein is a protein arginine deiminase (PAD) treated soy or almond protein, wherein said coffee has a pH in the range of 5.0 to 5.2 and wherein said soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v); PAD treated soy or almond protein combined with a coffee pH in the range of 5.0 to 5.2 is most preferred herein a method for preparing soy protein comprising coffee, comprising adding soy protein to coffee and wherein said soy is proline-specific endoprotease (PSP) treated soy protein, wherein said coffee has a pH in the range of 4.8 to 6.0 and wherein said soy protein is part of a soy milk alternative and said method further comprises adding said soy milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy protein comprising coffee, comprising adding soy protein to coffee and wherein said soy protein is a proline-specific endoprotease (PSP) treated soy protein, wherein said coffee has a pH in the range of 5.0 to 6.0 and wherein said soy protein is part of a soy milk alternative and said method further comprises adding said soy milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); a method for preparing soy protein comprising coffee, comprising adding soy protein to coffee and wherein said soy protein is a proline-specific endoprotease (PSP) treated soy protein, wherein said coffee has a pH in the range of 5.0 to 5.2 and wherein said soy protein is part of a soy milk alternative and said method further comprises adding said soy milk alternative to coffee in a ratio of 1 :1 (v / v) to 1 :3 (v / v); The coffee as described in any of the above method claims is preferably espresso, filter coffee or cold brew.

[0077] The tea as described in any of the above method claims is preferably black tea, green tea or white tea.

[0078] The disclosure also provides coffee or tea comprising protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein. The coffee is preferably espresso, filter coffee or cold brew. The tea is preferably black tea, green tea or white tea.

[0079] Preferably, said coffee or tea does not comprise a buffer, for example does not comprise potassium phosphate, potassium citrate, sodium citrate, calcium phosphate, calcium carbonate or potassium carbonate.

[0080] The disclosure also provides use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) for improving stability of soy protein or almond protein in a low pH beverage, preferably coffee or tea. Preferably, also provided is the use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) for improving stability of soy protein or almond protein in coffee. The preferred aspects as described for the method claims equally apply to the use described herein, such as - but not limited to - pH range of the coffee and the ratio of soy or almond milk alternative to coffee.

[0081] The term stability refers to stability of soy protein or almond protein in the absence of a typically stabilizer, i.e. stability in the absence of a buffer, for example the coffee does not comprise potassium phosphate, potassium citrate, sodium citrate, calcium phosphate, calcium carbonate or potassium carbonate. The disclosure thus provides use of protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) for improving stability of soy protein or almond protein in coffee in the absence of a buffer.

[0082] The invention is further illustrated in the examples below.

[0083] EXAMPLES

[0084] Materials and methods

[0085] Soybean material

[0086] Soybean base material was prepared by: a) Preparing a 12% (w / w) dehulled soybean solution in SPA* water (Ca: 5mg / L). SPA* is a brand of light mineral Belgian mineral water produced by Spa Monopole. b) Wet grinding during 10min at 85°C; a Thermomix was used which was set at 85 degrees Celsius, 10 minutes and velocity 8. Smaller particles result in more efficient enzyme treatment. c) Cool down and then centrifuge at 1000G for 10min. d) Separating the recovered supernatant from the pellet and heat treating it at 95°C for 15min to inactivate endogenous enzymes. e) Cooling down, measuring the pH and store the base at 4°C.

[0087] Almond material

[0088] A commercial almond drink (Zonnatura) was used which comprises only water and 5% (w / w) almond.

[0089] Alternatively, an almond solution can be prepared by: a) Weighing 5% of almond paste, and mixing with water at 45°C b) Stirring until complete dissolution

[0090] PAD

[0091] Two different PADs were used in the experimental part herein.

[0092] The first one is the PAD as described in W02008 / 000714. The used sample had an activity of 56.6 PAD U / g.

[0093] The second used PAD is described above as BochPAD_02 and had an activity of 313 PAD U / g and 19.2 mg protein / mL.

[0094] PSP

[0095] Properties of DelvoOPIant PSP: 7.5 PSP U / g of product. Dosage used: 1200ppm.

[0096] Experimental set-up Table 1 provides and overview of the protein content of the used material as well as the used PAD dosage.

[0097] Table 1

[0098] The mentioned PAD dosage of 0.3% w / w refers to 0.3 gram formulated PAD product dosed per 100g of raw material protein.

[0099] The protein content of the soy base was determined by previous experimental studies. The nitrogen content was quantified by Dumas method. The conversion factor to quantify the protein content was 5.7, value stated for soyabeans. The protein content of the almond material was based on the label information of the commercial material used.

[0100] Table 2 provides an overview of the negative control and the positive control.

[0101] Table 2

[0102] The negative control is the soybean base material without K2HPO4 and PAD. This sample is used to demonstrate the instability of the plant protein solution in coffee.

[0103] The positive control is the solution with 0.5 % w / w K2HPO4, this sample is used to provide a stable coffee solution with no curdling.

[0104] The PAD treatment is the solution with 0.3% w / w of the formulated PAD enzyme added based on the protein content of the raw material (example: 0.3 g of the formulated PAD protein was added per 100g of soy base protein). The PAD treatment comprised the steps of: a) Treating each solution at 55°C for two hours in a thermomixer. b) Inactivation of PAD by treating for 15min at 95°C. c) For soy, the PAD treatment was performed after the grinding step. d) For almond, the commercial drink was treated based on the protein content mentioned on the package. Stability of plant protein solutions in coffee

[0105] To determine the acid stability of the plant protein solutions, a strong coffee solution was prepared by putting 50g of ground coffee (Supermarket brand: Perla, flavor intensity number s, Arabica and Robusta beans, medium roasted) in a French press, adding 600mL of boiled water, followed by stirring and waiting for 5 minutes.

[0106] For the analysis: a) A 1 :3 ratio solution of plant protein solution to coffee was used. b) First, warm coffee was added to a transparent container and subsequently the solution to be tested was added. c) The obtained mix was - after 4 minutes - visually assessed for any separation, sedimentation or curdling. If one of the solutions exhibited one of the previous behaviors, the samples were characterized as not okay. In case of a clear milky coffee appearance, then the solution was labeled as excellent.

[0107] Testing for stability (or lack thereof) at various pH’s a) Different acid solutions were prepared to analyze the response of the different plant protein solutions treated with PAD. b) The pH of coffee (as described above) is 5.2. The pH was adjusted to five different pH points to test the acid stability of the plant protein solutions treated with PAD. The 5 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 decrease pH) or 0.5 M dipotassium phosphate (to increase pH). d) A 1 :3 ratio solution of plant protein solution to coffee was used in small tubes to check the responses. To keep the coffee warm, a water bath was set at 55°C to keep the tubes with coffee emerged before adding the enzyme treated plant protein solutions. e) The pH was measured, and photos (not shown herein) were taken of the results of the results. Example 1

[0108] Acid stability of soy base in coffee - PAD

[0109] Table 3:

[0110] Legend: pHo- corresponds to the pH of each solution. pHf. corresponds to the pH of both solutions mixed (coffee + solution).

[0111] N / A - Not Applicable.

[0112] 'The negative control is the soy solution without 0.5% of K2HPO4 nor PAD.

[0113] AThe positive control is the soy solution with 0.5% of K2HPO4

[0114] It is concluded that the addition of 0.3% PAD to soy protein base has a positive effect on the acid stability of soy base in coffee.

[0115] PAD can be used to replace buffer solutions such as K2HPO4.

[0116] Example 2

[0117] Acid stability of soy base in coffee - PAD - different pHs

[0118] Table 4: It is concluded that

[0119] Without addition of foHPC or PAD (i.e. the negative control), the soy base is stable in coffee at pH > 5.4

[0120] Both the positive control K2HPO4 and PAD treatments resulted in stable coffee at pH > 5.0

[0121] PAD can be used to replace buffer solutions such as K2HPO4

[0122] Example 3

[0123] Acid stability of almond base in coffee - PAD

[0124] In Example 3, almond protein base was treated similar as described in Examples 1 and 2. It is concluded that:

[0125] Untreated (-control) almond became stable at pH 6,0.

[0126] PAD improved the stability of almond solutions at pH > 5,4. This indicates that the enzyme slightly increased the buffer capacity of the almond solution.

[0127] In further experiments, an increased PAD dosage (0.6% instead of 0.3%) was tested in a similar coffee (50 g / 600 ml water) which showed clear improved stability of the almond milk in coffee.

[0128] In yet a further experiment, a PAD dosage of 0.3% was tested in a coffee which was prepared by using 33 gram of ground coffee / 600 ml water which also showed stability of the almond drink in the coffee solution.

[0129] Example 4

[0130] Acid stability of soy base in coffee - PSP

[0131] Table 5:

[0132] Legend: pHo - corresponds to the pH of each solution. pHf. corresponds to the pH of both solutions mixed (coffee + solution).

[0133] N / A - Not Applicable.

[0134] 'The negative control is the soy solution without 0.5% of K2HPO4 nor PSP.

[0135] AThe positive control is the soy solution with 0.5% of K2HPO4

[0136] It is concluded that:

[0137] Soy base treated with 1200ppm of PSP was stable in strong coffee conditions (50g / 600mL).

[0138] 1200ppm of PSP can replace the addition of K2HPO4 in soy drinks for buffer capacity properties for coffee applications.

Claims

CLAIMS1 . A method for preparing soy or almond protein comprising coffee or tea, comprising adding soy or almond protein to coffee or tea and wherein said soy or almond protein is a protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein.

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

3. Treated soy protein or treated almond protein obtainable by the method of claim 2.

4. A method for preparing soy or almond protein comprising coffee or tea comprising adding treated soy protein or treated almond protein according to claim 3 to coffee or tea.

5. A method according to claim 1 or 4, wherein said 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, most preferably in the range of 5.0 to 5.2.

6. A method according to any previous claim, wherein the soy or almond protein is part of a soy or almond milk alternative and said method further comprises adding said soy or almond milk alternative to coffee or tea in a ratio of 1 :1 (v / v) to 1 :3 (v / v).

7. A method according to any previous claim, wherein a buffer is not added.

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

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

10. Coffee or tea comprising protein arginine deiminase (PAD) or proline-specific endoprotease (PSP) treated soy or almond protein.11 . Coffee or tea according to claim 11 , wherein said coffee or tea does not comprise a buffer, for example does not comprise 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) for improving stability of soy protein or almond protein in coffee or tea.

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