Edible products comprising alpha-s1-casein variants a and b and methods for the producing the same

Abstract

The instant invention pertains to edible products comprising alpha-S1-casein variants A and B of non-animal origin in specific weight ratios. The invention further relates to methods for producing such edible products. The edible products can be dairy products, in particular animal-free cheese substitutes. The specific weight ratios between the alpha-S1-casein variants A and B allow to produce cheese substitutes that have improvements in terms of their similarity to milk-derived cheese, in particular as regards their organoleptic / physical properties such as cohesiveness, firmness, elasticity, meltability and spreadability.

Description

[0001] Edible products comprising alpha-S1 -casein variants A and B and methods for the producing the same

[0002] Field of the invention

[0003] The present invention relates to the fields of food technology, molecular microbiology and fermentation technology. In particular, the invention relates to edible products comprising alpha-S1- casein variants A and B of non-animal origin and methods for producing the same.

[0004] Background of the invention

[0005] In 2050 the global population will be around 10 billion people. It is generally recognized that the production of food and its ingredients needs to change significantly to keep within the agreed sustainability development goals (SDGs) for the environment and climate.

[0006] Milk, and especially cow milk, is an important source of protein and is produced all around the world (total production in 2018: 843 million tons). However, dairy production has an enormous impact on the environment. Currently over two-thirds of the world's agricultural land is used for maintaining livestock, including beef and dairy cows. Dairy cows and their manure generate significant amounts of greenhouse gas (including methane, which is a much more harmful greenhouse gas than CO2) emissions which contribute to climate change. Water demand is very high as dairy operations consume large volumes of water to grow feed, water cows, manage manure and process products. Additionally, nitrogen emissions (from e.g. manure and fertilizer) cause worldwide major issues. Consequently, the carbon footprint and land-use factor of milk and cheese are high, even higher than that of pigs, fish and chicken. Next to these environmental and climatological aspects, also animal welfare is quite often compromised. Concerns about sustainability and animal-welfare of milk production are two important motivations for an increasing percentage of consumers to replace animal-based proteins by (vegan) plant-based protein sources such as soy, almond, pea and coconut.

[0007] Bovine milk contains around 35 g / L of caseins (i.e. 80% of the milk protein fraction) divided over alpha-S1-, alpha-S2-, beta- and kappa-casein within an approximate ratio of 40, 10, 40 and 10 % respectively. The four caseins are well studied in terms of amino acid composition, molecular weight, post-translational modifications (PTMs) and general physico-chemical properties. Due to the high content of prolyl residues, each casein molecule has an open and flexible conformation. Furthermore, hydrophobic and hydrophilic regions show a block distribution within the protein chain, giving each casein an amphiphilic character. Because of their nature and physico-chemical properties, caseins are unique proteins that, for many applications, cannot easily be replaced by plant-based alternatives.

[0008] Cheese is the third most unsustainable animal product globally (when measuring greenhouse gas emissions per kg of product), and the consumption of dairy cheese hasn’t been slowed down by plant-based alternatives introduced into the market in the last 10 years. On the contrary, mozzarella cheese consumption is growing year on year in the US and in developing markets. Current vegan cheese alternatives do not match the functionality, nutrition and taste of dairy cheese due to their lack of casein proteins.

[0009] More recently, several attempts have been published to produce cheese substitutes using caseins of non-animal origin that have been produced by fermentation of recombinant microorganism expressing mammalian casein coding sequences. WO 2020 / 219596 discloses recombinant milk proteins with non-native posttranslational modifications for the making of food products. WO 2020 / 223700 discloses cheese and yogurt compositions and methods of making the same using one or more recombinant proteins as well as purified bovine caseins. WO 2022 / 058573 discloses methods for producing cheese substitutes comprising only caseins of non-animal origin.

[0010] There is however a need in the art for edible products, such as cheese substitutes, comprising caseins of non-animal origin, with improvements in terms of their similarity to milk- derived cheeses, in particular improvements in their organoleptic / physical properties such as cohesiveness, firmness, elasticity, meltability and spreadability. It is an object of the instant invention to provide for such improved edible products and for methods for producing the same.

[0011] Summary of the invention

[0012] In a first aspect, there is provided a method for producing an edible product, the method comprising: a) emulsifying an aqueous solution comprising alpha-S1 -casein variants A and B of non-animal origin with an edible fat, to produce an oil-in-water emulsion, wherein the weight ratio of variant A to variant B ranges from 2 : 98 to 80 : 20; b) acidifying the emulsion to a pH that differs less than 0.5 pH unit from the average of the isoelectric points of variant A and variant B; c) incubation to allow curd clotting and syneresis; and, d) separation of the curd and serum fraction to obtain a fresh curd, wherein prior to step b), the emulsion contains or is brought to a concentration of soluble calcium, at the pH set in step b), in the range of 0.20 - 1 .65 mmol Ca2+per gram casein, wherein the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 or the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 7, and wherein the alpha-S1 -casein variant B comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with at least one of SEQ ID NO: 1 and 7.

[0013] In one embodiment of the method, the weight ratio of variant A to variant B is in the range from 5 : 95 to 45 : 55, preferably in the range from 5 : 95 to 40 : 60.

[0014] In one embodiment of the method, the emulsion contains less than 0.20 mmol Ca2+per gram casein and the weight ratio of variant A to variant B is in the range from 45 : 55 to 50 : 50.

[0015] In one embodiment of the method, the alpha-S1 -casein variant A of non-animal origin comprises a deletion of at least 4 amino acids in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 , preferably the alpha-S1 -casein variant A of non-animal origin comprises a deletion of amino acids 14 - 26 in SEQ ID NO: 1 . In one embodiment of the method, at least one of the alpha-S1 -casein variants A and B of non-animal origin lacks native post-translational modifications.

[0016] In one embodiment of the method, at least one of: i) at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by intracellular expression of a casein comprising an amino acid sequence as defined herein above, in a microbial host cell, preferably a bacterial host cell, more preferably an Escherichia coli host cell; and, ii) at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by extracellular expression of a casein comprising an amino acid sequence as defined in claims 1 - 4, in a microbial host cell, preferably a fungal host cell, more preferably a K. phaffii host cell.

[0017] In one embodiment of the method, in step a) the combined alpha-S1 -casein variants A and B of non-animal origin and the edible fat are emulsified in a weight ratio on dry matter in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1 .5, more preferably in the range of 0.8 to 1 .2, and most preferably in the range of 0.9 to 1 .1 , or in a weight ratio on dry matter of about 1 .0.

[0018] In one embodiment of the method, the edible fat is fat or oil of non-animal origin, preferably an edible vegetable oil, more preferably an edible vegetable oil selected from the group consisting of canola (rapeseed) oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil, safflower oil, soya bean oil, sunflower oil and high-oleic sunflower oil.

[0019] In one embodiment of the method, at least one of: i) the concentration of the alpha-S1 -casein variants A and B of non-animal origin in the emulsion is in the range of 30 - 70 weight % on dry matter, preferably 40 - 60 weight % on dry matter, more preferably 45 - 55 weight % on dry matter; ii) the emulsion comprises 3 to 65 weight % dry matter; iii) the emulsion comprises 1 - 8 mM of a food grade buffer, e.g. a phosphate buffer; iv) the incubation in step c) above, is at a temperature in the range of 20 - 65 °C, preferably 30 - 60 °C, more preferably 45 - 55 °C, and most preferably 48 - 52 °C or about 50 °C, for at least 15, 20, 25, 30, 40, 50 or 60 minutes; and, v) the separation of the curd and serum fraction in step d) above, is performed by filtration, preferably filtration through a cheese cloth.

[0020] In a second aspect, there is provided an edible product obtained or obtainable in any method according to the first aspect above.

[0021] In a third aspect, there is provided an edible product comprising alpha-S1 -casein variants A and B of non-animal origin in a weight ratio of variant A to variant B that ranges from 5 : 95 to 50 : 50, wherein the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 or the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 7, and wherein the alpha-S1 -casein variant B comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with at least one of SEQ ID NO: 1 and 7.

[0022] In one embodiment of the edible product, the product further comprises at least one of: i) an edible fat, wherein the weight ratio on dry matter between the combined alpha-S1 -casein variants A and B of non-animal origin and the edible fat is in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1 .5, more preferably in the range of 0.8 to 1 .2, and most preferably in the range of 0.9 to 1 .1 , or in a weight ratio on dry matter of about 1 .0; and, ii) 0.20 - 1 .65 mmol of a source of Ca2+per gram casein.

[0023] In one embodiment of the edible product, the ratio of variant A to variant B in weight percentages is in the range from 5 : 95 to 45 : 55 , preferably in the range from 5 : 95 to 40 : 60.

[0024] In one embodiment of the edible product, the ratio of variant A to variant B in weight percentages is in the range from 45 : 55 to 50 : 50 and the product contains less than 0.20 mmol of a source of Ca2+per gram casein.

[0025] In one embodiment of the edible product, at least one of: i) the alpha-S1 -casein variants A and B of non-animal origin are as defined above in the first aspect; and, ii) the edible fat is an edible vegetable oil, preferably a fat or oil as defined above in the first aspect.

[0026] In one embodiment of the edible product, the product is a cheese substitute, preferably a Mozzarella analogue.

[0027] Description of the invention

[0028] Definitions

[0029] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0030] “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article “a” or “an” thus usually means “at least one”. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0031] “About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0032] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0033] “Exemplary”: this term means “serving as an example, instance, or illustration,” and should not be construed as excluding other configurations disclosed herein.

[0034] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... ,etc.

[0035] “Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods. The terms “sequence identity” or “sequence similarity” means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over the entire length (of at least the shortest sequence in the comparison) and maximizing the number of matches and minimizes the number of gaps such as by the programs ClustalW (1.83), GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) I 8 (proteins) and gap extension penalty = 3 (nucleotides) I 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is BLOSUM62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences disclosed herein is ClustalW (1 .83) using a BLOSUM matrix and default settings (Gap opening penalty:10; Gap extension penalty: 0.05). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are BLOSUM62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

[0036] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below. Alternative conservative amino acid residue substitution classes.

[0037] Alternative physical and functional classifications of amino acid residues. A “nucleic acid construct” or “nucleic acid vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term “nucleic acid construct” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms “expression vector” or expression construct" refer to nucleic acid molecules that are capable of effecting expression of a nucleotide sequence or gene in host cells or host organisms compatible with such expression vectors or constructs. These expression vectors typically include regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed to effect its expression. Such regulatory elements usually at least include suitable transcription regulatory sequences and optionally, 3’ transcription termination signals. Additional elements necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism whereas an expression construct will usually integrate in the host cell’s genome for it to be maintained. Techniques for the introduction of nucleic acid into cells are well established in the art and any suitable technique may be employed, in accordance with the particular circumstances. The introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques. The introduction may be followed by expression of the nucleic acid to produce the encoded fusion protein. In some embodiments, host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded fusion protein polypeptide is produced, when an inducible promoter is used, expression may require the activation of the inducible promoter.

[0038] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.

[0039] The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term “reporter” may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional.

[0040] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

[0041] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin.

[0042] The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) e.g. comprising a polyadenylation- and / or transcription termination site. “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

[0043] The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and / or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98 / 81 / EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed earlier herein.

[0044] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

[0045] Detailed description of the invention

[0046] It was surprisingly found that small differences in amino acid sequence can greatly affect the interfacial functionality of caseins. Hence, specific recombinant casein variants can provide the flexibility to select a desired functionality based on the requirements for the product. In addition, the inventors have surprisingly found and show here that when producing cheese substitutes using alpha-S1 -casein of non-animal origin, the addition of alpha-S1 -casein variant A in certain weight ratios to the most commonly used alpha-S1 -casein variant B, curds are produced that have improvements in terms oftheir similarity to milk-derived cheese.. These improved properties include organoleptic / physical properties such as cohesiveness, firmness, elasticity, meltability and spreadability.

[0047] In a first aspect, there is provided a method for producing an edible product, which edible product preferably comprises an alpha-S1 -casein, preferably an alpha-S1 -casein of non-animal origin. A casein “of non-animal origin” is herein understood as a casein that is not obtained from an animal, such as a lactating mammal, but rather a casein that is produced by fermentation of host cells expressing the casein, whereby preferably the host cells are microbial host cells such as fungal or bacterial host cells.

[0048] In one embodiment, the method for producing the edible product comprises the steps of: a) emulsifying an aqueous solution comprising alpha-S1 -casein variants A and B of non-animal origin with an edible fat, to produce an oil-in-water emulsion; b) acidifying the emulsion to a pH that differs less than 0.5 pH unit from the average of the isoelectric points of variant A and variant B; c) incubation to allow curd clotting and syneresis; and, d) separation of the curd and serum fraction to obtain a fresh curd.

[0049] In one embodiment of the method, the weight ratio of the alpha-S1 -casein variant A to the alpha-S1 -casein variant B in the aqueous solution in step a), ranges from 2 : 98 to 80 : 20, preferably the weight ratio ranges from 3 : 97 to 70 : 30, more preferably the weight ratio ranges from 4 : 96 to 60 : 40, and most preferably the weight ratio ranges from 5 : 95 to 50 : 50. As shown in the Examples herein, an edible product comprising the alpha-S1 -casein variants A and B in these weight ratios produce coagulated curds with improved organoleptic / physical properties, such as cohesiveness, firmness, and elasticity.

[0050] In one embodiment of the method, the weight ratio of the alpha-S1 -casein variant A to the alpha-S1 -casein variant B in the aqueous solution in step a), ranges from 5 : 95 to 45 : 55, preferably in the range from 5 : 95 to 40 : 60, preferably in the range of 10 : 90 to 30 : 70, more preferably about 20 : 80. As discussed below and shown in the Examples herein, an edible product comprising the alpha-S1 -casein variants A and B in these weight ratios show improved spreadability indices.

[0051] In one embodiment of the method, the pH to which the emulsion is acidified in step b) differs less than (with increasing preference) 0.4 pH unit, 0.3 pH unit, 0.2 pH unit, 0.15 pH unit, 0.125 pH unit or 0.1 pH unit from the average of the isoelectric points of the alpha-S1 -casein variant A and the alpha-S1 -casein variant B. The isoelectric points of the alpha-S1 -casein variants can be determined experimentally, e.g. as described in the Examples, or can be based on a theoretical calculation based on the amino acid sequences of the alpha-S1 -casein variants.

[0052] In one embodiment of the method, prior to step b), the emulsion contains or is brought to a concentration of soluble calcium in the range of 0.20 - 1.65 mmol Ca2+per gram casein, whereby preferably the concentration of soluble calcium is the concentration of soluble calcium at the pH set in step b). In one embodiment of the method, prior to step b), the emulsion contains or is brought to a concentration of soluble calcium in the range of (with increasing preference) 0.25 - 0.825 mmol Ca2+per gram casein, 0.275 - 0.60 mmol Ca2+per gram casein, 0.30 - 0.36 mmol Ca2+per gram casein, whereby preferably the concentration of soluble calcium is the concentration of soluble calcium at the pH set in step b). In a preferred embodiment of the method, prior to step b), the emulsion contains or is brought to a concentration of soluble calcium of about 0.33 mmol Ca2+per gram casein. As shown in the Examples herein, an edible product comprising the alpha-S1 -casein variants A and B and calcium in these concentrations produce coagulated curds with improved organoleptic / physical properties, such as cohesiveness, firmness, and elasticity.

[0053] In one embodiment of the method, the emulsion contains less than 0.20, less than 0.15, less than 0.10, less than 0.05, less than 0.02, less than 0.01 mmol, or no detectable soluble Ca2+per gram casein, whereby preferably the concentration of soluble calcium is the concentration of soluble calcium at the pH set in step b), and the weight ratio of the alpha-S1 -casein variant A to the alpha- S1 -casein variant B in the aqueous solution in step a), ranges from 45 : 55 to 50 : 50.

[0054] In one embodiment of the method, the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion in the region of amino acids positions 14-26 of SEQ ID NO: 1 , or the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion in the region of amino acids positions 14-26 of SEQ ID NO: 7. In one embodiment, the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion of (at least) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 amino acids in the region of amino acids positions 14-26 of SEQ ID NO: 1 , or the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion of (at least) 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 amino acids in the region of amino acids positions 14-26 of SEQ ID NO: 7. The deletion of the 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 can be contiguous or non- contiguous. In a preferred embodiment, the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion of amino acids 14- 26 in SEQ ID NO: 1 , or the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion of amino acids 14-26 in SEQ ID NO: 7. More preferably, the alpha-S1 -casein variant A of non-animal origin comprises an amino acid sequence as depicted in SEQ ID NO: 2 or 8. SEQ ID NO: 2 represents the amino acid sequence of the mature Bos taurus alpha-S1 -casein variant A (i.e. without its signal sequence). SEQ ID NO: 8 represents the mature amino acid sequence of the Capra aegagrus hircus alpha-S1 -casein (i.e. without its signal sequence), modified to correspond to the bovine variant A by deletion of amino acids 14-26 of SEQ ID NO: 7. An alpha- S1 -casein with the amino acid sequence of SEQ ID NO:8 is also known as the mature goat alpha- S1 -casein variant G.

[0055] In one embodiment of the method, the alpha-S1 -casein variant B of non-animal origin comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with at least one of SEQ ID NO: 1 and 7. More preferably, the alpha-S1 -casein variant B of non- animal origin comprises an amino acid sequence as depicted in SEQ ID NO: 1 or 7. SEQ ID NO: 1 represents the mature amino acid sequence of the Bos taurus alpha-S1 -casein variant B (i.e. without its signal sequence). SEQ ID NO: 7 represents the mature amino acid sequence of the Capra aegagrus hircus alpha-S1 -casein (i.e. without its signal sequence).

[0056] In one embodiment of the method, at least one of the alpha-S1 -casein variants A and B of non-animal origin lacks native post-translational modifications. Native post-translational modifications are herein understood as post-translational modifications that can be present on native alpha-S1 -casein variants as present in the milk of lactating mammals.

[0057] In one embodiment of the method, the alpha-S1 -casein variants A and B of non-animal origin lack an N-terminal methionine at a position corresponding to position 1 of one of SEQ ID NO.’s: 3 and 4, for example as a result production by secretion from a microbial host cell using a signal sequence that is cleaved off, e.g. as exemplified in SEQ ID NO.’s: 1 , 2, 7 and 8.

[0058] In one embodiment of the method, at least one of the alpha-S1 -casein variants A and B of non-animal origin comprises at least one non-native post-translational modification. Non-native post-translational modifications are herein understood as post-translational modifications that differ from native post-translational modifications as defined above, and that are the result of heterologous expression of a casein in a microbial host cell.

[0059] In one embodiment of the method, the alpha-S1 -casein variants A and B of non-animal origin can comprise an N-terminal formyl methionine, for example at a position corresponding to position 1 of SEQ ID NO.’s: 3 and 4, or position -1 of SEQ ID NO.’s: 1 , 2, 7 and 8. Such N-terminal formyl methionine can be present as a result of bacterial intracellular expression. In one embodiment, N- terminal formyl methionine is (partially) deformylated. In one embodiment, N-terminal formyl methionine is (partially) cleaved off and / or (partially) deformylated.

[0060] Thus, in one embodiment of the method, at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by intracellular expression of a casein comprising an amino acid sequence as defined above, in a microbial host cell, preferably a bacterial host cell, more preferably an Escherichia coli host cell. In another embodiment of the method, at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by extracellular expression of a casein comprising an amino acid sequence as defined above, in a microbial host cell, preferably a fungal host cell, more preferably a K. phaffii host cell. For extracellular expression of the casein, the N-terminal methionine in position 1 is replaced by a suitable signal sequence for secretion of the casein from the microbial host cell. The signal sequence can be a signal sequence that is native to the alpha-S1 -casein, or can be heterologous to the alpha-S1 -casein, e.g. a signal sequence from a secreted protein that is endogenous to the host cell in question. In a specific embodiment of the method, at least one of: a) the alpha-S1 -casein variant A is obtained or obtainable by expression of SEQ ID NO: 6 in a bacterial host cell, whereby preferably the host cell is an E.coli host cell; and b) the alpha-S1 -casein variant B is obtained or obtainable by expression of SEQ ID NO: 5 in a bacterial host cell, whereby preferably the host cell is an E.coli host cell.

[0061] A suitable host cell for fermentative production of the alpha-S1 -casein variants A and B of non-animal origin for use in the methods provided herein, is thus a microbial host cell. The microbial host cell can be a bacterial or a fungal host cell.

[0062] A bacterial host cell can be Gram-negative and Gram-positive bacterial host cell. Examples of suitable bacterial host cells include host cells from the genera Escherichia, Bacillus, Lactobacillus, Lactococcus and Streptococcus, or preferably bacterial host cells of the species Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus coagulans, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri, Lactococcus lactis, Streptococcus salivarius and Streptococcus thermophilus. More preferably the bacterial cell is E. coli.

[0063] A fungal host cell can a filamentous fungal host cell or a yeast host cell. Examples of suitable filamentous fungal host cells include host cells from genera Alternaria, Apophysomyces, Aspergillus, Cladosphialophora, Fonsecaea, Fusarium, Lichtheimia, Mucor, Myceliophthora, Neurospora, Penicillium, Rhizopus, Rhizomucor, Trichoderma and Trichophyton, or preferably filamentous fungi cells of the species Alternaria alternata, Apophysomyces variabilis, Aspergillus spp., Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus sojae, Aspergillus terreus, Cladosphialophora spp., Fonsecaea pedrosoi, Fusarium spp., Fusarium oxysporum, Fusarium solani, Lichtheimia spp., Lichtheimia corymbifera, Lichtheimia ramosa, Myceliophthora spp., Myceliophthora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium simplicissimum, Penicillium brasilianum, Rhizopus spp., Rhizopus microsporus, Rhizomucor spp., Rhizomucor pusillus, Rhizomucor miehei, Trichoderma spp., Trichoderma reesei Trichophyton spp., Trichophyton interdigitale, and Trichophyton rubru, and most preferably a species selected from Aspergillus oryzae and Aspergillus niger. Examples of suitable yeast host cells include host cells from the genera Saccharomyces, Kluyveromyces, Candida, Komagataella, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Debaryomyces and Naumovia, or preferably yeast host cells of the species K. phaffii, K. pastoris, K. pseudopastoris S. cerevisiae, S. exiguus, S. bayanus, K. lactis, K. marxianus Y. lipolytica and S. pombe, of which K. phaffii is most preferred. The yeast genus Pichia has more recently been reassigned to the genus Komagataella (see e.g., Heistinger et al., Microbiology, 2020;166(7):614- 616), which genus was split into the species K. phaffii, K. pastoris, and K. pseudopastoris. The Pichia species P. pastoris, that has been widely used in biotech industries and as used herein, has been reassigned to the Komagataella species K. phaffii (Heistinger et al., Microbiology, 2020, supra). Hence, when reference is made herein to the yeast species K. phaffii this is to be understood as equally referring to the yeast species formerly known as Pichia pastoris, and vice versa.

[0064] In one embodiment of the method, in step a) the combined alpha-S1 -casein variants A and B of non-animal origin and the edible fat are emulsified in a weight ratio on dry matter in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1 .5, more preferably in the range of 0.8 to 1 .2, and most preferably in the range of 0.9 to 1.1 , or in a weight ratio on dry matter of about 1 .0.

[0065] In one embodiment of the method, the edible fat can comprise fats or oils of vegetable, microbial, algal or animal origin, or combinations thereof. In one embodiment, the edible fat comprises or is milk fat or milk-derived fat such as (dairy) cream, butter, clarified butter, or fractions thereof. In a preferred embodiment of the method, the edible fat comprises or is fat or oil of nonanimal origin, such as an edible vegetable oil. In one embodiment, the edible vegetable oil selected from the group consisting of canola (rapeseed) oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil, safflower oil, soya bean oil, sunflower oil and high- oleic sunflower oil.

[0066] In one embodiment of the method, the concentration of the alpha-S1 -casein variants A and B of non-animal origin in the emulsion is in the range of 30 - 70 weight % on dry matter, preferably 40 - 60 weight % on dry matter, more preferably 45 - 55 weight % on dry matter. It is understood that the concentration of the alpha-S1 -casein variants A and B of non-animal origin in the emulsion refers to the total concentration of the alpha-S1 -caseins of non-animal origin in the emulsion, i.e. the sum of the concentration of the alpha-S1 -casein variant A and the concentration of the alpha- S1 -casein variant B.

[0067] In one embodiment of the method, the emulsion comprises 3 to 65 weight % dry matter, preferably, the emulsion comprises 6 to 65 weight % dry matter, more preferably, the emulsion comprises 9 to 65 weight % dry matter, and most preferably, the emulsion comprises 9 to 60 weight % dry matter. It is understood herein that the edible fat is included in the dry matter.

[0068] In one embodiment of the method, the emulsion comprises 1 - 8 mM, preferably 2 - 6 mM, more preferably 3 - 5 mM and most preferably about 4 mM, of a food grade buffer, e.g. a phosphate buffer.

[0069] In one embodiment of the method, the incubation in step c) is at a temperature in the range of 20 - 65 °C, preferably 30 - 60 °C, more preferably 45 - 55 °C, and most preferably 48 - 52 °C or about 50 °C, and for at least 15, 20, 25, 30, 40, 50 or 60 minutes.

[0070] In one embodiment of the method, the separation of the curd and serum fraction in step d) is performed by filtration, preferably filtration through a cheese cloth. It is understood herein that the term “curd” means a composition wherein casein has coagulated or precipitated, and which in addition to the caseins also contains other components such as water and lipids. After coagulation, the curd can be separated from a liquid phase (i.e. the serum fraction) by any known means, including e.g. filtration, such as filtration (or draining) through a cheese cloth.

[0071] In some embodiments, the curd is further treated to create a cheese or cheese substitute product. In some cases, such as a mozzarella product, renneted curd may be heated and stretched. In other embodiments, the curd is aged, such as for brie, camembert, feta, halloumi, gouda, edam, Cheddar, manchego, swiss, colby, muenster, blue cheese or parmesan type cheese or cheese-like product.

[0072] In a second aspect, there is provided an edible product that is obtained or obtainable in a method as described herein. In further embodiments, the edible product has the features defined in the third aspect hereinbelow.

[0073] In a third aspect, there is provided an edible product comprising alpha-S1 -casein variants A and B of non-animal origin in a weight ratio of variant A to variant B that ranges from 2 : 98 to 80 : 20, preferably the weight ratio ranges from 3 : 97 to 70 : 30, more preferably the weight ratio ranges from 4 : 96 to 60 : 40, and most preferably the weight ratio ranges from 5 : 95 to 50 : 50. As shown in the Examples herein, an edible product comprising alpha-S1 -casein variants A and B in these weight ratios produce coagulated curds with improved organoleptic / physical properties, such as cohesiveness, firmness, and elasticity.

[0074] In one embodiment of the edible product, alpha-S1 -casein variants A and B of non-animal origin in the edible product are as (structurally) defined above in the first aspect.

[0075] In one embodiment of the edible product, the weight ratio on dry matter of the caseins (i.e. the combined alpha-S1 -casein variants A and B of non-animal origin) to the edible fat is in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1 .5, more preferably in the range of 0.8 to 1 .2, and most preferably in the range of 0.9 to 1 .1 , or in a weight ratio on dry matter of about 1 .0.

[0076] In one embodiment of the edible product, the edible fat is as defined above in the first aspect.

[0077] In one embodiment of the edible product, the product comprises a source of calcium in a concentration in the range of 0.20 - 1.65 mmol Ca2+per gram casein. In one embodiment, the edible product comprises a source of calcium in a concentration in the range of (with increasing preference) 0.25 - 0.825 mmol Ca2+per gram casein, 0.275 - 0.60 mmol Ca2+per gram casein, 0.30 - 0.36 mmol Ca2+per gram casein. In a preferred embodiment, the edible product comprises a source of calcium in concentration of about 0.33 mmol Ca2+per gram casein. As shown in the Examples herein, an edible product comprising alpha-S1 -casein variants A and B and calcium in these concentration produce coagulated curds with improved organoleptic / physical properties, such as cohesiveness, firmness, and elasticity.

[0078] In one embodiment of the edible product, the product contains less than 0.20, less than 0.15, less than 0.10, less than 0.05, less than 0.02, less than 0.01 mmol, or no detectable source of Ca2+per gram casein, and the weight ratio of alpha-S1 -casein variant A to alpha-S1 -casein variant B in the aqueous solution in step a), ranges from 45 : 55 to 50 : 50. In one embodiment of the edible product, the weight ratio of alpha-S1 -casein variant A to alpha-S1 -casein variant B in the product ranges from 5 : 95 to 45 : 55. An edible product comprising caseins of non-animal origin, such as a cheese analogue, comprising alpha-S1 -casein variants A and B in a weight ratio in the range of 5 : 95 to 45 : 55 has a good meltability, as shown in the Examples herein. The meltability of an edible product comprising caseins of non-animal origin, such as a cheese analogue, is herein understood as the loss of shape of the product upon heating, preferably to a temperature of at least 90 °C. The meltability of an edible product comprising caseins of non-animal origin can be determined as described in the Examples herein.

[0079] In one embodiment of the edible product, the weight ratio of alpha-S1 -casein variant A to alpha-S1 -casein variant B in the product ranges from 5 : 95 to 40 : 60, preferably in the range of 10 : 90 to 30 : 70, more preferably about 20 : 80. As shown in the Examples herein, an edible product comprising caseins of non-animal origin, such as a cheese analogue, comprising alpha-S1 -casein variants A and B in a weight ratio in the range of 5 : 95 to 40 : 60 has a spreadability index of at least 0.70 dL / g product. The spreadability index of an edible product comprising caseins of non- animal origin is herein understood as the proportional increase in length (i.e. A length = length after melting minus the original length before melting, divided by the original length before melting) per gram product, due to spreading / flowing out of shape upon heating to 90 °C, as determined as described in the Examples herein. In one embodiment, an edible product comprising caseins of non-animal origin, has spreadability index of at least 0.35, 0.50, 0.60, 0.70, 0.80, 0.90, 1 .00, 1 .20, 1.50, 1.58, 1.60, 1 .61 , 1.80, 2.00 or 2.17 dL / g product.

[0080] In one embodiment of the edible product comprising casein variants of non-animal origin in ratios as defined above, the product is for human consumption. The edible product can thus be any food product. In one embodiment, the edible product further comprises minerals, synthetic substances, ferments, flavouring substances (such as for examples herbs and spices), plant based proteins or proteins from microbial origin such as yeast proteins. Plant based proteins and yeast proteins suitable for the use in food products are known to the skilled person in the art.

[0081] In one embodiment, the edible product is a dairy product or dairy substitute product. In one embodiment, the dairy substitute product is a vegan product, i.e. lacking ingredients of animal origin. A wide variety of dairy substitute products can be made using the methods, compositions and products described herein. Methods for producing animal-free dairy substitute products are inter alia described in WO2016 / 029193, which is herein incorporated by reference. Such products include without limitation, milk, whole milk, buttermilk, skim milk, infant formula, condensed milk, dried milk, evaporated milk, butter, clarified butter, cream, cottage cheese, cream cheese, creme fraiche, skyr, yogurt and various types of cheese. The dairy substitute products can also be incorporated into various food applications as a replacement for dairy products, which include ice cream, frozen custard, frozen yogurt, cookies, chocolate and cakes.

[0082] In a preferred embodiment, the dairy substitute product is cheese substitute or analogue. The terms “cheese substitute” or “cheese analogue” are herein used interchangeably and refer to a food product having the essential features of cheese in terms of nutritional value, aspect, texture and taste but which comprise at least one milk protein, preferably a casein, of non-animal origin. Preferably, the cheese substitute comprises no milk proteins of animal origin or more preferably, no ingredients of animal origin, i.e. a vegan cheese substitute. In one embodiment, the cheese substitute should have the protein and moisture content of the category of cheese it substitutes for (fresh cheese, soft / semi soft cheese or firm / hard cheese). WO 2022 / 058573, which is hereby incorporated by reference, discloses methods for producing animal-free fresh, soft / semi soft and firm / hard cheese substitutes.

[0083] Thus, in one embodiment, the cheese substitute has the essential features of fresh cheese. The fresh cheese contains a moisture of 80% or higher, on a fat-free basis. The percentage of moisture on a fat-free basis is the mass of water present in the product, divided by the total mass minus the mass of fat (= mass water / total mass - mass of fat). In fresh cheese, protein content is usually lower than 15%, and can be as low as 7% of total weight or even lower.

[0084] In one embodiment, the cheese substitute has the essential features of soft cheese or semi- soft cheese. Soft cheese contains between 67 and 80% moisture, on a fat-free basis, and can be bloomy rind or washed rind. Their protein content is often in the range of 20% of total weight. The soft cheese, bloomy rind has a rich and creamy texture with a slight elasticity in the cheese. The aging process depends on its thickness. This cheese has a mixed coagulation with slow draining, inoculated with specific molds. The soft, washed rind cheese has a rich and creamy texture with a slight elasticity in the cheese. During the aging process, the cheese is turned over regularly and brushed orwashed in brine with beer, mead, wine or spirits. The semi-soft cheeses contain between 62% and 67% moisture, on a fat-free basis. The texture can be soft and creamy. When aging, the cheese can be washed (washed rind) in brine with red smear (with or without alcohol). The cheese can also be brushed and / or develop a natural rind.

[0085] In one embodiment, the cheese substitute has the essential features of firm cheese or hard cheese. Generally, a firm or hard Cheese is pressed to remove as much whey as possible after the curdling process. Firm cheese contains 50% and 62% moisture, on a fat-free basis. Their protein content can be in the range of 30% of total weight. The texture for this cheese is firm and elastic. Among the firm cheeses, you will find some cheeses that are not aged and are milder. Aging for this type of cheese can last from months or years. Hard cheeses have a moisture of less than 50%, on a fat-free basis, and can be aged and stored for several years. They also usually have high protein content (>30%).

[0086] In one embodiment, the cheese substitute is a substitute of a cheese selected from the group consisting of pasta-filata like cheese, paneer, cream cheese and cottage cheese. In one embodiment, the cheese substitute is a mozzarella analogue. In one embodiment, the cheese substitute is a substitute of a cheese selected from the group consisting of Cheddar, swiss, brie, camembert, feta, halloumi, gouda, edam, Cheddar, manchego, swiss, colby, muenster, blue cheese or parmesan. WO 2022 / 058573, which is hereby incorporated by reference, discloses methods for producing such cheese substitutes.

[0087] The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

[0088] Description of the figures

[0089] Figure 1 . Curd quality of the mozzarella analogues in the presence of calcium, as impacted by the recombinant casein variants used.

[0090] Figure 2. Curd quality of the mozzarella analogues in the absence of calcium, as impacted by the recombinant casein variants used.

[0091] Figure 3. Melting performance of the mozzarella analogues comprising calcium, as impacted by the recombinant casein variants used.

[0092] Figure 4. Melting performance of the mozzarella analogues prepared without calcium, as impacted by the recombinant casein variants used.

[0093] Figure 5. Spreadability of the mozzarella analogues prepared with and without calcium, as a function of the ratio between alpha-S1 -casein variants A and B.

[0094] Examples

[0095] 1. Production of alpha-S1 -casein variants A and B by expression in recombinant microbial host cells

[0096] 1.1 Sequences

[0097] Table 1.1. summary of sequences.

[0098] 1 .2 Strain construction

[0099] Plasmids were constructed for the intracellular production of the alpha-S1 -casein within E. coli BL21-AI (Thermo Fisher Scientific). BL21-AI™ is an E. coli B strain derivative and does not contain the Lon protease. It is also deficient in the outer membrane protease OmpT. The lack of these two key proteases reduces degradation of heterologous proteins expressed within the cells. BL21-AI™ cells contain an expression cassette for phage T7 RNA polymerase (T7 RNAP), driven by the arabinose-inducible pBAD (araBAD) promotor. The expression cassette is integrated into the chromosomal araBAD locus, resulting into the deletion of the araB gene. As a result, the expression of the T7 RNAP, and as such also the protein of interest, can be regulated by the sugars L-arabinose and glucose, by varying their concentrations during cultivation. In the absence of glucose, basal expression from the pBAD promotor is generally low, but can be additionally repressed by the addition of glucose. The genotype for strain BL21-AI™ is as follows: F ompT hsdSe (rB_RIB ) gal dem a / 'aB::T7RNAP-tefA. The tetA gene confers resistance to tetracycline and permitted the selection of correct transformants for the pBAD-T7 RNAP expression cassette. The full genome sequence of the strain is available via GenBank accession number CP010816.

[0100] Codon-optimized (for E. coli) synthetic DNA fragments for bovine alpha-S1 variant B (SEQ ID NO: 5) and variant A (SEQ ID NO: 6) were cloned as Ndel / Xhol fragments into pET26b(+), digested with these same enzymes, resulting into pET26b(+)-AlphaS1-casein(B) resp. pET26b(+)- AlphaS1-casein(A). Commercial protein expression plasmids from the pET-series contain a functional expression cassette for the lacl gene, encoding a repressor that will bind on the lac operator which is located downstream of the T7 promotor. As a result, the T7-based expression of the gene of interest (i.e. AlphaSI -casein) will be blocked, despite the presence of L-arabinose into the cultivation medium to drive the expression of the T7 RNAP. Binding of the lacl repressor onto the lac operator can be lifted by the addition of IPTG. However, to prevent the need of adding IPTG during the induction process, the lacl expression cassette was removed from pET26b(+)-AlphaS1- casein(B) and pET26b(+)-AlphaS1-casein(A) by treating the plasmid with Sphl, Fspl and T4 polymerase, followed by a self-ligation. The resulting plasmid lacking the lacl expression cassette was annotated as pT7-AlphaS1-casein(B) resp. pT7-AlphaS1-casein(B) and contains a kanamycin selection marker to allow selection of E. co / / transformants. Plasmid pT7-AlphaS1-casein(B) or pT7-AlphaS1-casein(A) was transformed to BL21-AI™ One Shot chemically competent E. coli cells according to standard procedures, as described in the BL21-AI™ User Manual (Thermo Fisher Scientific). Transformants were selected on LB agar plates containing 30 pg / mL of kanamycin, upon overnight incubation at 37 °C. It is generally assumed that each transformed cell can contain up to 15-20 copies of a pET-based plasmid. A selection of kanamycin-resistant clones was cultivated in a 24 deep-well format to evaluate the intracellular expression of the bovine AlphaSI -casein variant B or variant A. Clones were cultivated overnight at 37 °C and 200 rpm shaking in 2.0 mL standard LB medium (1 % tryptone; 0.5% yeast extract; 1 % NaCI) with 30 pg / mL kanamycin. The next day, these pre-cultivations were used to inoculate new wells (24 deep-well format) containing 2.5 mL LB medium (30 pg / mL kanamycin) at an ODeoo of 0.05. After ± 3 hours of incubation at 30 °C and 200 rpm, the ODeoo of the cultivations reached approximately 0.5 to 1.0 and the induction of the T7 RNAP (and thus the bovine AlphaSI -casein variant B or variant A) was initiated via the addition of arabinose at a final concentration of 0.02%. Samples of the cultivation broth were taken after 3 hours (ODeoo of ± 2) and 20 hours (ODeoo of ± 4) of arabinose induction. Gel loading dye and reducing agent was added to about 2,5.106cells, followed by a heat denaturation step and analysis via SDS-PAGE. Staining of the gel with Instant Blue showed the clear presence of intracellular AlphaSI -casein (either variant B or variant A) at the expected size (in reference towards a sample containing purified AlphaSI - casein from bovine milk). Selected production clones were labeled as Ec0029 (variant B) or Ec0034 (variant A) and used for fermentative production of alpha-S1 -casein variants B and A, respectively.

[0101] 1 .3 Fermentative production and downstream processing

[0102] Alpha-S1 -casein variants A and B were produced fermentation of E. coli strains Ec0029 (variant B) or Ec0034 (variant A), respectively, in a 10L scale bioreactor, essentially as described in WO 2024 / 188832A1 . The alpha-S1 -caseins were extracted from the washed E. coli biomass by thermolysis at 80°C, at pH 2, essentially as described in WO 2024 / 188832A1 . Subsequently, alpha- S1 -caseins were pH-neutralised, desalted and dried.

[0103] 2. Preparation of cheese analogue

[0104] 2.1 Materials & Methods

[0105] 2. 1. 1 Fixation of pH- and Calcium settings for cheesemaking

[0106] The interaction with calcium and the decrease of the pH towards the iso-electric point (pl) both induce the casein clotting and thus, the transition from emulsion to a cheese-like curd. Consequently, the coagulation behavior of alpha-S1 -casein variant A and B, produced by E. coli, was determined in function of their calcium dependency and iso-electric point.

[0107] Purified alpha-S1 -casein variants A and B, produced and purified as described in Example 1 above, was formulated to a 3% casein level by the use of high purity water. For the calcium dependency, a concentration range of calcium chloride was added on top of the separate casein suspensions at neutral pH (~7.5). The optical density at an absorbance of 500 nm (GD500) was logged every minute by the use of a Biotek microplate reader (Agilent Technologies, USA). The sample was shaken for 2 seconds prior to the first measurement and kept at ambient temperature during the full measurement. The optical densities after 0, 30 and 60 minutes were plotted in function of the calcium dosage (mmol calcium per gram casein protein). For cheesemaking, the target calcium is defined as the level at which the casein variants reach their first drop in optical density at 500 nm. This calcium level signifies the start of the calcium-dependent casein precipitation, important for the formation of a cheese-like curd. For the pH-dependency, the isoelectric point of the separate casein variants measured via iso-electric focusing (IEF), performed at a Novex IEF 3-7 and 12% BisTris gel of 1 mm.

[0108] 2. 1.2 Preparation of a dilute milk analogue

[0109] Stock solutions of casein-stabilised o / w emulsions were prepared. First, dried alpha-S1- casein variant A (83% casein in powder) and variant B (90% casein in powder) were suspended separately in high-purity water at a concentration of 3%wt to assure a homogeneous distribution. 10 mL of the suspension was mixed with sunflower oil (Boni, Belgium) at a casein-protein-to-fat ratio of 1 and homogenised at 17.500 rpm for 2 minutes via a rotor-stator homogeniser (T25 ULTRA TURRAX by IKA, Germany) equipped with an S 25 N - 18 G dispersing tool. Then, the emulsions were divided according to the required weight proportions (Table 2.1.2) and shaken vigorously for 1 minute. The emulsified samples were equilibrated at 4°C for 24h and buffered by the addition of 50% of an 8 mM phosphate buffer at pH 5.2. The final samples were re-homogenized by vortexing for 10 seconds.

[0110] Table 2.1 .2. Weight proportions of the 3% casein variants A and B fractions in the base emulsion.

[0111] 2. 1.3 Preparation of a mozzarella analogue

[0112] The final dilution, containing 1.5% wt protein, was divided into two parts. One half was preblended with calcium chloride at the target dosage of 0.33 mmol calcium per gram caseins, before acidification to the pl by the use of 0.1 N HCI. The other half was acidified directly, in order to assess the impact of calcium before clotting on the curd and cheese analogue quality.

[0113] The samples were incubated at 50°C for 30 minutes to enhance curd clotting and syneresis. Then, the curd was separated by static filtration through a cotton cheese cloth. The fresh curd was evaluated based on its visual appearance and its ability to be pressed manually into a cohesive and elastic ball with some firmness, while the final pH was measured on the serum fraction.

[0114] Similar to a classic mozzarella process, the curd ball was stretched at 50 ± 5°C in a 0.5%wt calcium chloride solution. The manual stretching and refolding was applied 3 times resulting in a ball-shaped mozzarella analogue. The mozzarella analogue was evaluated on its visual appearance, its cohesiveness, firmness and elasticity. Finally, the mozzarella analogue was placed into an eppendorf tube of 2 mL and incubated in a hot water bath at 90°C for 30min in a horizontal position (~90° angle, see Figures 3 and 4). The change in morphology indicates the meltability of the curd and was distinguished by two categories: limited to no shape loss (0) -liquefaction / spreading (+1). The change in length per grams of mozzarella analogue (dL / g) indicates the spreadability and was analysed via image analysis in the software program Imaged. During this analysis, the 2 mL tube length was used as a guide for length comparison.

[0115] 2.2 Results

[0116] 2.2. 1 Determination of calcium- and pH-target values for cheesemaking

[0117] For both variants A and B, a calcium concentration dosage of 0.33 mmol calcium per grams of protein was used.

[0118] Based on the lEF-results, both variants A and B appeared to have the same iso-electric point, i.e. pH 5.2.

[0119] Consequently, the calcium and pH-settings summarized in Table 2.2.1 were applied on the level of the diluted milk analogue.

[0120] Table 2.2.1 . pH and calcium levels used during the mozzarella analogue cheesemaking.

[0121] * in mmol Ca2+per gram caseins, corresponding to 0.013 gram Ca2+per gram caseins.

[0122] 2.2.2 Curd quality of the mozzarella analogues, as impacted by the recombinant casein variants used

[0123] Figure 1 reports the curd quality for curds coagulated in the presence of calcium, in terms of curd cohesiveness, firmness and elasticity for fresh curd after coagulation (left-hand panels) and for stretched curd (right-hand panels). Figure 2 reports the curd quality for curds coagulated in the absence of calcium, in terms of curd cohesiveness, firmness and elasticity for fresh curd after coagulation (left-hand panels) and for stretched curd (right-hand panels).

[0124] Figure 1 shows that in the presence of calcium, the substitution of 5 to 50% wt of variant B by variant A in the alpha-S1 -casein fraction enables the formation of a cohesive curd with sufficient elasticity for stretching the clotted milk into a mozzarella analogue. 2.2.3 Melting performance of the mozzarella analogues, as impacted by the recombinant casein variants used

[0125] Figure 3 reports the meltability and spreadability of the mozzarella analogues coagulated in the presence of calcium, as a function of the ratio between alpha-S1 -casein variants A and B. Figure 4 reports the meltability and spreadability of the mozzarella analogues coagulated in the absence of calcium, as a function of the ratio between alpha-S1 -casein variants A and B. Figure 5 compares the spreadability indexes for the mozzarella analogues coagulated in the presence or absence of calcium, as a function of the ratio between alpha-S1 -casein variants A and B.

[0126] Figure 5 shows that in the presence of calcium, the substitution of 5 to 20% wt variant B by variant A in the alpha-S1 -casein fraction increases the spreading of the mozzarella analogue upon heating at 90°C and that in the absence of calcium at least 45 to 50% wt substitution of variant B by variant A is required to obtain at least some spreading upon heating.

[0127] 2.2.4 Conclusions

[0128] The above results show that coagulation in the presence of calcium is preferred for achieving a cohesive curd with sufficient elasticity for stretching the clotted milk into a mozzarella analogue, which cheese analogue also has a visible meltability.

[0129] In the presence of calcium, substituting 5 to 20%wt variant B by variant A as alpha-S1 -casein, leads to a mozzarella analogue with improved cohesiveness and melting as compared to a mozzarella analogue, solely containing variant B alpha-S1 -casein. Without the addition of calcium, at least 45 to 50% wt substitution of variant B by variant A is required to obtain sufficient cohesiveness to allow for at least some spreadability of the mozzarella analogue.

[0130] The cohesiveness is important for easy handling during production, the melting spread is an important factor in the acceptance of consumers for cheese alternatives.

Claims

24Claims1 . A method for producing an edible product, the method comprising: a) emulsifying an aqueous solution comprising alpha-S1 -casein variants A and B of nonanimal origin with an edible fat, to produce an oil-in-water emulsion, wherein the weight ratio of variant A to variant B ranges from 2 : 98 to 80 : 20; b) acidifying the emulsion to a pH that differs less than 0.5 pH unit from the average of the isoelectric points of variant A and variant B; c) incubation to allow curd clotting and syneresis; and, d) separation of the curd and serum fraction to obtain a fresh curd, wherein prior to step b), the emulsion contains or is brought to a concentration of soluble calcium, at the pH set in step b), in the range of 0.20 - 1 .65 mmol Ca2+per gram casein, wherein the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 or the alpha-S1- casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 7, and wherein the alpha-S1 -casein variant B comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with at least one of SEQ ID NO: 1 and 7.

2. A method according to claim 1 , wherein the weight ratio of variant A to variant B is in the range from 5 : 95 to 45 : 55, preferably in the range from 5 : 95 to 40 : 60.

3. A method according to claim 1 , wherein the emulsion contains less than 0.20 mmol Ca2+per gram casein and wherein the weight ratio of variant A to variant B is in the range from 45 : 55 to 50 : 50.

4. A method according to any one of the preceding claims, wherein the alpha-S1 -casein variant A of non-animal origin comprises a deletion of at least 4 amino acids in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 , preferably the alpha-S1 -casein variant A of nonanimal origin comprises a deletion of amino acids 14 - 26 in SEQ ID NO: 1 .

5. A method according to any one of the preceding claims, wherein at least one of the alpha- S1 -casein variants A and B of non-animal origin lacks native post-translational modifications.

6. A method according to any one of the preceding claims, wherein at least one of: i) at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by intracellular expression of a casein comprising an amino acid sequence asdefined in claims 1 - 4, in a microbial host cell, preferably a bacterial host cell, more preferably an Escherichia coli host cell; and, ii) at least one of the alpha-S1 -casein variants A and B of non-animal origin is obtained or obtainable by extracellular expression of a casein comprising an amino acid sequence as defined in claims 1 - 4, in a microbial host cell, preferably a fungal host cell, more preferably a K. phaffii host cell.

7. A method according to any one of the preceding claims, wherein in step a) the combined alpha-S1 -casein variants A and B of non-animal origin and the edible fat are emulsified in a weight ratio on dry matter in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1 .5, more preferably in the range of 0.8 to 1 .2, and most preferably in the range of 0.9 to 1 .1 , or in a weight ratio on dry matter of about 1 .0.

8. A method according to any one of the preceding claims, wherein the edible fat is fat or oil of non-animal origin, preferably an edible vegetable oil, more preferably an edible vegetable oil selected from the group consisting of canola (rapeseed) oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil, safflower oil, soya bean oil, sunflower oil and high-oleic sunflower oil.

9. A method according to any one of the preceding claims, wherein at least one of: a) the concentration of the alpha-S1 -casein variants A and B of non-animal origin in the emulsion is in the range of 30 - 70 weight % on dry matter, preferably 40 - 60 weight % on dry matter, more preferably 45 - 55 weight % on dry matter; b) the emulsion comprises 3 to 65 weight % dry matter; c) the emulsion comprises 1 - 8 mM of a food grade buffer, e.g. a phosphate buffer; d) the incubation in step c) of claim 1 , is at a temperature in the range of 20 - 65 °C, preferably 30 - 60 °C, more preferably 45 - 55 °C, and most preferably 48 - 52 °C or about 50 °C, for at least 15, 20, 25, 30, 40, 50 or 60 minutes; and, e) the separation of the curd and serum fraction in step d) in claim 1 , is performed by filtration, preferably filtration through a cheese cloth.

10. An edible product obtained or obtainable in a method according to any one of claims 1 - 9.

11. An edible product comprising alpha-S1 -casein variants A and B of non-animal origin in a weight ratio of variant A to variant B that ranges from 5 : 95 to 50 : 50, wherein the alpha-S1- casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 1 and comprising a deletion in the region of amino acids positions 14 - 26 of SEQ ID NO: 1 or the alpha-S1 -casein variant A comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 7 and comprising a deletion in the region of amino acids positions 14 - 26of SEQ ID NO: 7, and wherein the alpha-S1 -casein variant B comprises an amino acid sequence having at least 90, 95, 96, 97, 98, 99, or 100% sequence identity with at least one of SEQ ID NO: 1 and 7.

12. An edible product according to claim 11 , wherein the product further comprises at least one of: i) an edible fat, wherein the weight ratio on dry matter between the combined alpha-S1- casein variants A and B of non-animal origin and the edible fat is in the range of 0.5 to 2.0, preferably in the range of 0.75 to 1.5, more preferably in the range of 0.8 to 1.2, and most preferably in the range of 0.9 to 1.1 , or in a weight ratio on dry matter of about 1 .0; and, ii) 0.20 - 1 .65 mmol of a source of Ca2+per gram casein.

13. An edible product according to claim 11 or 12, wherein the ratio of variant A to variant B in weight percentages is in the range from 5 : 95 to 45 : 55 , preferably in the range from 5 : 95 to 40 : 60.

14. An edible product according to any one of claims 11 - 13, wherein the ratio of variant A to variant B in weight percentages is in the range from 45 : 55 to 50 : 50 and wherein the product contains less than 0.20 mmol of a source of Ca2+per gram casein.

15. An edible product according to any one of claims 1 1 - 14, wherein at least one of: i) the alpha-S1 -casein variants A and B of non-animal origin are as defined in claims 4 - 6; ii) the edible fat is an edible vegetable oil, preferably a fat or oil as defined in claim 8; and, iii) the product is a cheese substitute, preferably a Mozzarella analogue.

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