rBLG COMPOSITIONS AND USES THEREOF

Abstract

The invention generally concerns compositions of rBLG having variable gelation properties and uses thereof in products such as food products.

Description

[0001] rBLG COMPOSITIONS AND USES THEREOF

[0002] TECHNOLOGICAL FIELD

[0003] The invention generally contemplates rBLG compositions and uses thereof, particularly in production of food products.

[0004] BACKGROUND

[0005] Beta-lactoglobulin (BLG) is a globular protein of the lipocalin family having a molecular weight of about 18 kDa and comprises 162 amino acid residues, including a relatively high proportion of branched-chain amino acids. While BLG has been proposed in a variety of substitute food products, there is a continuous need for the development of commercially viable methods for the preparation of alternative dairy, egg and meat analogue food products.

[0006] SUMMARY OF THE INVENTION

[0007] Gelation refers to an ability of a material to generate a matrix or a framework stabilized by agents known as “gelation agents”, and characterized by entrapped water molecules. Some proteins have gelation capabilities derived from their unique amino acid sequence and biochemical / biophysical properties. Where gelation is desired, protein gelation may be induced by applying specific conditions such as heat or acid conditions. Desired gel strengths may be achieved by modifying or selecting a specific type of gelation agent and by selecting a specific amount / concentration.

[0008] Most gelation agents are sugar polymers with low nutritional values. Proteins, however, have high nutritional values. Reducing a gel strength by lowering the protein concentration typically results in losing the nutritional value contributed by the protein; while increasing the protein concentration results in strengthening of the gel. However, this solution may be practically limited due to factors such as high cost, organoleptic properties of the application, astringency and others, as known in the art. Therefore, the ability to modulate gelation strength while keeping high nutritional value, on one hand, and maintain excellent organoleptic properties, on the other hand, is desirable to support a variety of applications.

[0009] One of the key limitations in controlling gel characteristics of recombinant P- lactoglobulin (rBLG) is the need to balance functionality with a high nutritional value, especially in food and dietary applications. To address this need, the inventors of the invention disclosed herein have developed a methodology using a class of materials, referred to herein as gelation modifiers, which addition to different rBLG compositions modifies or tunes gelation properties of the rBLG, rendering compositions comprising of rBLG with predetermined properties suitable for use in a vast variety of nutritional products. The gelation modifiers disclosed herein have been selected such that their composition and concentration induce controlled gelation, resulting in rBLG compositions having differing textural or mechanical properties, such as rupture strength, brittleness or viscoelasticity.

[0010] Thus, in a most general aspect, the invention concerns use of a gelation modifier for controlling gelation behavior of rBLG, or for endowing rBLG with modified or desired gelation properties different from those measured for rBLG alone. The type and amount of the gelation modifier may be varied to achieve rBLG gels with distinct mechanical properties while preserving the nutritional profile of the protein.

[0011] Further provided is a composition, in a form of a powder or a suspension, comprising a texturizing agent of the invention (rBLG and at least one gelation modifier), wherein the amount of the at least one gelation modifier is selected to modify a gelation property of the rBLG. The relative amount of each of the protein and gelation modifier may be selected as further disclosed herein.

[0012] Generally, the recombinant protein may be produced through the manipulation of gene expression in an organism by fusing sequences of foreign DNA into a host cell. The altered DNA is then inserted into the host genome, after which it can be replicated, transcribed, and translated to a recombinant protein and further purified. The selection of a suitable expression system and / or host organism depends on the desired production scale and the intended use of the recombinant protein, whereas the choice of vector is largely governed by the host.

[0013] Both cell-based and cell-free technologies are available for expressing recombinant proteins. Cell-based systems can be eukaryotic and prokaryotic systems. Recombinant protein production in mammalian cells can be conducted transiently or through stable cell lines. While stable cell lines can be used reproducibly, transient production offers higher-yields. Recombinant protein production in yeast is also an efficient strategy for producing large amounts of the recombinant proteins with high levels of purity. Proteins expressed have a certain degree of the post-translational processing capacity of mammalian proteins and are more stable than prokaryotic systems. Several yeast species can be used for recombinant protein expression, including Saccharomyces, Schizosaccharomyces pombe, Pichia pastoris and Hansanuela polymorpha. Fungal and bacterial expression systems for producing the recombinant protein are also used because of their simple operation, high expression level, and short culture period. For example, the rBLG in a highly purified form (>85% of total protein) may be produced by fermentation of a fungal strain Aspergillus oryzae.

[0014] In cell-free systems, protein synthesis may be carried out in vitro using translation-compatible extracts of whole cells that contain all the components needed for transcription, translation, and even post-translational modification. With additional supplements of cofactors, proteins of interest can even be formed in a matter of hours.

[0015] In addition to the aforementioned methods of production, the recombinant protein may be obtained commercially (available from a variety of sources such as evitria, ACRObiosymthesis and others).

[0016] The gelation modifier is a material selected to modulate the inherent gelation capabilities of rBLG and as such may be used in any amount and under conditions that generally contribute to achieving desired rheological and mechanical properties, as well as to achieving proper characteristics to support food products of desired attributes. Generally speaking, the gelation modifier is not selected amongst such agents which themselves are gels or which exhibit a degree of gel -like properties. The gelation modifier may be selected amongst materials having one or more carboxylate or one or more phosphate groups. The carboxylate or phosphate-based materials are anions or comprise at least one negatively charged center (i.e., in a form of a carboxylate or a phosphate ion) and may, in some cases, comprise two, three or more such charged centers. A suitable counterion is also present. The nature and number of counterions may change. Typically, the counterion may be selected from mono and divalent cations, such as but not limited to Li, Na, K, Mg, Zn, Ca and others.

[0017] In some embodiments, the counterion is not calcium.

[0018] The use of the class of gelation modifiers enables formation of solid powders or solid compositions which reconstitution in water or incorporation in a food product results in a final product of predesigned gelation properties, including for example rupture strength, brittleness, elasticity, water-holding capacity and others. The aforementioned predesigned properties may be achievable by combining the rBLG with an amount of at least one gelation modifier that is determined to result in a rBLG of a particular set of gelation properties.

[0019] Thus, in a first aspect, the present invention provides a solid composition, a powder or a suspension comprising recombinant beta-lactoglobulin (rBLG), as the sole protein, and at least one gelation modifier, in a combination referred to herein as a texturizing agent, for use in a manufacturing a variety of food products for generating products with proper texture and consistency.

[0020] In some embodiments, the gelation modifier is selected from carboxylate- and phosphate-based materials as defined herein.

[0021] Non-limiting examples of such modifiers include citrate salts, succinate salts, acetate salts, hydrogen phosphate salts and dihydrogen phosphate salts.

[0022] The citrate salts may be a mono-anion form thereof, a di-anion form thereof or a tri-anion form thereof; the succinate salts may be a mono-anion salt or a dianion salt; wherein in each of the aforementioned salts the cation may be any monovalent or divalent cation. The hydrogen or dihydrogen phosphate and acetate salts may similarly comprise at least one cation selected amongst monovalent and divalent cations.

[0023] Non-limiting examples of monovalent and divalent cations include Li, Na, K, Cs, Mg, Zn, Ca and others. In some cases, the cation is different than Ca. The salts may comprise one or more identical cations or a mixture of different cations.

[0024] In some cases, the gelation modifiers may be selected from tri-sodium citrate (TSC), acetate salts of alkali metal acetates (such as sodium and potassium acetates), succinate salts of alkali or alkaline metal succinates (such as sodium, potassium succinates), amino acid salts such as histidine and arginine (carboxylate salts with cations such as sodium and potassium), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di-sodium hydrogen phosphate, sodium dihydrogen phosphate, and others.

[0025] In some embodiments, the gelation modifier is TSC.

[0026] In some embodiments, a texturizing agent or a composition of the invention comprises or consists rBLG and TSC. The invention, thus, further provides a composition comprising rBLG and TSC.

[0027] By modifying gelation properties such as rupture strength, brittleness, elasticity, and water-holding capacity, the gelation modifier enables creation of tailored rBLG compositions for use in the food industry, e.g., in the manufacture of food products; in medicine, e.g., in the manufacture of therapeutic products, gels and other compositions; in the cosmetics industry, e.g., in the manufacture of cosmetic products, gels, lotions, powders, make-up products and others; in the bio-industry, e.g., in the manufacture of biomaterials.

[0028] The relative amounts of the rBLG and of the gelation modifier may vary. In some cases, the weight amount (wt%) of the rBLG in a composition of the invention, e.g., a powder form, may not be less than 35wt%. In some embodiments, the weight amount is at least 51wt%. In some embodiments, the weight amount is between 51 and 99.98wt%.

[0029] In some cases, the rBLG amount is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98 or 99wt% or more, based on the total weight of the composition. In case of a suspension or a solution, the amount may be determined accordingly.

[0030] The ratio amount rBLG:gelation modifier (mol / mol) may range between (1- 20):(0.1-20). In some embodiments, the ratio rBLG:gelation modifier (mol / mol) may be 1 :0.1, 1 :0.5, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 :1, 12: 1, 13: 1....200: 1.

[0031] In some embodiments, the (calculated, mathematical) molar ratio rBLG:gelation modifier (mol / mol) may be between 0.1 and 200, 0.1 and 150, 0.1 and 100, 0.1 and 90, 0.1 and 80, 0.1 and 70, 0.1 and 60, 0.1 and 50, 0.1 and 40, 0.1 and 30, 0.1 and 20, 0.1 and 10, 0.1 and 8, 0.1 and 6, 0.1 and 4, 0.1 and 2, 0.1 and 1 or 0.1, 0.5, 1, 1.5, 2, 2, 5....200. In some embodiments, the (calculated) molar ratio may be between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

[0032] In some embodiments, compositions of the invention are powder compositions. The powder composition may be regarded anhydrous, namely comprising no more than 10wt% water or moisture. Alternatively, the compositions may be provided as solutions, whereby the rBLG and the gelation modifier are solubilized in a liquid medium. In some embodiments, the solution is an aqueous solution. Other forms of the compositions may be prepared and utilized for manufacturing food products of the invention.

[0033] The invention further provides a composition, in a form of a powder or a suspension, comprising a texturizing agent (rBLG and at least one gelation modifier).

[0034] Also provided is a texturizing agent comprising or consisting rBLG and at least one gelation modifier. The texturizing agent comprises an amount of the rBLG and an amount of the at least one gelation modifier that is selected to modify at least one gelation property of the rBLG, when measured alone. The gelation property may be any one or more of rupture strength, brittleness, and elasticity. The texturizing agent may be added to any composition, e.g., used in the food industry, medicinal industry, cosmetic industry and other, and is designated to modify the overall texture or mouthfeel of a final product.

[0035] The invention further provides a composition, in a powder or a solution form, comprising rBLG, at least one gelation modifier, e.g., TSC, and at least one additive. The at least one additive may be selected amongst functional and inert additives. These may include salts, fats, carbonaceous materials, flavoring materials, sweetening agents, coloring materials (lycopene, burned sugar, beetroot powder), fillers, stabilizers, thickeners, antioxidants, proteins (e.g., textured and non-textured vegetable proteins) such as soy or pea protein, amino acids, stabilizing materials, solubilizing agents, pH adjusting agents, bases / acids, chelators, emulsifiers and others. Non-limiting examples of additives include without limitation salts such as calcium phosphate, tri-calcium dicitrate, citric acid, magnesium chloride, calcium carbonate, tricalcium phosphate, calcium chloride, calcium potassium phosphate citrate, calcium hydroxide, phosphoric acid, potassium hydroxide; amino acids such as histidine, arginine, and others; pectin; pH adjusting agents such as glucodelta lactone (GDL); textured proteins such as textured vegetable protein (TVP); cellulose materials such as methyl cellulose; oils such as vegetable oil, coconut oil; lecithin, phospholipids, starch, CMC (carboxymethylcellulose), HPMC (Hydroxypropyl methylcellulose) and others.

[0036] In some embodiments, the additive is trans glutaminase.

[0037] An exemplary composition is detailed in Table 1.

[0038] Table 1: Exemplary composition according to some embodiments of the invention. In Table 1, P-Lactoglobulin was produced via Fermentation of Aspergillus oryzae . Ash includes minerals such as sodium. In some embodiments, compositions of the invention may or may not comprise residual host cell proteins derived from the fermentation process, e.g., derived from Aspergillus.

[0039] Aqueous compositions may be formed by solubilizing a dry composition comprising a texturizing agent, as disclosed herein, in water, optionally in the presence of at least one additive, as disclosed herein. Stable gel compositions may be formed by heat-induced or acid-induced gelation in water under conditions known in the art and exemplified herein.

[0040] The invention further provides a hydrated composition, a water-based solution, a gel or a food product, each of which comprising a combination of rBLG and a gelation modifier, as disclosed herein.

[0041] The invention further provides a composition, a powder composition or a solution of a composition according to Table 1. In some cases, in a composition of Table 1, the gelation modified TSC may be replaced by any of acetate salts of alkali metal acetates, succinate salts of alkali or alkaline metal succinates, amino acid salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di -sodium hydrogen phosphate, or sodium dihydrogen phosphate. Thus, within the scope of the present invention, further provided are compositions according to Table 1, wherein TSC is replaced as aforementioned, wherein each gelation modifier represents an independent embodiment.

[0042] The invention further provides a kit or a commercial package or a commercial food ingredient labeled for manufacturing a food product as disclosed herein, the kit comprising an amount of a composition, in a powder, in a solution form or in any other form, and instructions of use.

[0043] In some embodiments, the kit or commercial package comprises an acceptable composition suitable for manufacturing a predetermined amount of a predefined food product.

[0044] The invention further provides a kit in a form of a therapeutic product or a cosmetic product comprising a composition comprising a texturizing agent according to the invention and instructions of use. The texturizing agent may be provided in a suitable carrier, such as a pharmaceutically acceptable or a cosmetically acceptable carrier.

[0045] Compositions of the invention are designed as components or as food ingredients to food products and other edible materials. Compositions of the invention may be used together with edible compositions to form food products that are safe for human or animal consumption. The food products may be substitute or alternative dairy products, vegetarian products, substitute or alternative egg products, substitute or alternative meat products, substitute or alternative fish products, or may be hybrid products comprising, in some cases, also a protein obtained from a plant or animal source.

[0046] In some embodiments, the food products are substitute dairy products or dairy alternative products. Such products may replace animal-based milk and milk-derived dairy products by mimicking the rheologic and / or organoleptic properties of traditional animal-milk-based products. Milk and milk-derived dairy alternative products include, but are not limited to milk substitute, yogurt substitute, cheese substitute, creams, ice cream substitute, butter substitute, and others.

[0047] In some embodiments, the food product is or comprises egg substitute.

[0048] In some embodiments, the egg substitute comprises trans glutaminase.

[0049] In some embodiments, the food product is a fish substitute.

[0050] In some embodiments, the food product is a meat substitute. The meat substitute is a meat alternative which does not comprise animal material, like animal protein. Typically, the meat substitute may be suitable for consumption by a vegetarian or vegan consumer, optionally after cooking and / or heating. The meat substitute may be a beef- like product, a pork-like product, a lamb-like product or a chicken-like product. The product may be in a form of a meat patty, a hamburger, a nugget, a meat chunk, a meat piece, a meat ball, etc.

[0051] The invention further provides a method for producing a composition according to the invention, the method comprising combining rBLG and at least one gelation modifier under conditions providing a composition suitable for the use in the food industry.

[0052] In some embodiments, the rBLG and the gelation modifier are blended and thereafter dried to provide the powder.

[0053] In some embodiments, each of rBLG and the gelation modifier are provided in a powder form and are mixed to provide the powder.

[0054] In some embodiments, a composition of rBLG and at least one additive in a liquid solution or medium is provided, such that prior to drying of the composition, an amount of the gelation modifier is added. In some embodiments, a composition of rBLG and at least one additive in a liquid solution or medium is provided, to which an amount of the gelation modifier is added.

[0055] In some embodiments, the powder composition is hydrated and maintained at a temperature between 40 and 95°C. In some embodiments, following hydration, the hydrated product is rapidly cooled to a temperature between 1 and 20°C permitting gelation.

[0056] The invention further provides:

[0057] A product comprising a texturizing agent consisting of a recombinant betalactoglobulin (rBLG) and at least one gelation modifier.

[0058] In some configurations of a product according to the invention, the amount of the at least one gelation modifier as a texturing agent is selected to achieve at least one gelation property.

[0059] In some configurations of a product according to the invention, the at least one gelation modifier is a carboxylate or phosphate-based material, having one or more carboxylate or phosphate groups, and one or more corresponding counterions.

[0060] In some configurations of a product according to the invention, the counterion is not calcium.

[0061] In some configurations of a product according to the invention, the at least one gelation modifier is selected amongst citrate salts, succinate salts, acetate salts, hydrogen phosphate salts and dihydrogen phosphate salts.

[0062] In some configurations of a product according to the invention, the at least one gelation modifier is selected from tri-sodium citrate (TSC), histidine salts, arginine salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di-sodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0063] In some configurations of a product according to the invention, the at least one gelation modifier is TSC.

[0064] In some configurations of a product according to the invention, the texturizing agent consists rBLG and TSC.

[0065] In some configurations of a product according to the invention, the product is a food product, a therapeutic gel, a cosmetic product, or a biomaterial.

[0066] In some configurations of a product according to the invention, a weight amount (wt%) of the rBLG in the texturizing agent is between 51 and 99.98wt%. In some configurations of a product according to the invention, a molar ratio amount rBLG:gelation modifier (mol / mol) in the texturing agent is between (l-20):(0.1- 20).

[0067] In some configurations of a product according to the invention, a (calculated) molar ratio rBLG:gelation modifier (mol / mol) in the texturizing agent is between 0.1 and 200.

[0068] In some configurations of a product according to the invention, the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

[0069] In some configurations of a product according to the invention, the product comprising at least one additive selected amongst salts, fats, carbonaceous materials, flavoring materials, sweetening agents, coloring materials, fillers, stabilizers, thickeners, antioxidants, proteins, amino acids, stabilizing materials, solubilizing agents, pH adjusting agents, bases / acids, chelators, and emulsifiers.

[0070] In some configurations of a product according to the invention, the at least one additive is trans glutaminase.

[0071] Also provided is a texturing agent comprising or consisting rBLG and at least one gelation modifier.

[0072] In some configurations of an agent according to the invention, the weight amount or molar amount of the at least one gelation modifier is selected to modify at least one gelation property of the rBLG.

[0073] In some configurations of an agent according to the invention, the at least one gelation modifier is a carboxylate or phosphate-based material, having one or more carboxylate or phosphate groups, and one or more corresponding counterions.

[0074] In some configurations of an agent according to the invention, the counterion is not calcium.

[0075] In some configurations of an agent according to the invention, the at least one gelation modifier is selected amongst citrate salts, succinate salts, acetate salts, hydrogen phosphate salts and dihydrogen phosphate salts.

[0076] In some configurations of an agent according to the invention, the at least one gelation modifier is selected from tri-sodium citrate (TSC), histidine salts, arginine salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di-sodium hydrogen phosphate, and sodium dihydrogen phosphate. In some configurations of an agent according to the invention, the at least one gelation modifier is TSC.

[0077] In some configurations of an agent according to the invention, the agent consists rBLG and TSC.

[0078] Also provided is a texturizing agent consisting rBLG and TSC.

[0079] In some configurations of an agent according to the invention, a weight amount (wt%) of the rBLG is between 35 and 90wt%, or between 51 and 99.98wt%.

[0080] In some configurations of an agent according to the invention, a molar ratio amount rBLG:gelation modifier (mol / mol) is between (l-20):(0.1-20).

[0081] In some configurations of an agent according to the invention, a (calculated) molar ratio rBLG:gelation modifier (mol / mol) is between 0.1 and 200.

[0082] In some configurations of an agent according to the invention, the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

[0083] In some configurations of an agent according to the invention, the agent is in a form or a powder, a gel, suspension or a solution.

[0084] In some configurations of an agent according to the invention, the agent is for use in manufacturing a product for human or animal consumption.

[0085] Also provided is a composition in a form of a powder composition or a solution comprising a texturing agent according to the invention.

[0086] A kit or a commercial package or a commercial food ingredient labeled for manufacturing a food product is provided, the kit comprising an amount of an agent according to the invention or a composition according to the invention and instructions of use.

[0087] Also provided is a substitute or alternative product selected from dairy products, vegetarian products, egg products, meat products, and fish products, said product comprising a texturizing agent according to the invention.

[0088] In some configurations of a product according to the invention, the product is a substitute dairy product or dairy alternative product, selected from a milk substitute, a yogurt substitute, a cheese substitute, a cream substitute, an ice cream substitute, and a butter substitute.

[0089] In some configurations of a product according to the invention, the product is an egg substitute, or a fish substitute, or a meat substitute, or a beef-like product, a pork-like product, a lamb-like product and a chicken-like product. A method is provided for producing a texturing agent according to the invention, the method comprising combining rBLG and an amount of the at least one gelation modifier, wherein the amount is selected to modify a gelation property of the rBLG.

[0090] In some configurations of a method according to the invention, a molar ratio amount rBLG:gelation modifier (mol / mol) is between (l-20):(0.1-20).

[0091] In some configurations of a method according to the invention, a (calculated) molar ratio rBLG:gelation modifier (mol / mol) is between 0.1 and 200.

[0092] In some configurations of a method according to the invention, the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

[0093] In some configurations of a method according to the invention, the gelation property is selected from rupture strength, brittleness, elasticity, and water-holding capacity.

[0094] BRIEF DESCRIPTION OF THE DRAWINGS

[0095] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0096] Figs. 1A-B. The effect of trisodium citrate (TSC) on rapture strength (A) and brittleness (B) of heat induced gels. Texture analyzer data was achieved at compression strain of 30% and speed of 0.5mm / sec

[0097] Figs. 2A-B. The effect of trisodium citrate (TSC) on rapture strength (A) and brittleness (B) of the acid induced gels. Texture analyzer data was achieved at compression strain of 30% and speed of 0.5mm / sec.

[0098] Figs. 3A-B. Alternative meat patties before frying (A) and after frying (B), wherein in each A and B, the patty on the left is a patty accoridng to the invention and the patty on the right is a pea-based patty.

[0099] DETAILED DESCRIPTION OF EMBODIMENTS

[0100] MATERIAL AND METHODS

[0101] Manufacture of rBLG

[0102] Host strain: A. oryzae strain RIB40, a filamentous fungus, is used as a base strain in the construction of the production strain of P-lactoglobulin (strain Ao_st0002). A. oryzae is a Biosafety Level 1 organism, as classified by the American Type Culture Collection, and has a long-history of safe use in the production of food enzymes. The production strain A. oryzae Ao_st0002 was constructed from the recipient host strain A. oryzae Ao_st0044. The recipient host strain A. oryzae Ao_st0044 was constructed from the base strain A. oryzae RIB40 by deletion of the endogenous orotidine-5'- monophosphate decarboxylase gene (pyrG) and LigD. Deletion of pyrG results in an uracil auxotrophic Strain Ao_st0044 as the gene pyrG is an established homolog to URA3 in Saccharomyces cerevisiae. Therefore, uracil was used as a selection marker for Strain Ao_st0044. The A. oryzae strain RIB40 has been deposited in an international culture collection, World Data Centre for Microorganisms (WDCM), as WDCM 139 and was obtained from a previous collection of microorganisms in 2019.

[0103] Construction of the Production Strain: Genetic modification practices that are commonly used and well defined were employed in order to obtain both a pure product and optimize expression of P-lactoglobulin. A. oryzae is frequently used as a production organism because of its characterized secretion capacity, genetic tractability, and fast growth. The Ao_st0002 production strain was constructed by transformation of 2 or more expression cassettes to the recipient strain Ao_st0044. The expression cassette consisted of a nucleic acid sequence of the P-lactoglobulin gene from the domestic cow (Bos taurus) fused to a secretion signal flanked by a promoter and a terminator sequence. The fusion of the secretion signal was designed with the addition of a cleavage site, removing the secretion signal with no trace of the signal or the cleavage site. Thus, the resulting amino acid sequence was identical to the bovine P-lactoglobulin isoform B protein. The nucleic acid sequence of P-lactoglobulin was optimized to induce maximal levels of expression without changing the native amino acid sequence of the protein. The expression cassette also contains the coding sequence of the Aspergillus niger orotidine-5 '-monophosphate decarboxylase gene (pyrA) homolog of the A. oryzae pyrG (GenBank: X96734.2). pyrA is utilized to regain the uracil production used as a selection marker from the pyrG deleted auxotrophic Ao_st0044 strain. In addition, the expression cassette contains a series of well characterized transcriptional elements such as promoters, terminators, and transcription factors to promote the expression of the P-lactoglobulin gene from endogenous elements of non-toxigenic relative species or synthetic sources. No antibiotic selection markers or origin of replication sequences were used in the construction process of the production strain Ao_st0002 or the recipient host strain Ao_st0044. Genomic Stability of the Production Strain: A multi -generational genomic stability study was conducted of the production strain. Genomic stability was assessed by growing the fungi from spores of the production strain on solid media plates until the production of new spores. Spores of Ao_st0002 were collected (representing a new generation of the fungi) and placed on a fresh solid media plate to grow. Genomic DNA was isolated from the spores and copy number of the P-lactoglobulin gene was evaluated by quantitative polymerase chain reaction (qPCR). The copy-number of the P- lactoglobulin gene in the fifth generation was evaluated and compared to the copynumber of the mother strain (generation 0) by the qPCR delta-delta (2-AACT ) method. The results show a consistent copy-number of the P-lactoglobulin gene after 5 generations of the fungi with RQ = 1.109 ± 0.031 (normalized relative quantity), establishing a genetic stability of Ao_st0002 production strain for 5 generations.

[0104] Manufacturing Process: the rBLG was manufactured by precision fermentation of genetically modified species of A. oryzae without expression induction by solvents such as methanol. The fermentation process began with the insertion of spore suspension stock vial seed into the seed fermentation stage to increase biomass amount. At the end of the seed fermentation stage, the culture was transferred to the main fermentation stage to produce P-lactoglobulin. Following the fermentation stage, the biomass was separated from the broth. The harvest then underwent a series of purification steps: pH, conductivity, and temperature adjustment (optional); centrifugation and / or filtration to remove impurities; concentration and dialysis using ultrafiltration / diafiltration; sterile filtration; and spray drying. The final product was a white to off-white to yellowish powder consisting of >60% total protein, of which rBLG was not less than 85%.

[0105] Recombinant BLG Composition

[0106] Recombinant BLG was formulated with tri-sodium citrate, potassium dihydrogen phosphate, di-potassium hydrogen phosphate, calcium phosphate, citric acid anhydrous, magnesium chloride hexahydrate, di-sodium hydrogen phosphate anhydrous, sodium dihydrogen phosphate dihydrate, tri-calcium dicitrate tetrahydrate, acetate, succinate, histidine, arginine, at different concentrations, in water or a water-based medium.

[0107] Typically, the weight amount of the rBLG was at least equal, if not greater to the amount of the other components, i.e., the gelation modifier. Spray Drying

[0108] Powder compositions were formed by spray drying an aqeous rBLG composition at an inlet temperature of between 150 and 200°C and an outlet temperature of between 50 and 90°C.

[0109] Heat induced gelation

[0110] A dry powder composition, as prepared herein, and Gellan Gum (1.5%), as a reference sample, were each separately dissolved in distilled water at different concentrations of trisodium citrate (Table 2). The samples were separately hydrated for 20 minutes followed by 30 minutes incubation at 95 °C in a hot water bath (Faithfull DK- 98-11 A, Cangzhou, Hebei Province, China). The samples were then rapidly cooled in an ice bath and left to set at 2-8°C overnight.

[0111] Table 2: Heat Induced Gelation tested Parameters

[0112] Acid induced Gelation

[0113] A powder composition, as disclosed herein, and Gellan Gum (1.5%), as a reference sample, were each separately dissolved in distilled water in different protein and trisodium citrate concentrations (Table 3). The samples were hydrated for 20 minutes followed by pasteurization in a digital electric stove (Siemenes) for 85°C, 5 minutes and then rapidly cooled in an ice bath. Once the samples reached 42°C, Glucodelta Lactone (GDL) was added to initiate acidification process (Concentration mentioned in Table 3). After 3h all samples were acidified to pH of 4.5-4.6 and left to cool overnight at 2-8°C.

[0114] Table 3: Acid Induced Gelation tested Parameters

[0115] Gelation Properties

[0116] For gel properties analysis, the sample containers were subjected to the texture analyzer TA.XT Plus (Stable Micro Systems, United Kingdom). The samples were placed in the middle of the base, and then compressed with a 7mm dia domical probe. The settings used for this test were single pressing the sample at compression strain of 30% at speed of 0.5mm / sec. Rupture strength / force and brittleness were recorded from this measurement.

[0117] Alternative Meat Patty Preparation Procedure

[0118] Preparation procedure was as follows: Texture vegetable protein (TVP) was mixed with dry ingredients (Table 4) besides methyl cellulose. Water was then added followed by Ih incubation at 2-8°C. Next, the mixture was placed in a mixer (KitchenAid, model 5KSM95) with a guitar hook attached, and mixed on low speed. Vegetable oil was added while mixing. Then, methyl cellulose was added, and mixer speed increased until the mass was cohesive. The mass was then shaped to a uniform burger shape and stored overnight (-20°C). The frozen patty was then fried on the electric stove until the inner temperature of the burger reached 75°C. The burger was left to rest for 2 min and 5 descriptors tasted hot and tasted again after the temperature decreased to 25°C. The recipe is listed in the following Table 4.

[0119] Table 4: Alternative meat patty ingredients

[0120] RESULTS

[0121] Heat / Acid Induced Gelation:

[0122] Heat induced gelation

[0123] Rupture Force is the force that is required to produce a major break / rupture in a sample. It is related to the firmness of the sample. As presented in Figs. 1A, when the concentration of trisodium citrate was increased, the rupture strength increased for 10%, 12% and 14% BLG samples. Gellan gum, however, did not show an increase in rupture force with increased concentration of TSC.

[0124] Brittleness is the tendency of a material to fracture, shatter or fail upon the application of a relatively small amount of force or impact. As presented in Fig. IB, when the concentration of trisodium citrate was increased, the brittleness also increased for 10% and 12% BLG. Gellan gum and 14% BLG , did not show an increase in rupture strength with increased concentration of TSC.

[0125] Acid induced gelation

[0126] Figs. 2A and B demonstrate similar behaviors.

[0127] The results are presented in comparison with those of Gellan Gum gels, which behavior did not depend on the concentration of TSC. As may be understood from the results, at a TSC concentration of between 1 and 10 mM different stable compositions of rBLG could be obtained each having a different consistency.

[0128] Alternative Meat Patty

[0129] Alternative meat patties were produced from a recombinant BLG powder and commercial Pea powder (Fig. 3). Meat alternative tasting was conducted after the patties were off the stove and rested for 2 minutes. Additional tasting was conducted after the temperature decreased to 25°C. After frying, the Pea burger was softer and juicer, while the burger formed with TSC and rBLG was stiffer and a bit drier. After cooling, the pea burger was very soggy and easly deconstructed, while the burger of the invention stayed firm.

Claims

CLAIMS:

1. A product comprising a texturizing agent consisting of a recombinant betalactoglobulin (rBLG) and at least one gelation modifier.

2. The product according to claim 1, wherein the amount of the at least one gelation modifier as a texturing agent is selected to achieve at least one gelation property.

3. The product according to claim 1 or 2, wherein the at least one gelation modifier is a carboxylate or phosphate-based material, having one or more carboxylate or phosphate groups, and one or more corresponding counterions.

4. The product according to claim 3, wherein the counterion is not calcium.

5. The product according to any one of the preceding claims, wherein the at least one gelation modifier is selected amongst citrate salts, succinate salts, acetate salts, hydrogen phosphate salts and dihydrogen phosphate salts.

6. The product according to any one of the preceding claims, wherein the at least one gelation modifier is selected from tri-sodium citrate (TSC), histidine salts, arginine salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di-sodium hydrogen phosphate, and sodium dihydrogen phosphate.

7. The product according to any one of the preceding claims, wherein the at least one gelation modifier is TSC.

8. The product according to claim 1, wherein the texturizing agent consists rBLG and TSC.

9. The product according to any one of the preceding claims, being a food product, a therapeutic gel, a cosmetic product, or a biomaterial.

10. The product according to any one of the preceding claims, wherein a weight amount (wt%) of the rBLG in the texturizing agent is between 51 and 99.98wt%.

11. The product according to any one of the preceding claims, wherein a molar ratio amount rBLG:gelation modifier (mol / mol) in the texturing agent is between (l-20):(0.1- 20).

12. The product according to any one of the preceding claims, wherein a (calculated) molar ratio rBLG:gelation modifier (mol / mol) in the texturizing agent is between 0.1 and 200.

13. The product according to claim 12, wherein the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

14. The product according to any one of the preceding claims, comprising at least one additive selected amongst salts, fats, carbonaceous materials, flavoring materials, sweetening agents, coloring materials, fillers, stabilizers, thickeners, antioxidants, proteins, amino acids, stabilizing materials, solubilizing agents, pH adjusting agents, bases / acids, chelators, and emulsifiers.

15. The product according to claim 14, wherein the at least one additive is trans glutaminase.

16. A texturing agent comprising or consisting rBLG and at least one gelation modifier.

17. The agent according to claim 16, wherein the weight amount or molar amount of the at least one gelation modifier is selected to modify at least one gelation property of the rBLG.

18. The agent according to claim 16 or 17, wherein the at least one gelation modifier is a carboxylate or phosphate-based material, having one or more carboxylate or phosphate groups, and one or more corresponding counterions.

19. The agent according to any one of claims 16 to 18, wherein the counterion is not calcium.

20. The agent according to any one of claims 16 to 18, wherein the at least one gelation modifier is selected amongst citrate salts, succinate salts, acetate salts, hydrogen phosphate salts and dihydrogen phosphate salts.

21. The agent according to any one of claims 16 to 19, wherein the at least one gelation modifier is selected from tri-sodium citrate (TSC), histidine salts, arginine salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, di-sodium hydrogen phosphate, and sodium dihydrogen phosphate.

22. The agent according to any one of claims 16 to 20, wherein the at least one gelation modifier is TSC.

23. The agent according to claim 16, consisting rBLG and TSC.

24. A texturizing agent consisting rBLG and TSC.

25. The agent according to any one of claims 16 to 24, wherein a weight amount (wt%) of the rBLG is between 51 and 99.98wt%.

26. The agent according to any one of claims 16 to 25, wherein a molar ratio amount rBLG:gelation modifier (mol / mol) is between (l-20):(0.1-20).

27. The agent according to any one of claims 16 to 26, wherein a (calculated) molar ratio rBLG:gelation modifier (mol / mol) is between 0.1 and 200.

28. The agent according to claim 27, wherein the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

29. The agent according to any one of claims 16 to 28, in a form or a powder, a gel, suspension or a solution.

30. The texturizing agent according to any one of claims 16 to 28, for use in manufacturing a product for human or animal consumption.

31. A composition in a form of a powder composition or a solution comprising a texturing agent according to any one of claims 16 to 30.

32. A kit or a commercial package or a commercial food ingredient labeled for manufacturing a food product, the kit comprising an amount of an agent according to any one of claims 16 to 30 or a composition according to claim 31, and instructions of use.

33. A substitute or alternative product selected from dairy products, vegetarian products, egg products, meat products, and fish products, said product comprising a texturizing agent according to any one of claims 16 to 30.

34. The product according to claim 33, being a substitute dairy product or dairy alternative product, selected from a milk substitute, a yogurt substitute, a cheese substitute, a cream substitute, an ice cream substitute, and a butter substitute.

35. The product according to claim 33, being an egg substitute.

36. The product according to claim 33, being a fish substitute.

37. The product according to claim 33, being a meat substitute.

38. The product according to claim 37, being selected from a beef-like product, a pork-like product, a lamb-like product and a chicken-like product.

39. A method for producing a texturing agent according to claim 16, the method comprising combining rBLG and an amount of the at least one gelation modifier, wherein the amount is selected to modify a gelation property of the rBLG.

40. The method according to claim 39, wherein a molar ratio amount rBLG:gelation modifier (mol / mol) is between (l-20):(0.1-20).

41. The method according to claim 39 or 40, wherein a (calculated) molar ratio rBLG:gelation modifier (mol / mol) is between 0.1 and 200.

42. The method according to claim 41 , wherein the (calculated) molar ratio is between 0.1 and 4, 0.1 and 3.5, 0.1 and 3, 0.1 and 2.5 or 0.1 and 2.

43. The method according to any one of claims 39 to 42, wherein the gelation property is selected from rupture strength, brittleness, elasticity, and water-holding capacity.

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