Cold gelling recombined beta-lactoglobulins and related food applications

Abstract

The present invention relates to a process for the preparation of cold gelling rBLG (recombinant beta-lactoglobulin), cold gelling rBLG obtained by said process and compositions thereof, and the use of such cold gelling rBLG for the preparation of animal and non-animal dairy products.

Description

Technical Field

[0001] This invention relates to a method for preparing cold-gelling rBLG (recombinant β-lactoglobulin), the cold-gelling rBLG obtained by said method and compositions thereof, and the use of such cold-gelling rBLG in the preparation of non-animal and animal dairy products. Background Technology

[0002] β-lactoglobulin (BLG) is a whey protein found in the milk of many mammals, particularly cow's and sheep's milk. Whey protein is widely recognized as a protein source and contains bioactive components that may be beneficial to human health.

[0003] Beta-lactoglobulin (BLG) is the main whey protein, accounting for more than 40% of total whey protein. Bovine beta-lactoglobulin (BLG) is a protein with 162 amino acid residues and a molecular weight of 18.4 kDa. Due to its beneficial effects on human health, as well as its physical properties and inherent characteristics, BLG has direct significance in the food industry. In fact, the amino acid content of BLG exceeds the nutritional intake recommendations of Food and Agriculture for both children aged 2 to 5 years and adults.

[0004] Various methods have been reported for enriching dairy products from conventional milk proteins and imparting them with texture. In general, whey proteins can be functionalized to improve their textural properties. As known from Mercade-Prieto, R. and Gunasekaran S. (Novel Food Processing: Effects on Rheological and Functional Properties, 2016, pp. 147–186, “Gelation and thickening with globular proteins at low temperatures”), whey proteins are denatured by heat treatment, followed by acid or salt treatment to induce the gelling properties of the resulting functionalized whey proteins.

[0005] Heat treatment of natural whey proteins causes denaturation of their natural secondary and tertiary structures, leading to aggregation and, under certain conditions, the formation of a protein gel. This process is known as thermogelation.

[0006] When the concentration of whey protein is below the critical gel concentration, heating the protein solution leads to the formation of stable and soluble aggregates. These aggregates can then form a protein gel at low temperatures (i.e., below the denaturation temperature). This process, also known as cold gelation, can only occur when the electrostatic repulsion between the aggregates is reduced. This can be achieved by adding a salt, such as calcium, after heat treatment (salt-induced gelation) or by lowering the pH to the isoelectric point (acid-induced gelation).

[0007] Methods for producing denatured whey proteins capable of cold gelation have been reported (US 2008 / 305235, US 5,217,741, WO2006 / 034856, and WO2018 / 011392). In contrast, natural whey proteins do not form gels at room temperature, i.e., at temperatures between 15 and 25°C, and more generally below 70°C, even after the addition of salt or under acidic conditions (pH near the isoelectric point).

[0008] Recombinant whey protein is a substitute for natural whey protein and is currently used in the dairy industry. Specifically, recombinant β-lactoglobulin (rBLG) is used in dairy products to increase the concentration of said β-lactoglobulin (US 5,795,611, US 2019 / 0216106, US 9,924,728, WO2022 / 251263, and WO2022 / 239000). Therefore, rBLG is often used as the sole source of milk protein in non-animal dairy analogue products.

[0009] However, to the applicant's knowledge, the natural form of recombinant β-lactoglobulin BLG (rBLG) does not possess cold gelling properties, which are essential for the production of fresh fermented products such as yogurt or cheese.

[0010] Therefore, there is a need to develop cold-gellable whey proteins, particularly cold-gellable recombinant β-lactoglobulin (rBLG) with the ability to form gels at low temperatures (i.e., below the denaturation temperature, i.e., below 75°C, and preferably below 70°C). Simultaneously, using such whey proteins to increase the concentration of said whey proteins in dairy products, and especially in non-animal dairy products, would be advantageous. Summary of the Invention

[0011] This invention relates to a method for preparing cold-gellable recombinant β-lactoglobulin (rBLG), comprising the following steps:

[0012] a) Provide an aqueous solution of rBLG containing 3% to 15% w / w rBLG and with a pH of 5 to 8, and

[0013] b) Heating the aqueous solution at 65 to 95°C for 1 to 30 minutes to obtain cool-gelling rBLG.

[0014] Percentages are expressed as weight relative to the total weight of the aqueous solution.

[0015] In one embodiment, the temperature of step a) according to the invention is 1 to 60°C.

[0016] In one embodiment, when carried out in batches, step b) according to the invention is performed under stirring at a stirring speed of 1 to 500 rpm.

[0017] In one embodiment, the method of the present invention further includes step c) cooling the heated aqueous solution at a temperature below 60°C.

[0018] In one embodiment, the method of the present invention further includes a step d) of concentrating the heated aqueous solution.

[0019] In one embodiment, the method of the present invention further includes the step of drying the heated aqueous solution.

[0020] In one embodiment, the aqueous solution of step a) contains rBLG and at least one polysaccharide.

[0021] In one embodiment, the polysaccharide / rBLG ratio of the aqueous solution in step a) is 1 / 3 to 1 / 30.

[0022] In one embodiment, rBLG is obtained from fungi, preferably from fungi of the genus Aspergillus.

[0023] Another object of the present invention is the cold-gellable rBLG obtained by the method according to the present invention.

[0024] In one embodiment, the cold-gellable rBLG is in the form of aggregates, which preferably have a particle size of 20 to 500 nm.

[0025] Another object of the present invention is a dry, cold-gellable rBLG composition comprising 45% to 95% w / w cold-gellable rBLG and at least one polysaccharide.

[0026] In one embodiment, the cold-gellable rBLG in the drying composition is in the form of aggregates, which preferably have a particle size of 20 to 500 nm.

[0027] Another object of the present invention is an aqueous solution of cold-gellable rBLG comprising 3% to 15% w / w cold-gellable rBLG, 0.1% to 5% w / w polysaccharide and water, the percentages being expressed relative to the total weight of the aqueous solution.

[0028] Finally, another object of the present invention is the use of cold-gelling rBLG or dried cold-gelling rBLG compositions or aqueous solutions of cold-gelling rBLG for the preparation of non-animal and animal dairy products.

[0029] definition

[0030] The present invention will now be described in more detail. This description is not intended to be a detailed catalogue of all the different ways in which the present invention can be implemented or of all the features that can be added to the present invention. For example, features exemplified with respect to one embodiment may be incorporated into other embodiments, and features exemplified with respect to a particular embodiment may be removed from that embodiment. Furthermore, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art based on this disclosure, and said variations and additions do not depart from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, and not to exhaustively describe all its permutations, combinations, and variations.

[0031] Unless otherwise stated in this document or clearly contradicted by the context, nouns without quantifiers as used herein refer to one / kind and / or more / kinds.

[0032] The range of values ​​recorded by endpoints in this document is intended to include all numbers contained within that range (for example, the recording of 1 to 5 includes 1, 1.25, 1.5, 1.75, 2, 2.45, 2.75, 3, 3.80, 4, 4.32 and 5).

[0033] The term “about” is used explicitly or implicitly herein. Each quantity given herein is intended to refer to an actual given value, and also to an approximation of such a given value based on reasonable deduction by one of ordinary skill in the art, including equivalent and approximate values ​​of such a given value due to experimental and / or measurement conditions. For example, the term “about” in the context of a given value or range refers to a value or range within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0034] As used herein, the expression “and / or” is considered to mean that each of the two specified features or components has or does not have a particular disclosure of the other. For example, “A and / or B” is considered to specifically disclose each of the following: (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein. The term “or” as used herein should generally be interpreted non-exclusively. For example, embodiments of “compositions comprising A or B” generally present aspects of compositions comprising both A and B. However, “or” should be interpreted to exclude those aspects presented that cannot be combined without contradiction (e.g., the composition has a pH of 9 to 10 or 7 to 8).

[0035] The terms “from…to…” and “between…and…” used in this document must be understood to include the stated boundaries.

[0036] In contrast, the expressions “greater than…”, “exceeding…”, “higher than…”, “less than…” and “lower than…” used in this document do not include the stated boundaries.

[0037] The abbreviation "% w / w" means weight / weight percentage. Other abbreviations can be used to define mass percentage, such as "wt%" (weight percentage).

[0038] The term “includes / contains” as used in this document is used in its non-restrictive sense to mean that the items following the word are included, but not excluding items not specifically mentioned.

[0039] The term "native protein" as used in this article refers to a protein in its native state, possessing an intact structure, meaning its structure has not been altered by chemical or physical processes. The native state of a protein is its correctly folded and assembled structure, which imparts stability and allows for functional activity.

[0040] As used herein, the term "recombinant protein" refers to a protein produced by a genetically modified organism that does not produce the protein unless genetically modified. The genetically modified organism, or "host" or "host cell," contains a DNA sequence encoding the protein, which is under the control of regulatory elements that allow the DNA to be transcribed and translated into the recombinant protein.

[0041] According to the technology of the present invention, a genetically modified organism or host or host cell is a DNA sequence encoding β-lactoglobulin (BLG) modified therein, and refers to a cell in which a recombinant nucleotide sequence has been introduced. It should be understood that such terms are intended not only to refer to a specific object cell, but also to the offspring of such cells. Because certain modifications can occur in offspring due to mutations or environmental influences, such offspring may actually differ from the parent cell, but are still included within the scope of the terms "host" or "host cell" as used herein.

[0042] In some preferred embodiments, the BLG can be a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, kangaroo, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth BLG.

[0043] Advantageously, BLG is bovine BLG. The protein sequence of BLG and the DNA sequence encoding said BLG are known and are publicly available at www.uniprot.org / uniprotkb / P0274 / entry.

[0044] As used in this article, "denatured protein" refers to a protein whose chemical structure has been disrupted. Proteins are highly organized structures with primary, secondary, tertiary, and quaternary structures. Any change in the arrangement of the secondary, tertiary, and / or quaternary structures corresponds to protein denaturation, which alters its structure and prevents the protein from performing its originally intended function.

[0045] As used in this article, "functionalized protein" refers to a protein whose properties have been altered through chemical or physical treatment. Methods for functionalizing proteins include chemical treatments such as salt washing, chemical hydrolysis, and precipitation, as well as physical treatments such as heat treatment, high / low pressure application, acoustic treatment, or shearing.

[0046] The term "cold-gellable" protein refers to a protein that has the ability to form a gel at temperatures below its denaturation temperature. As used in this paper, a gel refers to a semi-solid system in which a liquid is dispersed within a solid-like network. This network is capable of trapping liquid, giving the substance properties ranging from soft and fragile to hard and tough, or properties between liquid and solid states. Gels exhibit elasticity (solid-like) and fluidity (liquid-like) depending on the applied force. In the context of protein gels, a gel refers to a three-dimensional network of protein molecules that traps water and other molecules.

[0047] When used to characterize the temperature of a specific step in a method, the expression "room temperature" refers to the temperature within an enclosed space at which the reaction can be carried out without a device to maintain a constant temperature. In some embodiments, room temperature corresponds to a temperature of 15 to 25°C, preferably 18 to 25°C, and more preferably 20 to 25°C.

[0048] When used to characterize the properties of a compound, such as "gelatinizable at room temperature", the expression "room temperature" means 15 to 25°C.

[0049] As used herein, the term "food product" refers to a composition that can be ingested by humans or animals, including domesticated animals (e.g., dogs, cats), farm animals (e.g., cattle, pigs, horses), and wild animals (e.g., undomesticated predators). In several embodiments, the food products provided herein comply with food safety standards required by the U.S. Food and Drug Administration (FDA), the U.S. Department of Agriculture, the European Food Safety Authority, and / or other state or local food regulatory agencies. The term "food product" includes compositions that can be combined with or added to other ingredients to prepare compositions that can be ingested by humans or animals. According to the invention, some examples of food products are dairy products.

[0050] The term "dairy products" as used in this document, also known as "animal dairy products," refers to milk (e.g., whole milk [at least 3.25% milk fat], partially skimmed milk [1% to 2% milk fat], skimmed milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, steamed milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per cup], low-fat yogurt [2 to 5 grams of fat per cup], skimmed yogurt [less than 0.5% milk fat by weight], Greek yogurt [filtered yogurt with whey removed], whipped yogurt, stirred yogurt, goat milk yogurt, Labneh... [labne], sheep's milk yogurt, yogurt drinks [e.g., whole milk kefir, low-fat milk kefir], Lassi), cheese (e.g., whey cheese such as ricotta and mozzarella, semi-soft cheese such as Havarti and Munster, medium-hard cheese such as Swiss and Jarlsberg, hard cheese such as Cheddar and Gouda, soft mature cheese such as Brie and Camembert, cottage cheese, cream cheese, curd), cream (e.g., whipping cream, coffee whitening agent, coffee creamer, sour cream, crème brûlée). Frozen desserts (e.g., ice cream, smoothies, milkshakes, frozen yogurt, sundaes), fruit products, butter, infant formula, weight-loss drinks, nutritional drinks, puddings, buttermilk, milk protein concentrate, whey protein concentrate, whey protein isolate, casein concentrate, casein isolate, skim milk powder, whole milk powder, nutritional supplements, texturizing blends, flavoring blends, or coloring blends. As used herein, the terms "dairy products," "animal dairy products," or even "animal products" refer to products or dairy products that contain components of animal origin, or components of both animal origin and non-animal origin.

[0051] As used in this article, "high-protein dairy products" refers to dairy products rich in protein with a total protein concentration exceeding 5%, or dairy products with a protein concentration higher than that of commercially available products that are not considered high-protein standard products. Examples of such high-protein dairy products include (but are not limited to) high-protein yogurt, high-protein UHT (ultra-heat treatment) beverages, high-protein fruit products, high-protein cream cheese, high-protein snacks, high-protein bites, high-protein cereal bars, high-protein ready-to-drink milk, high-protein dairy beverages, or high-protein drinks.

[0052] As used herein, the term "non-animal derived" refers to components that are not naturally occurring in animal cells (e.g., proteins, lipids, carbohydrates), such as recombinant proteins produced, for example, by a microbial host or plant cells. In some embodiments, the term "non-animal derived" refers to components derived from naturally occurring or modified plants, algae, fungi, or microorganisms. Similarly, as used herein, the terms "non-animal product" and "non-animal dairy product" refer to products or dairy products that do not contain components of animal origin.

[0053] According to the present invention, "non-animal dairy products," also known as "dairy-like products," refer to products that do not contain milk or milk proteins of animal origin. Advantageously, dairy-like products contain milk of vegetable origin, such as plant-based milk, and preferably selected from soy milk, oat milk, almond milk, and coconut milk, or proteins extracted from plants, algae, fungi, or microorganisms.

[0054] As used herein, the term "animal-derived" refers to components produced by animals (e.g., cattle), such as milk proteins. In some embodiments, the term "animal-derived" refers to milk or dairy products produced by mammals. Detailed Implementation

[0055] This invention relates to a method for preparing cold-gellable rBLG, comprising the following steps:

[0056] a) Provide an aqueous solution of rBLG containing 3% to 15% w / w rBLG and with a pH of 5 to 8, and

[0057] b) Heating the rBLG aqueous solution at 65 to 95°C for 1 to 30 minutes to obtain cold-gellable rBLG.

[0058] Percentages are expressed as weight relative to the total weight of the aqueous solution.

[0059] In the specification, the terms “functionalized cold-gelling rBLG”, “functionalized rBLG”, and “cold-gelling rBLG” define an rBLG that has undergone treatment to modify its properties in order to obtain an rBLG with desired gelling properties according to the invention.

[0060] Recombinant β-lactoglobulin (rBLG)

[0061] Recombinant β-lactoglobulin (rBLG) is obtained by: a) culturing a genetically modified organism under conditions suitable for the production and / or secretion of rBLG, and b) isolating the recombinant protein from the culture.

[0062] Methods for generating rBLG are known and disclosed, for example, in Food Research International, 2023, 163, 112131, Mol. Biotechnol. 2016, 58, 10, 605-618 and Protein Eng; 1997, 10, 11, pp. 1339-1345.

[0063] The genetically modified organism or host used to produce rBLG is selected from yeast, bacteria, and fungi.

[0064] In one embodiment, the host is a yeast strain, and specifically yeasts selected from the following genera: Kluyveromyces sp., Pichia sp., Saccharomyces sp., Tetrahymena sp., Yarrowia sp., Hansenula sp., Blastobotrys sp., Candida sp., Zygosaccharomyces sp., Debaryomyces sp., Saccharomyces cerevisiae, and Pichia pastoris.

[0065] In another embodiment, the host is a bacterial cell. Some non-limiting examples of bacterial cells suitable for producing rBLG according to the invention are bacterial cells selected from the following species: *Acetobacter suboxydans*, *Acetobacter xylinum*, *Actinoplanemissouriensis*, *Bacillus cereus*, *Bacillus scoagulans*, *Bacillus licheniformis*, *Bacillus stearothermophilus*, *Bacillus subtilis*, *Escherichia coli*, *Lactobacillus bulgaricus*, *Lactococcus lactis*, *Lactococcus lactis* Lancefield N group, *Leuconostoc citrovorum*, and *Leuconostoc citrovorum*. * *Streptococcus dextranicum*, *Leuconostoc mesenteroides* strain NRRL B-512(F), *Micrococcus lysodeikticus*, *Streptococcus cremoris*, *Streptococcus lactis*, *Streptococcus diacetylactis*, *Streptococcus thermophilus*, *Streptomyces chattanoogensis*, *Streptomyces griseus*, *Streptomyces natalensis*, *Streptomyces olivaceus*, *Streptomyces olivochromogenes*, *Streptomyces rubiginosus*, and *Xanthomonas campestris*. Preferably, the host cell is selected from Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus subtilis. Advantageously, when the host is a bacterial cell, the host cell is Bacillus subtilis.

[0066] However, in another embodiment, the host is selected from fungi, and for example from Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida guilliermondii, Candidalipolytica, Candida pseudotropicalis, Candidautilis, Endothia parasitica, Eremothecium ashbyii, and Fusarium moniliforme.

[0067] In some preferred embodiments, the fungus is selected from filamentous fungi, and preferably belongs to the genera *Aspergillus* and *Trichoderma*. More preferably, the host is selected from species of the genus *Aspergillus*, particularly *Aspergillus niger*, *Aspergillus pumilus* var. *pumilus*, and *Aspergillus oryzae*.

[0068] In one specific implementation, rBLG is isolated from cultures of recombinant filamentous fungi (Microorganisms. 2022, 10, 4, 753 and US 9,924,728), and more specifically, from cultures of recombinant Aspergillus species selected from Aspergillus niger, Aspergillus niger var. avocado, and Aspergillus oryzae.

[0069] The recombinant β-lactoglobulin (rBLG) protein sequence may be identical or similar to BLG sequences from the following animals: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas, or mammoths. "Similar to BLG sequence" should be understood as the rBLG protein sequence having at least 50%, 60%, 70%, 80%, or 90% sequence homology with the stated BLG sequence.

[0070] Alternatively, the recombinant β-lactoglobulin (rBLG) protein sequence may be a fragment or variant having at least 70%, 80%, or 90% sequence identity with BLG sequences from the following animals: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas, or mammoths.

[0071] In some embodiments, recombinant β-lactoglobulin (rBLG) may be a blend of different rBLGs. In this case, the blend of different rBLGs advantageously comprises at least one full-length rBLG sequence identical to that of: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas, or mammoths. According to other embodiments, at least one of the rBLG sequences in the blend is a fragment or variant as defined above. However, in other embodiments, at least one of the rBLG sequences in the blend is modified, truncated, or pruned relative to a full-length rBLG sequence from: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas, or mammoths.

[0072] When recombinant β-lactoglobulin (rBLG) is a blend of different rBLGs, it should be understood that rBLGs can be produced by different genetically modified organisms. However, the genetically modified organisms, although different, can be cultured in the same cell culture medium (co-culture). Alternatively, the genetically modified organisms can be cultured in different media. Preferably, only one genetically modified organism produces the rBLG blend.

[0073] Preparation of Functionalized (Cold-Gelable) Recombinant β-Lactoglobulin (rBLG)

[0074] According to the present invention, the method for preparing cold-gellable rBLG includes several steps.

[0075] Step a)

[0076] The first step (step a) is to provide an aqueous solution of rBLG, the solution containing 3% to 15% w / w rBLG and having a pH of 5 to 8.

[0077] In one embodiment, the aqueous solution according to the invention is obtained by dissolving natural rBLG in water, preferably in purified water or demineralized water.

[0078] Advantageously, the dissolution of natural rBLG is achieved at a temperature of 1 to 60°C, preferably at a temperature of 10 to 50°C, and more preferably at a temperature of 15 to 40°C. In a preferred embodiment, the dissolution of natural rBLG in water is achieved at room temperature, i.e., at a temperature of 15 to 25°C.

[0079] Advantageously, the natural rBLG to be dissolved is purified, dehydrated rBLG. Such rBLG proteins can be obtained by culturing genetically modified organisms and isolating the recombinant protein, or they can be purchased.

[0080] In other embodiments, the aqueous solution of rBLG is a liquid solution obtained from the rBLG purification process after fermentation. In other words, the aqueous solution of rBLG is a liquid solution obtained from a downstream process. Therefore, according to these embodiments, the aqueous solution of rBLG is preferably the residue from the ultrafiltration process, that is, the residue obtained after concentration and percolation.

[0081] The aqueous solution according to the invention contains 3% to 15% w / w rBLG, the percentage being expressed by weight relative to the total weight of the aqueous solution. Those skilled in the art know that the amount of rBLG required to obtain such a concentration is determined based on the purity of the rBLG and the volume of the solution.

[0082] Advantageously, the aqueous solution of rBLG contains at least 1% w / w rBLG, preferably at least 2% w / w, more preferably at least 2.5% w / w, and even more preferably at least 3% w / w rBLG, the percentage being expressed by weight relative to the total weight of the aqueous solution.

[0083] Advantageously, the rBLG content in the aqueous solution is 1% to 20% w / w, preferably 2% to 20% w / w, more preferably 2% to 18% w / w, even more preferably 2% to 17% w / w, even more preferably 2% to 16% w / w, and even more preferably 2% to 15% w / w, the percentage expressed by weight relative to the total weight of the aqueous solution. More preferably, the aqueous solution of rBLG contains 3% to 15% w / w rBLG, preferably 4% to 15% w / w, more preferably 5% to 15% w / w. Even more preferably, the aqueous solution of rBLG contains 6% to 15% w / w rBLG, preferably 6% to 12% w / w, more preferably 6% to 10% w / w, even more preferably 6% to 9% w / w, and even more preferably 6% to 8% w / w rBLG, the percentage expressed by weight relative to the total weight of the aqueous solution.

[0084] Advantageously, the aqueous solution of rBLG contains 3% to 15% w / w, preferably 3% to 12% w / w, more preferably 3% to 10% w / w, preferably 3% to 9% w / w or preferably 3% to 8% w / w, and more preferably 3% to 7% w / w of rBLG, the percentage being expressed by weight relative to the total weight of the aqueous solution.

[0085] Advantageously, the aqueous solution contains about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, or about 9% w / w of rBLG, the percentages being expressed by weight relative to the total weight of the aqueous solution.

[0086] The pH of the aqueous solution is important and must be maintained between 5 and 8.

[0087] In one embodiment, the pH of the aqueous solution is greater than or equal to 5, preferably greater than or equal to 6.

[0088] In one embodiment, the pH of the aqueous solution is less than or equal to 8, preferably less than or equal to 7.

[0089] In one embodiment, the pH of the aqueous solution is 5 to 8, preferably 6 to 8, more preferably 6 to 7, and even more preferably 6.5 to 7. Advantageously, the pH of the aqueous solution containing rBLG is about 6.5, or about 6.6 or about 6.7.

[0090] The pH adjustment of the aqueous solution is performed according to techniques known in the art, particularly by adding an acid or base to the solution. It should be understood that the acid and base can be added in aqueous solution form or as a salt. Some examples of acids and bases that can be used to adjust the pH of an aqueous solution containing rBLG according to the invention are acidic acids, adipic acid, ammonium aluminum sulfate, ammonium bicarbonate, ammonium carbonate, diammonium citrate, ammonium citrate monobasic, ammonium hydroxide, diammonium hydrogen phosphate, ammonium sulfate monobasic, calcium acetate, calcium pyrophosphate, calcium carbonate, calcium chloride, calcium citrate, calcium fumarate, calcium gluconate, calcium hydroxide, calcium lactate, calcium oxide, calcium hydrogen phosphate, calcium dihydrogen phosphate, tricalcium phosphate, calcium sulfate, carbon dioxide, citric acid, cream of tartar. tartar), fumaric acid, gluconic acid, gluconic acid-δ-lactone, hydrochloric acid, lactic acid, magnesium carbonate, magnesium citrate, magnesium fumarate, magnesium hydroxide, magnesium oxide, magnesium phosphate, magnesium sulfate, malic acid, manganese sulfate, metatartaric acid, phosphoric acid, potassium hydrogen tartrate, potassium aluminum sulfate, potassium bicarbonate, potassium carbonate, potassium chloride, potassium citrate, potassium fumarate, potassium hydroxide, potassium lactate, dipotassium hydrogen phosphate, tripotassium phosphate, tetrapotassium pyrophosphate, potassium sulfate, potassium tartrate, potassium tripolyphosphate, sodium acetate, sodium acid pyrophosphate, sodium hydrogen tartrate, sodium aluminum phosphate, sodium aluminum sulfate, sodium bicarbonate, sodium bisulfate, sodium carbonate, sodium citrate, sodium fumarate, sodium gluconate, sodium hexametaphosphate, sodium hydroxide, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate Monobasic), trisodium phosphate, potassium sodium hexametaphosphate, potassium sodium tartrate, potassium sodium tripolyphosphate, tetrasodium pyrophosphate, sodium tripolyphosphate, sulfuric acid, sulfurous acid, and tartaric acid.

[0091] The aqueous solution containing rBLG may also contain one or more polysaccharides. According to the present invention, the term "polysaccharide" refers to a linear or branched polymer composed of monosaccharide units linked by glycosidic bonds. Monosaccharides have the general formula (CH2O). n Where n is the number of monosaccharides, and is at least three. Some examples of monosaccharides are triose, tetrose, pentose, hexose, and heptose. In particular, polysaccharides are polymers of galactose, glucose, and mannose in different proportions. Polysaccharides are preferably polymers of natural origin, and especially produced by plants, fungi, yeasts, or bacteria, and may be modified during or after extraction from the producing organism.

[0092] According to some implementation schemes, aqueous solutions containing rBLG do not contain polysaccharides.

[0093] According to other embodiments, the aqueous solution containing rBLG may also contain at least one polysaccharide.

[0094] When polysaccharides are present, they are advantageously fungal in origin, and preferably isolated from filamentous fungi selected from species of the genera Aspergillus and Trichoderma.

[0095] In another specific embodiment, the polysaccharide is produced by the same organism as the genetically modified organism that produces the rBLG as defined above.

[0096] However, in another specific embodiment, when rBLG is isolated from a culture of a genetically modified organism that produces rBLG as defined above, the polysaccharide and rBLG are obtained as a mixture.

[0097] In a preferred embodiment, the polysaccharide present in an aqueous solution of rBLG is a heteropolysaccharide. Preferably, the heteropolysaccharide comprises galactose and mannose, and after hydrolysis, the heteropolysaccharide is analyzed by spectrophotometry and chromatography. Advantageously, the mannose / galactose ratio in the polysaccharide present in the aqueous solution of rBLG is 0.5 / 1 to 5 / 1, preferably 0.5 / 1 to 4 / 1, more preferably 1 / 1 to 3 / 1, or even more preferably 1 / 1 to 2 / 1, and even more preferably 1.5 / 1 to 2 / 1.

[0098] In another preferred embodiment, when the polysaccharide is present in an aqueous solution of rBLG, it comprises the heteropolysaccharide as defined above, and also comprises glucose. Advantageously, when present, glucose may be present in free form or in the form of β-glucan.

[0099] The glucose content can be determined by spectrophotometry and chromatography, optionally after the hydrolysis of β-glucan. Advantageously, when the polysaccharide is present in an aqueous solution of rBLG, the glucose content in the polysaccharide is less than 30% w / w relative to the total weight of the polysaccharide, preferably less than 25% w / w, and more preferably less than 20% w / w. Preferably, when present, the glucose content is from 0.001% to 20% w / w relative to the total weight of the polysaccharide, preferably from 0.01% to 18% w / w, more preferably from 0.5% to 16% w / w, and even more preferably from 1% to 15% w / w. However, when present, the glucose content in the polysaccharide is from 3% to 15% w / w relative to the total weight of the polysaccharide, more preferably from 5% to 15% w / w, and even more preferably from 8% to 14% w / w.

[0100] The aqueous solution of rBLG may contain rBLG and one or more polysaccharides. Preferably, the polysaccharide content in the aqueous solution is 0 to 50% w / w relative to the dry matter content of the aqueous solution.

[0101] In one embodiment, the polysaccharide content in the aqueous solution is less than 40% w / w relative to the dry matter of the aqueous solution, and preferably less than 30% w / w.

[0102] In one embodiment, the polysaccharide content relative to the dry matter of the aqueous solution is 0 to 30% w / w, 2% to 30% w / w, or 3% to 30% w / w, or 5% to 30% w / w, or 8% to 30% w / w, or 8% to 25% w / w, preferably 9% to 22% w / w, and more preferably 10% to 20% w / w, or 10% to 17% w / w, or 10% to 15% w / w, or 10% to 12% w / w. Advantageously, the polysaccharide content in the rBLG aqueous solution is about 9% w / w, about 10% w / w, about 15% w / w, or about 20% w / w relative to the dry matter of the aqueous solution.

[0103] In one embodiment, the polysaccharide content is less than or equal to 3% w / w relative to the dry matter of the aqueous solution, preferably less than or equal to 2% w / w, and more preferably less than or equal to 1% w / w.

[0104] The polysaccharide content in rBLG aqueous solution can also be determined by the polysaccharide / rBLG ratio.

[0105] In one embodiment, the polysaccharide / rBLG ratio is less than or equal to 1 / 2, and preferably equal to or less than 1 / 3.

[0106] Advantageously, the polysaccharide / rBLG ratio of the aqueous solution containing rBLG and polysaccharide is 1 / 30 to 1 / 2, preferably 1 / 30 to 1 / 3, more preferably 1 / 25 to 1 / 3, even more preferably 1 / 20 to 1 / 3, and even more preferably 1 / 10 to 1 / 3, and even more preferably 1 / 15 to 1 / 3, or preferably 1 / 15 to 1 / 5.

[0107] Advantageously, the polysaccharide / rBLG ratio of the aqueous solution containing rBLG and polysaccharide according to the invention is about 1 / 6, about 1 / 5 or about 1 / 4.

[0108] The aqueous solution of rBLG may also contain sugars, which are different from the polysaccharides defined above. Adding sugars to the aqueous solution of rBLG can improve the controlled aggregation of rBLG during the preparation of functionalized rBLG. Additional sugars are particularly monosaccharides and / or disaccharides. Some non-limiting examples of monosaccharides and disaccharides are sucrose, glucose, fructose, and maltose.

[0109] In one embodiment, the aqueous solution of rBLG contains the sugars as defined above, and preferably the sugar concentration is less than 1% w / w, or even less than 0.5% w / w, the percentage being expressed by weight relative to the total weight of the aqueous solution. In a preferred embodiment, the aqueous solution containing rBLG does not contain any additional sugars other than residual sugars from rBLG isolated from cultures of genetically modified organisms that produce rBLG.

[0110] When present, the amount of additional sugar can be defined by the sugar / rBLG ratio. Preferably, the sugar / rBLG ratio in the aqueous solution of rBLG is less than 1 / 20, more preferably less than 1 / 50, more preferably less than 1 / 75, and even more preferably less than 1 / 100. Advantageously, the sugar / rBLG ratio is less than 1 / 110, less than 1 / 120, or even less than 1 / 130. In a preferred embodiment, the sugar / rBLG ratio is 1 / 120 to 1 / 130.

[0111] The aqueous solution of rBLG may ultimately contain one or more lipids. Such lipids are preferably selected from linoleic acid, oleic acid, stearic acid, and palmitic acid. In one embodiment, the aqueous solution of rBLG contains lipids, and preferably at a concentration of less than 1% w / w, or even less than 0.5% w / w, the percentage expressed as a weight relative to the total weight of the aqueous solution. In one embodiment, the aqueous solution does not contain additional lipids, except for residual lipids from rBLG isolated from cultures of genetically modified organisms that produce rBLG.

[0112] Step b)

[0113] After preparing an aqueous solution containing the rBLG as defined above, a heating step (step b) is performed to obtain functionalized, cold-gellable rBLG.

[0114] The heat treatment is performed at a temperature higher than or equal to the denaturation temperature of the rBLG. Therefore, according to the present invention, the temperature of the heating step (step b) is 65 to 95°C, preferably 70 to 95°C, and more preferably 75 to 95°C.

[0115] In one embodiment, the temperature of the heating step (also known as heat treatment) is higher than or equal to 60°C, preferably higher than or equal to 65°C, more preferably higher than or equal to 70°C, and even more preferably higher than or equal to 71°C.

[0116] In one embodiment, the heat treatment temperature is below or equal to 100°C, and preferably below or equal to 95°C.

[0117] In one embodiment, the heat treatment is performed at a temperature of 72 to 95°C, preferably 75 to 95°C, more preferably 78 to 95°C. In another embodiment, the heat treatment is performed at a temperature of 78 to 92°C, preferably 80 to 92°C, more preferably 80 to 91°C, or even more preferably 81 to 90°C, even more preferably 82 to 88°C, and even more preferably 83 to 86°C. Advantageously, the heat treatment in step b) is performed at a temperature of about 77°C, about 80°C, about 83°C, or about 85°C.

[0118] During heat treatment, the aqueous solution of rBLG is preferably stirred with moderate agitation, and more specifically, when the method is carried out in batches, at a speed of 1 to 500 rpm. In this case, stirring is achieved, for example, by using a rotary blade or a magnetic stirrer. It should be understood that those skilled in the art will know what a suitable stirring method is to obtain the desired speed based on the volume of the container and the aqueous solution.

[0119] In one embodiment, the method according to the invention is performed in batches, and the rotational speed is 10 to 500 rpm, preferably 20 to 500 rpm, more preferably 50 to 500 rpm. In some preferred embodiments, the rotational speed is 50 to 450 rpm, preferably 50 to 400 rpm, more preferably 50 to 350 rpm, even more preferably 100 to 300 rpm, and even more preferably 100 to 250 rpm. Advantageously, when the method is performed in batches, the rotational speed is about 120 rpm, 150 rpm, 170 rpm, or 200 rpm.

[0120] Rotational speed can also be defined as circumferential speed. Circumferential speed is defined by the linear velocity of the propeller at the tip of the blades. According to this embodiment, the circumferential speed is preferably 0.5 to 20 m / s, more preferably 1 to 18 m / s, even more preferably 2 to 15 m / s, even more preferably 5 to 12 m / s, and even more preferably 7 to 10 m / s.

[0121] In step b), the heat treatment of the rBLG aqueous solution according to the present invention lasts for 1 to 30 minutes.

[0122] Advantageously, the heat treatment in step b) is carried out for 1 to 20 minutes, preferably 5 to 30 minutes, more preferably 5 to 20 minutes, and even more preferably 2 to 20 minutes, 3 to 15 minutes, and even more preferably 5 to 15 minutes. Advantageously, the heat treatment of the aqueous solution containing rBLG is for about 5 minutes, about 8 minutes, about 10 minutes, about 12 minutes, or about 15 minutes.

[0123] In some embodiments, the heat treatment can be carried out as a continuous process. This is especially true when processing large quantities of rBLG on a technical scale. According to the invention, technical scale means processing more than 1 kg of rBLG per hour. When the heat treatment is a continuous process, plate heat exchangers and / or tubular heat exchangers are used. The reaction time corresponds to the residence time in the holding tube. Those skilled in the art are familiar with continuous processes and can determine suitable heating equipment and reaction times.

[0124] According to the two-step sequence of the present invention, namely step a) of providing an aqueous solution of rBLG and step b) of heating the aqueous solution, functionalized rBLG or cold-gellable rBLG, also known as functionalized cold-gellable rBLG, is allowed to form.

[0125] Other steps

[0126] The functionalized (cold-gellable rBLG) aqueous solution may further undergo several processing steps, such as a cooling step (step c) and / or a concentration step (step d) and / or a drying step (step e).

[0127] Step c)

[0128] The method for preparing the coolable gelling rBLG according to the present invention may further include an additional step c) of cooling the heated aqueous solution.

[0129] The cooling temperature of the aqueous solution of cold-gellable rBLG is advantageously lower than the denaturation temperature of rBLG, and preferably lower than or equal to 60°C.

[0130] In one embodiment, the heated aqueous solution is cooled to a temperature of 50°C or lower, preferably 45°C or lower, more preferably 40°C or lower, more preferably 35°C or lower, and even more preferably 30°C or lower.

[0131] In one embodiment, the aqueous solution of the cold-gellable rBLG is cooled to a temperature of 1 to 30°C, preferably 1 to 28°C, more preferably 2 to 25°C, even more preferably 5 to 25°C, and still more preferably 5 to 22°C, and even more preferably 5 to 20°C. More preferably, the aqueous solution of the cold-gellable rBLG is cooled to a temperature of 5 to 15°C, preferably 5 to 12°C, and even more preferably 5 to 10°C.

[0132] Step d)

[0133] The method for preparing the cold-gellable rBLG according to the present invention may further include an additional step d of concentrating the heated aqueous solution.

[0134] Heated aqueous solutions containing functionalized, cold-gellable rBLG can also be concentrated. Concentration of the heated aqueous solution is carried out according to methods known in the art. Such methods include centrifugation, dialysis, precipitation or salting out, filtration, or chromatography. Those skilled in the art will be able to select appropriate methods and apply suitable conditions.

[0135] Step e)

[0136] The method according to the invention may further include a drying step to obtain a dried, coolable gelling rBLG.

[0137] Suitable drying methods include, for example, freeze drying, spray drying, and supercritical drying. Those skilled in the art will know how to select the chosen drying method and appropriate parameters to obtain dried, cold-gellable rBLG without altering its properties, and in particular its gelling properties.

[0138] According to the present invention, the steps of cooling (step c), concentrating (step d), and drying (step e) of the aqueous solution containing functionalized (cold-gelling) rBLG are optional and can be performed independently of each other. When performed, steps c), d), and e) are preferably performed after the preparation of the cold-gelling rBLG, i.e., after step b).

[0139] The optional cooling step (c), concentration step (d), and drying step (e) can be performed individually or in combination. Therefore, it should be understood that step c) can be performed before or after step d). Furthermore, step d) can be performed before or after step c).

[0140] Methods of implementing the present invention

[0141] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0142] a) Provide an aqueous solution of rBLG containing 3% to 15% w / w rBLG and with a pH of 5 to 8, and

[0143] b) Heat the aqueous solution at a temperature of 65 to 95°C for 1 to 30 minutes.

[0144] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0145] a) Provide an aqueous solution of rBLG containing 3% to 9% w / w rBLG and with a pH of 5 to 8, and

[0146] b) Heat the aqueous solution at a temperature of 65 to 95°C, preferably 72 to 95°C, for 1 to 30 minutes.

[0147] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0148] a) Provide an aqueous solution of rBLG containing 3% to 15% w / w rBLG, preferably 3% to 9% w / w rBLG, and having a pH of 5 to 8, and

[0149] b) Heat the aqueous solution at a temperature of 75 to 90°C for 1 to 30 minutes, preferably 1 to 20 minutes.

[0150] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0151] a) Provide an aqueous solution of rBLG containing 6% to 15% w / w rBLG, and having a pH of 5 to 8, preferably pH 6 to 7, and

[0152] b) Heat the aqueous solution at a temperature of 72 to 95°C for 1 to 30 minutes.

[0153] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0154] a) Provides an aqueous solution of rBLG containing 3% to 7% w / w rBLG and having a pH of 5 to 8, preferably 6 to 7, and

[0155] b) Heat the aqueous solution at a temperature of 72 to 95°C for 1 to 30 minutes.

[0156] In one embodiment, a method for preparing cold-gellable rBLG includes the following steps:

[0157] a) Provide an aqueous solution of rBLG containing 3% to 7% w / w rBLG and with a pH of 5 to 8, and

[0158] b) Heat the aqueous solution at a temperature of 72 to 95°C for 1 to 30 minutes.

[0159] In one embodiment, the method for preparing cold-gellable rBLG includes only steps a) and b). In another embodiment, the method of the present invention includes steps a), b), and c). In another embodiment, the method of the present invention includes steps a), b), c), and d). However, in another embodiment, the method of the present invention includes steps a), b), c), d), and e). Furthermore, in another embodiment, the method of the present invention includes steps a), b), and d). In another embodiment, the method of the present invention includes steps a), b), and e). However, in another embodiment, the method of the present invention includes steps a), b), c), and e). Furthermore, in another embodiment, the method of the present invention includes steps a), b), d), and e).

[0160] Functionalized (cold-gelatinizable) recombinant β-lactoglobulin (rBLG)

[0161] The method of the present invention as defined herein yields a functionalized, cold-gelling rBLG. Another object of the present invention is to obtain a cold-gelling rBLG according to the present invention.

[0162] Cold-gellable rBLG exists in the form of aggregates. The preferred size of these aggregates is in the nanometer (nm) to micrometer (μm) range. It has been found that the size of the aggregates affects gelation properties, gelation kinetics, and the strength and intrinsic properties of the gel. When the aggregate size is too large, the aggregates become insoluble.

[0163] Preferably, the size of the cold-gellable rBLG aggregates is 20 to 500 nm. In fact, the size of the aggregates affects the gelation properties, gelation kinetics, and the strength and inherent properties of the gel. When the aggregate size is too large, the aggregates become insoluble. Preferably, the particle size of the cold-gellable rBLG aggregates is 20 to 450 nm, more preferably 20 to 400 nm, more preferably 50 to 350 nm, more preferably 50 to 300 nm, more preferably 50 to 250 nm, more preferably 50 to 225 nm, more preferably 50 to 200 nm, more preferably 50 to 175 nm, more preferably 50 to 150 nm, or more preferably 50 to 125 nm.

[0164] Functionalized (cold-gellable) recombinant β-lactoglobulin (rBLG) aqueous solution

[0165] Another object of the present invention is an aqueous solution comprising functionalized cold-gelling rBLG, and preferably one or more polysaccharides. In one specific embodiment, the aqueous solution of functionalized cold-gelling rBLG comprises 3% to 15% w / w cold-gelling rBLG, preferably 3% to 9% w / w cold-gelling rBLG, 0.1% to 5% w / w polysaccharide, and water.

[0166] The aqueous solution of cold-gelling rBLG according to the present invention has a composition similar to that of one of the rBLG aqueous solutions defined above. Therefore, the content of cold-gelling rBLG, polysaccharide, sugar, and lipids in the cold-gelling rBLG solution is the same as that in the rBLG aqueous solution. It should be understood that due to the treatment applied to the rBLG aqueous solution, the percentages of the cold-gelling rBLG, polysaccharide, sugar, and lipid content vary depending on the concentration of the cold-gelling rBLG aqueous solution. The above-described components, contents, and limitations for the rBLG aqueous solution are applicable to the cold-gelling rBLG aqueous solution after necessary modifications.

[0167] However, the ratio between the different components in the cold-gelling rBLG aqueous solution remains unchanged and is therefore the same as that in the rBLG aqueous solution.

[0168] In one embodiment, the polysaccharide / cold-gelling rBLG ratio is less than or equal to 1 / 2, and preferably equal to or less than 1 / 3.

[0169] According to the present invention, the polysaccharide / cold-gelling rBLG ratio of the aqueous solution containing cold-gelling rBLG and polysaccharide is 1 / 30 to 1 / 2, preferably 1 / 30 to 1 / 3, more preferably 1 / 25 to 1 / 3, even more preferably 1 / 20 to 1 / 3, and even more preferably 1 / 10 to 1 / 3, and even more preferably 1 / 15 to 1 / 3, or preferably 1 / 15 to 1 / 5.

[0170] Advantageously, according to the invention, the polysaccharide / cold-gelling rBLG ratio of the aqueous solution comprising cold-gelling rBLG and polysaccharide is about 1 / 6, about 1 / 5, or about 1 / 4.

[0171] According to the invention, the amount of additional sugar, when present, can be defined by the sugar / cold-gelling rBLG ratio. Preferably, the sugar / cold-gelling rBLG ratio in the aqueous solution is less than 1 / 20, more preferably less than 1 / 50, more preferably less than 1 / 75, and even more preferably less than 1 / 100. Advantageously, the sugar / cold-gelling rBLG ratio is less than 1 / 110, less than 1 / 120, or even less than 1 / 130. In a preferred embodiment, the sugar / cold-gelling rBLG ratio is 1 / 120 to 1 / 130.

[0172] Dry, functionalized (cold-gelatinizable) recombinant β-lactoglobulin (rBLG)

[0173] The present invention also relates to dried, functionalized, cold-gelling rBLG obtained according to the method defined above. In particular, the present invention relates to dried compositions comprising cold-gelling rBLG and at least one polysaccharide, as defined above. The compositions of the present invention preferably comprise 45% to 95% w / w of cold-gelling rBLG relative to the total weight of the composition.

[0174] In one embodiment, the dry composition according to the invention comprises, by weight, greater than 45% w / w, greater than 50% w / w, greater than 55% w / w, greater than 60% w / w, greater than 65% w / w, or even greater than 70% w / w of a cold-gelling rBLG relative to the total weight of the composition.

[0175] In one embodiment, the dried, cold-gelling rBLG composition comprises a cold-gelling rBLG content of 45% to 95% w / w, preferably 50% to 95% w / w, more preferably 55% to 95% w / w, even more preferably 60% to 95% w / w, and even more preferably 65% ​​to 95% w / w, and even more preferably 70% to 95% w / w, relative to the total weight of the dried composition. Preferably, the dried composition comprises 70% to 92% w / w rBLG, more preferably 72% to 91% w / w, more preferably 75% to 90% w / w, even more preferably 77% to 90% w / w, and even more preferably 80% to 90% w / w, of cold-gelling rBLG relative to the total weight of the dried composition. In another embodiment, the dried, cold-gelling rBLG composition contains 82% to 90% w / w of cold-gelling rBLG relative to the total weight of the dried, cold-gelling composition, preferably 85% to 90% w / w of cold-gelling rBLG.

[0176] According to the present invention, the dried, cold-gellable rBLG composition further comprises the polysaccharide as defined above. Preferably, the dried, cold-gellable rBLG composition comprises at least one polysaccharide, which is preferably of fungal origin and, in particular, isolated from filamentous fungi selected from the group consisting of species of the genera Aspergillus and Trichoderma.

[0177] When present, and according to the invention, the polysaccharide content in the dried, cold-gellable rBLG composition is 0 to 50% w / w relative to the total weight of the dried composition.

[0178] In one embodiment, the polysaccharide content in the dried, cold-gellable rBLG composition is less than 40% w / w relative to the total weight of the dried composition, and preferably less than 30% w / w.

[0179] In one embodiment, the polysaccharide content relative to the total weight of the dried composition is 0 to 30% w / w, 2% to 30% w / w, or 5% to 30% w / w, or 8% to 30% w / w, or 8% to 25% w / w, preferably 9% to 22% w / w, and more preferably 10% to 20% w / w. Advantageously, the polysaccharide content in the dried, cold-gelling rBLG composition is about 10% w / w, 15% w / w, or 20% w / w relative to the total weight of the dried composition.

[0180] In an alternative approach, when present, the polysaccharide content in the dried, cold-gelling rBLG composition may be limited to a polysaccharide / cold-gelling rBLG ratio.

[0181] In some embodiments, the dried, cold-gellable rBLG composition comprises cold-gellable rBLG and polysaccharide, wherein the polysaccharide / cold-gellable rBLG ratio is 1 / 30 to 1 / 2, preferably 1 / 30 to 1 / 3, preferably 1 / 25 to 1 / 3, preferably 1 / 20 to 1 / 3, or preferably 1 / 10 to 1 / 3, or preferably 1 / 15 to 1 / 3, or preferably 1 / 15 to 1 / 5.

[0182] Advantageously, the polysaccharide / cold-gelling rBLG ratio of the dried, cold-gelling rBLG composition is about 1 / 6, about 1 / 5, or about 1 / 4.

[0183] The dried, cold-gellable rBLG composition according to the invention may further contain sugars as defined above. When present, the dried, cold-gellable rBLG composition contains sugars at a preferably concentration of less than 1% w / w, or even less than 0.5% w / w, the percentage expressed as a weight relative to the total weight of the dried composition. More preferably, the dried, cold-gellable rBLG composition does not contain any additional sugars, except for residual sugars from rBLG isolated from cultures of genetically modified organisms that produce rBLG.

[0184] When present, the amount of additional sugar can also be defined by the sugar / rBLG ratio. Preferably, the sugar / rBLG ratio in the dried, cold-gelling composition is less than 1 / 20, more preferably less than 1 / 50, more preferably less than 1 / 75, and even more preferably less than 1 / 100. Advantageously, the sugar / rBLG ratio is less than 1 / 110, less than 1 / 120, or even less than 1 / 130. In a preferred embodiment, the sugar / rBLG ratio is 1 / 120 to 1 / 130.

[0185] Some lipids may also be present in the dried, cold-gellable rBLG composition according to the invention, the lipids being those defined above. When present, the lipid concentration in the dried, cold-gellable rBLG composition is less than 1% w / w, or even less than 0.5% w / w, the percentage expressed as a percentage relative to the total weight of the dried composition. More preferably, the dried, cold-gellable rBLG composition does not contain additional lipids.

[0186] Preferably, the dried, cold-gellable rBLG is in the form of aggregates, and the particle size of the aggregates is 20 to 500 nm. More preferably, the particle size of the dried, cold-gellable rBLG aggregates is 20 to 450 nm, more preferably 20 to 400 nm, more preferably 50 to 350 nm, more preferably 50 to 300 nm, more preferably 50 to 250 nm, more preferably 50 to 225 nm, more preferably 50 to 200 nm, more preferably 50 to 175 nm, more preferably 50 to 150 nm, or more preferably 50 to 125 nm.

[0187] Uses of Functionalized (Cold-Gelable) Recombinant β-Lactoglobulin (rBLG)

[0188] This invention relates to the use of the cold-gelling rBLG according to the invention as a dried cold-gelling rBLG composition as defined above or as an aqueous solution of cold-gelling rBLG for the preparation of gels. In fact, the cold-gelling rBLG possesses emulsifying and gelling properties, allowing its use in a variety of industries, and particularly the food industry.

[0189] Gel formation from the cold-gellable rBLG according to the invention is performed by: a) dissolving the cold-gellable rBLG in water to form an aqueous solution of functionalized rBLG, and b) gelling the cold-gellable rBLG under conditions that reduce electrostatic repulsion between the aggregates. These conditions can be obtained, for example, by adding a salt, such as calcium, to the cold-gellable rBLG solution (salt-induced gelation) or by lowering the pH to the isoelectric point (acid-induced gelation).

[0190] The dissolution of the dried, cold-gellable rBLG is carried out at a temperature of 1 to 60°C, and preferably at a temperature below 45°C, more preferably at a temperature equal to or below 30°C. Advantageously, the dissolution of the dried, cold-gellable rBLG composition according to the invention is carried out at a temperature of 10 to 30°C, preferably 15 to 30°C, and more preferably 15 to 25°C.

[0191] In some embodiments, the dried, cold-gellable rBLG may be part of a mixture of food ingredients such as starch, conditioning agents (e.g., pectin and carrageenan), vegetable oil, sugar, flavoring agents, or even milk and / or water, optionally pasteurizing, optionally homogenizing, or subjecting the mixture to any other food conversion treatment step.

[0192] After dissolving, and / or mixing, and / or treating the mixture, the solution is acidified to a pH of 4 to 6. Preferably, gelation occurs at a pH of 4.5 to 6, and advantageously at a pH of 4.5 to 5.5, and most preferably at about 4.60 or about 5.2.

[0193] In some preferred embodiments, acidification of the solution of cold-gellable rBLG or a mixture containing components of cold-gellable rBLG is obtained by using an appropriate amount of glucono-delta-lactone (GDL) and can take several hours at a temperature of 1°C to 60°C. In some embodiments, gelation takes place for several minutes or hours. Thus, the gelation step is preferably taken for 1 minute to 72 hours, 5 minutes to 48 hours, or preferably 5 minutes to 24 hours, or 5 minutes to 12 hours. In some embodiments, the gelation step takes place for 10 minutes to 1 hour, preferably 10 minutes to 30 minutes. Advantageously, gelation takes place for 5 minutes to 1 hour, preferably 10 to 30 minutes. The temperature of the gelation step is advantageously 1 to 60°C, preferably 1 to 50°C, and more preferably 5 to 30°C.

[0194] In other preferred embodiments, acidification of a solution of cold-gelling rBLG or a mixture containing components of cold-gelling rBLG is achieved by adding cultured microorganisms such as lactic acid bacteria. Acidification is then carried out for several hours, preferably 1 to 20 hours, at a temperature of 5°C to 55°C.

[0195] However, in some other preferred embodiments, acidification of the cold-gellable rBLG solution or a mixture containing cold-gellable rBLG components is achieved by adding an acid or a food-grade acidifying agent additive (e.g., citric acid or lemon juice concentrate). The solution is then placed at a temperature of 1 to 60°C for several minutes or hours, and particularly for 1 minute to 10 hours, to form an rBLG gel.

[0196] Furthermore, in some other preferred embodiments, and after dissolving the dried, cold-gellable rBLG, and / or mixing the dried, cold-gellable rBLG with other components and / or water, and / or treating the mixture, the solution containing the cold-gellable rBLG, upon addition of an appropriate amount of salt (e.g., calcium), results in the formation of an rBLG gel, the salt being added to the solution preferentially and gradually. Gelation occurs at a temperature of 1 to 60°C and lasts for a period of 1 minute to 10 hours.

[0197] Furthermore, in some other preferred embodiments, after dissolving the dried, cold-gelling rBLG and / or mixing the dried, cold-gelling rBLG with other food ingredients and water, the solution or mixture containing rBLG can be processed into a food product without acidification. Such food products include, for example, ice cream, cream desserts, processed cheeses, hard cheeses, etc.

[0198] This invention also relates to the use of the cold-gelling rBLG according to the invention, in dried form or as an aqueous solution, in the preparation of non-animal dairy products or animal dairy products. This invention is particularly applicable to the preparation of yogurt, cream cheese, fresh cheese, and ice cream.

[0199] Dairy products containing functionalized (cold-gellable) recombinant β-lactoglobulin (rBLG)

[0200] Dairy products containing the cold-gelling rBLG according to the invention advantageously have the texture and mouthfeel of "classic" dairy products, and in some embodiments, they do not contain milk produced by animals. These products may also be referred to as non-animal dairy analogues or non-animal dairy products (or dairy-like products).

[0201] In other embodiments, dairy products containing the cold-gelling rBLG according to the invention may contain animal-derived components. These products are referred to as dairy products or animal dairy products.

[0202] Animal or non-animal dairy products may contain one or more additional components selected from: proteins, texture agents, lipids, flavor compounds, sweeteners, color balancers, ash, vitamins, or any combination thereof. Some embodiments include animal and non-animal dairy products that contain animal-derived components (i.e., animal dairy products), non-animal-derived components (i.e., non-animal dairy products), or combinations thereof (animal dairy products).

[0203] As used in this article, the term "texturing agent" refers to any substance added to a food product to alter its physical properties, particularly its texture and mouthfeel, without significantly affecting its flavor or nutritional value. Texturing agents are widely used in the food industry to achieve desired textures in a variety of food products, stabilize formulations, thicken liquids, and form gels, thereby contributing to the overall sensory experience of the food. They can be derived from a variety of sources, such as plants, animals, and algae.

[0204] The animal or non-animal dairy products of the present invention may further comprise one or more texture agents. Some examples of texture agents are maltodextrin, natural starch, modified starch, cellulose derivatives (e.g., microcrystalline cellulose), carrageenan, xanthan gum, guar gum, locust bean gum, gum arabic, agar-agar, gelatin, pectin, alginate, or plant proteins (e.g., soy protein, pea protein), and mixtures thereof.

[0205] The term "lipid" refers to one or more molecules (e.g., biomolecules) that include fatty acyl groups (e.g., saturated or unsaturated acyl chains). For example, lipids include oils, phospholipids, free fatty acids, monoglycerides, diglycerides, and triglycerides, and mixtures thereof. Some non-limiting examples of lipids are described herein.

[0206] The lipids suitable for animal or non-animal dairy products of the present invention may be selected from sunflower seed oil, coconut oil, tributyric acid esters, monoglycerides and diglycerides, free fatty acids, phospholipids, and mixtures thereof. In some embodiments of any composition described herein, the free fatty acids are selected from the group consisting of butyric acid, caproic acid, caprylic acid, and capric acid, and mixtures thereof. In some embodiments of any composition described herein, the phospholipids are selected from soybean lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, or rapeseed lecithin phospholipids, and mixtures thereof. In some embodiments of any composition described herein, the monoglycerides and diglycerides are plant-derived monoglycerides or diglycerides, or bacterial-derived monoglycerides and diglycerides.

[0207] Lipids can also be plant-derived lipids, that is, lipids obtained from and / or produced by plants (such as monocots or dicots).

[0208] As used herein, the term "flavor" refers to the taste and / or aroma of a food or beverage. According to the present invention, the flavoring compound is preferably selected from: δ-decanolide, ethyl butyrate, 2-furanylmethyl ketone, 2,3-pentanedione, γ-undecanolide, and δ-undecanolide, and mixtures thereof.

[0209] "Sweetener" refers to sugars (e.g., monosaccharides, disaccharides, or polysaccharides) or artificial sweeteners (e.g., small molecule artificial sweeteners or protein artificial sweeteners) that, when added to a composition, make the composition taste sweet when ingested by a mammal (e.g., a human). Some non-limiting examples of sweeteners are described herein.

[0210] Some examples of sweeteners suitable for animal or non-animal dairy products according to the invention are sugars selected from glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose and mixtures thereof. In some embodiments, the sweetener is an artificial sweetener, and is preferably selected from: stevia, aspartame, cyclohexane, saccharin, sucralose, mogroside, brazzein, curculigo, erythritol, glycyrrhizin, inulin, isomaltitol, lactitol, mabinlin, maltitol, mannitol, miraculin, monosaccharide, monotriol, osladin, pentadine, sorbitol, thaumatin, xylitol, acesulfame potassium, advanceame, alitame, aspartame-acesulfame, sodium cyclohexane, glucin, neohesperidin dihydrochalcone, neotame, and P-4000 and mixtures thereof.

[0211] The terms "color balancer" or "colorant" refer to an agent added to a composition to adjust the color of the composition, for example, to make the color of the composition appear more similar to that of mammalian milk. Some non-limiting examples of color balancers or colorants include beta-carotene and annatto. Color balancers or colorants may be produced by or obtained from plants.

[0212] The term "ash" is a term known in the art and refers to one or more ions, elements, minerals, and / or compounds that may be present in mammalian milk. Non-limiting ions, elements, minerals, and compounds present in mammalian milk include, for example, calcium, phosphorus, potassium, sodium, citrates, and chlorides.

[0213] In some embodiments, the animal or non-animal dairy products of the present invention may further comprise one or more types of ash. The ash is preferably selected from minerals. In some embodiments of these dairy products, the minerals are preferably selected from sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, chloride, manganese, selenium, iodine, retinol, carotene, vitamins, vitamin D, vitamin E, vitamin B12, thiamine, and riboflavin, and mixtures thereof. In some embodiments of these animal or non-animal dairy products, the ash is preferably selected from anions. In some embodiments of these animal or non-animal dairy products, the ash is selected from phosphates, citrates, sulfates, carbonates, and chlorides, and mixtures thereof.

[0214] The term "vitamin" refers to an organic molecule (or a group of closely related molecules called vitamin-like molecules) in small amounts that are essential for the normal metabolic functions of an organism. According to the present invention, vitamins that can be used in dairy products are preferably selected from fat-soluble vitamins, water-soluble vitamins, thiamine [vitamin B1], riboflavin [vitamin B2], niacin O [vitamin B3], pantothenic acid [vitamin B5], vitamin B6 [pyridoxine], vitamin B12 [cobalamin], vitamin C, folic acid, vitamin A, vitamin D, vitamin E, vitamin K, and mixtures thereof.

[0215] In some embodiments, the animal or non-animal dairy products according to the invention may also contain fats. The fats include triglycerides and / or high-oleic oils. In some embodiments of these animal or non-animal dairy products, the high-oleic oils are selected from monounsaturated fatty acids, oleic acid, linoleic acid, linolenic acid, and saturated fatty acids and mixtures thereof. In some embodiments of these animal or non-animal dairy products, the fats contain short-chain fatty acids. In some embodiments of these animal or non-animal dairy products, the short-chain fatty acids are selected from butyric acid, caproic acid, caprylic acid, and capric acid and mixtures thereof. In some embodiments of these animal or non-animal dairy products, one or more fats contain trans-esterified fatty acids. In some embodiments of these animal or non-animal dairy products, one or more fats are isolated from plants. In some embodiments of these animal or non-animal dairy products, the plant is selected from one or more of the following: sunflower, corn, olive, soybean, peanut, walnut, almond, sesame, cottonseed, rapeseed, safflower, flaxseed, palm, palm kernel, palm fruit, coconut, babassu, shea butter, mango butter, cocoa butter, wheat germ, and rice bran oil.

[0216] In some embodiments, animal or non-animal dairy products may also contain sugars, including galactose, sucrose, glucose, fructose, and maltose, and mixtures thereof. In some embodiments of these animal or non-animal dairy products, dairy alternative food products are substantially lactose-free.

[0217] According to the present invention, animal or non-animal dairy products containing cold-gellable rBLG preferably have one or more characteristics selected from classic food dairy products: taste, aroma, appearance, handleability, mouthfeel, density, structure, texture, elasticity, resilience, coagulation, binding, fermentation, aeration, foaming, fat-like and emulsification.

[0218] Preferably, the animal or non-animal dairy products according to the invention, i.e., animal or non-animal dairy products containing cold-gelling rBLG, have improved or enhanced properties compared to classic dairy products. In some embodiments, these properties may be, for example, but not limited to, viscosity, foaming effect, cushioning effect, shelf life, opacity, odor, etc.

[0219] In some implementations, cold-gellable rBLG can be processed into high-protein food products. Such high-protein food products include, for example, high-protein yogurt, high-protein UHT (ultra-heat treated) beverages, high-protein fruit products, high-protein cream cheese, high-protein snacks, high-protein treats, high-protein cereal bars, high-protein ready-to-drink milk, high-protein milk beverages, or high-protein drinks.

[0220] In some embodiments, the non-animal dairy product is a vegan dairy product and may subsequently contain one or more of the following: (a) vegetable-derived oil; (b) vegetable-derived starch; (c) sugar; and (d) salt. In some embodiments, the dairy product also contains a flavoring agent selected from cheddar cheese flavoring, parmesan cheese flavoring, or mozzarella cheese flavoring.

[0221] In other embodiments, animal or non-animal dairy products may contain one or more of β-lactoglobulin, serum albumin, lactoferrin, and transferrin, which are of animal or non-animal origin. Preferably, in some embodiments of any of the compositions described herein, β-lactoglobulin is β-lactoglobulin from: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas, or mammoths; serum albumin is serum albumin from: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, Wolves, foxes, lions, tigers, echidnas, or mammoths; lactoferrin is found in the following animals: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, and tigers. Echidna or mammoth; and / or transferrin is the transferrin of the following animals: cattle, humans, sheep, goats, buffalo, camels, horses, donkeys, lemurs, pandas, guinea pigs, squirrels, bears, macaques, gorillas, chimpanzees, mountain goats, monkeys, apes, cats, dogs, wallabies, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orangutans, mandrills, pigs, wolves, foxes, lions, tigers, echidnas or mammoths.

[0222] In some embodiments, animal or non-animal dairy products comprising the cold-gellable rBLG according to the invention further comprise one or more of the following: κ-casein, β-casein, αS1-casein, and αS2-casein of animal or non-animal origin. The κ / β / αS1 / αS2-casein can be from the following sources: bovine, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboon, gibbon, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or mammoth.

[0223] Suitable non-animal proteins can be natural or recombinant non-animal proteins, or hydrolyzed natural or recombinant non-animal proteins, or combinations thereof. Non-animal components can also be derived from non-animal sources, including naturally occurring or modified plants, algae, fungi, or microorganisms.

[0224] Examples of suitable plants include, but are not limited to, seed plants (Seminophyta), terminal gymnosperms (acrogymnospermae), angiosperms (Angiospermae), Ginkgooidae, Pinidae, core angiosperms, cycads, Ginkgo (Ginkgo biloba), conifers, gynostemmas, and more. Biloba, cypress, juniper, golden cypress, cedar, pine, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, immortelle, marigold, artemisia, safflower, chamomile, lettuce, wormwood, calendula, lemongrass, sage, thyme, chia seeds, mustard greens, olives, coffee, chili peppers, eggplant, red chili peppers, cranberries, kiwifruit, vegetable plants (e.g., carrots, celery), marigold, tansy, tarragon, sunflower, holly, basil, hyssop, lavender, lemon verbena, marjoram, melissa, patchouli Pennyroyal, peppermint, rosemary, sesame, spearmint, primrose, winged fruit, pepper, pimento, potato, sweet potato, tomato, blueberry, nightshade plants, petunia, morning glory, clove, jasmine, honeysuckle, snapdragon, plantain, purslane, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, cypselea, chlorella, manila hemp, hazelnut, rapeseed, kale, bok choy choy), turnip, frankincense, myrrh, elemi, hemp, squash, squash, curcurbit, cassava, Dalbergia, legumes (e.g., alfalfa, lentils, common beans, clover, peas, fava coceira, frijole bola roja, frijole) Negroes, Lespedeza, Licorice, Lupins, Mesquite, Carob, Soybeans, Peanuts, Tamarind, Wisteria, Cinnamon, Chickpeas, Chickpeas, Fenugreek, Green Peas, Yellow Peas, Snow Peas, Lima Beans, Broad Beans), Geraniums, Flax, Pomegranates, Cotton, Okra, Neem, Figs, Mulberry, Cloves, Eucalyptus, Tea Trees, Melaleuca leucocephala, Fruit-bearing plants (e.g., Apples, Apricots, Peaches, Plums, Pears, Nectarines), Strawberries, Blackberries, Raspberries, Cherries, Prunes, Roses, Tangerines, Citrus fruits (e.g., Grapefruit, Lemons, Lime, Oranges, Bitter Oranges, Tangerines), Mangoes, Bergamot, Buchu, Grapes, Broccoli, BrusselsSprout, Camelina, Cauliflower, Rapeseed, Canola, Turnip, Cabbage, Cucumber, Watermelon, Honeydew Melon, Zucchini, Birch, Walnut, Cassava, Baobab, Sweet Pepper, Almond, Breadfruit, Sandalwood, Macadamia Nut, Taro, Tuberose, Aloe Vera, Garlic, Onion, Scallion, Herb, Yucca, Vetiver, Galangal, Barley, Corn, Curcuma Aromatica, Ginger, Lemongrass, Oat, Palm, Pineapple, Rice, Rye, Sorghum, Triticale, Turmeric, Dioscorea, Bamboo, Barley, Tea Tree, Canna, Cardamom, Corn, Oat, Wheat, Cinnamon, Sassafras, Lindera benzoin), laurel, avocado, ylang-ylang, nutmeg seed coat, nutmeg, moringa, horsetail, oregano, cilantro, chervil, chives, aggregate fruit, cereals, herbal plants, leafy vegetables, non-cereal legumes, nut-bearing plants, succulents, terrestrial plants, aquatic plants, dalbergia, chestnut, drupes, fissile fruits, flowering plants, non-flowering plants, cultivated plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, thickets, vines, cacti, green algae, tropical plants, subtropical plants, temperate plants, and their derivatives and crosses.

[0225] Some examples of suitable algae include, but are not limited to, green algae (e.g., Chlorella), brown algae (e.g., Alaria marginata, Analipus japonicus, Ascophyllum nodosum, Ecklonia sp., Eisenia bicyclis, Hizikia fusiforme, Kjellmaniella gyrata, Laminaria angustata, Laminaria longirruris, Laminaria Longissima, Laminaria ochotensis, Laminaria claustonia, Laminaria saccharina, Laminaria digitata, Laminaria japonica, Macrocystis pyrifera, Petalonia fascia, Scytosiphon lome), and red algae (e.g.,The following are listed: Gigartinaceae, Solieriaceae, Chondrus crispus, Chondrus ocellatus, Eucheuma cottonii, Eucheumaspinosum, Furcellaria fastigiata, Gracilaria bursa-pastoris, Gracilaria lichenoides, Gloiopeltis furcata, Gigartina acicularis, Gigartina bursa-pastoris, Gigartina pistillata, Gigartina radula, Gigartina skottsbergii, Gigartina stellata, Palmariapalmata, Porphyra columbina, Porphyra crispata, Porhyra *Porhyraperforata*, *Porhyra suborbiculata*, *Porphyra tenera*, *Porphyridium cruentum*, *Porphyridium purpureum*, *Porphyridium aerugineum*, *Rhodella maculate*, *Rhodella reticulata*, *Rhodella violacea*, Rhodophyceae, *Rhodymenia palmata*, and their derivatives and hybrids.

[0226] Examples of suitable fungi include, but are not limited to, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus niger* var. *amomori*, *Aspergillus oryzae*, *Candida albicans*, *Candida etchellsii*, *Candida humilis*, *Candida lipolytica*, *Candida tropicalis*, *Candida utilis*, *Candida versatilis*, *Chrysosporium lucknowense*, *Debaryomyces hansenii*, *Eremothecium ashbyii*, *Fusarium* sp., *Fusarium gramineum*, *Fusarium moniliforme*, *Fusarium venenatum*, and *Hansenula polymorpha*. Kluyveromyces polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces marxianus var. lactis, Kluyveromyces thermolones, Mortierella vinaceae var. raffinoseutilizer, Mucor miehei, and Mucormiehei var. Cooney et Emerson.Cooney et Emerson), Mucor pusillus Lindt Myceliophthorathermophile, Neurospora crassa, Penicillium roquefortii, Physcomitrella patens, Pichia, Pichia finlandica, Pichia trehalophila, Pichia kodakoclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijeppier Pichia stiptis, Pichia methanolica, Rhizopus niveus, and Rhodotorula spp.), genus *Saccharomyces*, *Saccharomyces bayanus*, *Saccharomyces beticus*, *Saccharomyces chevalieri*, *Saccharomyces diastaticus*, *Saccharomyces ellipsoideus*, *Saccharomyces exiguus*, *Saccharomyces florentinus*, *Saccharomyces fragilis*, *Saccharomyces pastorianus*, *Saccharomyces pombe*, *Saccharomyces sake*, *Saccharomyces uvarum*, *Sporidiobolus johnsonii*, *Sporidiobolus salmonicolor*, *Sporobolomyces roseus*, genus *Trichoderma*, *Trichoderma reesei* *Reesei*, *Xanthophyllomyces dendrorhous*, *Yarrowia lipolytica*, *Zygosaccharomyces rouxii*, and their derivatives and hybrids.

[0227] Examples of suitable microorganisms include, but are not limited to, firmicutes, cyanobacteria (blue-green algae), oscillatoriophcideae, bacillales, lactobacillales, oscillatoriales, bacillaceae, lactobacilli, weakly oxidizing acetic acid bacteria, xylitol, *Actinomyces milsuri*, *Arthrospira platensis*, *Arthrospira maxima*, *Bacillus cereus*, *Bacillus coagulans*, *Bacillus subtilus*, *Bacillus cerus*, *Bacillus licheniformis*, thermophilic steatobacterium, *Bacillus subtilis*, *Escherichia coli*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, *Lactococcus lactis*, *Lactococcus lactis* group N, and *Lactobacillus reuteri*. Leuconostoc reuteri, Leuconostoc citrate, Leuconostoc glucanum, Leuconostoc enterica strain NRRL B-512(F), Micrococcus lysate, Spirulina, Streptococcus lactis, Streptococcus lactis, Streptococcus lactis subspecies diacetyl lactic acid streptococcus, Streptococcus thermophilus, Streptococcus chatanuca, Streptococcus grayi, Streptococcus natalus, Streptococcus olivei, Streptococcus olivei, Streptococcus rust-brown, Tetrahymena thermophila, Tetrahymena hegewischi, Tetrahymena ayperangularis, Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena apyriformis and Tetrahymena vorax, and Xanthomonas violaceus, and their derivatives and hybrids.

[0228] Attached text

[0229] Figure 1 HPLC analysis of rBLG samples compared to animal BLG (WPI, whey protein isolated from milk).

[0230] Figure 2 TEM images at different magnifications show soluble aggregates of cold-gelled recombinant BLG produced according to conditions J listed in Table 2.

[0231] Figure 3Texture profiles during acid gelation analysis of cold-gellable rBLG protein: Hardness (g), consistency (g·s), and stickiness (g) were measured. Cold-gellable rBLG protein was produced according to condition J (Table 2).

[0232] Figure 4 Gel filtration chromatograms obtained for commercial natural bovine BLG (A), recombinant BLG (B), and thermally induced cold-gellable rBLG (C) according to the present invention.

[0233] Figure 5 Size distribution of recombinant BLG by volume (comparison).

[0234] Figure 6 Size distribution of cold-gelled recombinant BLG by volume (this invention).

[0235] Example

[0236] The present invention is further illustrated in a non-limiting manner through the following embodiments.

[0237] The rBLG used in the examples is rBLG obtained through precise fermentation and has the following composition (Table 1).

[0238]

[0239] Table 1: rBLG composition. "Total carbohydrates" corresponds to polysaccharides.

[0240] Purity was analyzed by HPLC and compared with WPI (bovine whey protein isolate) used as a reference. The analyzed chromatograms are shown in [the table / image]. Figure 1 The information is provided in the text.

[0241] The reference (WPI) chromatogram showed two peaks: one between 10 and 21 kDa, corresponding to α-lactalbumin, and another between 20 and 60 kDa, corresponding to dimeric β-lactoglobulin. The native rBLG sample showed a single main peak, corresponding to dimeric β-lactoglobulin. The rBLG sample can be considered pure β-lactoglobulin.

[0242] Example 1: Production of soluble aggregates that can be cold-gelled from rBLG and WPI

[0243] General Procedure

[0244] WPI (whey protein isolate) (refer to PRONATIV 95LL, Lactalis, France) contains 91% w / w protein and is produced by filtering cow's milk.

[0245] rBLG and WPI protein powders were rehydrated in demineralized water at room temperature to produce 5% and 7% w / w protein solutions. The pH of the solutions was 6.7 and remained constant. The production of cold-gellable soluble aggregates was then carried out under the conditions shown in Table 2 (conditions A to L). The solutions of WPI or rBLG at 5% or 7% w / w protein concentration were heated at 80°C with moderate stirring (150 rpm) for 5, 10, or 15 minutes. Finally, the heated protein solutions were rapidly cooled to 27°C.

[0246]

[0247] Table 2: Conditions for producing cold-gellable soluble aggregates from WPI and rBLG.

[0248] result

[0249] For WPI (bovine whey protein), all test conditions (A to F, Table 2) resulted in the formation of soluble protein aggregates, and the solution did not flocculate or gel during the heating, cooling and storage at room temperature, as assessed by visual observation.

[0250] The same results were obtained using rBLG. The test conditions (G to L, Table 2) all resulted in the formation of soluble protein aggregates, and the solution did not flocculate or gel, as assessed by visual observation, during the heating step, cooling step, and storage at room temperature.

[0251] Example 2: Microscopic observation (TEM) of soluble aggregates that can be cold-gelled from rBLG

[0252] The solutions of soluble aggregates that could be cold-gelled by rBLG under the conditions J shown in Table 2 were characterized by transmission electron microscopy (TEM). Figure 2 Sample preparation for TEM was as follows: a 200-mesh copper grid coated with a polymethyl methacrylate (formvar) / carbon film was hydrophilized; a ×10 diluted sample was deposited on the grid surface; the residue was absorbed after 1 minute (surplus); negative contrast was applied for 30 seconds with 2% sodium silicotungsticate; observation was performed using a TEM JEOL 1400 and images were acquired using a Gatan RIO 16 camera.

[0253] Figure 2 This indicates the presence of protein aggregates, which are small, curved chains with a size of approximately 20 to 200 nm.

[0254] Example 3: Gel formation from cold-gellable proteins

[0255] General Procedure

[0256] The following procedures were performed on solution samples of cold-gellable proteins generated under the conditions listed in Table 2 to evaluate their ability to form gels at acidic pH at near room temperature (27°C) (i.e., cold gelation):

[0257] All solutions were set at a 5% w / w protein concentration and diluted with demineralized water as necessary.

[0258] GDL (glucono-δ-lactone) (0.75% w / w) was added to a 5% w / w solution of cold-gellable proteins, which was then allowed to gel statically in a steam chamber at 27°C (without stirring) until pH 4.50 was reached, followed by storage at 5°C for 24 hours. Gel properties were then measured using a TAX t Plus texture analyzer (Stable Microsystems) with a cylindrical probe P / 5 (5 mm diameter). Measurement conditions were as follows: compression step up to 15 mm at 0.5 mm / s; then probe return to the surface (15 mm; 1 mm / s). Hardness was considered to be the maximum force (g) during compression. The area under the curve represents the consistency (g·s) during compression (consistency), and the maximum negative peak during probe return to the surface corresponds to viscosity. Figure 3 An example of such measurement was reported in the article, involving a solution of 5% w / w cold-gellable rBLG after acidification (produced under conditions J in Table 2).

[0259] result

[0260]

[0261] Table 3: Effect of different generation conditions (A to L) of cold-gellable protein aggregates on the physical properties of acid gel strength (5% protein).

[0262] Under all test conditions (A to F: initial protein concentration 5% to 7%, holding time at 80°C for 5 to 15 minutes, pH 6.7), the protein solution of cold-gelatinizable bovine WPI (5% w / ) produced during the heating step formed a strong gel after acidification to pH 4.5 at 27°C. The hardness of the acid gel varied from 23.6 g to 60.1 g with varying conditions, and the consistency varied from 456 to 1044 g·s. The viscosity varied from 5.6 to 16.3 g.

[0263] Under all test conditions (G to L: initial protein concentrations of 5% and 7%, holding times at 80°C for 5 to 15 minutes, pH 6.7), protein solutions of cold-gel-coagulating aggregates of rBLG (5% w / w) generated during the heating step, after acidification to pH 4.5 at 27°C, formed even stronger gels than cold-gel-coagulating aggregates of WPI generated under the same conditions. The hardness of the acid gel varied with holding time and initial protein concentration from 24.1 g to 315.9 g, and the consistency varied from 530.9 to 5719.8 g·s. Viscosity varied from 5.4 to 50.1 g.

[0264] The strength of acid gels produced by cold-gelling rBLG is much higher than that produced by cold-gelling borosilicate (WPI), indicating a much higher cold-gelling capability.

[0265] The results showed that it is feasible to use rBLG as a new raw material to generate cold-gellable protein aggregates, and its gelling ability is much higher than that of animal WPI.

[0266] Example 4: Size of the aggregate

[0267] General Procedure

[0268] Aggregate identification and conversion rate

[0269] Commercial natural bovine BLG, recombinant BLG (rBLG), and heat-induced cold-gelling rBLG (cBLG) as defined in Condition I (Example 3) of Table 2 from Sigma Aldrich (St. Louis, USA) were analyzed by Sephacryl® S-300 gel filtration chromatography (Cytiva) to obtain the conversion of rBLG to cold-gelling rBLG (cBLB) after heat treatment. All 0.5% w / w solutions were centrifuged at 14000 g over 10 minutes, and 450 mL of the supernatant was injected into the chromatogram (AKTA system). Peak height was used for conversion estimation.

[0270] Size distribution

[0271] Dynamic light scanning (DLS) analysis was performed on the Nanosizer Malvern at 20 °C. After centrifugation, 200 µL of sample was transferred to the UV cell. After equilibration in the UV cell, two runs of 10 replicates were performed. Polydispersity data processing was performed using DTS Nano software.

[0272] result

[0273] Commercial natural BLG bovine (A) and recombinant BLG (B) had the same elution volume (at approximately 75 mL). Figure 4 After heat treatment, approximately 90% of the rBLG was converted into the high molecular weight aggregates identified in chromatogram (C), corresponding to heat-induced cold-gellable rBLG (C), with an elution volume of 30 to 50 mL.

[0274] The conversion rate was estimated by considering the absorbance at approximately 75 mL in the thermally induced, cold-gelling rBLG solution (cBLG). The estimated conversion rate of rBLG to cBLG was calculated as the percentage ratio (%) between the absorbance (in mAu) of 0.5% w / w cBLG solution and 0.5% w / w rBLG solution at a 75 mL elution volume. The result was 90.5%, demonstrating an efficient method for producing cold-gelling rBLG from rBLG. The values ​​are shown in Table 4.

[0275]

[0276] Table 4: Absorbance measured at the elution peak of rBLG and cBLG solutions, and estimation of the conversion rate of rBLG to cBLG.

[0277] Dynamic light scanning (DLS) analysis showed that the rBLG solution contained non-aggregate particles with sizes ranging from 3.7 to 6.6 nm. Figure 5 This corresponds to the size of globular proteins in their native state. Two batches were analyzed. DLS analysis of the cold-gellable rBLG solution revealed the presence of aggregates ranging in size from 23 to 300 nm (average 82 nm). Figure 6 ).

[0278] Example 5: Gelation ability of rBLG according to the present invention relative to natural BLG

[0279] A) Based on the gelling ability of rBLG in existing technologies

[0280] The rBLG aqueous solution (9.5% w / w) according to the method disclosed in Food Hydrocolloids 2005, pages 269 to 278, was prepared and functionalized.

[0281] Powdered recombinant BLG (rBLG) was added to deionized water to obtain an aqueous solution with an rBLG concentration of 9.5% w / w as defined in this application. The solution was stirred for 1 hour, and the pH was adjusted to pH 7 with HCl or NaOH, followed by degassing.

[0282] The obtained solution was functionalized by heating at 85°C for 45 minutes in a water bath or in an MCR92 rheometer with CC10 coaxial geometry, and then cooled to 23°C for 2 hours.

[0283] Gelation of the functionalized rBLG was observed during heating at 85°C, and a self-supporting gel was obtained during the heating step. Therefore, this functionalization step via heating does not lead to cold-gellable rBLG aggregates in solution, but directly results in thermal gelation. No further cold-gelation step can be performed. In other words, cold-gellable rBLG cannot be obtained from this process.

[0284] B) Gelation properties of commercial natural bovine BLG and rBLG after functionalization were measured using a texture analyzer.

[0285] General Procedure

[0286] Aqueous solutions of commercial natural bovine BLG (BLG) from Sigma Aldrich, rBLG according to the invention, whey protein isolate (WPI), and milk protein concentrate have been prepared and functionalized to produce a cold-gellable protein solution.

[0287] Powdered BLG, rBLG, WPI, and milk protein concentrate were added to deionized water to produce 5% and 7% w / w protein solutions. The solutions were stirred for 10 minutes, and the pH was adjusted to 6.7 with HCl (1 N) or NaOH (1 N). The solutions were allowed to stand for 10 minutes. The solutions were then heated on a hot plate at 80°C for 5, 10, or 15 minutes (except for the milk protein concentrate, which was heated only for 15 minutes), and cooled to 27°C in an ice bath. After the heating and cooling steps, the solution containing 7% w / w protein was diluted with demineralized water to obtain a 5% w / w protein solution.

[0288] Glucono-δ-lactone (GDL) was added to the functionalized protein solution (GDL powder was added to achieve 1% w / w in solution). The resulting solution was stirred to dissolve the GDL, and then the solution was acidified in a steam chamber at 27°C without stirring until pH 4.50 was reached, and then stored at 5°C for 24 hours.

[0289] analyze

[0290] Gel properties were measured using a TAX t Plus texture analyzer (Stable Microsystems) with a cylindrical probe P / 5 (5 mm diameter). Measurement conditions were as follows: compression step up to 15 mm at 0.5 mm / s; then probe return to the surface (15 mm; 1 mm / s). Hardness was considered to be the maximum force (g) during compression. The area under the curve represents the consistency (g·s) during compression (consistency), and the maximum negative peak during probe return to the surface corresponds to viscosity. The analysis temperature was 5 °C.

[0291] result

[0292] Functionalized BLG and rBLG solutions were acidified to evaluate their cold gelling properties. Under all test conditions, the functionalized solutions (5% and 7% w / w BLG and rBLG, treated at 80°C for 5, 10, or 15 minutes) produced gels after acidification. However, the acid gel obtained from BLG was slightly more translucent than the acid gel obtained from the cold-gellable rBLG.

[0293] The textural properties of the acid gel were investigated, and the results are shown in Table 5.

[0294] Under all functionalization conditions tested, the gel hardness values ​​(586 to 1698 g) obtained from functionalized bovine Sigma BLG were significantly higher than those obtained from functionalized cold-gelling rBLG (22 to 315 g). Gels obtained from milk protein concentrate (total milk protein) showed even lower hardness values ​​(77 to 93 g) than commercial BLG, and acidic gels (23 to 60 g) obtained from functionalized WPI had even lower values ​​than gels obtained from milk protein concentrate.

[0295] C) The gelling properties of commercial natural bovine BLG and rBLG after functionalization were measured using rheological methods.

[0296] General Procedure

[0297] Aqueous solutions of commercial natural bovine BLG (BLG) from Sigma Aldrich, rBLG according to the invention, whey protein isolate (WPI), and milk protein concentrate have been prepared and functionalized to produce a cold-gellable protein solution.

[0298] Powdered BLG, rBLG, WPI, and milk protein concentrate were added to deionized water to produce a 5% w / w protein solution. The solution was stirred for 10 minutes, and the pH was adjusted to 6.7 with HCl (1 N) or NaOH (1 N). The solution was allowed to stand for 10 minutes. The solution was then heated on a hot plate at 80°C for 15 minutes and cooled to 27°C in an ice bath. After the heating and cooling steps, the solution containing 7% w / w protein was diluted with demineralized water to obtain a 5% w / w protein solution.

[0299] An appropriate amount of powdered glucono-δ-lactone (GDL) was added to the functionalized protein solution to achieve 1% w / w GDL in solution. The resulting solution was stirred over a period of 1 minute to dissolve the GDL and then poured into the cylindrical geometry of the rheometer.

[0300] analyze

[0301] The viscoelastic properties (elastic modulus G', viscous modulus G'', and loss factor Tan δ) during acidification with GDL were monitored in oscillating mode at 27°C using an Anton Paar MCR95 rheometer equipped with a coaxial cylindrical geometry (CC27). Deformation was set at 1%, and the frequency at 1 Hz to operate within the linear viscoelastic (LVE) range. The time required for the elastic modulus (G') to become higher than the viscous modulus (G'') represents the gelation time. G' and G'' were recorded after reaching plateau periods, and the results are shown in Tables 5 and 6.

[0302] result

[0303]

[0304]

[0305] Table 5: Gel hardness, gelation time, elastic modulus, and viscous modulus of acid gels obtained from commercially available natural bovine BLG, rBLG, WPI, and milk protein concentrates functionalized according to the present invention. NA indicates not analyzed.

[0306]

[0307] Table 6: Gel hardness, gelation time, elastic modulus, and viscous modulus of acid gels obtained from commercially available natural bovine BLG, rBLG, WPI, and milk protein concentrates functionalized according to the present invention. Tanδ is the ratio between G” and G’, and is an indicator of the relative contribution of liquid viscosity and solid elasticity to the gel texture.

[0308] Gelation time varied between different proteins. Functionalized cold-gelling rBLG showed the shortest gelation time (700 s). A high gelation time (4300 s) was observed in functionalized WPI. The gelation times for commercial BLG and total milk protein were 2100 s and 2800 s, respectively.

[0309] The elastic modulus of gels varies between components. As expected from the hardness values, the elastic modulus of acid gels derived from functionalized commercial BLG is much higher than that of cold-gellable rBLG (3240 Pa) (950 Pa), and also higher than that of gels obtained from total milk protein (250 Pa) and functionalized WPI (<100 Pa).

[0310] Tan δ, corresponding to the ratio between elastic modulus and viscous modulus, provides information about the relative contributions of liquid viscosity and solid elasticity to the gel texture. A lower Tan δ indicates a more elastic gel. Conversely, a higher Tan δ indicates a more viscous gel. The Tan δ at the end of gelation varies between one component and the other.

[0311] The acid gel obtained from commercial BLG had the lowest tan δ value (0.13), while the acid gel from total milk protein had the highest value (0.43). The gel produced from cold-gellable rBLG had a tan δ of 0.17, which was lower than that of the gel obtained from functionalized WPI (0.27).

[0312] Conclusion: The results show that the acid gel properties obtained from the functionalized cold-gelling rBLG according to the present invention are close to those of gels produced from functionalized WPI or total milk protein. In contrast, gels produced from commercial BLG have different properties.

Claims

1. A method for preparing cold-gellable recombinant β-lactoglobulin (rBLG), comprising the following steps: a) Provide an aqueous solution of rBLG containing 3% to 15% w / w rBLG and with a pH of 5 to 8, and b) Heating the solution at a temperature of 65 to 95°C for 1 to 30 minutes to obtain the cold-gelling rBLG. This percentage is expressed as a weight relative to the total weight of the aqueous solution.

2. The method according to claim 1, wherein the temperature in step a) is 1 to 60°C.

3. The method according to any one of claims 1 to 2, wherein, When performed in batches, step b) is carried out with stirring at a speed of 1 to 500 rpm.

4. The method according to any one of claims 1 to 3, wherein the method further comprises step c) cooling the heated aqueous solution at a temperature below 60°C.

5. The method according to any one of claims 1 to 4, wherein the method further comprises step d) concentrating the heated aqueous solution.

6. The method according to any one of claims 1 to 5, wherein the method further comprises step e) drying the heated aqueous solution.

7. The method according to any one of claims 1 to 6, wherein the aqueous solution of step a) comprises rBLG and at least one polysaccharide.

8. The method according to claim 7, wherein the polysaccharide / rBLG ratio is 1 / 3 to 1 / 30.

9. The method according to any one of claims 1 to 8, wherein the rBLG is obtained from fungi, preferably from fungi of the genus Aspergillus.

10. Cold-gellable rBLG, obtained by the method according to any one of claims 1 to 9.

11. The cold-gellable rBLG according to claim 10, wherein the cold-gellable rBLG is in the form of aggregates, the aggregates preferably having a particle size of 20 to 500 nm.

12. A dried, cold-gellable rBLG composition comprising 45% to 95% w / w cold-gellable rBLG and at least one polysaccharide.

13. The dry, cold-gellable rBLG composition according to claim 12, wherein the cold-gellable rBLG is in the form of aggregates, the aggregates preferably having a particle size of 20 to 500 nm.

14. An aqueous solution of cold-gellable rBLG, wherein the solution comprises 3% to 15% w / w cold-gellable rBLG, 0.1% to 5% w / w polysaccharide and water, the percentages being expressed relative to the total weight of the aqueous solution.

15. Use of the cold-gelling rBLG according to any one of claims 10 or 11, or the dried cold-gelling rBLG composition according to any one of claims 12 or 13, or an aqueous solution of the cold-gelling rBLG according to claim 14, for the preparation of animal and non-animal dairy products.

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