FORMULATED FOOD SYSTEMS

Field of the invention

[001] The present invention relates to novel plant derived protein compositions and to methods of their manufacture. The present invention also relates to the use of the novel plant derived protein compositions as functional and/or nutritional ingredient in formulated food systems.

Background art

[002] A large part of industrial food processing is dedicated to preparing so-called“formulated foods”. Formulated foods are ready-to-cook or ready-to-eat alimentary products, such as bakery or dairy products, which often contain specialist ingredients, e.g. partially or substantially purified/concentrated natural products. The majority of formulated food systems, can be classified as a foam, emulsion or gel.

[003] Proteins are often used as a functional ingredient in (formulated) food, especially because of their structuring ability, for example their ability to build or stabilize gels, foams, doughs, emulsions and/or fibrillar structures. The important functional properties of proteins that are relevant to formulated food systems are fundamentally related to their physicochemical and structural properties, such as size, shape, amino acid composition, net charge, charge distribution, apparent hydrophobicity, tertiary and quaternary structural arrangements, number of sub-domain structures and response to changes in environmental conditions. Many of these properties are influenced by the duration, intensity, and nature of the processing steps the protein is subjected to, such as the method of isolation, purification, storage, handling etc.

[004] In addition to functional properties, proteins can confer fundamental nutritional properties to food products. Certain classes of formulated food have been developed specifically with a view to enriching the protein content for nutritional purposes. It is generally known that in such products, such as protein fortified beverages and high protein bars, the physicochemical and structural properties of the protein can be detrimental to the organoleptic qualities of the formulated food product, e.g. by giving rise to undesirable mouthfeel attributes such as grittiness.

[005] To date, the relationships between protein structure, functionality and organoleptic attributes are not fully understood.

[006] Whey protein has been the major protein ingredient in formulated foods. Whey proteins are a by-product from processing of other dairy products. Whey proteins are important nutritionally as a rich source of essential amino acids. Whey proteins also have important functional benefits, particularly because of their gelling and water-binding capacity, and also because of their ability to stabilize interfaces in foams and emulsions. The long-term sustainability of using whey proteins as food ingredients is a matter of concern. Increased demand for animal-based protein is expected to have a negative environmental impact and to place increased pressure on the world’s resources.

[007] Significant research effort is thus being dedicated to developing techniques for the production and use of protein ingredients from plant-based sources. Such proteins should possess the physicochemical and structural properties to confer functional behavior, such as water-binding, emulsifying, gelling or foaming, in a formulated functional food. Flavor and the presence of anti- nutritional factors (such as protease inhibitors, trypsin inhibitors, haemagglutinins, tannins, phytic acid, saponins, polyphenols etc.) are typically major factors that limit the use of many plant-based proteins in foods. Many vegetable derived protein materials can have a strong bitter, off-setting flavor and odor. Processing steps aimed at the removal or conversion of off-flavor components may not lead to a protein product possessing the physicochemical and structural properties which yield the desired functional properties. The nutritional value of plant-derived protein can be quite different from that of animal derived protein, for example due to differences in digestibility and/or amino acid composition. Naturally, any process developed to obtain functional protein from a plant source should also be economically viable on a large scale and yield a protein product that is easily processed in subsequent food manufacturing steps.

[008] It is the object of the present invention to provide novel protein compositions that have distinctive functionality, adequate nutritional value and that can be derived from non-animal sources in a sustainable and economically viable manner.

Summary of the invention

[009] In accordance with the invention, this objective is realized by the provision of beet root derived protein compositions as described herein.

[010] As will be illustrated in the appending examples, beet root derived protein compositions of the invention have at least partially retained their“native” form, thus providing desirable properties such as excellent structuring (e.g. gelling) and nutritional properties, which set them apart from the plant-based proteins commonly used in present-day food manufacturing, as well as from proteins derived from beat leaf or denatured beet root protein. The beet root protein compositions typically have a high secondary structure content (in particular the a-helix content) and/or a high solubility.

[011] Furthermore, as will be illustrated in the examples, beet root derived protein compositions of the invention may be applied in food systems at the concentrations needed to confer functional behavior with a reduced impact on the organoleptic properties such as flavor and/or odor. The inventors have determined that the sugar beet root derived protein compositions of the invention can attribute to gel-forming, emulsification, water-binding, fat-binding, foam formation and/or stabilization etc. in a diverse range of food systems.

[012] Furthermore, the inventors have determined that the beet root derived protein compositions of the invention have distinctive nutritional value and do not seem to be associated with undesirable digestibility issues.

[013] The beet root derived protein compositions of the invention can be derived from fresh beet material, such as fresh (sugar) beet, without interfering with sugar manufacturing; as well as from (sugar) beet material from which sugar has been extracted, so-called‘spent sugar beet’. Hence, as will be understood by those skilled in the art, based on the present teachings, the beet root derived protein compositions can be derived from a side-stream of or integrated with conventional sugar production. Hence, the compositions of the invention have the advantage that they are plant-based rather than animal-based and in addition that they can be obtained without competing with current food production.

[014] The present invention further provides methods of producing the protein compositions of the invention from beet material, including fresh sugar beet as well as spent sugar beet. These methods have been designed to yield protein with the desired physicochemical and structural properties and in a form particularly suitable to be distributed, stored and processed in accordance with conventional food formulation practice. With the methods of the invention this can be accomplished in a manner that is economically viable.

[015] The present invention further provides formulated food compositions comprising the beet root derived protein compositions. As will be understood by those skilled in the art, based on the present teachings, (formulated) food products wherein the beet root derived protein compositions can beneficially be incorporated include beverages, doughs, processed meats, plant based meat replacements products, bakery products, confectionary products; cereal-based products, dairy products, dairy free replacements for dairy products, dressings, sauces, ice cream, et cetera. The present invention further provides methods of producing these formulated food compositions.

[016] These and other aspects will be described in more detail here below and they will be further illustrated by means of the appending examples.

Brief description of the drawings

[017] The present invention will be discussed in more detail below, with reference to the attached drawings.

[018] Figure 1 is a schematic representation of a beet root.

[019] Figure 2 is a picture of the SDS-PAGE test performed in example 2 on the beet root protein of example 1.

[020] Figure 3 is the calibration curve determined for the marker used in example 2.

[021] Figure 4 shows the solubility of the beet root protein of example 1A as a function of the pH, determined in example 3.

[022] Figure 5 shows the solubility of the beet root protein of example 1A as a function of the pH and ionic strength, determined in example 3.

[023] Figure 6 shows the CD spectrum of example 4.

[024] Figure 7 shows the gel strength of the dispersion of example 5 in demiwater and in buffer at pH 3, 5 and 7 (3 mS/cm conductivity).

[025] Figure 8 shows the enzymatic degradation kinetics tested in example 6 for pepsin.

[026] Figure 9 shows the enzymatic degradation kinetics tested in example 6 for pancreatin.

[027] Figure 10 shows the foams obtained in the foaming test of example 7.

[028] Figure 11 shows the results of the foaming test of example 7.

[029] Figure 12 shows the results of the foaming test of example 9.

[030] Figure 13 shows the results of the foaming test of example 9.

[031] Figure 14 shows the results of the rheology measurement of example 10. Description of embodiments

[032] A first aspect of the invention concerns a food ingredient composition comprising at least 3 wt.%, based on the total weight of the composition, beet root protein.

[033] As used herein, the term“beet root protein” refers to protein material obtainable from the root of plants of the species Beta Vulgaris, preferably from the root of plants of Beta Vulgaris subsp Vulgaris, most preferably from the root of Beta Vulgaris L. The term protein refers to a biomolecule comprising one or more polypeptide chains comprising more than 50 amino acid residues. As is known to the person skilled in the art, the polypeptide chain(s) in a protein may naturally occur in a modified state, such as glycosylated state. Unless explicitly indicated otherwise, the term protein includes modified (e.g. glycosylated) polypeptides comprising more than 50 amino acid residues.

[034] In highly preferred embodiments the beet root protein is obtainable from plants of the subspecies Beta Vulgaris subsp. Vulgaris, also referred to herein as“sugar beet root protein”.

[035] As will be shown in the appended examples, the present inventors have surprisingly found that it is possible to isolate a beet root protein wherein a significant part of the secondary structure and/or solubility of the beet root protein is preserved. Without wishing to be bound by any theory, the present inventors hypothesize that there is a relation between the secondary structure of the beet root protein and functional properties such as gelling properties.

[036] Thus, in highly preferred embodiments the beet root protein has:

• more than 30% secondary structure, preferably more than 40%, or more than 60% secondary structure, wherein the secondary structure is in the form of an a-helix and/or a b-sheet; and/or

• an aqueous solubility of more than 1 mg/ml, preferably more than 1 .5 mg/ml, wherein the solubility is determined at pH 7 and at an ionic strength of 8 mS/cm.

[037] In preferred embodiments the beet root protein has more than 30% secondary structure, preferably more than 40%, or more than 60% wherein the secondary structure is in the form of an a-helix and/or a b-sheet. In preferred embodiments at least 15%, preferably at least 20% or at least 30% of the secondary structure is in the form of an a-helix.

[038] In another highly preferred embodiment, the beet root protein has:

• more than 30% secondary structure, preferably more than 40%, or more than 60% secondary structure, wherein the secondary structure is in the form of an a-helix and/or a b-sheet and wherein at least 15%, preferably at least 20% or at least 30% of the secondary structure is in the form of an a-helix; and/or

• an aqueous solubility of more than 1 mg/ml, preferably more than 1 .5 mg/ml, wherein the solubility is determined at pH 7 and at an ionic strength of 8 mS/cm.

[039] A suitable way to determine the amount of secondary structure present in the beet root protein is using circular dichroism spectroscopy. A preferred way to determine the amount of secondary structure present in the beet root protein is in accordance with the“secondary structure test protocol” set out below.

[040] The secondary structure may be characterized by diluting the food ingredient composition with phosphate buffered water (pH ~7) to a concentration of 0.1 mg protein/mL, mixing the resulting sample and transferring it to a quartz cuvet with a 1 mm path length in a Peltier Temperature controlled cuvet-chamber (20 +/- 0.2°C); recording the ellipticity using a spectropolarimeter (Jasco Corporation, Japan; f.e. type J-815) in the spectral range from 185-260 nm, with a detector voltage of 600-850V at 200 nm, a spectral resolution of 0.2 nm, a scan speed of 100 nm/min, a time- constant of 0.125 sec and a band width on 1 nm; recording the spectrum as averages of 16 scans, and subsequently correcting for that of a corresponding protein-free sample; applying inverse Fourier transformation to reduce noise in the spectra and analyzing the far-UV CD spectrum for the secondary structure element content by comparing the digitized CD spectrum to a parametrized set of four reference spectra that represent 100% a-helical, b-stranded, b-turned and non-structured polypeptides, using a non-linear least-squares fitting procedure to fit the parameters of the four secondary structure types to the recorded spectrum such that the relative contributions of the four secondary structure types are obtained, as described by de Jongh et al. (de Jongh HHJ, Goormaghtigh E, Killian JA (1994) Analysis of circular dichroism spectra of oriented protein-lipid complexes: Toward a general application. Biochemistry 33: 14521-14528). In case the sample is not transparent at a concentration of 0.1 mg protein/mL, the sample is filtered through a 25 pm filter to isolate the soluble protein fraction and the CD spectrum of the resulting soluble protein fraction determined as described above, wherein the final result is adjusted to account for the insoluble protein fraction, which is considered to have a secondary structure content of 0%. In case the CD spectrum is distorted to such an extent that the secondary structure content cannot be determined, the sample is considered to have a secondary structure content of 0%. In an embodiment a food ingredient composition as defined herein is provided having the secondary structure characteristics as described above when determined using this method.

[041] In embodiments, the food ingredient composition comprises at least 5 wt.%, based on the total weight of the composition, preferably at least 10 wt.%, at least 30 wt.%, at least 50 wt.%, at least 70 wt.% or at least 90 wt.% beet root protein. A suitable way to determine the protein content of the composition in accordance with the invention is using the Kjeldahl method with a conversion factor of 6.25. It will be understood by the skilled person that; since the Kjeldahl method with a conversion factor of 6.25 is a theoretical calculation of the protein content based on the amount of Nitrogenous compounds in the composition; the beet root protein content may exceed 100% as determined using the Kjeldahl method with a conversion factor of 6.25. Unless indicated otherwise, protein contents referred to in this document are determined using the Kjeldahl method with a conversion factor of 6.25.

[042] In embodiments, the food ingredient composition comprises at least 30 wt.%, based on the dry matter content, preferably at least 50 wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.% or at least 95 wt.% beet root protein.

[043] In accordance with highly preferred embodiments, the food ingredient composition of the invention comprises the beet root protein in isolated form, which means that the protein is no longer part of the beet root. Hence, the food ingredient composition of the invention does not comprise or consist of whole or sliced beet, beet root or spent beet pulp. However, as will be understood by the skilled person, the beet root protein compositions of the invention may comprise one or more other plant based components such as pectin, hemicellulose, sugars, salts, betaine etc.

[044] In highly preferred embodiments the food ingredient composition of the invention is provided wherein the ratio (w/w) of beet root protein to galacturonic acid is more than 1 :1 , preferably more than 5:1 , more preferably more than 10:1 wherein the amount of galacturonic acid is determined after hydrolysis of any polysaccharides in the composition to sugar monomers. A sugar beet root typically contains about 8-12 wt.% (dry weight) protein, and about 20-25 wt.% (dry weight) pectin. Since galacturonic acid is the main constituent of pectin, it will be understood by the skilled person that the aforementioned ratios of beet root protein to galacturonic acid for the food ingredient composition of the invention reflects that the beet root protein is no longer part of the beet root. It further reflects that the food ingredient composition does not comprise or consist of whole or sliced beet, beet root or spent beet pulp and it furthermore reflects that pectin is not comprised in amounts which would significantly affect the functional properties of the beet protein. Determination of galacturonic acid content is known to the skilled person and is typically performed by Seaman Hydrolysis, which involves complete hydrolysation, preferably acid hydrolysation of the food ingredient composition followed by chromatographic analysis of the resulting sugar monomers in the hydrolysates. An exemplary method is by performing a prehydrolysis step using 72% w/w sulphuric acid at 30°C for 1 h, followed by hydrolysis with 1 M sulphuric acid at 100°C for 3h, derivatizing the sugars as alditol acetates and analyzing the gas chromatography.

[045] The food ingredient composition may be in the form of a liquid (e.g. a solution, suspension or an emulsion), a powder or a paste.

[046] In embodiments, the food ingredient composition has a dry-matter content of at least 50 wt.%, based on the total weight of the composition, preferably at least 70 wt.%, at least 80 wt.%, or at least 90 wt.%.

[047] In embodiments, the food ingredient composition has a dry-matter content of 10-50 wt.%, based on the total weight of the composition, preferably 10-30 wt.% or 15-25 wt.%.

[048] In embodiments, the food ingredient composition has a dry-matter content of less than 10 wt.%, based on the total weight of the composition, preferably less than 7 wt.%, less than 5 wt.% or less than 3 wt.%.

[049] A preferred method to measure dry matter content is in accordance with ICUMSA GS2/1 /3/9-15 (2007) which is known to the person skilled in the art.

[050] In preferred embodiments, said composition has the form of a powder or paste.

[051] As used herein, the term“paste” refers to a semi-liquid of high viscosity, which may be a colloidal suspension, emulsion and/or a dispersion of aggregated material.

[052] It will be understood by the skilled person that the dry-matter content range wherein the food ingredient composition has the form of a paste depends inter alia on the amount and type of other components present in the composition. It is within the capabilities of the person skilled in the art to adjust the dry-matter content such as to obtain a paste. [053] In embodiments, the composition is in the form of a paste, wherein the paste has a dry- matter content of 10-50 wt.%, based on the total weight of the composition, preferably 10-30 wt.% or 15-25 wt.%.

[054] In embodiments, the composition is in the form of a powder wherein at least 50 vol%, preferably at least 70 vol%, at least 90 vol% or at least 95 vol% of the granules have a particle size of more than 20 pm, preferably more than 50 pm or more than 100 pm. In embodiments, the composition is in the form of a powder wherein at least 50 vol%, preferably at least 70 vol%, at least 90 vol% or at least 95 vol% of the granules have a particle size of less than 500 pm, preferably less than 300 pm or less than 120 pm. In accordance with the invention, the particle size distribution is typically determined by measuring with a laser light scattering particle size analyzer utilizing the Mie theory of light scattering, such as the Malvern Mastersizer or another instrument of equal or better sensitivity and reporting the data using a volume equivalent sphere model.

[055] In embodiments, the composition is in the form of a powder wherein the powder has a bulk density of more than 400 g/l, preferably more than 500 g/l, most preferably more than 600 g/l. Bulk density measurements are known to the person skilled in the art.

[056] In an embodiment of the invention, the composition is provided as a free flowing powder. The term "free-flowing powder", as used herein, is well known to those skilled in the art and includes particulate materials that can be poured (e.g., from one vessel having an opening of from about 10 cm2 to 50 cm2 to another vessel of similar dimensions) without substantial clumping of the particles.

[057] In embodiments, there is provided the food ingredient composition of the invention, wherein more than 90%, preferably more than 95%, more than 98% or more than 99% of the beet root protein has a molecular weight of more than 10kDa, preferably more than 15 kDa.

[058] In embodiments, there is provided the food ingredient composition of the invention, wherein 20-50%, preferably 30-50%, preferably 35-45% of the beet root protein has a molecular weight within the range of 10-30 kDa.

[059] In embodiments, there is provided the food ingredient composition of the invention, wherein 30-70%, preferably 40-60%, preferably 45-55% of the beet root protein has a molecular weight within the range of 30-55 kDa.

[060] In embodiments, there is provided the food ingredient composition of the invention, wherein 5-30%, preferably 5-20%, preferably 10-20% of the beet root protein has a molecular weight within the range of 55-130 kDa.

[061] In embodiments, there is provided the food ingredient composition of the invention, wherein less than 20%, preferably less than 15%, preferably less than 10% of the beet root protein has a molecular weight of more than 200 kDa, preferably more than 150kDa, preferably more than 130 kDa.

[062] In embodiments, there is provided the food ingredient composition of the invention, wherein the beet root protein is characterized by two or more, such as three or four of the following properties:

• 5-15%, preferably 8-12% of the protein has a molecular weight within the range of 52-55 kDa, preferably 53 kDa; • 5-15%, preferably 8-12% of the protein has a molecular weight within the range of 41 -43 kDa, preferably 42 kDa;

• 10-20%, preferably 13-17% of the protein has a molecular weight within the range of 36-38 kDa, preferably 37 kDa;

• 7-17%, preferably 10-14% of the protein has a molecular weight within the range of 25-27 kDa, preferably 26 kDa; and/or

• 4-14%, preferably 7-1 1 % of the protein has a molecular weight within the range of 15-17 kDa, preferably 16 kDa.

[063] In embodiments, there is provided the food ingredient composition of the invention wherein the beet root protein is characterized by two or more, such as three or four of the following properties:

• 10-50%, preferably 20-40%, more preferably 25-35% of the protein has a molecular weight within the range of 10-30 kDa;

• 10-50%, preferably 20-40%, more preferably 25-35% of the protein has a molecular weight within the range of 30-55 kDa;

• 20-60%, preferably 30-50%, more preferably 35-45% of the protein has a molecular weight within the range of 55-130 kDa; and/or

• less than 20%, preferably less than 15%, preferably less than 10% of the protein has a molecular weight of more than 200 kDa, preferably more than 150kDa, preferably more than 130 kDa.

[064] A suitable way to characterize the molecular weight distribution of the beet root protein is using polyacrylamide gel electrophoresis (PAGE); preferably SDS-PAGE, more preferably SDS- PAGE performed under reducing conditions, for example using“Any kD Mini-Protean TGX gel” (Bio-Rad Laboratories), stained with coomassie stain; and analyzing the resulting gel using gel analysis software known to the skilled person; such as GelAnalyzer 2010a employed to scan the density of the electrophoretic bands. In an embodiment a food ingredient composition as defined herein is provided having the molecular weight distribution characteristics as described above when determined using this method.

[065] As is shown in the appended examples, the beet root protein in the food ingredient composition of the invention is characterized by a high solubility, especially when compared to thermally dried (e.g. oven dried) beet root protein compositions, which result in aggregated (insoluble) proteins. Hence, the beet root protein in the food ingredient composition of the invention is preferably not aggregated. Similarly, the beet root protein in the food ingredient composition of the invention has preferably not been subjected to a drying or dewatering step wherein the temperature exceeds 50°C. However, as will be explained herein elsewhere, a mild thermal protein extraction step is acceptable. Hence, in particular embodiments, the beet root protein in the food ingredient composition of the invention has not been subjected to a temperature of more than 50°C at a protein concentration of more than 5 wt.% (by total weight of the composition comprising beet root protein). [066] In accordance with highly preferred embodiments of the invention, the beet root protein in the food ingredient composition of the invention has an aqueous solubility of more than 1 mg/ml, such as more than 1 .2 mg/ml, more than 1 .3 mg/ml, more than 1 .4 mg/ml, preferably more than 1 .5 mg/ml, such as more than 1 .6 mg/ml, more than 1 .7 mg/ml, more than 1 .8 mg/ml, wherein the solubility is determined at pH 7 and at an ionic strength of 8 mS/cm.

[067] In embodiments, there is provided the food ingredient composition of the invention, wherein the composition is packaged in a suitable container, such as a closed and/or sealed container. In embodiments, the composition is packaged under an oxygen free environment. In embodiments, the composition is packaged under an inert atmosphere, such as a CO2 or nitrogen atmosphere. In embodiments, the packaging is an aseptic container. In embodiments, a single container comprises at least 1 g of beet root protein, preferably at least 50g, at least 200g, at least 500g or at least 1 kg.

[068] The present inventors have surprisingly found that beet root protein compositions as described herein can conveniently be provided which are microbially stable.

[069] Thus, in embodiments, there is provided the food ingredient composition of the invention, wherein the composition has an aerobic colony count (ACC), determined after 48 hours of incubation at 30°C, of less than 10 ⁶ cfu/g, preferably less than 10 ⁵ cfu/g, less than 10 ⁴ cfu/g, less than 10 ³ cfu/g, less than 10 ² cfu/g or less than 10 cfu/g.

[070] In embodiments, the food ingredient composition is packaged and has an ACC as defined herein before when sampled upon opening the package.

[071] In preferred embodiments, the food ingredient composition is packaged and has an ACC as defined herein before when sampled 1 day, preferably 2 days, most preferably 3 days after opening the package.

[072] In embodiments, there is provided the food ingredient composition of the invention, wherein the composition has a lactic acid bacteria count, determined after 48 hours of incubation at 30°C, of less than 10 ⁶ cfu/g, preferably less than 10 ⁵ cfu/g, less than 10 ⁴ cfu/g, less than 10 ³ cfu/g, less than 10 ² cfu/g or less than 10 cfu/g.

[073] In embodiments, the food ingredient composition is packaged and has a lactic acid bacteria count as defined herein before when sampled upon opening the package.

[074] In preferred embodiments, the food ingredient composition is packaged and has a lactic acid bacteria count as defined herein before when sampled 1 day, preferably 2 days, most preferably 3 days after opening the package.

[075] In embodiments, the food ingredient composition is a powder and has a water activity of less than 0.9, preferably less than 0.7, less than 0.6, or less than 0.5.

[076] The present inventors have surprisingly found that a beet root protein composition as described herein can be provided which may exhibit improved nutritional value and/or digestibility by animals or humans compared to other known plant-based protein sources.

[077] Thus, in embodiments, there is provided the food ingredient composition of the present invention, wherein the beet root protein has an Amino Acid Score (scored in accordance with Food and Agriculture Organization of the United Nations, Dietary protein quality evaluation in human nutrition: report of an FAO expert consultation; 2013; ISSN 0254-4725) of more than 100%, preferably more than 1 10%, more preferably more than 120% for adults and children over 1 years old. As is known to the skilled person, scores of more than 100% indicate that the protein is not limiting with regard to any essential amino acid.

[078] In embodiments, there is provided the food ingredient composition of the invention, wherein the composition has a digestibility of more than 70%, preferably more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%.

[079] In embodiments, there is provided the food-grade composition of the invention, wherein the composition has a Protein Digestibility Corrected Amino Acid Score (PDCAAS) of more than 0.5, preferably more than 0.6, more than 0.7, or more than 0.8.

[080] Suitable methods to determine the digestibility and the PDCAAS are known in the art and may include in vivo tests (e.g. in rats) or in vitro tests. A preferred method to determine the digestibility and the PDCAAS is in accordance with the method reported by Schaafsma (The Protein Digestibility-Corrected Amino Acid Score. The Journal of Nutrition, Volume 130, 2000, Pages 1865S-1867S). In an embodiment a food ingredient composition as defined herein is provided having the digestibility characteristics as described above when determined using this method.

[081] The present inventors have surprisingly found that a beet root protein composition as described herein can be provided which may be easier to dissolve or disperse compared to some other known plant-based protein sources.

[082] Thus, in embodiments, there is provided the food ingredient composition of the invention, wherein the composition can be homogeneously dissolved or dispersed in an aqueous phase at a concentration of more than 0.5% (w/v), preferably more than 1 % (w/v), preferably more than 2% (w/v) beet root protein, without visually distinguishable sedimentation and/or turbidity.

[083] In embodiments, the composition can be dissolved or dispersed as described above using only low-shear mixing equipment.

[084] In embodiments, the composition can be dissolved or dispersed as described above to yield an aqueous phase with a turbidity of less than 150 NTU, preferably less than 50 NTU, less than 10 NTU, less than 5 NTU or less than 1 NTU.

[085] In embodiments, the food ingredient composition provided herein is a food-grade composition.

[086] As used herein, the term“food-grade” means suitable for consumption by an animal or human, in particular a human. In an embodiment it means that the composition has been determined to be safe, functional and suitable for its intended use in animal or human food. For example, it is handled and labeled appropriately, and/or conforms to the appropriate regulations governing the use of the composition in animal or human food in the relevant jurisdiction, such as Europe.

[087] The present inventors have surprisingly found methods for isolating beet root protein compositions in accordance with the invention which may be performed on beet roots and which are economically viable. In another aspect the present invention thus provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material; b) processing the beet root material to obtain a liquid comprising beet root protein;

c) collecting the liquid;

d) separating the liquid into a protein-rich fraction and a protein-poor fraction and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C.

[088] It was found that by performing the method at a temperature of less than 70°C, beet root protein having the secondary structure characteristics according to the invention may be provided. As will be understood by the skilled person, performing the method at a temperature of less than X °C means that the temperature of the beet root protein containing product does not exceed this temperature during the different steps in the process, such as to avoid denaturation of the beet root protein. However, it will also be understood by the skilled person that shortly being subjected to a higher temperature is acceptable and within the scope of the present methods if this still results in beet root protein according to the invention. Hence, in preferred embodiments, wherein the method is performed at a temperature of less than X °C, the beet root protein containing product is not subjected to a temperature of more than X °C for more than 15 minutes, preferably the beet root protein containing product is not subjected to a temperature of more than X °C for more than 5 minutes, more preferably the beet root protein containing product is not subjected to a temperature of more than X °C for more than 1 minute, most preferably the beet root protein containing product is not subjected to a temperature of more than X °C at all.

[089] The present inventors have found that the methods to isolate beet protein provided herein may be performed as a standalone/dedicated beet protein production process, or integrated into a sugar manufacturing process.

[090] As will be illustrated in the examples, the present inventors have surprisingly found that it is possible to produce the beet root protein of the invention using mild extraction (up to 68°C) and even room or lower temperature extraction and low temperature isolation methods (e.g. freeze drying). Hence, in highly preferred embodiments the method described herein is performed at a temperature of less than 68°C, preferably less than 50°C, more preferably less than 30°C.

[091] The beet root material provided in step a) may be unsliced or may have been mechanically reduced in size, such as sliced or mashed. In embodiments, step a) comprises washing sliced or unsliced beet to remove adhering dirt and soil. In preferred embodiments, the method to isolate beet protein as described herein is provided wherein step a) comprises providing unsliced or whole beet and slicing the beet. As used herein, unsliced or whole beet root refers to beet devoid of stem and/or leaves (i.e. the taproot). Slicing the beet root may be performed by any suitable means known to the person skilled in the art, such as a drum slicer, a disc slicer, chopper or cutter. In embodiments, slicing the beet results in an average product thickness of 0.5-10 mm, preferably 1 - 5mm. In other embodiments, the method to isolate beet protein as described herein is provided wherein step a) comprises providing unsliced or whole beet and step a) does not comprise slicing beet. In embodiments, the beet root material provided in step a) is so-called‘spent’ sugar beet pulp, which is the pulp remaining after sugar has been at least partially extracted from a sugar beet root.

[092] As is known to those skilled in the art, a beet root, such as a sugar beet root, is often conically shaped comprising a broader upper part and a smaller lower part, as shown in Figure 1 . A beet root of total length‘a’ may thus be divided into an upper part of length‘b’ and a lower part of length‘c’, wherein the total length‘a’ is the sum of the length of the upper part‘b’ and the length of the lower part‘c’. The present inventors have found that step a) of the method for producing a beet root protein composition provided herein; preferably comprises or consists of providing the upper part of length‘b’ of a beet root, preferably a sugar beet root, wherein the length‘b’ is less than 70%, preferably less than 50%, less than 35% or less than 20% of the total length‘a’.

[093] In embodiments, the beet root material provided in step a) has been or is being subjected to a pre-treatment used in the sugar manufacturing industry, such as a pre-treatment selected from the group consisting of thermal cell disintegration, pulsed electric field (PEF) treatment, fermentation, acidification, freezing and/or enzymatic treatment. In preferred embodiments the beet root material provided in step a) has been or is being subjected to a pre-treatment selected from the group consisting of pulsed electric field (PEF) treatment, fermentation, acidification, freezing and/or enzymatic treatment.

[094] In embodiments, the beet root material provided in step a) has not been and is not being subjected to a pre-treatment commonly used in the sugar manufacturing industry, such as a pre- treatment selected from the group consisting of thermal cell disintegration, pulsed electric field (PEF), fermentation, acidification, freezing and/or enzymatic treatment.

[095] In embodiments, the extraction liquid of step b) has a pH within the range of 5-9, preferably a pH within the range of 6-8, preferably a pH within the range of 6.5-7.5, preferably a pH of approximately 7.

[096] In highly preferred embodiments, the extraction liquid of step b) has a pH within the range of 5-9, preferably a pH within the range of 5-7, preferably a pH within the range of 5.5-6.5, preferably a pH of approximately 6.

[097] The extraction liquid is generally water-based. Hence, according to the invention the extraction liquid is an aqueous composition comprising, preferably consisting of (i) at least 90% water, (ii) optionally acids and/or bases and (iii) conventional impurities such as minerals.

[098] Step b) may be performed by an extraction process. Thus, in embodiments, step b) comprises contacting the beet root material with an extraction liquid for an amount of time sufficient to at least partially extract the beet root protein and step c) comprises collecting the extraction liquid.

[099] In embodiments, the extraction of step b) is performed using a 1 :0.5 to 1 :4 by weight ratio of beet root materiafextraction liquid, preferably 1 :0.9 to 1 :3, preferably 1 :0.95 to 1 :2, preferably 1 :1 to 1 :1 .3. In embodiments, the extraction of step b) is performed using a counter-current extractor. In embodiments, the extraction of step b) is performed using a counter-current extractor and a 1 :0.9 to 1 :2 by weight ratio of beet root material: extraction liquid, preferably 1 :1 to 1 :1 .8, preferably 1 :1 to 1 :1 .5, preferably 1 : 1 .25, preferably 1 :1 .10. [0100] Generally, the extraction of step b) is performed at a temperature within the range of 0- 70°C. In embodiments, the extraction of step b) is performed at a temperature within the range of 50-68°C, more preferably 60-68 °C; most preferably 60-65°C. In embodiments, the extraction of step b) is performed at a temperature within the range of 0-50°C, preferably 0-25°C, preferably 0- 10°C.

[0101] In embodiments, the extraction of step b) is performed for at least 30 seconds, preferably at least 1 minute, preferably at least 10 minutes. In embodiments, the extraction of step b) is performed such that the extraction liquid collected in step c) comprises at least 1 wt.%, by total weight of the extraction liquid, preferably at least 3 wt.% beet root protein. In embodiments, the extraction of step b) is performed such that the extraction liquid collected in step c) has a dry matter content of at least 2 wt.%, preferably at least 5 wt.%.

[0102] In embodiments, step b) comprises or consists of contacting washed raw, untreated beet root which may be unsliced or sliced with an extraction liquid.

[0103] In embodiments, the extraction of beet protein coincides with the extraction of sugar in a sugar manufacturing process. Thus, in embodiments, the extraction of step b) is performed such that the extraction liquid collected in step c) comprises more than 10wt.%, by total weight of the extraction liquid, of sucrose, preferably more than. 14 wt.%, preferably more than 16 wt.%.

[0104] In embodiments, the extraction of beet protein is separate from the extraction of sugar in a sugar manufacturing process. Thus, in embodiments, the extraction of step b) is performed such that the extraction liquid collected in step c) comprises less than 10 wt.%, by total weight of the extraction liquid, preferably less than 7 wt.%, preferably less than 5 wt.%, preferably less than 1 wt.% of sucrose.

[0105] Step b) may be performed by directly collecting the beet root juice from beet root material. Thus, in embodiments, step b) comprises subjecting the beet root material to a mechanical treatment which releases beet root juice and step c) comprises collecting the beet root juice. As used herein, the term“beet root juice” refers to the juice released from beet roots when subjected to extensive mechanical treatment. In embodiments, the beet root juice collected in step c) comprises very little or no liquid (e.g. water) which does not originate from the beet root material, for example less than 50 vol%, less than 10 vol% or less than 1 vol% of the beet root juice collected in step c) is liquid which does not originate from the beet root material.

[0106] In embodiments, the mechanical treatment which releases beet root juice comprises pressing and/or mashing. In embodiments, pressing is performed using a screw press or basket press equipped with a screen with suitable pore size, such as 0.1 -10 mm, preferably 0.5-5 mm, preferably 1 -2 mm.

[0107] In embodiments, step a) may coincide with step b). For example, the beet root material provided in step a) may be subjected to a pre-treatment used in the sugar manufacturing industry, while the mechanical treatment which releases beet root juice is being performed.

[0108] In embodiments, step b) may coincide with step c). For example, the mechanical treatment which releases beet root juice may comprise collecting the beet root juice, for instance when filter pressing is used. [0109] In preferred embodiments, step a) may coincide with steps b) and c). For example, the beet root material provided in step a) may be subjected to a pre-treatment used in the sugar manufacturing industry, while the mechanical treatment which releases beet root juice is being performed, said mechanical treatment comprising collecting the beet root juice, for instance when filter pressing is used.

[0110] In embodiments, step c) comprises centrifugation, decantation and/or filtration.

[0111] In embodiments, step c) comprises centrifugation for at least 2 minutes, preferably at least 5 minutes, more preferably at least 10 minutes at more than 1500 g, preferably more than 2000g, preferably more than 3000g.

[0112] In embodiments, step c) comprises filtration using a membrane with a pore size of at least

30 micron, preferably at least 45 micron.

[0113] In embodiments, step c) comprises centrifugation followed by filtration.

[0114] In embodiments, step d) comprises pH precipitation and/or dialysis. In preferred embodiments step d) comprises dialysis. In highly preferred embodiments step d) comprises dialysis followed by pH precipitation. It will be understood by the skilled person that the dialysis product may be subjected to other process steps after dialysis and before the pH precipitation.

[0115] As used herein,‘pH precipitation’ refers to a process step wherein the pH of the (optionally dialysed) extraction liquid is lowered to a pH in the range of 2-7, preferably a pH within the range of 3-6, a pH within the range of 3-5 or a pH within the range of 3-4.5. In preferred embodiments pH precipitation comprises lowering the pH of the extraction liquid using acetic acid.

[0116] In embodiments, step d) comprises dialysis employing a membrane with a molecular weight cut-off value of 50 kDa, preferably 25 kDa or less, more preferably 10 kDa or less. In preferred embodiments step d) is performed such that the ratio of the protein content after dialysis (by dry weight) to the protein content before dialysis (by dry weight) is more than 1 .1 ; preferably more than 1 .3; preferably more than 1 .7; preferably more than 2.

[0117] In embodiments, step e) comprises concentrating the beet root protein composition using a membrane filtration process.

[0118] In preferred embodiments, step e) is performed. In embodiments, step e) comprises at least partially removing solvent from the beet root protein composition to obtain a beet root protein composition comprising at least 10 wt.%, based on the total weight of the composition, preferably at least 20 wt.%, at least 30 wt.%, at least 50 wt.%, at least 70 wt.%, at least 80 wt.% or at least 90 wt.% beet root protein.

[0119] In embodiments, step e) comprises at least partially removing solvent from the beet root protein composition to obtain a beet root protein composition in the form of a paste or powder.

[0120] In embodiments, step e) comprises at least partially removing solvent from the beet root protein composition to obtain a beet root protein composition with a dry-matter content of at least 50 wt.%, based on the total weight of the composition, preferably at least 70 wt.%, at least 80 wt.%, or at least 90 wt.%. In embodiments, step e) comprises at least partially removing solvent from the beet root protein composition to obtain a beet root protein composition with a dry-matter content of 10-50 wt.%, based on the total weight of the composition, preferably 10-30 wt.% or 15-25 wt.%. [0121] In embodiments, step e) coincides with step d), for example the dialysis of step d) may be performed such that the beet root protein composition is also concentrated.

[0122] In embodiments, step e) comprises spray drying, belt drying, drum drying, rotor drying, tray drying and/or freeze drying, preferably spray drying and/or freeze drying.

[0123] In embodiments, step e) comprises at least partially removing solvent from the beet root protein composition to obtain a beet root protein composition:

• in the form of a paste with a dry-matter content of 10-50 wt.%, based on the total weight of the composition, preferably 10-30 wt.% or 15-25 wt.%; or

• in the form of a powder with a dry-matter content of at least 50 wt.%, based on the total weight of the composition, preferably at least 70 wt.%, at least 80 wt.%, or at least 90 wt.%.

[0124] As will be understood by the skilled person, the methods of the present invention do not exclude a short, high temperature sterilization step after step e) in so far as the secondary structure characteristics of the beet root protein are not materially affected.

[0125] In embodiments, step e) of the methods to isolate beet protein provided herein is performed wherein the beet root protein composition obtained in step e) is the food ingredient composition of any of the embodiments described herein before.

[0126] In embodiments, the methods to isolate beet protein described herein further comprise a step f) wherein the beet protein composition is packaged in a suitable container, such as a closed and/or sealed container. In embodiments, the composition is packaged under an oxygen free environment. In embodiments, the composition is packaged under an inert atmosphere, such as a CO2 or nitrogen atmosphere. In embodiments, the packaging is an aseptic container. In embodiments, a single container comprises at least 1g of beet protein, preferably at least 50g, at least 200g, at least 500g or at least 1 kg.

[0127] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by contacting the beet root material with an extraction liquid for an amount of time sufficient to at least partially extract the beet root protein;

c) collecting the extraction liquid;

d) separating the extraction liquid into a protein-rich fraction and a protein-poor fraction and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein.

[0128] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by contacting the beet root material with an extraction liquid which has a pH within the range of 5-9, preferably a pH within the range of 6-8, preferably a pH within the range of 6.5-7.5, preferably a pH of approximately 7 for at least 30 seconds, preferably at least 1 minute, preferably at least 10 minutes;

c) collecting the extraction liquid;

d) separating the extraction liquid into a protein-rich fraction and a protein-poor fraction and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C.

[0129] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by contacting the beet root material with an extraction liquid which has a pH within the range of 5-9, preferably a pH within the range of 6-8, preferably a pH within the range of 6.5-7.5, preferably a pH of approximately 7 for at least 30 seconds, preferably at least 1 minute, preferably at least 10 minutes;

c) collecting the extraction liquid;

d) separating the extraction liquid into a protein-rich fraction and a protein-poor fraction by dialysis employing a membrane with a molecular weight cut-off value of 50 kDa or less, preferably 25 kDa or less, more preferably 10 kDa or less, and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C.

[0130] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by contacting the beet root material with an extraction liquid which has a pH within the range of 5-9, preferably a pH within the range of 6-8, preferably a pH within the range of 6.5-7.5, preferably a pH of approximately 7 for at least 30 seconds, preferably at least 1 minute, preferably at least 10 minutes;

c) collecting the extraction liquid, wherein the extraction liquid comprises less than 10 wt.%, by total weight of the extraction liquid, preferably less than 7 wt.%, preferably less than 5 wt.%, preferably less than 1 wt.% of sucrose;

d) separating the extraction liquid into a protein-rich fraction and a protein-poor fraction by dialysis employing a membrane with a molecular weight cut-off value of 50 kDa or less, preferably 25 kDa or less, more preferably 10 kDa or less, and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C.

[0131] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by contacting the beet root material with an extraction liquid which has a pH within the range of 5-9, preferably a pH within the range of 6-8, preferably a pH within the range of 6.5-7.5, preferably a pH of approximately 7 for at least 30 seconds, preferably at least 1 minute, preferably at least 10 minutes;

c) collecting the extraction liquid, wherein the extraction liquid comprises more than 10wt.%, by total weight of the extraction liquid, of sucrose, preferably more than 14 wt.%, preferably more than 16 wt.%;

d) separating the extraction liquid into a protein-rich fraction and a protein-poor fraction by dialysis employing a membrane with a molecular weight cut-off value of 50 kDa or less, preferably 25 kDa or less, more preferably 10 kDa or less, and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C.

[0132] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material;

b) processing the beet root material to obtain a liquid comprising beet root protein by subjecting the beet root material to a mechanical treatment which releases beet root juice;

c) collecting the beet root juice;

d) separating the beet root juice into a protein-rich fraction and a protein-poor fraction and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C. [0133] In embodiments, the present invention provides a method for producing a beet root protein composition, comprising the steps of:

a) providing a beet root material, preferably a sugar beet root material which has been or is being subjected to a pre-treatment used in the sugar manufacturing industry, such as a pre-treatment selected from the group consisting of thermal cell disintegration, pulsed electric field (PEF) treatment, fermentation, acidification, freezing and/or enzymatic treatment, preferably pulsed electric field treatment;

b) processing the beet root material to obtain a liquid comprising beet root protein by subjecting the beet root material to a mechanical treatment which releases beet root juice;

c) collecting the beet root juice;

d) separating the beet root juice into a protein-rich fraction and a protein-poor fraction and collecting the protein-rich fraction to obtain a beet root protein composition; and

e) optionally at least partially removing solvent from the beet protein composition to obtain a beet root protein composition comprising at least 5 wt.%, based on the total weight of the composition, beet root protein;

wherein the method is preferably performed at a temperature of less than 70°C, preferably of less than 50°C, most preferably less than 30°C.

[0134] In another aspect, the invention provides the beet protein compositions obtainable by the methods to isolate beet protein described herein.

[0135] As will be shown in the appended examples, the present inventors have surprisingly found that the beet root protein composition of the present invention has improved gelling properties. Such improved properties may include gelling at a lower concentration than protein compositions derived from other plants and/or a reduced influence of pH on the gelling behavior.

[0136] Thus, in another aspect of the invention, there are provided methods of forming a gel, comprising the steps of:

i) providing a beet root protein composition;

ii) providing an aqueous phase;

iii) blending the beet protein composition and the aqueous phase to obtain a gel-forming solution; and

iv) forming a gel from the gel-forming solution.

[0137] In preferred embodiments, the beet protein composition is the beet protein composition of the invention as described herein before.

[0138] In embodiments, step iv) comprises setting the pH of the gel-forming solution to more than pH 3.5, preferably more than pH 4, preferably more than pH 5.

[0139] In embodiments, step iv) comprises heating the gel-forming solution to a temperature of at least 60°C, preferably at least 70°C, more preferably at least 80°C for at least 5 seconds, preferably at least 30 seconds. In embodiments, the concentration of beet protein in the gel-forming solution is more than 1 % w/v, preferably more than 2% w/v, preferably more than 5% w/v.

[0140] In another aspect, the invention provides the gel obtainable by the methods of forming a gel provided herein. [0141] The present inventors have for the first time made available beet root protein in a form suitable/convenient for application in conventional food manufacturing. This beet root protein can be applied in various alimentary products for humans or feed products for animals to confer distinctive/beneficial functional and/or nutritional properties.

[0142] Hence, in another aspect, the invention thus provides a human alimentary product or an animal feed product comprising beet root protein.

[0143] In embodiments, the invention provides a human alimentary product or an animal feed product comprising more than 0.01 wt%, by total weight of the product, preferably more than 0.1 wt.%, or more than 0.5 wt.% beet root protein.

[0144] In embodiments, the human alimentary product or animal feed product comprises more than 0.01 wt.% (dry weight), preferably more than 0.1 wt.% (dry weight) or more than 0.5 wt.% (dry weight) beet root protein.

[0145] In a preferred embodiment the invention provides a human alimentary product or an animal feed product comprising the food ingredient composition of the invention. In embodiments, there is provided a human alimentary product or an animal feed product comprising more than 0.01wt%, by total weight of the product, preferably more than 0.1 wt.%, or more than 1 wt.% of the food ingredient composition of the invention. In embodiments, the human alimentary product or animal feed product comprises more than 0.1 wt.% (dry weight), preferably more than 0.5 wt.% (dry weight) or more than 1 wt.% (dry weight) of the food ingredient composition of the invention.

[0146] The human alimentary products and/or animal feed products described herein are preferably processed alimentary products or animal feed products, more preferably they are formulated alimentary products or animal feed products. In preferred embodiments the human alimentary products and/or animal feed products described herein do not comprise or consist of whole or sliced beet or beet root, spent beet pulp and/or molasses.

[0147] A highly preferred embodiment concerns processed, preferably formulated, human alimentary product or processed, preferably formulated, animal feed products comprising more than 0.01 wt%, by total weight of the product, preferably more than 0.1 wt.%, or more than 0.5 wt.% beet root protein; wherein the beet root protein has:

• more than 30% secondary structure, preferably more than 40%, or more than 60% secondary structure, wherein the secondary structure is in the form of an a-helix and/or a b-sheet and wherein at least 15%, preferably at least 20% or at least 30% of the secondary structure is in the form of an a-helix; and/or

• an aqueous solubility of more than 1 mg/ml, preferably more than 1 .5 mg/ml, wherein the solubility is determined at pH 7 and at an ionic strength of 8 mS/cm.

[0148] In embodiments, the human alimentary products and/or animal feed products described herein comprise less beet root pectin than beet root protein (by weight). In embodiments, they are substantially free or free of beet root pectin. Preferably the amount of beet root pectin is less than 0.5 times the amount of beet root protein (by weight), more preferably the amount of beet root pectin is less than 0.1 times the amount of beet root protein (by weight), most preferably the amount of beet root pectin is less than 0.01 times the amount of beet root protein (by weight). [0149] In highly preferred embodiments the human alimentary products and/or animal feed products comprise a structured aqueous phase comprising dispersed, preferably homogenously dispersed, beet root protein.

[0150] In highly preferred embodiments the human alimentary products and/or animal feed products comprise a matrix in the form of an emulsion, gel or foam, said matrix comprising the beet root protein as defined herein.

[0151] In embodiments, the human alimentary products and/or animal feed products are packaged.

[0152] In embodiments, the alimentary product is selected from the group consisting of beverages, doughs, processed meats, plant based meat replacements products, bakery products, confectionary products, cereal-based products, dairy products, dairy free replacements for dairy products, dressings, sauces and ice cream.

[0153] In embodiments, the alimentary product is a beverage, such as a protein shake.

[0154] In embodiments, the alimentary product is a solid food product, such as a protein bar.

[0155] In embodiments, the alimentary product is a dough.

[0156] In embodiments, the alimentary product is a bakery product, such as a cake or cookie.

[0157] In embodiments, the alimentary product comprises the beet protein composition of the invention and at least one food additive selected from the group consisting of preservatives, antioxidants, emulsifiers, (artificial) sweeteners, colourants, foaming agent, stabilizers and thickeners.

[0158] In embodiments, the alimentary product comprises the beet protein composition of the invention and at least one other plant derived protein source, preferably at least one other plant derived protein source selected from the group consisting of soybean protein, pea protein, bean protein, lupin protein and potato protein. In embodiments, the other plant derived protein source is provided in the form of optionally purified plant meal, or as an optionally coagulated protein isolate.

[0159] In embodiments, of the invention, the animal feed product comprises at least one further ingredient selected from the groups consisting of animal feed additives, animal feed grade formulating aids or excipients, and nutritional components.

[0160] In embodiments, of the invention, the animal feed product comprises at least one further ingredient selected from protein sources, energy sources, fat sources and mineral sources.

[0161] Suitable examples of protein sources include soybean meal, rapeseed meal, palm kernel meal, sunflower meal, peas, beans, lupins, fish meal, poultry meal (inclusive of feather meal) and blood plasma. Suitable examples of energy sources include corn, wheat, barley and rice. Suitable examples of fat sources include fish oil, tallow, corn oil, soy oil, rice bran oi, palm oil and canola oil. Suitable examples of mineral sources include calcium, magnesium, phosphorus, potassium, sodium, copper, selenium, zinc, iron, manganese, iodine, cobalt, chromium.

[0162] In embodiments, the animal feed product is a feed product adapted for a livestock animal, including avian species, aquatic species, and mammalian species. Examples of avian species include poultry species, such as turkey, duck and chicken. Examples of aquatic species include fish species, such as salmon, trout, tilapia, catfish and carp, and crustacean species, including shrimp and prawn. Preferably, the animal feed product is a feed product adapted for an aquatic species such as salmon, trout, tilapia, catfish, carp, shrimp or prawn.

[0163] As will be understood by the skilled person, the functional properties of the beet root protein composition of the present invention, and in particular the gelling properties, make it a suitable component of pet foods. In highly preferred embodiments the animal feed product is a pet feed product, preferably a wet pet feed product. In embodiments, the animal feed product is a pet feed product comprising a structured aqueous phase comprising dispersed, preferably homogenously dispersed, beet root protein, such as a gel.

[0164] The invention further provides the use of the food ingredient composition of the invention as an ingredient for a human alimentary product or animal feed product. Such uses may comprise the use of the food ingredient composition as a gelling agent, a viscosity modifying agent, a water binding agent, a fat binding agent, an emulsifying agent, a stabilizing agent or a foaming agent. Such uses may also comprise the use of the food ingredient composition for protein fortification. Examples

Example 1

[0165] This example describes the production of a food ingredient comprising sugar beet root protein in accordance with the invention from raw sugar beet root by a standalone process (i.e. not tied to a sugar manufacturing process).

[0166] Raw, washed sugar beet roots with a dry matter content of 23.2 wt.% and a protein content of 0.67 wt.% were provided and sliced into fries. The beet fries (2 kg) were added to 2 kg of extraction liquid (demi-water with 0.25% NaHSC>3) and blended for 1 minute in a kitchen blender (1 kg mixture at a time). The suspension was brought to pH 7 and left to stir for 1 hour, during which the pH was adjusted to keep it at pH 7. The extraction was performed at a temperature of 2-7°C (no active cooling was applied). The resulting mixture was filtered over a Buchner filter with a paper filter and the resulting filtrate (protein concentration of about 0.5 mg/ml) was centrifuged (30,000 g; 10 minutes, 20 °C) and filtered. The extracts were dialyzed over a 25 kDa membrane resulting in a liquid food ingredient comprising beet root protein in accordance with the invention.

[0167] Part of the liquid was freeze dried to provide a food ingredient comprising beet root protein in accordance with the invention in the form of a powder with a sugar beet root protein content of 29.5 wt.% by weight of dry matter (based on Kjeldahl analysis with a conversion factor of 6.25). This product is referred to as the beet root composition of example 1A in subsequent examples.

[0168] Another part of the liquid was subjected to pH precipitation by bringing the liquid to pH 4 using hydrochloric acid. The resulting liquid was centrifuged (12,400 g; 5 minutes) and the beet root protein collected by paper filtration; resuspended and freeze dried to provide a food ingredient comprising beet root protein in accordance with the invention in the form of a powder with a dry matter content of 74 wt.% by weight of dry matter (based on Kjeldahl analysis with a conversion factor of 6.25). This product is referred to as the beet root composition of example 1 B in subsequent examples.

Example 2:

[0169] As shown in Figure 2, the molecular weight distribution of the food ingredient comprising beet root protein composition of example 1A was characterized using reducing SDS-PAGE. As shown in Figure 3, a curve was fitted to the permeation measured for the marker (5 pL loaded of Precision Plus Protein™ All Blue Prestained Protein Standards #1610373) using GelAnalyzer 2010a software and used as a calibration curve for the other samples. The data for the points in Figure 3 is shown in the below table.

The beet root protein composition of example 1 A was characterized using the calibration curve as follows:

Example 3:

[0170] The solubility of the food ingredient comprising beet root protein composition of example 1 A was tested. The solubility experiment was performed in duplicate.

[0171] To test the effect of pH, the material was dispersed at 2 mg/ml_ protein (90.9 mg powder in 10 mL demi water) at 19 °C. The pH (6.6 ± 0.01 ) and conductivity (721 ± 16 pS/cm) of the solution were recorded. The pH was adjusted to pH 7 - 8 with 1 .5 M NaOH (addition of 3-5 pl_) and the conductivity was recorded. An aliquot was taken (0.5 mL). The solution was acidified to pH 6, 5, 4 and finally 3, using 0.1 M HCI (additions of 80 - 180 pL per step). After each pH adjustment, the conductivity and pH were recorded and an aliquot (0.5 mL) was taken. All aliquots were centrifuged in an Eppendorf centrifuge (14,000 rpm; 5 minutes). The supernatants were diluted (400 pL supernatant with 1 mL demi water) and the UV absorbance at 260 and 280 nm was recorded.

[0172] Figure 4 shows the solubility of the beet root protein of example 1A as a function of the pH, wherein 100% corresponds to 2 mg/ml and solubility above 2 mg/ml was not determined.

[0173] To test the effect of ionic strength and pH, the beet root protein of example 1A was dispersed in / solubilized in phosphate-citric acid buffers of pH 3/5/7 and ionic strengths of 3/8/13 mS/cm. This was performed in duplicate. Specifically, 4.8 - 6.1 mg powder was weighed in Eppendorf tubes, to which 517 - 616 pL buffer was added. The amount of buffer added was tailored to the amount of powder weighed in each Eppendorf tube, to reach a final protein concentration of 2 mg/mL in each tube. The protein dispersions were vortexed and subsequently stirred head-over- tail for 1 h. The final pH and conductivity of each dispersion/solution was recorded. The samples were then centrifuged in an Eppendorf centrifuge (14,000 rpm; 5 minutes). The supernatants were diluted (400 pL supernatant with 1 mL demi water) and the UV absorbance at 260 and 280 nm was recorded.

[0174] Figure 5 shows the solubility of the beet root protein of example 1A as a function of the pH and ionic strength, wherein 100% corresponds to 2 mg/ml and solubility above 2 mg/ml was not determined.

Example 4:

[0175] The structure of the beet root protein composition of example 1A was characterized at a concentration of 0.1 mg protein/mL in a quartz cuvet with a 1 mm path length in a Peltier Temperature controlled cuvet-chamber (20 +/- 0.2°C). The ellipticity was recorded using a spectropolarimeter (Jasco Corporation, Japan; f.e. type J-815) in the spectral range from 185-260 nm, with a detector voltage of 600-850V at 200 nm, a spectral resolution of 0.2 nm, a scan speed of 100 nm/min, a time-constant of 0.125 sec and a band width on 1 nm; spectra were recorded as averages of 16 scans, and subsequently corrected for that of a corresponding protein-free sample; subsequently inverse Fourier transformation was applied and the far-UV circular dichroism spectrum analyzed for the secondary structural element content using non-linear least-squares fitting procedure as described by de Jongh et al. (de Jongh HHJ, Goormaghtigh E, Killian JA (1994) Analysis of circular dichroism spectra of oriented protein-lipid complexes: Toward a general application. Biochemistry 33: 14521-14528).

[0176] The spectrum shows Zero-crossing around 202 nm; and distinct negative extremes around 208 and 222 nm. The spectrum contains predominantly globular proteins with (ensemble-averaged) structures of 35-40% b-strand, 25-30% a-helix and 30-40% random coil.

Example 5:

[0177] The gelling and foaming behavior of the beet root protein composition of example 1 A was evaluated. Tests were performed at 6% protein content (w/v). Beet protein was dispersed in 1 ml buffers or in 1 ml demi water. The following buffers were used: pH 3, 5 and 7; each pH at a conductivity of 3, 8 and 13 mS/cm. Buffers were made with Na ₂ HPC> ₄ , citric acid and NaCI.

[0178] After addition of the protein to demi water the conductivity was measured (5.7 mS/cm). After addition of the protein to demi water or buffer the pH was measured and is shown in the below table.

[0179] Samples were heated for 20 min at 90°C, after cooling they were stored in de fridge. After

24h the gelling and foaming behavior was evaluated, leading to the conclusions shown in the below table.

[0180] The gel strength of the dispersion in demiwater and in buffer at pH 3, 5 and 7 (3mS/cm conductivity) was measured with a TAXT analyzer (TA instruments), spindle TA8, 5 kg load, 1 mm/s test speed, 1 g trigger force and 5mm penetration depth. The results are shown in Figure 7.

Example 6:

[0181] The susceptibility of the beet root protein composition of the invention to enzymatic degradation reflecting stomach (pepsin) and digestive tract (pancreatin) conditions was evaluated. Whey protein and a pea protein isolate are included for comparison.

[0182] Production of sugar beet root protein: Food ingredient comprising sugar beet root protein in accordance with the invention was obtained according to the following procedure:

• 20 g sugar beet cossettes + 20 g demiwater + 0.75 wt% NaHSC>3;

• Blended in kitchen blender to a smooth slurry (pH ~6.2);

• pH adjusted to 7.0 using 0.1 N NaOH and stirred for 1 hour at RT;

· Filtration of slurry over Bitch ner-filter;

• Filtrate dialyzed in tubing (Molecular weight cut-off 10 kDa); 3 times dialysis in 1 L demi water; 5 hrs each at 4 °C.

[0183] The isolation was performed a second time in a similar manner as described above but with 60 g sugar beet cossettes. Both batches were combined resulting in a total of 85 mL of liquid food ingredient comprising sugar beet root protein in accordance with the invention (hereafter referred to as the beet root protein composition of example 6).

[0184] Pepsin stock-solution: 4 mL of a 0.5 mg/mL Pepsin (Sigma EC 3.4.231 - 2500-3500 units/mg) in demiwater was prepared and the pH adjusted to ~1 .9 using 10.1 M HCI.

[0185] Pancreatin stock-solution: 4 mL of a 5 mg/mL Pancreatin (ICN Nutritional Biochemicals, hog pancreas 5XUSP) in 10 mM phosphate-buffer pH 7.5.

[0186] Both enzyme-solutions were freshly prepared when used.

[0187] Experimental procedure Pepsin-digestion:

2 mL beet root protein composition of example 6 (~0.15 mg/mL) was adjusted to pH ~1 .9 using 0.1 M HCI (~40 pL)

• 0.2 mL pepsin stock-solution was added and the composition was mixed at RT

• Samples of 0.4 mL were taken at t=0, 1 , 5 and 20 minutes and immediately quenched by the addition of 50 pL 0.1 M NaOH resulting in a pH between 7.5-8.0 (checked by pH-paper)

• The quenched sample was transferred to a quartz cuvet (Hellma) with a path length of 1 mm and the ellipticity of the sample was recorded at a wavelength of 222 nm (band width 1 nm, time constant 4 sec, T=20°C) on a Jasco 815 spectropolarimeter.

[0188] Experimental procedure Pancreatin-digestion:

• 2 mL beet root protein composition of example 8 (~0.15 mg/mL) was adjusted to pH 7.5 using 0.1 M NaOH (~15 pL)

• 0.15 mL pancreatin stock-solution was added and the composition was mixed at RT

• Samples of 0.4 mL were taken at t=0, 1 , 2 and 5 hours

• The aliquots were transferred directly into a quartz cuvet (Hellma) with a path length of 1 mm and the ellipticity of the sample was recorded at a wavelength of 222 nm (band width 1 nm, time constant 4 sec, T=20°C) on a Jasco 815 spectropolarimeter.

[0189] For comparison the same digestion procedure was repeated with 0.15 mg/mL whey protein (Bipro; Davisco inc) and a 0.15 mg/mL pea protein isolate (Cosucra)

[0190] The Circular Dichroism-intensity shown in Figures 8 and 9 is normalized to the intensity at t=0.

[0191] The CD-intensity recorded correlates to the protein digestion as the CD-intensity is the result of the secondary structure content and the number of peptide bonds (as CD detects the chirality around the peptide bond) and when proteins become enzymatically degraded the number of peptide bonds decreases and secondary structure will disappear as smaller peptide fragments will lose their structural stability. 222 nm was selected as wavelength to monitor the secondary structure since the dominant secondary structure type a-helix and b-strand have profound (negative) intensity, whereas non-structured polypeptides have almost zero CD-intensity at this wavelength.

[0192] It can be observed for the beet root protein composition of example 6 that the half-time for pepsin-degradation (Figure 8) is about 12 minutes and that for pancreatin-digestion about 4 hours (Figure 9) under these conditions. It can be observed that it is slightly slower than observed for whey protein from cow’s milk , but significantly faster than observed for a relevant pea protein isolate.

[0193] In conclusion, these results show that the food ingredient compositions comprising beet root protein of the present invention are susceptible to stomach and intestinal degradation and thus has significant nutritional value; better than pea protein (which is a high-performing representative of plant-based protein).

Example 7:

[0194] The foam formation and stabilization characteristics of the beet root protein composition of example 6 was tested.

[0195] Experimental procedure foam formation and stabilization. 50 ml_ of beet root protein composition of example 8 (~0.15 mg/mL) was placed in a 3.5 cm diameter transparent tube and stirred at room temperature for 2 minutes using a fan-flutter at a rotation speed of 2500 rpm. Images were taken at t=0 and after 5 and 30 minutes and the foam height was evaluated and expressed as the fraction of foam relative to the volume of the beet root protein composition of example 8.

[0196] The used beet root protein composition of example 6 was diluted to ~0.10 mg/ml and ~0.05 mg/mL using demi water and the foaming experiment was repeated with a total volume of 50 mL.

[0197] It can be observed from the results presented in Figures 10 and 1 1 that a foam can be obtained using the food ingredient compositions comprising beet root protein of the present invention that has a profound stability over a long time period.

Example 8:

[0198] This example describes the production of a food ingredient comprising sugar beet root protein in accordance with the invention from raw sugar beet root by a proces integrated into a sugar manufacturing process.

[0199] Raw juice was collected from the sugar factory of Suiker Unie, Dinteloord. The process conditions in the sugar factory were adapted from conventional sugar manufacturing as follows. The diffusion temperature was set to 60-65 °C and the aqueous extraction was performed at pH 6. The resulting raw juice was collected and 1 % of bisulfite was added. The pH of the raw juice samples was adjusted to 7 and the samples were centrifuged to clarify the juice. From the clarified juice beet protein powder was made using two distinct methods: pH precipitation and membrane separation.

[0200] In the pH precipitation method the clarified juice was brought to pH 3.5. The acidified juice centrifuged (30020 g / 15 min / 4 °C). The pellet was harvested and washed with demi water. The washed pellet was resuspended, brought to pH 7 with 1 M NaOH and freeze dried. This product is referred to as the beet root composition of example 8A in subsequent examples.

[0201] In the membrane separation method the clarified raw juice was concentrated (CF 5.0) and dialyzed (W/F 5.5.) over a 25 kDa membrane (GR60PP Alfa Laval, 1 m cell). The concentrated and dialyzed raw juice (the retentate) was freeze dried. This product is referred to as the beet root composition of example 8B in subsequent examples.

[0202] Both methods yielded a beet protein powder containing approximately 35% protein (based on Kjeldahl analysis with a conversion factor of 6.25).

[0203] Furthermore, the beet root composition of example 8A was found to have a galacturonic acid content, determined after polysaccharide hydrolysis of about 1 .5% (by total weight of the sample).

Example 9:

[0204] The foaming properties of the beet root protein composition produced according to example 8B were investigated with a TURBISCANLAB (Formulaction, Toulouse, FR). Foams were created from a 0.5 % (w/v) beet root protein composition with 0.1 % (w/v) NaCI at pH 3, 4.5 and 7, using two different methods: the small syringes method and the utraturax method. In the small syringes method an aliquot of 3 ml_ protein composition were placed into a syringe (10 ml_, Henke-Sass Wolf, DE). A second syringe was filled with 6 ml_ of air. The syringes were connected via a short plastic tubing of 5 cm. Foaming was induced by pushing the plunger subsequently back and forth for 20 times and 40 seconds in total. After that, the foam was placed immediately inside a measurement vial and measured. In the ultraturax method a 15 ml_ protein dispersion was placed inside a measurement vial of 20 ml_. Then an S 25 N - 18 G dispersing tool (IKA, Staufen, DE) connected to a T 25 digital ULTRA-TURRAX ® was submerged in the protein dispersion. Protein dispersions were agitated at the speed of 12400 RPM for 20 seconds and immediately measured.

[0205] The results are displayed in Figures 12 and 13 and show the excellent foam stability obtained using beet root protein compositions according to the invention.

Example 10:

[0206] The gelling behaviour of the beet root protein composition produced according to example 8B was investigated with an Anton Paar 302 Rheometer . The gelling behavior test was performed with a 6 % (w/v) protein solution with 0.1 % (w/v) NaCI. The sample (1 ml) was placed in the cup of the rheometer. After lowering the inner cylinder, the top surface of the liquid was covered with paraffin oil to prevent sample dehydration during heating. The protein dispersion was heated from 20°C to 90°C at a rate of 1 C min. During the complete heating cycle, visco-elastic properties were measured at constant strain (1 %) and frequency (1 Hz).

[0207] Figure 14 shows the evolution of the complex viscosity with temperature, clearly indicating gel formation.

Example 11:

[0208] The gel strength of different gels prepared with the beet root protein composition produced according to example 8B was determined with a TAXT analyzer (TA instruments). The gel strength test were performed with a 4, 6, 8 and 10 % (w/v) protein solution with 0.1 % NaCI. The sample (1 ml_) was pipetted in a eppedorf tube (2ml_, inner diameter of 8.2 mm) and heated 20 min at 90°C. The sample was subsequently stored in the fridge and after 24h the gel strength was measured with a spindle TA8 (diameter of 6.35 mm), 5 kg load, 1 mm/s test speed, 1 g trigger force and 5mm penetration depth. The results are shown in the below table

Example 12:

[0209] The emulsification properties of the beet root composition produced according to example 8A were determined at different pHs; 3, 4.5 and 7. First, a 0.5 % (w/v) protein solution with 0.1 % (w/v) NaCI solution was prepared at pH 7. Sunflower oil was added to the protein solution to a volume fraction of 0.1 . A pre-emulsion was made in an Ultra-turrax Tube drive P control (IKA, Staufen, DE) at 6000 rpm for20 seconds. The pre-emulsion was then emulsified by passing through a high-pressure homogenizer (PandaPlus2000, GEA, Parma, IT) at a pressure of 1200/60 bar (First stage/second stage) one time. The pH of the emulsion was adjusted with 1 and 0.1 M HCI and the droplet size distribution of the emulsions was measured over time (after 0, 1 and 7 days) through laser diffraction by the with a Mastersizer 3000 (Malvern instruments, Malvern, UK).

Comparative example 13:

[0210] This comparative example describes the production of a food ingredient comprising sugar beet root protein not in accordance with the invention, using the method described in Ghoorchi, T., and S. Arbabi. "Study of protein characteristic of five feeds by CNCPS model." Asian Journal of Animal and Veterinary Advances 5.8 (2010): 584-591.

[0211] Spent sugar beet pulp was collected from the sugar factory of Suiker Unie in Dinteloord. The sugar beet pulp was dried in a 60°C oven to a dry matter content of over 95%. Subsequently, the dried sugar beet pulp was milled using a Pulverisette 14 with a 0.2 mm sieve. The dried sugar beet pulp had a protein content of approximately 9% (based on Kjeldahl analysis with a conversion factor of 6.25).

[0212] This composition was tested for functional properties employing the same methods as described in examples 10-13. Foam creation was not possible, emulsions were unstable and gelling was not possible. Attempts to produce a gel as described in example 11 resulted in the formation of a wet powder. Furthermore, the gel strength tests showed no correlation between protein concentration and strength, indicating that the protein were not responsible for gelation properties and primarily the hardness of the wet powder was being measured.

[0213] Determination of the secondary structure characteristics of the beet root protein was attempted. In order to extract the protein, 5 mg of the powder was dispersed in 1 ml 5 mM phosphate-buffer (PH 7.0) and extensively stirred on a vortex mixer and exposed to a micro-turrax for 5 minutes. After settling of the sediment the supernatant was filtered through a 25 micrometer filter to remove further insoluble particles. Only slightly over half of the proteins were extractable (on dry weight) and passed through a 25 micrometer filter and resulted in CD spectra typical for highly aggregated protein material. The CD spectrum shows a minimum at wavelengths above 222 nm indicating absorption flattening, caused by a non-ideal distribution of protein in the solution. As such the CD spectrum is distorted to such an extent that the secondary structure content cannot be determined and the sample is considered to have a secondary structure content of 0%.

[0214] Hence, it can be seen that prolonged exposure to elevated temperatures is detrimental to the functional properties and secondary structure characteristics of beet root protein.