High content yeast protein chilled products

Abstract

The present invention relates to a chilled product comprising both a milk and / or plant protein source and a yeast protein source.

Description

[0001] HIGH CONTENT YEAST PROTEIN CHILLED PRODUCTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a chilled product comprising both a milk and / or a plant protein source and a yeast protein source.

[0004] BACKGROUND TO THE INVENTION

[0005] Chilled dairy and / or plant-based products such as yoghurt and plant-based desserts are common products consumed globally. Depending on the particular product, various other ingredients such as sweeteners and / or other flavourings such as cocoa powder may be added to achieve the desired taste profile. Protein is also crucial for muscle health, mobility, and cognitive ability, and hence in recent years there has also been a trend in consumer preferences for products comprising high levels of protein, especially amongst health conscious consumers. Typically, the protein in chilled products has been provided by a milk protein source.

[0006] In recent years, the need to tackle urgent global challenges like food security and sustainability has driven food companies and academic groups to seek alternative protein sources that can replace animal-based ones in food products. This urgency arises from the projected increase in the world population from the current 8 billion to nearly 10 billion by 2050. The majority of efforts in this direction have primarily focused on plant proteins, resulting in the introduction of a range of liquid (e.g. ready- to-d rink beverages, coffee creamers), semi-solid (e.g. yogurts, cooking creams), and solid foods (e.g., meat and fish analogues) based on these ingredients in the market (D. J. McClements & Grossmann, 2021).

[0007] While increasing the use of plant proteins in human nutrition will be of paramount importance in the near future in order to ensure protein supply in a sustainable manner, processing of plant-based foods is often challenging, even more so when it comes to liquid products such as milk analogues (Qamar, Manrique, Parekh, & Falconer, 2020). This is due to the fact that most plant protein ingredients are characterized by highly ordered tertiary and quaternary structures, and thus low solubility in water, which often results in poor overall functionality, unless these are subjected to specific treatments, such as physical, chemical, enzymatic ones, or combinations thereof (Amagliani, Silva, Saffon, & Dombrowski, 2021). Furthermore, heat treatments of plant proteins above their denaturation temperature (e.g., UHT) may trigger the occurrence of phenomena such as sedimentation, fouling and / or gelation inside the heat exchangers, major hurdles towards the development of shelf-stable products (David Julian McClements, Newman, & McClements, 2019). It should also be taken into consideration that, with regard to plant-based products, meeting consumer expectations in terms of nutritional and sensory properties is a complex exercise, with only proteins derived from soy or potato displaying a biological value similar to that of animal proteins such as milk- and egg-based ones (Day, 2013), and with many commercial products having low levels of acceptance due to their poor flavour and mouthfeel (Moss et al., 2022). Indeed, the replacement of milk by plant protein sources, such as cereals proteins, may be perceived as bitter and / or astringent with slimy texture which impact the sensory experience.

[0008] In addition to plant proteins or milk protein, single cell proteins (SCPs) represent a promising option for the development of sustainable, nutrient-rich food products which could enable to cater for the growing world population. The term SCPs refers to proteins derived from microorganisms such as bacteria, fungi, yeasts or algae. These are obtained via fermentation and offer several advantages, including (i) rapid growth rates (typically 1 to 4 days), thus ensuring fast and efficient protein production; (ii) reduced land and water requirements compared to livestock farming, since fermentation is typically performed in bioreactors, as well as lower greenhouse gas emissions; (iii) ability to grow on diverse feedstocks, including a variety of agri-food by-products, which enables the valorization and efficient utilization of resources that might otherwise go to waste, thus reducing environmental impact and promoting sustainability; (iv) high protein concentration, with values which in most cases range from about 40 to >70 wt% on a dry weight basis, depending on microorganism, species and fermentation conditions used (Ritala, Hakkinen, Toivari, & Wiebe, 2017).

[0009] Concerning human nutrition, yeasts are by far the most interesting among SCPs, due to (i) their established history of food use (e.g., spreads, dietary supplements, as processing aids in alcoholic fermentation and baking, and as flavouring agents), which facilitates consumer acceptance; (ii) regulatory aspects, with two species (i.e., Saccharomyces cerevisiae and Candida utilis or Cyberlindnera jadinii, commonly known as Torula) being approved for use in human food in both Europe and the United States; (iii) their good commercial availability.

[0010] The preparation of chilled dairy and / or plant-based products comprising yeast protein ingredients have not to our knowledge been reported in the literature. However, there is a significant interest in developing such products due to the advantages associated with the use of yeast protein described above. One potential challenge with the use of yeast protein is, however, ensuring that the product remains stable and acceptable in taste to consumers, who are accustomed to a taste profile provided by proteins of animal and plant origin.

[0011] In view of the above, it would be desirable to prepare chilled products comprising yeast proteins that are stable and exhibit an acceptable taste profile to consumers. As discussed above, such products would be cheaper to produce whilst also being more environmentally friendly due to the reduced land and water requirements associated with the production of yeast protein.

[0012] Against the above background, the inventors unexpectedly found that the chilled products of the present invention comprising both a milk protein source and a yeast protein source were stable and had an acceptable taste profile, thus providing a more environmentally friendly product that is also acceptable to consumers. In addition, the inventors unexpectedly found that the plant-based products of the present invention comprising both a plant protein source and a yeast protein source were stable and had an acceptable taste profile, thus providing a more environmentally friendly product that is also acceptable to consumers.

[0013] SUMMARY OF THE INVENTION

[0014] According to one aspect of the invention, a chilled product is provided, the product comprising a milk and / or a plant protein source and a yeast protein source, optionally wherein the total protein content of the chilled product is 4 wt.% or higher.

[0015] The milk protein source may be any milk protein source suitable for a chilled product. In preferred embodiments, the milk protein source is chosen from the group consisting of milk, milk powder, milk protein concentrate, milk protein isolate, and mixtures thereof. In a particularly preferred embodiment, the milk protein source comprises skimmed milk, milk protein isolate, and optionally skimmed milk powder.

[0016] The milk protein source may provide any suitable amount of the total protein content of the chilled product. In preferable embodiments, the milk protein source may provide at least about 10 wt.%, preferably at least about 30 wt.%, more preferably at least about 50 wt.%, yet more preferably at least about 70 wt.%, most preferably at least about 80 wt.% of the total protein content of the chilled product.

[0017] The plant protein source may be any plant-based protein source suitable for a chilled product. In preferred embodiments, the plant protein is chosen from the group consisting of pulse protein, cereal protein, leguminous protein, single cell protein, or a combination thereof. In a particularly preferred embodiment, the plant protein is chosen from the group consisting of pea protein, fava bean protein, soy protein, canola protein, lentil protein, or a combination thereof. Advantageously, the plant protein is soy protein.

[0018] According to the invention, the plant proteins are available as hydrolysates, native protein, concentrates and isolates. The yeast protein source may similarly be any yeast protein source suitable for use in a chilled product. In preferable embodiments, the the yeast protein source is selected from the list consisting of yeast biomass, yeast protein concentrate, yeast protein isolate, or mixtures thereof. The yeast protein source is preferably derived from Saccharomyces cerevisae, Candida utilis, or combinations thereof.

[0019] The yeast protein source may be present in any suitable amount. Preferably, it is present in an amount of no more than 15 wt.%, preferably of no more than 10 wt.%, more preferably of no more than 5wt% based on the total weight of the chilled product. Preferably, it is present in an amount from 1 to 15 wt.%, preferably from 1 to 10 wt.% based on the total weight of the chilled product.

[0020] The total protein content of the chilled product may be any suitable amount. In some embodiments, the total protein content is greater than about 4 wt.%. Preferably, the total protein content is 5 wt.% or higher, more preferably 6 wt.% or higher, even more preferably 7 wt.% or higher, yet more preferably 8 wt.% or higher, still more preferably 9 wt.% or higher, and most preferably 10 wt.% or higher.

[0021] The chilled product preferably also comprises at least one non-proteic thickening agent. The non-proteic thickening agent may be any suitable thickening agent. In preferable embodiments, the thickening agent is selected from the list consisting of xanthan gum, carrageenan, starch, modified starch, and mixtures thereof. Preferably the starch, if present, is corn starch or modified corn starch.

[0022] The chilled product may contain further ingredients depending on the nature of the particular product. For example, in some embodiments, the chilled product may further comprise cocoa powder to impart a chocolate flavour. In further embodiments, the chilled product may comprise one or more plant-based liquid ingredient chosen from the group consisting of plantbased cream alternative, plant-based milk alternative, plant-based water or a combination thereof, preferably plant-based cream alternative to enrich the texture of the plant-based product.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Protein profile (ten most abundant proteins) of yeast protein concentrate (YPC; a, b) and yeast biomass (YB; c, d), as determined by UPLC-MS / MS.

[0025] Figure 2: Confocal laser scanning microscopy images of yeast protein concentrate (YPC) and yeast biomass (YB) dispersions (3 wt% protein) before and after homogenization. Proteins (green) were fluorescently labelled with Fast Green FCF. Figure 3: Confocal laser scanning microscopy images of yeast protein concentrate (YPC; a, b) and yeast biomass (YB; c, d) dispersions (3 wt% protein). Proteins (green) (e.g. cf. arrow labelled “prot.”), lipids (red) (e.g. cf. arrow labelled “fat”) and fibers (chitin and glucans, blue) (e.g. cf. arrow labelled “fib.”) were fluorescently labelled with Fast Green FCF, Nile Red and Calcofluor White, respectively. Images of the dispersion with the filters for the three stains combined (a, c) and the filter for Calcofluor White only (b, d) are shown.

[0026] Figure 4: Solubility of yeast protein concentrate (YPC) and yeast biomass (YB) dispersions (1 wt% protein) in the pH range 2-9.

[0027] Figure 5: Pictures assessing the texture of plant-based chilled set yogurt comprising high content of yeast protein in view of a plant-based chilled set yogurt not comprising yeast protein.

[0028] DETAILED DESCRIPTION

[0029] Definition of terms

[0030] As used herein, the term “amino acid” as used herein includes free form amino acids, or bound form of amino acids in molecules between 2 and 20 amino acids (referenced herein as “peptides”), and also in longer chains of amino acids (i.e. proteins). Small peptides, i.e., chains of 2 to 10 amino acids, are suitable for the product alone or in combination with other proteins. The “free form” of amino acid means the monomeric form of the amino acid. When the term “free amino acid” is used, it refers exclusively to “free form”, i.e. the monomeric form of the amino acid.

[0031] Each amino acid disclosed herein can be present in the product as only one type of the amino acid or as a mixture of one or more types of the amino acid, for example one or more (i) peptides containing the amino acid, (ii) longer chains of amino acids (i.e. proteins) including the amino acid, or (iii) free form of the amino acid. For example, a disclosure of “composition comprising an aromatic amino acid” or “product comprising an aromatic amino acid” constitutes a disclosure of aromatic amino acids only in free form, a disclosure of aromatic amino acids only bound to other amino acids, and a mixture of aromatic amino acids in free form and aromatic amino acids bound to other amino acids. Similarly, in embodiments where the referenced amino acid is in peptides or proteins, optionally the product can have substantially no free form of the referenced amino acid.

[0032] As used herein, the term “an essential amino acid (EAA)” or an indispensable amino acid as used means an amino acid that cannot be synthesized de novo by the organism at a rate commensurate with its demand, and thus must be supplied in its diet. Of the twenty-one amino acids common to all life forms the following nine amino acids are considered essential amino acids in the human diet which include phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine. Six other amino acids which are considered conditionally essential in the human diet are arginine, cysteine, glycine, glutamine, proline and tyrosine. There are six amino acids that are non-essential (dispensable) in human diet, and these six non-essential amino acids are alanine, aspartic acid, asparagine, glutamic acid, serine and selenocysteine.

[0033] As used herein, the term “an aromatic amino acid (AAA)” refers to an amino acid that includes an aromatic ring. Examples of aromatic amino acids include: Phenylalanine (symbol Phe or F); Tryptophan (symbol Trp or W); Tyrosine (symbol Tyr or Y); and Histidine (symbol His or H).

[0034] As used herein, the term “a branched chain amino acid (BCAA)” means an amino acid having an aliphatic side-chain with a branch (a central carbon atom bound to three or more carbon atoms). Among the proteinogenic amino acids, there are three BCAAs: leucine (Leu or L), isoleucine (lie or I), and valine (Vai or V). Non-proteinogenic BCAAs include 2- aminoisobutyric acid.

[0035] The product according to the invention is a chilled storage product. By “chilled storage”, it is understood a product which has a shelf-life of several days when stored under chilled conditions. The term “chilled conditions” refers to temperatures ranging from 2°C to 14°C, preferably from 2°C to 10°C, more preferably from 4°C to 8°C. In particular, a chilled storage product has a shelf-life of at least 25 days, preferably of at least 30 days when stored under chilled conditions. These storage temperatures relate to the storage of the product before being commercially obtained by an end consumer. Generally, the end consumer is advised to store the product under the same chilled conditions until consumption, for example in a refrigerator.

[0036] As used herein, the chilled product is selected from the group consisting of a plant-based fermented yogurt, a plant-based ready-to-drink beverage, a plant-based fermented beverage, a plant-based fermented ready-to-drink beverage, a plant-based dessert, a plant-based fermented dessert, an hybrid fermented dessert, a fermented dessert, an hybrid fermented yogurt, an hybrid ready-to-drink beverage, an hybrid fermented beverage, an hybrid fermented ready-to-drink beverage, a yoghurt, a plant-based mousse, a dairy-based mousse, an hybrid mousse or a dairy-based dessert. Preferably, the chilled product is a dairy-based dessert.

[0037] The term “hybrid” as mentioned above refers to a product comprising both dairy product and plant-based product, especially a product comprising milk and plant-based milk. As used herein, “plant based” refers to parts of plants that are consumed by humans or other animals as food, for example cereals, fruits and / or vegetables.

[0038] As used herein, "added sugar" refers to caloric mono- and disaccharides added during manufacture of a product, such as glucose, sucrose, maltose, fructose, which are not naturally found in the dairy component. For instance, lactose is naturally found in milk, therefore, for the purpose of this disclosure, lactose is not taken into account in "added sugar".

[0039] As used herein, the term "modified starch" refers to a native starch which has been treated physically, enzymatically, or chemically to change its properties. For example, “modified corn starch” refers to corn starch that has been treated physically, enzymatically, or chemically to change its properties.

[0040] As used herein, the term “non-proteic thickening agent” refers to ingredients other than proteins that increase the viscosity of the product. In a preferred embodiment, the non-proteic thickening agent is a polysaccharide.

[0041] As used herein, the term “protein” as used herein includes molecules between 2 and 20 amino acids (referenced herein as “peptides”), and also includes longer chains of amino acids (i.e. molecules having more than 20 amino acids). Small peptides, i.e., chains of 2 to 10 amino acids, are suitable for the products alone or in combination with other proteins. In a preferred embodiment, the term “protein” refers to molecules having more than 20 amino acids only.

[0042] The “free form” of an amino acid or “free amino acid” is the monomeric form of the amino acid. Suitable amino acids include both natural and non-natural amino acids.

[0043] As used herein, the term “vegan” refers to an edible composition which is entirely devoid of animal products, or animal derived products.

[0044] As used herein, the term “vegetarian” refers to an edible composition which is devoid of meat, including fish.

[0045] As used herein, the term “yeast protein concentrate” refers to an ingredient comprising whole yeast cells in a non-living state and / or components / fragments derived from said yeast cells and comprising from 70.0 to 90.0wt.% yeast proteins.

[0046] As used herein, the term “yeast protein isolate” refers to an ingredient comprising whole yeast cells in a non-living state and / or components / fragments derived from said yeast cells and comprising from 90.1 to 99.9wt.% yeast proteins. As used herein, the term “yeast biomass” refers to an ingredient comprising whole yeast cells in a non-living state and / or components / fragments derived from said yeast cells and comprising from 40.0 to 69.9wt.% yeast proteins.

[0047] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0048] Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. All publications mentioned in the specification are herein incorporated by reference.

[0049] As used in this specification, the words “comprises”, “comprising”, and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean “including, but not limited to”. The terms “comprises”, “comprising”, and similar words also include the term “consisting of”.

[0050] As used herein, the term “and / or” used in the context of “X and / or Y” should be interpreted as “X,” or “Y,” or “X and Y.”. Similarly, “at least one of X or Y” should be interpreted as “X,” or “Y,” or “both X and Y.”. For example, “pulses and / or cereals” means “pulses” or “cereals” or “both pulses and cereals”.

[0051] The practice of the present invention will employ, unless otherwise indicated, conventional techniques which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0052] Numeric ranges are inclusive of the numbers defining the range and all percentages disclosed herein are on a w / w basis, unless stated otherwise. Where components of the chilled product are given a wt.% value, these are relative to the total weight of the product.

[0053] As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0054] Milk protein source The chilled product may comprise a milk protein source. Any milk protein source may suitably be used.

[0055] Examples of suitable milk protein sources include milk, milk powder, milk protein concentrate, milk protein isolate, whey protein (i.e. whey protein isolate, whey protein concentrate, whey protein hydrolysate), milk protein, milk protein hydrolysate, microparticulated whey, caseinate, micellar casein, acid whey, sweet whey, modified sweet whey, fractions of whey protein, betalactoglobulin concentrate, beta-lactoglobulin isolate, and mixtures thereof. “Modified sweet whey” refers to sweet whey from which the caseino-glycomacropeptide has been removed. For example, the milk powder may be skimmed milk powder.

[0056] In preferable embodiments, the milk protein source is chosen from the group consisting of milk, milk powder, milk protein concentrate, milk protein isolate, whey protein and mixtures thereof. Advantageously, the milk protein source is chosen from the group consisting of skimmed milk, skimmed milk powder, milk protein isolate, whey protein and mixtures thereof.

[0057] In particularly preferred embodiments, the milk protein source comprises skimmed milk, milk protein isolate, whey protein and optionally skimmed milk powder.

[0058] The milk protein source may provide any percentage of the total protein content of the chilled product. The total protein content of the product may be measured by any suitable method known in the art. Preferably, the total protein content may be measured by the Kjeldahl method according to the AOAC Official Method 930.29 (AOAC, 2005) using the nitrogen-protein conversion of 6.25 as is described in further detail below.

[0059] In some embodiments, the milk protein source provides at least about 10 wt.%, preferably at least about 30 wt.%, more preferably at least about 50 wt.%, yet more preferably at least about 70 wt.%, most preferably at least about 80 wt.% of the total protein content of the chilled product. Preferably, a higher percentage of the total protein content is provided by the milk protein source than the yeast protein source.

[0060] In further preferred embodiments, the chilled product comprises milk, preferably skimmed milk, in an amount of from about 50 wt.% to about 99 wt.%, preferably from about 60 wt.% to about 99 wt.%, more preferably from about 70 wt.% to about 95 wt.%, yet more preferably from about 80 wt.% to about 95 wt.% and most preferably from about 85 wt.% to 95 wt.% based on the total weight of the chilled product.

[0061] In further preferred embodiments, the chilled product comprises milk protein isolate in an amount of from about 1 wt.% to about 20 wt.%, preferably from about 5 wt.% to about 15 wt.%, more preferably from about 5 wt.% to about 10 wt.%, and most preferably from about 6 wt.% to 8 wt.% based on the total weight of the chilled product.

[0062] Plant protein source

[0063] The chilled product may comprise a plant protein source. Any plant protein source may suitably be used.

[0064] In one embodiment, the plant protein is selected in the group consisting of pulse protein, cereal protein, leguminous protein, single cell protein, or a combination thereof.

[0065] In one preferred embodiment, the plant protein is selected in the group consisting of pea protein, fava bean protein, soy protein, canola protein, lentil protein, oat protein, wheat protein, rice protein, corn protein or a combination thereof. Advantageously, the plant protein is soy protein.

[0066] According to the invention, the plant proteins are available as hydrolysates, native protein, concentrates and isolates.

[0067] The term “isolate” means that other non-protein components have been partially removed to "isolate" the protein. Isolates are typically around 80% protein (dry basis). This is calculated using the Kjeldahl method.

[0068] In one more preferred embodiment, the plant protein is soy protein isolate.

[0069] In one embodiment, the chilled product comprises from 1 to 10wt%, preferably from 1 to 8wt%, more preferably from 1 to 5 wt.% of plant protein with respect to the total weight of chilled product.

[0070] Yeast protein source

[0071] The chilled product further comprises a yeast protein source. Any yeast protein source may suitably be used.

[0072] In some embodiments, the yeast protein source comprises at least 40wt.%, preferably at least 50wt.%, more preferably at least 60wt.%, even more preferably at least 70wt.% yeast proteins.

[0073] In preferred embodiments, the yeast protein source is selected from the list consisting of yeast biomass, yeast protein concentrate, yeast protein isolate, and mixtures thereof.

[0074] In a particularly preferred embodiment, the yeast protein source is yeast protein concentrate.

[0075] In some embodiments, the yeast protein concentrate comprises 70 wt.% to 90wt.% yeast proteins. Compared to biomass, yeast protein concentrates are advantageous as their protein content is higher, meaning that less ingredient is required for stabilization minimizing undesirable viscosity increase and as their flavour is more neutral. Compared to yeast protein isolates, yeast protein concentrates are advantageous due their higher availability.

[0076] The yeast protein source may be derived from any suitable yeast species. In preferred embodiments, the yeast protein source is derived from yeast from the genus Saccharomyces, Candida, and / or combinations thereof. In a particularly preferred embodiment, the yeast protein source is derived from Saccharomyces cerevisae, Candida utilis and / or combinations thereof.

[0077] Likewise, the yeast proteins of the yeast protein source and / or the chilled product are derived from yeast from the genus Saccharomyces, Candida and combination thereof. In some further preferred embodiment, the yeast proteins of the yeast protein source and / or the chilled product are derived from Saccharomyces cerevisae, Candida utilis and combination thereof.

[0078] Yeast protein rich ingredients are advantageous as they provide beneficial nutrients: minerals, amino acids and so on. In addition, they were unexpectedly found to impart limited sensory defects in the product following reconstitution and hence provided good sensory properties.

[0079] In preferred embodiments, the yeast protein source is very low in or is free from mannoprotein content. The yeast protein source preferably comprises less than 20wt.%, preferably less than 15wt.%, more preferably less than 10wt.%, even more preferably less than 5wt.% mannoproteins by weight of the yeast protein source. In some embodiment, the yeast protein source may comprise at least 0.05wt.%, preferably at least 1wt.% mannoproteins.

[0080] Likewise, the chilled product preferably comprises less than 5wt.%, preferably less than 4wt.%, more preferably less than 3wt.%, even more preferably less than 2wt.% mannoproteins by weight of the chilled product. Less than 20%, less than 10%, preferably less than 5%, more preferably less than 4% of the proteins of the yeast protein source and / or the chilled product are mannoproteins.

[0081] It was unexpectedly observed by the inventors that it was possible to stabilize the chilled product, preferably liquid emulsions, and in particular stabilize their fat component even when using yeast protein source with limited or no mannoproteins.

[0082] In preferred embodiments, the yeast protein source comprises an insoluble yeast proteins to soluble yeast proteins ratio of 95:5 to 70:30, more preferably of 95:5 to 80:20. Likewise, the chilled product preferably comprises an insoluble yeast proteins to soluble yeast proteins ratio of 95:5 to 70:30, more preferably of 95:5 to 80:20. The insoluble yeast proteins to soluble yeast proteins ratio is a concentration ratio. In particular, the insoluble yeast proteins to soluble yeast proteins ratio corresponds to the ratio between the insoluble yeast protein concentration to soluble yeast protein concentration of a composition (e.g. yeast protein source, chilled product etc.).

[0083] The soluble protein concentration, such as soluble yeast protein concentration or protein solubility of a composition, such as the yeast protein source or chilled product may be measured as follows. The protein-containing composition (e.g. yeast protein source, chilled product etc.) is reconstituted in ultrapure water at a concentration of 1wt.% protein under low- speed magnetic stirring for 1 hour at room temperature and the pH is adjusted to 6.8 using 0.1-1 M HCI and / or NaOH, as required. Samples are centrifuged at 1000 rpm for 15 min using a Sorvall evolution RC centrifuge (Thermo Fischer, Waltham, MA) equipped with a fixed angle rotor SS-34. The protein concentration of the supernatant is determined by the Kjeldahl method according to the AOAC Official Method 930.29 (AOAC, 2005) using the nitrogenprotein conversion of 6.25. Solubility or soluble protein concentration is calculated as the protein concentration of the supernatant expressed as a percentage of the protein concentration of the initial dispersion.

[0084] The insoluble protein concentration, such as insoluble yeast protein concentration corresponds to the total protein content minus the soluble protein content, such as soluble yeast protein content of a given composition (e.g. yeast protein source, chilled product etc.).

[0085] It has been observed that the use of yeast protein source(s) comprising a higher fractions of insoluble proteins compared to soluble proteins are unexpectedly particularly effective in stabilizing a chilled product, preferably liquid emulsions, in particular their fat component (found in, for example, milk fat or cocoa powder) by limiting fat coalescences.

[0086] In preferred embodiments, the yeast protein source comprises at most 30wt.%, preferably at most 18wt.% of soluble yeast proteins.

[0087] The low solubility may be explained that part of the proteins is within yeast cells structure, either within the yeast cells cytoplasm and / or within their cell wall. Without wishing to be bound by theory, it is believed that the yeast cells structure and the proteins they contain can participate in fat droplet stabilization by limiting their coalescences.

[0088] Accordingly, in some embodiments, part of the yeast proteins, in particular insoluble yeast proteins of the yeast protein source are contained within yeast cells, in particular yeast cells cytoplasm and / or entrapped in the cell wall of yeast cells, in particular in the inner cell of yeast cells. Accordingly, in some embodiments, part of the yeast proteins, in particular insoluble yeast proteins of the chilled product are contained within yeast cells, in particular yeast cells cytoplasm and / or entrapped in the cell wall of yeast cells, in particular in the inner cell of yeast cells.

[0089] In some embodiments, in view of the above, the yeast protein source may have low solubility as the proteins are embedded / contained within insoluble yeast cell structure. In particular, the yeast protein source has a solubility of 5 to 30%, preferably of 5 to 25%, more preferably of 5 to 20% at a pH of 2-9, preferably at a pH of 6.8.

[0090] The chilled product is a source of protein. In preferred embodiments, the yeast protein source is present in the chilled product in an amount of no more than about 6 wt.% based on the total weight of the chilled product. In such embodiments, the yeast protein source may be present in an amount of from about 0.1 wt.% to about 6 wt.%, preferably from about 0.3 wt.% to about 6 wt.%, more preferably from about 0.5 wt.% to about 5.5wt.%, even more preferably from about 0.8 wt.% to about 5 wt.% based on the total weight of the chilled product.

[0091] The total protein content of the chilled product is not particularly limited. In preferable embodiments, the total protein content of the chilled product may be 5 wt.% or higher, preferably, 6 wt.% or higher, more preferably 7 wt.% or higher, even more preferably 8 wt.% or higher, yet more preferably 9 wt.% or higher, and most preferably 10 wt.% or higher.

[0092] In other preferred embodiments, the total protein content of the chilled product is from about 5 wt.% to about 30 wt.%, preferably from about 5 wt.% to about 20 wt.%, more preferably from about 5 wt.% to about 20 wt.%, yet more preferably from about 5 wt.% to about 15 wt.%, still more preferably from about 8 wt.% to about 12 wt.%, most preferably about 10 wt.%.

[0093] In some embodiments, the yeast protein source preferably has a good protein quality. In particular, the yeast protein source may have a minimum PDCAAS of 0.80, preferably of 0.90, more preferably of 0.95, most preferably of 1. Likewise, the chilled product may have a minimum PDCAAS of 0.80, preferably of 0.90, more preferably of 0.95, most preferably of 1 .

[0094] The protein digestibility-corrected amino acid score (PDCAAS) is a method of evaluating the quality of a protein based on both the amino acid requirements of humans and their ability to digest it. PDCAAS compares the amount of the essential amino acids in a food to a reference (scoring) pattern based on the essential amino acid requirements of a preschool-age child to determine its most limiting amino acid (amino acid score). This approach is recommended by the Food and Drug Administration (FDA) and is described in the 1991 FAO / WHO Protein Quality Report. In an embodiment, the yeast protein source may comprise one or more conditionally essential amino acids (e.g., amino acids conditionally essential in illness or stress) selected from the group consisting of arginine, cysteine, glutamine, glycine, proline, ornithine, serine and tyrosine. In particular, the yeast protein source may comprise 15 to 30g of said conditionally essential amino acids per 100 g yeast protein in the yeast protein source.

[0095] In an embodiment, the yeast protein source may comprise one or more essential amino acids selected from the group consisting of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In particular, the yeast protein source may comprise 35 to 50g, preferably 40 to 50g of said essential amino acids per 100 g yeast protein in the yeast protein source.

[0096] In an embodiment, the yeast protein source may comprise one or more branched chain amino acids selected from the group consisting of leucine, Isoleucine and valine. In particular, the yeast protein source may comprise 15 to 50g, preferably 15 to 30g, more preferably 15 to 25g of said branched chain amino acids per 100 g yeast protein in the yeast protein source.

[0097] In an embodiment, the yeast protein source may comprise one or more autophagy-inducing amino acids selected from the group consisting of Glycine, Cysteine, Proline, Glutamate, Valine, Tyrosine and any precursors thereof. In particular, the precursors may be selected from the Serine (as a precursor to Glycine), N-Acetyl Cysteine, Methionine (as a precursor to Cysteine). In particular, the yeast protein source may comprise 25 to 50g, preferably 30 to 40g of said one or more autophagy-inducing amino acids per 100 g yeast protein in the yeast protein source.

[0098] In an embodiment, the yeast protein source may comprise one or more anabolic amino acids selected from the group consisting of Leucine, Isoleucine and arginine. In particular, the yeast protein source may comprise 10 to 30g, preferably 15 to 25g of said one or more anabolic amino acids per 100 g yeast protein in the yeast protein source.

[0099] In an embodiment, the yeast protein source may comprise one or more acidic amino acids selected from the group consisting of aspartic acid and glutamic acid. In particular, the yeast protein source may comprise 15 to 30g, preferably 20 to 30g of said one or more acidic amino acids per 100 g yeast protein in the yeast protein source.

[0100] In an embodiment, the yeast protein source may comprise one or more aromatic amino acids selected from the group consisting of Tryptophan, Tyrosine, Phenylalanine, and Histidine. In particular, the yeast protein source may comprise 5 to 30g, preferably 10 to 18g of said one or more aromatic amino acids per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 5 to 6g of Alanine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 5 to 6.5g of Arginine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 5 to 15g, preferably 8 to 12g, more preferably 10 to 11 ,5g of Aspartic acid per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 0.5 to 3g, preferably 0.5 to 2g, more preferably 0.5 to 1g of Cysteine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 5 to 15g, preferably 8 to 12g, more preferably 10 to 11 ,5g of Glutamic acid per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 4 to 5g of Glycine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 3 to 4g of Proline per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 5 to 6g of Serine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 4 to 5g of Tyrosine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 1 to 10g, preferably 1 to 6g, more preferably 2 to 3g of Histidine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 5 to 6.5g of Isoleucine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 5 to 15g, preferably 6 to 10g, more preferably 8 to 9.5g of Leucine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 5 to 15g, preferably 7 to 12g, more preferably 9 to 10g of Lysine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 0.5 to 5g, preferably 0.5 to 3g, more preferably 1 to 2g of Methionine per 100 g yeast protein in the yeast protein sopoturce. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 3 to 8g, more preferably 5 to 6g of Threonine per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 0.5 to 5g, preferably 0.5 to 3g, more preferably 1 to 2g of Tryptophan per 100 g yeast protein in the yeast protein source. In an embodiment, the yeast protein source may comprise 2 to 10g, preferably 5 to 8g, more preferably 6 to 7g of Valine per 100 g yeast protein in the yeast protein source.

[0101] In certain embodiments, the chilled product may further comprise at least one plant protein source and / or at least one collagen source and / or at least one collagen peptide source and / or at least one gelatin source and / or at least one fungal protein source and / or at least one bacterial protein source.

[0102] In some embodiments, the plant protein source may be selected from the list consisting of plant flour, plant protein concentrate, plant protein isolate and mixture thereof.

[0103] In some embodiments, the protein of said plant protein source may comprise or consist of protein coming from any one of pulses, nuts, oilseeds, cereals, coconut and mixture thereof.

[0104] Examples of pulses include bean, chickpea, faba, lentil, lupine, pea, soy, peanut and mixture thereof. For example, pea may be selected from split pea, cow pea, yellow pea, green pea and mixture thereof. For example, the bean may be selected from the list consisting of navy bean, black bean, butter bean, red bean, green bran, kidney bean, pinto bean, lima bean, cannellini bean, adzuki bean, mung bean, cranberry bean, Great Northern bean, yellow eye bean, black turtle bean, calypso bean, Jacob’s cattle bean, tongue of fire bean, and mixture thereof.

[0105] Examples of nuts include almond, cashew nut, hazelnut, macadamia nut, pecan nut, pine nut, pistachio, tiger nut, walnut and mixture thereof.

[0106] Examples of oilseeds include chia seed, Curcubitaceae seed, cotton seed, flaxseed, linseed, grape seed, hemp seed, rapeseed, sesame seed, sunflower seed, and mixture thereof. For example, the Cucurbitaceae seed may be selected from egusi seed, pumpkin seed, squash seed, watermelon seed, winter melon seed, cucumber seed, calabash seed and mixture thereof.

[0107] Examples of cereal include barley, buckwheat, maize, millet, oat, rice, rye, spelt, teff, quinoa, wheat and mixture thereof.

[0108] In particularly preferred embodiments, the plant protein source is protein obtained from cocoa solids.

[0109] In some embodiments, the chilled product further comprises at least one plant protein source, wherein the proteins of said plant protein source come from pulses and / or cereals and / or oilseeds.

[0110] The combination of yeast protein sources with plant protein sources, in particular cereal protein sources (e.g. protein sources from oat) or pulse protein sources (e.g. protein sources from soy) may be advantageous. Indeed, the compositions, in particular emulsions based on yeast protein sources with said plant protein sources have improved properties compared to compositions, in particular emulsions based on plant protein sources only. In particular, yeast protein sources can improve protein quality, foam stability, foamability and / or coffee stability of plant protein-containing compositions, in particular emulsions as shown in the example.

[0111] In particular, the pulses may be selected from the list consisting of bean, chickpea, faba, lentil, lupine, pea, soy and mixture thereof. For example, the pea may be selected from split pea, cow pea, yellow pea, green pea and mixture thereof. For example, the bean may be selected from the list consisting of navy bean, black bean, butter bean, red bean, green bran, kidney bean, pinto bean, lima bean, cannellini bean, adzuki bean, mung bean, cranberry bean, Great Northern bean, yellow eye bean, black turtle bean, calypso bean, Jacob’s cattle bean, tongue of fire bean, and mixture thereof.

[0112] In particular, the oilseeds are selected from the list consisting of chia seed, Curcubitaceae seed, cotton seed, flaxseed, linseed, grape seed, hemp seed, rapeseed, sesame seed, sunflower seed, and mixture thereof. The Cucurbitaceae seed may be selected from the list consisting of egusi seed, pumpkin seed, squash seed, watermelon seed, winter melon seed, cucumber seed, calabash seed and mixture thereof.

[0113] In particular, the cereals are selected from the list consisting of barley, buckwheat, maize, millet, oat, rice, rye, spelt, teff, quinoa, wheat and mixture thereof.

[0114] In a further preferred embodiment, the cereals are selected form the group consisting of oat, rice, wheat, maize and combinations thereof.

[0115] In a further preferred embodiment, the oilseeds are pumpkin seeds.

[0116] In a further preferred embodiment, the pulses are soy. In another further preferred embodiment, the pulses are pea. In a further preferred embodiment, the pulses are faba. In a further preferred embodiment, the pulses are chickpea.

[0117] The use of a yeast protein source in combination with other protein source such as plant protein source is advantageous because it provides a chilled product with good protein quality. In particular, the yeast protein source has a good PDCAAS. It can also improve the protein quality of other protein sources, such as plant protein sources by bringing amino acids that are absent in such other protein sources, in particular plant protein sources. For example, the yeast proteins may bring for example lysine that is missing in plant proteins coming from some cereal or for example methionine and cysteine that is missing in plant proteins coming from some pulse. In some embodiments, the chilled product has a minimum PDCAAS of 0.80, preferably of 0.90, more preferably of 0.95. In some preferred embodiment, the chilled product has PDCAAS of 1.

[0118] In some embodiment, the collagen source may be any composition comprising more than 50wt.% collagen, preferably more than 80% collagen, more preferably consisting of collagen. The collagen of the collagen source may be derived from any animal, for example, from mammals such as cows, pigs, chickens, and the like, or fish.

[0119] In some embodiments, the collagen peptides source may be any composition comprising more than 50wt.% collagen peptides, preferably more than 80% collagen peptides, more preferably consisting of collagen peptides. The collagen peptides of the collagen peptides source may be derived from any animal, for example, from mammals such as cows, pigs, chickens, and the like, or fish. The type of collagen peptide is not particularly limited, and may be, for example, I type, II type, or the like.

[0120] In some embodiments, the gelatin source may be any composition comprising more than 50wt.% gelatin, preferably more than 80% gelatin, more preferably consisting of gelatin. The gelatin of the gelatin source may be derived from any animal, for example, from mammals such as cows, pigs, chickens, and the like, or fish.

[0121] The fungal protein source is an ingredient comprising proteins derived from fungi. In some embodiment, the fungal protein source may be selected from fungi biomass, fungal protein concentrate, fungal protein isolate and mixture thereof.

[0122] The bacterial protein source is an ingredient comprising proteins derived from bacteria. In some embodiment, the fungal protein source may be selected from bacteria biomass, bacterial protein concentrate, bacterial protein isolate and mixture thereof.

[0123] The proteins of the yeast protein source and / or the milk protein source and / or the plant protein source and / or the collagen source and / or the gelatin source and / or at and / or fungal protein source and / or bacterial protein source may be unhydrolyzed, partially hydrolyzed (i.e., peptides of molecular weight 3 kDa to 10 kDa with an average molecular weight less than 5 kDa) or extensively hydrolyzed (i.e., peptides of which 90% have a molecular weight less than 3 kDa), for example in a range of 5% to 95% hydrolyzed. In some embodiments, the peptide profile of hydrolyzed protein of the of the yeast protein source and / or the milk protein source and / or the plant protein source and / or the collagen source and / or the gelatin source and / or fungal protein source and / or bacterial protein source can be within a range of distinct molecular weights. For example, the majority of peptides (>50 molar percent or >50 wt.%) can have a molecular weight within 1-5 kDa, or 5-10 kDa, or 10-20 kDa.

[0124] In preferred embodiments, the chilled product may be vegetarian.

[0125] Other ingredients

[0126] The chilled product may further comprise additional ingredients to those discussed above.

[0127] In a particularly preferred embodiment, the chilled product further comprises cocoa solids. The cocoa solids may comprise chocolate, cocoa powder, cocoa mass, and / or cocoa butter, preferably cocoa powder.

[0128] The amount of the cocoa powder present in the chilled product is not particularly limited and depends on the taste profile desired. In some embodiments, the cocoa powder may be present in an amount of from 0.1 wt.% to 10 wt.%, preferably in an amount of from 0.5 wt.% to 5 wt.%, more preferably in an amount of from 1 wt.% to 5 wt.%, even more preferably in an amount of from 1 wt.% to 3 wt.%, and most preferably about 2.2 wt.%, based on the total weight of the chilled product.

[0129] In a particularly preferred embodiment, the chilled product further comprises a plant-based liquid ingredient selected in the group consisting of plant-based cream alternative, plant-based milk alternative, plant-based water or a combination thereof, preferably plant-based cream alternative. Examples of plant-based cream alternative include almond cream, cashew cream, coconut cream, hazelnut cream, peanut cream, sunflower cream, and mixtures thereof. Examples of plant-based milk alternative include almond milk, banana milk, cashew milk, chestnut milk, coconut milk, hazelnut milk, flaxseed milk, hemp seed milk, lupine milk, oat milk, peanut milk, pine nut milk, pistachio milk, rice milk, sesame seed milk, sunflower seed milk, walnut milk and mixtures thereof. Examples of plant-based water include coconut water.

[0130] Advantageously, the plant-based liquid ingredient is coconut cream.

[0131] In one preferred embodiment, the chilled product comprises from 1 to 20wt%, preferably from 1 to 12wt%, more preferably from 2 to 10wt.% of plant-based liquid ingredient with respect to the total weight of the chilled product.

[0132] In a particularly preferred embodiment, the chilled product is a fermented chilled product. In one preferred embodiment, the fermented chilled product comprises at least one lactic acidproducing bacteria selected from the group consisting of: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Bifidobacterium, Lactiplantibacillus, preferably selected from the group consisting of Lactobacillus, Lactococcus, Streptococcus, and Bifidobacterium, further preferably selected from the group Lactobacillus, Streptococcus and Lactococcus, most preferably Streptococcus and Lactobacillus.

[0133] In a particularly preferred embodiment, the chilled product further comprises water. In one preferred embodiment, the chilled product further comprises from 50 to 90wt%, preferably from 60 to 90wt%, more preferably from 70 to 90wt.% of water with respect to the total weight of the fermented food composition.

[0134] In further embodiments, the chilled product may comprise one or more flavoured ingredients. Any suitable flavoured ingredient may be used, depending on the particular chilled product. In preferred embodiments, the one or more flavoured ingredients are selected from the group consisting of vegetable powder, fruit powder, fruit puree, fruit preparation, nuts powder, seed powder, cereal powder, powdered flavours, a caramel flavoured ingredient, a coffee flavoured ingredient, a mint flavoured ingredient, a vanilla flavoured ingredient and mixtures thereof.

[0135] In some embodiments, the chilled product may comprise one or more spices. Non-limiting examples of spices include cinnamon, vanilla, curry, cumin, pepper, paprika, tonka, cardamom, saffron, ginger, nutmeg, chili pepper, allspice, cloves and mixtures thereof. A preferred examples of a spice that may be included in the chilled product is vanilla. In a particularly preferred embodiment, the chilled product accordingly comprises vanilla flavour.

[0136] In some embodiments, the chilled product may comprise herbs. Non-limiting examples of herbs include mint, thyme, coriander, basil, verbena, fennel, chervil, rosemary, lemon balm, sage, oregano, and mixtures thereof.

[0137] In preferable embodiments, the chilled product comprises at least one non-proteic thickening agent. Any suitable non-proteic thickening agent may be used. Examples of suitable non- proteic thickening agents are alginate, xanthan gum, pectin, locust bean gum, gellan gum, carrageenan, guar gum, cellulose, carboxymethylcellulose, agar, gum arabic, starch, modified starch, konjac gum, tara gum, chitosan, tragacanth gum, psyllium husk and mixtures thereof. In particularly preferred embodiments, the one or more non-proteic thickening agent is selected from the list consisting of xanthan gum, carrageenan, starch, modified starch and mixtures thereof. It is preferred that the starch, where present, is corn starch and the modified starch, where present, is modified corn starch.

[0138] The chilled product may also comprise one or more sweeteners. Examples of suitable sweeteners are sugars and / or artificial sweeteners. Particular examples of suitable sugars are monosaccharides (e.g. galactose, fructose, glucose), disaccharides (e.g. sucrose, lactose, isomaltulose, maltose), oligosaccharides (e.g. fructooligosaccharides, galacto-oligosaccharides, gluco-oligosaccharides, maltodextrines), polysaccharides (e.g. resistant starches), and honey. In preferred embodiments, the one or more sugars are selected from the group consisting of sucrose, lactose, glucose, fructose, and dextrose. The one or more sugars may be present in any amount suitable for the particular chilled product being manufactured.

[0139] Particular examples of suitable artificial sweeteners are aspartame, acesulfame, sucralose, saccharin, neotame, and mixtures thereof. In particularly preferred embodiments, the artificial sweeteners are acesulfam K, sucralose, and mixtures thereof.

[0140] In preferred embodiments, the chilled product is low or free from sucrose. That is, the chilled product comprises less than 10 wt.%, preferably less than 8 wt.%, more preferably less than 5 wt.%, even more preferably less than 3 wt.% yet more preferably less than 1 wt.% of sucrose based on the total weight of the chilled product.

[0141] In particularly preferred embodiments, the chilled product is low or free from added sugars. That is, the chilled product comprises less than 10 wt.%, preferably less than 8 wt.%, more preferably less than 5 wt.%, even more preferably less than 3 wt.% yet more preferably less than 1 wt.% of added sugars based on the total weight of the chilled product. Most preferably, the chilled product is free from added sugars.

[0142] Such embodiments according to the invention that are free of sucrose or added sugars described above provide a particularly suitable product for health conscious consumers who are looking to increase intake of protein whilst also reducing caloric intake.

[0143] In further embodiments, the chilled product may further comprise at least one ingredient selected from the list consisting of vitamin, mineral, free amino acid, carbohydrate, prebiotic, probiotic, postbiotic, synbiotic, low molecular weight surfactant, pharmaceutically acceptable carrier, bioactive agent, flavour agent, colorant, coffee, and combinations thereof.

[0144] In some embodiments, the chilled product may comprise one or more vitamin(s). Non-limiting examples of vitamins include Vitamin A, Vitamin E, Vitamin C, Vitamin B1 , Vitamin B2, Pantothenic Acid, Vitamin B6, Vitamin B12, Niacin, Folic Acid, Biotin and Choline or any combination thereof.

[0145] In some further embodiment, the vitamins may comprise or consist of added vitamins, i.e. vitamins that do not come from the yeast protein source. In some embodiment, the chilled product may comprise one or more mineral(s). Non-limiting examples of minerals include sodium, potassium, calcium, phosphorus, magnesium, chloride, iron, zinc, copper, manganese, fluoride, chromium, molybdenum, selenium, iodine or any combination thereof. The minerals may be provided in the form of salts. In some further embodiment, the minerals may comprise or consist of added minerals, i.e. minerals that do not come from the yeast protein source. In some embodiment, the minerals may comprise or be any one or more of calcium, potassium and salts thereof. The minerals may be positively or negatively charged, in particular for use as electrolytes.

[0146] In some embodiments, the chilled product may comprise one more free amino acid(s). Nonlimiting examples of free amino acids include Alanine, Arginine, Asparagine, Aspartate, Citrulline, Cysteine, Glutamate, Glutamine, Glycine, Histidine, Hydroxyproline, Hydroxyserine, Hydroxytyrosine, Hydroxylysine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Taurine, Threonine, Tryptophan, Tyrosine, Valine, HICA (Alpha- Hydroxyisocaproic Acid), HIVA (Alpha- Hydroxyisovaleric Acid), HIMVA (alphahydroxymethylvaleric acid) or any combination thereof. In some further embodiment, the free amino acids may comprise or consist of added free amino acids, i.e. free amino acids that do not come from the yeast protein source. The free amino acid(s) may be added to the chilled product to achieve a desired amino acid profile / content.

[0147] The chilled product may comprise one or more prebiotics. The prebiotics that may be used are not particularly limited and include all food substances that promote the growth of probiotics or health beneficial micro-organisms in the intestines. They may be selected from the group consisting of oligosaccharides, optionally containing fructose, galactose, and mannose; dietary fibers, in particular soluble fibers, soy fibers; inulin; or mixtures thereof. Nonlimiting examples of prebiotics are alpha glucan, beta glucan, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS), mannan-oligosaccharides (MOS), soyoligosaccharides, gentiooligosaccharides, glucooligosaccharides, inulin, polydextrose, glycosylsucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), pecticoligosaccharides, malto-oligosaccharides, sugar alcohols, gums and / or hydrolysates thereof, pectins and / or hydrolysates thereof, or any combination thereof. In a particular embodiment, the prebiotics may be fructooligosaccharides and / or inulin. Suitable commercial products that can be used include combinations of FOS with inulin such as the product sold by BENEO under the trademark Orafti, or polydextrose sold by Tate & Lyle under the trademark STA-LITE®. The prebiotics can also be a BMO (bovine’s milk oligosaccharide) and / or a HMO (human milk oligosaccharide) such as N-acetylated oligosaccharides, sialylated oligosaccharides, fucosylated oligosaccharides and any mixtures thereof.

[0148] A particular example of prebiotic is a mixture of galacto-oligosaccharide(s), N-acetylated oligosaccharide(s) and sialylated oligosaccharide(s) in which the N-acetylated oligosaccharide(s) represent 0.5 to 4.0 wt% of the oligosaccharide mixture, the galacto- oligosaccharide(s) represent 92.0 to 98.5 wt% of the oligosaccharide mixture and the sialylated oligosaccharide(s) represent 1.0 to 4.0 wt% of the oligosaccharide mixture. For example, a chilled product according to the invention can contain from 2.5 to 15.0 wt% CMOS- GOS on a dry matter basis with the proviso that the product comprises at least 0.02 wt% of an N-acetylated oligosaccharide, at least 2.0 wt% of a galacto-oligosaccharide and at least 0.04 wt% of a sialylated oligosaccharide. W02006087391 and W02012160080 provide some examples of production of such an oligosaccharide mixture.

[0149] In some embodiments, the chilled product may comprise one or more probiotic(s). As probiotics are preferably microorganisms (alive, including semi-viable or weakened) that could confer health benefits on the host when administered in adequate amounts, more specifically that beneficially affect a host by improving its intestinal microbial balance, leading to effects on the health or well-being of the host. In general, it is believed that these probiotics inhibit and / or influence the growth and / or metabolism of pathogenic bacteria in the intestinal tract. The probiotics may also activate the immune function of the host. Non-limiting examples of probiotics include Aspergillus, Rhizopus, Mucor, Penicillium, Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus, Lactobacillus or a combination thereof.

[0150] In some embodiments, the chilled product may comprise one or more postbiotic(s). Postbiotics include all substances, in particular metabolites or fragments derived from microorganisms that could confer health benefits on the host. Examples of postbiotics include short-chain fatty acids, microbial lysates, cellular wall fragments of microbial origin, supernatant of microbial original or any combination thereof.

[0151] In some embodiments, the chilled product may comprise one or more synbiotic(s). The synbiotic is a supplement that contains both prebiotic(s) and probiotic(s). The prebiotic(s) and the probiotic(s) work together to improve the micro flora of the intestine. The synbiotic comprises any combination of the prebiotic(s) and the probiotic(s) referred to above. In some embodiments, the chilled product may comprise one or more low molecular weight surfactant(s). Non-limiting examples of low molecular weight surfactant include lecithin, mono- and diglycerides, polysorbate 80, sorbitan monostearate, sodium stearoyl lactylate, glycerol monostearate, polyglycerol esters of fatty acids, propylene glycol monostearate, sodium lauryl sulfate (SLS), sodium oleate or any combination thereof.

[0152] In some embodiments, the chilled product may comprise one or more pharmaceutically acceptable carrier(s).

[0153] In some embodiments, the chilled product may comprise one or more bioactive agent(s).

[0154] A chilled product according to the invention may be produced by any suitable method known in the art. For example, the dry ingredients may first be mixed together followed by addition of the liquid ingredients and further mixing until a homogenous product has been obtained.

[0155] EXAMPLES

[0156] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0157] Example 1: Nutrient composition and physicochemical properties of the protein ingredients

[0158] Yeast protein ingredients- The yeast protein ingredients used in the examples included a commercial protein concentrate (spray dried) from Saccharomyces cerevisiae, referred to hereinafter as YPC, and a commercial biomass from Candida utilis (also known as Torula), referred to hereinafter as YB.

[0159] Other ingredients- The protein ingredient used to produce the reference milk analogue was a commercial soy protein isolate (SPI). Other ingredients used included commercial high oleic sunflower oil, white sugar and gellan gum.

[0160] The proximate composition of the protein ingredients was determined using the standard methods of the Association of Analytical Chemists (AOAC), 2023 as described below.

[0161] Protein concentration measurement- Total nitrogen was determined by the Kjeldahl method and a nitrogen-protein conversion factor of 6.25 was used to calculate the protein concentration of the protein ingredients.

[0162] Moisture concentration measurement- Moisture concentration was determined by oven drying at 103°C for 5 hours. Ash concentration measurement- Ash concentration was determined by dry ashing in a muffle furnace at 500°C for 5 hours.

[0163] Fat concentration measurement- Fat concentration was determined by acid hydrolysis using a Hydrotherm (Gerhardt Analytical Systems, Kdnigswinter, Germany) followed by extraction with petroleum ether using a Soxtherm (Gerhardt Analytical Systems, Kdnigswinter, Germany).

[0164] Total dietary fiber measurement- Total dietary fiber was determined using the enzymatic kit K- TDFR (Megazyme, Bray, Co. Wicklow, Ireland).

[0165] Total carbohydrate measurement- Total carbohydrate (excluding fibre) was calculated by difference (100 - sum of protein, moisture, ash, fat and fibre).

[0166] PDCAAS measurement- The Protein Digestibility-Corrected Amino Acid Score (PDCAAS) was determined using the Megazyme K-PDCAAS assay kit, an in vitro digestion method, according to the protocol suggested by the supplier (Megazyme, Megazyme, Bray, Co. Wicklow, Ireland). The proximate composition and PDCAAS of the protein ingredients are reported in Table 1.

[0167] Table 1: Proximate composition (g / 100 g) and PDCAAS of yeast protein concentrate (YPC), yeast biomass (YB) and soy protein isolate (SPI).

[0168] The YPC had a considerably higher protein concentration when compared to the YB (i.e. , 79.7 vs 51.5 g / 100 g), which is due to the removal of most of the cell wall material during the extraction process, as can be inferred from the considerably lower fiber concentration (i.e., 5.80 vs 39.8 g / 100g). Interestingly, while the YPC was characterized by a PDCAAS of 1.0, that is the same as that of animal proteins such as those derived from milk and eggs, as well as soy proteins, the value displayed by the YB was significantly lower (i.e., 0.86) which, in addition to differences in the amino acid profile, could be ascribed to its higher concentration of fiber, which is known to hinder protein digestibility.

[0169] Amino acid profile determination- The amino acid profile of the protein ingredients was determined by acid hydrolysis followed by ion-exchange chromatography.

[0170] Tryptophan concentration measurement- Tryptophan concentration was determined by alkaline hydrolysis followed by ion-exchange chromatography (I EC). The non-essential and essential amino acid profiles of the protein ingredients are reported in Table 2 and 3, respectively.

[0171] Table 2: Non-essential amino acid profile (g / 100 g protein) of yeast protein concentrate (YPC), yeast biomass (YB) and soy protein isolate (SPI).

[0172] Table 3: Essential amino acid profile (g / 100 g protein) of yeast protein concentrate (YPC), yeast biomass (YB) and soy protein isolate (SPI). The two yeast protein ingredients displayed a similar amino acid profile, the main exceptions being represented by the lower concentration of glutamic acid and higher concentration of the essential amino acids leucine and lysine in YPC than in YB. Interestingly, when compared to SPI, both yeast protein ingredients displayed a significantly lower concentration of glutamic acid and higher concentration of various essential amino acids, including isoleucine, lysine, threonine and valine.

[0173] Mineral profile assessment- The mineral profile of the protein ingredients was determined using inductively coupled plasma-emission spectroscopy (ICP-ES). The mineral profile of the protein ingredients is reported in Table 4.

[0174] Table 4: Mineral profile (mg / 100 g) of yeast protein concentrate (YPC), yeast biomass (YB) and soy protein isolate (SPI).

[0175] A particularly striking difference between the two yeast protein ingredients in terms of mineral profile was the markedly higher concentration of potassium in YB than in YPC. Both ingredients displayed a relatively high concentration of phosphorous, the main component of the yeast biomass ash, and which exerts several important physiological functions, being a constituent of phospholipids, coenzymes and nucleic acids. Interestingly, SPI displayed a considerably higher concentration of sodium when compared to the yeast protein ingredients, which can be attributed to the sodium hydroxide used for the alkaline treatment during the extraction process to obtain plant protein isolates.

[0176] Protein profile analysis- The protein profile of the yeast protein ingredients was determined by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC- MS / MS). The analysis was performed using a Vanquish LIPLC system coupled with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, US). The yeast protein ingredients were reconstituted in ultrapure water (1 wt% protein) under low-speed magnetic stirring for 1 hour at room temperature followed by high pressure homogenization using an Emulsiflex C5 (Avestin, Mannheim, Germany) operating at 1000 bars (two passes), as this treatment was shown to facilitate complete protein solubilization upon sample preparation before UPLC-MS / MS. The protein dispersions were diluted five times with a solution of urea (final concentration 6 M), mixed with ammonium bicarbonate buffer (pH 8.5, final concentration 100 mM), reduced with dithiothreitol (final concentration 4 mM) for 30 min at 60°C in a ThermoMixer C (Eppendorf, Hamburg, Germany), and alkylated with iodoacetamide (final concentration 10 mM) for 30 min in the dark at room temperature. A further dilution of the dispersions with 100 mM ammonium bicarbonate buffer (pH 8.5) was performed to obtain a urea concentration of 2 M. Yeast proteins were then digested using sequencing grade trypsin (Promega Corporation, Madison, Wl, US) (enzyme-to-substrate ratio 1 :50, w / w) for 4 h at 37°C. An aliquot of each digesta (6 pg protein) was then loaded onto an Acquity BEH C18 column (130 A, 1.7 pm, 3 mm X 100 mm) (Waters Corporation, Milford, MA, US) for peptide separation. Elution was performed at 0.75 mL min-1. A mobile phase of two solvents was used, i.e., solvent A, consisting of 0.1 % v / v formic acid and 2% v / v acetonitrile in ultrapure LC-MS grade water, and solvent B, consisting of 0.1 % v / v formic acid and 80% v / v acetonitrile in ultrapure LC-MS grade water. The samples were eluted with a gradient from 2 to 50% B over 45 min. The mass spectrometer operated in a data-dependent Top10 setup to acquire full scan (MS1) and peptide fragment (MS2) spectra over the entire chromatographic run. The raw data were processed with the PEAKS X+ software (Bioinformatics Solutions Inc., Waterloo, Canada) using the Swiss-Prot database for protein identification. The most abundant proteins in both yeast protein ingredients were enzymes involved in functions such as metabolism of carbohydrates (G3P2, G3P3, ENO1 , ENO2, ICL1 , TDH1 , ACO2), amino acids (MET6), proteins (EF2), fatty acids (FAS1 , FAS2), acetate (ACS1) and aldehydes (ALD1 , ALD5), as well as ATP production (ATP1 , ATP2) (Figure 1). The mannoproteins represent a minor fraction in YPC and YB and are present at a content much lower than 20wt.%.

[0177] Microstructure analysis- The microstructure of the yeast protein dispersions (3 wt% protein) was analyzed with a LSM 710 confocal laser scanning microscope (CLSM) upgraded with an Airyscan detector (Carl Zeiss, Oberkochen, Germany) and using Plan-APOCHROMAT objectives (10x / 0.45, 20x / 0.8, 63x / 1.4). Proteins were fluorescently labelled by adding 10 pL of 1% (w / v) Fast Green FCF (Sigma-Aldrich, Saint Louis, MO, USA) in deionized water into 1 mL of heated dispersion. The fluorescently labelled samples (100 pL) were placed inside a 1 mm deep plastic chamber closed by a glass slide coverslip to prevent compression and drying artefacts. Imaging of the proteins was performed at an excitation wavelength of 633 nm and an emission wavelength of 645 nm. Acquisition and treatment of the images were performed using the Zen 2.1 software (Carl Zeiss, Oberkochen, Germany). The protein dispersions were analyzed before and after homogenization using a PandaPLUS 2000 (GEA, Parma, Italy) with first and second stage pressures of 250 and 50 bars, respectively. The CLSM images are shown in Figure 2. The two yeast protein ingredients displayed a similar microstructure, both consisting of roughly spherical, densely packed aggregates, which might have been induced by the spray drying process, with diameters ranging from ~10 to >20 pm. These were broken down by high pressure homogenization into their individual subunits, also roughly spherical and with diameters of ~3 pm.

[0178] In order to further understand the composition and microstructure of the yeast particles, CLSM analysis of non-homogenized YPC and YB dispersions (3 wt% protein) was performed upon fluorescent labelling of proteins, lipids and fibers (i.e. , chitin and glucans) by adding 10 pL of 1 % Fast Green FCF (Sigma-Aldrich, Saint Louis, MO, USA) in Milli-Q water, 10 pL of 2.5% Nile Red (Sigma-Aldrich, Saint Louis, MO, USA) in ethanol and 10 pL of Calcofluor White (Sigma-Aldrich, Saint Louis, MO, USA), respectively, into 1 mL of heated dispersion. The fluorescently labelled samples (100 pL) were placed inside a 1 mm deep plastic chamber closed by a glass slide coverslip to prevent compression and drying artefacts. Imaging of the proteins was performed at an excitation wavelength of 633 nm and an emission wavelength of 645 nm, imaging of the lipids was performed at an excitation wavelength of 488 nm and an emission wavelength of 570-620 nm, while imaging of the fibers was performed at an excitation wavelength of 405 nm and an emission wavelength of 475 nm. The images show that while proteins were dominant (green), lipid inclusions (red) and fibers (blue) were also present in the outer shell of the particles (Figure 3a and 3c). The presence of fibers, potentially both chitin and glucans, was even more evident when only the filter for Calcofluor White was used (Figure 3b and 3d), particularly for the YB. Therefore, it is safe to assume that the particles observed were yeast cells comprising at least the inner cell wall portion, where chitin is found, which could explain their resistance to high pressure homogenization.

[0179] Protein solubility For the determination of protein solubility, the protein ingredients were reconstituted in ultrapure water (1 wt% protein) under low-speed magnetic stirring for 1 hour at room temperature and their pH was adjusted to values in the range 2-9 (at 1 pH unit intervals) using 0.1-1 M HCI and / or NaOH, as required. Samples were centrifuged at 1000 rpm for 15 min using a Sorvall evolution RC centrifuge (Thermo Fischer, Waltham, MA) equipped with a fixed angle rotor SS-34. The protein concentration of each supernatant was determined by the Kjeldahl method according to the AOAC Official Method 930.29 (AOAC, 2005) using the nitrogen-protein conversion of 6.25. Solubility was calculated as the protein concentration of each supernatant expressed as a percentage of the protein concentration of the initial dispersion. The protein solubility curves are shown in Figure 4. Both yeast protein ingredients were characterized by low protein solubility across the whole pH range investigated, with values of ~8-12% and -14-18% for YPC and YB, respectively.

[0180] The YPC has a soluble yeast protein to insoluble yeast protein concentration ratio of 10:90. The YB has a soluble yeast protein to insoluble yeast protein concentration ratio of 17:83.

[0181] Example 2: Example chilled dairy desserts comprising a yeast protein source

[0182] Two different chilled dairy desserts comprising a yeast protein source were produced according to conventional methods. Two chilled dairy desserts not comprising a yeast protein source were also prepared for the purposes of comparison. The ingredients of the desserts are indicated in Table 5 below.

[0183] Table 5: Ingredients of example dairy desserts It was found that the chilled products produced according to Inventive Examples 1 and 2 comprising yeast protein were stable and acceptable in taste to consumers. It was also found that the chilled products of Examples 1 and 2 exhibited acceptable viscosity properties when compared to the reference products not comprising yeast protein.

[0184] The chilled products of the present invention are therefore more economical and environmentally friendly, as well as providing an acceptable taste, due to the inclusion of the yeast protein.

[0185] Example 3: Example chilled plant-based fermented set dessert comprising a yeast protein source

[0186] Three different chilled plant-based set fermented desserts comprising a yeast protein source were produced according to conventional methods. One chilled plant-based set fermented dessert not comprising a yeast protein source was also prepared for the purpose of comparison. The ingredients of the products are indicated in Table 6 below.

[0187] The preparation of strains diluted in plant-based milk such as soy milk is as follows: 100 II of Culture Vega Mild Fro Hansen were diluted in 1 L of soy milk for obtaining a strain diluted at 0,2g / L.

[0188] Table 6: Ingredients of example plant-based fermented set dessert It was found that the chilled products produced according to reference product 3 having a high content of soy protein had a compact and chalky texture while having a bitter and pronounced off-note taste.

[0189] It was found that the chilled products produced according to Inventive Products 4-6 comprising yeast protein were stable and acceptable in taste to consumers. It was also found that the chilled products of Inventive Products 4-6 exhibited acceptable viscosity properties when compared to the reference products not comprising yeast protein.

[0190] The chilled products of the present invention are therefore more economical and environmentally friendly, as well as providing an acceptable taste, due to the inclusion of the yeast protein.

[0191] BIBLIOGRAPHY

[0192] Amagliani, L., Silva, J. V. C., Saffon, M., & Dombrowski, J. (2021). On the foaming properties of plant proteins: Current status and future opportunities. Trends in Food Science & Technology.

[0193] Day, L. (2013). Proteins from land plants - Potential resources for human nutrition and food security. Trends in Food Science & Technology, 32(1), 25-42. doi: 10.1016 / j.tifs.2013.05.005

[0194] McClements, D. J., Newman, E., & McClements, I. F. (2019). Plant-based Milks: A Review of the Science Underpinning Their Design, Fabrication, and Performance. Comprehensive Reviews in Food Science and Food Safety, 18(6), 2047-2067. doi: 10.1111 / 1541-4337.12505

[0195] Moss, R., Barker, S., Falkeisen, A., Gorman, M., Knowles, S., & McSweeney, M. B. (2022). An investigation into consumer perception and attitudes towards plant-based alternatives to milk. Food Res Int, 159, 111648. doi:10.1016 / j.foodres.2022.111648

[0196] Qamar, S., Manrique, Y. J., Parekh, H., & Falconer, J. R. (2020). Nuts, cereals, seeds and legumes proteins derived emulsifiers as a source of plant protein beverages: A review. Crit Rev Food Sci Nutr, 60(16), 2742-2762. doi: 10.1080 / 10408398.2019.1657062

[0197] Ritala, A., Hakkinen, S. T., Toivari, M., & Wiebe, M. G. (2017). Single Cell Protein-State-of- the-Art, Industrial Landscape and Patents 2001-2016. Front Microbiol, 8, 2009. doi: 10.3389 / fmicb.2017.02009

Claims

CLAIMS1 . A chilled product comprising: a milk and / or a plant protein source; and a yeast protein source; optionally wherein the total protein content of the chilled product is 4 wt.% or higher.

2. A chilled product according to claim 1 , wherein the milk protein source is chosen from the group consisting of skimmed milk, skimmed milk powder, milk protein isolate, whey protein and mixtures thereof.

3. A chilled product according to any preceding claim, wherein the milk protein source comprises skimmed milk, milk protein isolate, whey protein, and optionally skimmed milk powder.

4. A chilled product according to any preceding claim, wherein the milk protein source provides at least about 10 wt.%, preferably at least about 30 wt.%, more preferably at least about 50 wt.%, yet more preferably at least about 70 wt.%, most preferably at least about 80 wt.% of the total protein content of the chilled product.

5. A chilled product according to any preceding claim, wherein the plant protein is chosen from the group consisting of pulse protein, cereal protein, leguminous protein, single cell protein, or a combination thereof.

6. A chilled product according to any preceding claim, wherein the plant protein is chosen from the group consisting of pea protein, fava bean protein, soy protein, canola protein, lentil protein, oat protein, rice protein, maize protein, wheat protein or a combination thereof, preferably soy protein.

7. A chilled product according to any preceding claim, wherein the chilled product comprises from 1 to 10wt%, preferably from 1 to 8wt%, more preferably from 1 to 5wt.% of plant protein with respect to the total weight of chilled product.

8. A chilled product according to any preceding claim, wherein the yeast protein source is selected from the list consisting of yeast biomass, yeast protein concentrate, yeast protein isolate, or mixtures thereof.

9. A chilled product according to any preceding claim, wherein the yeast protein source comprises less than 20 wt.%, preferably less than 15 wt.%, more preferably less than 10 wt.%, even more preferably less than 5 wt.% mannoproteins.

10. A chilled product according to any preceding claim, wherein the yeast protein source is derived from Saccharomyces cerevisae, Candida utilis, and / or combinations thereof.

11. A chilled product according to any preceding claim wherein the yeast protein source is present in an amount of no more than about 15 wt.% based on the total weight of the chilled product.

12. A chilled product according to any preceding claim, wherein the total protein content of the chilled product is 5 wt.% or higher, preferably, 6 wt.% or higher, more preferably 7 wt.% or higher, even more preferably 8 wt.% or higher, yet more preferably 9 wt.% or higher, and most preferably 10 wt.% or higher.

13. A chilled product according to any preceding claim, wherein the chilled product further comprises a plant-based liquid ingredient selected in the group consisting of plant-based cream alternative, plant-based milk alternative, plant-based water or a combination thereof, preferably plant-based cream alternative.

14. A chilled product according to any preceding claim, wherein the chilled product further comprises coconut cream.

15. A chilled product according to any preceding claim, wherein the chilled product further comprises from 1 to 20wt%, preferably from 1 to 12wt%, more preferably from 2 to 10wt.% of plant-based liquid ingredient with respect to the total weight of the chilled product.

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