FIELD OF THE INVENTION

The present disclosure relates to the field of food technology. The disclosure concerns an improved processing and purification method for production of plant-based protein ingredient with neutral colour and taste as well as greatly improved functional properties, which are valued in production of numerous dairy analogous and other food products. Especially, the disclosure relates to a plant-based high protein ingredient, a process for the manufacture thereof and uses in dairy-alternative products.

BACKGROUND OF THE INVENTION

The use of vegetable proteins in food and beverage products has increased tremendously during the past ten years. Changing consumer trends have attracted people towards healthier and climate friendly choices, and plant-based products are considered as such. Moreover, at the same time protein rich products have become more and more popular. Plant based protein products are consumed by both athletes and normal consumers, because plant-based protein products are considered to be healthy, safe and highly nutritious.

However, the poor solubility of plant proteins, off-flavours caused by them and tendency to precipitate in sour products have caused challenges to food manufacturers. Further, the characteristic "beany flavour" of faba bean has been reduced by thermal pre-treatments thus minimizing the activity of enzymes that are detrimental to product flavor. The solubility of plant proteins has also been improved for example by extracting plant protein source with aqueous calcium salt solution.

Document EP 2566346 A4 discloses production of soluble protein solutions from pulses wherein pulses are extracted with calcium salt at pH 1.5 - 4.4, and thereafter the extracted protein is concentrated by filtration and optionally spray dried.

Olsen (1978) described a continuous pilot plant production of bean protein by extraction, centrifugation combination of decanter centrifuge and separator, ultrafiltration and spray drying. Berot et al., (1987) described three different methods to extract protein from fava beans; a) ultrafiltration, b) alkaline extraction and acid precipitation combined with ultrafiltration and c) wet extraction method without concentration step.

Patent Application WO 2020051622 Al describes a production process for legume protein ingredients with high protein content of at least 80 %, preferably 85 %, on dry weight basis. The extraction of said high protein food product comprises of: a) milling a supply of legumes to form a fine powder, b) hydrating said fine powder to form a liquid slurry, c) separation of solids from the liquid slurry to form a milk-like fluid; d) pasteurizing said milk-like fluid to remove unwanted organisms therefrom; e) filtrating said pasteurized milk-like fluid to remove permeates therefrom to form a substantially liquid product and f) removing moisture from the substantially liquid product to generate a high protein food product in the form of a powder.

Patent Application US 20160309732 Al describes a production process for legume, non-soy, based ingredient with elevated protein and lowered starch content for use in cultured, dairy alternative, food products. Said process is comprised of following steps: a) hydrating non-soy legume material in water b) treating said aqueous solution with amylases, c) heat treating the solution, d) filtering the legume slurry to reduce starch content, d) adjusting the temperature of filtered legume slurry to add bacterial culture and e) holding the filtered legume slurry at the adjusted temperature for a period sufficient to acidify the filtered legume slurry to a pH of 4.7 or below to produce a cultured non-dairy product.

Patent US 10,143,226 Bl, discloses yellow pea protein compositions with high digestibility and amino acid scores, wherein bitterness causing peptides and glucose from hydrolysed starch are separated by ultrafiltration. It describes a production of protein product from yellow pea flour, which consists of following steps: alkaline extraction and proteolytic treatment of yellow pea flour slurry, extracted protein rich water-soluble fraction is treated with amylases to reduce starch concentration. Starch reduced protein rich slurry is concentrated with ultrafiltration and diafiltration step, and after concentration step concentrated protein rich slurry is evaporated to remove excess water and spray dried to produce protein product with at least 80% protein in dry weight basis.

A problem with the disclosures described above is that plant protein raw materials tend to affect adversely on the structure, taste and colour of the final product. This causes challenges especially in milk mimetic products wherein milk-like neutral taste, colour and structure is required. Plant based protein products, and in particularly pulse products have typically bitter taste and dark colour that ranges from brown to black.

As described above, there are several challenges in producing plant-based food products and completely new methods are needed. There is still a constant need to provide new and cost- effective alternatives for producing various plant-based dairy-alternative products.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome problems related to producing plant-based dairy-alternative products. Especially, an object of the present invention is to provide a process for producing a high protein ingredient, a high protein ingredient, and use of the high protein ingredient in a product selected from the group consisting of plant-based dairy alternatives.

Another object with the present invention, is to provide a plant-based high protein ingredient that has a protein content greater than about 70 % protein/dry matter, preferably the high- protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably at least about 100 % protein/dry matter, (N x 6.25) dry weight basis. Nitrogen is converted to protein percent by using coefficient 6,25. In an embodiment the high protein ingredient is obtainable by the process for producing a high protein ingredient.

It was surprisingly found that taste and functional properties of legume protein preparation can be efficiently improved in a simple and economic industrially applicable process involving low number of successive processing steps. Typical bitter or unpleasant taste of legume raw material is eliminated, and the structure formation characteristics of the high protein ingredient are improved in the same, simple process of the present invention.

An essential part of the present invention is utilizing a process by which leguminous protein raw material, such as protein concentrate is enzymatically modified in the presence of antioxidants, preferably ascorbic acid and Na2SOs. The protein concentrate is fractionated into different fractions, such as to a fraction comprising soluble protein, a fraction comprising other components than soluble protein (insoluble fraction) and a permeate fraction and the protein is concentrated by a membrane process and/or diafiltration. The aim of the claimed process is to prepare a high protein ingredient, such as a protein isolate, which can be used as a liquid or powder in vegan products, such as vegan gurt, vegan cheese or vegan drink.

A major challenge for commercially available plant protein raw materials is their varying characteristics related to the structure, taste and color of the final product. This is particularly highlighted in the case of products imitating dairy products, where a particularly neutral taste and color is required from the raw material as well as the ability to form structures similar to dairy products. Above all, the formation of the structure is disturbed by the polysaccharides that are impurities in commercial products and the insoluble form of the protein in them. Protein solubility is a prerequisite for achieving a smooth and strong structure. When organoleptic properties of dairy products are imitated, the strong bean content and bitterness of available raw materials are the main challenges. In addition to this, the brown to black colors of legume protein products are not suitable for imitating light dairy products.

In the present process a reduction or removal of bitterness through enzymatically assisted extraction of protein fraction (protein concentrate) is carried out. As a result, a light colored plant protein ingredient with a neutral flavor, in which the protein is in soluble form, is obtained.

For example, oxidizing enzymes contained by a broad bean cause unpleasant flavors or off- tastes by cleaving fatty acids (lipoxygenases), and/or discoloration (polyphenol oxidases).

Discoloration may be controlled by combination of antioxidants ascorbic acid and sodium sulfate. Bean flavor and side flavors of antioxidants are removed by membrane filtration, such as ultramembrane filtration. The effect can be further enhanced by rinsing the concentrate during filtration with water.

The bitterness can be removed by using at least one enzyme or enzyme mix that contains enzyme activity capable of modifying polyphenols originating from leguminous plant raw material. At least one enzyme capable of modifying polyphenols may comprise carbohydrase, cellulase and/or mixture thereof, preferably further comprising tannase activity. The combination of enzymes may be such as tannase with beta-glucanase, pectinase, hemicellulase or xylanase, or any combination thereof. One combination may be a mixture of tannase, pectinase and cellulase, or tannase and pectinase, or tannase and cellulase. Thus, the present invention concerns a process for producing a plant-based high-protein ingredient having a protein content greater than about 70 wt% protein/dry matter, wherein the process comprises the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension and an insoluble fraction, c. treating said clarified aqueous protein suspension with at least one enzyme capable of modifying polyphenols originating from leguminous plant raw material to obtain an enzyme- treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. optionally washing the concentrated aqueous protein suspension by diafiltration, g. optionally, further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

The present disclosure also relates to a high protein ingredient obtainable with the described process.

The present disclosure also concerns a high protein ingredient that has a plant-based protein content greater than about 70 % protein/dry matter, preferably, the high protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably at least about 100 % protein/dry matter, (N x 6.25) dry weight basis. The plant-based protein ingredient has improved organoleptic and functional properties, such as reduced bitterness and improved gelation properties in dairy product analogues. The improved organoleptic properties were achieved by reduced concentration of polyphenolic compounds. Polyphenolic compounds can be for example tannins. Polyphenolic concentration of ingredient is significantly lower than in the starting raw material. Thereto, the present invention concerns use of the high protein ingredient obtained with the process in a product selected from the group consisting of plant-based dairy alternatives such as gurt, yoghurts, drinkable yoghurt, creme fraiche, sour cream, sour milk, pudding, set-type yoghurt, smoothie, quark, cheese, cream cheese, ice creams, and meat analogues.

The high protein ingredient can also be used in nutritional powders, such as protein powders for athletes, and in food supplements intended for elderly or people suffering from malabsorption.

In a further embodiment of the preset disclosure, the separation of soluble proteins can be used to produce a protein ingredient with standardized concentration of plant-based protein, and/or the concentration of soluble plant-based protein, and/or concentration of non-soluble non-protein dry matter. Soluble proteins and non-soluble dry matter are key components in determining the texture and properties of food products, in which functionalities, such as gel formation or foaming are required. By standardizing previously mentioned components according to the present process, an ingredient of consistent quality can be produced and used in various food products to ensure their invariable product quality. Selection of raw material can thus be considered more flexible, and interchangeable, e.g. raw material with lower protein content can be processed according to our invention to produce an ingredient with similar properties as one made from another raw material of different composition or quality.

The characteristic features of the invention are defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGNS

Figure 1 is a process diagram presenting an embodiment of the process for production of leguminous-based (pulse-based) protein isolate.

Figure 2 is a diagram presenting gel hardness of yogurt analogue samples made with fava bean protein measured with TA. XT texture analyser.

Figure 3 is a picture showing the appearance of yogurt analogue samples: a) Yogurt analogue fermented with glucono delta-lactone, b) Yogurt analogue fermented with glucono delta- lactone, c) Yogurt analogue fermented with bacterial starter, d) Yogurt analogue fermented with bacterial starter and glucono delta-lactone.

Figure 4 is a picture showing the appearance of a) 8% fava bean protein enzyme treated suspension after heat-treatment step, as compared to the b) 10% resolubilized fava bean protein isolate produced according to the invention. The color of the suspension presented in figure a) is greyish and darker than the protein isolate presented in b), which is lighter, pale yellow, in color.

Figure 5 presents the appearance of a) the retentate and b) the permeate from the process having air classified and enzyme (Viscozyme L) treated fava bean concentrate as a starting material. The appearance of c) the retentate and d) the permeate from the process having enzyme (Viscozyme L) treated fava bean flour as a starting material. Non-dairy cheese blocks produced from e) the air classified and enzyme (Viscozyme L) treated fava bean concentrate and f) the enzyme (Viscozyme L) treated fava bean flour are presented. Non-dairy cheese block in e) is lighter in colour than the cheese block in f).

DEFINITIONS

In the present description and claims, the following words and expressions have meanings as defined below:

A "high-protein ingredient" refers to a protein rich ingredient that has a protein content greater than about 70 % protein/dry matter. Preferably the high-protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably at least about 100 % protein/dry matter, (N x 6.25).

The terms "protein isolate" and "protein concentrate" differ in terms of protein quantity. These differences are caused by the processing methods. "Protein concentrate" powder consists of up to 80% protein by weight. The remaining, such as 20%, of the concentrate powder contains carbohydrates and fats. If different processing steps are used to reduce the fat and carbohydrate content, a "protein isolate" powder containing 90% or more protein by weight can be produced. Overall, the processing steps used in the production of isolate result in higher protein content and lower fat and carbohydrate content. However, the types of amino acids found in both forms of whey are virtually identical, since they are derived from the same proteins.

The term "air classification" refers to separation of materials by a combination of size, shape and density. The separation is carried out with an industrial machine, an air classifier, which works by injecting the material stream to be sorted into a chamber which contains a column of rising air. Inside the separation chamber, air drag on the objects supplies an upward force which counteracts the force of gravity and lifts the material to be sorted up into the air. Due to the dependence of air drag on object size and shape, the objects in the moving air column are sorted vertically and can be separated in this manner. Air classifiers are commonly employed in industrial processes where a large volume of mixed materials with differing physical characteristics need to be separated quickly and efficiently. Air classification is carried out e.g. in food processing. Typically, the protein concentration of the protein concentrate produced by air classification is between 48 and 65% protein. The rest consisting of starch, fat and other polysaccharides, as well as ash.

The term "membrane process" or "membrane filtration" or "membrane filtration process" refers to microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) or reverse osmosis (RO). The membrane process or membrane filtration may contain one membrane filtration. Alternatively, the membrane process or membrane filtration may contain several i.e. more than one membrane filtrations.

Microfiltration (MF) refers to separation of macromolecules. For example, if the raw material contains a large amount of fat, MF may be used to separate fat from the raw material, or to clarify the product.

Ultrafiltration (UF) refers to concentration of large and macromolecules, for example proteins.

Nanofiltration (NF) refers to concentration of organic components by removal of part of monovalent ions like sodium and chlorine (partial demineralization).

Reverse osmosis (RO) refers to concentration of solutions by removal of water. RO is applied for example if a protein-free fraction is to be recovered from the permeate, or if an aqueous fraction is to be recycled. Said recycled fraction may for example be used in diafiltration.

Diafiltration refers to a design to obtain better purification. Water is added to the feed during membrane filtration to wash out the low molecular feed components that will pass through the membranes, such as lactose and minerals. Washing means that water is added once or several times. Washing can be done as many times and as much as necessary to remove undesired components.

A "starter culture" is a microbiological culture, which performs fermentation. The starters usually consist of a cultivation medium, such as nutrient liquids that have been well colonized by the microorganisms used for the fermentation. A "plant-based food product" may refer to fermented, acidified or non-acidic (neutral) food products, such as traditional dairy-based products like yoghurt, drinkable yoghurt, creme fraiche or sour cream, sour milk, quark, cream cheese (Philadelphia-type soft cheese), settype yoghurt, smoothie or pudding.

"Plant-based" refers to originating from plants, which are suitable for manufacturing edible food products in food technology applications. The plant-based raw material suitable for the product and process of the present invention may be from at least one plant selected from leguminous plants, such as dry and fresh beans, soybeans, dry and fresh peas, lentils, chickpeas and peanuts, more preferably selected from broad bean and pea, most preferably from broad bean.

A "legume" or leguminous plant" refers to a plant belonging to the family Fabaceae (or Leguminosae), which family is commonly known as the legume, pea, or bean family. Said family is a large family of flowering plants. A legume also refers to the fruit or seed of a leguminous plant. The seed is also called a pulse. Legumes include for example alfaalfa (Medicago sativa), clovers (Trifolium spp.), peas (Pisum), beans (Phaseolus spp., Vigna spp., Vicia spp.), chickpeas (C/cer), lentils (Lens), lupins (Lupinus spp.), mesquites (Propsis spp.), carob (Ceratonia siliqua), soybeans (Glycine max), peanuts (Arachis hypogaea), vetches (Vicia), tamarind (Tamarindus indica), kudzu (Pueraria spp.) and rooibos (Aspalathus linearis). Legumes produce a botanically unique type of fruit - a simple dry fruit that develops from a simple carpel and usually dehisces (opens along a seam) on two sides.

DETAILED DESCRIPTION OF THE INVENTION

Commercially available plant-based protein ingredients have limitations due to their variety in quality. For example, commercial pulse protein may have unwanted taste, such as bitterness and beany flavour. Additionally, colour changes and loss of functional properties resulting in poor texture in the final product. These qualities are emphasized when producing products that mimic dairy type products, naturally neutral in colour and flavour, and whose texture is typically achieved through protein interactions. Furthermore, pulse protein ingredients may contain impurities, such as polysaccharides and insoluble proteins that interferes the structure forming properties of plant-based proteins. Overall, good functionality, neutral colour and clean taste are prerequisites developing plant-based dairy alternatives. The present disclosure concerns a process for producing a high protein ingredient having a protein content greater than about 70 wt% protein/dry matter, wherein the process comprises the steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble non-suspended solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension and an insoluble fraction, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from leguminous plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. optionally washing the concentrated aqueous protein suspension by diafiltration, g. optionally, further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

The above-mentioned steps a. to g. may be performed in succession.

In an embodiment, the plant protein is selected from dry and fresh beans, soybeans, dry and fresh peas, lentils, chickpeas and peanuts, more preferably selected form broad bean and pea, most preferably from broad bean.

In an embodiment of the present process the first step of the process involves the solubilization of leguminous or pulse protein material from a raw material. The pulse raw material may be pulses or any pulse product or by-product derived from the processing of pulses, such as pulse flour. Pulse protein source material may also be referred to as a grain legume. Suitable leguminous plants or sources for pulse raw material include e.g.

1. Dry beans (Phaseolus') such as kidney bean, navy bean, pinto bean, haricot bean (Phaseolus vulgarisy, lima bean, butter bean (Phaseolus lunatusy, azuki bean (Vigna angularis mung bean, golden gram, greengram (Vigna radiatay black gram, urad bean (Vigna mungo); Scarlet runner bean (Phaseolus coccineus); ricebean (Vigna umbellatay moth bean (Vigna aeon iti folia); and tepary bean (Phaseolus acutifoliusy

2. Dry broad beans (Vicia faba) such as horse bean (Vicia faba equina); broad bean (Vicia faba); and field bean (Vicia faba),

3. Dry peas (Pisum) such as garden pea (Pisum sativum), protein pea (Pisum sativum),

4. Chickpea, garbanzo, Bengal gram (Cicer arietinum),

5. Dry cowpea, black-eyed pea, blackeye bean (Vigna unguiculata),

6. Pigeon pea, Arhar/Toor, cajan pea, Congo bean, gandules (Cajanus Cajan),

7. Lentil Lens culinaris),

8. Bambara groundnut, earth pea (Vigna subterranea),

9. Vetch, common vetch (Vicia sativa),

10. Lupins (Lupinus), and

11. Minor pulses such as lablab, hyacinth bean (Lablab purpureus); jack bean (Canavalia ensiformis); Sword bean (Canavalia gladiata); winged bean (Psophocarpus tetragonolobus); Velvet bean, cowitch (Mucuna pruriens); and yam bean (Pachyrhizus erosus).

According to an embodiment, the leguminous plant protein in step a. is air classified protein concentrate, or air classified protein isolate. The air classification can be performed with an industrial machine which separates plant protein material by a combination of size, shape, and density.

According to a further embodiment, the plant protein in step a. is air classified protein concentrate comprising 48 - 65 wt% of protein, the rest of said concentrate being starch, fat, polysaccharides and ash. The air classified protein concentrate may comprise 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 ,59, 60, 61, 62, 63, 64, or 65 wt% of protein, the rest of said concentrate being starch, fat, polysaccharides and ash. In air classification, most of the fibers are separated from proteins. By using air classified protein raw material, the formation of gray color may be avoided, and a pale-yellow final product is obtained.

Further, according to an embodiment the plant protein in step a. is in powder form, preferably having a particle size in the range of from 5 pm to 300 pm, more preferably in the range of from 10 pm to 275 pm.

In an embodiment, the aqueous protein suspension in step a. comprises about 1 to 40 wt.%, preferably 3 to 40 wt%, or about 5 to about 30 wt% or about 5 to 50 wt% plant protein, preferably about 6 to about 15 wt% plant protein, such as 3 to 20 wt%, even more preferably 4.5 to 10 wt% plant protein, such as 5 to 8 wt.% or 6 to 9 wt% plant protein, or 8 wt% plant protein. The aqueous protein suspension may contain such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 wt% plant protein.

In an embodiment the aqueous protein suspension is obtained by preparing plant protein suspension by mixing plant protein, at least two antioxidants, and water.

In an embodiment, the preparation in step a. and the enzyme treatment in step c. are carried out at a temperature of between 10°C and 60°C, preferably between 15°C and 50°C, more preferably between 20°C and 40°C, most preferably between 20°C and 25°C. The enzymatic treatment may be carried out at a temperature of 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, or 60°C, or in the range defined by any two of these values.

In the present disclosure, protein preparation from the plant protein source material, such as leguminous or pulse material, is affected by suitable additives, such as antioxidants. To achieve said effect, any convenient antioxidant can be chosen, preferably sulphites or sulphates and vitamins, more preferably sodium sulphite (Na2SOs) and ascorbic acid.

Further, in an embodiment, the at least one antioxidant is selected from the group consisting of sulphites, sulphates and vitamins, preferably sulphites and ascorbic acid, more preferably sodium sulphite and ascorbic acid. Other antioxidants that are suitable for use in food products may also be used alone or in any combinations. According to an embodiment the aqueous protein suspension in step a. comprises 0.001 - 1.0 % wt%, preferably 0.01 - 0.1 wt% of at least two antioxidants, such as 0.01 - 1.0 wt% sulphite salt or sulphate salt, preferably 0.02% wt% sulphite salt or sulphate salt, and/or 0.01 - 0.25 wt% ascorbic acid, preferably 0.1% ascorbic acid. In a preferred embodiment, the sulphite salt is sodium sulphite (Na2SOs). In a preferred embodiment, the combination of sodium sulphite (Na2SOs) and ascorbic acid is used. The amount of the antioxidant may be such as 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06. 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 wt%.

Antioxidants are known to inhibit internal enzyme activity, such as lipoxygenase, polyphenol oxidase and lipase present in plants, such as leguminous plants, and off-colouring.

According to an embodiment, the preparation of suspension in step a. and the enzyme treatment in step c. are carried out at a pH of about pH 4.5 to about pH 11, preferably from about pH 6.0 to about pH 7.0, such as at pH 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or in the range defined by any two of these values. For pH adjustment, any food grade alkali can be used, e.g. sodium or potassium hydroxide, as required.

Still in an embodiment, the preparation in step a. is carried out from 10 minutes to 4 hours, preferably from 20 minutes to 3 hours, more preferably from 30 minutes to 2 hours, most preferably 90 minutes. The preparation is carried out for a time sufficient to ensure a homogeneous suspension is obtained. The preparation time may be 10, 20, 30, 40, 50, 60, or 90 minutes, 1, 2, 3, or 4 hours.

Typically, in step b. the aqueous phase resulting from the extraction step a. may be separated from the insoluble residual protein source, in any convenient manner. Such as a decanter centrifuge, followed by disc centrifugation and/or filtration, to remove pulse protein source material from the aqueous phase containing soluble proteins may be employed. In the separation step b. 80-100% of insoluble non-suspended solids are separated from the clarified aqueous proteins suspension. In the further clarification step residual insoluble nonsuspended solids can be removed that the concentration of insoluble non-suspended solids is at least less than 0.2%. The separation step b. can be conducted at the same temperature as the protein suspension preparation step a.

In an embodiment the clarified aqueous phase resulting from the separation step b. is enzyme treated with at least one suitable enzyme capable of modifying polyphenols originating from plant raw material. The at least one enzyme may be an enzyme mix that contains hydrolase enzyme main or side activity, such as carboxylic-ester hydrolase or naringinase, which contains alpha-L-rhamnosidase and beta-D-glucosidase activities. Carboxylic-ester hydrolase hydrolases polyphenolic compounds, such as tannins and saponins. Alpha-L-rhamnosidase and naringinase hydrolyses naringin, rutin, quercitrin, hesperidin, dioscin, terpenyl glycosides and many other natural glycosides containing terminal alpha-L-rhamnose. To remove off- tastes, such as bitterness. The quantity of enzyme dosage employed in the enzyme treatment phase depends on the pulse protein source material. Optionally enzyme or enzyme mix can include other main or side activity such as pectinases, hemicellulose, xylanase, beta- glucanase, mannase, glucanase and amylases for example glucoamylase, isoamylase, alphaamylase and beta-amylase.

In an embodiment at least one enzyme capable of modifying polyphenols originating from plant raw material is used.

The bitterness can be removed by using an enzyme or enzyme mix that contains hydrolase enzyme activity, such as carboxylic ester hydrolase, such as tannase (EC 3.1.1.20, tannin acylhydrolase) or naringinase (E.C. 3.2.1.40) activity. For example, a multienzyme complex, containing wide range of carbohydrates including beta-glucanase(s), pectinase(s), cellulase(s), hemicellulose(s) and/or xylanase(s) can be used in the process of the present disclosure. Preferably, the enzyme mixture comprises tannase activity. The combination of enzymes may be such as tannase with beta-glucanase, pectinase, hemicellulase or xylanase, or any combination thereof. In an embodiment an enzyme mixture which comprises a mixture of a cellulase and carbohydrase, or mixture of a cellulase and carbohydrase type enzyme is used.

A carbohydrase that can be used in the present invention is Viscozyme® L available from Novozymes. Viscozyme® L is a blend, or a multienzyme complex, containing wide range of carbohydrases including arabinose, cellulase, beta-glucanases, pectinases, hemicellulases and xylanases and is generally derived from Aspergillus. Viscozyme® L also has tannase activity.

Surprisingly, by using said multienzyme complex, such as Viscozyme® L, two desired results are obtained, namely the cleavage of carbohydrate structures which releases proteins and the cleavage of tannins which improves taste and color. Viscozyme® L degrades for example long carbohydrate and/or polyphenol structures. In one preferred embodiment the enzyme or the enzyme mixture comprises tannase, such as 0.1 wt% tannase is used. For example, Viscozyme L enzyme mixture having tannase activity can be used.

One combination may be a mixture of tannase, pectinase and cellulase, or tannase and pectinase, or tannase and cellulase.

According to an embodiment, in step c. the enzyme treatment is carried out from 5 minutes to 2 hours, preferably from 10 minutes to 1 hour, more preferably for 30 minutes. The enzymatic treatment may be carried out for 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 ,45, 50, 55 or 60 minutes, or for 1 or 2 hours.

In an embodiment, the enzymatic treatment is carried out after the separation step, such as after centrifugation.

According to an embodiment, in step c. the enzyme or enzyme mix further includes activity of enzymes, as main or said activity, selected from the group consisting of enzyme activities of pectinases, hemicellulose, xylanase, beta-glucanase, mannase, glucanase and amylases for example glucoamylase, isoamylase, alpha-amylase and beta-amylase.

Typically, in step c. the enzyme is used in amount of 0.0001 - 10 wt% on dry matter basis, preferably 0.001 - 5 wt% on dry matter basis, more preferably 0.01 - 2 wt% on dry matter basis, most preferably O.lwt % on dry matter basis. The amount of enzyme may be 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, or 9 wt% on dry matter basis.

The enzyme treated aqueous protein solution is subjected to a heat treatment to inactivate the enzyme and heat labile anti-nutritional factors, such as trypsin inhibitors, present in the solution. Heating step also provides the additional benefit of reducing the microbial load. Generally, the protein solution is heated to a temperature of about 50°C to about 160°C, preferably about 60°C to about 120°C, more preferably about 75°C to about 80°C, for about 10 seconds to about 60 minutes, preferably about 10 seconds to about 5 minutes, more preferably about 5 minutes. The heat-treated pulse protein solution then may be cooled for further processing. The heat treatment may be carried out at a temperature of 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, or 160°C, or in the range defined by any two of these values. The heat treatment may be carried out for 10 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 ,35, 40, 45, 50, 55, or 60 minutes, or in the range defined by any two of these values.

Further, in step d. the heat-treatment may be carried out at a temperature of about 60°C to about 120°C, preferably about 75°C to about 80°C, for about 10 seconds to about 60 minutes, preferably about 10 seconds to about 5 minutes, more preferably about 5 minutes.

In an embodiment the heat-treatment is carried out at a temperature of about 60°C to about 135°C, preferably from about 60°C to about 120°C, more preferably from about 75°C to about 80°C, for about 2 seconds to about 60 minutes, preferably from about 10 seconds to about 60 minutes, preferably about 10 seconds to about 5 minutes, more preferably about 5 minutes.

In an embodiment the heat-treatment is carried out at a temperature of about 135°C for about 2 to 5 seconds.

Heating in step d. may be carried out by heating the suspension, by adding hot water to the suspension, or by using conventional techniques known in the art, such as a plate heat exchanger, tubular heat exchanger or jacket.

Optional cooling step may be carried out after the heating step d. A suitable temperature of the cooling step depends on how the following concentration step e. is performed, whether acidification is performed or not. If concentration is performed with a membrane process, using heat sensitive membranes, the suitable cooling temperature can be 5 to 60 °C. For other membrane types, such as ceramic ones, or further concentration methods, such as evaporation, higher temperatures may be applied.

In an embodiment acidification microbiologically or chemically may be carried out for the aqueous protein suspension.

If acidification or fermentation is performed after concentration, the suitable cooling temperature depends on the starter culture. For example, 38 to 45 °C for thermophilic cultures and for example 28 to 32 °C for mesophilic cultures. Other temperatures may also be suitable. According to an embodiment, in step e., the aqueous solution can be concentrated by suitable membrane process, such as microfiltration, ultrafiltration, nanofiltration or reverse osmosis. Said membrane process can be used to separate certain components from aqueous protein solution and the membrane type can be chosen depending on the desired composition of the final product. For example for high purity protein product, with low amount of small molecular weight impurities, e.g. salts, an ultrafiltration membrane with molecular weight cut-off (MWCO) of 1 to 100 kDa, preferably 5 to 20 kDa, more preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa or a range defined by any two of these values is preferred. Or the membrane type having nominal pore size below 0.1 pm, more preferably below 0.01 pm, would be preferred. Different membrane types, such as spiral wound, hollow fiber, flat sheet, etc. can be applied. Likewise, said membrane process can be operated in a way deemed suitable to reach the desired outcome, e.g. batchwise, semi-batchwise, continuously, etc.

In one preferred embodiment heat-treated suspension is concentrated with ultrafiltration. In a further preferred embodiment heat-treated suspension is concentrated with ultrafiltration using 10 kDa spiral-wound membrane and rinsed with diafiltration.

Diafiltration can be applied to further assist in separation of permeable compounds from concentrate produced in a membrane process of previous description. The concentrated retentate has a dry matter content of 5 - 30 wt%, preferably at least 10 - 20 wt%, more preferably at least 12 - 18 wt%. The dry matter content of the concentrated retentate may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 wt%.

In an embodiment the diafiltration step f. may contain one or more diafiltrations and/or diafiltration steps.

The concentrated retentate has a protein content greater than about 70 wt% in dry matter. Preferably, the concentrated retentate has a protein content 80 to 100 wt% protein in dry matter. The protein content of the concentrated retentate has a protein content of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 wt%.

Still in an embodiment, in step e., optionally other concentration methods can be used, such as evaporation or centrifugation. In an embodiment, in step e. the membrane filtration or membrane process is microfiltration, ultrafiltration, nanofiltration or reverse osmosis.

According to an embodiment, in step g. a further concentration may be carried out using evaporation or centrifugation.

Typically, in step f. concentration and washing steps are carried out to separate a retentate and a permeate.

According to an embodiment, the process further comprises after step f. a pasteurization step, which is carried out at a temperature of about 55°C to about 70°C, preferably about 60°C to about 65°C, for about 30 seconds to about 60 minutes, preferably about 10 minutes to about 15 minutes.

Heat treatment step may be pasteurization, which may be carried out at a temperature of about 75°C to about 105°C for about 30 seconds to about 5 minutes, preferably the pasteurization is carried out at a temperature of about 75°C for about 30 seconds to about 5 minutes, preferably for about 5 minutes. The pasteurized concentrated plant protein suspension then may be cooled for drying, preferably to a temperature of about 25° to about 40°C.

Typically, the process further comprises after step f. and after optional pasteurization and cooling steps drying the obtained aqueous protein slurry, preferably using spray drying. In a preferred embodiment protein solution or protein concentrate is spray dried to produce protein isolate or high protein ingredient.

The concentrated and diafiltered aqueous plant protein suspension may be dried by any convenient technique, such as spray drying, drum drying or freeze drying. A pasteurization step may be applied on the plant protein suspension prior to drying, to ensure good microbiological quality. Such heat treatment may be applied under any desired time and temperature conditions. Generally, the concentrated and diafiltered plant protein suspension is heated to a temperature of about 55°C to about 70°C, preferably about 60°C to about 65°C, for about 30 seconds to about 60 minutes, preferably about 10 minutes to about 15 minutes.

In an embodiment, the process further comprises after step f. and after optional pasteurization step cooling of the aqueous protein suspension to a temperature of about 25°C to about 40°C. The cooling temperature may be 25, 30, 35, or 40, or in the range defined by any two of these values.

Still, in an embodiment the process further comprises after step f. and after optional pasteurization and cooling steps drying the obtained aqueous protein suspension, preferably using spray drying.

In an embodiment, the present disclosure concerns a process for producing a plant-based high protein ingredient having a protein content greater than about 70 wt% protein/dry matter, comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating the insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme capable of modifying polyphenols originating from plant raw material to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. optionally washing the concentrated aqueous protein suspension by diafiltration, g. obtaining a high protein ingredient as a retentate from the membrane filtration process, h. optionally, further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

In another embodiment, the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate.

In another embodiment, the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

In another embodiment, the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. washing the retentate by diafiltration, g. further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

In another embodiment, the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. pasteurizing the high protein ingredient, g. further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

In another embodiment, the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, at least one antioxidant, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with at least one enzyme or enzyme capable of modifying polyphenols originating from plant raw material, to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate, f. pasteurizing the high protein ingredient, g. cooling the pasteurized high protein ingredient, h. drying the high protein ingredient, i. further concentrating the high protein ingredient into a protein concentrate or isolate in the form of suspension or powder.

In a preferred embodiment the present disclosure concerns a process for producing a plantbased high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing leguminous plant protein raw material, sodium sulphite (Na2SOs), ascorbic acid, and water to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with an enzyme mixture of a carbohydrase and cellulase and comprising tannase activity to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using membrane filtration to obtain a high protein ingredient as a retentate. In another preferred embodiment the present disclosure concerns a process for producing a plant-based high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing 5 - 30 wt%, preferably 6 - 15 wt%, more preferably 8 wt% of leguminous plant protein raw material, 0.01 - 1.0 wt%, preferably 0.02 wt% sodium sulphite (Na2SOs), 0.01 - 0.25 wt% , preferably 0.1 wt% ascorbic acid, and water at a pH from about pH 4.5 to pH about 11, preferably from about pH 6.0 to about pH 7.0, at a temperature of between 10°C and 60°C, preferably between 15°C and 50°C, more preferably between 20°C and 40°C, most preferably between 20°C and 25°C, from 10 minutes to 4 hours, preferably from 20 minutes to 3 hours, more preferably from 30 minutes to 2 hours, most preferably 90 minutes to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension by centrifugation to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with an enzyme mixture of a carbohydrase and cellulase and comprising tannase acticity in an amount of 0.0001 - 10 wt%, preferably 0.001 - 5 wt%, more preferably 0.01 - 2 wt%, most preferably 0.1 wt% at a pH from about pH 4.5 to pH about 11, preferably from about pH 6.0 to about pH 7.0 at a temperature of between 10°C and 60°C, preferably between 15°C and 50°C, more preferably between 20°C and 40°C, most preferably between 20°C and 25°C from 5 minutes to 2 hours, preferably from 10 minutes to 1 hour, more preferably for 30 minutes to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 50°C to about 160 °C, preferably at about 60° to about 120°C, preferably at about 75° to about 80°C, for about 10 seconds to about 60 minutes, preferably for about 10 seconds to about 5 minutes, more preferably about for 5 minutes to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using ultramembrane filtration to obtain a high protein ingredient as a retentate.

In another preferred embodiment the present disclosure concerns a process for producing a plant-based high-protein ingredient comprising the following steps of a. preparing a plant protein suspension by mixing 6 - 15 wt%, preferably 8 wt% of leguminous plant protein raw material, 0.01 - 1.0 wt%, preferably 0.02 wt% sodium sulphite (Na2SOs), 0.01 - 0.25 wt% , preferably 0.1 wt% ascorbic acid, and water at a pH from about pH 6.0 to about pH 7.0, at a temperature between 15°C and 50°C, more preferably between 20°C and 40°C, most preferably between 20°C and 25°C, from 10 minutes to 4 hours, preferably from 20 minutes to 3 hours, more preferably from 30 minutes to 2 hours, most preferably 90 minutes to obtain an aqueous protein suspension, b. separating insoluble solids from the aqueous protein suspension by centrifugation to obtain a clarified aqueous protein suspension, c. treating said clarified aqueous protein suspension with an enzyme mixture of a carbohydrase and cellulase in an amount of 0.0001 - 10 wt%, preferably 0.001 - 5 wt%, more preferably 0.01 - 2 wt%, most preferably 0.1 wt% and the enzyme mixture comprising tannase activity at a pH from about pH 6.0 to about pH 7.0 at a temperature of 15°C and 50°C, more preferably between 20°C and 40°C, most preferably between 20°C and 25°C from 5 minutes to 2 hours, preferably from 10 minutes to 1 hour, more preferably for 30 minutes to obtain an enzyme-treated aqueous protein suspension, d. subjecting the enzyme-treated aqueous protein suspension to a heat treatment at a temperature of about 75° to about 80°C, for about 10 seconds to about 5 minutes, more preferably about for 5 minutes to obtain a heat-treated aqueous protein suspension, e. concentrating the heat-treated aqueous protein suspension using ultramembrane filtration to obtain a high protein ingredient as a retentate.

The dry pulse protein product has a protein content greater than about 70 wt%. Preferably, the dry plant protein product is an isolate with a protein content in excess of about 90 wt% protein, preferably at least about 100 wt%, (N x 6.25) dry weight basis.

In an embodiment, the process results in the high protein ingredient that has a plant-based protein content greater than about 70 % protein/dry matter, preferably, the high protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably at least about 100 % protein/dry matter, (N x 6.25) dry weight basis. The plantbased protein ingredient has improved organoleptic and functional properties, such as reduced bitterness and improved gelation properties in dairy product analogues. The improved organoleptic properties were achieved by reduced concentration of polyphenolic compounds. Polyphenolic compounds can be for example tannins. Polyphenolic concentration of ingredient is significantly lower than in the starting raw material.

According to another embodiment, the high protein ingredient is obtainable with the process according to the specification.

In an embodiment a high protein ingredient having a protein content greater than about 70 % protein/dry matter, preferably the high-protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably at least about 100 % protein/dry matter, (N x 6.25) dry weight basis is obtained, and the high protein ingredient has neutral color and no perceived bitterness.

In an embodiment a high-protein ingredient is an isolate with a protein content in excess of about 90 % protein/dry matter, preferably about 100 % protein/dry matter, (N x 6.25) dry weight basis is obtained, and the high protein ingredient has neutral color and no perceived bitterness.

In an embodiment the high protein ingredient obtained with the above process is suitable for use in a product selected from the group consisting of plant-based dairy alternatives such as gurt, yoghurts, drinkable yoghurt, creme fraiche, sour cream, sour milk, pudding, set-type yoghurt, smoothie, quark, cheese, cream cheese, ice creams, and meat analogues.

The protein isolate retains native functional properties, such as high solubility, neutral colour and with little, or no, perceived bitterness, making the product ideal raw material for numerous food products and applications, such as yogurts, cheeses, meat analogues, ice creams and other plant-based dairy alternatives.

The texture of a product, such as cheese, can be measured by TA. XT texture analyzer, performing a compression test. A compression test is the most simple and popular test of instrumental texture measurement. A sample is placed on a flat surface and a flat platen is lowered onto the sample to a given force or distance. Sample is deformed and the extent of the deformation and/or the resistance offered by the sample is recorded. Hardness, springiness (elasticity) and gumminess are measured. Hardness is the force required to penetrate the sample to depth of 1 cm. For example, P05 probe can be used.

The raw material in step a., when providing a suspension containing protein, may be in meal or in powder form. The particle size of the powder is typically in the range of 5 to 300 pm, preferably 10 to 275 pm. Meal preferably has a particle size with a D90 value of 150 pm, i.e. 90% of the particles are smaller than 150 pm. In one embodiment, 100% of the particles have a particle size below 275 pm. In one embodiment, 90% of the particles have a particle size below 150 pm and in one embodiment, 50% of the particles have a particle size below 10 pm. The appropriate particle size will also ensure processability of the powder and the suspension formed in step a. of the process. The powder should not form lumps, because that would cause problems in the production line and reduce the quality of the plant-based food product.

Thus, according to one embodiment, the plant-based raw material is in powder form. According to one embodiment of the process of the invention, the plant-based raw material is a powder having a particle size of 5 to 300 pm, preferably 10 to 275 pm. In one embodiment, 90% of the particles are smaller than 150 pm.

Other pre-treatment steps may be required or useful depending on the raw material.

The present invention is further illustrated with the following examples.

EXAMPLES

Example 1

The protein extractability from fava bean and the effect of enzymatic treatment on the clarity and taste of protein solutions resulting from the concentration step was evaluated.

0.02 wt% sodium sulphite (Na2SOs) was solubilized in water with 8 wt% air classified fava bean protein concentrate flour after mixing, 0.1 wt% ascorbic acid was solubilized into the suspension. pH of the suspension was adjusted to pH 7.0 using sodium hydroxide and suspension was then mixed at room temperature for 90 minutes. The suspension was clarified by removal of insoluble solids with a decanter centrifuge and nozzle-bowl separator. The clarified suspension was enzyme treated by adding 0.1 wt% of a commercial enzyme with known tannase activity (Viscozyme L, Novozymes) and incubated for 30 minutes at room temperature under constant mixing. After this enzyme was inactivated by heat-treatment at 80°C for 5 minutes. Heat-treated suspension was then concentrated with ultrafiltration using 10 kDa spiral-wound membrane and rinsed with diafiltration. Subsequently concentrated fava bean protein retentate was then spray dried to produce fava bean protein isolate with protein content of 90 wt% /dry matter.

Example 2

In order to evaluate decreased perceived bitterness of fava bean protein isolate described in Example 1 sensory analysis was conducted using two-alternative forced choice test method (ISO 5495:2005). Nineteen (19) individuals tasted and compared the samples.

Fava bean protein isolate produced according to the method of the present invention was tested to study the impact of the processing method to the sensory quality of the samples. Special attention was paid on the sensed bitterness of the samples.

The processed fava bean isolate was resuspended in water at 8% concentration. This sample A was compared to 8% fava bean protein concentrate water suspension (sample B) and centrifugated clarified 8% fava bean protein concentrate water suspension (sample C).

Samples A, fava bean protein isolate suspended in water and processed according to the invention, were compared to samples B and samples C prepared without the relevant purifying process steps of the invention. Sample B was fava bean protein suspended into water. Sample C was broad bean protein preparation purified in a centrifugation step in accordance with the separation step of the process of the present invention. Separation step contains separating the aqueous protein suspension from insoluble non-suspended solids to obtain a clarified aqueous protein suspension.

The test persons evaluated the difference in bitterness of the samples. The difference between the samples A and, B and C was clear. Samples A were sensed as smooth, velvety and pleasant with clearly less bitterness compared to samples B and C.

Results of the tests showed that the test samples prepared according to the process of the invention were sensed also statistically clearly better in taste, less bitter, in their organoleptic and sensory properties among the test group. Table 1. Results of two-alternative forced choice test, sensory appraisals evaluated bitterness between the tested samples.

Example 3

In order to determine structural forming properties of the fava bean isolate described in Example 1, protein isolate was further processed with fermentation, this was done with combination of bacterial and chemical fermentation to set-type produce yogurt analogue.

Set-type yogurt analogue was produced as follows. 400 grams batch of pre-mix was prepared with following recipe (Table 2) 390 grams of fava bean retentate was mixed with 376 grams of tap water as well as 10 grams of coconut oil and 24 grams table sugar were mixed into the suspension. Fava bean protein suspension was heated to 50°C and homogenized with lab homogenizer at 150 to 160 bars and pasteurized at 85°C for 5 minutes in a water bath. After pasteurization fava bean protein suspension was cooled to 40°C and divided 150 grams batches and 0.08% microbial starter culture and 1% of glucono delta-lactone were added to the suspension. The fermentation was conducted at 38°C for 2 hours until target pH was achieved, which was < pH 5. The produced yogurt analogues had specific characteristics, such as white colour resembling of milk and spoonable texture. Gel hardness of yogurt analogue samples was measured by TA. XT texture analyser and results thereof are illustrated in Figure 2. The probe P05 was used.

Table 2. Yogurt analogue made with fava bean protein isolate described in Example 1.

Total 380 g

Total volume % Mass (g) Fava bean retentate 51 192

Water 44 168

Coconut oil 2 8

Sugar 3 12

Total 100 380

To fermentation 150 g

Bacterial starter 0.12 g

0.08 %

Glucono delta- 1 % lactone 1.5 g

Example 4

0.02% sodium sulphite (Na2SOs) was solubilized in water with 8% pea protein concentrate flour after mixing, 0.1% ascorbic acid and 0,05M NaCI were solubilized into the suspension. pH of the suspension was adjusted to 7.0 using sodium hydroxide and suspension was then mixed at room temperature for 90 minutes. The suspension was clarified by removal of insoluble solids with a lab centrifuge (4200 rpm, 10 minutes). The clarified suspension was enzyme treated by adding 0.1% of a commercial enzyme with known tannase activity (Viscozyme L, Novozymes) and incubated 30 min at room temperature under constant mixing. After this enzyme was inactivated by heat-treatment at 80°C for 5 minutes. Heat-treated suspension was then concentrated with ultrafiltration using 10 kDa spiral-wound membrane and rinsed with diafiltration.

In order to determine structural forming properties of pea protein isolate, that was processed same way as described above, concentrated pea protein retentate was further processed with fermentation, this was done with combination of bacterial and chemical fermentation to produce set-type yogurt analogue. Set-type yogurt analogue was produced as follows. 400 grams batch of pre-mix was prepared with following recipe 390 grams of pea protein retentate was mixed with 376 grams of tap water as well as 10 grams of coconut oil and 24 grams table sugar were mixed into the suspension. Pea protein suspension was heated to 50°C and homogenized with lab homogenizer at 150 to 160 bars and pasteurized at 85°C for 5 minutes in a water bath. After pasteurization pea protein suspension was cooled to 40°C and divided 150 grams batches and 0.08% microbial starter culture and 1% of glucono delta-lactone were added to the suspension. The fermentation was conducted at 38°C for 2 hours until target pH was achieved, which was < pH 5.

Example 5

Removal of bitterness

A pilot scale test for purifying fava bean concentrate in accordance with the present invention was performed.

Moisture and ash contents were measured using a Prepash device from Precisa. Protein content was measured with an automated equipment (Foss) based on the Kjeldahl method. A nitrogen conversion factor of 6.25 was used.

The fava bean concentrate was fine milled, air classified protein concentrate in a form of flour. The dry matter content of the fava bean flour was 92.3 w/w %. The protein content was 67 w/w % of the dry matter and ash 6.4 w/w % of the dry matter.

Fava bean slurry was prepared by dispersing in soft water (25 °C) 150 g of sodium sulfite, 60 kg of fava bean concentrate flour, 750 g of ascorbic acid. The pH of the slurry was adjusted to pH 7 using IM NaOH. The slurry was incubated for 90 minutes under agitation.

Following incubation at pH 7 and in the presence of antioxidants, the insoluble content was 10 -12% (volume). Insoluble content was separated with decanter centrifuge and disc stack centrifuge resulting in less than 0.2 w/w % insoluble content in the overflow.

The clarified process liquor from the centrifuge separation step was treated for fiber hydrolysis in a double jacketed tank. The protein content and ash per dry matter content of the clarified liquor were 78 w/w % and 8.8 w/w %, respectively, after insoluble solid separation.

The clarified protein extract received from the centrifugation step was hydrolyzed using 0.1% of a commercial enzyme with known tannase activity (Viscozyme L). Following the enzyme addition, the extract was incubated under agitation at 25 °C for 30 minutes. The enzyme/substrate ratio was 0.1%. Hydrolysis reaction was stopped treating the product at 85 °C for 5 minutes using a tubular heat exchanger.

The heat-treated extract was cooled to 50 °C and ultrafiltered in Skid TIA 150 L equipment. The membrane was organic lOkDa filter. Diafiltration followed the concentration step in the same membrane filter equipment. Two dia-volumes were used. The protein content of the retentate was 93 w/w % of dry matter and ash was 3.9 w/w % per dry matter. The permeate contained some protein and soluble sugars and minerals. The dry matter content of the retentate was 9.97 w/w %.

The retentate from ultrafiltering was spray dried to powder.

Example 6

Measurement of polyphenolic compounds

Polyphenolic compounds, specifically condensed tannins (proanthocyanidins), were analyzed from the samples of fava bean protein isolate using an HPLC method according to Mattila et al. 2018. Processed fava bean flour was produced according to the process described in Example 5. The results are presented in Table 3.

Table 3. Proanthocyanidins measured from the fava bean flour samples. Average ± standard deviation (n = 3).

Hydrolyzed polyphenolic compounds are removed by ultrafiltration.

References

Berot S, Gueguen J, Berthaud C. 1987. Ultrafiltration of faba bean protein extracts: Process parameters and functional properties of the isolates. Lebensm Wiss Tech 20: 143-150. Mattila P H, Pihlava J-M, Hellstrom J, Nurmi M, Eurola M, Makinen S, Jalava T, Pihlanto A. 2018. Contents of phytochemicals and antinutritional factors in commercial protein-rich plant products. Food Quality and Safety 2:213-219.

Olsen H.S. 1978. Continuous pilot plant production of bean protein by extraction, centrifugation, ultrafiltration and spray drying. Lebensm Wiss Tech 11 :57-64.

EP 2566346 A4

US 20160309732 Al

US 10,143,226 Bl

WO 2020051622 Al