Spreads comprising fermented plant proteins

Abstract

The present invention relates to methods for producing plant-based spreads that mimic the texture, flavour, and sensory properties of traditional dairy spreads. The invention involves the use of plant proteins that are fermented and / or treated with specific enzymes, such as protein deamidase, to enhance the butter-like flavour of plant-based spreads.

Description

[0001] P36861 PCOO / DHT / WZO

[0002] Title: Spreads comprising fermented plant proteins

[0003] FIELD OF THE INVENTION

[0004] The present invention pertains to the field of food technology and, more specifically, to methods for producing plant-based spreads that mimic the texture, flavour, and sensory properties of traditional dairy spreads. The invention involves enzymatic treatments and fermentation processes, with a focus on protein glutaminase treatment and bacterial fermentation of ingredients, to improve the organoleptic and functional characteristics of plantbased spreads and butter.

[0005] BACKGROUND OF THE INVENTION

[0006] Edible fat-containing spreads, such as butter or margarine, are well-known food products that typically have a fat continuous phase and a dispersed aqueous phase. The fat phase usually includes both liquid oils (which remain liquid at ambient temperature) and solid fats (which are solid at ambient temperature). Solid fats, often referred to as structuring or hard stock fats, play a critical role in forming a fat crystal network, giving the spread its structure and stabilizing the emulsion. This network of fat crystals encloses the water droplets, preventing coalescence and ensuring that the product remains stable.

[0007] In addition to fats and water, edible fat spreads may include other ingredients such as emulsifiers, stabilizers, salt, flavourings, and preservatives. These components work together to create product structure, provide desired texture, flavours, and product stability, particularly in processed dairy-based spreads like margarine or butter.

[0008] In recent years, there has been a growing demand for plant-based alternatives to traditional dairy products, driven by consumer preferences for healthier, environmentally sustainable, and allergen-free food options. However, plant-based spreads often face challenges in order to match texture, flavour, mouthfeel and performance of dairy butter.

[0009] Dairy-based spreads primarily get their structure, buttery flavours and melting profile from animal fats and proteins, particularly milk fats and proteins, which contribute to their creamy texture and rich flavour. In contrast, plant-based spreads may experience issues such as fat separation (emulsion instability), not optimal melting behaviour compared to the dairybased benchmark, or off-flavours due to the functionality gap between dairy and plant proteins. Typically, plant butter (i.e. a spread containing plant-based proteins) is distinguished from dairy-based spread (containing dairy proteins). Plant butter is typically composed of 60-80% oil phase and 20-40% aqueous phase, and its microstructure is similar to margarine. The water is present as finely dispersed droplets which have a size of a few micrometres andare covered by a shell of fat crystals. While the water droplet size distribution can be influenced by processing: intensive shear during processing results in a finer emulsion, and the shells of fat crystals are interconnected with the 3-D fat crystal network. The consistency of the plant butter is majorly derived from the continuous fat phase rather than from the dispersed water phase. The nature of the fat crystalline network i.e. the shape, size and aggregation of the fat crystals network determines the microstructure of plant butter.

[0010] In traditional spreads, protein content is typically low and the focus is on the fat and aqueous phases, with the structure being maintained by fats. The key role of proteins is to maintain emulsion stability by emulsification. In addition, in certain plant-based spreads or protein-fortified spreads, proteins may be included for nutritional enhancement. The addition of plant-based proteins, also in small amounts, unfortunately has led to flavour issues, such as bitterness, beany notes, or off-flavours due to the inherent characteristics of the plant protein containing products that are used, typically concentrates or isolate.

[0011] Consumer research has shown that off-flavours are the key limiting factor of the appeal of plant-based alternatives compared to traditional dairy-based products. Therefore, the challenge to provide plant-based spreads with dairy like flavour, texture and performance remains.

[0012] As off-flavours limiting the appeal of plant-based alternatives compared to traditional dairy-based products, it remains an ongoing purpose to provide plant-based spreads with improved flavour, texture and savoury evaluations. It desirable to provide a plant-based product with a butter-like flavour and a process for a plant-based product with a butter-like flavour similar to that of dairy products to meet the increasing demand for plant-based food alternatives with sensory characteristics similar to those of dairy products.

[0013] SUMMARY OF THE INVENTION

[0014] The inventors surprisingly found that by providing a plant-based spread including plant proteins that have been treated with specific enzymes (like protein deamidase) and / or fermented and / or lipase treated vegetable oil, the texture and buttery flavours of the spread is enhanced and the plant-based off-flavour is reduced. It is unexpected that such treated plant-based proteins can provide a butter flavour and mouthfeel that more closely resembles traditional dairybased butter flavour as in traditional dairy-based spreads.

[0015] Accordingly, in one aspect, the present invention provides a plant-based spread comprising:

[0016] a water phase;

[0017] 50 to 99 wt.% of a fat phase;

[0018] 0.1-10 wt.% of an enzyme-treated, fermented protein mixture as disclosed herein.In one aspect, the invention provides a method for providing a plant-based spread comprising the steps of:

[0019] (a) providing an aqueous mixture comprising a plant protein;

[0020] (b) treating the aqueous mixture with a protein deamidase;

[0021] (c) fermenting the mixture using lactic acid bacteria during or after the enzymatic treatment to provide an enzyme-treated, fermented protein mixture;

[0022] (d) blending the enzyme-treated, fermented protein mixture with a fat phase and an aqueous phase to prepare the plant-based spread.

[0023] Preferably, the method further comprises the step of combining the aqueous mixture with 0.1 to 10 wt.% of a hydrolysed vegetable oil prior to fermenting the mixture in step (c), thereby obtaining a mixture comprising the plant protein and the hydrolysed vegetable oil.

[0024] In one aspect, the invention provides an enzyme-treated, fermented protein mixture. The plant-based spread according to the present invention has an improved texture and flavour, in particular an improved freshness and / or butter-like flavour and reduced off-notes that are commonly experienced with plant-based products. Other advantages include an increased amounts of diacetyl, acetoin and / or lactones in plant-based spreads and enzyme-treated, fermented protein mixture of the present invention which improve taste and consumer perception. A further advantage of the present invention is its time efficiency. The method described here significantly reduces fermentation times compared to prior art processes.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] In one aspect, the invention provides a method for providing a plant-based spread comprising the steps of:

[0027] (a) providing an aqueous mixture comprising a plant protein;

[0028] (b) treating the aqueous mixture comprising a plant protein with a protein deamidase;

[0029] (c) fermenting the mixture using lactic acid bacteria during or after the enzymatic treatment to provide an enzyme-treated, fermented protein mixture;

[0030] (d) blending the enzyme-treated, fermented protein mixture with a fat phase and an aqueous phase to prepare the plant-based spread.

[0031] Preferably, the method further comprises the step of combining the aqueous mixture with 0.1 to 10 wt.% of a hydrolysed vegetable oil prior to fermenting the mixture in step (c), thereby obtaining a mixture comprising the plant protein and the hydrolysed vegetable oil. The hydrolysed vegetable oil is preferably obtained by treating a vegetable oil with a lipase.While enzymatic treatments involving protein deamidase and lipase can be performed in a stepwise manner, it is preferable that the lipase treatment and protein deamidase treatment be carried out simultaneously before or during fermentation, preferably before fermentation. This approach ensures efficiency and enhances the flavour development of the plant-based spread. However, a step-by-step approach can also be used depending on the specific process requirements. In either case, the timing and sequence of these treatments can be adjusted to optimize both the texture and flavour of the final product.

[0032] Thus, the step of combining the aqueous mixture with 0.1 to 10 wt.% of a hydrolysed vegetable oil may be performed by:

[0033] mixing the aqueous mixture of step (a) with a vegetable oil and treating the aqueous mixture with a lipase before, during, or after step (b), preferably during step (b); or

[0034] mixing the mixture of step (b) with a vegetable oil and subsequently treating the mixture with a lipase;

[0035] treating a vegetable oil with a lipase and adding the hydrolysed vegetable oil to the aqueous mixture of step (a) or the mixture of step (b);

[0036] preferably wherein step (c) is executed after the enzymatic treatment.

[0037] Preferably, the step of combining the aqueous mixture with 0.1 to 10 wt.% of a hydrolysed vegetable oil is performed by

[0038] mixing the aqueous mixture of step (a) with a vegetable oil and treating the aqueous mixture with a lipase during step (b),

[0039] wherein step (c) is executed after the enzymatic treatment.

[0040] Thus, preferably, the method for providing a plant-based spread comprises the steps of:

[0041] (a) providing an aqueous mixture comprising a plant protein and a vegetable oil; (b) treating the aqueous mixture comprising a plant protein and a vegetable oil with a protein deamidase and a lipase;

[0042] (c) fermenting the mixture using lactic acid bacteria after the enzymatic treatment to provide an enzyme-treated, fermented protein mixture;

[0043] (d) blending the enzyme-treated, fermented protein mixture with a fat phase and an aqueous phase to prepare the plant-based spread.

[0044] Plant ProteinPlant protein, also referred to as a plant-based protein herein, refers to proteins that are extracted, isolated, or derived from plant sources. These proteins can be obtained from a variety of plant species, including but not limited to legumes (such as peas, beans, lentils), cereals (such as oat, wheat, rice), seeds (such as sunflower, pumpkin, chia), and nuts. The protein may be used in a variety of forms, such as concentrates or isolates.

[0045] Preferably, the plant protein is a vegetable protein. More preferably, the plant protein is selected from the group consisting of lentil (Lens culinaris), broad bean (Vicia faba), oat (Avena spp.), soy, pea, almond (Prunus dulcis), cashew, chickpea (Cicerarietinum), canola (Brassica napus subsp. napus), and potato protein and mixtures thereof. These plant proteins, particularly those obtained from broad beans, lentils, peas, oat, almonds, and soy, impart an enhanced texture and flavour profile to plant-based spreads obtained by a method according to the present invention. Even more preferably, the plant protein is selected from the group consisting of lentil, broad bean (fava bean),, , oat, pea, , almond, and soy. Most preferably, the plant protein is broad bean (fava bean) or oat protein.

[0046] The plant protein can be isolated from the plant or parts of the plant. The isolate can be in the form of a plant-based protein isolate or concentrate. A plant-based protein isolate or concentrate contains plant-based proteins, but can contain impurities as well, such as other plant-based components. The impurities or plant-based components present depend on the manner of obtaining the isolate or concentrate and on the source of the plant material itself. The plant protein in the spread according to the invention can be a plant-based protein isolate or concentrate. Typically, the plant-based protein isolate or concentrate contains 20-80 wt.% plant protein, wherein wt.% is calculated based on the total weight of the protein isolate or concentrate. The amount of plant-based protein in the isolate or concentrate can vary, depending on the plant species and the manner of isolating and / or concentrating. The plantbased protein isolate or concentrate may contain 30-80 wt.% of plant protein, 40-70 wt. % of plant protein (oil seeds), 50-80 wt. % of plant protein (legumes, nuts), 50-70 wt. % of plant protein, 20-60 wt. % of plant protein (cereals), wherein wt.% is calculated based on the total weight of the protein isolate or concentrate.

[0047] The amount of actual plant protein in the protein ingredient (whether isolate or concentrate) in the plant-based spread may be from 0.05 - 2 wt.%, and preferably from 0.08 -1.6 wt.%, wherein the weight percentages are based on the total weight of the product. Thus, for a legume plant-based protein, a legume plant-based protein isolate that contains 70 wt.% of legume plant-based protein, the legume plant-based protein is present in the spread in an amount of from 0.035 wt.% (0.05wt.%*70wt.%) to 1.4 wt.% (2wt.%*70wt.%) . For other plantbased proteins and plant-based protein isolates analogous calculations are within the abilities of the average skilled person.The plant protein ingredient (in the form of a concentrate or isolate) may be provided in an amount of 0.1-10 wt. % calculated on the total weight of the plant-based spread composition.

[0048] In one embodiment, the method of providing a plant-based spread comprises the step of providing an aqueous mixture comprising a plant protein. The plant protein is preferably a vegetable protein as disclosed herein.

[0049] Preferably, the aqueous mixture comprising a plant protein comprises 0.1 to 10 wt.% plant protein, preferably 0.5 to 8 wt.%, more preferably 1-5 wt.%, wt.% based on total weight of the aqueous mixture.

[0050] Throughout the description, the terms 'protein mixture' and 'protein emulsion' are used interchangeably.

[0051] Enzymatic treatment of plant protein

[0052] In one embodiment, the method of providing a plant-based spread comprises the step of treating the aqueous mixture comprising a plant protein with a protein deamidase before and / or during the fermentation process to obtain an enzyme-treated, fermented plant protein mixture. A deamidating enzyme catalyzes the removal of an amide group from amino acid residues, specifically glutamine and asparagine, within proteins. This process converts glutamine into glutamic acid and asparagine into aspartic acid, altering the structure and properties of the protein.

[0053] The deamidating enzyme may be selected from the group consisting of protein glutaminase, peptidylglutaminase, transglutaminase, glutaminyl cyclase and asparaginase. Preferably the deamidating enzyme is a protein glutaminase as it provides deamidation of glutamine side chains without substantial proteolysis or crosslinking, and further improves the functionality of the protein in the plant-based spread. Protein glutaminase (EC 3.5.1.44) deamidates glutamine residues in proteins, converting them into glutamic acid. The protein glutaminase may as disclosed in, for example, WO 2015 / 133590, but is not limited thereto. Asparaginase deamidates asparagine residues, converting them into aspartic acid.

[0054] Deamidation of a plant protein provides a plant protein having a particular protein deamidation degree. The protein deamidation degree (PDD) refers to the level of enzymatic deamidation of a plant protein by a deamidating enzyme, relative to the total deamidation achieved through acid treatment of the same protein. During deamidation, ammonia is released. The amount of released ammonia may be determined using an ammonia assay (e.g. a colorimetric assay), with a corresponding blank prepared under identical conditions but in the absence of the deamidating enzyme. The PDD is calculated by dividing the amount of ammonia released by enzymatic deamidation by the amount released by acid-induceddeamidation, then multiplying by 100%. Unless stated otherwise, the PDD is determined on the total protein present in the enzyme-treated protein mixture.

[0055] Preferably, the plant protein in the enzyme-treated protein mixture treated with a deamidating enzyme has a deamidation degree of at least 30%, more preferably 40-80%, most preferably 55-65%. At these ranges of protein deamidation degree (PDD), plant-based spreads with increasingly improved texture were obtained compared to those made with untreated plant protein.

[0056] The deamidated plant protein may be obtained by treating an aqueous mixture comprising the plant protein with a protein deamidase under suitable reaction conditions. The reaction conditions depend, inter alia, on the selected protein deamidase, the temperature, the treatment time, and the type and concentration of the plant protein. Suitable reaction conditions can be selected and adjusted by the skilled person on the basis of routine experimentation.

[0057] Protein deamidating enzyme activity is expressed in units (U) and may be measured according to a method as described in WO2022 / 102723, as set out below. A reaction solution is prepared by adding 0.1 mL of a sample solution comprising the protein deamidating enzyme to 1.0 mL of 0.2 M phosphate buffer (pH 6.5) comprising 30 mM Z-GIn-Gly (benzyloxycarbonyl-L-glutaminylglycine; Cbz-GIn-Gly), followed by incubation at 37°C for 10 minutes. The reaction is stopped by addition of 1.0 mL of 0.4 M trichloroacetic acid (TCA) solution. A blank is prepared by adding 1.0 mL of 0.4 M TCA solution to 1.0 mL of 0.2 M phosphate buffer (pH 6.5) comprising 30 mM Z-GIn-Gly, followed by addition of 0.1 mL of the sample solution comprising the protein deamidating enzyme. The amount of ammonia generated is determined using an ammonia assay and by reference to a calibration curve prepared using an ammonia standard solution (e.g. ammonium chloride). One unit (1 U) of protein deamidating enzyme activity is defined as the amount of enzyme that produces 1 pmol of ammonia per minute under the assay conditions described above.

[0058] Preferably, the aqueous mixture comprising the plant protein is treated with a protein deamidase in an amount of 0.1 to 1000 U per g of plant protein, more preferably 1 to 1000 U / g, even more preferably 2 to 100 U / g, still even more preferably 3 to 50 U / g, and most preferably 4 to 20 U / g. When the plant protein is obtained from beans (e.g. soybean), the amount of protein deamidase is preferably 1 to 40 U per gram plant protein, more preferably 2 to 20 U / g, and most preferably 4 to 16 U / g. For plant proteins from cereals, like oat protein, the amount of the protein deamidase per 1 gram of plant protein is preferably 0.5 to 15 U, more preferably 2 to 10 U, and most preferably 4 to 7 U. For plant proteins from nuts, like almond protein, the amount of the protein deamidase per 1 gram of plant protein is preferably 5 to 40 U, more preferably 10 to 20 U, and most preferably 12 to 17 U.Preferably, the treating of the aqueous mixture comprising the plant protein is carried out at a temperature between 5 to 80° C. more preferably 20 to 70° C, and most preferably 30 to 65° C.

[0059] Preferably, the aqueous mixture comprising the plant protein is treated for 10 seconds to 48 hours, more preferably 1 minute to 24 hours, even more preferably 5 minutes to 2 hours, still even more preferably 5 to 60 minutes, and most preferably 10 to 30 minutes.

[0060] Preferably, the aqueous mixture comprising the plant protein has a pH of 5 to 10, more preferably 5.5 to 8, even more preferably 6 to 7.5, and most preferably 6.5 to 7.

[0061] In preferred embodiments, the treating of the aqueous mixture comprising a plant protein with a protein deamidase is carried out at a temperature of 40-70 ° C for 5 to 60 minutes using protein glutaminase. Preferably, the temperature is 50-65 ° C for 10 to 30 minutes.

[0062] In embodiments, the plant protein is present in an amount of 0.1 to 10 wt.% plant protein, preferably 0.5 to 8 wt.%, more preferably 1-5 wt.% wt.% based on total weight of the aqueous phase during enzyme treatment.

[0063] Lactic acid bacterial fermentation

[0064] In the fermentation step, microorganisms that are both considered safe for food production and capable of fermenting plant proteins may be used. Preferably the microorganisms are bacteria, more preferably lactic acid bacteria (LAB).

[0065] In one embodiment, the microorganisms are lactic acid bacteria. Lactic acid bacteria (LAB) are a group of bacteria that convert sugars (mainly glucose) into lactic acid through fermentation, a process that preserves food by preventing harmful microorganisms from growing. In addition to food preservation, LAB are known for their role in improving digestion and supporting the immune system when consumed as probiotics. Examples of lactic acid bacteria are Lactobacillus, Lacticaseibacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus thermophilus. Lactic acid bacteria (LAB) can be categorized into mesophilic and thermophilic lactic acid bacteria based on their preferred temperature ranges for optimal growth and activity. Mesophilic lactic acid bacteria are lactic acid bacteria that have an optimal growth temperature at moderate temperatures, preferably between 20°C and 40°C. Thermophilic lactic acid bacteria are lactic acid bacteria having an optimal growth temperature at higher temperatures than moderate temperatures, preferably between 40°C and 60°C. Optimal growth temperatures may be determined through controlled experiments that track bacterial growth, i.e. , the increase in the number of bacteria per unit of time, at various temperatures. Common methods involve measuring changes in bacterial population over time, for example using methods like optical density (which measures turbidity) orcolony-forming units (CFU) e.g., on agar plates, while incubating the bacteria at a particular temperature.

[0066] Preferably, the lactic acid bacteria are mesophilic bacteria, preferably selected from the group consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Leuconostoc mesenteroides. Such lactic acid bacteria can produce butter-like flavour compounds during fermentation, for example diacetyl and / or acetoin, thereby contributing to a buttery flavour profile of the plant-based spread.

[0067] Thus, preferably, the mesophilic bacteria have the ability to produce butter-like flavours during fermentation, more preferably to generate key flavour compounds such as diacetyl and acetoin.

[0068] In one embodiment, the lactic acid bacteria are a bacterial composition comprising mesophilic and thermophilic bacteria. Preferably, the mesophilic bacteria are selected from the group consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Leuconostoc mesenteroides. Preferably, the thermophilic bacteria are selected from the group consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.

[0069] Bulgaricus. It was found that enzyme-treated plant protein, preferably deamidated plant protein, fermented using a bacterial composition comprising mesophilic and thermophilic bacteria enhanced both the texture and flavour characteristics of a plant-based spread produced therefrom. Furthermore, the plant-based spread may comprise higher levels of diacetyl and acetoin per weight of the spread compared to a plant-based spread, wherein the plant-based protein is fermented using a bacterial composition comprising solely mesophilic or thermophilic bacteria.

[0070] In one embodiment, the lactic acid bacteria are a bacterial composition comprising mesophilic bacteria and thermophilic bacteria, wherein the mesophilic bacteria are selected from the group consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Leuconostoc mesenteroides’, and the thermophilic bacteria are selected from the group consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus. The mesophilic bacteria are obtainable, obtained from, or derived from a bacterial culture, preferably Lyofast V M01 (Sacco System). Lyofast V M01 is a mesophilic culture comprising bacteria from the taxa Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Lactococcus lactis ssp. lactis biovar. diacetylactis, and Leuconostoc mesenteroides ssp. Mesenteroides. The thermophilic bacteria are obtainable, obtained from, or derived from a bacterial culture, preferably Vege33 (Danisco). Vege 33 is a thermophilic culture comprising bacteria belonging to the taxa Streptococcus thermophilus and subspecies Lactobacillus delbrueckii subsp. Bulgaricus. A bacterial composition comprising the mesoophilic bacterial culture LyofastVMOI and the thermophilic bacterial culture Vege33 cultures provided aparticularly enhanced improvement of flavour in the plant-based spread compared to other mesophilic and thermophilic cultures.

[0071] In one embodiment, fermenting the enzyme-treated plant protein mixture using lactic acid bacteria comprises a step of combining the enzyme-treated plant protein mixture with lactic acid bacteria as disclosed herein. Combining the enzyme-treated plant protein mixture with lactic acid bacteria may be performed by inoculating the enzyme-treated plant protein mixture with the lactic acid bacteria.

[0072] In one embodiment, a fermentation stimulating agent is added to the enzyme-treated plant protein mixture prior to fermentation, i.e. prior to inoculation with the lactic acid bacteria, such that fermentation is carried out in the presence of the fermentation stimulating agent. Additionally or alternatively, further fermentation stimulating agents, such as pyruvate and / or citric acid may be added.

[0073] In one embodiment, the fermentation stimulating agent is one or more sugars. These sugars may serve as a substrate for the lactic acid bacteria, further aiding in the production of key flavour compounds. Preferably, the one or more sugars is a monosaccharide or a disaccharide, preferably selected from glucose, dextrose, sucrose, fructose, galactose, lactose, and maltose; or combinations thereof.

[0074] Preferably, the protein mixture to be fermented comprises between 0.1 and 20 wt.% of the one or more sugars, more preferably 0.1 and 10 wt.%, even more preferably between 0.1 and 5 wt.%, yet even more preferably 0.1 and 2.0 wt.%, most preferably between 0.1 and 1 wt.%, depending on the desired fermentation intensity and flavour outcome, wherein wt.% is calculated based on the total weight of the protein mixture to be fermented. Particularly preferred is the addition of 0.1 to 1 wt.% glucose and / or sucrose. The total sugar content may be measured by Brix analysis and specific sugars can be determined by chromatographical methods, e.g. ion chromatography. In the present context sugar is to be understood as saccharides, but may also include starch and cellulose, as some lactic acid bacteria may be able to degrade starch. Sugars preferably include monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0075] Preferably, at least part of the one or more sugars is provided in the form of a fruit and / or vegetable juice, i.e., the source of the one or more sugars is a vegetable juices and fruit juices; or a combination thereof. Preferably, the fruit and / or vegetable juice is selected from citrus juices (lemon, lime, orange, mandarin, grapefruit); red fruit juices (raspberry, strawberry, blueberry, blackberry, Amelanchier (juneberry), rose hip, cranberry, plum, prune, red and black currant, cherry, tomato); yellow and stone fruit juices (peach, apricot, nectarine, banana); tropical fruit juices (mango, passion fruit, pineapple, lychee); pome fruit juices (apple, pear, quince, medlar); and vegetable juices (beet, carrot, cucumber, onion). The fruitand / or vegetable juice is most preferably selected from apple juice, carrot juice, and beet juice; or a combination thereof. Preferably, the enzyme-treated plant protein mixture to be fermented comprises 0.1 and 10 wt.% one or more sugars, even more preferably between 0.1 and 5 wt.%, yet even more preferably 0.1 and 2.0 wt.%, most preferably between 0.1 and 1 wt.%, wherein the one or more are sugars are obtained from fruit and / or vegetable juices and wt.% is calculated based on the total weight of the protein mixture to be fermented. The fruit and / or vegetable juices are preferably added in a total amount of 0.1 to 10 wt.%, more preferably 0.2 to 5 wt.%, most preferably 0.5 to 3 wt.%, wherein wt.% is calculated based on the total weight of the protein mixture to be fermented. The fruit and / or vegetable juice may be provided as a single-strength juice or as a juice concentrate, optionally in reconstituted form. In some embodiment, all sugars may be obtained from fruit and / or vegetable juices.

[0076] In some embodiments, fruit and / or vegetable juices, such as apple juice, carrot juice, or beet juice, may be added as the fermentation stimulating agent. These juices not only provide sugars but also contribute to the overall sensory profile, enhancing the flavour complexity of the plant-based spread. Surprisingly, addition of fruit and / or vegetable juices resulted in an overall more positively perceived flavor profile as compared to addition of sugars only. The inclusion of natural juices appears to help balance the flavour and adds subtle taste notes that complement sugar fermentation-produced notes. Fruit and / or vegetable juices are preferably added in an amount of 0.1 to 10 wt.%, more preferably 0.2 to 5 wt.%, most preferably 0.5 to 3 wt.%, based on the total weight of the mixture to be fermented.

[0077] Preferably, the plant protein mixture is inoculated with lactic acid bacteria in an amount effective to initiate fermentation. Preferably, the lactic acid bacteria are provided in the form of a direct-vat-set culture and dosed according to a supplier-recommended dosage. For example, a mesophilic culture such as Lyofast V M01 may be used at 0.5-2.0 UC / 100 L of the protein mixture, wherein UC is a supplier-defined dosage unit (Sacco System). A thermophilic culture such as Danisco VEGE 033 may be used at 10-20 DCU / 100 L of the protein mixture, wherein DCU refers to the supplier-defined Danisco Culture Unit.

[0078] Alternatively, inoculation of the protein mixture may be carried out by providing at least 5*10A4 colony-forming units (i.e. , 50000 CFU) per gram of the protein mixture.

[0079] Preferably, inoculation of the protein mixture is carried out by providing 1*10A5 to 5*10A8 CFU / g of the protein mixture, more preferably 5*10A5 to 5*10A7 CFU / g, and most preferably 1*10A6 to 1*10A7 CFU / g.

[0080] In one embodiment, fermenting the plant protein using lactic acid bacteria is performed at a temperature between 25°C to 50°C, preferably 30°C to 45°C, for a period of 1 to 24 hours, preferably 2 to 12 hours, even more preferably 2.5 to 8 hours, and most preferably 3 to 5 hours. During fermentation, the LAB metabolize carbohydrates present inthe plant protein mixture matrix, producing organic acids and modifying the protein structure. Preferably fermenting the plant protein using lactic acid bacteria is performed at a temperature between 35°C to 45°C for a period of 2 to 8 hours, more preferably 4 to 8 hours, under anaerobic or semi-anaerobic conditions, preferably 30°C to 40°C for a period of 6 hours. In some embodiments, the fermentation is continued for 8-12 hours at 28 °C. The fermented protein emulsion may cooled down and be stored at 4 °C. Fermenting deamidated plant protein under such conditions provided fermented, enzyme-treated protein emulsions with excellent properties for plant-based spreads.

[0081] Preferably, fermentation is terminated when the pH of the protein mixture decreases by at least 0.5 pH units, preferably by at least 0.7 pH units and most preferably at least 1 pH units, relative to the pH of the protein mixture immediately after inoculation with the lactic acid bacteria. The pH may be measured at 20-25°C using a calibrated pH meter.

[0082] Preferably, the protein mixture has a pH of 5.5 to 10, more preferably 5.8 to 8, even more preferably 6.2 to 7.5, and most preferably 6.5 to 7 prior to fermentation. The pH may be adjusted using a food-grade acid and / or base, for example citric acid and / or sodium hydroxide.

[0083] In preferred embodiments, fermentation is terminated when the enzyme-treated, fermented protein mixture has a pH of 4.0 to 6.0, preferably 4.5 to 6.0, more preferably 5.0 to 6.0, and most preferably 5.4 to 5.8. The pH may be measured at 20-25°C using a calibrated pH meter.The fermentation with lactic acid bacteria, may result in the advantageous formation of diacetyl and / or acetoin and other flavour components.

[0084] Hydrolysed oil

[0085] In preferred embodiments of the present invention, a hydrolysed vegetable oil is used in the preparation of the plant-based spread. The hydrolysed vegetable oil may be obtained by treating a vegetable oil with a lipase, to at least partially hydrolyse triacylglycerols. Such treatment can increase the content of free fatty acids and / or partial glycerides (mono- and / or diacylglycerides) relative to the untreated oil.

[0086] Preferably, the hydrolysed vegetable oil is selected from the group consisting of coconut oil, rapeseed oil, linseed oil, soybean oil, palm oil, palm stearin, palm kernel oil, and sunflower oil; preferably, coconut oil or sunflower oil. Enzymatically treating the vegetable oil, particularly coconut oil, with lipase can aid in the development of taste and flavour, in particular when provided in an enzyme-treated protein mixture prior to fermentation.

[0087] Preferably, the hydrolysed vegetable oil comprises 5 to 50 wt.% free fatty acids, preferably 10 to 40 wt.%, more preferably 20 to 30 wt.%, based on the total weight of the hydrolysed vegetable oil. The free fatty acid content may be determined according to ISO 660:2020 (or if unsuitable, an equivalent AOCS method such as Cd 3d-63 or Ca 5a-40) andexpressed as wt.% free fatty acids as an equivalent fatty acid (e.g. as oleic acid; for coconut oil, as lauric acid).

[0088] Preferably, the lipase is selected from a triacylglycerol lipase, a phospholipase, and a lysophospholipase. Most preferably, the lipase is a triacylglycerol lipase (EC 3.1.1.3), preferably obtainable, obtained or derived from Rhizopus delemar. Such a lipase may be particularly beneficial for the flavour profile of the plant-based spread. Alternatively, a lipase derived from the genus of Penicillium, Burkholderia, Aspergillus, Candida, Pichia, Chromobacterium, Pseudomonas, Mucor.Thermomyces, or Geotrichum can be used.

[0089] Suitable enzymes are commercially available and may be used according to supplier instructions.

[0090] In one embodiment, the lipase-hydrolysed vegetable oil may be obtained by treating the vegetable oil with a lipase under suitable reaction conditions. Suitable reaction conditions can be selected and adjusted by the skilled person on the basis of routine experimentation.

[0091] Lipase activity may be expressed in units (II) and may be determined using a titrimetric assay using olive oil as substrate at pH 7.7 and 37°C. A reaction mixture is prepared by combining 2.50 mL deionized water, 1.00 mL of 200 mM Tris-HCI buffer (pH 7.7 at 37°C), and 3.00 mL olive-oil emulsion substrate, followed by equilibration to 37°C. The reaction is initiated by adding 1.00 mL of a lipase enzyme solution (optionally diluted in deionized water) and mixing vigorously. The mixture is incubated at 37°C for exactly 30 minutes. A blank is prepared under identical conditions, except that the enzyme solution is not present during the incubation period and is added only after termination of the incubation. The reaction is stopped by adding 3.00 mL of 95% ethanol. Thymolphthalein indicator is added and the mixture is titrated with standardized 50 mM NaOH to a (light) blue endpoint. The NaOH volume consumed for the blank is subtracted from that consumed for the sample to obtain the net NaOH consumption corresponding to free fatty acids released during the incubation. Lipase activity may be calculated from the net NaOH consumption, taking into account the molarity of the NaOH, the incubation time (normalized to one hour), the enzyme solution volume used (1.00 mL) and any dilution factor. One unit (1 U) is defined as the amount of enzyme that hydrolyzes 1.0 microequivalent of fatty acid from a triglyceride in one hour at pH 7.7 and 37°C. For monocarboxylic fatty acids, 1 peq corresponds to 1 pmol.

[0092] Preferably, the vegetable oil is treated with a lipase in an amount of 0.01 to 2000 U per gram of vegetable oil, more preferably 0.1 to 1000 U / g, even more preferably 1 to 500 U / g, still even more preferably 5 to 400 U / g, and most preferably 10 to 250 U / g.

[0093] Alternatively, the vegetable oil is treated with 0.001 to 1 wt.% of a lipase preparation, preferably 0.01 to 0.2 wt.%, based on the weight of the vegetable oil.

[0094] Preferably, the treating of the vegetable oil with a lipase is carried out at a temperature between 10 to 70° C. more preferably 20 to 70° C, and most preferably 30 to 65°C. Preferably, the vegetable oil is treated with the lipase for 10 seconds to 48 hours, more preferably 1 minute to 24 hours, even more preferably 5 minutes to 2 hours, still even more preferably 5 to 60 minutes, and most preferably 10 to 30 minutes. In preferred embodiments, the lipase treatment is carried out at a temperature of 40-70 ° C for 5 to 60 minutes, preferably, at 50-65 ° C for 10 to 30 minutes.

[0095] Preferably, the lipase treatment is carried out in parallel (i.e. , simultaneously) with treatment of the plant protein mixture with the protein deamidase, for example by adding the lipase and the protein deamidase to a plant protein material comprising a protein and a fat and oil, preferably an emulsion.

[0096] In preferred embodiments, the hydrolysed vegetable oil is combined with the protein mixture prior to fermentation (i.e., prior to inoculation with lactic acid bacteria), such that fermentation is carried out in the presence of the hydrolysed vegetable oil. Without being bound by theory, the presence of hydrolysed vegetable oil during fermentation can contribute to the development of a butter-like flavour profile, for example through the formation of flavour compounds such as diacetyl, acetoin and / or lactones.

[0097] Preferably, the hydrolysed vegetable oil is present in an amount up 25 wt. %, preferably between 0.1 and 10 wt.%, more preferably about 5 wt.% vegetable oil, wt.% calculated on the total weight of the plant protein mixture prior to fermentation (i.e., prior to inoculation with lactic acid bacteria).

[0098] In one embodiment, a hydrolysed vegetable oil can be provided and may be added to the fermented, plant-based protein emulsion separately or added to the fat phase or the mixture when the spread is blended. The hydrolysed oil can be obtained by treatment of vegetable oil with enzyme(s) such as a lipase. Being "treated with an enzyme" refers to the process of combining an enzyme with a vegetable oil so that the enzyme catalyzes the chemical reaction of (partially) breaking down triglycerides (fats) into free monoacylglycerides, diacylglycerides, free fatty acids and glycerol, known as (partial) hydrolysis. The lipase enzyme may be selected from the group consisting of triacylglycerol lipase, phospholipase, or lysophospholipase. Suitable lipase enzymes are commercially available and can be used following the suppliers instructions. Enzymatically treating the vegetable oil, particularly coconut oil, with lipase separately result in improving lactone production which can aid in the development of taste and flavour.

[0099] Lactones are cyclic esters formed when a hydroxyl group within a fatty acid reacts with a carboxyl group, creating a stable ring structure. This reaction is often facilitated by reaction conditions, including moderate heat and low water activity, which favor cyclization over further oxidation or polymerization.Precursors for lactones can be formed by the conversion of vegetable oils through hydrolysis which can be catalyzed by lipase enzymes. Lipases exhibit specificity towards the ester bonds in triglycerides, facilitating their breakdown into free fatty acids of varying chain lengths. Preferably the lipase is obtainable, obtained or derived from Rhizopus delemar.

[0100] In the case of vegetable oils, the fatty acid composition depends on the source oil, such as palm, coconut, or soybean oil, which contain varying proportions of saturated and unsaturated fatty acids. Hydrolysis can be tailored by adjusting factors such as temperature, pH, enzyme concentration, and the presence of water to optimize the production of shortchain fatty acids (SCFAs),

[0101] Without being bound by theory it is believed that the enzyme treatment of oil / fat provides an increase in short chain fatty acids (SCFAs) such as including butyric acid, caproic acid, and caprylic acid saturated fatty acids that can contribute to taste and flavour as they may serve as precursors for the formation of lactones and other flavour enhancing compounds.

[0102] The type of lactone formed depends on the carbon chain length and hydroxyl position of the SCFA precursor. For example, gamma-lactones (five-membered rings) and delta-lactones (six-membered rings) are the most common structures, with distinct sensory profiles, gamma-decalactone, derived from 4-hydroxydecanoic acid, is notable for its peach-like aroma, while delta-dodecalactone, from 5-hydroxydodecanoic acid, imparts a creamy, coconut-like flavor.

[0103] Treating a vegetable oil with a lipase enzyme thus may result in the formation of reaction products of the enzymatic treatment which may contribute to a more butter-like flavour. The enzymatically treated vegetable oil may be prepared separately and combined with the enzyme-treated plant protein emulsion prior to fermentation such that the fermentation with lactic acid bacteria is with the hydrolysed oil present. In an alternative embodiment, the lipase treated vegetable oil can be combined with the enzyme-treated, fermented plant protein emulsion. Alternatively, the enzymatically treated vegetable oil may be added to the enzyme treated, fermented protein emulsion, i.e. after the fermentation with lactic acid bacteria and / or may be incorporated in the preparation method of the spread when the fat and aqueous phase are blended. It is preferred that the hydrolysed oil is added prior to the fermentation step with lactic acid bacteria such that the hydrolysed oil is present during the fermentation. The production of diacetyl and lactones may be further enhanced or stimulated by including glucose, sucrose and / or citrate during fermentation.

[0104] Thus, in one embodiment of the invention a lipase- treated vegetable oil is present in the enzyme treated, fermented plant protein emulsion or the spread.

[0105] The enzyme-treated, fermented plant-based protein mixtureIn one aspect of the invention, the enzyme-treated, fermented plant protein emulsion, with or without the hydrolysed vegetable oil as disclosed herein is pasteurized and / or dried (for example spray-dried or freeze-dried) before being used to provide a plantbased spread or plant butter according to the present invention.

[0106] The enzyme-treated, fermented plant-based protein mixture (preferably an enzyme-treated, fermented plant-based protein emulsion comprising the hydrolyzed vegetable oil) may be an independent aspect of the present invention.

[0107] Thus, in one aspect, the invention relates to an enzyme-treated, fermented plantbased protein mixture obtainable by

[0108] providing a mixture comprising water and a plant protein, preferably fava and / or oat protein;

[0109] treating the protein mixture with a deamidating enzyme as disclosed herein; fermenting the mixture using lactic acid bacteria during or after the enzymatic treatment as disclosed herein to provide the enzyme-treated, fermented protein mixture.

[0110] In a preferred embodiment, the fermentation mixture further contains a (lipase) hydrolysed oil fraction as disclosed herein. The hydrolysed vegetable oil may be present in an amount up 25 wt. %, preferably between 0.1 and 10 wt.%, more preferably about 5 wt.% vegetable oil, wt.% calculated on the total weight of the enzyme-treated, fermented plantbased protein mixture.

[0111] Preferably, the enzyme-treated, fermented protein mixture has a pH of 4.0 to 6.0, preferably 4.5 to 6.0, more preferably 5.0 to 6.0, and most preferably 5.4 to 5.8. The pH may be measured at 20-25°C using a calibrated pH meter.

[0112] In a preferred embodiment, the hydrolysed oil fraction comprises a lipase-treated vegetable oil. Preferably the lipase-treated vegetable oil amounts to 10-50 wt.% of the oil fraction, more preferable 20-40 wt.%. Thus, when the oil fraction in the emulsion is 5 wt.%, calculated on the weight of the emulsion, the lipase-treated vegetable oil amounts to 1-2 wt.% calculated on the weight of the emulsion.

[0113] The emulsion obtained from the enzymatic treatment of protein with enzymes, subjected to bacterial fermentation and, optionally, combined with the lipase-treated oil can be used in the preparation of a plant-based spread.

[0114] Diacetyl and / or acetoin

[0115] Diacetyl (butanedione) is known as one of the molecules responsible for a butter-like flavour. Lactic acid bacterial fermentation may result in the formation of diacetyl as a byproduct. Lactic acid bacteria have been used in food fermentation for centuries and lactic acidbacteria are widely used in the food industry in fermentation of food products such as dairy products. In addition to extending the shelf-life of food products, lactic acid fermentation is often applied to affect the sensory properties of food products, smell, texture, flavor, and appearance are seen as such sensory properties. Diacetyl may be formed during fermentation, but it still remains a challenge to mimic the buttery flavor or taste sensation of dairy based butter.

[0116] Acetoin (3-hydroxybutanone), along with diacetyl, is one of the compounds that gives butter its characteristic flavor. Diacetyl and acetoin can be measured through various mass spectrometric analytical techniques, such as headspace Gas chromatography / Mass Spectrometry. Acetoin is less volatile than diacetyl and as such has a tendency to remain longer in the product, thus providing a longer lasting contribution to the flavor. Thus, one embodiment relates to the process and / or product as described herein, wherein the resulting product contains more acetoin than diacetyl. In embodiments, the ratio of acetoin to diacetyl is more than 1 , more than 1.1, more than 1.2, 1.3, 1.4 or even more than 1.5.

[0117] Thus, one embodiment relates to the process and / or product (spread, butter) as described herein, wherein the resulting plant-based product (spread, butter) comprises 1 ppm or more diacetyl. There is a preference for higher levels of diacetyl such as 2, 3 or 4 ppm diacetyl or more. Thus, in one embodiment, the fermented plant-based product (spread, butter) comprises diacetyl in the range of 1 to 100 ppm such as 5-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30-70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, such as 45-55 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably 20-40 ppm, and even more preferred 25-35 ppm diacetyl. In yet an embodiment the fermented plant based product (spread, butter) comprises in the range of 0.1 to 100 ppm, such as 1-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30- 70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably 20-40 ppm diacetyl and even more preferred 25-35 ppm diacetyl. In a preferred embodiment the fermented plant based product (spread, butter) comprising at least 4 ppm diacetyl, at in an even more preferred embodiment at least 10 ppm diacetyl.

[0118] Thus, one embodiment relates to the process and / or product (spread, butter) as described herein, wherein the fermented plant-based product comprises 1 ppm or more acetoin. There is a preference for higher levels of acetoin such as 2, 3 or 4 ppm acetoin or more . Thus, in one embodiment the fermented plant-based product (spread, butter) comprises acetoin in the range of 1 to 100 ppm such as 5-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30-70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, such as 45-55 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably20-40 ppm. A further aspect of the present invention relates to a fermented plant based product (spread, butter) comprising at least 0.1 ppm acetoin. In yet an embodiment the fermented plant based product (spread, butter) comprises acetoin in the range of 0.1 to 100 ppm, such as 1-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30- 70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably 20-40 ppm acetoin and even more preferred 25-35 ppm acetoin. In a preferred embodiment the fermented plant based product (spread, butter) comprising at least 4 ppm acetoin, at in an even more preferred embodiment at least 10 ppm acetoin.

[0119] Lactones

[0120] Thus, one embodiment relates to the process and / or product (spread, butter) as described herein, wherein the fermented plant-based product comprises 1 ppm or more lactones. There is a preference for higher levels of lactones such as 2, 3 or 4 ppm lactones or more . Thus, in one embodiment the fermented plant-based product (spread, butter) comprises lactones in the range of 1 to 100 ppm such as 5-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30-70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, such as 45-55 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably 20-40 ppm. A further aspect of the present invention relates to a fermented plant based product (spread, butter) comprising at least 0.1 ppm lactones. In yet an embodiment the fermented plant based product (spread, butter) comprises lactones in the range of 0.1 to 100 ppm, such as 1-95 ppm, e.g. 10-90 ppm, such as 15-85 ppm, e.g. 20-80 ppm, such as 25-75 ppm, e.g. 30- 70 ppm, such as 35-65 ppm, e.g. 40-60 ppm, e.g. 35-55 ppm, such as 30-50 ppm, e.g. 25-45 ppm, preferably 20-40 ppm acetoin and even more preferred 25-35 ppm acetoin. In a preferred embodiment the fermented plant based product (spread, butter) comprising at least 4 ppm lactones, at in an even more preferred embodiment at least 10 ppm acetoin

[0121] The plant-based product (spread, butter) of the invention is distinguished from plantbased products that do not contain ingredients that have been treated with enzymes or bacteria by the relative levels of diacetyl, acetoin and lactones. Typically, products (spread, butter) that do not contain ingredients that have been treated with enzymes or bacteria as mentioned herein contain levels of diacetyl and / or acetoin and / or lactones below 0.01 ppm, preferably below 0.1 ppm, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1 2 or even below 3 ppm.

[0122] Products (spread, butter) of the invention that contain ingredients that have been treated with enzymes or bacteria contain are characterised by increased levels of diacetyland / or acetoin and / or lactones compared to products (spread, butter) that do not contain ingredients that have been treated with enzymes or bacteria.

[0123] Typically, products (spread, butter) that contain ingredients that have been treated with enzymes or bacteria as mentioned herein contain levels of diacetyl above 1 ppm, preferably above 2 ppm, 3, 4, 5 or even 10 ppm.

[0124] Typically, products (spread, butter) that contain ingredients that have been treated with enzymes or bacteria as mentioned herein contain levels of acetoin above 1 ppm, preferably above 4 ppm, 6, 8, 10 or even 20 ppm.

[0125] Typically, products (spread, butter) that contain ingredients that have been treated with enzymes or bacteria as mentioned herein contain levels of lactones above 10 ppm, preferably above 20, 30, 40, 50 or even 100 ppm.

[0126] In dairy based products (spread, butter), conventional levels are diacetyl: 0.1-5 ppm, acetoin: 5-10 ppm (Journal of Dairy Science 1979, 62(5), 802), and lactones: 10-30 ppm (JOACS 1975, 52, 252-255).

[0127] Plant-based spread

[0128] In one aspect, the present invention provides a plant-based spread comprising: a water phase;

[0129] 40 to 99 wt.% of a fat phase, wt. % calculated based on total weight of the plantbased spread; and

[0130] 0.1-10 wt.% of an enzyme-treated, fermented protein mixture, wt. % calculated based on the total weight of the plant-based spread,

[0131] wherein the enzyme-treated, fermented protein mixture is obtainable by a method comprising the steps of:

[0132] o providing an aqueous mixture comprising a plant protein;

[0133] o treating the aqueous mixture with a protein deamidase to obtain an enzyme-treated protein mixture;

[0134] o fermenting the enzyme-treated protein mixture using lactic acid bacteria, preferably in the presence of 0.1 to 10 wt.% of a hydrolysed vegetable oil, wt. % calculated based on the total weight of the protein mixture, to provide the enzyme-treated, fermented protein mixture.

[0135] Preferably, the plant-based spread according to the present invention comprises:

[0136] 1 to 50 wt.% of a water phase; preferably 10 to 40 wt.%, wt. % calculated on total weight of the plant-based spread;

[0137] 50 to 99 wt.% of a fat phase; preferably 65 to 98 wt.%, more preferably 60 to 90 wt.%, wt. % calculated on total weight of the plant-based spread;0.1-10 wt.% of an enzyme-treated, fermented protein emulsion obtained by a method as disclosed herein

[0138] preferably, 0.1-10 wt.% of a hydrolysed vegetable oil is added as disclosed herein.

[0139] Preferably, the plant-based spread comprises 0.2 to 5 wt.%, most preferably 0.3 to 3 wt.%, of the enzyme-treated, fermented protein mixture, wt.% calculated based on total weight of the plant-based spread.

[0140] Preferably, the plant-based spread of the present invention comprises 0.01 - 6 wt.% plant protein, preferably 0.02 - 3 wt.%, more preferably 0.05 - 2 wt.%, and most preferably 0.08 - 1.6 wt.%, wherein wt.% is calculated based on the total weight of the plant-based spread. The plant protein is provided by the enzyme-treated, fermented protein mixture.

[0141] Preferably, the plant-based spread comprises one or more selected from: at least 5 ppm diacetyl, at least 10 ppm acetoin or at least 10 ppm lactones.

[0142] Preferably, the plant-based spread comprises 50 to 95 wt.%, more preferably 60 to 90 wt.% of a fat phase, preferably 65 to 80 wt.%, wt. % calculated on the total weight of the plant-based spread, the fat phase preferably comprises a vegetable oil and / or fat, preferably the vegetable oil and / or fat is selected from the group consisting of coconut oil, rapeseed oil, linseed oil, soybean oil, palm oil, palm stearin, palm kernel oil, and sunflower oil. The fat phase comprises a structuring fat, preferably 10-75 wt.% structuring fat, more preferably 12-55 wt.%, wt.% calculated on the total weight of the fat phase.

[0143] Spread is defined as a fat-continuous emulsion that is spreadable at refrigeration and room temperatures. Preferably, the present plant-based spreads contain between 50% and 99% fat, more preferably 65 to 98 wt.%, with water or aqueous phases dispersed within the fat. The present plant-based spreads further comprise fermented and enzyme-treated plant proteins as disclosed herein and / or the enzymatically treated oil, preferably in combination. These spreads are often used as alternatives to butter and margarine, and the formulations are optimized for stability, mouthfeel, and nutritional properties.

[0144] In one embodiment, the present plant-based spread comprises 0.1 to 6 wt.% of the fermented and enzyme- treated plant protein emulsion as disclosed herein, preferably 0.2 to 2 wt.%, more preferably 0.3 to 1 wt.%, based on the total weight of the spread.

[0145] There are low-protein spreads (0.1-2 wt.% protein) and moderate-protein spreads (3-6 wt.% protein).

[0146] In one embodiment, the plant-based spread comprises 50 to 99 wt.% vegetable oil or fat, preferably 60 to 98 wt.%, more preferably 65 to 90 wt.%, wt.% of the total weight of the plant spread. When an enzyme-treated oil is used, as such or in combination with the protein emulsion, the amount of oil is included in the total fat balance of the spread.Alternatively, the spread may be a very low fat spread (between 15 and 40 wt.% fat), a low fat spread (40-60% fat) , a full fat spread (60-80% fat) or a wrapper having a 80-99% fat. There is a preference for a full fat spread (60-80% fat) or a wrapper having a 80-99% fat.

[0147] Fats and oils

[0148] Fat Phase

[0149] The fat phase is an essential element of the edible fat-containing product (spread, butter) of the invention. Such a fat phase typically comprises edible fats and oils. There is a strong preference for non-hydrogenated fats and oils. Non-hydrogenated means that the fat or oil has not undergone any hydrogenation treatment. Preferably, the oils and fats in the fat phase contains at most 0.01 wt.% hydrogenated fat (wt.% drawn on the total fat phase), preferably no hydrogenated fat. This entails the starting fats and oils as well as blends and interesterified mixtures and even fractions of fats. Non-hydrogenated fats are defined fats having essentially no trans-fatty acids.

[0150] The fat phase preferably comprises a sufficient amount of solid fat at low temperatures in order to yield a desired composition. Simultaneously, in order to instil desirable organoleptic properties in terms of mouthfeel and appearance, the fat phase preferably essentially melts in the mouth upon consumption.

[0151] The fat phase is an essential element of the edible fat-containing product (spread, butter) of the invention and is typically present in an amount of 50 to 99 wt.% of a fat phase; preferably 65 to 98 wt.%, more preferably 60 to 90 wt.%, wt. % calculated on total weight of the plant-based spread.

[0152] Sources of fat and oils

[0153] A suitable fat phase may be derived from many different fat sources. The fat phase of the edible oil-in-water emulsion composition according to the present invention preferably comprises vegetable oil or vegetable fat or a combination thereof. It is preferred that the fat phase consists of vegetable oils and fats. The fat phase of a product according to the invention may comprise a liquid oil fraction and a (solid) structuring fat.

[0154] Liquid oil fraction

[0155] The fat phase may comprise from 0 (absent) up to 99 wt.% of liquid oil, drawn on the fat phase. The liquid oil fraction can be an element of the fat phase of the fat-containing product of the invention.

[0156] Preferably, the vegetable oil and / or fat is selected from the group consisting of coconut oil, palm (kernel) oil, rapeseed oil, soybean oil, sunflower oil, linseed oil, olive oil, corn oil, cottonseed oil, carinata oil, groundnut oil, safflower oil, peanut oil, rice oil, shea butterand camelina oil. Preferably, the vegetable oil is selected from the group consisting of coconut oil, palm (kernel) oil, rapeseed oil, soybean oil, sunflower oil, and fractions and blends thereof.

[0157] Structuring Fat

[0158] The structuring fat (hard stock) is an element of the fat phase of the edible composition (spread). The fat phase may comprise from 1 to 100 wt.% of structuring fat (wt.% calculated on the total weight of the fat phase). In certain embodiments from 20 to 60 wt.% (low fat spreads) and in certain other embodiments (high fat spreads) from 5 to 75 wt.%. In wrappers the amount of structuring fat may vary from 50 to 90 wt.%. Suitable structuring fats are known in the art and can be (interesterified) blends of originating (fractions of) from palm, palm kernel, shea or as described in WO2022162026.

[0159] Process

[0160] The invention further relates to a process for manufacturing the present plant-based spread or butter, comprising the steps of:

[0161] treating a plant protein mixture as disclosed herein with at least one enzyme as disclosed herein;

[0162] fermenting the plant protein mixture using lactic acid bacteria as disclosed herein during or after the enzymatic treatment to obtain an enzyme-treated, fermented protein;

[0163] preparing the fermented plant-based spread comprising the enzyme-treated, fermented protein.

[0164] The fermentation may be performed in the presence of a lipase-hydrolysed vegetable oil in the way and in the amounts as disclosed herein. Preferably, the fermentation is performed in the presence of one or more sugars as disclosed herein, further aiding in the production of flavour compounds. Preparing the plant-based spread is performed according to known processes that encompass the steps of mixing of the liquid oil, the structuring fat and if present the aqueous phase. In certain embodiments mixing (or blending) can be done at a temperature at which the structuring fat is (partly) liquid. The next step can be cooling of the mixture under high shear. This may induce crystallization of the structuring fat to create an emulsion. The emulsion may be stabilised to allow formation of a fat crystal network and confer the product some degree of firmness. The process may further comprise kneading or working to produce the desired firmness, for instance to confer plasticity and reduce the water droplet size.

[0165] In a preferred embodiment, the method for providing a plant-based spread as disclosed herein comprises the steps of:providing an enzyme-treated, fermented plant-based protein emulsion as disclosed herein;

[0166] optionally, providing an enzyme treated vegetable oil as disclosed herein providing an aqueous phase;

[0167] providing a fat phase;

[0168] blending the enzyme-treated, fermented plant-based protein emulsion, optionally the enzyme-treated vegetable oil, aqueous phase and the fat phase to form a blend;

[0169] processing the blend to form a plant-based spread.

[0170] The process according to the present invention may also include a pasteurisation or sterilisation process, to enhance the shelf life of the product. Homogenisation may also include a high-pressure homogenisation process. After preparation, the edible emulsion may for instance be filled into suitable containers.

[0171] Additional ingredients

[0172] The plant-based spread according to the present invention may also comprise further ingredients, for instance selected from sugars, sweeteners, flavourings, aroma compounds and combinations thereof, to improve the organoleptic properties and any other properties. The spreads may further comprise conventional emulsifiers and stabilizers. Additionally, the plantbased spread may also comprise antioxidants or preservatives to improve shelf-life.

[0173] Examples

[0174] The invention is exemplified by, but not limited to, the following examples.

[0175] Characterization of the starting components and products

[0176] The ingredients used are all commercially free available compounds and compositions.

[0177] The products were analysed using the following (common) analytical methods:

[0178] Diacetyl and acetoin

[0179] The levels of diacetyl and acetoin in dairy-based butter vary depending on the production method, fermentation process, and storage conditions. These compounds are natural by-products of the fermentation process used to create cultured butter and are responsible for its buttery flavor and aroma. The levels of diacetyl and acetoin can be determined with gas chromatography based on AOAC Official Method 966.11, HPLC (Jelen, H., & Wlazly, K. (2002), Journal of Chromatographic Science, 40(2), 96-101), GCMS (Keeney, M., & Day, E. (1951). Journal of Dairy Science, 34(9), 929-936.) or headspaceanalysis coupled with GCMS (Curioni, P. M., & Bosset, J. O. (2002). Journal of Agricultural and Food Chemistry, 50(1), 143-150.).

[0180] Lactones

[0181] Lactones were analyzed essentially as described in Yoshinaga et al. in Journal of Oleo science 2019, 68(12), 1298-1301 using GC-MS. The lactone contents expressed as the total amount of lactones. The total amount of lactones is defined as the sum of delta-lactones and gamma-lactones. Delta-lactones are delta-hexalactone, delta-octalactone , delta-decalactone, delta-dodecalactone delta-tetradecalactone, delta-hexadecalactone. Gammalactones are gamma- hexalactone, gamma- octalactone , gamma- decalactone, gammadodecalactone, gamma- tetradecalactone, gamma- hexadecalactone.

[0182] FA analysis

[0183] For starting fat or fat-containing products, the overall fatty acid analysis and the triglyceride composition are determined using conventional procedures in the art such as FAME analysis, GLC / Carbon number method and HPLC silver phase method such as described for example in EP78568, EP652289, JOACS (19914), 68(5), 289-293 and Hammond E.W.J., Chromatography, 203, 397, 1981.

[0184] Solid Fat Content (SFC) measurements

[0185] The solid fat content (SFC) in this description and claims is expressed as N-value, as defined in Fette, Seifen Anstrichmittel 80180-186 (1978). The stabilization profile applied is heating to a temperature of 80 degrees Celsius, keeping the oil for at least 10 minutes at 60 degrees Celsius or higher, keeping the oil for 1 hour at 0 degrees Celsius and then 30 minutes at the measuring temperature (tempered). An alternative method is described in IUPAC 2.150 method, serial, non-tempered.

[0186] Water Droplet Size Distribution of Spreads (D3,3 Measurement)

[0187] The normal terminology for Nuclear Magnetic Resonance (NMR) is used throughout this method. On the basis of this method the parameters D3,3 and exp(o) of a lognormal water droplet size distribution can be determined. The D3,3 is the volume weighted mean droplet diameter and o is the standard deviation of the logarithm of the droplet diameter. A D3,3 < 6 is acceptable for a low fat spread, but a D3,3 < 4 is preferred. A e-sigma of < 3 is desired.

[0188] The NMR signal (echo height) of the protons of the water in a water-in-oil emulsion are measured using a sequence of 4 radio frequency pulses in the presence (echo height E) and absence (echo height E*) of two magnetic field gradient pulses as a function of the gradientpower. The oil protons are suppressed in the first part of the sequence by a relaxation filter. The ratio (R=E / E*) reflects the extent of restriction of the translational mobility of the water molecules in the water droplets and thereby is a measure of the water droplet size. By a mathematical procedure — which uses the log-normal droplet size distribution — the parameters of the water droplet size distribution D3,3 (volume weighed geometric mean diameter) and o (distribution width) are calculated. A Bruker magnet with a field of 0.47 Tesla (20 MHz proton frequency) with an air gap of 25 mm is used (NMR Spectrometer Bruker Minispec MQ20 Grad, ex Bruker Optik GmbH, DE).

[0189] Spreadability

[0190] Spreadability is determined according to the following protocol.

[0191] A flexible palette knife is used to spread a small amount of the spread on to fat free paper. The spreading screen is evaluated according to standardized scaling. A score of 1 represents a homogeneous and smooth product without any defects, a 2 refers to the same product but then with small remarks as slightly inhomogeneous or some vacuoles, a 3 refers to the level where defects become almost unacceptable, like loose moisture or coarseness during spreading. A score of 4 or 5 refers to unacceptable products, where the 4 refers to a product still having some spreading properties, but an unacceptable level of defects.

[0192] Free Water

[0193] After spreading a sample of a fat spread, the stability of the emulsion after spreading is determined by using indicator paper (Wator, ref 90610, ex Machery-Nagel, DE) which develops dark spots where free water is adsorbed. A stable product does not release any water and the paper does not change. Very unstable products release free water easily and this is indicated by dark spots on the paper.

[0194] A six point scale is used to quantify the quality of fat spread (DIN 10311):

[0195] 0 (zero) is a very stable and good product;

[0196] 1 (one) is showing some loose moisture (one or two spots, or the paper changes a little in color as a total);

[0197] 2 (two) as one but more pronounced;

[0198] 3 (three) as one but to an almost unacceptable level;

[0199] 4 (four) indicator paper is almost fully changing into a darker color;

[0200] 5 (five) the paper changes completely and very fast into the maximum level of color intensity.

[0201] Spreads with a score of 4 or 5 are rejected for their stability. Spreads with a score of 0 or 1 show an acceptable quality with respect to free water.Preparation of a spread or wrapper

[0202] The fat phase and the aqueous phase were mixed and kept at 55-65 degrees Celsius. The mixtures was then passed through a series of scraped surface heat exchangers (A -units) and stirred crystallisers (C-units) at various speeds. The product leaving the last unit had a temperature of below 20 and in some occasions below 5-7 degrees Celsius. The product was filled in tubs or wrappers and stored at 5 degrees Celsius. A stable spread was obtained. Various products were prepared.

[0203] Stability testing

[0204] Products prepared are tested using conventional cycling protocols and conditions to determine stability.

[0205] Stevens value

[0206] Stevens values give an indication about the hardness (also called firmness) of a product at a given temperature. The Stevens value at a given temperature is determined according to the following protocol.

[0207] Freshly prepared products are stored at 5 degrees Celsius. To determine the hardness at a given temperature the sample is stored at the given temperature for at least 24 hours after stabilization at 5 degrees Celsius for at least one week. The hardness of the product is then measured with a Stevens penetrometer (Brookfield LFRA Texture Analyser (LFRA 1500), ex Brookfield Engineering Labs, UK) equipped with a stainless steel probe with a diameter of 4.4 mm (or 6.35 mm for softer products) and operated in "normal" mode. The probe is pushed into the product at a speed of 2 mm / s, a trigger force of 5 gram from a distance of 10 mm. The force required is read from the digital display and is expressed in gram.

[0208] Example 1. Preparation of hydrolysed oil

[0209] Coconut oil was treated with a Lipase following the guidance of the supplier (Southern Biological, Australia). The Lipase enzyme was added in an amount of 0.1 wt.% to coconut oil and warmed to 55 degrees Celsius for 2 hours under vacuum. The enzyme was deactivated by heat and the resulting emulsion of hydrolyzed oil was cooled, stored and used as such. The hydrolyzed oil contained about 20-40 wt.% of hydrolysed oil, calculated on the total weight (100%) of the oil. Sunflower oil was also used in the preparation of hydrolyzed oil.

[0210] Example 2. Preparation of protein dispersion

[0211] A 3 wt. % protein mixture was dispersed in water under stirring in pre-heated water at 60 degrees Celsius. Protein dispersions were prepared from commercially available Fababean (broad bean) protein, Lentil protein and pea protein (both supplied from AGT foods, Canada) and oat protein (obtainable from LantMannen Sweden) and used as such.

[0212] Example 3. Preparation of an enzyme-treated, fermented protein dispersion The protein dispersion of Example 2 was enzymatically treated using protein glutaminase PG500 at 60 degrees Celsius for 20 minutes. The enzyme was inactivated by heat treatment by 75-85 degrees Celsius for 5 minutes and the solution was homogenised and pasteurised. The resulting product was cooled until 38 degrees Celsius. The enzyme-treated protein dispersion was inoculated with a lactic acid bacterial culture (1-150 mg per Liter) of VEGE 33 and Sacco VM01. Glucose and sucrose (each 0.5 wt.%) were added and the pH adjusted with citric acid until pH 6.5-6.9. The solution was fermented in a Stefan cooker at 38 degrees Celsius for 4 hours until pH was 5.6. The resulting product was cooled and stored.

[0213] Example 4. Preparation of an enzyme-treated, fermented protein dispersion in hydrolysed oil

[0214] The protein dispersion of Example 2 was combined with a hydrolysed oil from example 1. The hydrolysed oil was added in an amount of about 10 wt. % on the total of the dispersion. The resulting dispersion was enzymatically treated using protein glutaminase PG500 at 60 degrees Celsius for 20 minutes. The enzyme was inactivated by heat treatment and the solution was emulsified, homogenised and pasteurised. The resulting product was cooled until 38 degrees Celsius. The emulsion was inoculated with a lactic acid bacterial culture (1-150 mg per Liter) of VEGE 33 and Sacco VM01. Dextrose (0.5 wt.%) was added and the pH adjusted with citric acid until pH 6.5-6.9. The solution was emulsified and still fermented in a Stefan cooker at 38 degrees Celsius for 4 hours until pH was 5.6. The resulting product was cooled and stored.

[0215] Example 5-9. Preparation of a plant-based spread

[0216] The fat phase, the aqueous phase and the (enzyme treated, fermented) protein emulsions of examples 3 and 4, respectively were blended and processed to obtain a spread (80% fat, full fat margarine) under conventional circumstances. Protein emulsions were prepared from fava bean protein, lentil protein, pea protein and oat protein. Different fat blends of SF and CN were tested. An interesterified blend of 20GTS / 25SF / 55CN (GTS: glycerol tristearate, SF: Sunflower oil; CN: Coconut fat / oil) was used as structuring fat as disclosed in WO2022 / 162026. The composition of the spread is presented below:

[0217]

[0218]

[0219] As a comparative experiment, an untreated (no enzymatic treatment, no fermentation, no addition of hydrolysed oil) plant butter was used (C. Ex. 1).

[0220] Results

[0221] Taste evaluation of the spread

[0222] Taste was assessed by a taste panel. The samples were evaluated for plant off-taste that is characteristic for plant-based products and compared to the reference product.

[0223] Taste characteristics were quantified using a 5-point scale. For butter flavour, 5 indicates a strong butter taste, while 1 represents no butter taste. Similarly, for plant off-taste, 5 represents a strong plant off-taste and 1 no plant off-taste.

[0224]

[0225] The spreads were analysed for the content of diacetyl, acetoin and lactones. Ex 6 to 9 were found to have increased amounts of diacetyl, acetoin and / or lactones compared to C1, Ex 5, Ex 10 and Ex 11.

[0226] The best performing spreads (Ex 6 and Ex 9) were analysed for Stevens value, D33, E-sigma, free water content, pH and solids (SFC). Tests were performed at production, after 2 weeks and after 4 weeks. The spreads were found to have acceptable product characteristics, similar to the reference (C. Ex 1), with a notable improvement in butter flavour and reduction of plant-protein derived off-taste.

[0227]

Claims

CLAIMS1. Plant-based spread, such as a plant butter, comprising:a water phase;50 to 99 wt.% of a fat phase, wt. % calculated based on total weight of the plantbased spread; and0.1-10 wt.% of an enzyme-treated, fermented protein mixture, wt. % calculated based on the total weight of the plant-based spread,wherein the enzyme-treated, fermented protein mixture is obtainable by a method comprising the steps of:o providing an aqueous mixture comprising a plant protein;o treating the aqueous mixture with a protein deamidase to obtain an enzyme-treated protein mixture;o fermenting the enzyme-treated protein mixture using lactic acid bacteria in the presence of 0.1 to 10 wt.% of a hydrolysed vegetable oil, wt. % calculated based on the total weight of the protein mixture, to provide the enzyme-treated, fermented protein mixture.

2. Plant-based spread according to claim 1, wherein the plant-based spread comprises 0.2 to 5 wt.%, most preferably 0.3 to 3 wt.%, of the enzyme-treated, fermented protein mixture, wt.% calculated based on total weight of the plant-based spread.

3. Plant-based spread according to any one of the preceding claims, wherein the plant protein is a vegetable protein, preferably selected from the group consisting of lentil, fava bean, oat, soy, pea, almond, cashew, chickpea, canola, and potato protein; more preferably lentil, fava bean, oat, or pea protein; most preferably fava bean protein or oat protein.

4. Plant-based spread according to any one of the preceding claims, wherein the plant-based spread comprises one or more selected from: at least 5 ppm diacetyl, at least 10 ppm acetoin or at least 10 ppm lactones.

5. Plant-based spread according to any one of the preceding claims, wherein the plant-based spread comprises 1 to 50 wt.% of a water phase; preferably 10 to 40 wt.%, wt. % calculated based on the total weight of the plant-based spread.

6. Plant-based spread according to any one of the preceding claims, wherein the plant-based spread comprises 0.05 to 2 wt.% plant protein provided by the enzyme-treated,fermented protein mixture, wt. % calculated based on the total weight of the plant-based spread.

7. Plant-based spread according to any one of the preceding claims, wherein the plant-based spread comprises 60 to 90 wt.% of a fat phase, preferably 65 to 80 wt.%, wt. % calculated on the total weight of the plant-based spread.

8. Plant-based spread according to any one of the preceding claims, wherein the fat phase comprises 10-75 wt.% structuring fat, more preferably 12-55 wt.%, wt. % calculated on the total weight of the fat phase.

9. Plant-based spread according to any one of the preceding claims, wherein the fat phase comprises a vegetable oil and / or fat, preferably the vegetable oil and / or fat is selected from the group consisting of coconut oil, rapeseed oil, linseed oil, soybean oil, palm oil, palm stearin, palm kernel oil, and sunflower oil.

10. Plant-based spread according to any one of the preceding claims, wherein the protein deamidase is a protein glutaminase.

11. Plant-based spread according to any one of the preceding claims, wherein the plant protein in the enzyme-treated protein mixture has a deamidation degree of at least 30%, preferably 50 - 80%.

12. Plant-based spread according to any one of the preceding claims,, wherein the lactic acid bacteria are mesophilic bacteria, preferably selected from the group consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Leuconostoc mesenteroides.

13. Plant-based spread according to any one of the preceding claims, wherein the lactic acid bacteria are thermophilic bacteria, preferably selected from the group consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

14. Plant-based spread according to any one of the preceding claims, wherein the fermentation is carried out for 2 to 12 hours, preferably 3 to 5 hours, at a temperature between 25°C to 50°C, preferably 30°C to 45°C.

15. Plant-based spread according to any one of the preceding claims, wherein the fermentation is terminated when the enzyme-treated, fermented protein mixture has a pH of 5 to 6, preferably 5.4 and 5.8.

16. Plant-based spread according to any one of the preceding claims, wherein the hydrolysed vegetable oil comprises 5 to 50 wt.% free fatty acids, wt.% calculated based on the total weight of the hydrolysed vegetable oil.

17. Plant-based spread according to any one of the preceding claims, wherein the hydrolysed vegetable oil is a lipase-hydrolysed vegetable oil, preferably obtained by treating a vegetable oil with a triacylglycerol lipase (EC 3.1.1.3), preferably a triacylglycerol lipase from Rhizopus delemar.

18. Plant-based spread according to any one of the preceding claims, wherein the hydrolysed vegetable oil is selected from the group consisting of coconut oil, rapeseed oil, linseed oil, soybean oil, palm oil, palm stearin, palm kernel oil, and sunflower oil; preferably, selected from coconut oil and sunflower oil.

19. Method for providing a plant-based spread according to any one of the preceding claims, the method comprising the steps of:(a) providing an aqueous mixture comprising a plant protein;(b) treating the aqueous mixture with a protein deamidase;(c) combining the aqueous mixture with 0.1 to 10 wt.% of a hydrolysed vegetable oil, thereby obtaining a mixture comprising the plant protein and the hydrolysed vegetable oil;(d) fermenting the mixture obtained in step (c) using lactic acid bacteria, wherein the fermentation is carried out during or after step (b) to obtain an enzyme-treated, fermented protein mixture;(e) blending the enzyme-treated, fermented protein mixture with a fat phase and an aqueous phase to provide the plant-based spread.

20. Method for providing a plant-based spread according claim 19, wherein the step of fermenting the aqueous mixture using lactic acid bacteria is performed after the enzymatic treatment of step (b) to obtain an enzyme-treated, fermented protein.

21. Method according to claim 19 or 20, wherein a fermentation stimulating agent is added to the enzyme-treated plant protein mixture prior to fermentation, such that fermentation is carried out in the presence of the fermentation stimulating agent, preferably wherein the fermentation stimulating agent is chosen from a) sugars, preferably glucose, dextrose, sucrose, fructose, galactose, lactose, maltose and / or combinations thereof and b)fruit and vegetable juices, preferably apple juice, carrot juice, beet juice, and / or combinations thereof.

22. Method according to claim 21, wherein the fermentation stimulating agent is a fruit or vegetable juice.

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