Process for preparing a plant-based acidified dairy alternative food product with protein deamidase and transglutaminase

Abstract

The present invention relates to the use of a protein deamidase and a transglutaminase for obtaining a plant-based fermented dairy alternative food product with improved organoleptic and techno-functional properties.

Description

[0001] PROCESS FOR PREPARING A PLANT-BASED ACIDIFIED DAIRY ALTERNATIVE FOOD PRODUCT WITH PROTEIN DEAMIDASE AND TRANSGLUTAMINASE

[0002] Reference to sequence listing

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the use of a protein deamidase and a transglutaminase for obtaining a plant-based fermented dairy alternative food product with improved organoleptic and techno-functional properties.

[0006] BACKGROUND OF THE INVENTION

[0007] Vegetarian diets in general, and vegetarian sources of protein in particular, have increased in popularity as consumer interest in healthier and more eco-friendly eating habits has grown. Plant-based fermented dairy alternatives, such as plant-based dairy-alternative yogurts, offer an interesting alternative to traditional animal-derived yogurts, also because of their reduced level of cholesterol and saturated fat and because they are free of lactose.

[0008] Therefore, there is a significant commercial interest in providing plant-based dairy alternatives such as plant-based fermented dairy alternatives.

[0009] The use of transglutaminase enzymes for modification of food proteins, including dairy proteins, is known from the prior art. For instance, EP0671885 describes a method for production of a milk-like product comprising transglutaminase treatment followed by acidification. Herein, a transglutaminase-treated milk-like product, obtained by acidification through a biological fermentation, is shown to exhibit consistency of a semi-solid yoghurt. Treatment with transglutaminase during the manufacturing of fermented milk products is known to increase the viscosity of the product.

[0010] The use of protein deamidase in the production of plant-based dairy alternative food products, such as plant-based yoghurt alternatives, is also known from the prior art. For instance, WO2022063833 A1 describes a process for preparing a plant-based yoghurt analogue by enzymatically treating a suspension of a defatted oleaginous plant, such as a soy or peanut press cake, with a protein glutaminase, a glycosidase and optionally a transglutaminase, followed by homogenizing, heating and fermenting. WO2022063833 A1 further discloses the use of protein deamidase, amylase and transglutaminase for obtaining peanut press cake protein solutions with reduced viscosity.

[0011] W02017171601 A1 discloses a process for producing a non-dairy food product, such as a yogurt, sour cream or cheese, which process comprises providing a mixture of deamidated oat base and vegetable protein isolate, treating the protein isolate and oat protein comprised by the oat base with a transglutaminase, and fermenting the mixture by means of a starter culture.

[0012] CN 115 736 022 A discloses a preparation method of oat puree for plant yogurt fermentation. In the disclosed preparation methods, the oat puree is prepared by treating a centrifugal insoluble matter of oat slurry with a protein glutaminase and the yogurt is obtained by treating the oat puree with a transglutaminase and an inoculation of Bacillus bulgaricus and Streptococcus thermophilus.

[0013] It is an object of the present invention to identify further improved processes for producing plant-based fermented dairy alternative food products, such as plant-based dairy alternative yogurts, with improved organoleptic and techno-functional properties..

[0014] SUMMARY OF THE INVENTION

[0015] In a first aspect, the present invention relates to a process for preparing a plant-based fermented dairy alternative food product, the process comprising:

[0016] (a) treating a plant-based substrate with a protein deamidase;

[0017] (b) treating the plant-based substrate of step (a) with a transglutaminase; and

[0018] (c) fermenting the plant-based substrate of step (b) by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative food product; wherein step (a) is performed before and / or during step (b), and step (b) is performed before and / or during step (c), and wherein the fermented dairy alternative food product has increased viscosity compared to a fermented dairy alternative food product obtained using the same process but without addition of protein deamidase and transglutaminase.

[0019] The present inventor has surprisingly found that in the production of a plant-based fermented dairy alternative food product, such as a plant-based yogurt alternative, treatment of a plant-based substrate, from which the fermented dairy alternative food product is produced, with a protein deamidase and a transglutaminase results in an improved quality of the final product. The treatment with the protein deamidase may be performed either as a pre-treatment step prior to fermentation, or it may be performed essentially at the same time as the fermentation.

[0020] In particular, the use of a protein deamidase and a transglutaminase has been found to result in a fermented dairy alternative food product having overall better texture, including increased cohesiveness, hardness and viscosity, compared to when treatment is performed with either the transglutaminase or the protein deamidase alone. Furthermore, the visual appearance of the fermented dairy alternative food product is improved and a less grainy, less lumpy and a smoother and thicker texture is observed and reported in a sensory test. Superior results are obtained for plant-based substrates derived from legumes, such as pea, faba bean and lentils. Also, good results could be seen for oat-based substrates, in particular oat beverages and fortified oat beverages. Most particular, leguminous protein isolates showed particularly good results when treated with both protein deamidase and transglutaminase and yielded a plant-based fermented dairy alternative product with improved smoothness, increased viscosity and improved firmness.

[0021] For the plant-based fermented dairy alternative food products, such as, e.g., plant-based yogurts, an additional benefit observed when treating with the protein deamidase and transglutaminase was a pH buffering capability of fermented dairy alternative food product. As shown in Example 6 herein, it was surprisingly found that even after storage for, e.g., 7 days, the pH of the fermented dairy alternative food product obtained using the processes disclosed herein, had dropped significantly less than those fermented dairy alternative food products obtained using the same process but without the protein deamidase and transglutaminase. This phenomenon was observed irrespective of whether the fermentation was terminated at the same pH (pH~4.5) for the food products compared. This additional property of the enzyme- treated plant-based fermented dairy alternative food product is highly beneficial for, e.g., manufacturers of yogurts since it can mediate the risk of post-acidification, a phenomenon which can result in very sour yogurts, a trait which is generally undesirable. Thus, using the processes of the invention, a more stable plant-based fermented dairy alternative food product can be obtained which retains a satisfactory taste and texture profile even after longer storage time. In particular, a plant-based yogurt alternative can be obtained which meets both the demand of the consumers in terms of taste and texture and which provides a more stable product with higher resilience to pH changes, also during prolonged storage.

[0022] In a second aspect, the invention relates to a plant-based fermented dairy alternative food product obtainable by any of the processes disclosed herein.

[0023] In a third aspect, the invention relates to the use of a protein deamidase and a transglutaminase in the production of a plant-based fermented dairy alternative food product to improve texture and / or stability of the plant-based fermented dairy alternative food product, in particular to increase its viscosity and / or improve stability against post-acidification..

[0024] SEQUENCES

[0025] SEQ ID NO: 1: Protein deamidase derived from Chryseobacterium viscerum (the strain has formerly been referred to as Chryseobacterium sp-62563) having the mature polypeptide sequence shown as SEQ ID NO: 2.

[0026] SEQ ID NO: 2: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium viscerum.

[0027] SEQ ID NO: 3: Protein deamidase derived from Chryseobacterium proteolyticum having the mature polypeptide sequence shown as SEQ ID NO: 4.

[0028] SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium proteolyticum. SEQ ID NO: 5: Protein deamidase derived from Chryseobacterium gambrini having the mature polypeptide sequence shown as SEQ ID NO: 6.

[0029] SEQ ID NO: 6: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium gambrini.

[0030] SEQ ID NO: 7: Protein deamidase derived from Chryseobacterium culicis having the mature polypeptide sequence shown as SEQ ID NO: 8.

[0031] SEQ ID NO: 8: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium culicis.

[0032] SEQ ID NO: 9: Protein deamidase derived from Chryseobacterium defluvii having the mature polypeptide sequence shown as SEQ ID NO: 10.

[0033] SEQ ID NO: 10: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium defluvii.

[0034] SEQ ID NO: 11: Transglutaminase derived from Streptomyces mobaraensis.

[0035] SEQ ID NO: 12: Transglutaminase derived from Streptomyces lyticus.

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] Fig. 1 shows the visual appearance of a reference faba bean protein based yogurt obtained using the method exemplified in Example 2.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0040] As used herein, the terms “drink", “milk” and "beverage" are used interchangeably and have the same meaning unless defined otherwise or clearly indicated by context.

[0041] Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”).

[0042] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] The present invention is directed to a process for preparing a plant-based fermented dairy alternative food product, the process comprising:

[0044] (a) treating a plant-based substrate with a protein deamidase;

[0045] (b) treating the plant-based substrate of step (a) with a transglutaminase; and

[0046] (c) fermenting the plant-based substrate of step (b) by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative food product; wherein step (a) is performed before and / or during step (b), and step (b) is performed before and / or during step (c), and wherein the fermented dairy alternative food product has increased viscosity compared to a fermented dairy alternative food product obtained using the same process but without addition of protein deamidase and transglutaminase.

[0047] The plant-based substrate may be derived or obtained from any plant, such as legumes (e.g., soy, pea, lentils, faba beans), cereals (e.g., wheat, oats), pseudocereals (e.g., quinoa), grasses, pasture legumes (e.g., alfalfa, clover), rapeseed, nuts, vegetables, fruits, mushrooms, cottonseed, or any combination thereof.

[0048] In an embodiment, the plant-based substrate is derived or obtained from almond, cashew, chickpea, coconut, faba bean, flax, hemp, lentil, lupin, macadamia, mung bean, oat, pea, peanut, pecan, pistachio, rice, soy, or any combination thereof, such as from faba bean, lentil, pea, oat or any combination thereof.

[0049] In a preferred embodiment, the plant-based substrate is derived or obtained from legumes, such as from pulses (e.g., peas, faba beans, lentils, chickpea) or from oil crops (e.g., soybean).

[0050] In a most preferred embodiment, the plant-based substrate is derived or obtained from pea, faba bean, lentil, oat or any combination thereof.

[0051] The plant-based substrate may be a plant-based milk alternative, such as a soy beverage, a pea beverage, a lentil beverage, a faba bean beverage or an oat beverage, optionally fortified with a plant-based milk alternative powder, such as pea milk powder, or with a concentrated or isolated protein, such as a leguminous protein isolate or concentrate. Or the plant-based substrate may be aqueous solution or suspension of a plant-based milk alternative powder, such as pea milk powder or faba bean milk powder. Or the plant-based substrate may be an aqueous solution or suspension of a plant protein preparation, such as a plant protein isolate or a plant protein concentrate, preferably a leguminous protein isolate or leguminous protein concentrate, more preferably a soy protein isolate, a soy protein concentrate, a pea protein isolate, a pea protein concentrate, a faba protein isolate, a faba protein concentrate, a lentil protein isolate or a lentil protein concentrate. Or the plant-based substrate may be any other suitable preparation obtained from a plant, such as, e.g., an aqueous suspension of a flour or the like obtained from a plant, such as from a part of a plant. The plant-based substrate may be a combination of any of the aforementioned substrates.

[0052] In an embodiment, the plant-based substrate is an oat-based beverage fortified with a leguminous protein isolate. In an embodiment, the plant-based substrate is an oat-based beverage fortified with a pea protein isolate. In the context of the invention, it is understood that for such fortified beverages, such as fortified oat-based beverages, both the oat base and the leguminous protein isolate or leguminous protein concentrate is subjected to enzymatic treatment. In particular, it is understood that both the leguminous protein isolate / concentrate and the oat-based beverage is subjected to protein deamidase treatment.

[0053] In an embodiment, the plant-based substrate is an oat-based beverage fortified with a leguminous protein to a total protein content in the range of 2-6% (w / w). In an embodiment, the plant-based substrate is an oat-based beverage fortified with a leguminous protein to a total protein content in the range of 3-5% (w / w).

[0054] In a preferred embodiment, the plant-based substrate is a pea- or faba-based beverage, optionally fortified with a leguminous protein isolate or leguminous protein concentrate. In a more preferred embodiment, the plant-based substrate is an aqueous solution or suspension of a pea protein isolate, a pea protein concentrate, a faba protein isolate, a faba protein concentrate, a lentil protein isolate, a lentil protein concentrate, or any combination thereof.

[0055] The plant-based substrate may be obtained from more than one plant, such as, e.g., faba bean milk alternative fortified with pea protein or vice versa.

[0056] Preferably, the plant-based substrate has a protein content of at least 1% (w / w), such as at least 2% (w / w).

[0057] Preferably, the plant-based substrate has a protein content of at most 8% (w / w), such as e.g., at most 6% (w / w).

[0058] Preferably, the plant-based substrate has a lipid content of at most 5% (w / w).

[0059] In one embodiment, the plant-based substrate is essential free of lipid. In an embodiment, “essentially free of" means 0% (w / w). In an embodiment, a plant-based fermented dairy alternative food product, such as a plant-based dairy alternative yogurt, which is substantially free of lipid is obtained. In another embodiment, the plant-based substrate has a lipid content of at least 0.1% (w / w), such as at least 2% (w / w).

[0060] In the context of the invention, the plant-based substrate is not a byproduct, in particular the plant-based substrate is not an industrial byproduct. As used herein, the terms “byproduct” and “industrial byproduct” refer to a secondary or incidental product generated during the processing of plant-based materials. Examples of such byproducts include in particular press cakes and meals, such as peanut or soy press cakes, which are byproducts from the oil extraction process, or oat byproducts characterized by a high fraction of insolubles or fibers, derived from the processing of oats. In an embodiment, the plant-based substrate is not a byproduct obtained from an oil-extraction process.

[0061] Dietary fiber, also referred to as roughage or bulk, refers to the indigestible portion of plant-based substrate which the body cannot digest or absorb and which can pass relatively intact through the stomach, small intestine and colon and out of the body. The dietary fiber may further be classified as a soluble fiber, which dissolves in water, or an insoluble fiber, which does not dissolve in water.

[0062] In an embodiment, the plant-based substrate has a dietary fiber content below 5% (w / w), such as below 4% (w / w), below 3% (w / w), below 2.5% (w / w), below 2% (w / w), below 1.5%

[0063] (w / w), or below 1% (w / w). In an embodiment, the plant-based substrate has a dietary fiber content below 2.5% (w / w). In an embodiment, the plant-based substrate has a dietary fiber content below 1.5% (w / w). In an embodiment, the plant-based substrate has a dietary fiber content below 1% (w / w). In an embodiment, the plant-based substrate is essentially free of dietary fibers. In an embodiment, the plant-based substrate has a dietary fiber content in the range of 0.1-3% (w / w). In an embodiment, the plant-based substrate has a dietary fiber content in the range of 0.5-2% (w / w).

[0064] Other ingredients may be added to the plant-based substrate, such as, e.g., oil, such as plant oil, e.g., a sunflower oil, sugar, such as sucrose, yeast extract, and / or peptone. In a preferred embodiment, sunflower oil is added to the plant-based substrate. In the processes of the invention, lipid may be added to the plant-based substrate, e.g., prior to or together with the addition of the protein deamidase, or prior to or together with the addition of the transglutaminase. Also possible is the addition of the lipid prior to or during the fermentation of the plant-based substrate. Finally, the lipid may be added to the plant-based fermented dairy alternative food product. Interestingly, it has been found that the addition of a lipid early in the process, such as, e.g., prior to or together with the addition of the protein deamidase, does not have a negative impact on the overall process for obtaining the plant-based fermented dairy alternative food product, nor does it influence the texture or taste of the plant-based dairy alternative food product.

[0065] The plant-based substrate may have been standardized and / or homogenized. The plantbased substrate may have been pasteurized or otherwise heat-treated.

[0066] The plant-based fermented dairy alternative food product of the invention is a plantbased food product which is produced by fermentation and which is a plant-based alternative to a fermented dairy product produced by fermentation of a milk substrate based on milk obtained from a mammal.

[0067] In one embodiment, fermentation is performed by incubating with a lactic acid bacterium, preferably of the genus Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Propionibacterium, Enterococcus, Brevibacterium, Bifidobacterium or any combination thereof. In the context of the present invention, a plantbased fermented dairy alternative food product may also be referred to as a fermented food product, a fermented dairy alternative, and similar.

[0068] In one embodiment, fermentation is performed by incubating with a thermophilic lactic acid bacterium.

[0069] In one embodiment, fermentation is performed by incubating with a mesophilic lactic acid bacterium.

[0070] In another embodiment, fermentation is performed by incubating with a lactic acid bacterium combined with yeast.

[0071] In a preferred embodiment, the plant-based fermented dairy alternative food product is a yoghurt alternative, a set-type yoghurt alternative, a stirred yoghurt alternative, a strained yoghurt alternative, a drinking yoghurt alternative, a fermented milk drink alternative, a kefir alternative, a sour cream alternative, a greek-style yoghurt alternative or a skyr alternative. A stirred yoghurt alternative may be produced by carrying out fermentation in fermentation tanks where the formed acid gel is disrupted, e.g., by agitation after fermentation when the desired pH has been obtained. The stirred product may be partially cooled to 20-30°C and flavoring ingredients may be added. The stirred product is pumped to filling line and filled in retail containers. The stirred yoghurt alternative may then be cooled and then stored.

[0072] A set-type yoghurt alternative may be fermented in retail container and not agitated after fermentation. After fermentation, a set yoghurt alternative may be cooled and then stored. The cooling may be carried out in blast chiller tunnel or in a refrigerated storage room.

[0073] The term "after fermentation" as used herein means when fermentation is ended and the desired pH obtained.

[0074] A strained yoghurt alternative, such as a Greek yoghurt alternative or a labneh alternative, is a yoghurt alternative that has been strained to remove part of its aqueous phase, thus resulting in a thicker consistency than an unstrained yoghurt alternative, while preserving yoghurt's distinctive sour taste.

[0075] When using fermentation with a microorganism, the pH after fermentation may preferably be between 3.5 and 5.5, most preferably between 4 and 5.

[0076] In one embodiment, the plant-based fermented dairy alternative food product is a stirred yoghurt alternative wherein agitation is performed during or following the fermentation step.

[0077] In one embodiment, the plant-based fermented dairy alternative food product is cooled, preferably immediately.

[0078] A stirred yoghurt alternative may be cooled down to approx. 20-25°C in the fermentation tank. Then, agitation, e.g., by stirring, may be performed to break the gel. The yoghurt alternative may then be pumped to the filling line followed by a second cooling step to storage temperature of approximately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

[0079] Alternatively, for a stirred yoghurt alternative, the fermented product may be first stirred to break the gel, then cooled down to approximately 20-25°C by heat exchanger in the line towards the filling station, and then in a second cooling step cooled down to storage temperature of approximately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

[0080] The process for a set yoghurt alternative may be: After fermentation in retail pot (carried out in tempered room), the yoghurt alternative is cooled down to storage temperature of approximately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

[0081] The process of the invention may further include a storage step after fermentation. This may be carried out after agitation, e.g., by stirring or pumping, and / or cooling (one or more times), preferably after both. Storage may be carried out at a low temperature, preferably at less than 10°C, more preferably at 0-10°C, such as at 4-6°C. In a preferred embodiment, the plant-based fermented dairy alternative food product is a spoonable plant-based fermented dairy alternative, such as a stirred yoghurt alternative, a settype yoghurt alternative or a strained yoghurt alternative, or a drinkable plant-based fermented dairy alternative, such as a drinking yoghurt alternative or a kefir alternative.

[0082] In a more preferred embodiment, the plant-based fermented dairy alternative food product is a spoonable plant-based fermented dairy alternative, preferably a spoonable yoghurt alternative.

[0083] In the process of the present invention, a pasteurization step is preferably performed before step (b). This may be to thermally inactivate microorganisms and / or to better control the fermentation. Pasteurization before fermentation may also give a better structure of the plantbased fermented dairy alternative food product.

[0084] Pasteurization may be performed, e.g., at 80-95°C for 1-30 minutes, such as at 80-85°C for 30 minutes or at 90-95°C for 2-15 minutes.

[0085] In steps (a) and (b) of the processes of the invention, the plant-based substrate is treated with a protein deamidase and a transglutaminase, respectively. Step (a) may be performed before and / or during step (b). I.e., the protein deamidase may be added first to the plant-based substrate and after incubation for some time, such as 0.5-20 hours, such as, e.g., 1-2 hours, the transglutaminase may be added with or without a fermenting agent. When the transglutaminase is added with the fermenting agent, the incubation is continued until the desired pH is reached for the plant-based fermented dairy alternative food product.

[0086] In the processes of the invention, step (a) may be performed before step (b). A pasteurization step may be performed before step (a). And / or a pasteurization step may be performed after step (a) and before step (b). In that case, the pasteurization will inactivate the enzyme(s) prior to the fermentation. A pasteurization step may be performed before step (a) and another one after step (a) but before step (c).

[0087] In the processes of the invention, step (b) may be performed before and / or during step (c). I.e., the transglutaminase may be added to the enzymatically deamidated plant-based substrate and after incubation for some time, such as 0.5-20 hours, such as, e.g., 1-2 hours, the fermenting agent is added and the incubation is continued until the desired pH is reached.

[0088] In a preferred embodiment, a pasteurization step is performed before step (a).

[0089] In a preferred embodiment, step (b) is performed before and during step (c) and a pasteurization step is performed before step (b).

[0090] In another preferred embodiment, steps (a), (b) and step (c) are performed simultaneously, i.e., the protein deamidase, transglutaminase and fermenting agent are added at the same time or essentially at the same time in the process, and a pasteurization step is performed before step (a).

[0091] In one embodiment, the plant-based substrate is treated with the protein deamidase prior to pasteurization and treated with the transglutaminase after pasteurization. In the context of the invention, what is meant is that the treatment with the protein deamidase and the transglutaminase is performed sequentially. E.g., the protein deamidase may be added to the plant-based substrate, which has optionally been pasteurized, and after some time, such as, e.g., 30-90 minutes, preferably 60 minutes, the transglutaminase is added. Or the treatment with the protein deamidase may be performed first, optionally followed by a pasteurization step, and then the transglutaminase is added, e.g., at the same time as the fermenting agent.

[0092] The enzyme treatment may be performed, e.g., at a temperature in the range of 20- 60°C, such as in the range of 40-60°C, for 15 minutes to 10 hours, such as for 30 minutes to 3 hours. In further embodiments, the enzyme treatment is performed at a temperature of about 20°C, 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

[0093] The fermentation in step (c) is performed until the desired pH is reached. It is well-known in the art how to choose the optimal temperature and incubation time for the fermentation. The fermentation may be performed, e.g., at 40-45°C for 3-12 hours, such as for 4-8 hours. Lower temperatures, such as down to 20-30°C, may be used for mesophilic cultures.

[0094] In the processes of the invention, the viscosity of the plant-based fermented dairy alternative food product is increased compared to a plant-based fermented dairy alternative obtained by the same process but without addition of a protein deamidase and a transglutaminase. The viscosity may be determined after six days of storage at 4°C by allowing a sample of the plant-based fermented dairy alternative to set for 1 hour at 4°C followed by viscosity measurement carried out at 20°C at 50 rpm and the viscosity value read after 70 seconds. The term “viscosity” denotes the resistance of a fluid, such as a liquid, to a change in shape, or movement of neighboring portions relative to one another. For liquids, the term "thickness" may also be used to describe viscosity. The viscosity of the plant-based fermented dairy alternative food product of the present invention may be varied according to desired end food product, e.g., for obtaining a spoonable yogurt and a drinking yogurt, different viscosities are desired. For example, in the processes of the invention, the concentration and ratio of transglutaminase and protein deamidase may be varied to achieve a desired viscosity of the plant-based fermented dairy alternative food product. The skilled person will know how to determine optimum parameters in the process for achieving a desired viscosity of the plantbased fermented dairy alternative food product.

[0095] In an embodiment, the plant-based fermented dairy alternative food product expels less liquid in a forced syneresis test compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of protein deamidase and transglutaminase. The amount of whey expelled from the food product, such as, e.g., a yoghurt, upon centrifugation (forced syneresis) is a measure of the water holding capacity (WHC) and correlates with the proneness to spontaneous syneresis occurring during storage. The forced syneresis test may be performed after six days of storage at 4°C by centrifugation of the plant- based fermented dairy alternative food product for 15 min at 2643 x g. The weight of remaining solid is recorded after removal of supernatant and the amount of expelled liquid is calculated using the formula: (weight of fermented dairy alternative food product sample - weight of solid phase) / (weight of fermented dairy alternative food product sample) * 100%.

[0096] In an embodiment, the plant-based fermented dairy alternative food product has an increased hardness compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of a protein deamidase and a transglutaminase. The hardness may be determined after six days of storage at 4°C by allowing a sample of the plant-based fermented dairy alternative food product to set for 1 hour at 4°C followed by hardness measurements using texture analyzer with cylinder probe, depth 10 mm, analysis time 10 seconds.

[0097] In a preferred embodiment, the plant-based fermented dairy alternative food product has a more smooth texture and less grainy texture compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of a protein deamidase and a transglutaminase. The texture may be visually evaluated after six days of storage at 4°C by placing a sample of the plant-based fermented dairy alternative food product on the backside of a black plastic spoon. For the visual evaluation, the evaluated parameters may be one or more of: flakiness / graininess, syneresis, initial homogeneity, adhesiveness, and homogeneity after stirring. A score system from 1 to 7 may be used to distinguish samples on each parameter. The definitions of visual evaluated parameters defined here are:

[0098] Flakiness / graininess: visual inspection of lumps, flakes on the surface, or grainy texture by placing a plant-based fermented dairy alternative food product sample on the back side of black plastic.

[0099] Syneresis: the water amount observed once the plastic jar containing the plant-based fermented dairy alternative food product is opened.

[0100] Initial homogeneity: the homogeneity of the plant-based fermented dairy alternative food product after it is flipped from the bottom of the plastic jar by a spoon.

[0101] Homogeneity after stirring: the homogeneity after the whole sample in the plastic jar is stirred 30 times manually by a spoon.

[0102] Adhesiveness: the amount and shape of plant-based fermented dairy alternative food product sample on the back of a spoon after the spoon is pressed and lifted quickly on the surface of fully stirred plant-based fermented dairy alternative food product sample.

[0103] In a preferred embodiment, the plant-based fermented dairy alternative food product has a less grainy texture compared to a plant-based fermented dairy alternative food product obtained by the same process but without addition of a protein deamidase and a transglutaminase. The texture may be visually evaluated after six days of storage at 4°C by placing a sample of the plant-based fermented dairy alternative food product on the backside of a black plastic spoon. In another preferred embodiment, the plant-based fermented dairy alternative food product has a higher resilience against deformation as measured using a rheological method to quantify thixotropy compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4. In an embodiment, the plant-based fermented dairy alternative food product has an at least 15%, such as, e.g., 18%, 20%, 25%, 30%, 35%, 40% or 50%, reduction in percentage structure loss as measured using a rheological method to quantify thixotropy compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4.

[0104] The increased resistance against deformation observed for the plant-based fermented dairy alternative food product obtained using a combination of protein deamidase and transglutaminase increases the versatility of the plant-based fermented dairy alternative food product, e.g. a yogurt alternative, obtained, since the end product has an increased ability to restructure itself and not lose viscosity following mechanical stress, e.g. as a result of stirring the fermented dairy alternative food product, compared to a plant-based fermented dairy alternative food product obtained using the same processes as disclosed herein, but without the use of a protein deamidase and a transglutaminase.

[0105] Without wishing to be bound by any particular theory, the inventor believes that the specific combination of the protein deamidase and transglutaminase in the production of a plant-based fermented dairy alternative food product, in particular a plant-based yogurt alternative, is responsible for the observed improvements in not only smoothness of the final product, but also the superior overall textural properties of the food product. In particular, in a sensory evaluation (see Example 5) of a pea-based yogurt alternative produced according to the processes disclosed herein, the yogurt alternative obtained a higher score in perceived thickness and had improved smoothness (i.e. , less grainy appearance and feel) compared to a yogurt alternative produced without the combination of protein deamidase and transglutaminase. Also interesting was that the findings were irrespective of concentration of the protein deamidase and irrespective of whether the protein deamidase was used in a pretreatment prior to fermentation or if the protein deamidase was added during the fermentation. This further strengthens the versatility of the processes disclosed herein.

[0106] In one embodiment, a hydrocolloid or stabilizer such as pectin is added to the plantbased fermented dairy alternative food product, in which case addition of a protein deamidase will likely not result in further reduction of syneresis, since syneresis will already be very low. However, treatment with a protein deamidase will still confer other benefits. In another embodiment, no hydrocolloid or stabilizer is added to the plant-based fermented dairy alternative food product. In another embodiment, no pectin is added to the plant-based fermented dairy alternative food product. From a clean-label perspective, avoidance of hydrocolloid or stabilizer, such as pectin, is preferred.

[0107] The plant-based substrate is fermented with a lactic acid bacterium in step (c) and the plant-based fermented dairy alternative food product is a plant-based fermented dairy alternative food product. Preferably in such process of obtaining a plant-based fermented dairy alternative food product, the addition of the protein deamidase further results in a pH buffering capacity in the plant-based fermented dairy alternative food product. Thus, the plant-based fermented dairy alternative food product produced according to the processes of the invention has improved storage stability compared to a plant-based fermented dairy alternative food product obtained using the same process but without the addition of a protein deamidase and a transglutaminase. The improved storage stability is observed, e.g., by the plant-based fermented dairy alternative food product having a stable pH over time, i.e., the pH does not change significantly in the time between production and consumption, also referred to as a higher resilience of the food product to post-acidification. The improved storage stability of the plant-based fermented dairy alternative food obtained according to the methods of the invention has been shown at least in Example 6. In particular, in Example 6 it has been shown that the use of protein deamidase alone and in combination of with the transglutaminase, gives rise to a better resilience to post-acidification of the exemplified pea protein-fortified oat yogurt than when the transglutaminase is used alone or when no enzyme is used to produce the plantbased fermented dairy alternative food. Thus, an embodiment of the invention relates to a process for preparing an oat-based yogurt, the process comprising:

[0108] (a) treating an oat base, optionally fortified with a leguminous protein, with a protein deamidase;

[0109] (b) treating the oat base, optionally fortified with a leguminous protein, of step (a) with a transglutaminase; and

[0110] (c) fermenting the oat base, optionally fortified with a leguminous protein, of step (b) by incubating with a lactic acid bacterium to produce the oat-based yogurt; wherein step (a) is performed before and / or during step (b), and step (b) is performed before and / or during step (c), and wherein the oat-based yogurt has increased resilience to post-acidification compared to an oat-based yogurt obtained using the same process but without addition of protein deamidase and transglutaminase.

[0111] The term “post-acidification” or “post-fermentation acidification” is understood as an undesirably process which may occur in fermented dairy / non-dairy products due to continued acidification beyond the optimal range of the fermented product. This may occur due to a persistent metabolic activity of product’s microflora during its shelf-life. Apart from shortening the shelf-life, it can result in severe acidity, whey syneresis, unclean flavor, decreased lactic acid bacteria count or even gas production. The person of skill in the art knows how to determine post-acidification in fermented dairy alternative food products. One such method of determining post-acidification is exemplified in Example 6.

[0112] Without wishing to be bound by any particular theory, the inventor believes that the beneficial effects of the protein deamidase and transglutaminase on the overall texture and taste and the resilience to post-acidification of the plant-based fermented dairy alternative food product enables manufacturers to produce the food products without the need for added preservatives, flavorings and additives in general. I.e., a clean-label plant-based fermented dairy alternative food product can be obtained using the processes as claimed herein.

[0113] Protein deamidase

[0114] In the processes of the invention, a plant-based substrate is treated with a protein deamidase to obtain the plant-based fermented dairy alternative food product.

[0115] In the present invention, a protein deamidase refers to an enzyme having an effect of directly acting on an amide group of a side chain of an amino acid that constitutes a protein to cause deamidation and release ammonia without cleaving a peptide bond of the protein and without crosslinking the protein.

[0116] The term “deamidase” means a protein-glutamine glutaminase (also known as glutaminylpeptide glutaminase) activity, as described in EC 3.5.1.44, which catalyzes the hydrolysis of the gamma-amide of glutamine substituted at the carboxyl position or both the alpha-amino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L- glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidase. Deamidases comprise a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al. “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691- 38702) and belong to the InterPro entry IPR041325.

[0117] Deamidase may also include a protein asparaginase that directly acts on an amide group of a side chain of an asparagine residue contained in a protein to release ammonia and thus converts the asparagine residue into an aspartate residue. In the present invention, as a protein deamidase, any one of the protein glutaminase and the protein asparaginase can be used, or both can be used in combination. One example of the protein deamidase used in the present invention is a protein glutaminase.

[0118] A protein deamidase to be used in the processes of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one embodiment, the polypeptide obtained from a given source is secreted extracellularly. The types or origins of the protein deamidase used in the present invention are not particularly limited. Examples of the protein deamidase includes protein deamidases derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus.

[0119] In some embodiments, the protein deamidase may be derived from Chryseobacterium genus, such as Chryseobacterium viscerum, C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase used in the processes of the invention is derived from or obtained from Chryseobacterium proteolyticum or Chryseobacterium viscerum. In an embodiment, the deamidase used in the processes of the invention is derived from or obtained from Chryseobacterium viscerum.

[0120] EP1839491 discloses cloning of a protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. Deamidases are also commercially available, e.g., protein glutaminases derived from Chryseobacterium genus, for example, "Amano PG500” (manufactured by Amano Enzyme Inc.).

[0121] The term “derived” means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e., the polypeptide sequence of the protein deamidase is identical to a native polypeptide. The term “derived” also means that the enzyme may have been produced recombinantly in a host organism, the recombinantly produced enzyme having either a polypeptide sequence which is identical to a native enzyme or having a modified polypeptide sequence, e.g., having one or more amino acids which are deleted, inserted and / or substituted, i.e., a recombinantly produced enzyme which is a mutant of a native polypeptide sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term “derived” includes enzymes produced synthetically by, e.g., peptide synthesis. The term “derived” also encompasses enzymes which have been modified e.g., by glycosylation, phosphorylation etc., whether in vivo or in vitro. With respect to recombinantly produced enzymes the term “derived from” refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.

[0122] The protein deamidase may be obtained from a microorganism by use of any suitable technique. For instance, an enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a protein deamidase preparation from the resulting fermented broth or microorganism by methods known in the art. The protein deamidase may also be obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the protein deamidase and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the enzyme in a culture medium under conditions per-mitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.

[0123] The protein deamidase may be purified. The term "purified" as used herein covers protein deamidase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers protein deamidase enzyme protein essentially free from insoluble components from the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography.

[0124] Preferably, the protein deamidase is purified from its production organism. More preferably, the protein deamidase is purified from its production organism meaning that the protein deamidase preparation does not comprise living production organism cells.

[0125] Accordingly, the protein deamidase may be purified, viz. only minor amounts of other proteins being present. The expression “other proteins” relate in particular to other enzymes. The term "purified" as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the protein deamidase. The protein deamidase may be "substantially pure", i.e., free from other components from the organism in which it is produced, i.e., e.g., a host organism for recombinantly produced protein deamidase. Preferably, the protein deamidase is an at least 40% (w / w) pure enzyme protein preparation, more preferably at least 50%, 60%, 70%, 80% or even at least 90% pure.

[0126] The term protein deamidase includes whatever auxiliary compounds may be necessary for the enzyme's catalytic activity, such as, e.g., an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system.

[0127] The protein deamidase may be in any form suited for the use in question, such as, e.g., in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme.

[0128] For example, protein deamidases can be obtained from a culture broth of the abovedescribed microorganisms.

[0129] Protein deamidases are produced by microbial cells in an inactive proform, which comprises a propeptide domain tightly bound to a deamidase domain. The proform is expressed as a fusion protein, which has reduced deamidase activity to protect the viability of the host cell. In nature, the fusion protein is post- processed to remove the propeptide and release the active deamidase outside of the host cell. However, in recombinant expression systems, the fusion protein is secreted outside of the host cell as an inactive proform comprising the propeptide. The propeptide may then be enzymatically cleaved off to separate it from the mature deamidase. The protein deamidases of the processes and compositions of the present invention are mature deamidases where the propeptide has been removed. In some embodiments, the propeptide was cleaved enzymatically by an endopeptidase. In some embodiments, the propeptide may still be present in the composition comprising the mature deamidase.

[0130] The recombinant mature protein deamidases used in the processes of the invention comprises the polypeptide of SEQ ID NOs: 2, 4, 6, 8, and 10. Each mature protein deamidase is derived from a deamidase proform polypeptide, which comprises the polypeptide of SEQ ID NO: 1 , 3, 5, 7, and 9, respectively. The proform polypeptide comprises a propeptide at the N- terminal end, fused to a deamidase which is the same as that of the polypeptide of SEQ ID NOs: 2, 4, 6, 8, or 10. The propeptide may be enzymatically cleaved from the proform polypeptide to release the mature deamidase. Naturally occurring propeptide sequences are provided in the proform polypeptide.

[0131] For purposes of the present invention, the sequence identity between two polypeptide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labelled “longest identity” is calculated as follows:

[0132] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0133] The processes and compositions of the invention include a mature deamidase and optionally a second polypeptide which is derived from the propeptide of a deamidase. The second polypeptides described herein are mutated variants of the naturally occurring propeptides. These variant propeptide sequences have been found to bind less strongly to their corresponding deamidase, so that they are more easily enzymatically cleaved off after recombinant expression and secretion from of the host cell. The polypeptides of SEQ ID NOs: 1 to 10 are derived from Chryseobacterium spp. and are described in PCT application PCT / EP2023 / 055936; filed March 8, 2023, herein incorporated by reference.

[0134] After expression of the proform polypeptide in a recombinant expression system, a sitespecific endopeptidase is used to cleave off the propeptide, leaving an active, mature deamidase. In some embodiments, the cleaved propeptide is not purified away from the mature deamidase. Therefore, the propeptide may be present in the composition with the mature deamidase.

[0135] According to a preferred embodiment the protein deamidase applied in the process of the invention is derived from or obtained from a Chryseobacterium species, e.g., Chryseobacterium proteolyticum or Chryseobacterium viscerum. In the context of the present invention, the term “mature polypeptide” means a polypeptide in its mature form following N terminal processing (e.g., removal of signal peptide). A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0136] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 2.

[0137] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 4.

[0138] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 6.

[0139] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 8.

[0140] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 10.

[0141] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 1.

[0142] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 3.

[0143] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 5.

[0144] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 7.

[0145] In one embodiment the deamidase is selected from a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a mature polypeptide of SEQ ID NO: 9.

[0146] A protein deamidase to be used in the processes of the invention may be added at a concentration of 0.01-20 IPA(U) / g substrate protein, such as 0.1-15 IPA(U) / g substrate protein, such as 1-13 IPA(U) / g substrate protein, such as 3-12 IPA(U) / g substrate protein. Deamidase (protein glutaminase) activity may be measured using the assay described in Example 1. The activity assay consists of two separate de-coupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non- enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

[0147] Transglutaminase

[0148] In the processes of the invention, the plant-based substrate is treated with a transglutaminase to obtain the plant-based fermented dairy alternative food product.

[0149] The transglutaminase to be used in the processes of the invention may be obtained from any source, in particular from a filamentous fungus or yeast, or from a bacterium.

[0150] The transglutaminase may, e.g., be derived from a strain of Agaricus, e.g. A. bisporus Ascovaginospora; Aspergillus, e.g. A. niger, A. awamori, A. foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia; Dictyostelium, e.g. D. discoideum; Fusarium, e.g. F. oxysporum, F. solani, F. sp.; Kluveromyces, e.g. K. fragilis, K. lactis; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhinocladiella; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S. libertiana; Torula; Torulopsis; Trichoderma, e.g. T. reesei; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sole roti o rum, Actinosynnema, e.g. A mirunr, Bacillus, e.g. B. sp. B. coagulans, B. circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B. stearothermophilus, B. thuringiensis', Bifidobacterium, e.g. B. animalis, B. bifidum, B. breve, B. infantis, B. lactis, B. longum; Chryseobacterium; Citrobacter, e.g. C. freundii; Clostridium, e.g. C. perfringens; Diplodia, e.g.

[0151] D. gossypina; Enterobacter, e.g. E. aerogenes, E. cloacae Edwardsiella, E. tarda; Erwinia, e.g.

[0152] E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae; Kribbella, e.g., K. flavida; Kutzneria, e.g., K. albida; Miriococcum; Myrothesium; Mucor; Neurospora, e.g. N. crassa; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii; Pycnoporus, e.g. Pycnoporus cinnabarinus, Pycnoporus sanguineus; Ruminococcus, e.g. R. torques; Salmonella, e.g. S. typhimurium', Serratia, e.g. S. liquefasciens, S. marcescens; Shewanella, e.g. S. woodyi; Shigella, e.g. S. flexneri; Streptomyces, e.g. S. antibioticus, S. castaneoglobisporus, S. violeceoruber; Trametes; Trichoderma, e.g. T. reesei, T. viride; Yersinia, e.g. Y. enterocolitica.

[0153] In a preferred embodiment, the transglutaminase is derived from a fungus. In another preferred embodiment, the transglutaminase is derived from a bacterium.

[0154] In a preferred embodiment, the transglutaminase is derived from a strain of Streptomyces, preferably Streptomyces mobaraensis or Streptomyces lydicus, more preferably Streptomyces mobaraensis.

[0155] In a preferred embodiment, the transglutaminase to be used in a process of the invention has an amino acid sequence which is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 12 of the present invention or a transglutaminase active fragment hereof.

[0156] In a preferred embodiment, the transglutaminase to be used in a process of the invention has an amino acid sequence which is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the mature polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12.

[0157] In a more preferred embodiment, the transglutaminase to be used in a process of the invention has an amino acid sequence which is at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

[0158] The transglutaminase may be extracellular. It may have a signal sequence at its N- terminus, which is cleaved off during secretion.

[0159] The transglutaminase may be derived from any of the sources mentioned herein. The term “derived” means in this context that the enzyme may have been isolated from an organism where it is present natively, i.e. , the amino acid sequence of the transglutaminase is identical to a native polypeptide. The term “derived” also means that the enzyme may have been produced recombinantly in a host organism, the recombinantly produced enzyme having either an amino acid sequence which is identical to a native enzyme or having a modified amino acid sequence, e.g. having one or more amino acids which are deleted, inserted and / or substituted, i.e. a recombinantly produced enzyme which is a mutant of a native amino acid sequence. Within the meaning of a native enzyme are included natural variants. Furthermore, the term “derived” includes enzymes produced synthetically by, e.g., peptide synthesis. The term “derived” also encompasses enzymes which have been modified e.g., by glycosylation, phosphorylation etc., whether in vivo or in vitro. With respect to recombinantly produced enzymes the term “derived from” refers to the identity of the enzyme and not the identity of the host organism in which it is produced recombinantly.

[0160] The transglutaminase may be obtained from a microorganism by use of any suitable technique. For instance, an enzyme preparation may be obtained by fermentation of a suitable microorganism and subsequent isolation of a transglutaminase preparation from the resulting fermented broth or microorganism by methods known in the art. The transglutaminase may also be obtained by use of recombinant DNA techniques. Such method normally comprises cultivation of a host cell transformed with a recombinant DNA vector comprising a DNA sequence encoding the transglutaminase and the DNA sequence being operationally linked with an appropriate expression signal such that it is capable of expressing the enzyme in a culture medium under conditions permitting the expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be incorporated into the genome of the host cell. The DNA sequence may be of genomic, cDNA or synthetic origin or any combinations of these, and may be isolated or synthesized in accordance with methods known in the art.

[0161] The transglutaminase may be purified. The term "purified" as used herein covers transglutaminase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers transglutaminase enzyme protein essentially free from insoluble components from the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography.

[0162] Preferably, the transglutaminase is purified from its production organism. More preferably, the transglutaminase is purified from its production organism meaning that the transglutaminase preparation does not comprise living production organism cells.

[0163] Accordingly, the transglutaminase may be purified, viz. only minor amounts of other proteins being present. The expression “other proteins” relate in particular to other enzymes. The term "purified" as used herein also refers to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the transglutaminase. The transglutaminase may be "substantially pure", i.e., free from other components from the organism in which it is produced, i.e., e.g., a host organism for recombinantly produced transglutaminase. Preferably, the transglutaminase is an at least 40% (w / w) pure enzyme protein preparation, more preferably at least 50%, 60%, 70%, 80% or even at least 90% pure.

[0164] The term transglutaminase includes whatever auxiliary compounds may be necessary for the enzyme's catalytic activity, such as, e.g., an appropriate acceptor or cofactor, which may or may not be naturally present in the reaction system.

[0165] The transglutaminase may be in any form suited for the use in question, such as, e.g., in the form of a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme.

[0166] The transglutaminase may be added at a concentration of 0.1 - 5 TGHU(A) / g plant protein, preferably 0.2 - 1 TGHU(A) / g plant protein. The dosage will depend on parameters such as the temperature and the incubation time. The skilled person will know how to determine the optimal enzyme dosage.

[0167] According to the present invention, transglutaminase activity may be determined by any method known in the art. For example, analysis of transglutaminase activity may be done by quantitation of the released ammonia resulting from the formation of an iso-peptide bond between a free amino group (6-aminohexanoic acid) and an acyl group from a glutamine (Z- GLN-GLY) like described below:

[0168] Chemicals and enzymes used: Z-GLN-GLY. Eg. Sigma C6154

[0169] 6-aminohexanoic acid. Eg. Sigma 07260

[0170] L-Gluthatione reduced. Eg. Sigma G4251 a-Ketoglutarate. Eg. Sigma K3752

[0171] NADH

[0172] L-GLDH. Eg. Roche 107735

[0173] MOPS. Eg. Sigma M-1254

[0174] Transglutaminase standard

[0175] Method:

[0176] To 75 myl of an enzyme solution, dissolved in 0.1 M MOPS / 5 mM L-Gluthatione reduced pH 7.0, is added 50 myl of 1% 6-aminohexanoic acid, and 75 myl of 1% Z-GLN-GLY, and 75 myl of (0.44 g / L NADH, 2.5 g / L a-Ketoglutarate in 0.1 M MOPS pH 7.0).

[0177] The absorbance at 340 nm is followed by kinetic measurement for 5 min at 30°C.

[0178] The enzyme activity is determined relative to a transglutaminase standard that has been aligned to match the transglutaminase Unit Definition (Folk, J. E. and Cole, P. W. (1966) Biochim. Biophys. Acta.241, 5518-5525). Result are expressed as TGHU(A).

[0179] The invention is further defined by the following numbered embodiments:

[0180] Embodiment 1. A process for preparing a plant-based fermented dairy alternative food product, the process comprising:

[0181] (a) treating a plant-based substrate with a protein deamidase;

[0182] (b) treating the plant-based substrate of step (a) with a transglutaminase; and

[0183] (c) fermenting the plant-based substrate of step (b) by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative food product; wherein step (a) is performed before and / or during step (b), and step (b) is performed before and / or during step (c), and wherein the fermented dairy alternative food product has increased viscosity compared to a fermented dairy alternative food product obtained using the same process but without addition of protein deamidase and transglutaminase.

[0184] Embodiment 2. The process of embodiment 1, wherein the plant-based substrate is derived or obtained from almond, cashew, chickpea, coconut, faba bean, flax, hemp, lentil, lupin, macadamia, mung bean, oat, pea, peanut, pecan, pistachio, rice, soy, or any combination thereof, such as from faba bean, lentil, pea, oat or any combination thereof.

[0185] Embodiment 3. The process of any of the preceding embodiments, wherein the plantbased substrate is derived or obtained from legumes, preferably from soy, pea, chickpea, mung bean, lentils, faba bean or lupin, more preferably from pea, lentil or faba bean.

[0186] Embodiment 4. The process of any of embodiments 1-2, wherein the plant-based substrate is derived or obtained from oat, almond or coconut. Embodiment 5. The process of any of embodiments 1-2, wherein the plant-based substrate is derived or obtained faba bean, lentil, pea, oat or any combination thereof.

[0187] Embodiment 6. The process of any of the preceding embodiments, wherein the plantbased substrate is not a byproduct, preferably wherein the plant-based substrate is not an industrial byproduct of an oil-extraction process.

[0188] Embodiment 7. The process of any of the preceding embodiments, wherein the plantbased substrate has a dietary fiber content below 5% (w / w), such as below 4% (w / w), below 3% (w / w), below 2.5% (w / w), below 2% (w / w), below 1.5% (w / w), or below 1% (w / w).

[0189] Embodiment 8. The process of any of the preceding embodiments, wherein the plantbased substrate has a dietary fiber content below 2.5% (w / w)

[0190] Embodiment 9. The process of any of the preceding embodiments, wherein the plantbased substrate has a dietary fiber content below 1.5% (w / w), such as below 1% (w / w).

[0191] Embodiment 10. The process of any of the preceding embodiments, wherein the plantbased substrate is essentially free of dietary fibers.

[0192] Embodiment 11. The process of any of embodiments 1-7, wherein the plant-based substrate has a dietary fiber content in the range of 0.1-3% (w / w), such as in the range of 0.5- 2% (w / w).

[0193] Embodiment 12. The process of any of the preceding embodiments, wherein the plantbased substrate is (i) a plant-based milk alternative, such as a soy beverage, pea beverage, a lentil beverage, a faba bean beverage or oat beverage, optionally fortified with a plant-based milk alternative powder, such as pea milk powder, or with a concentrated or isolated protein, such as a leguminous protein isolate or concentrate; or (ii) an aqueous solution or suspension of a plant-based milk alternative powder, such as pea milk powder or a faba bean milk powder, or of a plant protein isolate or concentrate, such as a leguminous protein isolate or concentrate, preferably a soy protein isolate, soy protein concentrate, pea protein isolate, pea protein concentrate, faba protein isolate, faba protein concentrate, lentil protein isolate or lentil protein concentrate.

[0194] Embodiment 13. The process of any of the preceding embodiments, wherein the plantbased substrate is (i) an oat-, coconut-, or almond-based beverage, optionally an oat-, coconut-, or almond-based beverage fortified with a pea protein concentrate, a pea protein isolate, a faba protein concentrate, a faba protein isolate, a lentil protein concentrate, a lentil protein isolate, or any combination thereof; or (ii) an aqueous solution or suspension of a pea protein isolate, a faba protein isolate, a lentil protein isolate, or any combination thereof.

[0195] Embodiment 14. The process of any of the preceding embodiments, wherein the plantbased substrate is an aqueous solution or suspension of a pea protein isolate, a faba protein isolate, a lentil protein isolate, or any combination thereof. Embodiment 15. The process of any of embodiments 1-13, wherein the plant-based substrate is an oat beverage fortified with a leguminous protein, preferably a leguminous protein isolate or a leguminous protein concentrate.

[0196] Embodiment 16. The process of the preceding embodiment, wherein the leguminous protein isolate is selected from a pea protein isolate, a faba protein isolate, a lentil protein isolate, or any combination thereof.

[0197] Embodiment 17. The process of the preceding embodiment, wherein the oat beverage fortified with pea protein isolate, a faba protein isolate, a lentil protein isolate, or any combination thereof, has a protein content in the range of 2-6 % (w / w), preferably a protein content in the range of 3-5% (w / w).

[0198] Embodiment 18. The process of any of the preceding embodiments, wherein the plantbased substrate has a protein content of at least 2% (w / w).

[0199] Embodiment 19. The process of any of the preceding embodiments, wherein the plantbased substrate has a protein content of at most 8% (w / w), such as at most 6% (w / w).

[0200] Embodiment 20. The process of any of the preceding embodiments, wherein the plantbased substrate has a lipid content of at most 10% (w / w), such as at most 5% (w / w).

[0201] Embodiment 21. The process of any of the preceding embodiments, wherein the plantbased substrate is essentially free of lipids.

[0202] Embodiment 22. The process of any of the preceding embodiments, wherein the treatment of step (a) is carried out at a temperature in the range of 20-60°C, such as in the range of 40-60°C.

[0203] Embodiment 23. The process of any of the preceding embodiments, wherein the treatment of step (a) is carried out at a temperature in the range of 50-60°C

[0204] Embodiment 24. The process of any of the preceding embodiments, wherein the treatment of step (a) is carried out for 15 minutes to 10 hours, such as for 30 minutes to 3 hours.

[0205] Embodiment 25. The process of any of the preceding embodiments, wherein the treatment of step (a) is carried out for 30-60 minutes.

[0206] Embodiment 26. The process of any of the preceding embodiments, wherein pasteurization is performed after step (a) and before step (b).

[0207] Embodiment 27. The process of any of the preceding claims, wherein steps (b) and (c) are performed simultaneously.

[0208] Embodiment 28. The process of any of the preceding embodiments, wherein heat treatment, preferably at a temperature of 95-120°C, is performed after step (c).

[0209] Embodiment 29. The process of any of the preceding embodiments, further comprising the addition of a lipid during step (a) or (b), or after step (b), preferably during step (a) or (b).

[0210] Embodiment 30. The process of the preceding embodiment, wherein the lactic acid bacterium is of the genus Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Propionibacterium, Enterococcus, Brevibacterium, Bifidobacterium or any combination thereof.

[0211] Embodiment 31. The process of any of the preceding embodiments, wherein the protein deamidase is derived from or obtained from a Chryseobacterium species, more preferably from Chryseobacterium proteolyticum or Chryseobacterium viscerum.

[0212] Embodiment 32. The process of any of the preceding embodiments, wherein the protein deamidase comprises a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0213] Embodiment 33. The process of any of the preceding embodiments, wherein the protein deamidase comprises the polypeptide sequence of SEQ ID NOs: 2, 4, 6, 8, or 10.

[0214] Embodiment 34. The process of any of the preceding embodiments, wherein the transglutaminase is a bacterial endopeptidase, preferably derived from Streptomyces, more preferably from Streptomyces mobaraensis.

[0215] Embodiment 35. The process of any of the preceding embodiments, wherein the transglutaminase is selected from a polypeptide having at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 or SEQ ID NO: 12 or a transglutaminase active fragment hereof.

[0216] Embodiment 36. The process of any of the preceding embodiments, wherein the transglutaminase is selected from a polypeptide having at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide of SEQ ID NO: 11 or SEQ ID NO: 12.

[0217] Embodiment 37. The process of any of the preceding embodiments, wherein the transglutaminase is selected from a polypeptide having at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0218] Embodiment 38. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative is a yoghurt alternative, a set-type yoghurt alternative, a stirred yoghurt alternative, a strained yoghurt alternative, a drinking yoghurt alternative, a fermented milk drink alternative, a kefir alternative, a sour cream alternative, a greek-style yoghurt alternative, or a skyr alternative.

[0219] Embodiment 39. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative is a spoonable plant-based fermented dairy alternative, such as a stirred yoghurt alternative, a set-type yoghurt alternative or a strained yoghurt alternative, or a drinkable plant-based fermented dairy alternative, such as a drinking yoghurt alternative or a kefir alternative. Embodiment 40. The process of any of the preceding embodiments, wherein the viscosity of the plant-based fermented dairy alternative food product is increased compared to a plant-based fermented dairy alternative obtained using the same process but without addition of a protein deamidase and a transglutaminase, where the viscosity is determined after six days storage at 4°C by allowing a sample of the plant-based fermented dairy alternative food product to set for 1 hour at 4°C followed by viscosity measurement carried out at 20°C at 50 rpm and the viscosity value read after 70 seconds.

[0220] Embodiment 41. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative food product has an increased hardness compared to a plantbased fermented dairy alternative food product obtained using the same process but without addition of a protein deamidase and a transglutaminase, where the hardness is determined after six days storage at 4°C by allowing a sample of the plant-based fermented dairy alternative food product to set for 1 hour at 4°C followed by Texture Profile Analysis using texture analyzer with cylinder probe, after which only the hardness value was utilized.

[0221] Embodiment 42. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative food product has a more smooth texture and less grainy texture compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of a protein deamidase and a transglutaminase, wherein the texture is visually evaluated by placing a sample of the plant-based fermented dairy alternative food product on the backside of a black plastic spoon.

[0222] Embodiment 43. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative food product has a higher resilience against deformation as measured using a rheological method to quantify thixotropy compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4.

[0223] Embodiment 44. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative food product has an at least 15%, such as an 18%, 20%, 25%, 30%, 35%, 40% or 50%, reduction in percentage structure loss as measured using a rheological method to quantify thixotropy compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4.

[0224] Embodiment 45. The process of any of the preceding embodiments, wherein the plantbased fermented dairy alternative food product has a higher resilience to post-acidification compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase. Embodiment 46. The process of the preceding embodiment, wherein post-acidification is determined using a method as exemplified in Example 6.

[0225] Embodiment 47. The process of any of embodiments 45-46, wherein the plant-based substrate is an oat-, coconut-, or almond-based beverage, optionally an oat-, coconut-, or almond-based beverage fortified with a pea protein concentrate, a pea protein isolate, a faba protein concentrate, a faba protein isolate, a lentil protein concentrate, a lentil protein isolate, or any combination thereof.

[0226] Embodiment 48. The process of any of embodiments 45-47, wherein the plant-based substrate is an oat-based beverage, optionally an oat-based beverage fortified with a pea protein concentrate, a pea protein isolate, a faba protein concentrate, a faba protein isolate, a lentil protein concentrate, a lentil protein isolate, or any combination thereof.

[0227] Embodiment 49. The process of any of embodiments 45-48, wherein the plant-based fermented dairy alternative food product has improved water holding capacity compared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase.

[0228] Embodiment 50. The process of any of embodiments 45-49, wherein water holding capacity is determined using a method as exemplified in Example 7.

[0229] Embodiment 51. A plant-based fermented dairy alternative food product obtainable by a process as embodied in any of the preceding embodiments.

[0230] Embodiment 52. The plant-based fermented dairy alternative food product of embodiment 51 , further comprising a protein deamidase comprising a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0231] Embodiment 53. The plant-based fermented dairy alternative food product of embodiment 51 or 52, further comprising a transglutaminase comprising a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the mature polypeptide of SEQ ID NOs: 11 or 12.

[0232] Embodiment 54. The plant-based fermented dairy alternative food product of any of the embodiment 51-53, which has an improved texture and / or higher resilience to post-acidification compared to a plant-based fermented dairy alternative food product obtained using a process as embodied in any of the preceding embodiments but without the use of a protein deamidase and transglutaminase.

[0233] Embodiment 55. Use of a protein deamidase and a transglutaminase in the production of a plant-based fermented dairy alternative food product to improve texture and / or resilience to post-acidification.

[0234] Embodiment 56. Use according to embodiment 55, wherein the plant-based fermented dairy alternative food product has increased viscosity compared to a plant-based fermented dairy alternative food product obtained using a process as embodied in embodiments 1-50 but without the use of a protein deamidase and a transglutaminase.

[0235] Embodiment 57. Use according to any of embodiments 55-56, wherein the plant-based fermented dairy alternative product is a spoonable plant-based fermented dairy alternative, such as a stirred yoghurt alternative, a set-type yoghurt alternative or a strained yoghurt alternative, or a drinkable plant-based fermented dairy alternative, such as a drinking yoghurt alternative or a kefir alternative, preferably a spoonable plant-based fermented dairy alternative, preferably a spoonable plant-based fermented dairy alternative.

[0236] Embodiment 58. Use according to any of embodiments 55-57, wherein the plant-based fermented dairy alternative food product has a more smooth texture and a less grainy texture compared to a plant-based fermented dairy alternative food product prepared without the use of a protein deamidase and a transglutaminase, wherein the texture is visually evaluated by placing a sample of the plant-based fermented dairy alternative food product on the backside of a black plastic spoon; and / or wherein the plant-based fermented dairy alternative food product has a higher resilience against deformation as measured using a rheological method to quantify thixotropy compared to a plant-based fermented dairy alternative food product prepared without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4.

[0237] Embodiment 59. Use according to any of the embodiments 55-58, wherein the plantbased fermented dairy alternative food product has a higher resilience to post-acidification compared to a plant-based fermented dairy alternative food product obtained using the same process as embodied in any of the preceding embodiments 1-50 but without the use of a protein deamidase and transglutaminase.

[0238] Embodiment 60. Use according to embodiment 59, wherein the plant-based fermented dairy alternative food product is an oat-based yogurt, such as a leguminous protein-fortified oatbased yogurt.

[0239] Embodiment 61. Use of a protein deamidase and a transglutaminase in the production of an oat-based fermented dairy alternative food product to improve texture, resilience to postacidification and / or water holding capacity.

[0240] Embodiment 62. Use according to embodiment 61 , wherein the oat-based fermented dairy alternative food product has been fortified with a leguminous protein, such a pea protein isolate, a faba protein isolate, a lentil protein isolate, or any combination thereof, before treatment with the protein deamidase and transglutaminase.

[0241] EXAMPLES

[0242] Throughout the examples, the terms “yogurt” and “milk” mean a plant-based yogurt alternative and milk alternative, respectively, unless otherwise specified. Materials

[0243] Enzymes

[0244] The following enzymes are used throughout the examples:

[0245] Protein deamidase’. Protein glutaminase derived from Chryseobacterium viscerum having the mature polypeptide sequence shown as SEQ ID NO:2. Cleavage of the propeptide was achieved by treating the deamidase of SEQ ID NO: 1 with a site-specific endopeptidase. The site-specific endopeptidase used is a glutamyl endopeptidase from Bacillus licheniformis. The resulting active deamidase after maturation was the polypeptide shown in SEQ ID NO: 2.

[0246] Transglutaminase'. Transglutaminase derived from Streptomyces mobaraensis having the polypeptide sequence of SEQ ID NO: 11.

[0247] Methods

[0248] Viscosity measurement

[0249] Viscosity was measured using Rapid Visco Analyzer (RVA) 4500 (Perten Instruments, Sweden). 30 g yoghurt sample was transferred into an RVA cup and allowed to set in the refrigerator for 1 hour before the measurement. The measurement was carried out at 25°C at 50 rpm for 130 sec. For simplicity, the viscosity reading after 70 seconds is presented.

[0250] Evaluation of spontaneous syneresis

[0251] 50 g fresh yoghurt sample was transferred into a 50 ml NUNC centrifuge tube and stored refrigerated for 16 days. The free whey separated on top of the yoghurts upon storage was weighed out and the amount was expressed as % of yoghurt weight.

[0252] Forced syneresis test

[0253] 25 g of yoghurt sample was centrifugated for 15 min at 3500 rpm (= 2643 x g) and the weight of remaining solid was recorded after removal of supernatant (whey). Amount of expelled whey is calculated using the formula: (Weight of yoghurt sample - weight of solid phase) / (weight of yoghurt sample) * 100%.

[0254] Visual evaluation

[0255] Visual evaluation was performed by an experienced technician following a standard procedure established for evaluating plant-based yoghurt analogues. The visual appearance of the yoghurt samples was evaluated by placing a yoghurt sample on the back side of black plastic spoons, where lumps or graininess and runny / thin texture could be easily observed.

[0256] The evaluated parameters are syneresis, shrinkage, flakiness, setting, firmness and cohesiveness. A score system from 1 to 7 was used to distinguish samples on each parameter. The definitions of visual evaluated parameters defined here are: • Syneresis - the water amount observed once the plastic jar is opened

[0257] • Shrinkage - the gap between the body of yoghurt and plastic jar

[0258] • Flakiness - flakes on the surface of the yoghurt body

[0259] • Setting - the shape and its change of a spoon of yoghurt sample which is spooned out from the plastic jar and put it on the table

[0260] • Firmness - the force sensed by assessor when he / she uses back of a spoon to press the spooned-out yoghurt sample on a table

[0261] • Cohesiveness - the deformation of macro-structure and integrity of the whole piece of yoghurt sample when the assessor uses back of a spoon to press the spooned-out yoghurt sample on a table. The more difficult to be deformed and the higher integrity of the edge of the sample, the higher cohesiveness.

[0262] • Homogeneity - the homogeneity after spreading out the pressed yoghurt sample from evaluation of firmness and cohesiveness.

[0263] Sensory evaluation

[0264] Sensory evaluation was carried out by 9 assessors that had participated in a basic sensory screening and were familiarized with evaluation of yoghurt texture. Samples were anonymized by 3-digit codes. The assessors were asked to rank the yogurts form thinnest to thickest, ties allowed. Data was collected and analyzed using EyeQuestion® and the statistical significance was analyzed using Friedman test (p<0.1 ).

[0265] Texture analyzer measurement

[0266] The stored yoghurt was measured on Texture Analyzer (TA. XT plus, Stable Micro System, UK) equipped with a 25 mm diameter acrylic cylinder probe. The yoghurt was measured immediately after it was taken out from 4°C storage condition. Adjusted Texture Profile Analysis (TPA) procedure was carried out at pretest speed 2 mm / s, test speed 2 mm / s, waiting time 5s and posttest speed 5 mm / s. Thickness (positive area integrated to peak force during first compression), adhesiveness (negative area between two compressions), stirring cohesiveness (adhesiveness divided by thickness) were calculated. The higher value of stirring cohesiveness, the higher homogeneity would be obtained after stirring due to higher cohesiveness during stirring. Measures were performed with duplicated samples.

[0267] Thixotropy measurements

[0268] Thixotropy testing was performed on the samples after three days of storage under cool conditions to determine the ability of the gel to recover, or lose viscosity, under shear measurement. Thixotropy is thus a valuable measurement to determine the ability of the sample to restructure itself following mechanical stress. The three interval thixotropy test was performed on the samples after three days of storage under cool conditions to determine the ability of the gel to recover, or lose viscosity, under shear measurement. The first, oscillatory, interval was performed at a strain of 0.1% and a frequency of 1 Hz for 120 s, followed by an interval of rotational deformation at a shear rate of 200 s-1 for 120 s. The third interval was performed at the same conditions as the first interval, but lasted for 300 s. Structure loss by this intense shearing was calculated as follows:

[0269] AG’ [%] = 100% - (GO end x 1OO%) / G’ start)

[0270] AG” [%] = 100% - (GOO end x 100%) / G” start)

[0271] Example 1 : Protein deamidase activity assay

[0272] The protein deamidase activity assay consists of two separate de-coupled parts:

[0273] 1) An enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and

[0274] 2) A non-enzymatic detection step wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm.

[0275] In step (1), the ammonia is developed by the deamidating action of the protein deamidase. In step (2), the generated ammonia reacts with phenol to form dioxyphenylamine under alkaline conditions. The reaction is catalyzed by sodium pentacyanonitrosylferrate(lll) (sodium nitroprusside). “Color Reagent solution A” contains phenol and sodium nitroprusside. “Color Reagent Solution B” provides alkaline reaction conditions. The intermediate is then oxidized by addition of sodium hypochlorite (“Color Reagent Solution C”) to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0276] Assay Procedure:

[0277] Step (1) Enzymatic step with ammonia formation

[0278] Reagents:

[0279] Assay dilution solution: 0.2 M Na-phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0280] Assay buffer: Same as above. Used to prepare stock solution and diluted sample of protein deamidase (referred to in the following as “enzyme”).

[0281] Substrate solution: 30 mM Z-GIn-Gly (Merck C6154-1G) in assay dilution solution (check pH after dissolution).

[0282] Stop solution: 0.4M TCA. Standard: NH4CI (Ammonium Standard for IC, Merck 59755-100ML, 1000 mg / L NH4+in water) diluted in assay dilution solution (see also “Standard curve” section).

[0283] Dissolve / dilute enzyme product in assay buffer and prepare suitable dilution resulting in a linear assay response.

[0284] Incubation:

[0285] 1. Add 10 pL of diluted enzyme samples in triplicates to the wells of a 96-well microtiter plate (MTP).

[0286] 2. Add 100 pL of substrate solution to each well.

[0287] 3. For blank samples add 100 pL 0.4M TCA solution.

[0288] 4. Seal the plate using transparent plate sealer.

[0289] 5. Incubate the plate for 10 minutes at 37°C, 500 rpm, on a thermomixer equipped with a lid heating function.

[0290] 6. To stop the reaction, carefully add 100 pL 0.4M TCA solution (except for the blank samples, which already contain TCA).

[0291] Total reaction volume: 210 pL

[0292] Step (2) Ammonia detection step

[0293] Reagents:

[0294] Color reagent A: 4% (w / v) Phenol, 0.015% (w / v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO] 2H2O).

[0295] Color reagent B: 5% (w / v) Potassium hydroxide.

[0296] Color reagent C: 28% (w / v) Potassium carbonate, 6% (v / v) sodium hypo-chlorite (Sigma- Aldrich 239305-25ml, < 5% available CI2).

[0297] Incubation:

[0298] 1. Transfer 15 pL from each well from step (1) into a new 96-well MTP.

[0299] 2. Transfer 45 pL Milli-Q water to each well.

[0300] 3. To each well, add 30pL of color reagent B (on lab table, shake gently by hand to mix).

[0301] 4. To each well, add 60pL of color reagent A (on lab table, shake gently by hand to mix).

[0302] 5. To each well, add 60pL of color reagent C (on lab table, shake gently by hand to mix).

[0303] 6. Color development: Carefully seal the plate and leave it on lab table for 30 minutes.

[0304] 7. Carefully transfer the MTP to a plate reader and measure absorbance at 630 nm.

[0305] Total reaction volume: 210 pL

[0306] Standard curve:

[0307] Standard stock solution: 1000 mg NH4+ / L. The standard curve is prepared by adding dilutions of the ammonium standard in the assay dilution buffer in the ammonia detection step. That is, mixing 15 pL diluted ammonia standard with 45 pL water and then add the color reagents in the order given above; B, A, and C.

[0308] The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)): which can be shortened to , mlL180.04 ml * min. where

[0309] • CNH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

[0310] • 18.04 is the molecular mass of ammonium used for the standard solution.

[0311] • Vreaaion is the reaction volume in the well when ammonia is generated (210 pL).

[0312] • V enzyme is the volume of enzyme solution added to the well when ammonia is generated

[0313] (10 pL).

[0314] • VNH3 detection is the reaction volume in the well when ammonia is detected (210 pL).

[0315] Example 2: Process of producing a faba-based yogurt with improved textural properties in laboratory scale

[0316] Materials:

[0317] Faba protein isolate (AGT foods) containing about 80% by weight of protein dispersed in water to which rapeseed oil had been added to a fat content of about 2% by weight; yeast extract (0.45%); granulated sugar; ABY-3 yogurt culture (Chr. Hansen); Transglutaminase (TG); and Protein Deamidase (PD).

[0318] Methods:

[0319] Faba protein isolate, sugar and yeast extract was added to water to a final protein content of 3.5% in the milk. The mixture was stirred for 45 minutes at 40°C. Pre-emulsification was done by incorporating the rapeseed oil, while applying high shear for 10 minutes, after which the mixture was homogenized in a lab homogenizer (GEA, Panda Plus) at 500 bars. This mixture was then called ‘milk’ and was used as a base for fermentation.

[0320] Variety 1: Pre-treatment of the milk with protein deamidase.

[0321] Protein deamidase solution (3.7 - 7.5 IPA(U) / g protein) was added to the milk. The mixture was heated to 60°C and held for 30 minutes while mixing. After 30 minutes the mixture was heated to 90°C and held for 10 minutes to terminate the reaction and inactivate the enzymes, after which the mixture was cooled rapidly on melting ice. The obtained milk was either inoculated with the yoghurt culture the same day or kept in the fridge for a maximum of one (1) night before inoculation. At inoculation, the milk was brought to a temperature of 43°C after which it was inoculated with the yoghurt culture and 0.5 TGHU(A) / g protein of transglutaminase was added to the milk. The milk was incubated at 43°C for 6 hours or until the pH reached 4.5, at which point a yoghurt was obtained. The obtained yogurt was stirred after fermentation by applying low shear for 1 minute and the samples were then stored at cool conditions for 5-7 days until evaluation.

[0322] Variety 2: Protein deamidase added simultaneous with yoghurt culture and transglutaminase.

[0323] The mixture was heated to 90°C and held for 10 minutes to pasteurize the milk, after which the mixture was cooled rapidly on melting ice. The obtained milk was either inoculated the same day or kept in the fridge for a maximum of one (1) night. At inoculation, the milk was brought to 43°C after which it was inoculated with yoghurt culture and 0.5 TGHU(A) / g protein of transglutaminase and the protein deamidase solution (tested at two different concentrations, 3.7 - 7.5 IPA(U) / g protein) was added. The milk was incubated at 43°C for 6 hours or until the pH reached 4.5, at which point a yoghurt was obtained. The yogurt was stirred after fermentation by applying low shear for 1 minute and the samples were then stored cool for 5-7 days until evaluation.

[0324] The yogurts obtained according to Variety 1 and 2 were evaluated for viscosity and hardness. The value for hardness was obtained by performing a Texture Profile Analysis (TPA) on the yogurts. Viscosity was measured using a Rapid Viscosity Analyzer (RVA). The results are illustrated in Table 1.

[0325] Table 1 Viscosity and hardness of faba protein isolate yogurt prepared with or without transglutaminase and / or protein deamidase (low and high referring to the concentration of the protein deamidase). PD, protein deamidase; TG, transglutaminase.

[0326] The results shown in Table 1 clearly confirm that the combination of transglutaminase and protein deamidase increases the viscosity and hardness of 3.5% protein plant-based yogurt, irrespective of whether the protein deamidase has been added as a pre-treatment (Variety 1) or during fermentation (Variety 2).

[0327] Example 3: Variety in plant protein functionality

[0328] A selection of different plant protein isolates was tested to show the solutions performance across protein from various sources and different suppliers. The nature of the protein isolates can influence the gelling and emulsifying properties and thus also the characteristics of a plant-based yogurt made from such an isolate. The yogurts were evaluated for viscosity and hardness using the same methods as described in Example 2. The results are illustrated in Table 2.

[0329] Table 2 Viscosity and hardness of yogurts prepared from different plant-based protein isolate and with or without transglutaminase and protein deamidase (low and high referring to the concentration of the protein deamidase). PD, protein deamidase; TG, transglutaminase.

[0330] As seen from Table 2, it was shown firstly that variations in raw materials of the plant protein source affects the plant-based yogurt texture parameters, such as viscosity and hardness. However, the enzymatic solution can for all raw materials, all be it not to the same extent, increase the viscosity and the hardness of plant-based yogurts. This confirms the robustness and versatility of the processes disclosed herein.

[0331] Example 4: Effect of enzymatic solution on resilience against deformation

[0332] During their manufacture, some yogurts undergo a further treatment of stirring and pumping to break down the protein network. Such treatment results in a smooth and homogeneous yogurt which can be poured into a cup or carton. However, too harsh pumping results in total structure loss in the yogurt which have a low gel strength due to a weak protein network.

[0333] Thixotropy is a rheological method which can be used to evaluate the resilience of a gel, such as that of yoghurt, against deformation. First, the loss modulus (G’) and the storage modulus (G”) are measured by oscillation, then a rotational shear is applied at high speed, and then a second oscillation measurement is applied. The difference between the loss modulus and storage modulus before and after the rotational shear can be expressed as the percentage of structure loss (% structure loss) in the gel.

[0334] A thixotropy test was applied to faba protein isolate yogurt samples prepared according to Example 2 and the results from the test are shown in Table 3.

[0335] Table 3 Results of thixotropy measurements on faba protein isolate yogurt prepared with or without transglutaminase and / or protein deamidase (low and high referring to the concentration of the protein deamidase). PD, protein deamidase; TG, transglutaminase.

[0336] As can be seen from the results shown in Table 3, the samples treated with a combination of the transglutaminase and protein deamidase had a lower AG’ and AG”, i.e., lower percentage of structure loss, compared to the non-treated sample (‘No enzyme’). These results indicate that the samples treated with transglutaminase and protein deamidase were more resilient to deformation by high shear than non-enzymatically treated samples. This can be a great advantage in large scale yogurt production, in which the treatment of stirring and pumping to break down the protein network is of a key process step to achieve a desired final texture of the yogurt.

[0337] Example 5: Sensory evaluation of a pea-based dairy alternative yogurt

[0338] Dairy-alternative yogurts were evaluated by a sensory panel for textural attributes. The dairy-alternative yogurts were produced according to the method of Example 2, except Faba protein isolate was exchanged with pea protein isolate (PPI, BULK). The sensory panel consisted of nine trained panelists who were screened and trained in the evaluation of dairy and plant-based dairy alternative food products. Evaluation occurred on days 10 and 12 after production of the dairy-alternative yogurt. Yogurts treated with transglutaminase and protein deamidase in different setups (deamidase in two concentrations; deamidase added during fermentation or pre-treatment, i.e., in total six samples) were compared in terms of perceived thicknesses. The panelists were asked to rank the yogurts from thinnest to thickest with ties allowed. Data was collected and analyzed using EyeQuestion® and the statistical significance was analyzed using Friedman test (p<0.1). The results are shown in Table 4. The Rank Sum Thickness is the sum of the rank given to each sample by the different panelists.

[0339] Table 4 Rank Sum Thickness and viscosity of yogurts prepared from pea protein isolate and with different concentrations and addition times of protein deamidase (low and high referring to the concentration of the protein deamidase). PD, protein deamidase; TG, transglutaminase. The results depicted in Table 4 show that the sample prepared without transglutaminase and protein deamidase (‘No Enzymes’) stands out as the sample being significantly less thick (Rank Sum Thickness of 16, p=0.049) compared to the other yogurt samples included in the sensory evaluation. All other yogurts were perceived to have a similar thickness (Rank Sum Thickness values of 33-38.5) and were not significantly different from each other in terms of viscosity and overall appearance. These results clearly show that the yogurts treated with a combination of protein deamidase and transglutaminase do not only have textural benefits that can be measured with instruments, but are also perceived as significantly thicker by the consumer.

[0340] Example 6: Effect of enzymatic solution on post-acidification in plant-based yogurt

[0341] Post-acidification is the drop in pH which occurs in a finished fermented product, such as a plant-based yogurt, during packaging, storage, and distribution. The drop in pH can be up to 0.5 pH units. It is preferable to keep such drop in pH to a minimum to avoid the fermented food products becoming too sour which can be undesirable for the consumer. In this example, the pH of the tested plant-based yogurt was measured at the point where fermentation was stopped (for all yogurts about pH 4.5), and again after 7 days of storage. The results for a pea- protein-fortified oat-based yogurt (total protein content 4.5% (w / w)) are shown in Table 5 below.

[0342] Table 5 Post-acidification of a pea protein-fortified oat yogurt (total protein content 4.5% (w / w)) following 7 days’ storage. PD, protein deamidase; TG, transglutaminase.

[0343] As seen from the results shown at least in Table 5, protein deamidase alone and in combination with transglutaminase decreases post-acidification more significantly than if transglutaminase was used alone or when compared to a yogurt sample which was not enzyme treated.

[0344] Example 7: Effect of enzymatic solution on water holding capacity of plant-based yogurt

[0345] Water holding capacity (WHC), or syneresis, is the expelling of liquid from the yogurt matrix, causing a visible layer of aqueous liquid on top of the yogurt gel. Syneresis is known to increase over shelf-life and is an important quality aspect of yogurts and plant-based yogurt alternatives. Consumers can perceive this as a negative aspect and may believe that the yogurt has expired or “gone bad”, and will discard it.

[0346] Water holding capacity was measured by the forced-syneresis method, in which a yogurt sample was exposed to centrifugal force, and the expelled water discarded. Then the WHC was calculated by expressing the yogurt gel left as a percentage of the initial weight. As seen from the data presented at least in Table 6, WHC or syneresis is highly influenced by the enzymatic solution offered with the present invention. In particular, it was confirmed that the combination of protein deamidase and transglutaminase was effective in increasing water holding capacity of the obtained plant-based yogurt included in the study. The results for a pea- protein-fortified oat-based yogurt (total protein content 4.5% (w / w)) is shown in Table 6 below.

[0347] Table 6 Water holding capacity (WHC) of pea protein-fortified oat yogurt (total protein content 4.5% (w / w)). PD, protein deamidase; TG, transglutaminase

Claims

CLAIMS1. A process for preparing a plant-based fermented dairy alternative food product, the process comprising:(a) treating a plant-based substrate with a protein deamidase;(b) treating the plant-based substrate of step (a) with a transglutaminase; and(c) fermenting the plant-based substrate of step (b) by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative food product; wherein step (a) is performed before and / or during step (b), and step (b) is performed before and / or during step (c), and wherein the fermented dairy alternative food product has increased viscosity compared to a fermented dairy alternative food product obtained using the same process but without addition of protein deamidase and transglutaminase.

2. The process of claim 1 , wherein the plant-based substrate is derived or obtained from almond, cashew, chickpea, coconut, faba bean, flax, hemp, lentil, lupin, macadamia, mung bean, oat, pea, peanut, pecan, pistachio, rice, soy, or any combination thereof, such as from faba bean, lentil, pea, oat or any combination thereof.

3. The process of any of the preceding claims, wherein the plant-based substrate is(i) a plant-based milk alternative, such as a soy beverage, a pea beverage, a lentil beverage, a faba bean beverage or an oat beverage, optionally fortified with a plantbased milk alternative powder, such as a pea milk powder, or with a concentrated or isolated protein, such as a leguminous protein isolate or concentrate; or(ii) an aqueous solution or suspension of a plant-based milk alternative powder, such as a pea milk powder or a faba bean milk powder, or of a plant protein isolate or concentrate, such as a leguminous protein isolate or concentrate, preferably a soy protein isolate, soy protein concentrate, pea protein isolate, pea protein concentrate, faba protein isolate, faba protein concentrate, lentil protein isolate or lentil protein concentrate.

4. The process of any of the preceding claims, wherein the plant-based substrate has a protein content of 1-8% (w / w), such as 2-6% (w / w).

5. The process of any of the preceding claims, wherein the plant-based substrate has a dietary fiber content below 5% (w / w), such as a dietary fiber content in the range of 0.1-3% (w / w), such as 0.5-2% (w / w).

6. The process of any of the preceding claims, wherein the treatment of step (a) is carried out at a temperature in the range of 20-60°C, such as in the range of 40-60°C.

7. The process of any of the preceding claims, wherein pasteurization is performed after step (a) and before step (b), and / or wherein step (b) and step (c) are performed simultaneously.

8. The process of any of the preceding claims, further comprising the addition of a lipid during step (a) or (b), or after step (b), optionally wherein the plant-based substrate or the plant-based fermented dairy alternative food product has a lipid content of at most 10% (w / w), such as at most 5% (w / w).

9. The process of any of the preceding claims, wherein the protein deamidase is derived from or obtained from a Chryseobacterium species, preferably from Chryseobacterium proteolyticum or Chryseobacterium viscerum.

10. The process of any of the preceding claims, wherein the transglutaminase is a bacterial endopeptidase, preferably derived from Streptomyces, more preferably from Streptomyces mobaraensis.

11. The process of any of the preceding claims, wherein the plant-based substrate is incubated with a lactic acid bacterium, preferably of the genus Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Propionibacterium, Enterococcus, Brevibacterium, Bifidobacterium or any combination thereof.

12. The process of any of the preceding claims, wherein the plant-based fermented dairy alternative food product is a spoonable plant-based fermented dairy alternative food product, such as a stirred yoghurt alternative, a set-type yoghurt alternative, or a strained yoghurt alternative, or a drinkable plant-based fermented dairy alternative food product, such as a drinking yoghurt alternative or a kefir alternative.

13. The process of any of the preceding claims, wherein a. the plant-based fermented dairy alternative food product has a more smooth and less grainy texture compared to a plant-based fermented dairy alternative food product obtained using the same process but without treating with the protein deamidase and transglutaminase, wherein the texture is visually evaluated by placing a sample of the plant-based fermented dairy alternative food product on the backside of a black plastic spoon; and / or b. the plant-based fermented dairy alternative food product has a higher resilience against deformation as measured using a rheological method to quantify thixotropycompared to a plant-based fermented dairy alternative food product obtained using the same process but without addition of the protein deamidase and transglutaminase, wherein the rheological method to quantify thixotropy is performed using a method as exemplified in Example 4.

14. A plant-based fermented dairy alternative food product obtainable by the process as claimed in any of the preceding claims.

15. Use of a protein deamidase and a transglutaminase in the production of a plant-based fermented dairy alternative food product to improve texture and / or stability of the plant-based fermented dairy alternative food product, such as to increase viscosity.

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