PROCESS FOR PREPARING A PLANT-BASED FERMENTED DAIRY ALTERNATIVE

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer reada ble form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to preparation of a plant-based fermented dairy alternative where the plant-based substrate is treated with an enzyme.

BACKGROUND OF THE INVENTION

There is an increasing consumer demand for plant-based alternatives to animal-based tradi tional foods such as meat and dairy products.

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 yoghurt alternatives, e.g., so- called soy yoghurt, have appeared as an interesting alternative to traditional animal yoghurts also because of their reduced level of cholesterol and saturated fat and because they are free of lactose.

Therefore, there is a huge commercial interest in providing plant-based dairy alternatives such as plant-based fermented dairy alternatives.

It is an object of the present invention to improve the quality of plant-based fermented dairy al ternatives such as yoghurt alternatives produced from soy, pea or other legumes.

WO2018/049853A1 discloses production of a fermented dairy milk product wherein a small amount of a proliferating agent produced from hydrolysed soy protein is added to dairy milk prior to fermentation.

WO2010/033985 discloses production of a frozen confection composition by mixing a protein hydrolysate, e.g. a soy protein hydrolysate or a combined soy/dairy protein hydrolysate, and an edible material, e.g. yoghurt, and freezing the composition.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that in the production of plant-based fermented dairy alternatives such as yoghurt alternatives, treatment of the plant-based substrate from which the fermented dairy alternative is produced with an endopeptidase improves the quality of the product. The treatment with the endopeptidase may be performed either as a pre-treatment step prior to fermentation, or it may be performed essentially at the same time as the fermenta tion.

Treatment with an endopeptidase resulted in quality improvements such as decreased synere- sis, decreased viscosity or both - properties which can be inversely correlated in fermented dairy products produced from milk. Further, the visual appearance was improved, and a less grainy, less lumpy and/or smoother texture was observed.

For fermented dairy alternatives, an additional benefit may be a faster fermentation time. Such processing benefit can be used to increase the production capacity and it also reduces the risk of contamination since the fermented dairy alternatives are exposed for a shorter time to neutral pH and raised temperatures.

The present invention therefore relates to a process for preparing a plant-based fermented dairy alternative, the process comprising:

(a) treating a plant-based substrate with an endopeptidase; and

(b) fermenting the plant-based substrate by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative; wherein step (a) is performed before and/or during step (b).

The present inventors have further found that inclusion of a phospholipase treatment resulted in an even more creamy and smooth texture and an even more appealing appearance. It resulted in improved parameters such as high cohesiveness and homogeneity which are important for the mouthfeel of the resulting product. Inclusion of a phospholipase treatment may also be used to increase the viscosity compared to treatment with an endopeptidase alone, thus enabling a tailoring of the viscosity to fit the desired product.

Therefore, in a preferred embodiment, the plant-based substrate is further treated with a phos pholipase before, during or after step (a) and before or during step (b).

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the visual appearance of reference pea-yogurt 10% (from Example 3)

Fig. 2 shows the visual appearance of pea-yogurt 10% with 100 KPRU TL1 (from Example 3)

Fig. 3 shows the visual appearance of pea-yogurt 10% with 450 KPRU TL1 (from Example 3)

Fig. 4 shows the effects of TL1 and Galaya Enhance on soy set yoghurt 7% protein (from Ex ample 10). From left to right is seen soy set yoghurt produced with TL1 0.4, GE 0.52, TL1 0.4 + GE 0.52, Control. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a process for preparing a plant-based fermented dairy alter native, the process comprising:

(a) treating a plant-based substrate with an endopeptidase; and

(b) fermenting the plant-based substrate by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative; wherein step (a) is performed before and/or during step (b).

The plant-based substrate may be obtained from any plant, such as legumes, cereals (e.g. wheat, oats), pseudocereals (e.g. quinoa), grasses, pasture legumes (e.g. alfalfa, clover), rape- seed, nuts, almonds, vegetables, fruits, mushrooms, cottonseed, or any combination thereof.

The plant-based substrate may be obtained from more than one plant.

In a preferred embodiment, at least part of the plant-based substrate is obtained from legumes, such as from pulses (e.g. peas, lentils, faba bean, chickpea) or from oil crops (e.g. soybean, peanuts). In a more preferred embodiment, the plant-based substrate is obtained from soy, pea, chickpea, mung bean, lentils, faba bean or lupin, preferably from soy, pea, faba bean or lentils.

The plant-based substrate may be a plant-based milk alternative, such as soy milk or soy bev erage, optionally fortified with a plant-based milk alternative powder such as soy milk powder or with concentrated or isolated protein such as soy protein isolate or soy protein concentrate. Or the plant-based substrate may be another plant-based milk alternative, such as coconut milk, oat milk or almond milk, preferably coconut milk, fortified with soy milk powder or with concen trated or isolated legume protein, preferably with soy protein, pea protein, lentil protein or faba bean protein, preferably in the form of an isolate or a concentrate. Or the plant-based substrate may be an aqueous solution or suspension of a plant-based milk alternative powder such as soy 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, prefera bly a legume protein isolate or legume protein concentrate, more preferably a soy protein iso late, a soy protein concentrate, a pea protein isolate, or a pea 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 above.

The plant-based substrate may be oat milk, coconut milk, almond milk or another plant-based milk alternative, optionally fortified with plant protein, preferably legume protein, e.g., in the form of a flour, an isolate or a concentrate. Or it may be a plant-based milk alternative, such as, e.g., almond milk, which has been concentrated to increase the protein content.

In a preferred embodiment, the plant-based substrate is soy milk or soy beverage, optionally fortified with soy milk powder or with soy protein isolate or soy protein concentrate. In another preferred embodiment, the plant-based substrate is an aqueous solution or suspension of soy milk powder. In another preferred embodiment, the plant-based substrate is an aqueous solu tion or suspension of a soy protein isolate, a soy protein concentrate, a pea protein isolate, or a pea protein concentrate.

In another preferred embodiment, the plant-based substrate is (i) soy milk or soy beverage, op tionally fortified with soy milk powder or with soy protein isolate or soy protein concentrate, or (ii) an aqueous solution or suspension of soy milk powder, soy protein isolate, soy protein concen trate, pea protein isolate, pea protein concentrate, or any combination thereof.

In another preferred embodiment, the plant-based substrate is (i) soy milk or soy beverage, op tionally fortified with soy milk powder or with concentrated or isolated legume protein, (ii) anoth er plant-based milk alternative, such as coconut milk, oat milk or almond milk, preferably coco nut milk, fortified with soy milk powder or with concentrated or isolated legume protein, or (iii) an aqueous solution or suspension of soy milk powder or of isolated or concentrated legume pro tein. The legume protein is preferably soy protein, pea protein, lentil protein or faba bean pro tein, preferably in the form of an isolate or a concentrate.

The plant-based substrate may be obtained from more than one plant, such as, e.g., soy milk alternative fortified with, e.g., pea protein, or coconut milk, oat milk or almond milk fortified with, e.g., pea protein or soy protein.

Preferably, the plant-based substrate has a protein content of at least 2% (w/w). In one embod iment, the plant-based substrate has a protein content of at least 3% (w/w). In another embodi ment, the plant-based substrate has a protein content of at least 5% (w/w).

Preferably, the plant-based substrate has a protein content of at most 12% (w/w), more prefera bly at most 10% (w/w).

Preferably, the plant-based substrate is 100% plant-based.

Preferably, all of the protein in the plant-based substrate is plant protein.

Preferably, at least 90% (w/w), preferably at least 95% (w/w), more preferably all of the protein in the plant-based fermented dairy alternative is plant protein.

Preferably, the protein in the plant-based substrate constitutes at least 50% (w/w), preferably at least 80% (w/w), more preferably at least 90% (w/w), even more preferably at least 95% (w/w), such as 100%, of the protein in the plant-based fermented dairy alternative.

Preferably, the plant-based substrate which has been treated with the endopeptidase and fer mented by incubating with a lactic acid bacterium constitutes at least 50% (w/w), preferably at least 80% (w/w), more preferably at least 90% (w/w), even more preferably at least 95% (w/w), such as 100%, of the plant-based fermented dairy alternative. Other ingredients may be added to the plant-based substrate, e.g., oil, such as plant oil, sugar, sucrose, fruit, yeast extract and/or peptone. Plant oil may be added to provide fat to the plant- based fermented dairy alternative. Sugar, sucrose or fruit may be added to sweeten the plant- based fermented dairy alternative. Yeast extract or peptone may be added to speed up fermen tation.

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

A plant-based fermented dairy alternative in the context of the present invention is a plant- based 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.

Fermentation is performed by incubating with a lactic acid bacterium, preferably of the genus Streptococcus, Lactococcus, Lactobacilllus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Propionibacterium, Enterococcus, Brevi bacterium, or Bifidobacterium or any combination there of.

In one embodiment, fermentation is performed by incubating with a thermophilic lactic acid bac terium.

In one embodiment, fermentation is performed by incubating with a mesophilic lactic acid bacte rium.

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

In a preferred embodiment, the plant-based 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, a skyr alternative or a cream cheese 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 ingre dients 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.

A set yoghurt alternative may be fermented in retail container and not agitated after fermenta tion. 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.

The term "after fermentation" as used herein means when fermentation is ended and the de sired pH obtained. 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 distinc tive sour taste.

The pH after fermentation may preferably be between 3.5 and 5.5, most preferably between 4 and 5.

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

In one embodiment, the plant-based fermented dairy alternative is cooled, preferably immedi ately.

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 ap proximately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

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 approxi mately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

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 approximately 5°C by blast chilling in cooling tunnels or slower in a refrigerated storage room.

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), pref erably after both. Storage may be carried out at a low temperature, preferably less than 10°C, more preferably 0-10°C, such as 4-6°C.

In a preferred embodiment, the plant-based fermented dairy alternative is a spoonable plant- based fermented dairy alternative, such as a stirred yoghurt alternative, a set-type yoghurt al ternative or a strained yoghurt alternative, or a drinkable plant-based fermented dairy alterna tive, such as a drinking yoghurt alternative or a kefir alternative.

In a more preferred embodiment, the plant-based fermented dairy alternative is a spoonable plant-based fermented dairy alternative, preferably a spoonable yoghurt alternative.

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 fermen- tation. Pasteurization before fermentation may also give a better structure of the plant-based fermented dairy alternative.

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.

In step (a), the plant-based substrate is treated with an endopeptidase. Step (a) may be per formed before and/or during step (b).

In the process 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 enzymes prior to the fer mentation. A pasteurization step may be performed before step (a) and another one after step (a) but before step (b).

In the process of the invention, step (a) may be performed before and during step (b). I.e., the endopeptidase may be added to the plant-based substrate and after incubation for some time, e.g., 0.5-20 hours, the lactic acid bacterium is added and the incubation is continued until the desired pH is reached.

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

In a preferred embodiment, step (a) is performed before and during step (b) and a pasteuriza tion step is performed before step (a).

In another preferred embodiment, step (a) and step (b) are performed simultaneously, i.e., the endopeptidase and the lactic acid bacterium are added at the same time or essentially at the same time, and a pasteurization step is performed before step (a).

If step (a) is performed before step (b), the enzyme treatment may be performed, e.g., at 40- 55°C, such as at 45-55°C, for 15 minutes to 10 hours, such as for 30 minutes to 3 hours.

If step (a) is performed before step (b), the enzyme treatment may be performed, e.g., at 4- 10°C, such as at 4-6°C, for 3 hours to 20 hours, such as for 5 to 15 hours.

The fermentation in step (b) 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 fer mentation 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.

In a preferred embodiment, the viscosity of the plant-based fermented dairy alternative is re duced by at least 25%, preferably at least 40%, compared to a plant-based fermented dairy al ternative prepared by the same process but without addition of a endopeptidase. The viscosity may be determined after six days storage at 4°C by allowing a sample of the plant-based fer- merited 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.

Reduction in viscosity is often desired, in particular for fermented dairy alternatives having a high protein content. For fermented dairy alternatives having a low protein content, a reduction in viscosity may not be desired.

In a preferred embodiment, the plant-based fermented dairy alternative expels at least 10%, preferably at least 20%, less liquid in a forced syneresis test compared to a plant-based fer mented dairy alternative prepared by the same process but without addition of an endopepti- dase. The forced syneresis test may be performed after six days storage at 4°C by centrifuga tion of the plant-based fermented dairy alternative for 15 min at 2643 x g. The weight of remain ing solid is recorded after removal of supernatant and the amount of expelled liquid is calculated using the formula: (weight of fermented dairy alternative sample - weight of solid phase)/(weight of fermented dairy alternative sample) * 100%.

In one embodiment, a hydrocolloid or stabilizer such as pectin is added to the plant-based fer mented dairy alternative, in which case addition of an endopeptidase will likely not result in fur ther reduction of syneresis, since syneresis will already be very low. However, treatment with an endopeptidase will still confer other benefits. In another embodiment, no hydrocolloid or stabi lizer is added to the plant-based fermented dairy alternative. In another embodiment, no pectin is added to the plant-based fermented dairy alternative. From a clean-label perspective, avoid ance of hydrocolloid or stabilizer such as pectin is preferred.

In a preferred embodiment, the plant-based fermented dairy alternative has a smoother texture compared to a plant-based fermented dairy alternative prepared by the same process but with out addition of an endopeptidase. The texture may be visually evaluated after six days storage at 4°C by placing a sample of the plant-based fermented dairy alternative on the backside of a black plastic spoon.

In a preferred embodiment, the plant-based fermented dairy alternative has a less grainy texture compared to a plant-based fermented dairy alternative prepared by the same process but with out addition of an endopeptidase. The texture may be visually evaluated after six days storage at 4°C by placing a sample of the plant-based fermented dairy alternative on the backside of a black plastic spoon.

In a preferred embodiment, the plant-based substrate is fermented with a lactic acid bacterium in step (b) and the plant-based fermented dairy alternative is a plant-based fermented dairy al ternative. Preferably in such process according to the invention, the fermentation time until the desired pH is reached is reduced by at least 10%, more preferably at least 20%, compared to the same process but without addition of an endopeptidase. In a preferred embodiment of the process of the invention, the plant-based substrate is further treated with a phospholipase before, during or after step (a) and before or during step (b).

The treatment with the endopeptidase and the phospholipase may be performed sequentially. E.g., the phospholipase may be added to the plant-based substrate, which has optionally been pasteurized, and after some time, such as, e.g., 30-60 minutes, the endopeptidase is added. Or the treatment with the phospholipase may be performed first, optionally followed by a pasteuri zation step, and then the endopeptidase is added, e.g., at the same time as the lactic acid bac terium.

Alternatively, the endopeptidase may be added to the plant-based substrate, which has option ally been pasteurized, and after some time, such as, e.g., 30-60 minutes, the phospholipase is added. Or the treatment with the endopeptidase may be performed first, optionally followed by a pasteurization step, and then the phospholipase is added, e.g., at the same time as the lactic acid bacterium.

Alternatively, both enzymes and the lactic acid bacterium may be added at the same time or essentially at the same time. Or the phospholipase may be added first, then the endopeptidase, then the lactic acid bacterium. Or the phospholipase may be added first, then the lactic acid bacterium, then the endopeptidase. Or the endopeptidase may be added first, then the phos pholipase, then the lactic acid bacterium. Or the endopeptidase may be added first, then the lactic acid bacterium, then the phospholipase. Or the lactic acid bacterium may be added first, then the enzymes.

In a preferred embodiment, the plant-based substrate is treated with the phospholipase before step (a). In another preferred embodiment, the plant-based substrate is treated with the phos pholipase followed by pasteurization before step (a). In another preferred embodiment, the plant-based substrate is treated with the phospholipase followed by pasteurization before step (a), and step (a) and step (b) are performed simultaneously.

Endopeptidase

In the process of the invention, a plant-based substrate is treated with an endopeptidase.

In a preferred embodiment, the endopeptidase is a specific endopeptidase.

A specific endopeptidase may be defined as an endopeptidase having a preference, preferably a strong preference, for cleaving before or after one or two specific amino acids. The skilled person will know whether a certain endopeptidase is specific or not.

In a preferred embodiment, the specific endopeptidase has a preference for cleaving before or after, preferably after, a non-hydrophobic amino acid.

In a preferred embodiment, the endopeptidase is selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; and ii) a variant of the polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 comprising a substitution, deletion, and/or insertion at one or more positions.

In a more preferred embodiment, the endopeptidase is selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to any of SEQ ID NOs: 1, 13 or 14; and ii) a variant of the polypeptide of any of SEQ ID NOs: 1, 13 or 14 comprising a substitution, dele tion, and/or insertion at one or more positions.

The endopeptidase may be a trypsin-like endopeptidase, preferably a trypsin-like endopepti dase selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; and ii) a variant of the polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 com prising a substitution, deletion, and/or insertion at one or more positions.

A trypsin-like endopeptidase is an endopeptidase having specificity for cleaving after Lys and/or Arg.

In a more preferred embodiment, the endopeptidase is a trypsin-like endopeptidase selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably 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: 1; and ii) a variant of the polypeptide of SEQ ID NO: 1 comprising a substitution, deletion, and/or inser tion at one or more positions.

The trypsin-like endopeptidase is preferably derived from a strain of Fusarium, more preferably from Fusarium oxysporum.

The endopeptidase may be a lysine-specific endopeptidase, preferably a lysine-specific endo peptidase selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably 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: 13; and ii) a variant of the polypeptide of SEQ ID NO: 13 comprising a substitution, deletion, and/or in sertion at one or more positions.

The lysine-specific endopeptidase is preferably derived from a strain of Achromobacter, more preferably from Achromobacter lyticus.

The endopeptidase may be a glutamyl-specific endopeptidase, preferably a glutamyl-specific endopeptidase selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably 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: 14; and ii) a variant of the polypeptide of SEQ ID NO: 14 comprising a substitution, deletion, and/or in sertion at one or more positions.

The glutamyl-specific endopeptidase is preferably derived from a strain of Bacillus, more prefer ably from Bacillus licheniformis.

The endopeptidase may be a proline-specific endopeptidase.

For purposes of the present invention, the sequence identity between two amino acid 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 ai, 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 labeled “longest identity” is calculated as follows:

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

In the context of the present invention, a trypsin-like endopeptidase is an endopeptidase which specifically cleaves on the carboxy terminal side of arginine and/or lysine. I.e., it specifically cleaves on the carboxy terminal side of arginine or lysine or both. In a preferred embodiment, the trypsin-like endopeptidase specifically cleaves on the carboxy terminal side of arginine and lysine.

In the context of the present invention, a lysine-specific endopeptidase is an endopeptidase which specifically cleaves on the carboxy terminal side of lysine. A lysine-specific endopepti dase may also be termed a lysyl-specific endopeptidase.

Preferably, the trypsin-like or lysine-specific endopeptidase has a specificity for cleaving after Arg or Lys (whichever is the larger) which is at least 100-fold higher than its specificity for cleav ing after any one of Ala, Asp, Glu, lie, Leu, Met, Phe, Tyr or Val (whichever is the larger).

In an embodiment, the trypsin-like or lysine-specific endopeptidase has a specificity for cleaving after Arg or Lys (whichever is the larger) which is at least 10-fold, such as at least 20-fold or at least 50-fold, higher than its specificity for cleaving after any one of Ala, Asp, Glu, lie, Leu, Met, Phe, Tyr or Val (whichever is the larger). In another embodiment, the trypsin-like or lysine- specific endopeptidase has a specificity for cleaving after Arg or Lys (whichever is the larger) which is at least 200-fold, such as at least 500-fold or at least 1000-fold, higher than its specifici ty for cleaving after any one of Ala, Asp, Glu, lie, Leu, Met, Phe, Tyr or Val (whichever is the larger).

Preferably, such determination of specificities should be performed at a pH-value where the ac tivity of the endopeptidase is at least half of the activity of the endopeptidase at its pH optimum. Preferably, any such relative specificities are to be determined using Suc-AAP-X-pNA sub strates as described in Example 3 of WO 2008/125685 which is incorporated by reference.

In the context of the present invention, a glutamyl-specific endopeptidase is an endopeptidase which has a strong preference for glutamic acid in the P1 position and which releases peptides with a glutamic acid in the C-terminal.

In an embodiment, the glutamyl-specific endopeptidase has a specificity for cleaving after Glu which is at least 10-fold, such as at least 20-fold or at least 50-fold, higher than its specificity for cleaving after any one of Ala, Arg, Asp, lie, Leu, Lys, Met, Phe, Tyr or Val (whichever is the larger).

Preferably, a trypsin-like endopeptidase to be used in the process of the invention is classified in EC 3.4.21.4.

Preferably, a lysine-specific endopeptidase to be used in the process of the invention is classi fied in EC 3.4.21.50.

Preferably, a glutamyl-specific endopeptidase to be used in the process of the invention is clas sified in EC 3.4.21.19.

Any endopeptidase, in particular any specific endopeptidase, such as any trypsin-like or lysine- specific or glutamyl-specific or proline-specific endopeptidase, can be used in the process of the invention. The origin of such endopeptidase to be used in the process of the invention is not im portant for a successful outcome.

The endopeptidase to be used in the process of the invention may be derived from any source. It may be derived from an animal, e.g., it may be a porcine or a bovine trypsin. Such porcine or bovine trypsin may have been extracted, e.g., from porcine or bovine pancreas, or it may have been expressed in a microorganism, such as in a filamentous fungus or yeast, or in a bacterium.

The endopeptidase to be used in the process of the invention may be derived from a microor ganism, such as from a filamentous fungus or yeast, or from a bacterium.

In a preferred embodiment, the endopeptidase is derived from a fungus. In another preferred embodiment, the endopeptidase is derived from a bacterium. The endopeptidase may be extracellular. It may have a signal sequence at its N-terminus, which is cleaved off during secretion.

The endopeptidase may be derived from any of the sources mentioned herein. The term “de rived” 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 endopeptidase is identical to a native polypeptide. The term “derived” also means that the enzyme may have been produced recom- binantly 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 recombi nantly produced enzyme which is a mutant of a native amino acid sequence. Within the mean ing 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 encom passes enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro. Wth respect to recombinantly produced enzymes the term “derived from” re fers to the identity of the enzyme and not the identity of the host organism in which it is pro duced recombinantly.

The endopeptidase 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 microorgan ism and subsequent isolation of an endopeptidase preparation from the resulting fermented broth or microorganism by methods known in the art. The endopeptidase 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 en dopeptidase 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.

The endopeptidase may be purified. The term "purified" as used herein covers endopeptidase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers endopeptidase enzyme protein essentially free from insoluble com ponents 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.

Preferably, the endopeptidase is purified from its production organism. More preferably, the en- dopeptidase is purified from its production organism meaning that the endopeptidase prepara tion does not comprise living production organism cells.

Accordingly, the endopeptidase may be purified, viz. only minor amounts of other proteins being present. The expression “other proteins” relate in particular to other enzymes. The term "puri fied" 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 endopeptidase. The endopep tidase 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 endopeptidase. Preferably, the endopeptidase 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.

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

The endopeptidase 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.

A trypsin-like or lysine-specific or glutamyl-specific endopeptidase to be used in the process of the invention may be added at a concentration of 0.1-1000 mg enzyme protein per kg substrate protein, preferably 0.5-500 mg enzyme protein per kg substrate protein, more preferably 1-100 mg enzyme protein per kg substrate protein.

The dosage will depend on parameters such as the temperature, the incubation time and the dairy alternative recipe. The skilled person will know how to determine the optimal enzyme dos age.

A trypsin-like or lysine-specific endopeptidase to be used in the process of the invention may be added at a concentration of 1-3000 KPRU/kg substrate protein, preferably 5-2000 KPRU/kg substrate protein, more preferably 25-600 KPRU/kg substrate protein.

Trypsin-like and lysine-specific endopeptidases hydrolyse the chromophoric substrates Ac-Arg- p-nitro-anilide (Ac-Arg-pNA) and/or Ac-Lys-p-nitro-anilide (Ac-Arg-pNA). The liberated pNA pro duces an absorption increase at 405 nm, which is proportional to enzyme activity. One KPRU is equivalent to the amount of enzyme that produces 1 micromole p-nitroaniline per minute, when Ac-Arg-pNA or Ac-Lys-pNA is incubated with the enzyme at pH 8.0 at 37°C. The activity may be determined relative to a standard of declared strength.

Phospholipase

In a preferred embodiment of the process of the invention, the plant-based substrate is further treated with a phospholipase. In a preferred embodiment, the phospholipase is a phospholipase A1 or a phospholipase A2, preferably a phospholipase A1.

In a preferred embodiment, the phospholipase is selected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably 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: 15; and ii) a variant of the polypeptide of SEQ ID NO: 15 comprising a substitution, deletion, and/or in sertion at one or more positions.

The phospholipase is preferably derived from a strain of Fusarium, more preferably from Fusarium venenatum.

Preferably, a phospholipase to be used in the process of the invention is classified in EC 3.1.1.32.

Any phospholipase, such as any phospholipase A1 or A2, can be used in the process of the in vention. The origin of such phospholipase to be used in the process of the invention is not im portant for a successful outcome.

The phospholipase to be used in the process of the invention is preferably derived from a mi croorganism, such as from a filamentous fungus or yeast, or from a bacterium.

In a preferred embodiment, the phospholipase is derived from a fungus. In another preferred embodiment, the phospholipase is derived from a bacterium.

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

The phospholipase may be derived from any of the sources mentioned herein. The term “de rived” 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 phospholipase is identical to a native polypeptide. The term “derived” also means that the enzyme may have been produced recom- binantly 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 recombi nantly produced enzyme which is a mutant of a native amino acid sequence. Within the mean ing 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 encom passes enzymes which have been modified e.g. by glycosylation, phosphorylation etc., whether in vivo or in vitro. Wth respect to recombinantly produced enzymes the term “derived from” re fers to the identity of the enzyme and not the identity of the host organism in which it is pro duced recombinantly. The phospholipase 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 microorgan ism and subsequent isolation of a phospholipase preparation from the resulting fermented broth or microorganism by methods known in the art. The phospholipase 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 phos pholipase and the DNA sequence being operationally linked with an appropriate expression sig nal 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 se quence 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 syn thesized in accordance with methods known in the art.

The phospholipase may be purified. The term "purified" as used herein covers phospholipase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers phospholipase enzyme protein essentially free from insoluble com ponents 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.

Preferably, the phospholipase is purified from its production organism. More preferably, the phospholipase is purified from its production organism meaning that the phospholipase prepara tion does not comprise living production organism cells.

Accordingly, the phospholipase may be purified, viz. only minor amounts of other proteins being present. The expression “other proteins” relate in particular to other enzymes. The term "puri fied" 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 phospholipase. The phospho lipase 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 phospholipase. Preferably, the phospholipase 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.

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

The phospholipase 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. A phospholipase to be used in the process of the invention may be added at a concentration of 0.0001-1 EEU/g of plant-based substrate. The plant-base substrate is inclusive of the water content.

The dosage will depend on parameters such as the substrate, the temperature, the incubation time and the dairy alternative recipe. The skilled person will know how to determine the optimal enzyme dosage.

PREFERRED EMBODIMENTS

1. A process for preparing a plant-based fermented dairy alternative, the process compris ing:

(a) treating a plant-based substrate with an endopeptidase; and

(b) fermenting the plant-based substrate by incubating with a lactic acid bacterium to produce the plant-based fermented dairy alternative; wherein step (a) is performed before and/or during step (b).

2. The process of embodiment 1, wherein at least part of the plant-based substrate is ob tained from legumes, preferably from soy, pea, chickpea, mung bean, lentils, faba bean and/or lupin, more preferably from soy, pea, lentils and/or faba bean, most preferably from soy and/or pea; preferably where at least 50%, such as at least 80% or at least 90%, of the protein in the plant-based substrate is obtained from legumes, preferably from soy, pea, chickpea, mung bean, lentils, faba bean and/or lupin, more preferably from soy, pea, lentils and/or faba bean, most preferably from soy and/or pea.

3. The process of any of the preceding embodiments, wherein the plant-based substrate is obtained from legumes, preferably from soy, pea, chickpea, mung bean, lentils, faba bean or lupin, more preferably from soy, pea, lentils or faba bean, most preferably from soy or pea.

4. The process of any of the preceding embodiments, wherein the plant-based substrate is (i) soy milk or soy beverage, optionally fortified with soy milk powder or with concentrat ed or isolated legume protein, (ii) another plant-based milk alternative, such as coconut milk, oat milk or almond milk, preferably coconut milk, fortified with soy milk powder or with concentrated or isolated legume protein, or (iii) an aqueous solution or suspension of soy milk powder or of isolated or concentrated legume protein.

5. The process of the preceding embodiment, wherein the legume protein is soy protein, pea protein, lentil protein and/or faba bean protein, preferably in the form of an isolate or a concentrate. 6. The process of any of the preceding embodiments, wherein the plant-based substrate is (i) a plant-based milk alternative, preferably soy milk or soy beverage, optionally fortified with plant-based milk alternative powder such as soy milk powder or with concentrated or isolated protein such as soy protein isolate or soy protein concentrate, or (ii) an aque ous solution or suspension of a plant-based milk alternative powder such as soy milk powder or of a plant protein isolate or concentrate, preferably a legume protein isolate or concentrate, more preferably a soy protein or pea protein isolate or concentrate.

7. The process of any of the preceding embodiments, wherein the plant-based substrate has a protein content of at least 2%, preferably at least 3% (w/w).

8. The process of any of the preceding embodiments, wherein the plant-based substrate has a protein content of at least 5% (w/w).

9. The process of any of the preceding embodiments, wherein the plant-based substrate has a protein content of at most 12%, preferably at most 10% (w/w).

10. The process of any of the preceding embodiments, wherein the plant-based substrate has a protein content of 2-12%, preferably 3-12% (w/w).

11. The process of any of the preceding embodiments, wherein the plant-based substrate has a protein content of 5-12%.

12. The process of any of the preceding embodiments, wherein the plant-based substrate is 100% plant-based.

13. The process of any of the preceding embodiments, wherein all of the protein in the plant- based substrate is plant protein.

14. The process of any of the preceding embodiments, wherein at least 90% (w/w), prefera bly at least 95% (w/w), more preferably all of the protein in the plant-based fermented dairy alternative is plant protein.

15. The process of any of the preceding embodiments, wherein the protein in the plant- based substrate constitutes at least 50% (w/w), preferably at least 80% (w/w), more preferably at least 90% (w/w), even more preferably at least 95% (w/w), such as 100%, of the protein in the plant-based fermented dairy alternative.

16. The process of any of the preceding embodiments, wherein the plant-based substrate which has been treated with the endopeptidase and fermented by incubating with a lactic acid bacterium constitutes at least 50% (w/w), preferably at least 80% (w/w), more pref erably at least 90% (w/w), even more preferably at least 95% (w/w), such as 100%, of the plant-based fermented dairy alternative. 17. The process of any of the preceding embodiments, wherein the plant-based 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, a skyr alternative or a cream cheese alternative.

18. The process of any of the preceding embodiments, wherein the plant-based 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 alterna tive, or a drinkable plant-based fermented dairy alternative, such as a drinking yoghurt alternative or a kefir alternative.

19. The process of any of the preceding embodiments, wherein the plant-based fermented dairy alternative is a spoonable plant-based fermented dairy alternative, such as a stirred yoghurt alternative or a set-type yoghurt alternative.

20. The process of any of the preceding embodiments, wherein pasteurization is performed before step (b).

21. The process of any of the preceding embodiments, wherein pasteurization is performed before step (a).

22. The process of any of the preceding embodiments, wherein heat treatment, preferably at a temperature of 95-120°C, is performed after step (b).

23. The process of any of the preceding embodiments, wherein step (a) and step (b) are performed simultaneously.

24. The process of the preceding embodiment, wherein the lactic acid bacterium is of the genus Streptococcus, Lactococcus, Lactobacilllus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Propionibacterium, Enterococcus, Brevi bacterium, or Bifidobacterium or any combination thereof.

25. The process of any of the preceding embodiments wherein the fermentation time is re duced by at least 10%, preferably at least 20%, compared to the same process but with out addition of an endopeptidase.

26. The process of any of the preceding embodiments, wherein the endopeptidase is a spe cific endopeptidase, preferably a specific endopeptidase having a preference for cleav ing before or after one or two specific amino acids.

27. The process of the preceding embodiment, wherein the specific endopeptidase has a preference for cleaving before or after, preferably after, a non-hydrophobic amino acid. 28. The process of any of the preceding embodiments, wherein the endopeptidase is select ed from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; and ii) a variant of the polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 comprising a substitution, deletion, and/or insertion at one or more positions.

29. The process of any of the preceding embodiments, wherein the endopeptidase is select ed from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, identical to any of SEQ ID NOs: 1, 13 or 14; and ii) a variant of the polypeptide of any of SEQ ID NOs: 1, 13 or 14 comprising a substitu tion, deletion, and/or insertion at one or more positions.

30. The process of any of the preceding embodiments, wherein the endopeptidase is a tryp- sin-like endopeptidase, a lysine-specific endopeptidase, a glutamyl-specific endopepti dase, or a proline-specific endopeptidase.

31. The process of any of the preceding embodiments, wherein the endopeptidase is a tryp- sin-like endopeptidase, preferably derived from a strain of Fusarium, more preferably from Fusarium oxysporum, a lysine-specific endopeptidase, preferably derived from a strain of Achromobacter, more preferably from Achromobacter lyticus, or a glutamyl- specific endopeptidase, preferably derived from a strain of Bacillus, more preferably from Bacillus licheniformis.

32. The process of the preceding embodiment, wherein the trypsin-like or lysine-specific en dopeptidase has a specificity for cleaving after Arg or Lys (whichever is the larger) which is at least 100-fold higher than its specificity for cleaving after any one of Ala, Asp, Glu, lie, Leu, Met, Phe, Tyr or Val (whichever is the larger).

33. The process of any of the two preceding embodiments, wherein the glutamyl-specific endopeptidase has a strong preference for glutamic acid in the P1 position releasing peptides with a glutamic acid in the C-terminal.

34. The process of any of the three preceding embodiments, wherein the glutamyl-specific endopeptidase has a specificity for cleaving after Glu which is at least 10-fold, such as at least 20-fold or at least 50-fold, higher than its specificity for cleaving after any one of Ala, Arg, Asp, lie, Leu, Lys, Met, Phe, Tyr or Val (whichever is the larger). 35. The process of any of the preceding embodiments, wherein the viscosity of the plant- based fermented dairy alternative is reduced by at least 25%, preferably at least 40%, compared to a plant-based fermented dairy alternative prepared by the same process but without addition of an endopeptidase.

36. The process of any of the preceding embodiments, wherein the viscosity of the plant- based fermented dairy alternative is reduced by at least 25%, preferably at least 40%, compared to a plant-based fermented dairy alternative prepared by the same process but without addition of an endopeptidase, where the viscosity is determined after six days 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.

37. The process of any of the preceding embodiments, wherein the plant-based fermented dairy alternative expels at least 10%, preferably at least 20%, less liquid in a forced syn- eresis test compared to a plant-based fermented dairy alternative prepared by the same process but without addition of an endopeptidase, where the forced syneresis test is per formed after six days storage at 4°C by centrifugation of the plant-based fermented dairy alternative for 15 min at 2643 x g, and where the weight of remaining solid is recorded after removal of supernatant and the amount of expelled liquid calculated using the for mula: (weight of fermented dairy alternative sample - weight of solid phase)/(weight of fermented dairy alternative sample) * 100%.

38. The process of any of the preceding embodiments, wherein the plant-based fermented dairy alternative has a more smooth texture compared to a plant-based fermented dairy alternative prepared by the same process but without addition of an endopeptidase, where the texture is visually evaluated after six days storage at 4°C by placing a sample of the plant-based fermented dairy alternative on the backside of a black plastic spoon

39. The process of any of the preceding embodiments, wherein the plant-based substrate is further treated with a phospholipase before, during or after step (a) and before or during step (b).

40. The process of the preceding embodiment, wherein the phospholipase is a phospho lipase A1 or a phospholipase A2, preferably a phospholipase A1.

41. The process of any of the two preceding embodiments, wherein the phospholipase is se lected from the group consisting of: i) a polypeptide having an amino acid sequence which is at least 60%, preferably 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: 15; and ii) a variant of the polypeptide of SEQ ID NO: 15 comprising a substitution, deletion, and/or insertion at one or more positions.

42. The process of any of the three preceding embodiments, wherein the phospholipase is a fungal phospholipase, preferably derived from a strain of Fusarium, more preferably from Fusarium venenatum.

43. A plant-based fermenteded dairy alternative obtainable by the process of any of the pre ceding embodiments.

44. Use of an endopeptidase, preferably a specific endopeptidase, in the production of a plant-based fermented dairy alternative.

45. Use of an endopeptidase, preferably a specific endopeptidase, and a phospholipase in the production of a plant-based fermented dairy alternative.

Examples

Throughout the examples, the terms “yoghurt” and “milk” mean a plant-based yoghurt alterna tive and milk alternative, respectively, unless otherwise specified.

Method 1

Trypsin-like and lysine-specific endopeptidases hydrolyse the chromophoric substrates Ac-Arg- p-nitro-anilide (Ac-Arg-pNA) and/or Ac-Lys-p-nitro-anilide (Ac-Arg-pNA). The liberated pNA pro duces an absorption increase at 405 nm, which is proportional to enzyme activity. One KPRU is equivalent to the amount of enzyme that produces 1 micromole p-nitroaniline per minute, when Ac-Arg-pNA or Ac-Lys-pNA is incubated with the enzyme at pH 8.0 at 37°C. The activity may be determined relative to a standard of declared strength.

Method 2

EEU may be determined as follows: Lecitin is used as the substrate and the amount of free fatty acids generated is quantified colorimetrically using a Wako NEFA-HR kit at 37°C and at pH 6.9. A Galaya Enhance sample of known activity may be used to make a standard curve and quanti fy the activity.

Materials

The following enzymes are used throughout the examples:

TL1: Trypsin-like peptidase from Fusarium oxysporum having the sequence of SEQ ID NO: 1.

Lysine-specific peptidase: Lysine-specific endopeptidase from Achromobacter lyticus having the sequence of SEQ ID NO: 13. Glutamyl-specific peptidase: glutamyl-specific endopeptidase from Bacillus licheniformis having the sequence of SEQ ID NO: 14.

Galaya Enhance: Phospholipase A1 from Fusarium venenatum having the sequence of SEQ ID NO: 15.

Example 1

Production of high-protein (6%) stirred soy-yogurt in laboratory scale

Commercial, non-sweetened soymilk was purchased from local supermarket and fortified with a commercial soy protein isolate until a protein content of 6 wt% protein, which was used as base for production of soy-yogurt. The soy suspension was homogenized (500 bar), pasteurized (90°C for 10 min), and subsequently cooled (43° C). A commercial dairy starter culture (0.4 U/kg), sucrose (1 wt%), yeast extract (0.045 wt%), and TL1 were added, and fermentation car ried out (43°C for 4-6 hours), until the pH reached 4.5.

TL1 was dosed at 0, 20, 200, 400 or 600 KPRU/kg protein.

After ended fermentation, the soy-yogurt gel was broken using a shear mixer (Ultra Turrax, IKA, Germany) until smoothened (0-300 sec). The soy-yogurt was stored refrigerated until evaluation after 1 week.

The soy-yoghurts were analyzed according to common industry practices:

• Viscosity was measured using Rapid Visco Analyzer (RVA) 4500 (Perten Instruments, Swe den). 30 g yogurt sample was transferred in RVA cup and allowed to set in the refrigerator for 1h before the measurement. The measurement was carried out at 20° C at 50 rpm and the vis cosity value read after 70 seconds

• Forced syneresis test was conducted on 30 g of yogurt sample by centrifugation for 15 min at 2643 x g. The weight of remaining solid is recorded after removal of supernatant, and amount of expelled liquid, called Syneresis, is calculated using the formula: (Weight of yogurt sample - weight of solid phase)/(weight of yogurt sample) * 100% (given in wt%). Water-holding capacity = 100% - Syneresis.

Additionally, the visual appearance of the soy-yogurt samples was evaluated by placing yogurt sample on the back side of black plastic spoons, where lumps or graininess and runny/thin tex ture are easily observed. The results are summarized in Table 1.

Table 1. Soy-yogurt properties after 1-week storage.

The reference soy-yogurt prepared using no enzyme had the longest fermentation time of 5.3 h. Addition of TL1 resulted in faster fermentations, around 1 h shorter.

The reference soy-yogurt prepared using no enzyme had a high viscosity of 5,502 cP. Addition of TL1 resulted in a continuous decrease in viscosity with increasing enzyme dose, down to 2,118 cP or a viscosity reduction of more than 60%.

The reference soy-yogurt prepared using no enzyme had a high level of syneresis of 31.4 wt%. Addition of TL1 resulted in a continuous decrease in viscosity with increasing enzyme dose, down to 21.1 wt%.

The reference soy-yogurt prepared using no enzyme had a grainy, lumpy, and solid appear ance. Addition of TL1 resulted in an improvement of the appearance and at certain dosage lev els were smooth without visible graininess.

Addition of endoprotease TL1 resulted in overall best yoghurt properties, evaluated by several parameters: The soy-yogurts prepared with TL1 had a smoother texture than those prepared without. Interestingly, TL1 lowered both the amount of syneresis and the level of viscosity - which are otherwise often inversely correlated. An additional benefit is a faster fermentation time.

Example 2

Production of high-protein (9%) stirred soy-yogurt in laboratory scale

This example demonstrates the applicability of TL1 in soy-yoghurts with even higher protein contents than in Example 1: Commercial, non-sweetened soymilk, purchased from local super market, fortified with a commercial soy protein isolate until a protein content of 9% protein, was used as base for soy-yogurt production, as described in Example 1.

TL1 was dosed 0, 40, 200, 500, or 800 KPRU/kg protein. After ended fermentation, the soy- yogurt gel was broken using a shear mixer (Ultra Turrax, IKA, Germany) until smoothened (0- 300 sec). The soy-yogurts were stored refrigerated until evaluation after 1 week.

Viscosity, forced syneresis test and visual appearance of the soy-yogurt samples were evaluat ed according to protocols described in Example 1. The results are summarized in Table 2.

Table 2. Soy yogurt properties after 1-week storage.

The reference soy-yogurt prepared using no enzyme had the longest fermentation time of 5.9 h, the highest viscosity of 13,752 cP, and highest level of syneresis of 20.2 wt%. The reference soy-yogurt was very firm and grainy. Addition of TL1 resulted in slightly faster fermentation rates, and a significant, continuous de crease in viscosity (to 5,944 cP or close to 60% viscosity reduction) and syneresis (to 11.3 wt%). Further, the enzymatic treated soy-yogurts were visually more appealing, as the lumpi ness disappeared and texture became smooth, shiny and softer.

Example 3 Production of high-protein yogurt in laboratory scale from 10% pea protein hydrolysate

This example demonstrates the applicability of TL1 a) in other legume yoghurts, exemplified by pea, and b) as a pre-treatment before fermentation: Commercial pea protein isolate was used as base for pea-yogurt production. The pea protein suspension was homogenized (800 bar), pasteurized (90°C for 10 min), and subsequently cooled (45° C). TL1 was dosed at 0, 100, or 450 KPRU/kg protein and incubated overnight (16 h at 45° C), before heat inactivation (90°C for

10 min). A commercial dairy starter culture (0.2 U/kg) and sucrose (1 wt%) was added, and fer mentation carried out (43° C for 4-20 hours), until the pH reached 4.5.

After ended fermentation, the pea-yogurt gel was broken using a shear mixer (Ultra Turrax, IKA, Germany) until smoothened (0-300 sec). The yogurt was stored refrigerated until evaluation af- ter 4 days.

Viscosity, forced syneresis test and visual appearance of the pea-yogurt samples were evaluat ed according to protocols described in Example 1. The degree of hydrolysis was determined spectrophotometrically (340 nm) from the formed complexes of o-phthaldialdehyde and the free a-amino groups generated during proteolysis (given as difference from unhydrolyzed pea pro- tein). The results are summarized in Table 3.

Table 3. Soy yogurt properties after 4 days of storage.

The reference pea-yogurt prepared using no enzyme had the longest fermentation time (data not shown), the highest viscosity (11,967 cP), and highest level of syneresis (19 wt%). The ref erence pea-yogurt was very firm and grainy (see also Figure 1). Pre-treatment of pea protein isolate with TL1 at 100 or 450 KPRU/kg protein caused an in creased degree of hydrolysis of 2.9 and 3.4, respectively. The yogurts produced from the pea protein hydrolysates had significantly lower viscosities (to 2,973 cP) and less syneresis (to 9 wt%). Further, the enzymatic treated pea-yogurts were visually more appealing, as the lumpi ness disappeared and texture became smooth, shiny and softer, as is evident from the images (see also Figure 2 and Figure 3).

Example 4

TL1 effect in fermentation of a pea-based yogurt

Here a pea yogurt made from pea isolate and rape seed oil was made where TL1 was added together with the culture showing that a protease treatment can be done before pasteurization of a fermentation base (as in Example 3) or in the fermentation step.

A commercial pea protein isolate (80% protein) was mixed with water, refined rape seed oil, sugar and peptone to a fermentation base containing 3.5% protein, 1.5% fat, 1% sucrose and 0.2 g/L peptone. The fermentation base was homogenized at 300 bar and pasteurized for 10 min at 90°C and then cooled to 43°C before TL1 at 200 KPRU/kg protein and a starter culture was added. Fermentation and post fermentation treatment was done as in Example 1. Analysis of the yogurts was performed as in Example 1.

Table 4. Yogurt characteristics based on the average of two individual yogurt samples The product made in this example resembled a drinking yogurt with low viscosity and showed that TL1 had the ability of reducing viscosity significantly without destroying the stability of the product. In fact, just as seen in other examples the syneresis in the protease treated drinking yogurt was lower than for the untreated pea yogurt. The examples in pea also demonstrate that protease can be added either in fermentation or in a pretreatment step before pasteurization.

Example 5

Dose response in commercial soy beverage 3.7% protein for a stirred fermented product

Commercial, non-sweetened soymilk was purchased from local supermarket and 0.4% sucrose added before pasteurization (95°C 8 min) followed by cooling to 43°C. Protease and yogurt starter (Yoflex L811) culture was added at the same time. The pH was monitored until the pH reached below pH 4.5 and the samples were treated post fermentation as in Example 1.

TL1 was tested at 20, 40, 100, 200, 300, 400 and 500 KPRU/kg. The glutamyl-specific pepti dase was tested at 0.01, 0.1, 0.5, 1, 5, 10, and 20 mg EP (Enzyme Protein)/kg soy protein and the lysine-specific peptidase was tested at 2 and 25 mg EP/kg soy protein. All samples were made in duplicates and the average value of these two individual yogurts are reported.

Viscosity and forced syneresis test of the yogurt samples were evaluated according to Example 1. The results are summarized in Tables 5-7. A commercial dairy yogurt with 1.5 % fat was also included in the viscosity and syneresis analysis for comparison.

Table 5. Characteristics of stirred yogurts treated with TL1

Table 6. Characteristics of stirred yogurts treated with glutamyl-specific peptidase

Table 7. characteristics of stirred yogurts treated with lysine-specific peptidase

Compared to a commercial dairy yogurt comprising 3.5% protein and 1.5% fat, the viscosity measurements of the control soy yogurts showed a significantly higher viscosity at a similar pro tein content (Table 5). All tested proteases had a dose dependent reduction on viscosity of the soy yogurts. They also showed a potential to reduce fermentation time. Protease treatment also had a weak positive impact on the amount of syneresis. This allows tailoring the viscosity of a soy yogurt to fit the desired product and to make the final viscosity more dairy-like. Example 6

Dose response in commercial soy beverage fortified with soymilk powder to a final concentra tion of 8% protein for a stirred fermented product

Commercial, non-sweetened soymilk was purchased from local supermarket and fortified by addition of a commercial soymilk powder to a final protein concentration of 8%. The beverage was mixed at room temperature for 5 min and sucrose (0.8%) and yeast extract (0.045% to speed up fermentation) was added and the mix was agitated for 20 min. The beverage was then pasteurized (95°C 5 min) before being cooled to 43°C. Protease and yogurt starter (Yoflex L811) culture was added at the same time and the beverage was held at 43°C. The pH was monitored and until it reached below pH 4.5 and the samples were treated post fermentation as in Example 1.

TL1 was tested at 200 and 400 KPRU/kg. The glutamyl-specific peptidase was tested at 2 and 4 mg EP/kg soy protein and the lysine-specific peptidase was tested at 25 and 50 mg EP/kg soy protein. All samples were made in duplicates and the average value of these two individual yo gurts are reported. Viscosity and forced syneresis test of the yogurt samples were evaluated according to Example 1. The results are summarized in Table 8.

Table 8. Characteristics of finished stirred yogurts using various specific proteases in a high pro tein fermented soy product At the higher protein content of 8%, the blank sample is very grainy and does not resemble a yogurt product. All three proteases tested had the capacity to reduce the graininess and make a smoother soy yogurt. The proteases treated yogurts also had shorter fermentation time and a lowered viscosity of the final stirred yogurt. Protease treatment also has a positive impact on the structure making the soy yogurts smoother and reducing syneresis.

The data in these examples clearly shows that proteases can be used to improve legume-based yogurts exemplified by soy and pea, not only by reducing viscosity but also by making a smoother product and reducing syneresis. The proteases also show processing benefits by re ducing the fermentation time which could be used to increase the production capacity and re duce the risk of contamination since the yogurts are exposed for a shorter time to neutral pH and raised temperatures.

Example 7

Sensory evaluation of TL1 and lipase in high protein soy yogurts

Commercial, non-sweetened soymilk was purchased from local supermarket and fortified by addition of commercial soymilk powder to a final protein concentration of 8%. The beverage was mixed at room temperature for 5 min and sucrose (0.8%) and yeast extract (0.045% to speed up fermentation) was added and the mix was kept with agitation for 20 min. In samples treated with a phospholipase product, Galaya Enhance (3800 EEU/g) was added at a dose of 0.01% weight / volume before pasteurization and it was incubated for 30 min before pasteurization at 40°C. The beverage was then pasteurized (95°C 5 min) before being cooled to 43°C. Peptidase and yogurt starter culture (ABY-3) was added at the same time and the beverage was held at 43°C. The pH was monitored until it reached below pH 4.5 and the samples were treated post fermen tation as in Example 1.

A sensory evaluation was performed according to the description below:

Sensory evaluation of plant-based yoghurt alternatives

• Initial stir. A spoon was used to stir the sample for one round and then turned. Samples are scored from 1- grainy and separated to 7- smooth and coherent.

• Amount of stir. Samples are stirred to full stir and the amount of stir was scored from: 1- required a lot of stirring to 7- samples quickly became smooth.

• Full stir. Samples are scored from 1- grainy, matte to 7- smooth, glossy

• Astringency. 1- very astringent, 7- not astringent

• Acidity. 1- very sour, 7- low acidity

• Bitterness·. 1- very bitter, 7- not bitter

• Mouthfeel·. 1- watery, 7- rich and creamy

• Preference·. Samples are ranked based on preference and panelist are asked to de scribe why they ranked the samples in the order they did.

The sensory panel consisted of untrained panelists and in the sensory evaluations each panelist was given four anonymous samples per tasting session. They were asked to give all samples a score on several parameters both by visual inspection as well as from tasting the samples. Fi nally, the panelists were asked to rank the samples based on preference and comment on why they preferred the sample they did.

Table 9. Analytical results of the yogurts used for sensory evaluation Table 10. Preference scores based on overall sensory in set containing TL1 and Galaya En hance. Number of panelists n=6

Table 11. Visual evaluation of first sensory set: TL1 and Galaya Enhance

Table 12. Taste evaluation of first sensory set: TL1 and Galaya Enhance

At 8% protein it was apparent that the structural defects were primarily solved by the addition of a TL1. Not only did TL1 lower the viscosity of the soy yogurts but more importantly TL1 im proved all three sensory parameters related to the visual appearance of the yogurts. “Initial stir” relates to the early homogeneity of the product, “amount of stir” relates to the difficulty to stir the yogurt and “full stir” to how appealing the visual product was, once completely stirred. These attributes correspond to the first impression a consumer would have when opening a soy yogurt and spoon it from the package and the TL1 had a significant positive impact on all three param eters. However, peptidases are known to create bitter off notes when hydrolyzing proteins. In the sensory evaluation there was no difference in bitterness between the peptidase treated samples compared to control. The peptidase also improved some of the in-mouth aspects of the soy yogurts such as reduced astringency and improved mouthfeel. Surprisingly, the Galaya Enhance treated soy yogurt was the least preferred sample (5 out of 6 panelists) in the set with TL1 and Galaya Enhance and while the differences between TL1 and the combination was relatively small in the sensory evaluation there was a clear preference for the combination in the preferred samples ranking (4.5 out of 6 panelists preferred the combina tion). The comments connected to the preference related to a creamy and smooth texture and that it was the most appealing looking sample.

Example 8

Sensory evaluation of glutamyl-specific peptidase in high protein soy yogurts Commercial, non-sweetened soymilk was purchased from local supermarket and fortified by addition of commercial soymilk powder to a final protein concentration of 8%. The beverage was mixed at room temperature for 5 min and sucrose (0.8%) and yeast extract (0.045% to speed up fermentation) was added and the mix was kept with agitation for 20 min. The beverage was then pasteurized (95°C 5 min) before being cooled to 43°C. Peptidase and yogurt starter culture (ABY-3) was added at the same time and the beverage was held at 43°C. The pH was moni tored until it reached below pH 4.5 and the samples were treated post fermentation as in Exam ple 1.

A sensory evaluation was performed according to Example 7. The sensory panel consisted of untrained panelists and in the sensory evaluations each panelist was given four anonymous samples per tasting session. They were asked to give all samples a score on several parame ters both by visual inspection as well as from tasting the samples. Finally, the panelists were asked to rank the samples based on preference and comment on why they preferred the sam ple they did. Table 13. Analytical results of the yogurts used for sensory evaluation

Table 14. Visual evaluation of glutamyl-specific peptidase

Table 15. Taste evaluation of glutamyl-specific peptidase

Table 16. Preference scores based on overall sensory in sample treated with glutamyl-specific peptidase. Number of panelists n=7 Like in Example 7, with TL1 , the samples treated with glutamyl-specific peptidase solved many of the negative aspects caused by the high protein content. The viscosity of the TL1 treated yo gurt and the glutamyl-specific peptidase were also close to each other. Glutamyl-specific pepti dase treated high protein soy yogurts also showed similar improvements in the visual evaluation (Table 14) and reduced astringency while increasing mouthfeel without generating any bitter- ness (Table 15). Form a production point of view, the shortened fermentation time was seen again (Table 13). In the preference data (Table 16) all panelists preferred the glutamyl-specific peptidase treated samples mentioning that it looked better and had a smoother texture and was without a floury mouthfeel.

Example 9 Effects of TL1 and Galaya Enhance on high protein soy (7%) stirred yoghurt via Texture Ana lyzer parameters including a new parameter: stirring cohesiveness

Texture of yoghurt can be measured by sensory and instrument analysis. Texture Analyzer (TA) is well used for measuring texture of food products. The parameters measured by TA are de fined according to measurement conditions and subjected food categories. In our previous stud- ies, we found thickness extracted from Texture Analyzer correlated well with viscosity measured by Rapid Visco Analyzer (RVA) and stirring cohesiveness extracted from Texture Analyzer cor related well with homogeneity measured by sensory. Stirring cohesiveness is a new identified parameter. We also found optimal dosage of TL1, and its combination with Galaya Enhance in other studies (Example 1, 2 and 7). The objective of this example was to demonstrate effects of TL1, Galaya Enhance and their combination on thickness and homogeneity via stirring cohe- siveness and thickness measured by Texture Analyzer (TA) as well as Rapid Visco Analyzer (RVA) and visual evaluation.

Commercial soybean milk (Naturli’, no sugar added version, protein content 3.7%) was preheat ed to 55°C. Soybean milk powder was added into the preheated soybean milk for obtaining soybean milk base with protein content at 7%. The enriched soybean milk was subsequently kept at 55°C for 30 minutes. The enriched soybean milk was then divided into two portions. Su crose and yeast extract were then added at 0.4% (w/w) and 0.02% (w/w) respectively into one portion of the enriched soybean milk base. This portion was heated up to 90°C and maintained at temperature for 10 minutes. The other portion of soybean milk was cooled down to 40°C and Galaya Enhance was added at 0.52 EEU/g based on soybean milk weight. After 30 minutes in cubation, the Galaya Enhance treated portion was subjected to the same addition of sucrose and yeast extract and heat treatment (90°C for 10 minutes) as non-enzyme treated portion. The two portions of heat-treated soybean milk were cooled down and stored under 5°C as high pro tein (7%) soybean yoghurt fermentation base. Soybean yoghurt fermentation base (protein content 7%) was preheated to 43°C. Protease TL1 and starter culture (YF-L811, Chr. Hansen, Denmark) was subsequently added into fermenta tion base and stirred for 2 minutes. The final enzyme treatment of each sample is listed in Table 17. Fermentation was kept at 43°C. Coagulated yoghurt gel was stirred by high shear mixer (Ul tra Turrax, IKA, Germany) at 9000 rpm for approximately 40 seconds when pH dropped around 4.45. Approximately 80 g of the yoghurt was kept in one closed plastic jar of 100 ml. The yo ghurt samples were stored under 5°C for 8 days before evaluation.

Table 17. Enzyme treatment in example 9. Galaya Enhance is abbreviated GE in the table

*Phospholipase Galaya Enhance was dosed at pretreatment step (40°C, 30 min) and denatured by heat treatment. Protease TL1 was dosed at fermentation step. 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 5°C storage condition. Adjusted Texture Profile Analysis (TPA) pro cedure was carried out at pretest speed 2 mm/s, test speed 2 mm/s, waiting time 5s and post- test speed 5 mm/s. Thickness (positive area integrated to peak force during first compression), adhesiveness (negative area between two compressions) and stirring cohesiveness (adhesive ness divided by thickness) were calculated. The higher value of stirring cohesiveness, the high er homogeneity would be obtained after stirring due to higher cohesiveness during stirring. Measures were performed with duplicated samples.

Viscosity measurement was performed as in Example 1.

Visual evaluation was performed by an experienced technician following a standard procedure established for evaluating plant-based protein yoghurt. The evaluated parameters are synere- sis, initial homogeneity, adhesiveness, and homogeneity after stirring. A score system from 1 to 7 was used to distinguish samples on each parameter. The definitions of visual evaluated pa rameters defined here are:

• Syneresis - the water amount observed once the plastic jar is opened

• Initial homogeneity - the homogeneity of yoghurt after it is flipped from the bottom of the plastic jar by a spoon

• Homogeneity after stirring - the homogeneity after the whole sample in the plastic jar is stirred 30 times manually by a spoon

• Adhesiveness - the amount and shape of yoghurt sample on the back of a spoon after the spoon is pressed and lifted quickly on the surface of fully stirred yoghurt sample

The yoghurts treated with TL1 (0.4 KPRU/g protein) turned to be thinner and less adhesive compared to blank control. The yoghurts had increased stirring cohesiveness which indicated a better homogeneity. The yoghurts treated with Galaya Enhance (0.52 EEU/g yoghurt fermenta tion base) turned to be thicker and more adhesive compared to blank control. The yoghurts had reduced stirring cohesiveness which indicated an inferior homogeneity. The yoghurts treated with the combination of the two enzymes had thicker and more adhesive texture compared to the yoghurts with single TL1. Surprisingly, stirring cohesiveness of these yoghurts was also higher than that of yoghurts with only TL1, which indicates homogeneity was further improved.

Table 18. Texture analyzer data from stirred yoghurts. Galaya Enhance is abbreviated GE in the table

TL1 (0.4 KPRU/g protein) reduced viscosity obviously. Galaya Enhance (0.52 EEU/g yoghurt fermentation base) increased viscosity. The combination of these two enzymes still reduced vis cosity obviously compared to blank control. Table 19. Viscosity of stirred yoghurts. Galaya Enhance is abbreviated GE in the table

There was no syneresis noticed from all samples after 8 days storage even on samples having low viscosity (sample ID: TL1 0.4, TL1 0.4+GE0.52). Both initial homogeneity and homogeneity after stirring were improved by TL1 (0.4 KPRU/g protein). Galaya Enhance (0.52 EEU/g yoghurt fermentation base) reduced initial homogeneity which means that intact samples after storage looked less homogeneous than blank control. However, the same yoghurt sample did not show inferiority on homogeneity after stirring. The combination of GE (0.52 EEU/g yoghurt fermenta tion base) with TL1 (0.4 KPRU/g protein) even further improved homogeneity after stirring com pared to yoghurt with TL1 alone. This indicates Galaya Enhance had effect on microstructure of yoghurts.

Table 20. Visual evaluation of stirred yoghurts. Galaya Enhance is abbreviated GE in the table

The enzyme effects highlighted in this example are

• Compared to blank control, TL1 alone (0.4 KPRU/g protein) increased homogeneity while reduced thickness, adhesiveness and viscosity. • Compared to blank control, Galaya Enhance alone (0.52 EEU/g yoghurt fermentation base) increased thickness, adhesiveness and viscosity while it reduced homogeneity of high protein (7%) soy stirred yoghurt.

• Compared to TL1 alone, a combination of TL1 (0.4 KPRU/g protein) and Galaya En- hance (0.52 EEU/g yoghurt fermentation base) surprisingly further improved homogenei ty and increased thickness of high protein (7%) soy stirred yoghurt.

Example 10

Effects of TU and Galaya Enhance on high protein (7%) soy set yoghurt

The objective of this example was to demonstrate effects of TL1, Galaya Enhance and their combination on high protein soy yoghurt via measures of Texture Analyzer and visual evalua tion.

Yoghurt was made as in Example 9 up to fermentation. The yoghurts were fermented directly in closed plastic jars. The yoghurts were transferred directly to storage condition (5°C) instead of being stirred, when the targeted pH range was observed. The yoghurt samples were stored at 5°C for 8 days before evaluation.

Table 21. Enzyme treatment in example 10. Galaya Enhance is abbreviated GE in the table

*Phospholipase Galaya Enhance was dosed at pretreatment step (40°C, 30 min) and inactivat ed by heat. Protease TL1 was dosed in the fermentation step.

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 5°C storage condition. Texture Profile Analysis (TPA) procedure was carried out at pretest speed 2 mm/s, test speed 1 mm/s, waiting time 5 s and posttest speed 5 mm/s. The following parameters were calculated:

• Fracturability, g - the force value of first breaking point greater than 0.5 g. The higher value, the more difficult for a yoghurt gel to break.

• Elasticity, g/mm - the ratio between force and distance at first breaking point. The higher value, the higher force is needed for the same extent of deformation of a yoghurt gel. • Thickness, g.s - the value of positive area from the point probe starts compression to the point the probe finishes the first compression. The higher value, the thicker of a yoghurt gel.

• Adhesiveness, g.s - the absolute value of negative area between two compressions of the probe. The higher value, the more chance of a yoghurt sample to stick to another subject.

• Cohesiveness, % - the ratio between the two positive areas from the point probe starts compression to the point probe finishes compression. The higher value, the more chance of a yoghurt gel stays as a continuous body during compression

• Stirring cohesiveness, % - the ratio between adhesiveness and thickness. The higher value, the more chance of a yoghurt stays as a continuous body during repeating com pressions

Visual evaluation was performed by an experienced technician following a standard procedure established for evaluating plant-based protein yoghurt. The evaluated parameters are synere- sis, 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

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

• Flakiness - flakes on the surface of the yoghurt body

• 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

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

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

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

Compared to blank control, the yoghurt treated with TL1 (0.4 KPRU/ g protein) was easier to break, less elastic, thinner and less adhesive. This yoghurt had higher value on normal cohe siveness and stirring cohesiveness. This texture profile indicates the yoghurt melts away easily and coat more evenly in mouth, but it is thin.

Compared to blank control, the yoghurt treated with Galaya Enhance (0.52 EEU/g yoghurt fer mentation base) was slightly harder to break, more elastic, thicker and higher value on normal cohesiveness. While this yoghurt had less adhesiveness and lower value on stirring cohesive ness. This texture profile indicates a firm and unsmooth mouth feeling.

Compared to the yoghurt with TL1 alone, the yoghurt treated with the combination of the TL1 and Galaya Enhance needed higher stress to break and was thicker, more adhesive and cohe sive. However, the elasticity reduced a lot. This texture profile indicates the yoghurt melts away very easily, coats mouth more smoothly while keeping a relative thick mouth feel.

Table 22. Texture profile of high protein (7%) set soy yoghurt measured by texture analyzer. Galaya Enhance is abbreviated GE in the table

Syneresis, shrinkage or flakiness was found in all yoghurt samples even on samples with single TL1.

Compared to blank control, the yoghurt with TL1 (0.4 KPRU/g protein) showed a little lower set ting. During spoon pressing, this yoghurt was found softer but more cohesive and homogene ous. A spoon pressing is used to mimic pressing yoghurt with tongue in the sensory. Therefore, this visual evaluation also indicated a cohesive and smooth mouth feeling of this yoghurt.

Compared to blank control, the yoghurt with Galaya Enhance (0.52 EEU/g yoghurt fermentation base) showed the same setting as control. During spoon pressing, this yoghurt was found a little softer, more cohesiveness but had poor homogeneity. The reason for a softer gel during spoon pressing compared to the result of texture analyzer is due to no limited boundary for a sample to deform during the spoon pressing test.

Compared to the yoghurt with TL1 alone, the yoghurt treated with the combination of the TL1 and Galaya Enhance had higher setting, slightly firmer gel, higher cohesiveness and the same homogeneity. The combination was the one with highest cohesiveness according to the visual evaluation. This visual evaluation indicated that the yoghurt would coat mouth smoothly and would have relatively thicker mouth feeling.

This is further illustrated in Figure 4.

Table 23. Visual evaluation of set yoghurts. Galaya Enhance is abbreviated GE in the table

• Compared to blank control, high protein (7%) soy set yoghurt with TL1 alone (0.4 KPRU/g protein) was more homogeneous and cohesive while it turned to be softer and less set.

• Compared to blank control, high protein (7%) soy set yoghurt with Galaya Enhance alone (0.52 EEU/g yoghurt fermentation base) had even worse homogeneity although the cohesiveness was increased.

• Compared to TL1 alone, a combination of TL1 (0.4 KPRU/g protein) and Galaya En hance (0.52 EEU/g yoghurt fermentation base) further improved cohesiveness and miti gated reduced setting. Therefore, the combination of these two enzymes resulted in a set yoghurt with good setting and smoothness in the visual evaluation and highly proba bly a smooth and thick mouth coating due to high cohesiveness and homogeneity measured by both TA and visual evaluation.

Example 11

Effect of TL1 on high-protein (10%) stirred legume-yoghurt

In this example, yoghurts were made from protein from lentil and faba bean, to demonstrate the applicability of TL1 in high-protein yoghurts of other legume sources: Suspensions of commer cial lentil and faba bean protein isolate were prepared in water to a final protein content of 10%, and 2% sunflower oil, 1% sugar and 0.045% yeast extract added. The mixture was pre homogenized on overhead stirrer (8,000 rpm, 2 min), before high-pressure homogenization (first pass 250/50 bar, second pass 750/50 bar). After pasteurization (90° C, 10 min) and cooling, TL1 was dosed 0, 50, 100, 150, or 200 KPRU/kg protein, together with 0.4 U/L commercial starter culture (ABY-3, Chr. Hansen). Fermentation was carried out at 43° C until pH reached 4.5, and the legume-yoghurt gels were broken using a shear mixer (Ultra Turrax, IKA, Germany) until smoothened (0-120 sec). The yoghurts were stored refrigerated until evaluation after 1 week.

Viscosity and forced syneresis test of the legume-yoghurt samples were evaluated according to protocols described in Example 1. Texture was assessed visually, according to protocol in Ex ample 7, on ‘initial stir’, ‘amount of stir’, and ‘full stir’, graded 1-7, according to procedure de scribed in Example 7. The results are summarized in Tables 24-25.

Table 24. Lentil yoghurt properties after 1-week storage.

Table 25. Faba bean yoghurt properties after 1-week storage.

The reference lentil- and faba bean-yoghurts prepared without enzyme had the highest viscosi- ties, highest level of syneresis, and inferior visual textural properties, such as most grainy, inco herent, and matte.

Addition of TL1 resulted in a significant, continuous decrease in viscosities and syneresis. Fur ther, the enzymatic treated lentil- and faba bean-yoghurts were visually more appealing, as the lumpiness disappeared and texture became smooth, shiny, and softer. Example 12

Sensory effect of TL 1 on pea protein stirred yoghurt

Commercial pea protein isolate was dispersed in water to obtain a 5% protein content and stirred for 30 min at room temperature. 2 wt% rapeseed oil were added while high-sheer mixing (8,000 rpm, 2 min) and the sample homogenized (first pass 250/50 bar, second pass 750/50 bar). Sucrose (1 wt%) and yeast extract (0.045%) were added to aid the later fermentation. The mixtures were pasteurized (90°C 10 min) and rapidly cooled on ice. TL1 (0 or 20 KPRU/kg pea protein) was added to the substrate along with 0.4 U/L yogurt starter culture (ABY-3) and the samples were fermented at 43°C until pH reached below pH 4.5. Samples were stirred by high- sheer mixing, according to Example 1 , and stored refrigerated until sensory evaluation 10 days later.

Sensory evaluation was performed by an internal panel consisting of 14 panelists, having some yoghurt evaluation experience and a training session prior to the evaluation. Each panelist was given anonymous samples labeled with 3-digit random code, presented in random order. Panel ists were asked to rate samples on 9-point scale according to level of smoothness of texture. Afterwards, they were asked to rank the samples according to preferred texture.

14 out of 14 assessors ranked the untreated sample as the least preferred texture. The TL1 was scored significantly higher (8 points vs. 4 for untreated) with regards to smoothness.

Example 13

Synergistic effect of TL 1 and pectin on pea protein yoghurt texture

Pea protein yoghurts with TL1, pectin, their combination, or no additives, were compared based on viscosity, water-holding capacity and visual texture: Commercial pea protein isolate was dis persed in water at a 5 % protein content. Sugar (1 wt%), and yeast extract (0.045 wt%) were added to aid fermentation. The mixture was homogenized (800 bar) and pasteurized (90° C, 10 min).

TL1 was dosed 0 or 20 KPRU/kg protein, together with 0.4 U/L commercial starter culture (ABY- 3). Fermentation was carried out at 43°C until pH reached 4.5. 0% or 0.5 wt% pectin was add ed, and the pea-yoghurt gels were broken using a shear mixer (4,000 rpm) until smoothened (0- 120 sec). The yoghurts were stored refrigerated until evaluation after 1 week.

Viscosity and forced syneresis test of the pea yoghurt samples were evaluated according to pro tocols described in Example 1. Texture was assessed visually by a trained laboratory personnel, scoring the samples 1-7 on the following parameters:

• Coherence: how coherent the structure of the yoghurt gel is upon first turn with a spoon. Separation gives low score.

• Homogeneity: how homogeneous the texture is when it has been stirred with the spoon. Presence of grains/lumps gives low score, while uniform texture gives high score

• Glossiness: how glossy is the surface of the yoghurt. High reflectance gives high score, while matte surface gives low score.

The results are summarized in Table 26.

Table 26

The results from the visual evaluation showed pectin mainly improved coherence of the yoghurt, while TL1 improves the homogeneity. By combining the pectin and the TL1, both coherence and homogeneity is ensured. Further, the water-holding capacity of the gel is far superior. Example 14 Applicability of TL1 in stirred, fermented product based on other plant milk- analogues

As will be apparent to those skilled in the art, the enzyme solutions will work on any plant base where protein is present, here exemplified by coconut milk and pea.

Commercial coconut milk comprising 1.5% protein and 17% fat was purchased from a super- market and fortified with a commercial pea protein isolate to a final pea protein content of 9 % (after dilution) and diluted with tap-water to a coconut fat content of 5%. The mixture was ho mogenized (800 bar) and sucrose (2 wt%), and yeast extract (0.045 wt%) (to speed up fermen tation) were added before pasteurization (90° C, 10 min). After cooling, 0.4 U/L commercial starter culture (ABY-3) was added, along with TL1 dosed at 0 (‘Reference’) or 300 KMTU/kg pea protein. The inoculated mixture was held at 43°C, and the pH monitored during fermenta tion until it reached pH<4.5. The samples were stirred and analyzed after 1-week cold storage as described in Example 1, and texture assessed visually by a trained laboratory personnel, scoring the samples according to Example 13.

Table 27. Coconut-pea protein yogurt properties after 1-week storage.

RVA measurements show that TL1 has a similar lowering effect on viscosity as in the previous examples, regardless of the plant base source. A thickness compared to a Greek-style dairy product could be obtained. TL1 again decreased the level of water expelled from the yoghurts during forced syneresis test. Finally, the yoghurts pre-treated with TL1 scored better on visual parameters, being more coherent, having no grains/particulate matter, and being shinier.