Methods for foaming and / or stabilizing foam in a milk or milk-based product with aid of phospholipases

Abstract

The present disclosure generally relates to a method for foaming and / or stabilizing foam in a milk or milk- based product, wherein a phospholipase is used to promote foam and / or its stabilization by acting on phospholipids and degrading the same and a use of a phospholipase-treated milk or milk-based product for creating milk foam.

Description

[0001] P8184PC00 (17965-WO-PCT)

[0002] 1

[0003] METHODS FOR FOAMING AND / OR STABILIZING FOAM IN A MILK OR MILK-BASED PRODUCT

[0004] TECHNICAL FIELD

[0005] The present disclosure generally relates to a method for foaming and / or stabilizing foam in a milk or milkbased product, wherein a phospholipase is used to promote foam and / or its stabilization by acting on phospholipids and degrading the same and a use of a phospholipase-treated milk or milk-based product for creating milk foam.

[0006] BACKGROUND

[0007] Foaming of milk is a trend and a need in the coffee industry. The demand for foamed milk where the foam is stable over time and pleasant to consumers continues to grow and has extended, as well, to the homes of the consumers.

[0008] Several alternatives exist to promote the foamability of milk. As a way of exemplifying, one of these alternatives is to add cyclodextrins or emulsifiers to milk. However, cyclodextrins or emulsifiers contribute to the increase of carbohydrates in milk, which is undesirable from a nutritional perspective. Another alternative is to foam milk by frothing (or mechanical whipping), which works with milk having a low-fat content.

[0009] However, despite the existent alternatives, there is a constant and continuous need for developing new and improved methods for foaming and / or stabilizing foam in a milk or milk-based product or for developing alternative solutions to the methods currently available.

[0010] SUMMARY

[0011] The present disclosure provides a method for foaming and / or stabilizing foam in a milk or milk-based product according to the claims.

[0012] DEFINITIONS

[0013] The following definitions are herein used and apply to the entire disclosure.

[0014] The term "foaming" refers or can refer to the technique of aerating milk by whipping it cold or warm (frothing) or introducing steam into it (steaming), creating a smooth, creamy foam. The latter process involves using the steam wand of an espresso machine to heat and inject air into the milk, which results in a textured micro foam. P8184PC00 (17965-WO-PCT)

[0015] 2

[0016] The term "steam" refers or can refer to the hot vapor produced by an espresso or other coffee machine's steam wand, which is used to heat and aerate milk. When the steam wand is submerged in a pitcher of milk and activated, it releases steam under pressure, causing the milk to heat up and expand as air is incorporated.

[0017] The term "whipping" refers or can refer to the process of vigorously mixing a liquid, such as cream or milk, to incorporate air and create a light, airy texture.

[0018] The term "denaturation" or "whey protein denaturation" refers or can refer to the structural transformation of whey proteins when they are exposed to heat. Whey proteins, which are naturally present in milk, undergo denaturation when the milk is heated during the steaming or frothing process. This means that the proteins unfold from their native structure and form new bonds, leading to changes in their physical and functional properties.

[0019] The term "barista milk" refers or can refer to a type of milk used in coffee beverages, particularly espressobased drinks like lattes and cappuccinos. It can contain higher protein content, which helps create a more stable micro foam when steamed, achieving a creamy texture. Barista milk often has enhanced fat content to enrich the mouthfeel of the coffee beverage, and it is typically homogenized to ensure consistency in texture and performance when steamed. It can also present improved frothing and consistency.

[0020] The term "milk" refers or can refer to cow milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk and / or camel milk.

[0021] The term "non-dairy milk" refers or can refer to almond milk, cashew milk, coconut milk, rice milk, rye milk, soy milk, hemp milk, oat milk, pea milk, peanut milk, spelt milk, barley milk, fonio milk, maize milk, millet milk, sorghum milk, teff milk, triticale milk and wheat milk.

[0022] The term "milk-based product" refers or can refer to any product that primarily contains milk or milk derivatives as a key ingredient. This can include products made from whole, skimmed, or partially skimmed milk, as well as those containing milk components such as whey, casein, lactose, or milk fat.

[0023] The term "fat" of "milk fat" refers or can refer to the fat content of a milk or milk-based substrate, wherein the milk may be a non-dairy milk and / or wherein the milk-based substrate may be a non-dairy milk-based Substrate. This can be expressed as % Wfa / Wmilk or milk-based product or non-dairy milk or non dairy milk-based substrate-

[0024] "Phospholipase" is an enzyme that plays a role in the metabolism of phospholipids. The phospholipase which may be used for the present application includes phospholipase A such as phospholipase Al (EC 3.1.1.32) and phospholipase A2 (EC 3.1.1.4), phospholipase B (EC 3.1.1.5), phospholipase C (EC 3.1.4.3) and phospholipase D (EC 3.1.4.4). Phospholipases Al (EC 3.1.1.32), A2 (EC 3.1.1.4) and B (EC 3.1.1.5) are considered Carboxylic Ester Hydrolases (EC 3.1.1), meaning that they cleave the same bond C-0 in R1-O=C-O-R2 but they do it in different parts of the substrate. Phospholipases C (EC 3.1.4.3) and D (EC 3.1.4.4) are considered Phosphoric Diester Hydrolases (EC 3.1.4). P8184PC00 (17965-WO-PCT)

[0025] 3

[0026] The phospholipase may have two or more types of phospholipase activity such as phospholipase Al and phospholipase A2 activity, phospholipase Al and phospholipase B activity, or phospholipase A2 phospholipase B activity.

[0027] The phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian), e.g., from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom. Alternatively, the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g. A. niger; Dictyostelium, e.g. D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa; Rhizomucor, e.g. R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, e.g. S. libertiana; Trichophyton, e.g. T. rubrum; Whetzelinia, e.g. W. sclerotiorum; Bacillus, e.g. B. megaterium, B. subtilis; Citrobacter, e.g. C. freundii; Enterobacter, e.g. E. aerogenes, E. cloacae; Edwardsiella, e.g. E. tarda; Erwinia, e.g. E. herbicola; Escherichia, e.g. E. coli; Klebsiella, e.g. K. pneumoniae; Proteus, e.g. P. vulgaris; Providencia, e.g. P. stuartii; Salmonella, e.g. S. typhimurium; Serratia, e.g. S. liguefasciens, S. marcescens; Shigella, e.g. S. flexneri; Streptomyces, e.g. S. violeceoruber; Yersinia, e.g. Y. enterocolitica. The phospholipase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, F. venenatum, or a strain of F. oxysporum. The phospholipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae. A preferred phospholipase is derived from a strain of Fusarium, particularly from F. venenatum or F. oxysporum, e.g., from strain DSM 2672 as described in WO 98 / 26057, especially described in claim 36 of WO 98 / 26057. In further embodiments, the phospholipase is a phospholipase as disclosed in WO 00 / 32758 (Novozymes A / S, Denmark). Another preferred phospholipase A is phospholipase A2 from Streptomyces, such as e.g., PLA2 from S. violaceoruber such as the one described in JP2013252077A or its mature sequence or even a phospholipase as described in EP3078741.

[0028] Phospholipases are commercially available under different names, such as YieldMax® (Chr. Hansen A / S, Denmark), Lecitase® Ultra, Lecitase® Novo, Lipopan® F, Lipopan® Xtra, Quara® LowP and Quara® Boost (from Novozymes A / S, Denmark), Purafine® PLA1, Purafine® LM, Maxapal® A2, Gumzyme®, CakeZyme® and BakeZyme® (DSM Food Specialties, The Netherlands), Rohalase® F and Rohalase® PL-Extra (AB Enzymes, Germany), LysoMax® (Genencor, USA), and Polirase® Neo from Nagase (Nagase ChemteX Corporation).

[0029] Other food-grade phospholipases are well known to a skilled person in the art and can be found for example in Casado, Victor, et al. "Phospholipases in food industry: a review." Lipases and Phospholipases: Methods and Protocols (2012): 495-523.

[0030] "Phospholipase activity", such as "phospholipase Al activity", is measured relative to a phospholipase standard using lecithin as a substrate. The enzyme catalyzes the hydrolysis of lecithin to lyso-lecithin and a free fatty acid. The liberated fatty acid is titrated with 0.1 N sodium hydroxide under standard conditions (pH=8.0; 40 °C ± 0.5). The activity of the enzyme is determined as the rate of sodium hydroxide consumption P8184PC00 (17965-WO-PCT)

[0031] 4 during neutralization of the fatty acid and is expressed in Lecitase units (Leu) relative to a Lecitase (phospholipase) standard.

[0032] "1 Leu" is defined as the amount of enzyme that under standard conditions (pH=8.0; 40 °C ± 0.5) results in the same rate of sodium hydroxide consumption (in microeq / min) as the Lecitase standard diluted to a nominal activity of 1 Leu / g. The quantification limit of the method is approximately 1,5 Leu / mL The method can be carried out using either an automated system or standard laboratory equipment for carrying out titration experiments. Procedures and calculations for both the automated and manual versions are described. Further details can be found in the Specifications prepared at the 65th JECFA (2005), published in the Combined Compendium of Food Additive Specifications, Food and Agriculture Organization of the United Nations (FAO) JECFA Monographs 1 (2005). Alternatively, details can also be found in "Safety evaluation of the food enzyme phospholipase Al from the genetically modified Aspergillus oryzae strain NZYM-PP" published on EFSA Journal 2023; 21(2):7835. Alternatively, and as explained in par. 47 or in par. 120-122, both passages disclosed in US2004 / 0253680 Al, phospholipase activity may e.g., be measured in the LEU assay by hydrolyzing soy lecithin (L-alfa-phosphatidyl-choline) at pH 8.0 and 40° C for 2 minutes. Phospholipase activity is expressed as the rate of titrant consumption (0.1 M NaOH) necessary for keeping constant pH, relative to a standard. Identical passages can also be found in W02004 / 097012. Finally, determining the phospholipase activity is well known in the art.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] Fig. 1 - Foam volume (in mL) over time (sec) of samples of milk having 0.1% of fat, treated at 20 °C, 35 °C, 45 °C, 55 °C and 65 °C, with and without phospholipase.

[0035] Fig. 2 - Foam volume (in mL) over time (sec) of samples of milk having 0.4% of fat, treated at 20 °C, 35 °C, 45 °C, 55 °C and 65 °C, with and without phospholipase.

[0036] Fig. 3 - Foam volume (in mL) over time (sec) of samples of milk having 1.5% of fat, treated at 20 °C, 35 °C, 45 °C, 55 °C and 65 °C, with and without phospholipase.

[0037] Fig. 4 - Foam volume (in mL) over time (sec) of samples of milk having 3.5% of fat, treated at 20 °C, 35 °C, 45 °C, 55 °C and 65 °C, with and without phospholipase.

[0038] Fig. 5 - Foam volume (in mL) over time (sec) of samples of milk having 3.5% of fat, treated at 20 °C and 66 °C, with and without phospholipase.

[0039] Fig. 6 - Foam volume (in mL) over time (sec) of samples of milk having 3.5% of fat, treated at 65 °C, with and without phospholipase. P8184PC00 (17965-WO-PCT)

[0040] 5

[0041] Fig. 7 - Foam volume (in mL) over time (sec) of samples of milk having 1.5% of fat, treated at 75 °C and 85 °C, with and without phospholipase.

[0042] Fig. 8 - Foam volume (in mL) over time (sec) of samples of milk having 3.5% of fat, treated at 75 °C and 85 °C, with and without phospholipase.

[0043] Fig. 9 - Maximum foam volume (in mL) of samples of milk having 5% of fat, treated at 35 °C, 45 °C, 55 °C, 65 °C, 75 °C and 85 °C, with and without phospholipase.

[0044] Fig. 10 - Foam stability (sec) of samples of milk having 5% of fat, treated at 35 °C, 45 °C, 55 °C, 65 °C, 75 °C and 85 °C, with and without phospholipase.

[0045] Fig. 11 - Foam volume at 400 seconds (in mL) of samples of milk having 5% of fat, treated at 35 °C, 45 °C, 55 °C, 65 °C, 75 °C and 85 °C, with and without phospholipase.

[0046] DETAILED DESCRIPTION

[0047] The present disclosure relates to a method for foaming and / or stabilizing foam in a milk or milk-based product, comprising the following steps:

[0048] I) submitting the milk or milk-based product to a temperature between 35 °C - 80 °C, such that denaturation of whey protein and / or fat takes place; ii) steaming or whipping or frothing the milk or milk-based product such that foaming and / or stabilized foam in the milk or milk-based product is obtained; wherein the milk or milk-based product comprises a phospholipase and has 0.2% - 5% of preferably wherein the phospholipase degrades (or converts) phospholipids found in the milk or milk-based substrate into lyso-phospholipids which will stabilize the foam created by this method.

[0049] It should be understood that by "submitting ... to a temperature" is meant that the milk or the milk-based product is brought to a temperature between 35 °C - 80 °C, e.g., by heating the milk or the milked-based product.

[0050] The step of ii) may be carried out during or after the step of I), such that when the step of ii) is performed, the temperature of the milk or milk-based product is the same or close to the temperature that the milk was submitted to in step I). Preferably, when the step of ii) is performed, the temperature of the milk or milkbased product is within the same range of temperatures that are possible submission temperatures of step I). I.e., the step of ii) may preferably be carried out when the milk or milk-based product has a temperature between 35 °C - 80 °C, such as 35 °C - 75 °C, preferably 45 °C - 68 °C, even more preferably 55-66 °C. Alternatively, the step of ii) may be preferably carried out when the milk or milk-based product has a P8184PC00 (17965-WO-PCT)

[0051] 6 temperature between 45 °C - 75 °C, even more preferably 55-68 °C. In another embodiment, the step of ii) may be preferably carried out when the milk or milk-based product has a temperature between 45 °C - 80 °C, preferably 55 °C - 75 °C.

[0052] In a preferred embodiment the step of ii) may be performed during the step of i). In another embodiment, the step of ii) may be performed after the step of i), such that the temperature of the milk or milk-based product has a temperature within the submission temperature range of step i).The examples of the present disclosure show, indeed, that more foam, which is stable for longer periods of time, can be achieved when a phospholipase is used, preferably a phospholipase as herein disclosed either in the definition section or in one of the herein preferred embodiments.

[0053] In a preferred embodiment, the milk or milk-based product may have 0.4% - 4% Wfat / wmiik preferably 1 that more foam, which is stable for longer periods of time, can be achieved with at least 0.4% to least 3.5% milk fat, more preferably 0.4% to 3.5% milk fat, even more preferably with 1.5% to 3.5% milk fat. In another preferred embodiment, the milk or milk-based product may have 0.3% - 6% Wfat / wmiik preferably 0.4% - 5% Wfat / wmiik In another preferred embodiment the milk or milk-based product may have

[0054] In a preferred embodiment, the phospholipase comprised in the milk or milk-based product may be added to the milk or milk-based product before or at the same time or after submitting the milk or milk-based product to the temperature between 25 °C - 80 °C.

[0055] In an embodiment, the phospholipase is allowed to react with phospholipids of the milk or milk-based product before the step of i). In a preferred version of this embodiment, the phospholipase may be allowed to react with the phospholipids for at least 2 minutes, preferably at least 5 minutes, more preferably at least 10 minutes, even more preferably at least 20 minutes or at least 30 minutes, most preferably at least 24 hours or until the reaction has substantially reached equilibrium. In an embodiment according to any of the previous two embodiments, the phospholipase may be allowed to react with the milk or milk-based product when the milk or milk-based product is at a temperature of 4 °C - 30 °C, preferably 5 °C - 28 °C, more preferably 10 °C - 25 °C, most preferably 15 °C - 20 °C.

[0056] For longer phospholipase treatment times, e.g. at least 24 hours, it is preferred that the temperature of the milk or milk-based product be below 30 °C, more preferably below 25°C, most preferably below 22°C. As such, denaturation of whey proteins are minimized.

[0057] In a preferred embodiment, the phospholipase comprised in the milk or milk-based product may be added to the milk or milk-based product at least 2 minutes, preferably at least 5 minutes, more preferably at least 10 minutes, even more preferably at least 20 minutes or at least 30 minutes, most preferably at least 24 P8184PC00 (17965-WO-PCT)

[0058] 7 hours, before submitting the milk or milk-based product to the temperature between 25 °C - 80 °C, thereby contributing to improve or further improve foam stability of the milk or milk-based product. In a preferred embodiment, the phospholipase comprised in the milk or milk-based product may be added to the milk or milk-based product when the milk or milk-based product is at a temperature of 4 °C - 30 °C, preferably 10 °C - 25 °C, more preferably 15 °C - 20 °C, thereby contributing to improve or further improve foam stability of the milk or milk-based product.

[0059] In a preferred embodiment, the step of submitting the milk or milk-based product may be carried out at the temperature between 35 °C - 66 °C, preferably 45 °C - 66 °C, even more preferably 55-66 °C. Indeed, examples show that more foam, which is stable for longer periods of time, can be achieved when temperatures of at least 35 °C to least 66 °C are used, more preferably 35 °C to 66 °C, even more preferably 45 °C to 66 °C, even more preferably 55 °C to 66 °C.

[0060] In a preferred embodiment, the step of submitting the milk or milk-based product may be carried out at the temperature between 55 °C - 85 °C, preferably 65 °C - 75 °C, and the milk or milk-based product has a fat content fat content o or milk-based product has a fat content of 1.5% - 5% Wfat / wmiik a preferred embodiment, the milk or milk-based product may comprise 0.001-0.250 mg of phospholipase / L of milk, preferably 0.010-0.180 mg of phospholipase / L of milk, more preferably 0.050-0.150 mg of enzyme / L of milk.

[0061] In a preferred embodiment, the phospholipase may be selected from a list consisting of a phospholipase A, such as phospholipase Al or A2, a phospholipase B, a phospholipase C, a phospholipase D, and a combination thereof.

[0062] In a preferred embodiment, the phospholipase may be selected from a list consisting of a phospholipase Al, a phospholipase A2, a phospholipase C, and a combination thereof.

[0063] In an even more preferred embodiment, the phospholipase may be selected from a list consisting of a phospholipase Al, a phospholipase A2 and a combination thereof. This even more preferred embodiment is supported by Examples 1-4 and Example 5. The phospholipase is most preferably a phospholipase A2, which is supported by example 6.

[0064] In a preferred embodiment the phospholipase may either have phospholipase Al or A2 as its main activity.

[0065] In a preferred embodiment the phospholipase may have phospholipase A2 as its main activity.

[0066] In another preferred embodiment the phospholipase may have phospholipase Al activity and phospholipase A2 activity. In a more preferred version of this embodiment, the phospholipase may have more phospholipase Al activity than phospholipase A2 activity.

[0067] In a preferred embodiment, the phospholipase may have a sequence identity of at least 80% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, preferably at least 90% or at least 91%, or at least 92%, or P8184PC00 (17965-WO-PCT)

[0068] 8 at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 3 or SEQ ID NO: 4.

[0069] In a preferred embodiment, the phospholipase may have a sequence identity of at least 80% to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 SEQ or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, more preferably at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 SEQ or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16.

[0070] In a most preferred embodiment, the phospholipase may have a sequence identity of at least 80% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7, or SEQ ID NO: 8, such as at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7, or SEQ ID NO: 8.

[0071] In an embodiment, the milk may be a dairy milk or a non-dairy milk, preferably wherein the dairy milk may be selected from a list consisting of: cow milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk and camel milk or wherein the non-dairy milk is selected from a list consisting of: almond milk, cashew milk, coconut milk, rice milk, rye milk, soy milk, hemp milk, oat milk, pea milk, peanut milk, spelt milk, barley milk, fonio milk, maize milk, millet milk, sorghum milk, teff milk, triticale milk and wheat milk.

[0072] In another aspect the present invention relates to a use of a phospholipase-treated milk or milk-based product for creating milk foam. In particular, the use comprises steaming or whipping or frothing the phospholipase-treated milk or milk-based product at a temperature in the range of 35 °C - 80 °C.

[0073] The steaming or whipping or frothing may preferably be carried out when the phospholipase-treated milk or milk-based product has a temperature between 35 °C - 75 °C, preferably 45 °C - 68 °C, even more preferably 55-66 °C. Alternatively, the phospholipase-treated milk or milk-based product has a temperature between 45 °C - 75 °C, even more preferably 55-68 °C. In another embodiment, the milk or milk-based product has a temperature between 45 °C - 80 °C, preferably 55 °C - 75 °C.

[0074] The use is preferably for creating milk foam in a coffee beverage.

[0075] The phospholipase-treated milk may preferably have been treated with a phospholipase selected from a list consisting of a phospholipase A, such as phospholipase Al or A2, a phospholipase B, a phospholipase C, a phospholipase D, and a combination thereof.

[0076] The phospholipase-treated milk may preferably have been treated with a phospholipase selected from a list consisting of a phospholipase Al, a phospholipase A2, a phospholipase C, and a combination thereof. P8184PC00 (17965-WO-PCT)

[0077] 9

[0078] In an even more preferred embodiment, the phospholipase-treated milk may preferably have been treated with a phospholipase selected from a list consisting of a phospholipase Al, a phospholipase A2 and a combination thereof.

[0079] In a preferred embodiment the phospholipase-treated milk may preferably have been treated with a phospholipase that has either phospholipase Al or A2 as its main activity.

[0080] In a preferred embodiment the phospholipase may have phospholipase A2 as its main activity.

[0081] In another preferred embodiment the phospholipase-treated milk may preferably have been treated with a phospholipase that has phospholipase Al activity and phospholipase A2 activity. In a more preferred version of this embodiment, the phospholipase used may have more phospholipase Al activity than phospholipase A2 activity.

[0082] In a preferred embodiment, the phospholipase used may have a sequence identity of at least 80% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, preferably at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 3 or SEQ ID NO: 4.

[0083] In a preferred embodiment, the phospholipase used may have a sequence identity of at least 80% to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 SEQ or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, more preferably at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 SEQ or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16.

[0084] In a most preferred embodiment, the phospholipase used may have a sequence identity of at least 80% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7, or SEQ ID NO: 8, such as at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 7, or SEQ ID NO: 8.

[0085] EXAMPLES

[0086] The examples illustrate the foaming properties of milks with different fat content (0.1%, 0.4%, 1.5%, 3.5%, 5%) at different temperatures (20 °C, 35 °C, 45 °C, 55 °C, 65 °C, 75 °C, 85 °C), with and without the addition of a phospholipase, and different enzyme treatment time (in the range of 2-30 minutes, about 1 hour, about 24 hour). P8184PC00 (17965-WO-PCT)

[0087] 10

[0088] Enzymes

[0089] The phospholipase used in the Examples are listed below in table (i).

[0090] Table (i)

[0091] EXAMPLE 1

[0092] Samples (10 in total) were prepared as follows. Each sample was made with 200 mL of milk having 0.1% of fat and placed in a 500 mL bottle. Half of the samples was treated with a phospholipase, while the remaining half was left untreated. The dosage of phospholipase used was 0.144 mg of enzyme / L of milk (in particular 0.144 mg of active enzyme / L of milk), per treated sample. Samples were placed at approximately 20 °C, for a given period which may go from a few seconds to more than 1 hour, for example, for least 2 minutes, at least 5 minutes, at least 10 minutes or at least 20 minutes or at least 30 minutes.

[0093] Every two samples, one treated and one untreated, was submitted to 20 °C, 35 °C, 45 °C, 55 °C or 65 °C, respectively. Once said temperature was reached, the sample was analyzed immediately for foaming properties using FOAMSCAN™ from Teclis Scientific (apparatus number F2023.00.15 and software

[0094] FOAMSCAN for windows version 3.15.0.0) - see Table 1. P8184PC00 (17965-WO-PCT)

[0095] 11

[0096] The phospholipase used was YieldMax® from Chr. Hansen A / S, herein named as enzyme A, which is or can be represented by SEQ ID NO: 1 or 2 (full or mature sequence, respectively).

[0097] Foam was generated by gas injection, such as nitrogen gas or compressed atmospheric air, into each sample. Gas sparging of app. 60 mL test sample (milk) by nitrogen gas injecting through a porous glass filter frit with a suitable pore size at a gas flow rate of 100 mL / min and injected for 90 seconds giving a total gas injection volume as shown in the respective figure. Video monitoring up to 1800 seconds with measurement of final foam volume immediately after gas sparging and foam volume stability as the time for 50% foam volume settlement.

[0098] The results are shown in Fig. 1. Table 1. List of samples tested in example 1.

[0099] Fig. 1 shows that all samples foam, regardless of the presence of phospholipase or temperature tested.

[0100] Sample 1.1, prepared without phospholipase and heated to 20 °C, foams less than the remaining samples.

[0101] Further this sample has about 25% less foam after 180 sec than the remaining samples at the same point in time, showing that the sample has less foam stability than the other ones.

[0102] EXAMPLE 2

[0103] Example 2 was performed as example 1 except that a milk with 0.4% of fat was used. The results are shown in Fig. 2.

[0104] Table 2. List of samples tested in example 2. P8184PC00 (17965-WO-PCT)

[0105] 12

[0106] Fig. 2 shows that samples with phospholipase and submitted to temperatures of at least 35 °C, such as a temperature of 35 °C (sample 2.4), 45 °C (sample 2.6), 55 °C (sample 2.8) and 65 °C (sample 2.10), foam and can maintain foam volume without significant losses for at least 100 sec after reaching the max of foam volume. Temperatures of 45 °C (sample 2.6), 55 °C (sample 2.8) and 65 °C (sample 2.10) are preferred. The ability to foam and maintain foam volume is also observed for samples without phospholipase and submitted to temperatures of 55 °C and 65 °C (samples 2.7 and 2.9).

[0107] On the other hand, samples at 20 °C (with or without phospholipase) and at 35 °C without phospholipase (sample 2.3), are no longer able to foam when a milk with 0.4% fat is used. In addition, samples at 45 °C without phospholipase (sample 2.5) have their ability to foam significantly impaired when compared with samples 2.4 and 2.6 - 2.10.

[0108] EXAMPLE 3

[0109] Example 3 was performed as the previous examples except that a milk with 1.5% of fat was used. The results are shown in Fig. 3. Table 3. List of samples tested in example 3. P8184PC00 (17965-WO-PCT)

[0110] 13

[0111] Fig. 3 shows that samples with phospholipase and submitted to a temperature of 45 °C, 55 °C and 65 °C (samples 3.6, 3.8 and 3.10), preferably 55 °C and 65 °C (samples 3.8 and 3.10), foam better than samples without phospholipase and submitted to the same temperature range, while simultaneously maintaining foam stability. On the other hand, all the remaining samples have their foaming ability impaired.

[0112] EXAMPLE 4

[0113] Example 4 was performed as the previous examples except that a milk with 3.5% of fat was used. The results are shown in Fig. 4.

[0114] Table 4. List of samples tested in example 4.

[0115] Fig. 4 shows a similar result to the one of example 3, that is samples with phospholipase and submitted to a temperature of 45 °C, 55 °C and 65 °C (samples 4.6, 4.8 and 4.10), preferably 55 °C and 65 °C (samples 4.8 and 4.10), foam well and betterthan samples without phospholipase and submitted to the same temperature P8184PC00 (17965-WO-PCT)

[0116] 14 range, while simultaneously maintaining foam stability. On the other hand, all the remaining samples have their foaming ability impaired.

[0117] The above examples show the unexpected effect that temperature and fat content of milk (or milk-based substrate) have on foam volume and foam stability when a phospholipase is present.

[0118] Preferably, the examples show that more foam, which is stable for longer periods of time, can be achieved with at least 0.4% to least 3.5% milk fat, more preferably 0.4% to 3.5% milk fat, even more preferably with 1.5% to 3.5% milk fat.

[0119] Preferably, the examples show that more foam, which is stable for longer periods of time, can be achieved with a phospholipase.

[0120] Preferably the examples show that more foam, which is stable for longer periods of time, can be achieved when temperatures of at least 35 °C to least 65 °C (or 66 °C) are used, more preferably 35 °C to 65 °C (or 66 °C), even more preferably 45 °C to 65 °C (or 66 °C), even more preferably 55 °C to 65 °C (or 66 °C).

[0121] The above examples illustrate that samples with phospholipase, submitted to temperatures in a range of at least 35 °C to at least 65 °C (or 66 °C), preferably 35 °C to 65 °C (or 66 °C), more preferably 45 °C to 65 °C (or 66 °C), even more preferably 55 °C to 65 °C (or 66 °C), perform as well or even better than samples without a phospholipase and at the same temperature ranges when the milk fat content is above 0.4% to at least 3.5%, preferably 0.4% to 3.5% milk fat, more preferably with 1.5% to 3.5% milk fat.

[0122] In other words, examples 3 and 4 show that adding a phospholipase to a milk (or milk-based substrate) with a fat content above 0.4% and at least 3.5%, preferably with a fat content between 1.5% and 3.5%, and at a temperature of above 35 °C and at least 65 °C (or 66 °C), preferably 45 °C and 65 °C (or 66 °C), leads to more foam volume than when a temperature of 65 °C (for example) is used without any phospholipase. Thus, examples 3 and 4 show that it is possible to improve the method of foaming milk currently used by a Barista if the milk (or milk-based substrate used comprises a phospholipase, has 0.2% - 5% of Wfat / wmiik or milk-based product and is submitted to a temperature above 25 °C.

[0123] In other words, example 2 show that adding a phospholipase to a milk (or milk-based substrate) with a fat content above 0.1% and at least 0.4%, preferably with a fat content of 0.4%, and at a temperature of above 35 °C and at least 65 °C (or 66 °C), preferably 45 °C and 65 °C (or 66 °C), leads to the same (or identical) foam volume than when a temperature of 65 °C (for example) is used without any phospholipase. Thus, example 2 shows that it is possible to provide an alternative method of foaming milk currently used by a Barista if the milk (or milk-based substrate used comprises a phospholipase, has 0.2% - 5% of Wfat / wmiik or milk-based product and is submitted to a temperature above 25 °C. P8184PC00 (17965-WO-PCT)

[0124] 15

[0125] In addition, the above examples also illustrate the possibility of adding a phospholipase (like YieldMax® from Chr. Hansen A / S or a similar one) to cold unhomogenized whole-milk (3.5% fat) or semi-skimmed milk (1.5% fat) at the dairy letting the hydrolysis of phospholipids, preferably letting the hydrolysis of phospholipids and formation of lyso-phospholipids, to take place at cold storage. This will lead to the generation of more voluminous, longer standing and more creamy milk foam than when steam foaming by barista coffee making is used. Adding a phospholipase (like YieldMax® from Chr. Hansen A / S or a similar one) to skimmed milk (0.1% fat) or semi-skimmed milk (0.4% fat) may facilitate for the same beneficial effects, also by cold milk frothing, e.g., in household barista coffee making.

[0126] EXAMPLE 5

[0127] Example 5 was performed as previous example 4, i.e., with milk having 3.5% of fat. Enzyme and dosage used for samples 5.3 and 5.4 were the same as in Examples 1-4. The dosage of enzyme for samples 5.5 and 5.6 was 0.130 mg of enzyme / L of milk (in particular 0.130 mg of active enzyme / L of milk). The enzyme used was a phospholipase A2 which can be selected from any commercial phospholipase A2 on the market, e.g., it can be Polirase® Neo, herein labelled as enzyme B. The sequence of a phospholipase A2 is or can be represented by SEQ ID NO: 3 or 4 (full or mature sequence, respectively).

[0128] Table 5. List of samples tested in example 5.

[0129] Example 5 shows that 2 different phospholipases perform similarly under the same conditions, regardless of if they are described in the literature as phospholipase Al or A2. Therefore, similar results are expected for other phospholipases Al and A2. A similar result is also expected for other phospholipases, for example, phospholipase B.

[0130] EXAMPLE 6

[0131] Example 6 was performed as previous example 5, i.e., with milk having 3.5% of fat. Enzyme used are listed in table (i). Enzymes were dosed at 0.144 mg protein / L, wherein the protein is the total protein in the enzyme material used. The milk was treated with enzyme for about 24 hours at room temperature (20 °C). Every two samples, one treated and one untreated, was submitted to 65 °C. Once said temperature was reached, the P8184PC00 (17965-WO-PCT)

[0132] 16 sample was analyzed immediately for foaming properties using FOAMSCAN™ from Teclis Scientific (software

[0133] FOAMSCAN for windows version 4.3.9.0). The results are shown in Fig. 6.

[0134] EXAMPLE 7

[0135] Example 7 was performed as previous example 3, i.e., with milk having 3.5% of fat.

[0136] Every two samples, one treated and one untreated, was submitted to 75 °C or 85 °C, respectively. Once said temperature was reached, the sample was analyzed immediately for foaming properties using FOAMSCAN™ from Teclis Scientific (software FOAMSCAN for windows version 4.3.9.0). The results are shown in Fig. 7.

[0137] EXAMPLE 8

[0138] Example 8 was performed as previous example 7, albeit with milk having 3.5% of fat. The results are shown in Fig. 8.

[0139] EXAMPLE 9

[0140] Example 9 was performed as previous example 8, albeit with milk having 5% of fat and at different temperatures. Every two samples, one treated and one untreated, was submitted to 75 °C or 85 °C, respectively. Once said temperature was reached, the sample was analyzed immediately for foaming properties using FOAMSCAN™ from Teclis Scientific. The results are shown in Figs. 9-11.

[0141] "Noise floor" or "NF" as mentioned in Figs. 9-11 is defined as a foam volume that is below 20 mL. Below the 20 mL, artefacts and signal noise are observed (data not shown).

[0142] "Max foam volume" as mentioned in Fig. 9. is defined as the maximal foam volume reached during the 1800 seconds of measurement.

[0143] "Foam stability" as mentioned in Fig. 10. is defined as the time during the 1800 seconds of measurement that the foam volume was higher than the noise floor.

[0144] Foam volume observed at 400 seconds are shown in Fig. 11.

[0145] The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

[0146] All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any preferred embodiment(s) disclosed. P8184PC00 (17965-WO-PCT)

[0147] 17

[0148] SEQUENCE LISTING

[0149] SEQID NO: 1

[0150] MKFSATILSLLPAVLALPTGEDASVSKRQSVNTVTDQLLFSVTLPQFTARRNARDPPTVDWTSDGCTSSPDNPFGFPFIPAC NRHDFGYHNYRAQSRFTVSAKSRIDNNFKTDLYFQCQSSSVSGVCRALADVYFAAVRAFGGDDATPGKRDEALVKEYEKKV EVYNKLVEEAQKKGDLPRLD

[0151] SEQID NO: 2

[0152] LPTGEDASVSKRQSVNTVTDQLLFSVTLPQFTARRNARDPPTVDWTSDGCTSSPDNPFGFPFIPACNRHDFGYHNYRAQSR FTVSAKSRIDNNFKTDLYFQCQSSSVSGVCRALADVYFAAVRAFGGDDATPGKRDEALVKEYEKKVEVYNKLVEEAQKKGDL PRLD

[0153] SEQID NO: 3

[0154] MRTTTRTRTTLAAVGAALALGVAAAPAQAAPADKPQVLASFTQTSASSQNAWLAANRNQSAWAAYEFDWSTDLCTQAP DNPFGFPFNTACARHDFGYRNYKAAGSFDANKSRIDSAFYEDMKRVCTGYTGEKNTACNSTAWTYYQAVKIFG

[0155] SEQID NO: 4

[0156] AYEFDWSTDLCTQAPDNPFGFPFNTACARHDFGYRNYKAAGSFDANKSRIDSAFYEDMKRVCTGYTGEKNTACNSTAWT YYQAVKIFG

[0157] SEQID NO: 5

[0158] MKFLVLAVLLTVGAAQEGISSRALWQFRSMIKCAIPGSHPLMDFNNYGCYCGLGGSGTPVDELDRCCETHDNCYRDAKNL DSCKFLVDNPYTESYSYSCSNTEITCNSKNNACEAFICNCDRNAAICFSKAPYNKEHKNLDTKKYC

[0159] SEQID NO: 6

[0160] QEGISSRALWQFRSMIKCAIPGSHPLMDFNNYGCYCGLGGSGTPVDELDRCCETHDNCYRDAKNLDSCKFLVDNPYTESYS YSCSNTEITCNSKNNACEAFICNCDRNAAICFSKAPYNKEHKNLDTKKYC

[0161] SEQID NO: 7

[0162] MKFLVLAVLLTVGAAQEGISRRALWQFRSMIKCAIPGSHPLMDFNNYGCYCGLGGSGTPVDELDRCCETHDNCYRDAKNL DSCKFLVDNPYTESYSYSCSNTEITCNSKNNACEAFICNCDRNAAICFSKAPYNKEHKNLDTKKYC

[0163] SEQID NO: 8

[0164] ALWQFRSMIKCAIPGSHPLMDFNNYGCYCGLGGSGTPVDELDRCCETHDNCYRDAKNLDSCKFLVDNPYTESYSYSCS NTEITCNSKNNACEAFICNCDRNAAICFSKAPYNKEHKNLDTKKYC

[0165] SEQID NO: 9

[0166] MKKKVLALAAAITLVAPLQSVAFAHENDGGSKIKIVHRWSAEDKHKEGVNSHLWIVNRAIDIMSRNTTLVKQDRVAQLNE WRTELENGIYAADYENPYYDNSTFASHFYDPDNGKTYIPFAKQAKETGAKYFKLAGESYKNKDMKQAFFYLGLSLHYLGDV NQPMHAANFTNLSYPQGFHSKYENFVDTIKDNYKVTDGNGYWNWKGTNPEDWIHGAAVVAKQDYSGIVNDNTKDWF

[0167] VKAAVSQEYADKWRAEVTPMTGKRLMDAQRVTAGYIQLWFDTYGDR

[0168] SEQID NO: 10

[0169] HENDGGSKIKIVHRWSAEDKHKEGVNSHLWIVNRAIDIMSRNTTLVKQDRVAQLNEWRTELENGIYAADYENPYYDNSTF ASHFYDPDNGKTYIPFAKQAKETGAKYFKLAGESYKNKDMKQAFFYLGLSLHYLGDVNQPMHAANFTNLSYPQGFHSKYE NFVDTIKDNYKVTDGNGYWNWKGTNPEDWIHGAAVVAKQDYSGIVNDNTKDWFVKAAVSQEYADKWRAEVTPMTGK

[0170] R LM DAQRVTAG Yl QLW F DTYG D R P8184PC00 (17965-WO-PCT)

[0171] 18

[0172] SEQID NO: 11

[0173] MTHLIGFAPRLLAFSALLLSQTAFSQESPAFIDPASWNTPFNGIAQVACHNCYEKQYANTFSSVLDSVRTLELDFWDQRDAV

[0174] SGGSPHHWFVRHNPGTLFQSGNDNNCTGDGTGKNDLEACLNDVKNWSDKHPGHFPITLILDKKQGWSKESSGRTPKDF DELVARVFQGKLFTPQDLATHIGSGAGALQGNLKGKSWPTANDLQGKVLLVLNHSENQKLSQYAEARTSKAKVFISPVTNG

[0175] QNDISGKVSGMSSQSSGYVAMNNMGKGDKSWAKQAFAYSHIGRVWGDDEVSFAQHINQKINLSAYYRFAAQSAGGYRI RPF

[0176] SEQID NO: 12

[0177] QESPAFIDPASWNTPFNGIAQVACHNCYEKQYANTFSSVLDSVRTLELDFWDQRDAVSGGSPHHWFVRHNPGTLFQSGN DNNCTGDGTGKNDLEACLNDVKNWSDKHPGHFPITLILDKKQGWSKESSGRTPKDFDELVARVFQGKLFTPQDLATHIGS GAGALQGNLKGKSWPTANDLQGKVLLVLNHSENQKLSQYAEARTSKAKVFISPVTNGQNDISGKVSGMSSQSSGYVAMN

[0178] NMGKGDKSWAKQAFAYSHIGRVWGDDEVSFAQHINQKINLSAYYRFAAQSAGGYRIRPF

[0179] SEQID NO: 13

[0180] WSAEDKHNEGINSHLWIVNRAIDIMSRNTTIVNPNETALLNEWRADLENGIYSADYENPYYDDSTYASHFYDPDTGTTYIPF AKHAKETGAKYFNLAGQAYQNQDMQQAFFYLGLSLHYLGDVNQPMHAASFTDLSYPMGFHSKYENFVDTIKNNYIVSDS NGYWNWKGANPEDWIEGAAVAAKQDYPGVVNDTTKDWFVKAAVSQEYADKWRAEVTPVTGKRLMEAQRVTAGYIHL

[0181] WFDTYVNR

[0182] SEQID NO: 14

[0183] MASSINVLENWSRWMKPINDDIPLARISIPGTHDSGTFKLQNPIKQVWGMTQEYDFRYQMDHGARIFDIRGRLTDDNTIV LHHGPLYLYVTLHEFINEAKQFLKDNPSETIIMSLKKEYEDMKGAESSFSSTFEKNYFRDPIFLKTEGNIKLGDARGKIVLLKRYS GSNESGGYNFFYWPDNETFTSTINGNVNVTVQDKYKVSLDEKINAIKDTLNETINNSEDVNHLYINFTSLSSGGTAWTSPYY

[0184] YASRINPEIANYIKQKNPTRVGWIIQDFINEKWHPLLYQEVINANKSLVK

[0185] SEQID NO: 15

[0186] MVQFKSVRTLAVAAFAALGAAAPAGLAERDVSASVLQKLSLFAQYSAAAYCTNNINSTGTKLTCSAGNCPLVEAANTKTLAE FYEAGSSESFGDTAGFLVADTTNKLLVVSFRGSRTIDNWIANLDFVLDSVSDICSGCAAHGGFWKSWEVVANSLTTELNSAV NTYPGYTIVFTGHSLGAALATLGATTLRKAGIPIQLYNYGSPRVGNTALATYITAQGPNYRVTHTNDIVPRLPPQAFGFSHLSP

[0187] EYWITSGDNVPVTTSDITVIQGIDSDAGNSGEDITSIEAHNWYLGDIDACQ

[0188] SEQID NO: 16

[0189] ERDVSASVLQKLSLFAQYSAAAYCTNNINSTGTKLTCSAGNCPLVEAANTKTLAEFYEAGSSESFGDTAGFLVADTTNKLLVV SFRGSRTIDNWIANLDFVLDSVSDICSGCAAHGGFWKSWEVVANSLTTELNSAVNTYPGYTIVFTGHSLGAALATLGATTLR KAGIPIQLYNYGSPRVGNTALATYITAQGPNYRVTHTNDIVPRLPPQAFGFSHLSPEYWITSGDNVPVTTSDITVIQGIDSDA

[0190] G NSG E D ITSI EAH N WYLG D I DACQ

Claims

P8184PC00 (17965-WO-PCT)19CLAIMS1. Method for foaming and / or stabilizing foam in a milk or milk-based product, comprising the following steps: i) submitting the milk or milk-based product to a temperature between 35 °C - 80 °C; ii) steaming or whipping or frothing the milk or milk-based product such that foaming and / or stabilized foam in the milk or milk-based product is obtained; wherein the milk or milk-based product comprises a phospholipase and has 0.2% - 5% of2. Method according to the previous claim, wherein the milk or milk-based product has 0.4% - 4%3. Method according to any of previous claims, wherein the phospholipase comprised in the milk or milk-based product is added to the milk or milk-based product before the step of i).

4. Method according to previous claim, wherein the phospholipase is allowed to react with phospholipids of the milk or milk-based product before the step of i).

5. Method according to previous claim, wherein in the step of allowing the phospholipase to react with phospholipids of the milk or milk-based product, the phospholipase is allowed to react with the phospholipids for at least 2 minutes, preferably at least 5 minutes, more preferably at least 10 minutes, even more preferably at least 20 minutes or at least 30 minutes, most preferably at least 24 hours or until the reaction has substantially reached equilibrium.

6. Method according to previous claims 4 or 5, wherein the phospholipase is allowed to react with the milk or milk-based product when the milk or milk-based product is at a temperature of 4 °C - 30 °C, preferably 5 °C - 28 °C, more preferably 10 °C - 25 °C, most preferably 15 °C - 20 °C.

7. Method according to any of previous claims, wherein the step of ii) is carried out at the temperature between 35 °C - 70 °C, preferably 45 °C - 68 °C, even more preferably 55-66 °C.

8. Method according to any of previous claims, wherein the step of ii) is preferably carried out when the milk or milk-based product has a temperature between 35 °C - 80 °C, such as between 35 °C -70 °C, preferably between 45 °C - 68 °C, even more preferably between 55-66 °C.P8184PC00 (17965-WO-PCT)209. Method according to any of the previous claims, wherein the milk or milk-based product comprises 0.001-0.250 mg of phospholipase / L of milk, preferably 0.010-0.180 mg of phospholipase / L of milk, more preferably 0.050-0.150 mg of enzyme / L of milk.

10. Method according to any of the previous claims, wherein the phospholipase is selected from a list consisting of a phospholipase A, such as phospholipase Al or A2, a phospholipase B, a phospholipase C, a phospholipase D, and a combination thereof.

11. Method according to the previous claim, wherein the phospholipase is selected from a list consisting of a phospholipase Al, a phospholipase A2, a phospholipase B, and a combination thereof.

12. Method according to claim 10, wherein the phospholipase is selected from a list consisting of a phospholipase Al, a phospholipase A2, a phospholipase C, and a combination thereof.

13. Method according to previous claim, wherein the phospholipase is selected from a list consisting of a phospholipase Al and a phospholipase A2 or a combination thereof, preferably a phospholipase A2.

14. Method according to any previous claim, wherein the phospholipase has phospholipase Al activity and phospholipase A2 activity.

15. Method according to previous claim, wherein the phospholipase has more phospholipase Al activity than phospholipase A2 activity.

16. Method according to any of the previous claims, wherein the phospholipase has a sequence identity of at least 80% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, preferably at least 90% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, more preferably at least 95% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, even more preferably 100% to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.

17. Method according to any of the previous claims, wherein the phospholipase has a sequence identity of at least 80% to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16, more preferably at least 90% or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 SEQ or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15, or SEQ ID NO: 16.P8184PC00 (17965-WO-PCT)2118. Method according to any of previous claims, wherein the milk is dairy milk or non-dairy milk or wherein the milk-based product is a dairy milk-based product or a non-dairy milk-based product.

19. Method according to previous claim 11, wherein the dairy milk is selected from a list consisting of: cow milk, sheep milk, goat milk, buffalo milk, yak milk, lama milk and camel milk.

20. Method according to previous claim 12, wherein the non-dairy milk is selected from a list consisting of: almond milk, cashew milk, coconut milk, rice milk, rye milk, soy milk, hemp milk, oat milk, pea milk, peanut milk, spelt milk, barley milk, fonio milk, maize milk, millet milk, sorghum milk, teff milk, triticale milk and wheat milk.

21. Use of a phospholipase-treated milk or milk-based product for creating milk foam, wherein the phospholipase-treated milk or milk-based product is steamed or whipped or frothed at a temperature in the range of 35 °C - 80 °C.

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