A technology for modifying animal milk using a food-grade enzyme

Abstract

The present invention relates to a milk product, a UHT milk, and a dairy product obtainable form said milk product or UHT milk and methods for producing the same. The present invention relates in particular to a method for producing a UHT milk, comprising the step of (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase (PG), and further comprising the step of ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

Description

A technology for modifying animal milk using a food-grade enzyme

[0001] The present invention relates to methods for producing milk products, such as a UHT milk, from a raw milk including an enzymatic treatment step with a protein-glutaminase.

[0002] The market for dairy products which includes liquid milk, yogurt, cheese, milk drinks is increasing worldwide and there is an interest in improving the quality of the dairy products. The use of enzymes for modifying solubility, emulsification, foaming, and gelation properties of milk proteins has been described. For dairy products enzymatic modification is preferred because of its specificity, mild reaction conditions and safe modification. For example, a protein-glutaminase, also known as PG, catalyzes the deamidation of glutamine residue within proteins by converting the glutamine residue into glutamic acid. The deamination increases the negative charge, the electrostatic repulsion, the hydrating power, and decreases the isoelectric point of the protein. In addition, PG has no protease or glutamine transaminase activity. As a result, various functional characteristics of the protein may be changed, including solubility and dispersibility. PG has been previously used, e.g., for manufacturing of dairy products such as cheese or yogurt (see, e.g., EP 1 839 491), where PG may be added during various stages of processing. However, the effect of enzymatic treatment during the processing of a raw milk and the resulting effects have not yet been explored.

[0003] Raw milk provides an ideal environment for the multiplication of pathogenic microorganisms. To reduce the growth of microorganisms and improve shelf-life, raw milk is subjected to a pasteurization or sterilization treatment. Said treatments include low-temperature long time (LTLT) or a high-temperature short-time (HTST) pasteurization, as well as ultra-high temperature (UHT) sterilization. UHT sterilization involves heating milk to between 135 and 145℃ for less than 10 seconds. In comparison to more moderate treatments (such as LTLT or HTST pasteurization), a notable advantage of UHT sterilization is that it eliminates not only living microorganisms but also spore-forming pathogenic bacteria, thereby extending the shelf life of the raw milk and eliminating the necessity of refrigeration. For instance, milk subjected to UHT processing, called UHT milk, can have a shelf life exceeding six months when stored at room temperature. Consequently, UHT milk is popular. However, UHT processing can cause alterations in the physicochemical characteristics of the resulting UHT-milk, and their severity extends during storage and with increased temperatures. This can result in the development of off-flavors, undesirable browning, separation of fat, sediment formation, or age-gelation during subsequent storage (Krishna et al., 2021).

[0004] Hence, there is a need for a method of producing a UHT milk which maintains beneficial physiochemical properties after UHT processing, such as taste and textures, and is safe for consumption even when stored for a prolonged amount of time. Moreover, there is a need for a method of producing milk and milk-based products employing conditions that do not impair food safety and storage quality.

[0005] EP 2 332 421JP-A-2000-50887JP-A-2001-218590EP 1 839 49WO 2022 / 039281US 2011 / 0064847A1Non-Patent Literature

[0006] NPTL 1

[0007] Krishna, T.C.; Najda, A.; Bains, A.; Tosif, M.M.; Papliński, R.; Kapłan, M.; Chawla, P. Influence of Ultra-Heat Treatment on Properties of Milk Proteins. Polymers 2021, 13, 3164. doi: 10.3390 / polym13183164. PMID: 34578063.NPTL 2

[0008] Singh R, Rathod G, Meletharayil GH, Kapoor R, Sankarlal VM, Amamcharla JK. Invited review: Shelf-stable dairy protein beverages-Scientific and technological aspects. J Dairy Sci. 2022 Nov;105(12):9327-9346. doi: 10.3168 / jds.2022-22208. Epub 2022 Oct 4. PMID: 36207179.NPTL 3

[0009] Yamaguchi S, Jeenes DJ, Archer DB. Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins. Purification, characterization and gene cloning. Eur J Biochem. 2001 Mar;268(5):1410-21. doi: 10.1046 / j.1432-1327.2001.02019.x. PMID: 11231294.

[0010] The present invention meets or addresses at least some of the above needs and aims at solving the above problems in the art by providing a method for producing a milk product in which milk is subjected to an enzymatic treatment step with PG prior to UHT processing. In addition, the inventors have found that the enzymatic treatment of raw milk with PG can be carried out at low temperatures such as temperatures below 20 ℃. Lastly, the inventors have found that an enzymatic treatment of raw milk with PG at temperatures below 20 ℃ (low temperature condition) can be combined with subsequent UHT processing.

[0011] The invention, hence, provides several method steps that each individually achieves an improvement over the art.

[0012] In a first aspect, the present invention provides a method for producing a UHT milk comprising the steps of (a) subjecting a raw milk to enzymatic treatment with a protein glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0013] The inventors have found that in UHT milk obtained by the method for producing UHT milk of the present invention, undesired physicochemical changes that are otherwise associated with UHT processing and the following prolonged storage of the UHT milk can be reduced. In addition, sensory (organoleptic) properties of the UHT milk can be improved. Improved shelf storage of UHT milk helps extend its freshness and reduce spoilage, ensuring a safer and more reliable product for consumers. It also facilitates inventory management, minimizing waste, and improving cost efficiency associated with storage.

[0014] In a second aspect, the present invention provides a method for producing a milk product, comprising a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃.

[0015] In particular, the inventors have found that the enzymatic treatment step can be carried out at a temperature below 20 ℃, e.g., the enzymatic treatment step is preferably carried out at temperatures below 15 ℃, below 10 ℃, most preferably at temperatures between 2 and 8 ℃. In addition, the enzymatic treatment step may be performed on a raw milk without the necessity of any upstream processing steps.

[0016] This was rather surprising, since PG treatment of a raw milk has been previously described to be inefficient due to the presence of an inhibitor of PG in a raw milk material (see, e.g., EP 2 332 421). The inhibitor of PG may be reduced or removed, e.g., by adding a calcium chelating agent, an alkaline, or a reducing agent, or by removing a low molecule fraction via filtration and by carrying out the enzymatic reaction at above 50 ℃. In this context, temperatures exceeding 50 ℃ are preferable for enzymatic treatment with PG, given that the optimal temperatures for PG have been documented to be within the range of 50 to 60 ℃ (Yamaguchi et al., 2001).

[0017] The decrease in temperature employed in the enzymatic treatment step of the second aspect of the present invention confers certain advantages. First, lower temperatures (such as temperatures below 20 ℃) may be preferable for quality control purposes, as the risk of bacterial growth in milk during enzymatic treatment is reduced. Unpasteurized milk may contain a multitude of microorganisms. Even after undergoing pasteurization, which effectively eliminates viable microbial life, spores remain. Therefore, the incubation of milk at elevated temperatures, such as those exceeding 20 ℃, may provide conditions that promote bacterial proliferation, which may affect the characteristics of the obtained milk product (e.g., quality and stability). For instance, this may result in the souring of the milk or a reduction in its storage stability. Hence, an enzymatic treatment step using a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃, is beneficial, as it reduces the growth of microorganism, resulting in a milk product which complies with quality control standards.

[0018] In a third aspect, the present invention provides a method for producing a UHT milk comprising the steps of: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0019] The inventors have surprisingly found that a UHT milk obtained by the method for producing a UHT milk of the present invention showed less undesired physicochemical changes such as less amount of sedimentation. For instance, the amount of sedimentation, which may be caused by the release of κ-casein from casein micelles due to heating and the settling of these aggregated / depleted κ-casein micelles to the bottom of a storage container during storage, was reduced.

[0020] These beneficial effects could even be observed when storing the UHT milk at elevated temperatures such as 45 ℃. Storage at an elevated temperature (45 ℃) enhances storage-related effects in comparison to storage at room temperature in order to perform an ‘accelerated shelf-life study’ aiming to reproduce the effect of several months of storage at room temperature. Hence, the need to store the UHT milk at a low temperature (e.g., refrigerator temperature such as about 4 ℃) was eliminated and shelf-storage stability at room temperature was enhanced, thus facilitating storage and reducing the costs associated therewith for a UHT milk obtained by the method for producing UHT milk of the present invention.

[0021] Moreover, by using a sensory (organoleptic) evaluation, the inventors found that the texture of a UHT milk of the present invention was improved, and the development of off-flavors during shelf-storage of the UHT milk was decreased. Hence, the method of the present invention provides a UHT milk having improved sensory properties (e.g., freshness, mouthfeel) and improved storage stability (e.g., shelf-life) even after prolonged storage at elevated temperatures.

[0022] The inventors have surprisingly found that the method for producing a UHT milk of the present invention eliminates the need of additional processing steps that were previously taken to increase the heat stability and thus the storage stability of UHT milk such as mechanical treatments including the combination of ultra-high-pressure homogenization (UHPH) and microfiltration to the UHT milk (Krishna et al., 2021), or the addition of stabilizers or emulsifiers, the adjustment of the pH via pH modulators or the modulation of the calcium content via calcium chelators (Singh et al., 2022).

[0023] Preferred embodiments are set out in the respective dependent claims, as well as in the embodiments described below.

[0024] 1. A method for producing a UHT milk, comprising the steps of: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0025] 2. The method according to item 1, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein.

[0026] 3. The method according to item 1 or 2, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g.

[0027] 4. The method according to any one of items 1 to 3, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.5 U / g, 1 U / g, or 2 U / g.

[0028] 5. The method according to any one of items 1 to 4, wherein in step (a), the enzymatic treatment is performed for 10 min to 24 hours, preferably for 1 to 24 hour(s), even more preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours.

[0029] 6. The method according to any one of items 1 to 5, wherein in step (a), the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium.

[0030] 7. The method according to any one of items 1 to 6, wherein in step (a), the enzymatic treatment is performed at a temperature below 65 ℃, below 60 ℃, below 55 ℃, below 50 ℃, below 45 ℃, below 40 ℃, below 35 ℃, below 30 ℃, below 25 ℃, below 20 ℃, below 14 ℃, below 10 ℃, or below 8 ℃.

[0031] 8. The method according to any one of claims 1 to 7, wherein in step (a), the enzymatic treatment is performed at a temperature of 2 to 65 ℃, 2 to 60 ℃, 2 to 55 ℃, 2 to 50 ℃, 2 to 45 ℃, 2 to 40 ℃, 2 to 35 ℃, 2 to 30 ℃, 2 to 25 ℃, 2 to 20 ℃, or 2 to 15 ℃.

[0032] 9. The method according to item 8, wherein in step (a), the temperature is a temperature of 2 to 14 ℃, preferably 2 to 10 ℃, more preferably 2 to 8 ℃, even more preferably 4 to 8 ℃, such as 4 ℃.

[0033] 10. The method according to any one of items 1 to 9, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g.

[0034] 11. The method according to any one of items 1 to 10, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours.

[0035] 12. The method according to any one of items 1 to 11, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g.

[0036] 13. The method according to any one of items 7 to 12, wherein in step (a), the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

[0037] 14. The method according to any one of items 1 to 13, wherein in step (b), the UHT processing includes a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step.

[0038] 15. The method according to item 14, wherein in step (b), the heating step is performed at at least 135℃, preferably at 135 to 150 ℃.

[0039] 16. The method according to item 14 or 15, wherein in step (b), the heating step is performed for at least 2 seconds, preferably for 2 to 10 seconds, such as 5 seconds.

[0040] 17. The method according to any one of items 14 to 16, wherein in step (b), a further pre-heating step is performed; optionally wherein the further pre-heating step is performed by pre-heating at 90℃ for 60 seconds followed by the heating step at 135℃ for 5 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating.

[0041] 18. The method according to any one of items 14 to 17, wherein in step (b), the homogenization step is performed prior to the heating step and / or after the heating step, preferably wherein the homogenization step is performed after the heating step.

[0042] 19. The method according to any one of items 14 to 18, wherein in step (b), the homogenization step is carried out in two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar).

[0043] 20. The method according to any one of items 1 to 19, wherein the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk.

[0044] 21. The method according to any one of items 1 to 20, wherein the raw milk is skimmed milk, semi skimmed milk or whole milk.

[0045] 22. The method according to any one of items 1 to 21, wherein the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step of at least 70 ℃, further optionally wherein the pasteurization step is performed at 72 to 75 ℃ for 15 to 30 seconds, such as 75 ℃ for 15 seconds.

[0046] 23. The method according to any one of items 1 to 22, wherein prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk.

[0047] 24. A UHT milk obtainable by the method according to any one of items 1 to 23.

[0048] 25. A method for producing a dairy product, by using the UHT milk obtainable by the method according to any one of items 1 to 23.

[0049] 26. The method according to item 25, wherein the dairy product is a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.

[0050] 27. A method for producing a milk product, comprising a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃.

[0051] 28. The method according to item 27, wherein the temperature is below 14 ℃, below 10 ℃, or below 8 ℃.

[0052] 29. The method according to item 27 or 28, wherein the temperature is a temperature of 2 to 14 ℃, preferably 2 to 10 ℃, more preferably 2 to 8 ℃, even more preferably 4 to 8 ℃, such as 4 ℃.

[0053] 30. The method according to any one of items 27 to 29, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein.

[0054] 31. The method according to any one of items 27 to 30, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g.

[0055] 32. The method according to any one of items 27 to 31, wherein the protein-glutaminase is added to the raw milk in an amount of 0.5 U / g, 1 U / g, or 2 U / g.

[0056] 33. The method according to any one of items 27 to 32, wherein the enzymatic treatment is performed for 10 min to 24 hours, preferably for 1 to 24 hour(s), even more preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours.

[0057] 34. The method according to any one of items 27 to 33, wherein the protein-glutaminase is a cream, a protein-glutaminase derived from Chryseobacterium.

[0058] 35. The method according to any one of items 27 to 34, wherein the enzymatic treatment is performed at a temperature below 10 ℃, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g.

[0059] 36. The method according to any one of items 27 to 35, wherein the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours.

[0060] 37. The method according to any one of items 27 to 36, wherein the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g.

[0061] 38. The method according to any one of items 27 to 37, wherein the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

[0062] 39. The method according to any one of items 27 to 38, further comprising a step (b) of ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0063] 40. The method according to item 39, wherein in step (b), the UHT processing includes a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step.

[0064] 41. The method according to item 40, wherein in step (b), the heating step is performed at at least 135 ℃, preferably at 135 to 150 ℃.

[0065] 42. The method according to item 40 or 41, wherein in step (b), the heating step is performed for at least 2 seconds, preferably for 2 to 10 seconds, such as 5 seconds.

[0066] 43. The method according to any one of items 40 to 42, wherein in step (b), a further pre-heating step is performed; optionally wherein the further pre-heating step is performed by pre-heating at 90 ℃ for 60 seconds followed by the heating step at 135 ℃ for 5 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating.

[0067] 44. The method according to any one of items 40 to 43, wherein in step (b), the homogenization step is performed prior to the heating step or after the heating step, preferably wherein the homogenization step is performed after the heating step.

[0068] 45. The method according to any one of items 40 to 44, wherein in step (b), the homogenization step is carried out in two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar).

[0069] 46. The method according to any one of items 27 to 45, wherein the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk.

[0070] 47. The method according to any one of items 27 to 46, wherein the raw milk is skimmed milk, semi skimmed milk or whole milk.

[0071] 48. The method according to any one of items 27 to 47, wherein the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step of at least 70 ℃, further optionally wherein the pasteurization step is performed at 72 to 75 ℃ for 15 to 30 seconds, such as 75 ℃ for 15 seconds.

[0072] 49. The method according to any one of items 27 to 48, wherein prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk.

[0073] 50. The method for producing a milk product according to any one of items 39 to 49, comprising the steps of: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 2 to 8 ℃ (such as 4 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 8 to 24 hours (such as 20 hours); and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a), wherein the UHT processing comprises or consists of steps: (i) a pre-heating step by indirect heating (such as 90 ℃ for 60 seconds), (ii) a heating step by indirect heating (such as 135 ℃ for 5 seconds), (iii) a cooling step (such as cooling to a temperature below 65 ℃), (iv) a homogenization step carried out in two steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa (240 bar)), (v) a further cooling step (such as cooling to a temperature below 8 ℃), and (vi) a packaging step (such as filling into a container); wherein the steps (i) to (vi) are executed consecutively; and wherein the milk product is a UHT milk.

[0074] 51. A milk product obtainable by the method according to any one of items 27 to 50, wherein the milk product is a UHT milk.

[0075] 52. A method for producing a dairy product, by using the milk product obtainable by the method according to any one of items 27 to 50.

[0076] 53. The method according to item 52, wherein the dairy product is a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.

[0077] In order to best describe the manner in which the above-described embodiments are implemented, as well as define other advantages and features of the disclosure, a more particular description is provided below and is illustrated in the appended drawings. Understanding that these drawings depict only exemplary embodiments of the invention and are thus not to be considered to be limiting in scope, the examples will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0078] Figure 1 depicts an exemplary method for producing a UHT milk. In some embodiments, the UHT processing comprises a heating step and a cooling step, and optionally pre-heating, homogenisation and / or packaging step(s).Figure 2 depicts an exemplary method for producing a milk product. Further embodiments are marked with the term “opt.” in the figure. The method comprises an enzymatic treatment of a raw milk with a protein-glutaminase (PG) at a temperature below 20 ℃ (e.g., below 10 ℃, such as 2-8 ℃) for a long reaction time (e.g., 8-24 hours). In addition, the enzymatic treatment may be performed at a low PG dosage (e.g., 0.1 U / g to 4.5 U / g; such as 0.5 U / g, 1 U / g, or 2 U / g, wherein U / g stands for Units per gram of protein). In some embodiments, the obtained milk product may be further processed to a dairy product such as a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.Figure 3 depicts an exemplary method for producing a milk product. Further embodiments are marked with the term “opt.” in the figure. The method comprises an enzymatic treatment of a raw milk (e.g., LTLT or HTST pasteurized milk) with PG at a temperature below 20 ℃ (e.g., below 10 ℃, such as 2-8 ℃) for a long reaction time (e.g., 8-24 hours). In addition, the enzymatic treatment may be performed at a low PG dosage (e.g., 0.1 U / g to 4.5 U / g; such as 0.5 U / g, 1 U / g, 1.5 U / g, or 2 U / g). The UHT processing comprises a heating step and a cooling step, and optionally pre-heating, homogenisation and / or packaging step(s).Figure 4 depicts an exemplary method for producing a UHT milk. Further embodiments are marked with the term “opt.” in the figure. The method comprises an enzymatic treatment of a raw milk (e.g., LTLT or HTST pasteurized milk) with PG at a temperature below 10 ℃ (e.g., 2-8 ℃) for at least 8 hours (e.g., 8-24 hours). In addition, the enzymatic treatment may be performed at a low PG dosage (e.g., 0.1 U / g to 4.5 U / g; such as 0.5 U / g, 1 U / g, or 2 U / g). The UHT processing of the obtained mixture comprises a pre-heating step (e.g., at 75-100℃ for 20-120 seconds), a heating step (e.g., at 135-150℃ for 2-10 seconds), a cooling step (e.g., to 65 ℃), and a homogenization step (e.g., comprising two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar)), preferably performed in the stated order.Figure 5 depicts an exemplary method for producing a UHT milk process according to example 1. A bovine raw milk was subjected to enzymatic treatment with PG at a dosage of 0.1-2.5 U / g (e.g., 0.5 U / g, 1 U / g, 2 U / g) at a temperature of 2-8 ℃ (e.g., 4 ℃) for a reaction time of 8-24 hours (e.g., 20 hours). A control was performed without enzymatic treatment with PG (0 U / g). Next, the obtained mixture was subjected to UHT processing comprising a pre-heating step at 90 ℃ for 60 seconds, a heating step at 135 ℃ for 5 seconds, a first cooling step to 65 ℃, a homogenization step comprising two consecutive homogenization steps at 30 bar and 240 bar, a second cooling step to 4 ℃, and a packaging step. The UHT milk was stored at 4 ℃ or 45 ℃ for 14 days. The stored UHT milk was analysed by pH measurement, sedimentation analysis and / or sensory analysis.Figure 6A depicts a sensory evaluation (taste deviation) between a UHT milk (enzymatic treatment at the indicated PG dosage of 0.5 U / g, 1 U / g, or 2 U / g) and a UHT milk without enzymatic treatment with PG (control). The sensory evaluation of the UHT milk samples which had a temperature of 13 ℃ for tasting was performed after storage at 4 ℃ for 14 days. Samples were rated based on their deviation from control with the following notation: “0.0”: no deviation, taste is similar to control; “1.0”: small deviation, there is small difference between rated sample and the control; “2.0”: medium deviation, there is a medium deviation between rated sample and control; “3.0”: large deviation, there is a large deviation between rated sample and control. The panellist reported a positive taste deviation from the control, which was attributed to an enhanced freshness and mouthfeel.Figure 6B depicts a sensory evaluation (taste deviation) between a UHT milk (enzymatic treatment at the indicated PG dosage of 0.5 U / g, 1 U / g, or 2 U / g) and a UHT milk without treatment with PG (control). The sensory evaluation of the UHT milk samples (at 13 ℃) was performed after storage at 45 ℃ for 14 days. Temperatures above 45 ℃ allow for an ‘accelerated shelf-life study’ aiming at reproducing the effect of several months’ storage at room temperature. Samples were rated based on their deviation from control with the following notation: “0.0”: no deviation, taste is similar to control; “1.0”: small deviation, there is small difference between rated sample and the control; “2.0”: medium deviation, there is a medium deviation between rated sample and control; “3.0”: large deviation, there is a large deviation between rated sample and control. The panellist reported a positive taste deviation from the control, which was attributed to an enhanced freshness and mouthfeel.

[0079] Definitions All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0080] Unless context clearly indicates otherwise, each occurrence of the term “comprise”, and variations such as “comprising”, may optionally be substituted with the term “consist of”, and variations such as “consisting of”.

[0081] Unless context clearly indicates otherwise, the articles “a”, “an” and “the” are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0082] Unless context clearly indicates otherwise, the term “or” is used herein to mean, and is used interchangeably with, the term “and / or”. The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements” connected by said term.

[0083] Unless context clearly indicates otherwise, the term “about”, as used herein, refers to a deviation of ± 10 % from the recited value. When the word “about” is used herein in reference to a number, it should be understood that still another embodiment includes that number not modified by the presence of the word “about”. In the absence of the term “about” and unless the context dictates otherwise, generally accepted rounding rules apply to the specified values.

[0084] Unless context clearly indicates otherwise, the term “similar”, as used herein, is interchangeable for alike, analogous, comparable, corresponding, and -like, and is meant to have the same or common characteristics, and / or in a quantifiable manner to show comparable results i.e., with a variation of maximum 20 %, 10 %, more preferably 5 %, or even more preferably 1 %, or less.

[0085] Unless context clearly indicates otherwise, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0086] “Enzymatic treatment”, as used herein, refers to a step wherein a protein-glutaminase is used.

[0087] “Protein-glutaminase” or “PG”, as used herein, refers to a protein-glutaminase (EC 3.5.1.44) which catalyses the deamidation of glutamine residues within proteins, and glutamine is converted to glutamic acid. The type of protein-glutaminase in the present invention is not particularly limited as long as it acts directly on an amide group of a protein to deamidate it without cleaving a peptide bond or cross-linking the protein. Examples of such an enzyme include, but are not particularly limited to, protein-glutaminases derived from Chryseobacterium, Flavobacterium or Empedobacter disclosed in JP-A-2000-50887, JP-A-2001-218590, EP 1 839 491, commercially available protein-glutaminase derived from Chryseobacterium and the like. For example, protein-glutaminase may be derived from Chryseobacterium proteolyticum (see, e.g., example 1 of EP 1 839 491 and sequences disclosed therein).

[0088] There are several commercially available protein-glutaminase enzyme preparations that are suitable for use in the invention. In a preferred embodiment, a protein-glutaminase is a protein-glutaminase derived from Chryseobacterium (e.g., Chryseobacterium proteolycum). Preparations of protein-glutaminase are commercially available. Protein-glutaminase that will be developed and found in the future can also be used in the invention as long as they have the transglutaminase activity. The use of such transglutaminases and protein-glutaminase is, as a matter of course, included in the present invention. The protein-glutaminase can be prepared from a culture liquid for microorganism that produces the protein-glutaminase, for example, using known methods for protein separation and purification (e.g., centrifugation, ultrafiltration (UF) concentration, salting out, various chromatography methods using an ion exchange resin, etc.). For example, a culture medium may be centrifuged to remove bacteria, after which salting out, chromatography, and the like, may be combined to give the object enzymes. When collecting PG enzymes from the interior of bacterial cells, the bacterial cells may be recovered from a culture liquid by filtration or centrifuge, etc., and then the bacterial cells can be crushed by, for example, a pressure processing or ultrasonic processing technique. Protein separation and purification methods such as those described above may then be performed to obtain the target PG enzymes. The enzymes may be powdered by a drying method such as a freeze drying or vacuum drying, etc., with optional use of an appropriate diluent or drying aid. In a preferred embodiment, a protein-glutaminase is of a quality suitable for animal and / or human consumption, or for use in food production or storage (e.g., food grade enzyme).

[0089] “Raw milk”, as used herein, means a milk which has not experienced ultra-high temperature heating sterilization by heating the milk to a temperature above 100 ℃ (also referred to as “non-heat-treated milk”). In some embodiments, the raw milk includes an edible milk obtainable from an animal (e.g., an animal such as a cow, a buffalo, a goat, a sheep, a horse, a camel, a yak, a donkey etc.). Hence, the raw milk may be bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk etc. In some embodiments, the raw milk includes a pasteurized milk (composition) which experienced heating at a temperature below 100 ℃ (such as a low temperature and long sterilization time (LTLT) milk or a high temperature and short sterilization time (HTST) milk). In some embodiments, the raw milk includes an edible milk composition including milk proteins and milk fat (such as a dried powdered milk suspended and dissolved in water).

[0090] In some embodiments, the raw milk is a pasteurized raw milk (composition) or pasteurized raw milk. In some embodiments, the raw milk is a pasteurized raw milk (composition) or pasteurized raw milk which did not experience heating at a temperature above 100 ℃ (such as a low temperature and long sterilization time (LTLT) milk or a high temperature and short sterilization time (HTST) milk). Hence, the term “pasteurized raw milk” as used herein, refers to raw milk subjected to mild heat treatment such as low-temperature sterilization (such as LTLT sterilised milk), or a high temperature short time sterilisation (such as HTST sterilised milk), but excludes raw milk subjected to ultra-high temperature sterilization. In other words, pasteurized raw milk is raw milk subjected to pasteurization treatments below 100 ℃. Preferably, the pasteurized raw milk may be raw milk obtainable by a pasteurization step with a temperature below 75 ℃. The pasteurized raw milk is further characterised in that living microorganisms are destroyed.

[0091] In some embodiments, the pasteurized raw milk is a LTLT (low-temperature long-time) pasteurized milk. LTLT (low-temperature long-time) pasteurized milk, as referred to herein, is a pasteurized milk (composition) obtainable by a low-temperature long-time pasteurization step, for instance, the LTLT pasteurization step is performed at a temperature between 62 ℃ and 66 ℃ for a duration of 30 minutes or at 75 ℃ or more for 15 minutes or more. In some embodiments, the pasteurized raw milk may be HTST (high-temperature short-time) pasteurized milk. HTST (high-temperature short-time) pasteurized milk, as referred to herein, is a pasteurized milk (composition) obtainable by a pasteurization step performed at a temperature between 72 ℃ and 75 ℃ for a duration of 15 seconds. In some embodiments, the pasteurized raw milk is obtainable by a pasteurization step at a temperature of at least 70 ℃ and less than 100 ℃, preferably wherein the pasteurization step is performed at 72 to 75 ℃ for 15 to 30 seconds, such as 74 ℃ for 30 seconds or 75 ℃ for 15 seconds.

[0092] In some embodiments, a raw milk is obtained by homogenizing milk obtained from an animal (e.g., by pre-heating at 60 ℃, 30 kgf / cm2), pasteurizing (e.g., 72℃, 15 sec), and cooling to the reaction temperature of the enzymatic treatment step (e.g., to below 20 ℃).

[0093] Raw milk having a wide range of fat contents may be utilized herein. Typically, the raw milk has a fat content of up to 10 wt%, preferably up to 8 wt%, preferably up to 6 wt%, preferably up to 5 wt%, preferably up to 4 wt%, preferably up to 3 wt%, preferably up to 2 wt%, preferably up to 1.5 wt%, preferably up to 1 wt%, preferably up to 0.5 wt%, preferably up to 0.3 wt%, preferably up to 0.1 wt%, preferably 0 wt%, based on a total weight of the raw milk. As used herein, wt% means weight percent which is sometimes written as w / w i.e. [weight of solute / weight of solvent*100 = percent of solute in the solution].

[0094] In some embodiments, the raw milk is skimmed milk, semi skimmed milk or whole milk. Skimmed milk, semi skimmed milk and whole milk differ in their amount of fat. “Skimmed milk” is milk from which the cream has been removed. Semi skimmed milk is characterized in that it has a fat content of about 0.1 wt% to 0.3 w%. “Semi skimmed milk” or “reduced fat milk” or “low fat milk” is milk from which some of the cream has been removed. Semi skimmed milk is characterized in that it has a fat content of about 1 wt% to 2 wt%, such as 1.8 wt%. “Whole milk”, as used herein, is milk with no fat removed. Whole milk is usually characterized in that it has a fat content of about 2.5 to 7.5 wt%.

[0095] In some embodiments, the raw milk may be supplemented with ingredients generally used in the production of milk products, such as fat, protein, vitamins, minerals, stabilisers and / or sugar fractions or the like. For instance, a raw milk may be a “fortified raw milk”. The process of fortifying raw milk entails enriching it with additional vitamins and minerals. The most commonly added vitamins to raw milk are vitamin D and A, with the preference being for heat-resistant vitamin A and vitamin D3. However, other nutrients, such as zinc, iron, and folic acid, may also be included, resulting in the designation of "fortified raw milk”. In some embodiments, minerals such as calcium and phosphate may be added or removed to influence the heat stability of milk. In particular, stabilizers based on phosphate salts and / or citrate may be added to the raw milk. While raw milk having fat and protein contents outside of the above-mentioned ranges may be utilized in some circumstances, preferred raw milks are those commercial raw milks or raw milks that have been adjusted to fall within the above-mentioned ranges according to methods known to those of skilled in the art.

[0096] In some embodiments, a raw milk refers to a composition based on milk or milk components obtained from mammals which has not been subject to enzymatic treatment for alteration of proteins contained therein. Conversely, the term “milk product” as used herein refers to those compositions based on raw milk which have been subject to enzymatic treatment with PG.

[0097] “Dairy product”, as used herein, refers to a product produced from the UHT milk or the milk product of the present invention. In an embodiment, the dairy product is produced from UHT milk, wherein the UHT milk is obtainable by the following consecutive steps: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a). In an embodiment, the dairy product is produced from a milk product, wherein the milk product is obtained by a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃. Step (a) may be followed by a step of deactivating PG, e.g., in a step of subjecting the milk product to temperatures above 75 ℃, preferably by UHT processing.

[0098] In some embodiments, the dairy product is a cream, a yoghurt, a cheese, a cheese-like food, a pudding, or a milk-based drink.

[0099] “Cream”, as used herein, refers to a dairy product composed of a higher-fat composition obtainable by physical separation of the higher-fat composition from a milk composition. In some embodiments, a cream may be the higher-fat layer skimmed from the top of milk before homogenization (e.g., sweet cream). In some embodiments, a cream may be the higher fat-layer skimmed from whey during cheese making (e.g., sour cream). The obtained cream may be subjected to further processing including the addition of thickening agents or stabilizers, heat-treatment, and / or combinations thereof. A cream includes any cream that conforms to the requirements of the Food and Drug Administration for milk and cream (21 CFR part 131), and any cream that conforms to the global standards for the definition of cream according to the Codex Alimentarius (CODEX STAN (CXS) 288-1976).

[0100] “Yogurt”, as used herein, refers to a product produced by culturing a milk composition, such as the UHT milk or milk product of the present invention, with a characterizing bacterial culture that contains the lactic acid-producing bacteria, such as Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus and which conforms to the requirements of the Food and Drug Administration for cheeses and related cheese products (21CFR 131.200), and any yogurt that conforms to the global standards for the definition of cheese according to the Codex Alimentarius (CODEX STAN (CXS) 243-2003).

[0101] “Cheese”, as used herein, refers to the fresh or matured dairy product obtained after coagulation of a milk composition, such as the UHT milk or milk product of the present invention, optionally in combination with other milk products (such as cream), and including any cheese that conforms to the requirements of the Food and Drug Administration for cheeses and related cheese products (21 CFR part 133), and any cheese that conforms to the global standards for the definition of cheese according to the Codex Alimentarius (CODEX STAN (CXS) 283-1978). “Cheese-like food” refers to a food that has similar properties to a cheese.

[0102] “Pudding”, as used herein, refers to a milk pudding or custard that is made from a milk composition, such as the UHT milk or milk product of the present invention, sugar, and a thickening agent such as cornstarch, gelatine or flour.

[0103] “Milk-based drink”, as used herein, refers to drinks substantially based on a milk composition, such as the UHT milk or milk product of the present invention. For instance, milk-based drinks may be drinks comprising 50% or more milk composition (such as the UHT milk or milk product of the present invention). The milk composition may be the UHT milk or milk product of the present invention itself or may be further processed such as fermented.

[0104] “Milk protein” as referred to herein, is the total protein content of the milk, measured by any valid method. Such methods are known to the skilled person. The raw milk may have a wide range of protein contents, with typical protein content values being at least 0.5 wt.%, preferably at least 1 wt%, preferably at least 1.5 wt%, preferably at least 2 wt%, preferably at least 2.5 wt.%, preferably at least 3 wt%, and up to 10 wt%, preferably up to 8 wt%, preferably up to 6 wt%, preferably up to 5.5 wt%, preferably up to 5 wt%, preferably up to 4.5 wt%, preferably up to 4 wt%, preferably up to 3.5 wt%, based on a total weight of the raw milk.

[0105] “pH” or “pH value”, as used herein, is established by standard methods known to the skilled person. The pH of the raw milk, the UHT milk or the milk product is between 6.5 to 6.9, preferably between 6.7 to 6.9 at temperatures between 2 to 25 ℃. Temperatures above 40 ℃ may affect the pH by inducing physiochemical changes. For example, the pH change in the production method may be monitored by using a portable pH meter.

[0106] “Sedimentation”, as used herein, refers to the formation of a substance, the “sediment”, at the bottom of the storage container (e.g., the milk carton) during storage (e.g., at room temperature for 14 days or longer) of a UHT milk or milk product. The sediment may comprise a milk protein aggregate (e.g., containing aggregated / depleted κ-casein micelles and protein-lipid interactions) and further components (e.g., fat). The amount of sediment can be measured by, e.g., centrifugation.

[0107] “Dry weight” or “dry matter” as used herein, refers to the measure of the mass of a completely dried substance. The “dry weight of a sediment” may, hence, be obtained by drying the sediment obtained from a UHT milk or milk product, for example, after a centrifugation step and measuring the mass of a completely dried substance. Drying can be carried out using, e.g., a drying oven.

[0108] Detailed Description of Embodiments Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0109] In a first aspect (I), the present invention provides a method for producing a UHT milk comprising the steps of: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0110] In a second aspect (II), the present invention provides a method for producing a milk product, comprising a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃.

[0111] In a third aspect (III), the present invention provides a method for producing a UHT milk, comprising the steps of: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0112] In the following, the individual processing steps comprised in the methods of the present invention are described sequentially and may be combined by the skilled person to obtain a UHT milk or a milk product according to the present invention.

[0113] Step (a): Subjecting a raw milk to enzymatic treatment with a protein-glutaminase The methods of the present disclosure involve subjecting a raw milk to enzymatic treatment with a protein-glutaminase (PG). PG deamidates proteins contained in the raw milk thereby forming a mixture (step a).

[0114] The protein-glutaminase is added to the raw milk, preferably to raw milk already at the treatment temperature for carrying out the deamidation reaction. As the protein-glutaminase of the present disclosure, a commercially available PG or a PG prepared from a culture medium of a microorganism producing protein-glutaminase can be used - the kind of protein-glutaminase used is not particularly limited as long as it directly acts on glutamine residues in protein(s) contained in the raw milk to deamidate the same without peptide bond cleavage and / or crosslinking of the protein(s).

[0115] “Enzyme activity” may be measured in U (μmol / min) which is defined as the amount of enzyme that catalyses the conversion of one micromole of substrate per minute. The amount of protein-glutaminase may be adjusted depending on the enzyme activity. The activity of the protein-glutaminase may be measured by the following steps: (1) 100 μL of an aqueous solution containing the protein-glutaminase is added to 1 ml of 0.2 M phosphoric acid buffer solution (pH 6.5) containing 30 mM of Z-Gln-Gly (Peptide Institute, Inc.), and incubated for 10 minutes at 37 ℃, and then the reaction is ceased by adding 1 ml of 0.4 M trichloroacetic acid (TCA) solution. (2) An amount of ammonia produced by the reaction in a reacted solution of (1) is measured by using Ammonia-test-Wako (manufactured and retailed by Waka Pure Chemical Industries, Ltd.). (3) A blank solution is measured at the same time. The blank solution is prepared such that a solution without the enzyme is incubated for 10 minutes at 37 ℃, and then 1 ml of 0.4 M TCA solution and 100 μL of an aqueous solution containing the protein-glutaminase enzyme are added in this order. (4) The concentration of the enzyme is adjusted by diluting with a phosphoric acid buffer solution (pH 6.5) such that an absorbance at 630 nm becomes in the range of 0.2 to 0.8. (5) The activity is measured by a unit (U) which indicates the amount of the enzyme that can produce one μmol of ammonium in one minute. The enzyme activity is calculated by the following formula: Enzyme activity (U / ml) = (Es-Eb) *F*0.123xDf where: Es: absorbance of a solution enzyme reacted, Eb: absorbance of a blank, F: factor (the reciprocal of the slope of a straight line of a calibration curve for standard ammonium solution), and Df: dilution ratio of an enzyme solution.

[0116] The amount of protein-glutaminase to be added can be varied according to the composition of raw milk (e.g., protein content), a kind of protein to be modified, or a desired effect to be obtained. Preferably, the enzyme dosages are adjusted based on the grams (g) of milk protein in the raw milk.

[0117] The milk protein content may be determined by chemical analysis. For instance, the amount of protein may be measured in accordance with the International Standard (ISO 8968-1:2014) which specifies a method for the determination of the nitrogen content and crude protein calculation of milk and milk products using the Kjeldahl principle. This process relies on the digestion of food with a strong acid, which releases nitrogen and can then be quantified using a titration technique. The mass fraction of crude protein is then calculated from the total nitrogen concentration of the food using a conversion factor, e.g., 6.25.

[0118] Alternatively, the amount of protein in the raw milk may be measured by a spectroscopy method such as visible (Vis), and near-infrared (NIR) spectroscopy such as Fourier transform near-infrared spectroscopy (FT-NIR) (e.g., using MPA II-LSM, Bruker Optics France) in g / (100g total weigh of the coaglum).

[0119] The amount or dosage of PG added may be at least 0.01 unit of enzyme (U) per g of protein in the raw milk (as measured by a spectroscopy method such as visible (Vis) and near-infrared (NIR) spectroscopy), preferably at least 0.1 U / g, preferably at least 0.5 U / g, preferably at least 1 U / g, preferably at least 1.5 U / g, preferably at least 2 U / g, preferably at least 2.5 U / g of protein in the raw milk. The amount of PG added may be up to 10 U / g of protein in the raw milk, preferably up to 8 U / g, preferably up to 6 U / g, preferably up to 5 U / g, preferably up to 4 U / g, preferably up to 3 U / g of protein in the raw milk. Hence, preferably the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, such as 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g. Most preferably the protein-glutaminase is added to the raw milk in an amount of 0.2-2.5 U / g, such as 0.5 U / g, 1 U / g, 1.5 U / g or 2 U / g. For instance, the protein-glutaminase may be added to the raw milk in an amount of about 0.4 U / g, about 0.5 U / g, about 0.6 U / g, about 0.7 U / g, about 0.8 U / g, about 0.9 U / g, about 1 U / g, about 1.1 U / g, about 1.2 U / g, about 1.3 U / g, about 1.4 U / g, about 1.5 U / g, about 1.6 U / g, about 1.7 U / g, about 1.8 U / g, about 1.9 U / g, about 2 U / g, about 2.1 U / g, about 2.2 U / g, about 2.3 U / g, about 2.4 U / g, about 2.5 U / g, about 2.6 U / g, about 2.7 U / g, about 2.8 U / g, about 2.9 U / g, about 3 U / g, about 3.1 U / g, about 3.2 U / g, about 3.3 U / g, about 3.4 U / g, about 3.5 U / g, about 3.6 U / g, about 3.7 U / g, about 3.8 U / g, about 3.9 U / g, about 4 U / g, about 4.1 U / g, about 4.2 U / g, about 4.3 U / g, about 4.4 U / g, or about 4.5 U / g.

[0120] The amount of enzyme preparation in grams (g) to be added to the raw milk may be calculated in an exemplary embodiment as follows: ((([protein (g / 100 g)] / 100) *[batch volume] * [enzyme activity (target)]) / [enzyme activity (initial)])*1000, wherein: [protein (g / 100 g)] refers to the protein content of the raw milk; [batch volume] refers to the total weight (in kg) of the composition to be treated (raw milk); [enzyme activity (target)] in U / g protein refers to the final dosage of PG in the reaction, which is between 0.1 - 10 U / g protein; and [enzyme activity (initial)] refers to the PG activity in U / g in the enzymatic preparation / enzyme stock.

[0121] In some embodiments, the enzyme treatment step may further comprise additional components. For instance, the enzyme treatment step may comprise the addition of protein-glutaminase and transglutaminase (TG). However, at least in terms of lowering operational costs and reducing complexity, it is preferred that protein-glutaminase is the only enzyme used for protein modification, e.g., the only enzyme used that acts on glutamine residues of milk proteins contained in raw milk. Other types of enzymes that act on glutamine residues that may be used to treat the raw milk concurrently or in conjunction with the PG are preferably excluded from the methods of the present disclosure. Hence, preferably the enzymatic treatment step does not comprise the addition of crosslinking enzymes such as transglutaminase (TG). Transglutaminase is not a deamidating enzyme because it functionally cross-links proteins via glutamine and amino-containing residues (e.g., lysine) and provides almost no deamidation. Both calcium independent TG types (e.g., TG obtained from microorganisms such as an Actinomycetota or Bacillus subtilis, etc.) and calcium dependent TG types (e.g., those obtained from the liver of guinea pigs or the blood of cattle or pigs, human epidermal keratin cells, human blood coagulation factor XIII, microorganisms such as oomycete, those obtained from fish and oysters, etc.) may be used in addition to PG, but are preferably excluded in the disclosed methods, for example, those described in US 2011 / 0064847A1.

[0122] In a preferred embodiment, no additional agent or material is added to the raw milk and / or no further processing steps are performed prior to or during the enzymatic treatment step with PG. For instance, the method of the present invention does not comprise a step of treating a raw milk to reduce an inhibitor of a protein deamidating enzyme. Hence, in an embodiment, prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), and a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) is added to the raw milk. In an embodiment, the method of the present invention does not comprise a step of removing a low molecule fraction, a molecular weight of which is 10,000 or less, such as by using an ultrafiltration membrane, from the raw milk prior to step (a) and during step (a).

[0123] In addition, in some embodiments, the method of the present invention does not comprise a step of adding a stabilizer to the raw milk prior to or during treating the raw milk with the protein-glutaminase and / or a step of treating the raw milk with a reducing agent prior to, or concurrently with, the treating of the raw milk with the protein-glutaminase. In some embodiments, the method of the present invention does not comprise a step of adding a stabilizer, such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, and modified food starch, as well as mixtures thereof, to the raw milk prior to, or concurrently with, the treating of the raw milk with the protein-glutaminase. In some embodiments, the method of the present invention does not comprise a step of treating the raw milk with a reducing material or agent, such as thiol compounds such as a glutathione, cysteine, γ-glutamilcysteine, yeast extract containing at least one such thiol compound, thiosulfuric acid, sulfurous acid, ascorbic acid, erythorbic acid and salts thereof which are allowed to be used as food additives; and tocopherols; prior to, or concurrently with, the treating of the raw milk with the protein-glutaminase.

[0124] Hence, in a preferred embodiment, prior to step (a) and during step (a), no stabilizer (such as pectin, agar, carrageenan, gelatine, whey protein concentrates, chicory root fiber or modified food starch) or reducing agent (such as thiol compounds such as a glutathione, cysteine, γ-glutamilcysteine, yeast extract containing at least one such thiol compound, thiosulphuric acid, sulphurous acid, ascorbic acid, erythorbic acid and salts thereof which are allowed to be used as food additives; and tocopherol) is added to the raw milk.

[0125] According to step (a) of the present methods, the raw milk may be treated with the protein-glutaminase at an enzymatic treatment temperature below 65 ℃, preferably below 60 ℃, even more preferably below 50 ℃, even more preferably below 20 ℃, such as below 14 ℃, below 10 ℃, or below 8 ℃. The raw milk may be treated with the protein-glutaminase at an enzymatic treatment temperature that is at least 0 ℃, more preferably at least 2 ℃. Hence, the enzymatic treatment temperature is preferably between 0 ℃ to 65 ℃, more preferably between 2 ℃ to 55 ℃, even more preferably between 2℃ to 20 ℃, even more preferably between 2 ℃ to 14 ℃, even more preferably between 2 ℃ to 10 ℃, even more preferably between 2 ℃ to 8 ℃, such as about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, or about 7 ℃, most preferably at about 4 ℃

[0126] According to step (a) of the present methods, the raw milk may be treated with the protein-glutaminase for an enzymatic treatment time of up to 24 hours. For example, the raw milk may be treated with the protein-glutaminase for a duration of at least 10 min, such as at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 18 hours, and up to 24 hours, preferably up to 22 hours, preferably up to 20 hours. Hence, the enzymatic treatment may be performed for about 10 min to about 24 hour(s), preferably for about 8 to about 24 hours, even more preferably for about 18 to about 22 hours, such as about 18, about 19, about 20, about 21 or about 20 hours, most preferably for about 22 hours.

[0127] According to step (a) of the present methods, an enzymatic treatment with a PG dosage below 4.5 U / g (e.g., as described herein), an enzymatic treatment temperature of below 65 ℃ (e.g., as described herein), and an enzymatic treatment time below 24 hours (e.g., as described herein) may be suitably combined. For instance, lower temperatures (e.g., below 20 ℃) may be combined with longer enzymatic treatment time (e.g., above 8 hours); higher temperatures (e.g., above 30 ℃) may be combined with shorter enzymatic treatment time (e.g., below 8 hours).

[0128] Enzymatic treatment according to step (a) of aspect (I) In an embodiment of step (a) according to aspect (I), the enzymatic treatment temperature is preferably between 2℃ to 20 ℃, even more preferably between 2 ℃ to 14 ℃, even more preferably between 2 ℃ to 10 ℃, even more preferably between 2 ℃ to 8 ℃, and most preferably at 4 ℃ and the enzymatic treatment is performed for 1 to 24 h, more preferably 8 to 24 hours, even more preferably for 18 to 22 hours, most preferably for 22 hours. For instance, in step (a) the enzymatic treatment is performed at a temperature of at least 10 ℃, and the treatment is performed for at least 8 hours. In a preferred embodiment, in step (a) the enzymatic treatment is performed for 18 to 22 hours, such as 20 hours, at a temperature of 4 ℃ to 8 ℃, such as 4 ℃.

[0129] In an embodiment of step (a) according to aspect (I), the enzymatic treatment is performed at a temperature of more than 20 ℃, such as 30 ℃ to 60℃, and the treatment is performed for 15 min to 12 hours, preferably for 30 min to 8 hours, such as 30 min to 4 hours, or 30 min to 2 hours. For instance, in step (a) the enzymatic treatment may be performed at a temperature of about 30 ℃ for about 3 hours, at a temperature of about 40 ℃ for about 2 hours, at a temperature of about 50 ℃ for about 1 hour (e.g., 60 or 75 minutes), or at a temperature of about 55 ℃ for about 45 to about 60 min.

[0130] In some embodiments, the enzymatic treatment according to step (a) is performed at a PG dosage of about 0.1 U / g to 4.5 U / g (such as 0.5 U / g, 1 U / g, 1.5 U / g, 2 U / g, or 2.5 U / g) and at a temperature of about 30 ℃ for about 3 hours.

[0131] In some embodiments, the enzymatic treatment according to step (a) is performed at a PG dosage of about 0.1 U / g to 4.5 U / g (such as 0.5 U / g, 1 U / g, 1.5 U / g, 2 U / g, or 2.5 U / g) and at a temperature of about 40 ℃ for about 2 hours.

[0132] In some embodiments, the enzymatic treatment according to step (a) is performed at a PG dosage of about 0.1 U / g to 4.5 U / g (such as 0.5 U / g, 1 U / g, 1.5 U / g, 2 U / g, or 2.5 U / g) and at a temperature of about 50 ℃ for about 1 hour (e.g., 60 or 75 minutes).

[0133] In some embodiments, the enzymatic treatment according to step (a) is performed at a PG dosage of about 0.1 U / g to 4.5 U / g (such as 0.5 U / g, 1 U / g, 1.5 U / g, 2 U / g, or 2.5 U / g) and at a temperature of about 55 ℃ for about 45 to about 60 min.

[0134] Enzymatic treatment according to step (a) of aspects (I), (II) and (III) In step (a) according to aspects (I), (II) and (III), the raw milk may be treated with the protein-glutaminase at an enzymatic treatment temperature below 20 ℃, preferably below 14 ℃, more preferably below 10 ℃, even more preferably below 8 ℃ and further preferably at least 0 ℃, more preferably at least 2 ℃. Hence, the enzymatic treatment temperature is preferably between 0 ℃ to 20 ℃, more preferably between 2 ℃ to 14 ℃, even more preferably between 2 ℃ to 10 ℃, even more preferably between 2 ℃ to 8 ℃, such as about 2 ℃, about 3 ℃, about 4 ℃, about 5 ℃, about 6 ℃, or about 7 ℃, most preferably at 4 ℃.

[0135] In step (a) according to aspects (I), (II) and (III), the raw milk may be treated with the protein-glutaminase for up to 24 hours. For example, the raw milk may be treated with the protein-glutaminase for a duration of at least 1 hour, preferably at least 4 hours, preferably at least 8 hours, preferably at least 12 hours, preferably at least 16 hours, preferably at least 18 hours, and up to 24 hours, preferably up to 22 hours, preferably up to 20 hours. Hence, the enzymatic treatment may be performed for 1 to 24 hour(s), preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 18, 19, 20, 21 or 22 hours, most preferably for 22 hours. In an embodiment, in step (a) the enzymatic treatment is performed at a temperature of at least 10 ℃, and the treatment is performed for at least 8 hours. Hence, in a preferred embodiment, in step (a) the enzymatic treatment is performed for 18 to 22 hours, such as 20 hours, and the temperature is a temperature of 4 to 8 ℃, such as 4 ℃.

[0136] In a preferred embodiment of step (a), the enzymatic treatment is performed at a temperature below 10 ℃, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g. In another preferred embodiment, in step (a), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, such as 0.4 U / g, about 0.5 U / g, about 0.6 U / g, about 0.7 U / g, about 0.8 U / g, about 0.9 U / g, about 1 U / g, about 1.1 U / g, about 1.2 U / g, about 1.3 U / g, about 1.4 U / g, about 1.5 U / g, about 1.6 U / g, about 1.7 U / g, about 1.8 U / g, about 1.9 U / g, about 2 U / g, about 2.1 U / g, about 2.2 U / g, about 2.3 U / g, about 2.4 U / g, about 2.5 U / g, about 2.6 U / g, about 2.7 U / g, about 2.8 U / g, about 2.9 U / g, about 3 U / g, about 3.1 U / g, about 3.2 U / g, about 3.3 U / g, about 3.4 U / g, about 3.5 U / g, about 3.6 U / g, about 3.7 U / g, about 3.8 U / g, about 3.9 U / g, about 4 U / g, about 4.1 U / g, about 4.2 U / g, about 4.3 U / g, about 4.4 U / g, or about 4.5 U / g.

[0137] Hence, in a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature below 20 ℃, preferably wherein the temperature is a temperature of 4 ℃ to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, 1.5 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

[0138] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃ to about 8 ℃, preferably about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 0.5 U / g, 1 U / g, 1.5 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for about 8 to about 24 hours, preferably for about 20 hours.

[0139] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃ to about 8 ℃, preferably about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 0.5 U / g, and wherein the enzymatic treatment is performed for about 20 hours.

[0140] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 1 U / g, and wherein the enzymatic treatment is performed for about 20 hours.

[0141] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 1.5 U / g, and wherein the enzymatic treatment is performed for about 20 hours.

[0142] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 2 U / g, and wherein the enzymatic treatment is performed for about 20 hours.

[0143] In a preferred embodiment of step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, the enzymatic treatment is performed at a temperature of about 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of about 2.5 U / g, and wherein the enzymatic treatment is performed for about 20 hours.

[0144] Step (b): Ultra-high-temperature (UHT) processing of the mixture obtained in step (a) The deamidation reaction may optionally be monitored, for example, by measuring an amount of ammonium produced during the treatment operation with PG.

[0145] After sufficiently treating the raw milk with the protein-glutaminase, the milk may be subject to heat sterilization to cease protein modification by deactivating the protein-glutaminase enzyme, as well as any other enzymes or live organisms present. In some embodiments, the modified milk is heat pasteurized at a temperature of at least 70 ℃, preferably at least 75 ℃, preferably at least 80 ℃, preferably at least 85 ℃, and up to 150 ℃. Depending on the temperature employed, the heat sterilizing may be performed for e.g., at least 1 sec, and up to 30 minutes. This heat treatment is preferably ultra-high-temperature (UHT) processing.

[0146] UHT processing of the mixture obtained in step (a) according to step (b) may include a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step.

[0147] To obtain ‘commercial sterility’, which indicates that bacterial growth in the product under typical storage conditions is unlikely, UHT processing in the range of 135 ℃ to 150 ℃ is used in combination with a proper holding time. Hence, the heating step of step (b) may be performed at at least 135 ℃, and / or may be performed at less than 150 ℃, preferably at 135 ℃ to 150 ℃, such as 140 ℃. The holding time for the heating step may be at least 2 seconds and / or may be less than 10 seconds. Hence, heating step may be performed for 2 to 10 seconds, preferably between 2 to 5 seconds, more preferably for 5 seconds. Hence, preferably, the heating step of step (b) may be performed at 135 ℃ to 150 ℃, such as 140 ℃, for 2 to 10 seconds, such as 5 seconds.

[0148] The ultra-high-temperature (UHT) processing may further comprise a pre-heating step, prior to the heating step. The pre-heating step may be performed by pre-heating at at least 75 ℃, such as at least 85 ℃, such as at least 90 ℃ and at 100 ℃ or less, such as at temperatures between 75 ℃ to 100 ℃, preferably between 85 ℃ or 100 ℃, preferably between 85 ℃ or 95 ℃, such as 90 ℃. The pre-heating step may be performed for more than 20 seconds, preferably for more than 30 seconds, more preferably for more than 60 seconds and / or may be performed for less than 120 seconds. Preferably, the pre-heating step may be performed for 20 to 120 seconds, more preferably for 30 to 60 seconds. Preferably, the pre-heating step is performed at 90 ℃ for 60 seconds.

[0149] A heating step during UHT processing may contain direct heating, indirect heating or microwave heating. During direct heating, the saturated steam is directly mixed with milk, and with the help of a vacuum flash vessel, the steam (added water) is removed from the milk. During indirect heating, the heating medium, namely hot water or steam, is used to heat the milk indirectly by convection or conduction through a barrier in the form of a heat exchanger. The heating step during UHT processing according to step (b) may be performed by direct or indirect heating and is preferably performed by indirect heating. Likewise, a pre-heating step during UHT processing according to step (b) may be performed by direct or indirect heating and is preferably performed by indirect heating. In a preferred embodiment, the pre-heating and / or heating step(s) during UHT processing may be performed by indirect heating. For instance, the pre-heating and / or heating step(s) shown in FIG. 3, FIG. 4, and FIG. 5 may be performed by indirect heating.

[0150] Step (b) may further comprise a homogenization step. Homogenization is a mechanical treatment which disperses milk fat globules. In particular, high pressure is used to break up fat globules leading to reduction of their size, and an increase in the number and total surface area, of fat globules in the milk. This homogenization step can be performed with a variety of mixing equipment, which applies high shear forces to the product. Preferably the process of the present invention also comprises a homogenization step, suitably conducted using a conventional high-pressure two-stage homogenization plant. This step may be included at any convenient point in the process, for instance prior to pre-heating step, prior to the heating step, or after the heating step. In the latter case, in order to produce a sterile product, it is clearly necessary that the homogenization should be under aseptic conditions. The process of the present invention may comprise one, two or more homogenization steps. For instance, a homogenization step may be performed prior to the heating step and after the heating step. The homogenization step(s) may be performed by standard homogenization which is carried out at an upstream pressure of 20-60 MPa (200-600 bar) by high-pressure homogenization (HPH), which reaches 200 MPa (2000 bar) or by ultra-high-pressure homogenization (UHPH), which is carried out up to 400-600 MPa (4000-6000 bar). The homogenization step(s) may be performed in one or multiple sub-steps. Preferably, a homogenization step may be performed by using two steps using two different pressures. The homogenization steps may be consecutive steps or may be carried out before and after the heating step. The homogenization steps may be carried out at different homogenization pressures, for instance, the homogenization step may comprise a step at lower pressure followed by a step at a higher pressure. The higher pressure may more than 20 MPa (200 bar), such as between 20 MPa (200 bar) to 600 MPa (6000 bar), preferably between 20 MPa (200 bar) to 60 MPa (600 bar), such as 24 MPa (240 bar). For example, homogenization may be performed at 250 MPa prior to the UHT processing and 400 MPa post-UHT processing. Preferably, the homogenization step may be performed after the heating step only. Preferably, the homogenization step may be performed after the heating step and carried out in two consecutive steps: a first step at a homogenization pressure of 3 MPa (30 bar) and a second step at a homogenization pressure of 24 MPa (240 bar). Hence, in an embodiment, a homogenization step may be carried out in two consecutive steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa 32 (240 bar)), following the heating step. Other homogenization schemes applicable to milk products also may be used. The homogenization step may be performed at a temperature of below 90 ℃, such as below 80 ℃, such as below 70 ℃. For instance, the homogenization step may be performed at a temperature in the range of 30 to 90 ℃, preferably in the range of 55 to 80 ℃, more preferably in the range of 60 to 70 ℃, such as 65 ℃.

[0151] In a preferred embodiment of the invention, the UHT processing further includes a cooling step. The cooling step may be performed after the heating step and / or homogenization step. The cooling step may be a rapid cooling step such as by aseptic flash cooling. In the context of the present invention, the term “flash cooling” is the cooling obtained by introducing, e.g., spraying, a hot liquid or aerosol into a vacuum chamber, whereby parts of the liquid evaporate and rapidly cool the remaining liquid. In instances, where the cooling step is performed after the heating step and prior to homogenization, the cooling step preferably cools the UHT milk or milk product to a temperature of below 90 ℃, such as below 80 ℃, such as below 70 ℃. In this instance, the UHT milk or milk product is cooled to a temperature in the range of 30 to 90 ℃, preferably in the range of 40 to 80 ℃, and even more preferably in the range of 50 to 70 ℃, such as 65 ℃.

[0152] In instances, where the cooling step is performed after the heating step and / or after the homogenization step, and / or wherein the cooling step is the final step of the UHT processing, the cooling step preferably cools the UHT milk or milk product to a temperature of below 90 ℃, such as below 20 ℃, such as below 10 ℃. In an instance where the cooling step is the final step of the UHT processing, the UHT milk or milk product is cooled to a temperature in the range of 2 to 90 ℃, preferably in the range of 2 to 20℃, and even more preferably in the range of 2 to 10 ℃, such as 8 ℃.

[0153] In a preferred embodiment of the invention, the UHT processing further includes a packaging step. The packaging step may be performed after the heating step, and / or homogenization step, and / or cooling step. The packaging technique utilized is not inherently constrained, provided that it maintains the structural integrity of the milk composition (such as the UHT milk or milk product of the present invention) to an adequate extent, ensuring the applicable shelf life of the dairy product. Preferably the UHT milk or milk product is packaged under aseptic conditions. For example, the aseptic packaging may be performed by using an aseptic filling system, and it preferably involves filling the milk into one or more aseptic container(s), e.g., sterilized containers, presterilized bottles or foil-lined gable-top carton materials may be used. Food packaging systems designated as extended shelf life (ESL) or aseptic packaging systems may also be used, but the invention is not limited thereto. The packaging is preferably performed at or below room temperature. Thus, the temperature of the cooled milk or milk-related product is preferably at most 30 ℃ during the packaging, preferably at most 25 ℃ and even more preferably at most 20 ℃, such as at most 10 ℃.

[0154] Any commercially available UHT processing system can be used for the UHT processing. An example of commercially available UHT processing systems is the FT174X, ARMFIELD Ltd. Time (sec) and Temperature (℃) during UHT processing, as well as the homogenization pressures (bar / MPa) can be set up according to the manufacturer’s recommendations.

[0155] Milk composition obtained by the methods of the present invention In instances, where the method of the present invention comprises a step (b) of ultra-high-temperature (UHT) processing of the mixture obtained in step (a), a UHT milk is obtained. The UHT milk obtained by the method of the present invention may be characterized by a decrease of sedimentation, when compared to UHT milk produced with a traditional method lacking step (a), i.e., a method lacking a step of subjecting a raw milk to enzymatic treatment with a protein-glutaminase. For instance, the UHT milk obtained by the method of the present invention may have a 2-fold reduction in the amount of sedimentation compared to a UHT milk produced without an enzymatic treatment step (a).

[0156] The UHT milk obtained by the method of the present invention may further be characterized by an increased freshness and mouthfeel, when compared to UHT milk produced with a method lacking an enzymatic treatment step (a), i.e., a method lacking a step of subjecting a raw milk to enzymatic treatment with a protein-glutaminase. Hence, in an embodiment, the UHT milk of the present invention comprises one or more favourable dairy-like properties selected from the group consisting of mouthfeel and freshness.

[0157] In instances, where the milk product is not subjected to UHT processing, the method further comprises a step (b) of heat sterilization of the mixture obtained in step (a) to cease protein modification by deactivating the protein-glutaminase enzyme, preferably at a temperature of at least 70 ℃, preferably at least 75 ℃, preferably at least 80 ℃ preferably up to 150 ℃, for at least 1 second, and up to 30 minutes. For instance, the milk product may be subjected to a pasteurization step. The milk product obtained after heat sterilization may be further processed.

[0158] The UHT milk or milk product obtained by the methods of the present invention may be further processed. Hence, in an embodiment, the present invention further provides a method for producing a dairy product, by using the UHT milk or milk product obtainable by the method of the present invention. The dairy product may be a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink. The skilled person is aware of methods for producing a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.

[0159] Method for analysing a UHT milk during storage The present invention further provides a method for analysing a UHT milk during storage. In this method, the amount of sedimentation in UHT milk is measured by centrifugation and by measuring the dry weight of a sediment obtained after centrifugation. In particular, the inventors have found that this new analytical method allows to easily monitor physiochemical changes in UHT milk during storage. Prior to sedimentation analysis, the UHT milk may be stored at temperatures above 45 ℃ in an ‘accelerated shelf-life study’ aimed at reproducing the effect of several months’ storage at room temperature.

[0160] Hence, in an embodiment, the present invention provides a method for analysing a UHT milk during storage, a) wherein the UHT milk is stored at temperatures above 4 ℃ for 14 days or more; and b) wherein the amount of sedimentation is measured.

[0161] In a preferred embodiment, the present invention provides a method for analysing a UHT milk during storage, a) wherein the UHT milk is stored at temperatures above 4 ℃ for 14 days or more; and b) wherein the amount of sedimentation is measured by centrifugation and the weight of the resulting sediment is determined, optionally wherein the weight of the resulting sediment is the dry weight of said sediment.

[0162] In another preferred embodiment, the present invention provides a method for analysing a UHT milk during storage, a) wherein the UHT milk is stored at temperatures above 4 ℃ for 14 days or more; preferably at 40 to 50 ℃, preferably at 45 ℃ and b) wherein the amount of sedimentation is measured by centrifugation and the weight of the resulting sediment is determined, optionally wherein the weight of the resulting sediment is the dry weight of said sediment, preferably wherein the centrifugation is performed at at least 4000 RPM, and / or wherein the centrifugation is performed for at least 15 min.

[0163] Methods for producing the UHT milk or milk products according to the present invention Hence, the present invention described the following methods for producing a milk product:

[0164] In aspect (I), the method for producing a UHT milk comprises or consist of the following consecutive steps: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a)

[0165] In a preferred embodiment of step (a) of aspect (I), the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk. In a preferred embodiment of step (a) of aspect (I), the raw milk is skimmed milk, semi skimmed milk or whole milk. In a preferred embodiment of step (a) of aspect (I), the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step at 72 to 75 ℃ for 15 to 30 seconds, such as 74 ℃ for 30 seconds. In a preferred embodiment of step (a) of aspect (I), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein, preferably 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 2 U / g, and even more preferably 0.5 U / g, 1 U / g, or 2 U / g. In a preferred embodiment of step (a) of aspect (I), the protein-glutaminase is added to the raw milk in an amount of about 0.4 U / g, about 0.5 U / g, about 0.6 U / g, about 0.7 U / g, about 0.8 U / g, about 0.9 U / g, about 1 U / g, about 1.1 U / g, about 1.2 U / g, about 1.3 U / g, about 1.4 U / g, about 1.5 U / g, about 1.6 U / g, about 1.7 U / g, about 1.8 U / g, about 1.9 U / g, about 2 U / g, about 2.1 U / g, about 2.2 U / g, about 2.3 U / g, about 2.4 U / g, or about 2.5 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed for 1 to 24 hour(s), preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours. In a preferred embodiment of step (a) of aspect (I), the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature below 65 ℃, below 20 ℃, below 14 ℃, below 10 ℃, or below 8 ℃, preferably the temperature is a temperature between 2 and 65 ℃, more preferably between 2 and 14 ℃, even preferably between 2 and 10 ℃, even more preferably between 2 and 8 ℃, even more preferably between 4 and 8 ℃, such as 4 ℃. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature below 10 ℃, and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g. In a preferred embodiment of step (a) of aspect (I), the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours. In another embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of at least 20℃, such as 30 to 60 ℃, and the treatment is performed for 15 min to 12 hours, preferably for 30 min to 8 hours, such as 30 min to 4 hours, or 30 min to 2 hours, most preferably the enzymatic treatment is performed at a temperature of 45 to 50 ℃, and the treatment is performed for 45 min to 2 hours. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of at least 20 ℃ and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of at least 20 ℃ and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g. In a preferred embodiment of step (a) of aspect (I), the temperature is a temperature of 20 to 65 ℃, preferably 45 to 50 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 15 min to 22 hours, preferably for 45 min to 2 hours. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 30 ℃, and the treatment is performed for about 3 hours. In another preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 40 ℃, and the treatment is performed for about 2 hours. In another preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 50 ℃, and the treatment is performed for about 1 to about 1.25 hours. In another preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 55 ℃, and the treatment is performed for about 45 to about 60 min. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 30 ℃ for about 3 hours, and the protein-glutaminase is added to the raw milk in an amount of about 0.1 U / g to about 10 U / g, preferably about 0.1 U / g to about 4.5 U / g, even more preferably about 0.1 U / g to about 2.5 U / g, such as about 0.5 U / g, about 1 U / g, or about 2 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 40 ℃ for about 2 hours, and the protein-glutaminase is added to the raw milk in an amount of about 0.1 U / g to about 10 U / g, preferably about 0.1 U / g to about 4.5 U / g, even more preferably about 0.1 U / g to about 2.5 U / g, such as about 0.5 U / g, about 1 U / g, or about 2 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 50 ℃ for about 1 to about 1.3 hours, and the protein-glutaminase is added to the raw milk in an amount of about 0.1 U / g to about 10 U / g, preferably about 0.1 U / g to about 4.5 U / g, even more preferably about 0.1 U / g to about 2.5 U / g, such as about 0.5 U / g, about 1 U / g, or about 2 U / g. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment is performed at a temperature of about 55 ℃ for about 45 to about 60 min, and the protein-glutaminase is added to the raw milk in an amount of about 0.1 U / g to about 10 U / g, preferably about 0.1 U / g to about 4.5 U / g, even more preferably about 0.1 U / g to about 2.5 U / g, such as about 0.5 U / g, about 1 U / g, or about 2 U / g. In a preferred embodiment of step (a) of aspect (I), prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk. In a preferred embodiment of step (a) of aspect (I), prior to step (a) no step of removing a low molecule fraction, such as a low molecule fraction having a molecular weight of 10,000 or less is performed, such as by using an ultrafiltration membrane. In a preferred embodiment of step (a) of aspect (I), the enzymatic treatment step does not comprise the addition of crosslinking enzymes such as transglutaminase (TG).

[0166] In a preferred embodiment of step (b) of aspect (I), the UHT processing includes a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step. In a preferred embodiment of step (b) of aspect (I), the heating step is performed at at least 135 ℃, preferably at 135 to 150 ℃. In a preferred embodiment of step (b) of aspect (I), a further pre-heating step is performed prior to the heating step; optionally wherein the further pre-heating step is performed by pre-heating at 85 to 100 ℃ for 20 to 120 seconds; optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating. In a preferred embodiment of step (b) of aspect (I), a further pre-heating step is performed prior to the heating step; optionally wherein the further pre-heating step is performed by pre-heating at 90 ℃ for 60 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating. In a preferred embodiment of step (b) of aspect (I), a further pre-heating step is performed prior to the heating step; optionally wherein the further pre-heating step is performed by pre-heating at 90 ℃ for 60 seconds followed by the heating step at 135 ℃ for 5 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating. In a preferred embodiment of step (b) of aspect (I) the homogenization step is performed prior to the heating step and / or after the heating step. In a preferred embodiment of step (b) of aspect (I) a cooling step is performed after the heating step and / or prior to the homogenization step, optionally wherein the cooling is cooling to a temperature below 90 ℃, preferably to below 85 ℃, more preferably between 55 to 80 ℃, even more preferably between 60 and 70 ℃, such as 65 ℃.

[0167] In a preferred embodiment of step (b) of aspect (I) the homogenization step is performed after the heating step. In a preferred embodiment of step (b) of aspect (I), the homogenization step is carried out in two steps using two homogenization pressures. In a preferred embodiment of step (b) of aspect (I), the homogenization step is carried out after the heating step in two consecutive steps. In a preferred embodiment of step (b) of aspect (I), the homogenization step is carried out after the heating step in two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar). In a preferred embodiment of step (b) of aspect (I), the homogenization step is carried out after the heating step in two consecutive steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar). In a preferred embodiment of step (b) of aspect (I), the homogenization step is performed after the heating step and after a cooling step, wherein in the cooling step the UHT milk is cooled to a temperature below 90 ℃, or below 80 ℃, or below 70 ℃, such as 65 ℃. In a preferred embodiment of step (b) of aspect (I) the homogenization step is performed at a temperature in the range of 30 to 90 ℃, preferably in the range of 55 to 80 ℃, more preferably in the range of 60 to 70 ℃, most preferably at 65 ℃.

[0168] In a preferred embodiment of step (b) of aspect (I), a further cooling step is performed, optionally wherein the cooling is cooling to a temperature below 25 ℃, preferably below 10 ℃, more preferably below 8 ℃. In a preferred embodiment of step (b) of aspect (I), a further packaging step is performed, optionally wherein the packaging is aseptic packaging.

[0169] Hence, in an embodiment of aspect (I), the method for producing a UHT milk comprises or consist of the following consecutive steps: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 20 to 60 ℃ (such as 45 to 50 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 15 min to 2 hours (such as 45 to 60 minutes); (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a), wherein the UHT processing comprises or consists of steps: (i) a pre-heating step by indirect heating (such as 90 ℃ for 60 seconds), (ii) a heating step by indirect heating (such as 135 ℃ for 5 seconds), (iii) a cooling step (such as cooling to a temperature below 65 ℃), (iv) a homogenization step carried out in two steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa (240 bar)), (v) a further cooling step (such as cooling to a temperature below 8 ℃), and (vi) a packaging step (such as filling into a container, preferably under aseptic conditions); wherein the steps (i) to (vi) are executed consecutively.

[0170] Hence, in an embodiment of aspect (I), the method for producing a UHT milk comprises or consist of the following consecutive steps: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 2 to 8 ℃ (such as 4 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 8 to 24 hours (such as 20 hours); and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a), wherein the UHT processing comprises or consists of steps: (i) a pre-heating step by indirect heating (such as 90 ℃ for 60 seconds), (ii) a heating step by indirect heating (such as 135 ℃ for 5 seconds), (iii) a cooling step (such as cooling to a temperature below 65 ℃), (iv) a homogenization step carried out in two steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa (240 bar)), (v) a further cooling step (such as cooling to a temperature below 8 ℃), and (vi) a packaging step (such as filling into a container); wherein the steps (i) to (vi) are executed consecutively; and wherein the milk product is a UHT milk.

[0171] In aspect (II), the method for producing a milk product comprises a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃.

[0172] In a preferred embodiment of step (a) of aspect (II), the temperature is below 14 ℃, below 10 ℃, or below 8 ℃, preferably the temperature is a temperature of 2 to 14 ℃, preferably 2 to 10 ℃, more preferably 2 to 8 ℃, even more preferably 4 to 8 ℃, such as 4 ℃. In a preferred embodiment of step (a) of aspect (II), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein, preferably the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g, such as 0.5 U / g, 1 U / g, or 2 U / g. In a preferred embodiment of step (a) of aspect (II), the protein-glutaminase is added to the raw milk in an amount of about 0.4 U / g, about 0.5 U / g, about 0.6 U / g, about 0.7 U / g, about 0.8 U / g, about 0.9 U / g, about 1 U / g, about 1.1 U / g, about 1.2 U / g, about 1.3 U / g, about 1.4 U / g, about 1.5 U / g, about 1.6 U / g, about 1.7 U / g, about 1.8 U / g, about 1.9 U / g, about 2 U / g, about 2.1 U / g, about 2.2 U / g, about 2.3 U / g, about 2.4 U / g, or about 2.5 U / g. In a preferred embodiment of step (a) of aspect (II), the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium. In a preferred embodiment of step (a) of aspect (II), the enzymatic treatment is performed at a temperature below 10 ℃, and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g. In a preferred embodiment of step (a) of aspect (II), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours. In a preferred embodiment of step (a) of aspect (II), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g. In a preferred embodiment of step (a) of aspect (II), the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours. In a preferred embodiment of step (a) of aspect (II), prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk. In a preferred embodiment of step (a) of aspect (II), prior to step (a) no step of removing a low molecule fraction, such as a low molecule fraction having a molecular weight of 10,000 or less is performed, such as by using an ultrafiltration membrane. In a preferred embodiment of step (a) of aspect (II), the enzymatic treatment step does not comprise the addition of crosslinking enzymes such as transglutaminase (TG).

[0173] In a preferred embodiment of aspect (II), the method further comprises a step (b) of heat sterilization of the mixture obtained in step (a) to cease protein modification by deactivating the protein-glutaminase enzyme, preferably at a temperature of at least 70 ℃, preferably at least 75 ℃, preferably at least 80 ℃ preferably up to 150 ℃, for at least 1 sec, and up to 30 minutes. In a preferred embodiment of aspect (II), the method further comprises a step (b) of ultra-high-temperature (UHT) processing of the mixture obtained in step (a). The method of ultra-high-temperature (UHT) processing is as defined for aspect (I).

[0174] Hence, in an embodiment of aspect (II), the method for producing a milk product comprises or consist of the following consecutive steps: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 2 to 8 ℃ (such as 4 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 8 to 24 hours (such as 20 hours); and (b) heat sterilization of the mixture obtained in step (a) to cease protein modification by deactivating the protein-glutaminase enzyme (such as temperature of at least 70 ℃ for up to 30 minutes, preferably 95 ℃ for 5 min).

[0175] In aspect (III), the method for producing a UHT milk comprises or consist of the following consecutive steps: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

[0176] In a preferred embodiment of step (a) of aspect (III), the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk. In a preferred embodiment of step (a) of aspect (III), the raw milk is skimmed milk, semi skimmed milk or whole milk. In a preferred embodiment of step (a) of aspect (III), the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step at 72 to 75 ℃ for 15 to 30 seconds, such as 74 ℃ for 30 seconds. In a preferred embodiment of step (a) of aspect (III), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein, preferably 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 2 U / g, and even more preferably 0.5 U / g, 1 U / g, or 2 U / g. In a preferred embodiment of step (a) of aspect (III), the protein-glutaminase is added to the raw milk in an amount of about 0.4 U / g, about 0.5 U / g, about 0.6 U / g, about 0.7 U / g, about 0.8 U / g, about 0.9 U / g, about 1 U / g, about 1.1 U / g, about 1.2 U / g, about 1.3 U / g, about 1.4 U / g, about 1.5 U / g, about 1.6 U / g, about 1.7 U / g, about 1.8 U / g, about 1.9 U / g, about 2 U / g, about 2.1 U / g, about 2.2 U / g, about 2.3 U / g, about 2.4 U / g, or about 2.5 U / g. In a preferred embodiment of step (a) of aspect (III), the enzymatic treatment is performed for 1 to 24 hour(s), preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours. In a preferred embodiment of step (a) of aspect (III), the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium.

[0177] In a preferred embodiment of step (a) of aspect (III), the enzymatic treatment is performed at a temperature below 10 ℃, and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g. In a preferred embodiment of step, the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours. In a preferred embodiment of step (a) of aspect (III), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g. In a preferred embodiment of step (a) of aspect (III), the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

[0178] In a preferred embodiment of aspect (III), the method for producing a milk product comprises or consist of the following consecutive steps: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 2 to 8 ℃ (such as 4 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 8 to 24 hours (such as 20 hours); and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a), wherein the UHT processing comprises or consists of steps: (i) a pre-heating step by indirect heating (such as 90 ℃ for 60 seconds), (ii) a heating step by indirect heating (such as 135 ℃ for 5 seconds), (iii) a cooling step (such as cooling to a temperature below 65 ℃), (iv) a homogenization step carried out in two steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa (240 bar)), (v) a further cooling step (such as cooling to a temperature below 8 ℃), and (vi) a packaging step (such as filling into a container); wherein the steps (i) to (vi) are executed consecutively; and wherein the milk product is a UHT milk.Example

[0179] Hereinafter, the present invention will be more specifically described based on examples. It is understood that various other aspects may be practiced, given the general and detailed descriptions provided elsewhere herein.

[0180] Example 1Preparation of samples Raw milk was obtained from the local farm (La ferme de Launay, 95690 Nesle-la-Vallée, France). The raw milk was cooled down to 4 ℃ and stored in plastic containers (e.g., 20 L) at 4 ℃. The content of protein was measured using spectrophotometry (MPA II-LSM, Bruker Optics France).

[0181] PG was added to the raw milk to obtain 4 conditions: 0 units of PG / g of protein (U / gP), 0.5 units of PG / g of protein, 1 unit of PG / g of protein and 2 units of PG / g of protein was added. The composition was mixed until homogenous and stored at 4 ℃ for 20 hours.

[0182] After 20 hours, UHT processing was performed using heat-treatment pilot (FT174X, ARMFIELD Ltd) with the following setup: 1) Indirect pre-heating: 90 ℃ for 60s, 2) Indirect main heating: 135 ℃ for 5s, 3) Cooling: down to 65 ℃, 4) Downstream homogenization in two steps: 30 / 240 bar, 5) Cooling: down to 4 ℃, and 6) Packaging: 200 mL plastic bottles under sterile conditions.

[0183] The UHT milk samples were stored at 4 ℃ and 45 ℃ for 14 days. Temperatures above 45 ℃ allow for an ‘accelerated shelf-life study’ aiming at reproducing the effect of several months’ storage at room temperature. After 14 days, the analysis of the UHT milk samples was performed. The results are shown in Table 1 (pH analysis and sedimentation analysis) and Figures 6A and 6B (analysis of taste deviation).

[0184] pH measurement After 14 days, the pH of the UHT milk samples was measured using a METTLER TOLEDO pH meter prior to sedimentation analysis. The pH is shown in Table 1.

[0185] Sedimentation analysis In order to assess the effect of PG treatment at the exemplary concentration of 2 U / gP, each of the UHT milk samples obtained above (UHT milk samples stored at 4 ℃ and 45 ℃ for 14 days), was further subjected to the following sedimentation analysis. The UHT milk samples were shaken and then filled into 3x 50 mL tubes in triplicates each comprising 50g sample. The weight of each tube was determined prior to and after filling in the samples. Each of the tubes comprising the samples was centrifugated at 4,000 RPM at room temperature for 15 min. The supernatant was removed and each tube comprising the respective pellet was weighted separately. The tubes were put in an oven / drying oven (e.g., an oven able to reach a temperature high enough to dry the samples) at 50 ℃ for 24 hours (see Table 1, “Sediments (mg / 100g milk)after 24h”). The tubes comprising the pellets were weighted again and the total mass was noted. The tubes were subjected to a further drying step in the oven / drying oven at 50 ℃ for an additional 24 hours (see Table 1, “Sediments (mg / 100g milk)after 48h”). The tubes comprising the pellets were weighted again and the total mass was noted.

[0186] The results from the above Example are shown in Table 1. As shown in the Table, the amount of sediments in PG treated UHT milk was approximately half of the amount of sediments in the control UHT milk.

[0187]

[0188] Sensory analysis In order to assess the effect of PG treatment, each of the UHT milk samples obtained above (UHT milk samples stored at 4 ℃ and 45 ℃ for 14 days), was further subjected to sensory analysis with a panel of 5 people, trained for dairy products. The samples were tasted at 13 ℃.

[0189] The samples were rated based on their deviation from control with the following notation: ・0: no deviation, taste is similar to control. ・1.0: small deviation, there is small difference between rated sample and the control. ・2.0: medium deviation, there is a medium deviation between rated sample and control. ・3.0: large deviation, there is a large deviation between rated sample and control.

[0190] The results are shown in Figures 6A and 6B. As shown in Figures 6A and 6B, taste deviation compared to the control was measured in all samples treated with PG. The deviation between the rated sample and the control increased with an increasing amount of PG added to the raw milk prior to UHT processing, with the largest deviation when comparing a sample treated with 2 U / gP. According to panellists, the deviation came from increased freshness and mouthfeel of the UHT milk treated with PG compared to the nontreated milk.

[0191] The present invention is useful in a wide range of fields, including the field of milk products such a UHT milk. The milk products or UHT milk obtained by the methods of the present invention support nutrition and are useful as food products for human or animal consumption. In addition, the milk products or UHT milk may be further processed to obtain other dairy products (such as a cream, a yoghurt, a cheese, a cheese-like food, a pudding, or a milk-based drink) for consumption.

[0192] Thus, the present invention is industrially applicable.

[0193] Further applications and practical exploitation in industry may be derived from the present description by the skilled person’s general knowledge.

[0194] The preferred embodiments of the present invention have been described in detail above. It should be understood that many modifications and changes can be made in accordance with the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited times of experiments based on the concept of the present invention and the prior art should fall within the scope defined by the accompanying claims.

[0195] This application is based on a patent application No. 24307119.8 filed in European Patent Office, the contents of which are incorporated in full herein.

Claims

1. A method for producing a UHT milk, comprising the steps of: (a) subjecting a raw milk to enzymatic treatment with a protein-glutaminase; and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

2. The method according to claim 1, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein.

3. The method according to claim 1 or 2, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g.

4. The method according to any one of claims 1 to 3, wherein in step (a), the protein-glutaminase is added to the raw milk in an amount of 0.5 U / g, 1 U / g, or 2 U / g.

5. The method according to any one of claims 1 to 4, wherein in step (a), the enzymatic treatment is performed for 10 min to 24 hours, preferably for 1 to 24 hour(s), even more preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours.

6. The method according to any one of claims 1 to 5, wherein in step (a), the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium.

7. The method according to any one of claims 1 to 6, wherein in step (a), the enzymatic treatment is performed at a temperature below 65 ℃, below 60 ℃, below 55 ℃, below 50 ℃, below 45 ℃, below 40 ℃, below 35 ℃, below 30 ℃, below 25 ℃, below 20 ℃, below 14 ℃, below 10 ℃, or below 8 ℃.

8. The method according to any one of claims 1 to 7, wherein in step (a), the enzymatic treatment is performed at a temperature of 2 to 65 ℃, 2 to 60 ℃, 2 to 55 ℃, 2 to 50 ℃, 2 to 45 ℃, 2 to 40 ℃, 2 to 35 ℃, 2 to 30 ℃, 2 to 25 ℃, 2 to 20 ℃, or 2 to 15 ℃.

9. The method according to claim 7 or 8, wherein in step (a), the temperature is a temperature of 2 to 14 ℃, preferably 2 to 10 ℃, more preferably 2 to 8 ℃, even more preferably 4 to 8 ℃, such as 4 ℃.

10. The method according to any one of claims 1 to 9, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g.

11. The method according to any one of claims 1 to 10, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours.

12. The method according to any one of claims 1 to 11, wherein in step (a), the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g.

13. The method according to any one of claims 7 to 12, wherein in step (a), the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

14. The method according to any one of claims 1 to 13, wherein in step (b), the UHT processing includes a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step.

15. The method according to claim 14, wherein in step (b), the heating step is performed at at least 135℃, preferably at 135 to 150 ℃.

16. The method according to claim 14 or 15, wherein in step (b), the heating step is performed for at least 2 seconds, preferably for 2 to 10 seconds, such as 5 seconds.

17. The method according to any one of claims 14 to 16, wherein in step (b), a further pre-heating step is performed; optionally wherein the further pre-heating step is performed by pre-heating at 90℃ for 60 seconds followed by the heating step at 135℃ for 5 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating.

18. The method according to any one of claims 14 to 17, wherein in step (b), the homogenization step is performed prior to the heating step and / or after the heating step, preferably wherein the homogenization step is performed after the heating step.

19. The method according to any one of claims 14 to 18, wherein in step (b), the homogenization step is carried out in two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar).

20. The method according to any one of claims 1 to 19, wherein the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk.

21. The method according to any one of claims 1 to 20, wherein the raw milk is skimmed milk, semi skimmed milk or whole milk.

22. The method according to any one of claims 1 to 21, wherein the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step of at least 70 ℃, further optionally wherein the pasteurization step is performed at 72 to 75 ℃ for 15 to 30 seconds, such as 75 ℃ for 15 seconds.

23. The method according to any one of claims 1 to 22, wherein prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk.

24. A UHT milk obtainable by the method according to any one of claims 1 to 23.

25. A method for producing a dairy product, by using the UHT milk obtainable by the method according to any one of claims 1 to 23.

26. The method according to claim 25, wherein the dairy product is a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.

27. A method for producing a milk product, comprising a step (a) of subjecting a raw milk to enzymatic treatment with a protein-glutaminase, wherein the enzymatic treatment is performed at a temperature below 20 ℃.

28. The method according to claim 27, wherein the temperature is below 14 ℃, below 10 ℃, or below 8 ℃.

29. The method according to claim 27 or 28, wherein the temperature is a temperature of 2 to 14 ℃, preferably 2 to 10 ℃, more preferably 2 to 8 ℃, even more preferably 4 to 8 ℃, such as 4 ℃.

30. The method according to any one of claims 27 to 29, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g, wherein U / g means unit of enzyme (U) per grams (g) of milk protein.

31. The method according to any one of claims 27 to 30, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g, preferably 0.1 U / g to 2.5 U / g, more preferably 0.1 U / g to 1 U / g.

32. The method according to any one of claims 27 to 31, wherein the protein-glutaminase is added to the raw milk in an amount of 0.5 U / g, 1 U / g, or 2 U / g.

33. The method according to any one of claims 27 to 32, wherein the enzymatic treatment is performed for 10 min to 24 hours, preferably for 1 to 24 hour(s), even more preferably for 8 to 24 hours, even more preferably for 18 to 22 hours, such as 20 hours.

34. The method according to any one of claims 27 to 33, wherein the protein-glutaminase is a protein-glutaminase derived from Chryseobacterium.

35. The method according to any one of claims 27 to 34, wherein the enzymatic treatment is performed at a temperature below 10 ℃, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 10 U / g.

36. The method according to any one of claims 27 to 35, wherein the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours.

37. The method according to any one of claims 27 to 36, wherein the enzymatic treatment is performed at a temperature below 10 ℃ for at least 8 hours, and wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 4.5 U / g.

38. The method according to any one of claims 27 to 37, wherein the temperature is a temperature of 2 to 8 ℃, preferably 4 ℃, wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g, preferably 0.5 U / g, 1 U / g, or 2 U / g, and wherein the enzymatic treatment is performed for 8 to 24 hours, preferably for 20 hours.

39. The method according to any one of claims 27 to 38, further comprising a step (b) of ultra-high-temperature (UHT) processing of the mixture obtained in step (a).

40. The method according to claim 39, wherein in step (b), the UHT processing includes a heating step, a cooling step, optionally a homogenization step, and further optionally a packaging step.

41. The method according to claim 40, wherein in step (b), the heating step is performed at at least 135 ℃, preferably at 135 to 150 ℃.

42. The method according to claim 40 or 41, wherein in step (b), the heating step is performed for at least 2 seconds, preferably for 2 to 10 seconds, such as 5 seconds.

43. The method according to any one of claims 40 to 42, wherein in step (b), a further pre-heating step is performed; optionally wherein the further pre-heating step is performed by pre-heating at 90 ℃ for 60 seconds followed by the heating step at 135 ℃ for 5 seconds; further optionally wherein the pre-heating step and / or the heating step involve(s) indirect heating.

44. The method according to any one of claims 40 to 43, wherein in step (b), the homogenization step is performed prior to the heating step or after the heating step, preferably wherein the homogenization step is performed after the heating step.

45. The method according to any one of claims 40 to 44, wherein in step (b), the homogenization step is carried out in two steps at a first homogenization pressure of 3 MPa (30 bar) and a second homogenization pressure of 24 MPa (240 bar).

46. The method according to any one of claims 27 to 45, wherein the raw milk is selected from the group consisting of bovine milk, cow milk, buffalo milk, sheep milk, goat milk, camel milk, horse milk, yak milk, and donkey milk.

47. The method according to any one of claims 27 to 46, wherein the raw milk is skimmed milk, semi skimmed milk or whole milk.

48. The method according to any one of claims 27 to 47, wherein the raw milk of step (a) is a pasteurized raw milk, optionally wherein the pasteurized raw milk is obtainable by a pasteurization step of at least 70 ℃, further optionally wherein the pasteurization step is performed at 72 to 75 ℃ for 15 to 30 seconds, such as 75 ℃ for 15 seconds.

49. The method according to any one of claims 27 to 48, wherein prior to step (a) and during step (a), no agent or material selected from the group consisting of a calcium chelating agent (such as sodium citrate, sodium γ-polyglutamate or tetrasodium ethylenediamine-tetraacetate), an alkaline material (such as sodium hydroxide, potassium carbonate, sodium carbonate, trisodium phosphate, sodium hydrogencarbonate, calcined calcium or arginine), a reducing material (such as glutathione, cysteine or γ-glutamilcysteine) and a stabilizer (such as pectin, agar, carrageenan, gelatin, whey protein concentrates, chicory root fiber, or modified food starch) is added to the raw milk.

50. The method for producing a milk product according to any one of claims 39 to 49, comprising the steps of: (a) subjecting a raw milk (such as a bovine raw milk) to enzymatic treatment with a protein-glutaminase (such as a protein-glutaminase derived from Chryseobacterium), wherein the enzymatic treatment is performed at a temperature of 2 to 8 ℃ (such as 4 ℃), wherein the protein-glutaminase is added to the raw milk in an amount of 0.1 U / g to 2.5 U / g (such as 0.5 U / g, 1 U / g, or 2 U / g), and wherein the enzymatic treatment is performed for 8 to 24 hours (such as 20 hours); and (b) ultra-high-temperature (UHT) processing of the mixture obtained in step (a), wherein the UHT processing comprises or consists of steps: (i) a pre-heating step by indirect heating (such as 90 ℃ for 60 seconds), (ii) a heating step by indirect heating (such as 135 ℃ for 5 seconds), (iii) a cooling step (such as cooling to a temperature below 65 ℃), (iv) a homogenization step carried out in two steps at a first homogenization pressure (such as 3 MPa (30 bar)) and a second homogenization pressure (such as 24 MPa (240 bar)), (v) a further cooling step (such as cooling to a temperature below 8 ℃), and (vi) a packaging step (such as filling into a container); wherein the steps (i) to (vi) are executed consecutively; and wherein the milk product is a UHT milk.

51. A milk product obtainable by the method according to any one of claims 27 to 50, wherein the milk product is a UHT milk.

52. A method for producing a dairy product, by using the milk product obtainable by the method according to any one of claims 27 to 50.

53. The method according to claim 52, wherein the dairy product is a cream, a yoghurt, a cheese, a cheese-like food, a pudding or a milk-based drink.

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