Fermented milk and its manufacturing method, and dephosphorylated milk

Abstract

This invention provides a fermented milk with stable quality, a method for manufacturing the same, and a dephosphorylated milk. One fermented milk contains more than 0.8 g / 100 g of dephosphorylated casein.

Description

Technical Field

[0001] This invention relates to fermented milk and its manufacturing method, as well as dephosphated milk. Background Technology

[0002] Fermented milk is produced by fermenting raw milk using microorganisms such as lactic acid bacteria. In recent years, the physiological functions of extracellular polysaccharides (EPS) produced by lactic acid bacteria during fermentation have attracted attention.

[0003] It is known that the main protein in milk is casein. When fermentation lowers the pH, casein micelles aggregate to form a gel. Casein is mainly classified into α-cell protein. S1 α S2 These four types of phosphates—β, κ, and β—have phosphates bonded to 8, 11, 5, and 1 serine residues, respectively. Phosphate modification in casein contributes to the hydrophobicity of the casein micelle surface and hydrophobic core.

[0004] The texture (physical properties) of fermented milk change over time, and dehydration (whey separation) during storage is one of the important factors that reduces its commercial value. Therefore, to date, functional milk raw materials such as pectin and milk protein concentrates have been used to prevent dehydration. In addition, the utilization of enzymes has also been studied. For example, methods using enzymes with curdling activity such as transglutaminase and glucose oxidase have been reported (e.g., Patent Document 1).

[0005] However, the production of milk and fermented milk using enzymes that act on phosphoserine in casein is unknown.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: International Publication No. 2015 / 041194 Summary of the Invention

[0009] The subject of this invention is to provide fermented milk with stable quality, a method for manufacturing the same, and dephosphated milk.

[0010] The inventors conducted in-depth research focusing on phosphoserine in casein, and found that dephosphorylated casein, obtained by removing phosphate from serine residues of casein, is related to the physical properties of fermented milk. Fermented milk containing a specified amount of this dephosphorylated casein exhibits inhibited dehydration, increased viscosity and stress, and increased production of extracellular polysaccharides (EPS). Furthermore, it was discovered that fermenting raw milk in the presence of protein phosphatase, or fermenting raw milk treated with protein phosphatase, resulted in inhibited dehydration, increased viscosity and stress, and increased EPS production, thus completing this invention.

[0011] That is, the present invention provides the following [1] to [6].

[0012] [1] A fermented milk containing more than 0.8 g / 100 g of dephosphorylated casein.

[0013] [2] The fermented milk according to [1] contains 0.8 to 1.5 g of dephosphorylated casein per 100 g.

[0014] [3] The fermented milk according to [1] or [2], wherein the content of free phosphate is 15 mM or more.

[0015] [4] Fermented milk according to any one of [1] to [3], wherein the fermented milk is yogurt.

[0016] [5] A dephosphorylated milk containing dephosphorylated casein.

[0017] [6] The dephosphorylated milk according to [5], wherein the content of dephosphorylated casein is 0.8g / 100g or more.

[0018] In addition, the present invention provides the following [7] to

[13] .

[0019] [7] A method for manufacturing fermented milk, comprising any of the following steps:

[0020] (a) The process of fermenting raw milk in the presence of protein phosphatase.

[0021] (b) The process of fermenting raw milk produced by enzymatic treatment with protein phosphatase.

[0022] [8] The method for manufacturing fermented milk according to [7], wherein the protein phosphatase is a protein phosphatase derived from a microorganism belonging to the Trichoderma genus.

[0023] [9] In the method for manufacturing fermented milk according to [8], the microorganism belonging to the Trichoderma genus is Trichoderma viride.

[0024]

[10] A method for producing fermented milk according to any one of [7] to [9], wherein the protein phosphatase is any one of (i) to (iii) below.

[0025] (i) A protein consisting of the amino acid sequence represented by sequence number 2,

[0026] (ii) A protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted, or added in the amino acid sequence indicated by sequence number 2, and which has protein phosphatase activity.

[0027] (iii) A protein consisting of an amino acid sequence that has more than 80% sequence identity with the amino acid sequence represented by sequence number 2 and has protein phosphatase activity.

[0028]

[11] A method for manufacturing fermented milk according to any one of [7] to

[10] , wherein the fermented milk is yogurt.

[0029]

[12] An enzyme for fermented milk containing protein phosphatase.

[0030]

[13] A protein having protein phosphatase activity, which is any one of (i) to (iii) below.

[0031] (i) A protein consisting of the amino acid sequence represented by sequence number 2,

[0032] (ii) A protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted, or added in the amino acid sequence indicated by sequence number 2, and which has protein phosphatase activity.

[0033] (iii) A protein consisting of an amino acid sequence that has more than 80% sequence identity with the amino acid sequence represented by sequence number 2 and has protein phosphatase activity.

[0034] The fermented milk of this invention exhibits minimal dehydration, high viscosity, and a stable structure. Furthermore, it contains numerous in vitro polysaccharides (EPS), resulting in excellent physiological functions.

[0035] Furthermore, the method for manufacturing fermented milk according to the present invention can provide a fermented milk that is less dehydrated, has high viscosity, a stable structure, and contains many extracellular polysaccharides (EPS).

[0036] Furthermore, the dephosphated milk of the present invention alters the mouthfeel by increasing the hydrophobicity of casein molecules. This effect is also observed in dairy products using dephosphated milk. Attached Figure Description

[0037] Figure 1 This is a schematic diagram showing the electrophoresis of milk.

[0038] Figure 2 This is a diagram illustrating the method for determining phosphorylated and dephosphorylated casein.

[0039] Figure 3 This is a graph showing the a. temperature dependence, b. temperature stability, c. pH dependence, and d. pH stability of protein phosphatases derived from Trichoderma viride Gv29-8.

[0040] Figure 4 This is a graph showing the metal ion requirements of protein phosphatases derived from Trichoderma viride Gv29-8.

[0041] Figure 5 This is a graph showing the concentration of free phosphate in the fermented milk of Example 1.

[0042] Figure 6 This is a graph showing the concentration of free phosphoric acid in the dephosphorylated emulsion of Example 2.

[0043] Figure 7 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0044] Figure 8 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0045] Figure 9 This is a graph showing the amount of extracellular polysaccharide (EPS) (μg / g-fermented milk) in fermented milk supplemented with protein phosphatase derived from Trichoderma viride Gv29-8.

[0046] Figure 10 This is a graph showing the viscosity (cp) of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0047] Figure 11 This is a graph showing the analysis results of the destructive strength of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0048] Figure 12 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0049] Figure 13 This is a graph showing the amount of extracellular polysaccharide (EPS) (μg / g-fermented milk) in fermented milk supplemented with protein phosphatase derived from Trichoderma viride Gv29-8.

[0050] Figure 14This is a graph showing the viscosity (cp) of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0051] Figure 15 This is a graph showing the analysis results of the destructive strength of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0052] Figure 16 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0053] Figure 17 This is a graph showing the amount of extracellular polysaccharide (EPS) (μg / g-fermented milk) in fermented milk supplemented with protein phosphatase derived from Trichoderma viride Gv29-8.

[0054] Figure 18 This is a graph showing the viscosity (cp) of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0055] Figure 19 This is a graph showing the analysis results of the destructive strength of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0056] Figure 20 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk treated with Trichoderma viride Gv29-8 protein phosphatase.

[0057] Figure 21 This is a graph showing the pH shift of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0058] Figure 22 This is a graph showing the dehydration rate (%), free phosphate concentration (mM), and pH of fermented milk containing protein phosphatase derived from Trichoderma viride Gv29-8.

[0059] Figure 23 This is a graph showing the shifts in free phosphate and calcium ion concentrations in raw milk caused by treatment with protein phosphatase derived from Trichoderma viride Gv29-8. Detailed Implementation

[0060] In this specification, fermented milk refers to fermented milk and lactic acid bacteria beverages as defined in the Ministry Ordinance on Milk, etc. (Ministry Ordinance on standards for the composition of milk and dairy products, etc., Ministry of Health, Labour and Welfare Ordinance No. 52 of 1947). Fermented milk is defined as "a product made by fermenting milk or milk containing an equal or greater amount of skim milk solids using lactic acid bacteria or yeast, and producing it as a paste or liquid, or by freezing it." Lactic acid bacteria beverages are defined as "beverages (excluding fermented milk) obtained by processing substances obtained from fermenting milk using lactic acid bacteria or yeast, or by using such substances as a main ingredient."

[0061] Examples of fermented milk classified as "products made into a paste" include hard yogurt and soft yogurt. Examples of fermented milk classified as "products made into a liquid" include drinkable yogurt (ready-to-drink yogurt). Examples of fermented milk classified as "products made from frozen ingredients" include frozen yogurt. From the perspective of easily enjoying the effects of the present invention, hard yogurt and soft yogurt are preferred fermented milks of the present invention.

[0062] The fermented milk of this invention contains more than 0.8g / 100g of dephosphorylated casein. Dephosphorylated casein is generated by the removal of serine phosphate from casein in milk.

[0063] From the viewpoint of improving the dephosphorylated casein content in fermented milk, the content is preferably 0.9 g / 100g or more, more preferably 1.0 g / 100g or more, and preferably 1.5 g / 100g or less, more preferably 1.4 g / 100g or less, and even more preferably 1.3 g / 100g or less.

[0064] In the fermented milk of the present invention, the proportion of dephosphorylated casein, which is casein, is preferably 25-45%, more preferably 26-43%.

[0065] In this specification, the analysis of casein can be performed by subjecting known concentrations of each casein (α-casein, β-casein, and κ-casein) and the sample to polyacrylamide gel electrophoresis (SDS-PAGE), followed by Coomassie brilliant blue staining (CBB staining), and then using image analysis software to quantify the intensity of the bands to create a standard curve, thereby calculating the amount of casein in the sample.

[0066] In addition, the analysis of dephosphorylated casein in this specification is performed according to the methods described below.

[0067] Dephosphorylated casein can be quantified using electrophoresis. This method calculates the amounts of phosphorylated and dephosphorylated casein based on the electrophoresis results. Figure 1 The following explanation is provided. It should be noted that the electrophoresis method is as described in the examples described later.

[0068] Figure 1 The electrophoresis diagram of milk is shown schematically. Phosphorylated casein has three regions: α-casein 1, β-casein 2, and κ-casein 3. The amount of phosphorylated casein can be calculated by summing these regions. Although other phosphorylated casein exists, it is negligible due to its minute quantity. It should be noted that the sum of αs-1 casein, αs-2 casein, β-casein, and κ-casein in cow's milk accounts for approximately 97% of the total casein content.

[0069] Dephosphorylated casein exists adjacent to each casein band. Due to varying degrees of dephosphorylation, dephosphorylated casein is diffuse after electrophoresis. The concentrations of phosphorylated casein bands and dephosphorylated casein regions differ, allowing for visual identification. The amount of dephosphorylated casein can be calculated by summing the values ​​of dephosphorylated casein regions 11, 21, and 31. The terminus of dephosphorylated casein region 31 can be visually identified as the boundary between stained and unstained areas.

[0070] When using bovine milk, the order of increasing phosphorylated casein content is α-casein, β-casein, and κ-casein. The degree of dephosphorylated casein can be determined using the same method for milk from different sources. One or more specific caseins can be selected for determination, representing more than 90% of the total casein content.

[0071] Figure 1 The image shows a good band shape, but in reality, even when all conditions are kept consistent, the band shape will often be distorted. Utilizing... Figure 2 The methods for determining phosphorylated and dephosphorylated casein at this time are explained.

[0072] Figure 2 When a non-strip region exists between the leading edge 100 (the electrophoresis start side) and the base 110 of the leading edge 110, a cumulative start line A can be set at approximately 50% of the area of ​​the non-strip region, and the area of ​​the strip region can be calculated from this point. When setting the cumulative start line A, a graph obtained by plotting the electrophoresis pattern as a curve can be referenced.

[0073] Figure 2 When a non-strip region exists between the leading edge 200 (the end of electrophoresis) and the base 210 of the leading edge, a cumulative end line B can be set at approximately 50% of the area of ​​the non-strip region, and the area of ​​the strip region up to this point can be calculated. When setting the cumulative end line B, a graph obtained by plotting the electrophoresis pattern as a curve can be referenced.

[0074] By using the above method to set the cumulative start line and cumulative end line, the concentration of the band region existing between them can be quantified.

[0075] The concentration of the dephosphorylated region can be quantified using the same method as described above.

[0076] The fermented milk of the present invention preferably further contains free phosphoric acid.

[0077] From the perspective of the dehydration inhibition effect of fermented milk, the increase in viscosity and destructive stress, and the increase in EPS production, the content of free phosphate in fermented milk is preferably 15 mM or more, and more preferably in the range of 15 mM to 40 mM.

[0078] To ensure that fermented milk contains dephosphorylated casein, the raw milk used as the starter for fermentation can be fermented, for example, in the presence of protein phosphatase. Alternatively, the raw milk can be treated with protein phosphatase before fermentation, resulting in dephosphorylated milk that has undergone this treatment.

[0079] That is, the method for manufacturing fermented milk according to the present invention includes any of the following steps:

[0080] (a) The process of fermenting raw milk in the presence of protein phosphatase.

[0081] (b) The process of fermenting raw milk produced by enzymatic treatment with protein phosphatase.

[0082] Raw milk only needs to contain casein and can be made from common milk sources. Examples include raw milk, cow's milk, specialty cow's milk, raw goat's milk, pasteurized goat's milk, raw sheep's milk, formulated milk, low-fat milk, non-fat milk, processed milk, cream, butter, shortening, cheese, whey concentrate, ice cream, concentrated milk, skimmed concentrated milk, unsweetened condensed milk, unsweetened skimmed condensed milk, sweetened condensed milk, sweetened skimmed condensed milk, whole milk powder, skimmed milk powder, cream powder, whey powder, whey protein concentrate powder, and buttermilk powder.

[0083] The amount of casein contained in the raw milk is not particularly limited, but is preferably 0.01 to 10 g / 100 g, more preferably 0.1 to 8 g / 100 g, and even more preferably 1 to 5 g / 100 g.

[0084] The pH of the raw milk is preferably pH 4.0 to 8.0, more preferably pH 4.7 to 7.5, and even more preferably pH 5.3 to 6.8. The same applies to the pH of the raw milk containing other ingredients besides the raw milk itself.

[0085] The raw milk may contain other ingredients that can be added before fermentation or after fermentation, as needed, without impairing the effects of the present invention. Examples of such other ingredients include, for instance, sweeteners (monosaccharides, oligosaccharides, sugar alcohols, synthetic sweeteners, etc.), stabilizers (gelatin, pectin, carrageenan, xanthan gum, etc.), fruit juice, fruit pulp, and flavorings.

[0086] Protein phosphatase is an enzyme that dephosphorylates phosphorylated proteins. In this specification, any enzyme capable of removing serine phosphate from casein in raw milk is acceptable. Preferably, the protein phosphatase removes serine phosphate from casein during fermentation.

[0087] The optimal pH for protein phosphatase is preferably in the range of 4.0 to 7.5, more preferably 4.5 to 7.0, and even more preferably 5.0 to 6.0.

[0088] Protein phosphatase is preferably slowly inactivated during the production of fermented milk and is inactivated within the fermented milk itself. Specifically, it is preferable that it functions even under weakly acidic conditions at pH 5.0–6.0 and is inactivated at pH less than 4.5. During the fermentation of the raw milk, phosphate is slowly removed from casein. It is believed that the increase of free phosphate in the milk facilitates the formation of EPS during the metabolism of free phosphate by lactic acid bacteria. By inactivating protein phosphatase after fermentation, the quality of the fermented milk can be easily and stably maintained, and is therefore preferred.

[0089] The optimal temperature for protein phosphatase is preferably in the range of 1°C to 60°C, and more preferably in the range of 10°C to 55°C.

[0090] The protein phosphatase used in this invention preferably has the aforementioned optimal pH and optimal temperature. The type and origin of the protein phosphatase are not limited, but it is preferably a protein phosphatase derived from microorganisms belonging to the genera *Trichoderma*, *Aspergillus*, *Sccharomyces*, *Bacillus*, and *Streptomyces*, more preferably a protein phosphatase derived from *Trichoderma virens*, and even more preferably a hypothetical protein (XP_013951069.1 hypothetical protein TRIVIDRAFT_87714) derived from *Trichoderma virens* Gv29-8. This hypothetical protein (XP_013951069.1 hypothetical protein TRIVIDRAFT_87714) has had its protein phosphatase activity elucidated by the inventors and possesses the following properties.

[0091] (a) Optimal temperature: 50℃

[0092] (b) Temperature stability: More than 80% residual activity at 43°C for 2 hours.

[0093] (c) Optimal pH: pH 5.42

[0094] (d) pH stability: More than 80% residual activity is maintained under pH 4.7–6.8 conditions for 6 hours.

[0095] (e) Metal ion demand: from 2.5 to 5.0 mM Ca 2+ Mg 2+ Mn 2+ Co 2+ Activation by divalent metal cations

[0096] The base sequence of the DNA encoding the protein phosphatase (XP_013951069.1 hypothetical protein TRIVIDRAFT_87714) from Trichoderma viride Gv29-8 is shown in sequence number 1 of the sequence listing, and the amino acid sequence is shown in sequence number 2 of the sequence listing.

[0097] Besides the wild type, protein phosphatases can also be proteins with protein phosphatase activity, which are proteins whose gene encoding the protein phosphatase is expressed in hosts such as Escherichia coli, or whose gene expression is altered through various gene manipulations.

[0098] The protein phosphatase is preferably any one of the proteins (i) to (iii) below.

[0099] (i) Proteins composed of the amino acid sequence represented by sequence number 2

[0100] (ii) A protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted, or added in the amino acid sequence indicated by sequence number 2 and possessing protein phosphatase activity.

[0101] (iii) A protein consisting of an amino acid sequence having more than 80% sequence identity with the amino acid sequence represented by sequence number 2 and possessing protein phosphatase activity.

[0102] The number of amino acids missing, substituted, or added in the amino acid sequence represented by serial number 2 is not limited as long as it exhibits the same enzymatic activity as the protein phosphatase composed of the amino acid sequence represented by serial number 2. Preferably, it is 1 to 20, more preferably 1 to 10, and even more preferably 1 to 8.

[0103] Furthermore, the sequence identity with the amino acid sequence represented by sequence number 2 is 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and even more preferably 99% or more. This percentage of sequence identity can be calculated using publicly available or commercially available software equipped with an algorithm that compares the sequence with a reference sequence as the lookup sequence (reference matching). Examples include BLAST, FASTA, or GENETYX (manufactured by GENETYX Corporation).

[0104] The protein phosphatase used in this invention is useful as an enzyme for fermented milk because it stabilizes the quality of fermented milk and enhances its physiological functions.

[0105] In step (a), protein phosphatase only needs to be present during the fermentation of the raw milk, and there are no particular restrictions on when to add it.

[0106] The amount of protein phosphatase used in step (a) is sufficient to remove serine phosphate from casein in the raw milk. For example, it is preferably 0.1 to 25 U, more preferably 0.5 to 15 U, and even more preferably 1 to 10 U, relative to 1 mL of raw milk. Here, in this specification, 1 U refers to the amount of enzyme solution added to 1 / 10 of a substrate solution containing 20 mg of bovine casein dissolved in 20 mM MES-NaOH buffer (pH 6.0) containing 10 mM Tris-HCl, reacted at 37°C, and an equal amount of reaction stop solution was added, under which conditions 1 μmol of phosphate was released per minute.

[0107] The fermentation method in step (a) follows general procedures. For example, raw milk and other raw materials are mixed and dissolved, the resulting fermentation mixture is homogenized, sterilized by heating, cooled, and then microorganisms (starter) and protein phosphatase are added for fermentation. After cooling, it can be pulverized and homogenized as needed.

[0108] The fermentation temperature can be set at any temperature that allows microorganisms (starter) to grow without inactivating enzymes. It can be set appropriately according to the type of microorganisms, preferably 20℃~45℃, and more preferably 30℃~37℃.

[0109] The fermentation time is, for example, 1 to 48 hours, preferably 2 to 24 hours, more preferably 3 to 10 hours, even more preferably 3 to 6 hours, and particularly preferably 3 to 5 hours.

[0110] The optimal pH for fermented milk is 4.0–5.0.

[0111] In step (b), the raw milk (dephosphorylated milk) prepared by enzymatic treatment with protein phosphatase for fermentation only requires dephosphorylation of casein in the raw milk using protein phosphatase. Pre-treating the raw milk with protein phosphatase allows for the dephosphorylation of more casein and makes it easier to control the degree of dephosphorylation.

[0112] The amount of protein phosphatase used in step (b) is sufficient to remove serine phosphate from casein in the raw milk. For example, it is preferably 0.1 to 25 U relative to 1 part by weight of raw milk, more preferably 0.5 to 15 U, and even more preferably 1 to 10 U.

[0113] The enzyme treatment of raw milk is preferably carried out at a reaction temperature of 1°C to 60°C for 0.1 hours to 24 hours. The reaction temperature is preferably 10°C to 60°C, more preferably 30°C to 55°C, and even more preferably 40°C to 50°C. The reaction time is preferably 0.3 hours to 12 hours, more preferably 0.5 hours to 5 hours, and particularly preferably 1 hour to 4 hours.

[0114] Before enzymatically treating raw milk with protein phosphatase, the pH of the raw milk can be adjusted.

[0115] Enzyme treatment allows the raw milk to ferment without inactivating the enzyme, or it can be treated to inactivate the enzyme through heating or other methods before fermentation. Fermentation without inactivation is preferred. During fermentation, casein dephosphorylation also occurs slowly.

[0116] The heating conditions for inactivating the enzyme are preferably high-temperature heating for a short time, for example, preferably heating at 60-100°C for 1-30 minutes. Alternatively, the enzyme can be treated before the sterilization process of the raw milk, and then inactivated during the sterilization process of the raw milk.

[0117] The fermentation method in step (b) is the same as in step (a), following general methods. For example, the fermentation mixture containing raw milk treated with protein phosphatase and other raw materials is homogenized, sterilized by heating, cooled, and then microorganisms (fermenting agent) are added to induce fermentation. Alternatively, after cooling, it can be pulverized and homogenized as needed.

[0118] The fermentation temperature can be set appropriately in the same way as in step (a), preferably 20°C to 45°C, and more preferably 30°C to 37°C.

[0119] The fermentation time is, for example, 1 to 48 hours, preferably 2 to 24 hours, more preferably 3 to 10 hours, even more preferably 3 to 6 hours, and particularly preferably 3 to 5 hours.

[0120] The optimal pH for fermented milk is 4.0–5.0.

[0121] In this invention, from the viewpoint of easy formation of uniform curd and superior processability and cost, it is preferable to manufacture fermented milk through step (a).

[0122] By treating raw milk with protein phosphatase, casein in the raw milk is dephosphorylated, resulting in dephosphorylated milk containing dephosphorylated casein. The dephosphorylation of casein increases the hydrophobicity of casein molecules, thereby altering the texture of the milk. Utilizing this change in texture, the dephosphorylated milk of this invention can be used in various dairy products besides fermented milk, just like the raw milk.

[0123] From the viewpoint of achieving a good taste and obtaining fermented milk with stable quality, the content of dephosphorylated casein in the dephosphorylated milk of the present invention is preferably 0.8g / 100g or more, more preferably 0.9g / 100g or more, even more preferably 1.0g / 100g or more, and preferably 1.5g / 100g or less, more preferably 1.4g / 100g or less.

[0124] In the dephosphorylated milk of the present invention, the proportion of dephosphorylated casein in casein is preferably 45% to 65%, more preferably 48% to 63%.

[0125] Furthermore, dephosphated milk preferably contains more free phosphate. From the viewpoint of achieving good taste and obtaining fermented milk with stable quality, the content of free phosphate in dephosphated milk is preferably in the range of 1 mM to 100 mM, more preferably in the range of 5 mM to 50 mM, even more preferably in the range of 10 mM to 40 mM, and even more preferably in the range of 12 mM to 30 mM.

[0126] Common fermenting agents such as lactic acid bacteria and yeast are used in the fermentation of raw milk and in the fermentation of raw milk (dephosphated milk) produced by enzymatic treatment with protein phosphatase.

[0127] Examples of lactic acid bacteria include, for example, Lactobacillus (Lactobacillus casei, Lactobacillus acidophilus, etc.), Lactococcus (Lactococcus lactis, etc.), Leuconostoc mesenteroides (Leuconostoc mesenteroides, etc.), Enterococcus faecalis (Enterococcus faecalis, etc.), and Bifidobacterium (Bifidobacterium bifidum, Bifidobacterium breve, etc.).

[0128] Examples of yeasts include, for instance, yeasts of the genus *Saccharomyces cerevisiae*.

[0129] The number of live bacteria (lactic acid bacteria or yeast) in the starter culture is, for example, 10. 5 ~10 13 cfu / mL, preferably 10 6 ~10 12 cfu / mL, more preferably 10 7 ~10 11 cfu / mL, more preferably 10 8 ~1010 cfu / mL.

[0130] There is no particular limitation on the amount of starter culture used, which can be appropriately set according to its type. For example, it is 0.01 to 10% by mass of the raw milk and / or dephosphorylated milk, preferably 0.1 to 10% by mass, and more preferably 0.5 to 10% by mass.

[0131] The fermented milk of the present invention may contain other ingredients that can be added before fermentation or after fermentation, as needed, without impairing the effects of the present invention. Examples of such other ingredients include, for instance, sweeteners (monosaccharides, oligosaccharides, sugar alcohols, synthetic sweeteners, etc.), stabilizers (gelatin, pectin, carrageenan, xanthan gum, etc.), fruit juice, fruit pulp, and flavorings.

[0132] As shown in the embodiments described later, the fermented milk of the present invention has less dehydration, higher viscosity, and stable hardness.

[0133] For the viscosity of fermented milk, it is preferable that the viscosity is 3000 Pa·s or higher when the fermented milk is treated for 30 seconds using the measurement method described later. There is no particular upper limit, for example, 10000 Pa·s.

[0134] Furthermore, based on the destructive strength analysis value described in the examples below, the hardness of the fermented milk preferably has a destructive load of 0.41 N or more, more preferably 0.45 N or more, and even more preferably 0.5 N or more. There is no particular limitation on the upper limit value; for example, it is 1.0 N.

[0135] Furthermore, the fermented milk of the present invention contains a large amount of extracellular polysaccharides (EPS). The amount of EPS in the fermented milk can be 40 μg or more, 60 μg or more, 80 μg or more, 110 μg or more, or 180 μg or more per gram of fermented milk. There is no particular upper limit, for example, 300 μg.

[0136] Example

[0137] The present invention will be described in more detail below with examples, but the present invention is not limited in any way.

[0138] Example 1: Purification of protein phosphatase (hereinafter referred to as "PPase") derived from Trichoderma viride Gv29-8

[0139] Trichoderma virens strain NBRC6355 was inoculated onto potato dextrose agar (Reiken) and cultured aerobically for 3 days at 25°C. Colonies of the production strain grown on potato dextrose agar were cut into approximately 5 mm squares along with the agar medium. These colonies were then transferred to a production medium (pH 4.0) consisting of 5.0% sucrose, 2.0% TUBERMINE FV (Roquette Japan), 0.3% calcium chloride, 0.1% magnesium sulfate, and 0.001% dipotassium hydrogen phosphate, and cultured at 27°C and 220 rpm for 4 days using a rotating shaking method.

[0140] Approximately 700 mL of culture medium was subjected to solid-liquid separation using ADVANTEC No. 2 filter paper (ADVANTEC) and 3.0% KC-Flock (Nippon Paper). The resulting filtrate was concentrated to approximately 1 / 10 of its original volume using UF (Ahiru Kasei). Next, PEG4000 (Nacalai Tesque) was added to the concentrate at a final concentration of 15%, dissolved, and allowed to stand overnight at 4°C. Then, the solution was centrifuged at 8000 rpm and 4°C for 20 minutes (HIMAC CENTRIFUGECR20B2, HITACHI), and the supernatant was discarded. The resulting precipitate was dissolved in approximately 10 mL of 20 mM acetate-potassium acetate buffer (pH 4.8), and the solution was incubated at 50°C for 1 hour, followed by centrifugation at 14800 rpm and 4°C for 10 minutes (LEGEND MICRO 21R, Thermo Fisher Scientific). The supernatant was collected as purified protein.

[0141] The purified protein was identified as a hypothetical protein (XP_013951069.1 hypothetical protein TRIVIDRAFT_87714) from *Trichoderma viride* Gv29-8, based on its amino acid sequence. The function of this protein is unknown.

[0142] The activity of protein phosphatase (PPase) was confirmed by purifying the protein.

[0143] 1) PPase activity assay

[0144] Measure 0.2 g of bovine casein (prepared by CALBIOCHEM, casein, bovine milk, carbohydrate, and fatty acid free) into a 20 mL beaker, add 2.0 mL of 0.1 M Tris-HCl buffer (pH 9.0) and a small amount of distilled water, and dissolve using a magnetic stirrer. Next, add 2.0 mL of 0.2 M MES-NaOH buffer (pH 6.0) and approximately 10 mL of distilled water, adjust the pH to 6.0 using 1.0 equivalent of hydrochloric acid, and bring the volume to 20 mL. Use this as the substrate solution. Aliquot 450 μL of the substrate solution into a 1.5 mL centrifuge tube, add 50 μL of the enzyme sample, and incubate for 20 minutes. After the reaction, 500 μL of a reaction stop solution containing 18.0 g trichloroacetic acid, 18.0 g anhydrous sodium acetate, and 19.8 g acetic acid per 1 L was added, and the mixture was vigorously stirred and centrifuged at 15000 rpm and 4°C for 5 minutes. 200 μL of the appropriately diluted supernatant was added to 800 μL of distilled water, followed by 30 μL of a 2M sulfuric acid and 40 g / L ammonium molybdate aqueous solution, and stirred thoroughly. 50 μL of a 0.1M sodium ascorbate aqueous solution was added, and the mixture was stirred again and incubated at 40°C for 20 minutes. Subsequently, the absorbance at 880 nm was measured using a spectrophotometer (UV-1240, Shimadzu Corporation), and the phosphate concentration (μM) was calculated using a pre-prepared standard curve based on potassium dihydrogen phosphate. The activity value was calculated using the following formula.

[0145] Activity value (U / mL) = Phosphate concentration (μM) × Reaction volume (L) / Reaction time (min) / Enzyme amount (mL) × Dilution rate

[0146] a. Temperature dependence: The activity of PPase at various temperatures, including room temperature (23.5℃), 37℃, 43℃, 50℃, and 60℃, was determined.

[0147] b. Temperature stability: PPase samples were incubated at 4℃, 25℃, 37℃, 43℃, 50℃, and 60℃ for 2 hours, and then centrifuged at 15000r / min and 4℃ to determine the residual activity of the supernatant.

[0148] c. pH Dependence: The substrate solution with a 3-fold concentration of casein was diluted 3-fold with 0.2M acetate-potassium acetate buffer (pH 3.6, 4.2, 4, 7, 5.2, 5.6), MES-NaOH buffer (pH 5.3, 6.0, 6.8), and Tris-HCl buffer (pH 7.0, 7.5, 8.0, 9.0). After standing at 4°C for 6 hours, the residual activity of each sample was measured. It should be noted that the pH value after diluting the substrate with each buffer is used as the pH at the time of reaction.

[0149] pH stability: Each sample was diluted 10-fold with 0.2M acetate-potassium acetate buffer (pH 3.6, 4.2, 4, 7, 5.6, 6.0), MES-NaOH buffer (pH 5.3, 6.0, 6.8), and Tris-HCl buffer (pH 7.5, 8.0, 9.0). After standing at 4°C for 6 hours, the residual activity of each sample was measured.

[0150] e-Metal Ion Demand: Sodium, potassium, lithium, calcium, cobalt, and manganese chloride salts and disodium EDTA salt aqueous solutions were added to the reaction solution at concentrations of 2.5 mM, 5 mM, and 10 mM, and the PPase activity was measured at these concentrations.

[0151] The temperature dependence of PPase is shown in... Figure 3 In the figure 'a', temperature stability is shown as... Figure 3 b in the figure shows the pH dependence. Figure 3 c in the figure shows the pH stability. Figure 3 In the 'd', the metal ion demand is shown as... Figure 4 .according to Figure 3 From a to d in the data, we can see that for PPase, the optimal temperature is 50℃, the optimal pH is pH 5.42, and more than 80% of the residual activity is maintained at 43℃ for 2 hours and pH 4.7 to 6.8 for 6 hours.

[0152] In addition, according to Figure 4 It can be seen that PPase consists of 2.5–5.0 mM Ca 2+ Mg 2+ Mn 2+ Co 2+ Activated by divalent metal cations.

[0153] Example 1

[0154] (1) Preparation of fermented milk

[0155] Add 2% skim milk powder (Morinaga Milk Industry) to delicious milk (Meiji), mix thoroughly by shaking to dissolve, and sterilize in boiling water while stirring for 20 minutes. After sterilization, incubate the raw milk at 43°C for about 30 minutes, then add starter culture L812 (Chr. Hansen) and PPase or 20 mM acetate-potassium acetate buffer (pH 5.2) as shown in Table 1 to achieve a final enzyme activity concentration of 0–10 U / mL-milk. Ferment at 43°C for 4.5 hours to obtain fermented milk.

[0156] It should be noted that PPase was used as a solution appropriately diluted with 20 mM acetate-potassium acetate buffer (pH 5.2). The casein content in the fermented milk of Example 1 was 3.3 g / 100 g.

[0157] [Table 1]

[0158]

[0159] (2) Determination of dephosphorylated casein

[0160] Fermented milk was diluted 50-fold with distilled water and mixed with an equal volume of ×2 sample buffer. The mixture was then heated in boiling water for 5 minutes to prepare the assay sample. 10 μL of the sample was loaded onto a SuperSep™ Phos-tag (12.5%, Fujifilm film and photopurified reagent) and subjected to electrophoresis at a constant current of 25 mM. After staining and destaining the electrophoresis gel, the electrophoresis curve was plotted using ImageJ software. Next, regions varying based on the presence or absence of phosphate modification were defined, and the area of ​​each region was calculated. The proportion and absolute amount of dephosphorylated casein at each enzyme concentration were then calculated.

[0161] (3) Determination of free phosphoric acid concentration (mM)

[0162] The fermented milk prepared in (1) above was vigorously shaken and mixed to homogenize the curd. 1 mL of the homogenized fermented milk was injected into a 1.5 mL centrifuge tube and centrifuged at 15000 r / min and 4°C for 10 minutes. 200 μL of the appropriately diluted supernatant was added to 800 μL of distilled water, followed by 30 μL of 2M sulfuric acid and 40 g / L ammonium molybdate aqueous solution, and stirred thoroughly. 50 μL of 0.1M sodium ascorbate aqueous solution was added, and the mixture was stirred again and incubated at 40°C for 20 minutes. Then, the absorbance at 880 nm was measured using a spectrophotometer (UV-1240, manufactured by Shimadzu Corporation), and the phosphoric acid concentration (mM) was calculated using a pre-prepared standard curve of potassium dihydrogen phosphate.

[0163] (4) Results

[0164] The results of the determination of dephosphorylated casein are shown in Table 2. The results of the determination of free phosphate concentration are shown in Table 2. Figure 5 .

[0165] [Table 2]

[0166]

[0167] Example 2

[0168] (1) Preparation of dephosphorylated milk

[0169] Dephosphated milk was obtained by adding 300 U / mL of PPase as shown in Table 3 to delicious milk (Meiji brand) adjusted to pH 5.9 with 1N HCl and allowing it to stand at 43°C for 4 hours. The dephosphated milk in Example 2 contained 2.5 g / 100 g of casein.

[0170] [Table 3]

[0171]

[0172] (2) Determination of dephosphorylated casein

[0173] The dephosphorylated casein was determined using the same method as in Example 1, except that the dephosphorylated casein was determined using the dephosphorylated milk after PPase had been made to exert its effect.

[0174] (3) Determination of free phosphoric acid concentration (mM)

[0175] The free phosphoric acid concentration was determined using the dephosphorylated emulsion after PPase had been made to function, except that the method was the same as in Example 1.

[0176] (4) Results

[0177] The results of the determination of dephosphorylated casein are shown in Table 4. The results of the determination of free phosphate concentration are shown in... Figure 6 .

[0178] [Table 4]

[0179]

[0180] Example 3

[0181] (1) Preparation of fermented milk

[0182] Measure 392g of low-temperature sterilized milk (manufactured by Takanashi Dairy) and 8g of skim milk powder (manufactured by Morinaga Dairy) into a 500mL medium-sized bottle, mix thoroughly by shaking to dissolve, and sterilize in boiling water while stirring for 20 minutes. After sterilization, incubate the raw milk at 43°C for approximately 30 minutes, then add starter culture L812 (manufactured by Chr. Hansen, hereinafter the same) to a concentration of 0.01g / mL, and gently invert and mix about 10 times. Next, aliquot 10mL of the raw milk with added starter culture into 15mL screw-cap tubes containing the samples listed in Table 5, screw on the caps, gently invert and mix 2-3 times, and ferment at 43°C for 5 hours. After fermentation, cool at 4°C overnight to obtain fermented milk. Measure the pH of the fermented milk using a pH meter (F72-S, HORIBA).

[0183] [Table 5]

[0184] Various samples added to fermented milk

[0185]

[0186] (2) Determination of dehydration rate

[0187] The fermented milk sample prepared in (1) above was vigorously shaken and mixed to homogenize the curd. Approximately 5 mL was injected into a 15 mL graduated screw-cap tube and centrifuged at 3000 r / min (KOKUSAN, H-19F MR) and 8°C for 10 minutes. Then, the total sample volume and the volume of solid components were read, and the dehydration rate was calculated using the following formula.

[0188] Dehydration rate (%) = 100 - ((volume of solid components / volume of sample) × 100)

[0189] (3) Determination of free phosphoric acid concentration (mM)

[0190] One mL of the homogenized fermented milk sample was injected into a 1.5 mL centrifuge tube and centrifuged at 15000 rpm (TOMY MRX-150 micro-centrifuge) at 4°C for 10 minutes. 200 μL of the appropriately diluted supernatant was added to 800 μL of distilled water, followed by 30 μL of a 2M sulfuric acid and 40 g / L ammonium molybdate aqueous solution, and the mixture was stirred thoroughly. Then, 50 μL of a 0.1M sodium ascorbate aqueous solution was added, and the mixture was stirred again and incubated at 40°C for 20 minutes. Subsequently, the absorbance at 880 nm was measured using a spectrophotometer (UV-1240, Shimadzu Corporation), and the phosphoric acid concentration (mM) was calculated using a pre-prepared standard curve based on potassium dihydrogen phosphate.

[0191] (4) Results

[0192] The results are shown in Figure 7 .according to Figure 7 It can be seen that the dehydration rate of fermented milk with added PPase was lower than that of the unadded (blank) milk. On the other hand, the dehydration of fermented milk containing phosphoric acid, a reaction product of PPase, but without dephosphorylated casein, was not inhibited.

[0193] In addition, according to Figure 7 It can be seen that adding PPase during fermentation resulted in dehydration inhibition compared to no addition (blank). On the other hand, even the addition of phosphate, a reaction product of PPase, did not inhibit dehydration.

[0194] Example 4

[0195] (1) Preparation of fermented milk

[0196] Measure 392g of low-temperature sterilized milk (manufactured by Takanashi Dairy) and 8g of skim milk powder (manufactured by Morinaga Dairy) into a 500mL medium-sized bottle, shake thoroughly to dissolve, sterilize in boiling water for 20 minutes, and then incubate at 43°C for about 30 minutes. Add 40mg of starter culture L812 and gently invert 10 times. Then, measure 60g of raw milk into each of the following jam jars, each containing 1.46mL of appropriately diluted purified PPase (1.0U / mL-milk or 3.0U / mL-milk) or 20mM acetate-potassium acetate buffer (pH 5.2), invert 5 times, and ferment at 43°C for 4.5 hours. After fermentation, cool at 4°C overnight to obtain fermented milk. The pH of the fermented milk was measured using a pH meter (Thermo Fisher, ORION3STAR).

[0197] (2) Determination of dehydration rate

[0198] The determination was performed using the same method as in Example 3.

[0199] (3) Determination of free phosphoric acid concentration (mM)

[0200] The determination was performed using the same method as in Example 3.

[0201] (4) EPS measurement

[0202] Weigh 0.4 g of fermented milk sample, add 200 μL of 100% (w / v) TCA solution, mix by inversion, and centrifuge at 15000 rpm (TOMY, MRX-150 micro-centrifuge, hereinafter the same) at 4°C for 10 minutes. Collect all supernatant into a new centrifuge tube, add 500 μL of acetone, and let stand overnight at 4°C. Then, centrifuge at 15000 rpm at 4°C for 10 minutes, discard the supernatant, dissolve the precipitate in 400 μL of MilliQ water, add 400 μL of acetone, mix by inversion, and let stand overnight at 4°C. Then, centrifuge at 15000 rpm at 4°C for 10 minutes, discard the supernatant, and dissolve the precipitate in 300 μL of MilliQ water. The solution was centrifuged at 15000 rpm and 4°C for 10 minutes, and the supernatant was collected. 100 μL of this supernatant was used to determine the monosaccharide and disaccharide content using HPLC (Waters). The remaining 200 μL was used to determine the total sugar content using the phenol-sulfuric acid method. The amount of trisaccharides and higher in vitro polysaccharides was calculated by subtracting the monosaccharides and disaccharides from the total sugar content.

[0203] (5) Viscosity measurement

[0204] The fermented milk sample was vigorously shaken and homogenized. 13.5 g of the sample was dispensed into the disposable sample chamber (aluminum) of the viscometer (DV-I Prime, BROOK FIERD). The spindle was SC4-29, and the viscosity was measured at 10 rpm, with readings taken every 30 seconds and recorded. It should be noted that all operations were performed at 10°C.

[0205] (6) Analysis of destructive strength

[0206] The device uses GREEP METER RE2-33005C (Sanden Corporation), and the plunger is No. 3.

[0207] The measurements were performed in a mode with a load cell of 0.01, an amplifier magnification of 0.1, a storage pitch of 0.07 seconds, a strain rate of 60%, a measurement speed of 1 mm / s, and automatic sample thickness measurement.

[0208] (7) Results

[0209] The results of the determination of dehydration rate, free phosphate concentration, and post-fermentation pH are shown in the figure. Figure 8 The EPS concentration is shown in Figure 9 The viscosity measurement results are shown in Figure 10 The analysis results of the damage intensity are shown in Figure 11 .

[0210] Fermented milk containing dephosphorylated casein had a lower dehydration rate compared to milk without additives (blank). Figure 8 ), with a high content of extracellular polysaccharides (EPS). Figure 9 The viscosity of this fermented milk is more than 1000 cP higher than that of the unadded milk. Figure 10 Furthermore, the fermented milk exhibits higher destructive stress compared to the unadded variety, resulting in a decrease in stress reduction (i.e., reduced fragility) after destructive processes. Figure 11 ).

[0211] Example 5

[0212] (1) Preparation of fermented milk

[0213] Measure 147g of low-temperature sterilized milk (manufactured by Takanashi Dairy) and 3g of skim milk powder (manufactured by Morinaga Dairy) into five 250mL medium-sized bottles, and mix thoroughly by shaking to dissolve. Then, sterilize by heating in boiling water for 20 minutes, and incubate at 43°C for about 30 minutes. Add the various starter cultures shown in Table 6 (all manufactured by Chr. Hansen), and gently invert and mix 10 times. Subsequently, measure 60g of raw milk into each jam bottle that has been pre-filled with 0.488mL of 123U / mL purified PPase (Final 1.0U / mL-milk) or 20mM acetate-potassium acetate buffer (pH 5.2), invert and mix 5 times, and ferment at 43°C for 4.5 hours. After fermentation, cool at 4°C overnight to obtain fermented milk.

[0214] [Table 6]

[0215] Name and dosage of fermenting agent

[0216] Fermentation agent Added amount L811 15mg L812 15mg Harmony 30mg

[0217] (2) Determination of dephosphorylated casein, (3) Determination of dehydration rate, (4) Determination of free phosphate concentration (mM), (5) EPS determination, (6) Viscosity determination, (7) Analysis of destructive strength

[0218] The determination was performed using the same method as in Example 4 above.

[0219] (8) Results

[0220] The results of the determination of dehydration rate, free phosphate concentration, and post-fermentation pH are shown in the figure. Figure 12 The EPS concentration is shown in Figure 13 The viscosity measurement results are shown in Figure 14 The analysis results of the damage intensity are shown in Figure 15 .

[0221] Fermented milk containing dephosphorylated casein had a lower dehydration rate compared to milk without additives (blank). Figure 12 ), with a high content of extracellular polysaccharides (EPS). Figure 13 The viscosity of this fermented milk is 1000–2000 cP higher than that of the unadded milk. Figure 14 In addition, the destructive stress of this fermented milk is higher than that of milk without additives. Figure 15 ).

[0222] In addition, by adding PPase, dehydration inhibition, increased free phosphate content, and decreased pH were observed in all starter cultures compared to those without PPase (blank). Figure 12 The amount of extracellular polysaccharides (EPS) increased in all starter cultures compared to those without addition. Figure 13 Results of viscosity measurement of fermented milk: viscosity increased by 1000–2000 cP ( Figure 14 Furthermore, the results of the analysis of the destructive strength of fermented milk showed an increase in destructive stress across all starter cultures. Figure 15 ).

[0223] Example 6(1) Preparation of Fermented Milk

[0224] Measure 392g of low-temperature sterilized milk (manufactured by Takanashi Dairy) and 8g of skim milk powder (manufactured by Morinaga Dairy) into a 500mL medium-sized bottle, shake thoroughly to dissolve, sterilize in boiling water for 20 minutes, and then incubate at 43°C for approximately 30 minutes. Add 40mg of starter culture L812 and gently invert 10 times. Then, measure 60g of raw milk into each of two jam jars pre-filled with 1.46mL of appropriately diluted purified PPase or 20mM acetate-potassium acetate buffer (pH 5.2), invert 5 times, and ferment at 43°C for 4.5 hours. After fermentation, cool overnight at 4°C to obtain fermented milk. The pH of the fermented milk was measured using a pH meter (Thermo Fisher, ORION 3STAR).

[0225] (2) Determination of dehydration rate, (3) Determination of free phosphoric acid concentration (mM), (4) Determination of EPS

[0226] The determination was performed using the same method as in Example 4 above.

[0227] (5) Viscosity measurement

[0228] The fermented milk sample was vigorously shaken and homogenized. 13.5 g of the sample was dispensed into the disposable aluminum sample chamber of the viscometer (DV-I Prime, BROOK FIERD). The spindle was SC4-29, and the viscosity was measured at 10 rpm, with readings taken every 30 seconds and recorded. It should be noted that all operations were performed at 10°C.

[0229] (6) Analysis of destructive strength

[0230] The apparatus used was a GREEP METER RE2-33005C (Sanden), and the plunger was No. 3. Measurements were performed in a mode with a force sensor of 0.01, a magnification of 0.1, a storage interval of 0.07 seconds, a strain rate of 60%, a measurement speed of 1 mm / s, and automatic sample thickness measurement.

[0231] (7) Results

[0232] The results of the determination of dehydration rate, free phosphate concentration, and post-fermentation pH are shown in the figure. Figure 16 The EPS concentration is shown in Figure 17 The viscosity measurement results are shown in Figure 18 The analysis results of the damage intensity are shown in Figure 19 When PPase was added to fermented milk at a concentration ranging from 0.1 to 3.0 U / mL-milk, compared to the control group (without PPase), dehydration inhibition, increased free phosphate content, and decreased pH were observed. Figure 16 The amount of in vitro polysaccharides (EPS) increased compared to no addition in the addition of PPase at 1.0 and 3.0 U / mL-milk. Figure 17 The viscosity of fermented milk with added PPase was measured. The results showed that when PPase was added at concentrations of 0.1–0.5 U / mL-milk, the viscosity increased by approximately 1000 cP compared to the absence of added PPase. The viscosity further increased when PPase was added at concentrations of 1.0 and 3.0 U / mL-milk. Figure 18 Furthermore, analysis of the destructive strength of fermented milk with added PPase was conducted. The results showed that, although no concentration-dependent effect was observed, the destructive stress increased compared to the absence of added PPase. Additionally, when PPase was added at levels above 0.5 U / mL-milk, the reduction in post-destructive stress (i.e., fragility) decreased. Figure 19 ).

[0233] Example 7 (Enzyme Treatment of Raw Milk)

[0234] (1) Preparation of fermented milk

[0235] Measure 392g of ultra-high temperature (UHT) flash-sterilized milk (Meiji Delicious Milk) or low temperature (LTS) pasteurized milk (Takanashi Dairy) and 8g of skim milk powder (Morinaga Dairy) into a 500mL medium-sized bottle, and shake thoroughly to dissolve. Add 2mL of purified PPase (700U / mL, Final 23.3U / mL-milk) or 20mM acetate-potassium acetate buffer (pH 5.2), and mix by inverting 5 times. Next, measure 60g into each jam jar, mix by inverting 5 times, and let stand at 37°C for 3 hours to obtain enzyme-treated milk. Boil each jam jar in boiling water for 20 minutes to inactivate PPase. Then, after cooling to room temperature, add 20mg of starter culture L812 to each jam jar, gently invert 10 times, and ferment at 43°C for 4.5 hours. After fermentation, cool at 4°C overnight to obtain fermented milk. The pH of the fermented milk was measured using a pH meter (Thermo Fisher, ORION 3STAR).

[0236] (2) Determination of dehydration rate; (3) Determination of free phosphoric acid concentration (mM)

[0237] The determination was performed using the same method as in Example 4 above.

[0238] (4) Results

[0239] The results of the determination of dehydration rate, free phosphate concentration, and post-fermentation pH are shown in the figure. Figure 20 Fermentation of raw milk was treated with PPase, resulting in decreased dehydration, increased free phosphate content, and decreased pH compared to the control group (without PPase). Figure 20 The results suggest that casein in milk only needs to be dephosphorylated, and PPase does not need to be present in an active state during fermentation or in fermented milk.

[0240] Example 8

[0241] (1) Preparation of fermented milk

[0242] Measure 392g of ultra-high temperature (UHT) milk (Meiji Delicious Milk) and 8g of skim milk powder (Morinaga Milk Industry) into a 500mL medium-sized bottle. Shake thoroughly to dissolve the milk, then sterilize in boiling water while stirring for 20 minutes. Incubate the sterilized milk at 43°C for approximately 30 minutes, then add 0.571mL of purified PPase (350U / mL, Final 1U / mL-milk) or 20mM acetate-potassium acetate buffer (pH 4.5) and 20mg of starter culture L812. Gently invert and mix about 10 times. Measure 10g into each of eight 15mL screw-cap tubes, screw on the caps, gently invert and mix 2-3 times, and ferment at 43°C for 5 hours. After fermentation, cool at 4°C overnight to obtain fermented milk. The pH was measured using a pH meter (Thermo Fisher, ORION 3STAR) before fermentation and at 2.5, 3, 3.5, 4, and 4.5 hours after the start of fermentation.

[0243] (2) Results

[0244] The results are shown in Figure 21 It was confirmed that the pH of fermented milk with added PPase decreased more rapidly compared to the control group (without PPase). This is presumably due to the increased phosphate content caused by PPase. Additionally, it was confirmed that the fermented milk with added PPase curdled more quickly.

[0245] Example 9 (Adjusting fermentation time to achieve a post-fermentation pH of approximately 4.5)

[0246] (1) Preparation of fermented milk

[0247] 60g of the mixture was dispensed into the jam jar to replace the 15mL screw cap tube. To match the pH after fermentation, the fermentation time was set to 4.75 hours for the system with added buffer and 3.5 hours for the system with added PPase. Otherwise, the fermented milk was prepared in the same manner as in Example 8. The pH of the fermented milk was measured using a pH meter (Thermo Fisher, ORION3STAR).

[0248] (2) Determination of dehydration rate; (3) Determination of free phosphoric acid concentration (mM)

[0249] The determination was performed using the same method as in Example 4 above.

[0250] (4) Results

[0251] The results are shown in Figure 22Although the post-fermentation pH was approximately 4.5, compared to the control group (no additives), the addition of PPase during fermentation resulted in dehydration inhibition and an increase in free phosphate. This suggests that the reduction in dehydration rate and the increase in free phosphate were not affected by the post-fermentation pH, but rather achieved through the action of PPase.

[0252] Reference example (concentration of free phosphate and calcium ions in enzyme-treated milk)

[0253] (1) Preparation of enzyme-treated milk

[0254] Add 0.45 mL of ultra-high temperature instantaneous pasteurized milk (Meiji Delicious Milk, pH 6.76) or milk adjusted to pH 5.48 by adding 1 M hydrochloric acid to the milk, then add 0.05 mL of purified PPase (700 U / mL), and react at 37°C for the specified time. Finally, add 0.5 mL of 18% trichloroacetic acid to stop the reaction, thus obtaining enzyme-treated milk.

[0255] (2) Determination of free phosphoric acid concentration

[0256] The concentration of free phosphate (mM) before and after enzyme treatment was determined using the same method as in Example 3 above.

[0257] (3) Determination of calcium ion concentration

[0258] The milk before and after enzyme treatment was placed on a calcium ion sensor (LAQUAtwin manufactured by Horiba Corporation) in an undiluted state for measurement.

[0259] (4) Results

[0260] The results are shown in Figure 23 Milk adjusted to pH 5.48 showed higher PPase reactivity compared to milk at pH 6.76, with a temporary increase in free phosphate concentration that eventually converged to a certain value. Milk at pH 6.76 showed a gradual increase followed by a convergence to a certain value. In any milk with added PPase, the free phosphate concentration converged to a certain value. Since the calcium ion concentration also changed along with the enzymatic reaction, this suggests the formation of calcium phosphate in the milk, reaching an equilibrium at a certain concentration. sequence list <110> Contract Alcohol Co., Ltd. <120> Fermented milk <130> GD0015 <150> JP2020-071708 <151> 2020-04-13 <150> JP2020-071709 <151> 2020-04-13 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1379 <212> DNA <213> Trichoderma viride Gv29-8 <400> 1 atgctgtcca aggttgtctc tctgctggct gccgccggcc tggcctctgc cgccgccgtt 60 gacaagagcc actgcaagcc tcccaagcct tcctccacca tcactgtctg ggagtctcag 120 tacactgcca ccggcactgc tgctgttgct gctgctgctg ccacggccaa gaccagcagc 180 ccgaccagcc acgtcaaggg caaggccttt gaccgctttg ttgtcattta ctttgagaac 240 caggactacg acaaggctga tggtgaccgt aagtttgaat ctcgaatcct ttgtctaaag 300 aaggggaccc ggaagacgct gacagtattt gctttgcagc caacttcact tggttcgcca 360 agaagggtgt caagttgacc aactacttcg gtgtcactca cccttctgag cccaactata 420 tggccggtat tattggtgac tactttggca tgcagaacga tgacttcaac tctgccgact 480 tcaacgtgtc caccgtcatc gacctgctcg aggatcgtgg catctcttgg ggccactacc 540 aggaggacca gccctacacc ggctacgagg gcttcagcta cgtcaacacc aagaccggtg 600 ccaacgacta cgtccgcaag caaaccccg ccatcttggc caacagcgtc acccactacg 660 agcagcgcct gtcccaggtc aagaacctgt ccatgatcga caccggtcgc tccatgttcc 720 acaaggacct caaggacaac aagctgcctc agtggatgtt catcaccccc aacatgacct 780 ctgacggcca cgacaccaac gtcggcgttg ctggcaagtg gtgccgtgct ttcctcgagc 840 ccctcctgaa cgataagcac ttcatggaca acacccttgt cctcctcacc tgggatgaga 900 acgagaccta tgcccagcgc aaccagattc tcgcaatctt gctcggtgac gccgtcccca 960 agcaccttgt tggcaccacc gacaacaact tctataacca ctacagcgag atcgccaccacg 1020 tcgaggccaa ctggaacctc cacatctgg gccgctggga tgttggtgcc aacgtcttcg 1080 acctggttgc caaggagact ggcgaccgcc tccgcaagtg gtcttccacc agggctgcca 1140 acagccactt ctggaacgcc tcttacgccg gtgtcttcaa cgaggagact ggcaacccca 1200 aaaccagta ccctgccccc aacctggctc tcgaccgcag cttctctggc cgtaccatcc 1260 tgcccgccat taagaacc tgggagcaca gcaagtctgc cacctactac gccgacacca 1320 ttgagctccc cgacggcctg aaccctcccc agggatacaa gccgctctcc accactaa 1379 <210> 2 <211> 434 <212> PRT <213> Smoke Gv29‑8 <400> 2 Met Leu Ser Lys Val Val Ser Leu Ala Ala Ala Ser 1 5 10 15 Ala Ala Ala Val Asp Lys Ser His Cys Lys Pro Pro Lys Pro Ser Ser 20 25 30 Thr Ile Thr Value of Glu Ser Gln Tyr Ala Thr Gly Thr Ala Ala 35 40 45 Val Ala Ala Ala Ala Ala Thr Ala Lys Thr Be Be Pro Thr Be His 50 55 60 Val Lys Gly Lys Ala Phe Asp Arg Phe Val Val Ile Tyr Phe Glu Asn 65 70 75 80 Gln Asp Tyr Asp Lys Ala Asp Gly Asp Pro Asn Phe Thr Trp Phe Ala 85 90 95 Lys Lys Gly Val Lys Thr Asn Tyr Phe Gly Val Thr His Pro Ser Glu 100 105 110 Pro Asn Tyr Met Ala Gly Ile Ile Gly Asp Tyr Phe Gly Met Gln Asn 115 120 125 Asp Asp Phe Asn Ser Ala Asp Phe Asn Val Ser Thr Val Ile Asp Leu 130 135 140 Leu Glu Asp Arg Gly Ile Ser Trp Gly His Tyr Gln Glu Asp Gln Pro 145 150 155 160 Tyr Thr Gly Tyr Glu Gly Phe Ser Tyr Val Asn Thr Lys Thr Gly Ala 165 170 175 Asn Asp Tyr Val Arg Lys His Asn Pro Ala Ile Leu Ala Asn Ser Val 180 185 190 Thr His Tyr Glu Gln Arg Leu Ser Gln Val Lys Asn Leu Ser Met Ile 195 200 205 Asp Thr Gly Arg Ser Met Phe His Lys Asp Leu Lys Asp Asn Lys Leu 210 215 220 Pro Gln Trp Met Phe Ile Thr Pro Asn Met Thr Ser Asp Gly His Asp 225 230 235 240 Thr Asn Val Gly Val Ala Gly Lys Trp Cys Arg Ala Phe Leu Glu Pro 245 250 255 Leu Leu Asn Asp Lys His Phe Met Asp Asn Thr Leu Val Leu Leu Thr 260 265 270 Trp Asp Glu Asn Glu Thr Tyr Ala Gln Arg Asn Gln Ile Leu Ala Ile 275 280 285 Leu Leu Gly Asp Ala Val Pro Lys His Leu Val Gly Thr Thr Asp Asn 290 295 300 Asn Phe Tyr Asn His Tyr Ser Glu Ile Ala Thr Val Glu Ala Asn Trp 305 310 315 320 Asn Leu His Thr Leu Gly Arg Trp Asp Val Gly Ala Asn Val Phe Asp 325 330 335 Leu Val Ala Lys Glu Thr Gly Asp Arg Leu Arg Lys Trp Ser Ser Thr 340 345 350 Arg Ala Ala Asn Ser His Phe Trp Asn Ala Ser Tyr Ala Gly Val Phe 355 360 365 Asn Glu Glu Thr Gly Asn Pro Asn Asn Gln Tyr Pro Ala Pro Asn Leu 370 375 380 Ala Leu Asp Arg Ser Phe Ser Gly Arg Thr Ile Leu Pro Ala Ile Lys 385 390 395 400 Lys Thr Trp Glu His Ser Lys Ser Ala Thr Tyr Tyr Ala Asp Thr Ile 405 410 415 Glu Leu Pro Asp Gly Leu Asn Pro Pro Gln Gly Tyr Lys Pro Val Ser 420 425 430 Thr Asn

Claims

1. A method for manufacturing fermented milk, comprising any of the following steps: (a) A process of fermenting raw milk in the presence of 0.1–3 U of protein phosphatase per 1 mL of raw milk. (b) A process of fermenting raw milk prepared by enzymatic treatment with protein phosphatase at a concentration of 0.1 to 3 U relative to 1 part by mass of the raw milk; The protein phosphatase is an enzyme that dephosphorylates serine phosphorylate in casein from raw milk. The raw milk is a milk-derived raw material containing casein. The fermented milk contains more than 0.8g of dephosphorylated casein per 100g.

2. The method for producing fermented milk according to claim 1, wherein The milk source is any one of cow's milk, raw goat's milk, sterilized goat's milk, or raw sheep's milk.

3. The method for manufacturing fermented milk according to claim 1, wherein, The milk source is either blended milk or concentrated milk.

4. The method for manufacturing fermented milk according to claim 1, wherein, The milk source is either low-fat milk or non-fat milk.

5. The method for manufacturing fermented milk according to claim 1, wherein, The raw materials for the milk source are any one of whole milk powder, skim milk powder, cream powder, whey powder, and buttermilk powder.

6. The method for manufacturing fermented milk according to claim 1, wherein, Protein phosphatase is a protein phosphatase derived from microorganisms belonging to the genus Trichoderma.

7. The method for manufacturing fermented milk according to claim 6, wherein, The microorganism belonging to the Trichoderma genus is *Trichoderma viride*.

8. The method for producing fermented milk according to any one of claims 1 to 7, wherein, Protein phosphatases are as follows (i). (i) Proteins consisting of the amino acid sequence represented by sequence number 2.

9. The method for manufacturing fermented milk according to claim 1, wherein, Fermented milk is yogurt.

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