How to make starched yogurt

Abstract

A method for producing starched yogurt involves treating raw milk with protein glutaminase to prepare modified milk prior to fermentation with a starter culture. After fermentation, the gel structure of the resulting yogurt is disrupted to produce starched yogurt. This process results in a starched yogurt with a sufficiently high viscosity without the need for thickeners or cross-linking enzymes.

Description

[Technical Field]

[0001] Background of the Invention FIELD OF THE INVENTION The present invention relates to a method for producing stirred yogurt using protein glutaminase, and to stirred yogurt produced by said method. [Background technology]

[0002] Background information The "Background" section provided herein is intended to provide a general overview of the contents of the present disclosure. The inventor's work to the extent described in this Background section, and aspects of the description that may not otherwise be admitted as prior art at the time of filing, are not admitted, expressly or impliedly, as prior art to the present invention.

[0003] Enzymes are used in various areas of the food industry. The advantage of using enzymes in food is that they act only on specific substances under mild conditions and usually do not significantly affect the taste of food.

[0004] One enzyme that has recently attracted attention for the modification of dairy products such as yogurt is protein glutaminase (PG). PG is a catalytic enzyme that promotes the deamidation of glutamine residues in proteins by converting glutamine residues to glutamic acid (amide → carboxylate), thereby increasing the protein's negative charge, electrostatic repulsion, and hydration potential, and lowering its isoelectric point. This is known to result in improvements in various functional properties of proteins, such as improved solubility and dispersibility. Therefore, dairy products treated with PG have been shown to have a rich, creamy texture that consumers find desirable.

[0005] For example, US 7,947,315 B2 (incorporated herein by reference in its entirety) describes the production of set yogurt with a smooth texture, reduced firmness, and reduced sourness by treating raw milk with protein glutaminase.

[0006] Similarly, US 8,318,223B2 (incorporated herein by reference in its entirety) describes a set yogurt produced by modifying raw milk with protein glutaminase while simultaneously improving the effect of PG by removing natural PG inhibitors contained in the raw milk, and the set yogurt is said to have a soft, creamy texture.

[0007] Set yogurt has a gel-like, semi-solid structure, and viscosity is usually not an issue. In fact, the viscosity of set yogurt is often too high to be measured mechanically. On the other hand, stirred yogurt (also known as "Swiss-style" yogurt) is a fluid yogurt product formed as a result of breaking down the gel structure at the end of the fermentation (incubation) period and represents an entirely different class of yogurt product. Many consumers prefer the lighter, creamier consistency of stirred yogurt and the variety of stirred fruit and fruit puree options available in stirred yogurt products. However, unlike set yogurt, which is not sensitive to viscosity changes, viscosity changes in stirred yogurt are a major concern, and care must be taken to ensure an acceptable viscosity.

[0008] To date, attempts to produce stirred yogurt with a sufficiently high viscosity by modifying proteins using protein glutaminase alone have been unsuccessful. For example, US 2011 / 0064847A1 (incorporated herein by reference in its entirety) describes the production of stirred yogurt by adding PG to raw milk during the fermentation process (simultaneously with a lactic acid bacteria starter culture) and then filtering it. It was found that adding PG during fermentation resulted in stirred yogurt with a watery, soft texture and no improvement in viscosity compared to the control (no PG added).

[0009] In another example, US 2019 / 0021353A1 (incorporated herein by reference in its entirety) describes the production of low-fat stirred yogurt by adding protein glutaminase to non-fat milk during the fermentation process (simultaneously with a bacterial starter culture) followed by filtration. The resulting stirred yogurt was classified as "undesirable," and the addition of PG was found to significantly reduce viscosity.

[0010] Thus, in attempts to improve the rheological properties (e.g., viscosity) of starched yogurt, researchers have turned to other additives, such as cross-linking enzymes (e.g., transglutaminases, "TGs") (see US 2011 / 0064847A1, incorporated herein by reference in its entirety) or thickeners (e.g., starches) (see US 2019 / 0021353A1, incorporated herein by reference in its entirety), added together with PG (and starter cultures) during the fermentation process. Even when such additional measures are taken, controlling viscosity has proven difficult. For example, US 2011 / 0064847A1 reports that modification of raw milk with a combination of PG and TG added together with lactic acid bacteria starter cultures during fermentation results in a decrease in viscosity with increasing dosage of PG. Furthermore, US 2019 / 0021353A1 discloses that in many cases the combination of PG and starch does not result in an improvement in viscosity or actually reduces viscosity compared to yogurt prepared with starch thickener alone. Summary of the Invention

[0011] In view of the above, there is a need for a method for producing starched yogurt of a sufficiently high viscosity without relying on cross-linking enzymes or thickeners, while also maintaining the familiar thick and creamy texture provided by the action of protein glutaminase.

[0012] It is therefore one object of the present invention to provide a novel method for producing starched yogurt.

[0013] Another object of the present disclosure is to provide a novel starched yogurt produced by the method of the present invention.

[0014] These and other objects, which will become apparent in the detailed description below, have been achieved by the inventors' discovery that modified milk proteins in raw milk with protein glutaminase prior to fermentation produces started yogurt with excellent rheological properties (e.g., high viscosity and torque) and desirable organoleptic properties (e.g., smoothness, firmness, reduced acidity, and low levels of syneresis).

[0015] Thus, the present invention provides: (1) Treating raw milk with protein glutaminase to prepare modified milk; fermenting the modified milk in the presence of a starter culture to prepare yogurt; and breaking the gel structure of the yogurt to prepare a stirred yogurt. (2) The method according to (1), wherein the raw milk has a fat content of up to 10% by weight based on the total weight of the raw milk. (3) The method according to (1) or (2), wherein the raw milk has a protein content of 3 to 10% by weight based on the total weight of the raw milk. (4) The method according to any one of (1) to (3), wherein the raw milk is treated with 10 to 500 ppm of protein glutaminase based on the total weight of the raw milk. (5) The method according to any one of (1) to (4), wherein the raw milk is treated with protein glutaminase in an amount of 0.01 to 5 U per 1 g of protein in the raw milk. (6) A method according to any one of (1) to (5), wherein the raw milk is treated with protein glutaminase at a treatment temperature of up to 50°C. (7) The method according to any one of (1) to (6), wherein the raw milk is treated with protein glutaminase for 15 minutes to 24 hours. (8) The method according to any one of (1) to (7), further comprising heat sterilizing the modified milk after treating the raw milk with protein glutaminase and before fermenting the modified milk. (9) The method according to (8), wherein the heat sterilization is carried out at a temperature of 70 to 95°C for 1 to 30 minutes. (10) The method according to any one of (1) to (9), further comprising adding a stabilizer to the raw milk before treating the raw milk with protein glutaminase. (11) The method according to any one of (1) to (10), further comprising treating the raw milk with a reducing agent before or simultaneously with treating the raw milk with protein glutaminase. (12) The method according to (11), wherein the raw milk is treated simultaneously with a reducing agent and protein glutaminase. (13) The method according to (11) or (12), wherein the raw milk is treated with 10 to 500 ppm of a reducing agent based on the total weight of the raw milk. (14) The method according to any one of (11) to (13), wherein the reducing agent is yeast extract. (15) The method according to any one of (1) to (14), wherein the modified milk is fermented with 0.0001 to 1% by weight of a starter culture based on the total weight of the modified milk. (16) The method according to any one of (1) to (15), wherein no thickener is used. (17) The method according to any one of (1) to (16), further comprising treating the raw milk with a transglutaminase enzyme simultaneously with treating the raw milk with protein glutaminase. (18) The method according to any one of (1) to (16), wherein protein glutaminase is the only enzyme used that acts on glutamine residues of milk proteins contained in the raw milk. (19) The method according to any one of (1) to (18), wherein the stirred yogurt has a viscosity of 134,000 cP to 250,000 cP as measured at 4.0 rpm and 40°F (4.4°C) using a Brookfield viscometer. (20) Stirred yogurt produced by the method according to any one of (1) to (19).

[0016] BRIEF DESCRIPTION OF THE DRAWINGS The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the claims that follow. The described embodiments, and further advantages, will be best understood by reference to the following detailed description when considered in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0017] [Figure 1] 1A-1B are graphs showing the viscosity (FIG. 1A) and torque (FIG. 1B) of starched yogurt samples #1 and #2 after 7 days of refrigerated storage. [Figure 2] Figures 2A-2B are images of starched yogurt samples #1 (Figure 2A) and #2 (Figure 2B) after 7 days of refrigerated storage. [Figure 3] 3A-3B are graphs showing the viscosity (FIG. 3A) and torque (FIG. 3B) of starched yogurt samples #3-9 after 10 days of refrigerated storage. [Figure 4] Figures 4A-4G show images of starched yogurt samples #3 (Figure 4A), #4 (Figure 4B), #5 (Figure 4C), #6 (Figure 4D), #7 (Figure 4E), #8 (Figure 4F), and #9 (Figure 4G) after 10 days of refrigerated storage. [Figure 5] 5A-5C are graphs showing the viscosity of starched yogurt sample #10-17 after 1 day and 25 days of refrigerated storage. [Figure 6] 6A-6C are graphs showing the torque of starched yogurt sample #10-17 after 1 day and 25 days of refrigerated storage. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention In the following description, it is understood that other embodiments may be utilized and structural and operational changes may be made without departing from the scope of the present embodiments disclosed herein.

[0019] definition As used herein, unless otherwise specified, the phrase "substantially free" means a composition / ingredient that contains less than 1 wt.%, preferably less than 0.5 wt.%, preferably less than 0.3 wt.%, preferably less than 0.2 wt.%, preferably less than 0.1 wt.%, preferably less than 0.05 wt.%, preferably less than 0.03 wt.%, preferably less than 0.02 wt.%, preferably less than 0.01 wt.%, preferably less than 0.001 wt.%, preferably less than 0.0001 wt.%, preferably 0 wt.%, based on the total weight of the composition / ingredient.

[0020] As used herein, the term "optional" or "optionally" means that the subsequently described event may or may not occur, or that the subsequently described ingredient may or may not be present (e.g., 0 wt %).

[0021] The term "raw milk" is used broadly in this application to refer to a composition based on milk or milk components obtained from a mammal that has not been subjected to enzymatic treatment to modify the proteins contained therein and that can be used as a medium for the growth and fermentation of lactic acid bacteria. This includes compositions that have been skimmed, supplemented, pasteurized, homogenized, fortified, concentrated, diluted, or otherwise processed. Conversely, the term "modified milk" as used herein refers to a composition based on milk or milk components that has been subjected to enzymatic treatment to modify the proteins contained therein.

[0022] As used herein, "set yogurt" is a non-fluid yogurt product obtained after precipitation of milk proteins. Set yogurt is formed by leaving the dairy product undisturbed through a fermentation (incubation) and cooling period, which provides the set yogurt with a continuous, gel-like (semi-solid) structure.

[0023] "Starved yogurt," on the other hand, is a fluid yogurt product formed as a result of breaking down the gel structure at the end of the fermentation (incubation) period and before cooling and further processing. Starved yogurt therefore has different texture and rheological properties (e.g., lower viscosity) than set yogurt. Starved yogurt with a sufficiently high viscosity can actually be handled with a spoon ("spoonable") and is particularly highly valued.

[0024] The term "short texture" means the opposite of a sticky texture. Thus, the less sticky a yogurt is, the "shorter" its texture is. "Sticky" texture refers to the characteristic texture of yogurt as assessed by sensory evaluation, where a spoonful of yogurt is removed from a sample and the sticky properties are evaluated. The longer the threads can be before breaking, the stickier the product.

[0025] How to make starched yogurt The present disclosure relates to a method for producing a starched yogurt with excellent rheological properties (e.g., high viscosity) and favorable sensory properties (e.g., smoothness, firmness, mild acidity, low syneresis) by modifying milk proteins in raw milk with protein glutaminase prior to fermentation.

[0026] Thus, the method of the present disclosure typically comprises (at least) the following sequence: (i) Treating raw milk with protein glutaminase to prepare modified milk, and then (ii) fermenting the modified milk to prepare yogurt by adding a starter culture, and then (iii) Breaking the gel structure of yogurt to prepare starched yogurt.

[0027] raw milk In the present disclosure, the raw milk to be modified with protein glutaminase is not particularly limited, and may be any edible milk, for example, milk obtained from cows, buffalo, goats, sheep, horses, camels, yaks, etc. Any of the above, including pasteurized milk (e.g., low temperature long pasteurized milk - "LTLT milk", high temperature short time pasteurized milk - "HTST milk", ultra high temperature pasteurized milk - "UHT milk"), milk with adjusted components such as protein and / or milk fat, fortified milk containing additional vitamins and minerals, skim milk, homogenized milk, processed milk, diluted milk, concentrated milk, dried milk (powdered milk), skim milk powder, skim milk solution, dried (powdered) milk suspended or dissolved in water, low-lactose milk, etc., are included in this category.

[0028] In the present specification, raw milk having a wide range of fat contents can be used. Typically, the raw milk has a fat content of up to 10% by weight, preferably up to 8% by weight, preferably up to 6% by weight, preferably up to 5% by weight, preferably up to 4% by weight, preferably up to 3% by weight, preferably up to 2% by weight, preferably up to 1.5% by weight, preferably up to 1% by weight, preferably up to 0.5% by weight, preferably up to 0.3% by weight, preferably up to 0.1% by weight, or preferably 0% by weight, based on the total weight of the raw milk. Specific reference is made to commercially available dairy products such as whole milk (fat content about 3.25% by weight), reduced-fat milk (fat content about 2% by weight), low-fat milk (fat content about 1% by weight), and skim milk (fat content about 0-0.5% by weight). In some embodiments, it may be desirable to use raw milk modified with respect to fat content, such as low-fat milk or skim milk, to prepare a low-fat starched yogurt product.

[0029] The raw milk can also have a wide range of protein contents, with typical protein content values ​​being at least 0.5% by weight, preferably at least 1% by weight, preferably at least 1.5% by weight, preferably at least 2% by weight, preferably at least 2.5% by weight, preferably at least 3% by weight, and up to 10% by weight, preferably up to 8% by weight, preferably up to 6% by weight, preferably up to 5.5% by weight, preferably up to 5% by weight, preferably up to 4.5% by weight, preferably up to 4% by weight, preferably up to 3.5% by weight, based on the total weight of the raw milk. In some embodiments, it may be desirable to use raw milk that has been modified in terms of its protein content, for example protein-supplemented raw milk, to prepare starched yogurt products with a high protein content.

[0030] Although raw milk having fat and protein contents outside the above ranges may be utilized in some circumstances, preferred raw milks are commercially available raw milks or raw milks adjusted to fall within the above ranges according to methods known to those skilled in the art.

[0031] stabilizers The method of the present disclosure may optionally include the addition of a stabilizer, for example, to help prevent syneresis. The stabilizer can be added at any stage of the production of starched yogurt. For example, the stabilizer can be added to raw milk, i.e., before treating the raw milk with glutaminase; to modified milk, i.e., after treatment with PG and before fermentation; to yogurt, i.e., after fermentation; or the stabilizer can be added at two or more of these stages. It is particularly preferred to add the stabilizer to raw milk before treating the raw milk with glutaminase.

[0032] When a stabilizer is used, it may be added in an amount of at least 0.01 wt. %, preferably at least 0.05 wt. %, preferably at least 0.1 wt. %, preferably at least 0.2 wt. %, preferably at least 0.4 wt. %, preferably at least 0.6 wt. %, preferably at least 0.8 wt. %, preferably at least 1 wt. %, and up to 5 wt. %, preferably up to 4.5 wt. %, preferably up to 4 wt. %, preferably up to 3.5 wt. %, preferably up to 3 wt. %, preferably up to 2.5 wt. %, preferably up to 2 wt. %, preferably up to 1.5 wt. %, based on the total weight of the composition to which it is added, for example when added to raw milk, based on the total weight of the raw milk.

[0033] Examples of stabilizers that can be used herein include, but are not limited to, pectin, agar, carrageenan, gelatin, whey protein concentrate, chicory root fiber, and modified food starch, and mixtures thereof. Specifically, one or more of pectin, modified food starch, and gelatin, preferably two or more of pectin, modified food starch, and gelatin.

[0034] Pectin is a structural heteropolysaccharide rich in galacturonic acid found in the primary cell walls of terrestrial plants. A wide variety of pectins are suitable for use as stabilizers in the present disclosure, particularly commercially available pectins extracted from fruits such as citrus peel or apple peel. While high-methoxyl (HM) pectins (i.e., having more than 50% of the total galacturonic acid units methyl esterified) are contemplated, preferred pectins are those classified as low-methoxyl (LM) pectins (i.e., having 50% or less of the total galacturonic acid units methyl esterified). For example, pectins may have less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%, preferably less than 15%, preferably less than 10%, and preferably less than 5% of their total galacturonic acid content in the form of methyl esters. As used herein, the non-esterified galacturonic acid units of pectin may be either the free acid (carboxylic acid group) or a salt with sodium, potassium, calcium, and / or ammonium ions. Specific examples of pectin stabilizers that can be used herein include, but are not limited to, the UNIPECTIN products available from Cargill.

[0035] Modified Food Starch Modified food starch is a complex carbohydrate derived from grains, vegetables, root vegetables, legumes, and fruits, such as those derived from wheat, rice, corn, potato, taro, yam, pumpkin, beans, cassava, etc., and has one or more components that have been physically, chemically, or enzymatically modified to provide, for example, a more desirable texture, heat and / or acid resistance, and / or solubility / swelling profile compared to regular (unmodified) starch. Starch processing can be carried out by any method known to those skilled in the art, such as acid treatment, alkali treatment, bleaching, oxidation, enzyme treatment, phosphorylation, cross-linking (e.g., with sodium trimetaphosphate), acetylation, hydroxypropylation / hydroxyethylation, carboxymethylation, etc., and mixtures thereof (e.g., cross-linked and stabilized starches such as hydroxypropylated phosphate cross-linked starch). Specific examples of modified food starches that can be used herein include, but are not limited to, chemically modified starches derived from waxy corn or tapioca (e.g., THERMTEX and NATIONAL products, each available from Ingredion), and SHUR STAB Cultured Dairy Systems available from Denali.

[0036] Gelatin is a mixture of proteins and peptides produced by partial hydrolysis (breakdown) of collagen extracted from animal skin, bones, and / or connective tissue. Various gelatins can be useful as stabilizers herein, such as gelatin obtained from domesticated cattle, chicken, pigs, and marine sources, particularly cattle bones and hides. Typically, type A gelatin (obtained from acid-treated raw materials) or type B gelatin (obtained from alkali-treated raw materials) can provide acceptable stabilization for starched yogurt, and more specifically, type B gelatin, more preferably type B bovine (beef) gelatin. The gelatin of the present disclosure may be very low bloom gelatin (bloom value less than 50); low bloom gelatin (bloom value between 50 and 150); medium bloom gelatin (bloom value between 150 and 220); or high bloom gelatin (bloom value greater than or equal to 220); according to the Gelatin Manufacturers Institute of America Bloom Jelly Strength Testing Procedure (Standard Methods for Sampling and Testing Gelatin, Gelatin Manufacturers Institute of America, Inc., 1986, 501 Fifth Ave. New York, NY, and The Association of Analytical Communities (AOAC) international, AOAC Method 948.21 "Jelly Strength of Gelatin" - each of which is incorporated herein by reference in its entirety). Bloom value is the force (in grams) required to depress a standard AOAC plunger (a flat cylindrical probe with a sharp edge, 12.7 mm diameter) 4 mm into set gelatin (e.g., 7.5 g gelatin in 105 mL water) at a concentration of 6.66% (w / v) maintained for 16 hours at 10° C. In a preferred embodiment, the gelatin is a high-bloom gelatin with a Bloom value of at least 220, preferably at least 230, preferably at least 240, preferably at least 250, and up to 325, preferably up to 300, preferably up to 290, preferably up to 280, preferably up to 260.

[0037] In some embodiments, a mixture of two or more stabilizers may be used in the disclosed methods. Examples of mixtures include, but are not limited to, a mixture of pectin and modified food starch, a mixture of whey protein concentrate and pectin (e.g., VITEX AYS 08, available from Cargill), and a mixture of agar and pectin (e.g., VITEX AYS 10, available from Cargill). When a mixture of a first stabilizer and a second stabilizer (e.g., a mixture of pectin and modified food starch) is used, the weight ratio of the first stabilizer to the second stabilizer is at least 1:20, preferably at least 1:15, preferably at least 1:10, preferably at least 1:5, preferably at least 1:3, preferably at least 1:2, preferably at least 1:1, up to 20:1, preferably at most 15:1, preferably at most 10:1, preferably at most 5:1, preferably at most 3:1, preferably at most 2:1.

[0038] Protein glutaminase The method of the present disclosure includes treating raw milk, preferably raw milk to which a stabilizer has been added, with protein glutaminase (PG) to deamidate proteins contained in the raw milk, thereby preparing modified milk.

[0039] Before adding the protein glutaminase, the raw milk can be optionally brought to a temperature at which protein modification will occur (processing temperature). For example, before adding PG, the raw milk can be heated to a temperature of up to 60°C, preferably up to 50°C, for example, at least 10°C, preferably at least 15°C, preferably at least 20°C, preferably at least 25°C, preferably at least 30°C, and up to 60°C, preferably up to 55°C, preferably up to 50°C, preferably up to 45°C, preferably up to 40°C, preferably up to 35°C, and maintained at this temperature for at least 1 minute, preferably at least 5 minutes, preferably at least 10 minutes, preferably at least 15 minutes, preferably at least 20 minutes, and up to 60 minutes, preferably up to 45 minutes, preferably up to 40 minutes, preferably up to 35 minutes, preferably up to 30 minutes. In a preferred embodiment, before adding PG, the raw milk is heated to a temperature of about 45-50°C and maintained at this temperature for about 30-35 minutes.

[0040] Next, protein glutaminase is added to the raw milk, preferably the raw milk already at a treatment temperature for the deamidation reaction. The protein glutaminase of the present disclosure can be a commercially available PG or a PG prepared from a culture medium of a microorganism that produces protein glutaminase. The type of protein glutaminase used is not particularly limited, as long as it acts directly on glutamine residues in proteins contained in the raw milk and deamidates the glutamine residues without cleaving peptide bonds and / or cross-linking proteins. Examples of such protein glutaminases include, but are not limited to, protein glutaminases derived from Chryseobacterium, Flavobacterium, or Empedobacter (including commercially available protein glutaminases derived from Chryseobacterium), as disclosed in JP-A-2000-50887, JP-A-2001-218590, and WO 2006 / 075772 (each of which is incorporated herein by reference in its entirety). A specific example of a protein glutaminase that can be used in the methods of the present disclosure includes, but is not limited to, protein glutaminase (500 U / g, available from Amano Enzyme Inc.).

[0041] Protein glutaminase can be prepared from the culture medium of a microorganism that produces protein glutaminase using, for example, known protein separation and purification methods (e.g., centrifugation, ultrafiltration (UF) concentration, salting out, various types of chromatography using ion exchange resins, etc.). For example, the medium can be centrifuged to remove the bacteria, and then the target enzyme can be obtained by a combination of salting out, chromatography, etc. When collecting PG enzyme from inside the bacteria, the bacteria can be recovered from the culture medium by filtration or centrifugation, etc., and then disrupted by, for example, pressure treatment or ultrasonic treatment. The target PG enzyme can then be obtained by carrying out the protein separation and purification method described above. This enzyme can be powdered by a drying method such as freeze-drying or vacuum drying, optionally using an appropriate diluent or drying aid.

[0042] In some embodiments, the activity of a protein glutaminase can be measured by the following steps: (1) 100 μL of an aqueous solution containing protein glutaminase was added to 1 ml of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly (Peptide Institute, Inc.), and the mixture was incubated at 37°C for 10 minutes. The reaction was then stopped by adding 1 ml of 0.4 M trichloroacetic acid (TCA) solution. (2) The amount of ammonia produced by the reaction in the reaction solution of (1) is measured using Ammonia-Test-Wako (manufactured and sold by Wako Pure Chemical Industries, Ltd.). (3) Prepare a blank solution by incubating an enzyme-free solution at 37°C for 10 minutes, then adding 1 ml of 0.4 M TCA solution and 100 μL of an aqueous solution containing protein glutaminase enzyme, in that order. (4) The enzyme concentration is adjusted by diluting with phosphate buffer (pH 6.5) so that the absorbance at 630 nm falls within the range of Δ0.2 to 0.8. (5) Activity is measured by defining the amount of enzyme required to produce 1 μmol of ammonium per minute as 1 unit (U). Enzyme activity is calculated using the following formula: Enzyme activity (U / ml)=(Es-Eb)*F*0.123xDf During the ceremony; Es: absorbance of the enzyme reacted solution, Eb: blank absorbance, F: Factor (the inverse of the slope of the linear calibration curve of the standard ammonium solution), and Df: Dilution factor of the enzyme solution.

[0043] The degree of deamidation of the proteins contained in the raw milk, i.e., the degree of denaturation of the raw milk, can be adjusted by controlling the processing temperature, processing time, amount of PG used, etc. in order to obtain the desired physical properties of the starched yogurt.

[0044] With regard to the processing temperature, the raw milk is typically treated with protein glutaminase at a processing temperature of up to 60°C, preferably up to 50°C, for example at a processing temperature of at least 10°C, preferably at least 15°C, preferably at least 20°C, preferably at least 25°C, preferably at least 30°C, and up to 60°C, preferably up to 55°C, preferably up to 50°C, preferably up to 45°C, preferably up to 40°C, preferably up to 35°C, with 45-50°C being most preferred.

[0045] Regarding the treatment time, the raw milk can be treated with protein glutaminase for up to 24 hours. For example, the raw milk can be treated with protein glutaminase for at least 15 minutes, preferably at least 30 minutes, preferably at least 45 minutes, preferably at least 50 minutes, preferably at least 55 minutes, preferably at least 60 minutes, and up to 24 hours, preferably up to 12 hours, preferably up to 8 hours, preferably up to 4 hours, preferably up to 2 hours, preferably up to 1.5 hours, with 45 to 60 minutes being the most preferred.

[0046] The amount of protein glutaminase added can vary depending on the composition of the raw milk (e.g., protein content), the type of protein to be modified, or the desired effect to be obtained. For example, the amount of PG added can be at least 0.01U per 1g of protein in the raw milk, preferably at least 0.1U per 1g of protein in the raw milk, preferably at least 0.5U per 1g, preferably at least 1U per 1g, preferably at least 1.5U per 1g, preferably at least 2U per 1g, preferably at least 2.5U per 1g, and up to 10U per 1g of protein in the raw milk, preferably up to 8U per 1g of protein in the raw milk, preferably up to 6U per 1g, preferably up to 5U per 1g, preferably up to 4U per 1g, preferably up to 3U per 1g.

[0047] In terms of the weight of raw milk, the raw milk can be treated with protein glutaminase at least 10 ppm, preferably at least 20 ppm, preferably at least 30 ppm, preferably at least 40 ppm, preferably at least 50 ppm, preferably at least 60 ppm, preferably at least 70 ppm, preferably at least 80 ppm, preferably at least 90 ppm, preferably at least 100 ppm, and up to 500 ppm, preferably up to 450 ppm, preferably up to 400 ppm, preferably up to 350 ppm, preferably up to 300 ppm, preferably up to 250 ppm, preferably up to 200 ppm, preferably up to 150 ppm, preferably up to 125 ppm, based on the total weight of the raw milk.Of course, PG dosages outside this range can also be used as needed to obtain the desired level of deamidation / effect from protein modification.

[0048] While enzymes other than protein glutaminase may optionally be used for protein modification herein, one advantage of the present disclosure is that the use of other protein-modifying enzymes is not required to produce starched yogurt with a sufficiently high viscosity. At least from the perspective of reducing operational costs and complexity, it is preferred that protein glutaminase be the only enzyme used for protein modification, e.g., the only enzyme used that acts on glutamine residues of milk proteins contained in the raw milk. Another type of enzyme that acts on glutamine residues, which can be used simultaneously or in combination with PG to treat raw milk but is preferably excluded from the method of the present disclosure, is a cross-linking enzyme such as transglutaminase (TG). Transglutaminase functionally cross-links proteins via glutamine residues and amino-containing residues (e.g., lysine) and rarely performs deamidation, and therefore is not a deamidating enzyme. Both calcium-independent TG types (e.g., TG obtained from microorganisms such as actinomycetes or Bacillus subtilis) and calcium-dependent TG types (e.g., those obtained from microorganisms such as guinea pig liver or bovine or porcine blood, human epidermal keratinocytes, human blood coagulation factor XIII, oomycetes, those obtained from fish and oysters, etc.) can be used in addition to PG, but are preferably excluded in the disclosed methods (e.g., those described in US 2011 / 0064847A1, which is incorporated herein by reference in its entirety).

[0049] Specific examples of transglutaminase enzymes or preparations containing transglutaminase enzymes include, but are not limited to, ACTIVA MP (a transglutaminase enzyme designed for dairy applications) available from Ajinomoto Foods Europe; and ACTIVA YG (a transglutaminase preparation containing yeast extract designed for dairy applications) available from Ajinomoto Health & Nutrition North America, Inc. Combinations of transglutaminase enzymes and protein glutaminase enzymes are also commercially available, such as ACTIVA SYG (a combination of protein glutaminase enzyme and transglutaminase enzymes designed for dairy applications) available from Ajinomoto Foods Europe.

[0050] reducing agent The disclosed method can also optionally use a reducing agent to reduce / remove inhibitors present in the raw milk that may interfere with the protein glutaminase enzyme, for example. Thus, the use of a reducing agent can increase the effectiveness of the PG treatment, and in some cases, the PG dosage can be reduced to achieve the same level of effectiveness. Other benefits provided by the use of a reducing agent can include accelerating fermentation, replacing starters, and improving taste / texture.

[0051] The amount of reducing agent used can be adjusted based on, for example, the activity of the selected reducing agent, the type of raw milk to be processed, and the desired taste profile of starched yogurt.Usually, reducing agent can be added in an amount of up to 10,000 ppm.Those skilled in the art can determine the appropriate amount of reducing agent to be used and adjust it as needed.Most typically, when used, reducing agent can be added in an amount of at least 10 ppm, preferably at least 20 ppm, preferably at least 30 ppm, preferably at least 40 ppm, preferably at least 50 ppm, preferably at least 60 ppm, preferably at least 70 ppm, preferably at least 80 ppm, preferably at least 90 ppm, preferably at least 100 ppm, and up to 500 ppm, preferably up to 450 ppm, preferably up to 400 ppm, preferably up to 350 ppm, preferably up to 300 ppm, preferably up to 250 ppm, preferably up to 200 ppm, preferably up to 150 ppm, preferably up to 125 ppm, based on the total weight of raw milk.

[0052] Suitable reducing agents include, but are not limited to, thiol compounds such as glutathione, cysteine, and γ-glutamylcysteine, which are permitted for use as food additives; yeast extracts containing at least one such thiol compound; thiosulfate; sulfite; ascorbic acid; erythorbic acid and their salts; and tocopherols. Yeast extract is particularly preferred. Regarding yeast extract, any yeast extract containing glutathione can be applied to the disclosed method to improve the texture of PG-treated starched yogurt, among other advantages. Non-limiting examples include yeast extracts containing at least 4% by weight, preferably at least 6% by weight, preferably at least 8% by weight, and up to 25% by weight, preferably up to 20% by weight, and preferably up to 15% by weight, of glutathione. A suitable example includes, but is not limited to, AROMILD U, a yeast extract containing 8% by weight of natural glutathione, available from Kohjinsha.

[0053] Regarding the timing of addition, the raw material milk can be treated with the reducing agent before or simultaneously with the treatment with the protein glutaminase. From a practical standpoint and from the standpoint of maximizing the effect of the reducing agent, it is preferable to treat the raw material milk with the reducing agent and the protein glutaminase simultaneously, for example, by adding the reducing agent and the PG simultaneously or substantially simultaneously (for example, consecutively) to the raw material milk, and then subjecting the raw material milk to the above-mentioned treatment temperature and treatment time for protein modification with the PG.

[0054] Heat sterilization The deamidation reaction can be optionally monitored, for example, by measuring the amount of ammonium produced during the PG treatment operation. After the raw milk has been thoroughly treated with protein glutaminase to prepare the modified milk, and before the modified milk is fermented, the modified milk can be heat sterilized to inactivate the protein glutaminase enzyme and any other enzymes or living organisms present, thereby halting the protein modification. This heat treatment can also denature milk proteins (including modified milk proteins) to prevent them from forming curds.

[0055] Any heat sterilization conditions used in the production of dairy products can generally be used herein. In a preferred embodiment, the modified milk is heat sterilized at a temperature of at least 70°C, preferably at least 75°C, preferably at least 80°C, preferably at least 85°C, and up to 95°C, preferably up to 90°C. Depending on the temperature used, heat sterilization can be carried out for, for example, at least 1 minute, preferably at least 2 minutes, preferably at least 4 minutes, preferably at least 5 minutes, preferably at least 10 minutes, preferably at least 15 minutes, and up to 30 minutes, preferably up to 25 minutes, preferably up to 20 minutes. Of course, the effects of the present disclosure can be obtained without such heat sterilization treatment.

[0056] fermentation Fermentation of modified milk involves the addition of a starter culture to the modified milk, which is a culture of food-grade microorganisms, particularly thermophilic lactic acid bacteria (e.g., Streptococcus spp. and Lactobacillus spp.), that is responsible for the acidification of the modified milk. During the fermentation stage, the consumption of lactose by these bacteria results in the formation of lactic acid, a decrease in pH, and the formation of protein coagulum. Thus, the acidification and coagulation of this modified milk produces yogurt.

[0057] Specifically, the starter cultures used in the fermentation herein are Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus paracasei, or any microorganisms derived therefrom. Lactic acid bacteria strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus can optionally be included to impart various properties to the finished yogurt product, such as the property of promoting flora equilibrium. Starter cultures can be fresh, frozen, or freeze-dried.

[0058] Specific examples of starter cultures that can be used to ferment the modified milk of the present disclosure include, but are not limited to, YOFLEX products such as YC-X11, YC-180, YC-280, YC-370, YC-380, YC-381, and PREMIUM 1.0, and NU-TRISH products such as ABT-1, ABT-10, ABY-1, and ABY-10 (each available from Chr. Hansen). It should also be mentioned that yogurt containing residual live lactic acid bacteria can also be used as a starter culture for fermentation.

[0059] Typical amounts of starter culture range from at least 0.0001 wt.%, preferably at least 0.0005 wt.%, preferably at least 0.001 wt.%, preferably at least 0.005 wt.%, preferably at least 0.01 wt.%, preferably at least 0.015 wt.%, preferably at least 0.02 wt.%, and up to 1 wt.%, preferably up to 0.5 wt.%, preferably up to 0.2 wt.%, preferably up to 0.1 wt.%, preferably up to 0.05 wt.%, preferably up to 0.03 wt.%, based on the total weight of the modified milk.

[0060] If the modified milk is at a high temperature due to a heat sterilization operation or the like, the modified milk can first be cooled to an appropriate fermentation temperature before adding the starter culture. After the starter culture is added, fermentation can be carried out at a temperature of at least 22°C, preferably at least 26°C, preferably at least 30°C, preferably at least 34°C, preferably at least 38°C, preferably at least 42°C, and up to 45°C, preferably up to 44°C, and preferably up to 43°C. The modified milk can be fermented until it reaches a pH of at least 4.0, preferably at least 4.2, preferably at least 4.4, and up to 5.0, preferably up to 4.8, and preferably up to 4.6, which usually corresponds to a fermentation time of at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 4 hours, and up to 10 hours, preferably up to 8 hours, and preferably up to 6 hours. It should be understood that these conditions can be changed or adjusted as appropriate depending on the purity of the starter culture used, the type and purity of the protein in the modified milk, and the like.

[0061] Destruction of the gel structure To produce the stirred yogurt, the gel structure (coagulum) of the yogurt produced after fermentation is then broken down. The gel structure can be broken down by any technique known to those skilled in the art, such as by stirring / mixing (e.g., kitchen mixer), filtering / sieving (e.g., pore size of 200-500 μm), etc. to prepare the stirred yogurt. In a preferred embodiment, the gel structure of the yogurt is broken down by stirring. The production of the stirred yogurt may also include one or more of pumping, cooling (e.g., 4-15°C), and refrigerating and / or packaging the stirred yogurt for distribution / sale.

[0062] additives Additives commonly used in the production of yogurt can optionally be used in this method. Examples of additives include, but are not limited to, additional dairy ingredients (e.g., cream); sugars or sweeteners (e.g., sucrose, maltitol, sorbitol, lactose); oils and fats; emulsifiers; flavorings, including seasonings and spices; coloring agents; antioxidants; fruit (e.g., strawberry pulp and juice); grains; thickeners (e.g., starch); nutritional substances such as vitamins, minerals, and fiber (e.g., vitamin A, vitamin B, vitamin D, calcium, zinc, iron, folic acid, riboflavin, dextrin); animal and / or vegetable proteins (e.g., soy protein, wheat protein, etc.); and solid foods (e.g., chocolate). Regarding the timing of addition, if used, additives can be added at any suitable addition point known in the art. For example, if a starched yogurt product containing fruit is desired, fruit additives can be added by stirring the fruit additive into the yogurt during the stirring operation in a step that breaks the gel structure of the yogurt produced from fermentation.

[0063] Particular mention is made of thickeners. While the use of any suitable food-grade thickener known to those skilled in the art is contemplated (and may be used optionally), it is preferred that the sequence of operations specified in this disclosure is sufficient to provide the desired increase in viscosity to the stirred yogurt without the need for the addition of a thickener. Thus, in a preferred embodiment, no thickener is added during the production of the stirred yogurt. Examples of thickening agents include, but are not limited to, starches such as starches (including both pregelatinized and non-pregelatinized starches) derived from tubers (e.g., potato, sweet potato, etc.), grains (e.g., wheat, rice, corn, etc.), vegetables, root vegetables, or fruits, for example, the starches described in US 2019 / 0021353A1 (incorporated herein by reference in its entirety); xanthan gum; guar gum; tragacanth; alginates (e.g., sodium alginate, potassium alginate, ammonium alginate, and / or calcium alginate); karaya gum; carob; furcellaran; locust bean gum; tapioca; gum arabic (acacia); modified alginates (e.g., propylene glycol alginate); and modified cellulose polymers such as hydroxypropylmethylcellulose (HPMC), methylcellulose (MC), carboxymethylcellulose (CMC), hydroxymethylcellulose, hydroxypropylcellulose, and microcrystalline cellulose; and mixtures thereof. In preferred embodiments, starch is not utilized in the disclosed methods, for example, as a method of increasing viscosity.

[0064] A particularly preferred method for producing starched yogurt in the present disclosure may include preparing raw milk having a fat and protein content within the aforementioned ranges, adding one or more stabilizers (e.g., pectin and modified food starch) as needed, and preheating the raw milk to a desired processing temperature (e.g., 45-50°C). The raw milk can then be treated with protein glutaminase in the presence of a reducing agent (e.g., yeast extract) at a selected processing temperature (e.g., 45-50°C) for an appropriate processing time (e.g., 45-60 minutes) to produce modified milk. The PG treatment can be terminated by heat sterilization of the modified milk, for example, at a temperature of 80-95°C for 15-30 minutes. After conventional heat sterilization and cooling, a starter culture containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is added to the modified milk, and the modified milk is incubated in a tank at 38-45°C to ferment the modified milk and produce yogurt. After fermentation is deemed complete, the yogurt is preferably stirred to break up any coagulum, producing a starched yogurt that can be stored at low temperatures (e.g., 5-10°C).

[0065] As described above, the method of the present disclosure involves treating raw milk with protein glutaminase before fermenting with a starter culture to prepare modified milk. The inventors have unexpectedly discovered that only by following this series of processes can a stirred yogurt with suitable rheological properties (e.g., high viscosity and torque) be produced. For example, it has been found that modifying raw milk with protein glutaminase before fermentation increases the viscosity of stirred yogurt by up to 60% compared to the control (without PG treatment) (see, for example, Example 3, Sample #16 compared to Control Sample #10).

[0066] On the other hand, when protein glutaminase was added during the fermentation process, the resulting stirred yogurt was found to have a lower viscosity compared to the control (no PG treatment) (see, e.g., Example 2, Sample #9 compared to Control Sample #3), which is consistent with previous efforts to use protein glutaminase to modify stirred yogurt products.

[0067] Thus, the increased viscosity obtained by the disclosed method is particularly unexpected considering that previous efforts using only PG to produce stirred yogurt actually decreased the viscosity of the stirred yogurt product (see US 2011 / 0064847A1 and US 2019 / 0021353A1, each of which is incorporated by reference in its entirety), which in light of the present disclosure can now be understood to be due to the addition of PG at an inopportune time during fermentation.

[0068] Starched yogurt The present disclosure also relates, in one or more embodiments, to the stirred yogurt produced by the above-described method.

[0069] The fat and protein contents of the stirred yogurt produced by the method of the present disclosure are the same or substantially the same as the fat and protein contents of the raw milk used (described above), since the fat and protein contents are not substantially changed after lactic acid fermentation. The stirred yogurt of the present disclosure is not particularly limited, but is preferably a low-fat stirred yogurt, for example, a stirred yogurt having a fat content of 1.5 wt% or less, based on the total weight of the stirred yogurt. Furthermore, the stirred yogurt typically has a protein content of 5.5 wt% or less, preferably 5 wt% or less, preferably 4.5 wt% or less, and preferably 4.2 wt% or less, based on the total weight of the stirred yogurt.

[0070] The stirred yogurt of the present disclosure is made by modifying proteins contained in raw milk with protein glutaminase, and because at least a portion of the glutamine residues are deamidated by the protein glutaminase enzyme, the total number of glutamine residues contained in the stirred yogurt may be less than the total number of glutamine residues present in the raw milk used to make the stirred yogurt.

[0071] The stirred yogurt also contains live Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and optionally other live microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus, and Lactobacillus paracasei, or any microorganisms derived therefrom. In particular, co-cultured Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus may be found to be viable in the stirred yogurt product in an amount of at least 1 million, preferably at least 5 million, preferably at least 10 million colony forming units (CFU) / g.

[0072] According to the present disclosure, the stirred yogurt produced by the method herein has excellent rheological properties (e.g., high viscosity and torque) and favorable sensory properties (e.g., smoothness, firmness, mild acidity, low level of syneresis, etc.). These advantageous properties can be achieved by modifying the raw milk with protein glutaminase before fermentation without the need for cross-linking enzymes or thickeners; therefore, a preferred stirred yogurt is one that is substantially free, preferably completely free (0% by weight), of cross-linking enzymes / inactivated cross-linking enzymes (e.g., transglutaminase) and / or thickeners (e.g., starch).

[0073] The viscosity of the stirred yogurt can vary widely depending on the yogurt base used. However, desirable stirred yogurt will typically have a viscosity of at least 100,000 cP, preferably at least 110,000 cP, preferably at least 115,000 cP, preferably at least 120,000 cP, preferably at least 125,000 cP, preferably at least 130,000 cP, preferably at least 132,000 cP, preferably at least 134,000 cP, preferably at least 136,000 cP, as measured with a Brookfield DV-I viscometer (Helipath A spindle; 4.0 rpm; 40°F). The viscosity is at least 136,000 cP, preferably at least 138,000 cP, preferably at least 140,000 cP, and up to 250,000 cP, preferably up to 200,000 cP, preferably up to 180,000 cP, preferably up to 150,000 cP, preferably up to 148,000 cP, preferably up to 146,000 cP, preferably up to 145,000 cP, preferably up to 144,000 cP, preferably up to 142,000 cP. Particularly preferred is a stirred yogurt having a spoonable viscosity of at least 138,000 cP, preferably at least 140,000 cP, preferably at least 142,000 cP, preferably at least 144,000 cP.

[0074] A measurement related to viscosity is torque percent, or the amount of torque resistance (expressed in %) measured by a rotating spindle immersed in the material. In a preferred embodiment, the stirred yogurt has a torque (%) of at least 6.4, preferably at least 6.5, preferably at least 6.6, preferably at least 6.7, preferably at least 6.8, preferably at least 6.9, preferably at least 7.0, and up to 7.5, preferably up to 7.4, preferably up to 7.3, preferably up to 7.2.

[0075] Various additives can be optionally incorporated into the starched yogurt to provide starched yogurt products with various sweetnesses, flavors, and textures. For example, the starched yogurt of the present disclosure can optionally be formulated with one or more additives, including, but not limited to, additional dairy ingredients (e.g., cream); sugars or sweeteners (e.g., sucrose, maltitol, sorbitol, lactose); fats and oils; emulsifiers; flavorings, including seasonings and spices; coloring agents; antioxidants; fruit (e.g., strawberry pulp and juice); grains; thickeners (e.g., starch); nutritional substances such as vitamins, minerals, and fiber (e.g., vitamin A, vitamin B, vitamin D, calcium, zinc, iron, folic acid, riboflavin, dextrin); animal and / or vegetable proteins (e.g., soy protein, wheat protein, etc.); and solid foods (e.g., chocolate).

[0076] The following examples are intended to further illustrate the starched yogurts, their properties, and methods for their production, and are not intended to limit the scope of the claims. [Example]

[0077] Stirred yogurt samples were prepared according to the procedures described in the examples below and tested according to one or more of the test procedures described below.

[0078] The gelatin used was 250 bloom beef gelatin. The yeast extract used was AROMILD U, a yeast extract containing 8% by weight of natural glutathione, available from Kojinsha. ACTIVA SYG is a combination of protein glutaminase and transglutaminase enzymes designed for dairy applications, available from Ajinomoto Foods Europe. ACTIVA MP is a transglutaminase enzyme designed for dairy applications, available from Ajinomoto Foods Europe. ACTIVA YG is a transglutaminase preparation containing yeast extract designed for dairy applications, available from Ajinomoto Health & Nutrition North America, Inc. "PG" is a protein glutaminase enzyme (deamidation activity 500 U / g) available from Amano Enzyme Co., Ltd.

[0079] test The prepared starched yogurt samples were tested at specified time intervals (e.g., after 1 day, 25 days, etc.) after refrigeration at 5°C.

[0080] <Viscosity> Viscosity was measured with a Brookfield DV-I viscometer according to the following parameters: -Spindle; Helipath A -Speed: 4.0 rpm -Temperature; 40°F Viscosity results are expressed in centipoise (cP).

[0081] <torque> Torque was measured simultaneously with viscosity using the viscosity procedure described above (both viscosity and torque were selected as automatic outputs when reading the sample on a Brookfield DV-I viscometer). Torque results are expressed as a percentage (%).

[0082] <Sensory analysis> The prepared starched yogurt samples were evaluated by trained panelists with profiling experience and pre-screened for sensory acuity. The trained panelists evaluated the prepared starched yogurt samples for one or more of appearance (e.g., amount of syneresis), texture, and flavor.

[0083] Example 1 (Comparative Example) procedure: The raw milk was adjusted to a fat content of 1.5% by weight and a protein content of 4.2% by weight. Stabilizers added at specified levels. The raw milk was heated at 50°C for 30 minutes, and then sterilized by heating at 85°C for 20 minutes while stirring. The raw milk was then cooled to 43°C. -Then a starter culture (YOFLEX PREMIUM 1.0 available from Chr. Hansen) was added in an amount of 0.02% by weight. - Protein modifying enzymes were then added at the indicated levels and the raw milk was incubated (fermented) at 43°C until a pH of 4.6 was reached. The resulting yogurt was stirred. The starched yogurt was stored at 5°C for 7 days and then evaluated for viscosity, torque, and sensory properties.

[0084] result:

[0085] [Table 1]

[0086] The addition of 200 ppm transglutaminase enzyme (ACTIVA MP) was found to provide a slight increase in viscosity and torque, as shown in Table 1 and Figures 1A-1B. However, sensory analysis of Sample #2 revealed a poor, lumpy appearance compared to Sample #1 (control) (Figures 2A and 2B).

[0087] Example 2 (Comparative Example) procedure: The raw milk was adjusted to a fat content of 1.5% by weight and a protein content of 4.2% by weight. Stabilizers added at specified levels. The raw milk was heated at 50°C for 30 minutes, and then sterilized by heating at 85°C for 20 minutes while stirring. The raw milk was then cooled to 43°C. -Then a starter culture (YOFLEX PREMIUM 1.0 available from Chr. Hansen) was added in an amount of 0.02% by weight. - Protein modifying enzymes were then added at the indicated levels and the raw milk was incubated (fermented) at 43°C until a pH of 4.6 was reached. The resulting yogurt was stirred. The starched yogurt was stored at 5°C for 10 days and then evaluated for viscosity, torque, and sensory properties.

[0088] result:

[0089] [Table 2]

[0090] The results of enzymatic protein modification during fermentation are shown in Table 2 and Figures 3A-3B. With the notable exception of Sample #9 (prepared by adding glutaminase simultaneously with the starter culture during fermentation), all stirred yogurt samples were found to have higher viscosity and torque compared to the control (Sample #3). Sample #9 also showed a significant decrease in viscosity and torque compared to the control (Sample #3) and stabilized samples #4-6. Furthermore, the addition of a combination of glutaminase and transglutaminase enzymes (ACTIVA SYG, Sample #7) only resulted in a slight improvement over the control, with significantly lower viscosity / torque compared to the stirred yogurt modified with transglutaminase enzyme alone (Sample #8). These results indicate that the use of glutaminase during fermentation adversely affects the rheological properties of stirred yogurt.

[0091] Regarding the sensory analysis (Figures 4A-4G), Sample #3 (control) (Figure 4A) showed the most syneresis, followed by Sample #7 (prepared with ACTIVA SYG) (Figure 4E). The thinnest textured starched yogurts were Sample #3 (control) (Figure 4A) and Sample #9 (prepared with PG) (Figure 4G), which is consistent with the viscosity findings, but Sample #9 was found to have a smoother texture than the control.

[0092] Example 3 (invention) procedure: The raw milk was adjusted to a fat content of 1.5% by weight and a protein content of 4.2% by weight. Stabilizers added at specified levels. The raw milk was heated at 50°C for 30 minutes. -Protein-modifying enzymes and yeast extract were added at the levels specified. The temperature was maintained at 50°C for the specified treatment time. For samples without protein-modifying enzymes, this treatment time was omitted. The samples were then heat sterilized at 85°C for 20 minutes with stirring and cooled to 43°C. - Then a starter culture (YOFLEX PREMIUM 1.0 available from Chr. Hansen) was added in an amount of 0.02% by weight, followed by incubation (fermentation) at 43°C until a pH of 4.6 was reached. The resulting yogurt was stirred. The starched yogurt was stored at 5°C and evaluated for viscosity, torque, and sensory properties after 1 and 25 days of storage.

[0093] result: The results of enzymatic protein modification performed before fermentation are shown in Table 3 and Figures 5A-5C and 6A-6C. The addition of 200 ppm transglutaminase enzyme (ACTIVA YG) at a 15-minute treatment time (sample #12) resulted in the lowest viscosity closest to the control (sample #10), while increasing treatment time resulted in improved viscosity up to approximately 45 minutes of treatment time, after which only a slight improvement in viscosity was observed on day 1 (samples #13-15).

[0094] Surprisingly, the best results were obtained by adding protein glutaminase (sample #16) or protein glutaminase used in combination with transglutaminase (sample #17) before fermentation. Notably, the pre-fermentation treatment using protein glutaminase as the sole protein-modifying enzyme (sample #16) yielded the highest day-1 viscosity of all the samples tested, with an overall viscosity increase of 26–60% compared to the control over the 25-day refrigeration period. These results are in stark contrast to starved yogurts produced by adding protein glutaminase during fermentation (e.g., samples #7 and #9), where the addition of protein glutaminase was found to have a negative impact on the rheological properties of the starved yogurt.

[0095] [Table 3]

[0096] [Table 4]

[0097] Regarding the sensory analysis (Table 4), Sample #11 (prepared with stabilizer only) had the least favorable texture and flavor, with the modified food starch weakening the flavor. Sample #11 also had a lackluster appearance and was slightly grainy. Samples #16 and #17 had the firmest, full-bodied texture and the most favorable overall sensory attributes.

[0098] Where a numerical limit or range is stated herein, the endpoints are intended to be inclusive, and all values ​​and subranges within the numerical limit or range are intended to be specifically included as if expressly written out.

[0099] As used herein, words such as "a" and "an" mean "one or more."

[0100] Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.

[0101] All patents and other publications cited above are incorporated herein by reference as if fully set forth.

[0102] This application is based on U.S. Patent Application No. 16 / 997,279, filed August 19, 2020, the contents of which are incorporated herein by reference in their entirety.

Claims

1. The raw milk is treated with protein glutaminase to prepare modified milk; fermenting the modified milk in the presence of a starter culture to prepare yogurt; and breaking the gel structure of the yogurt to prepare a stirred yogurt.

2. 2. The method of claim 1, wherein the raw milk has a fat content of up to 10% by weight based on the total weight of the raw milk.

3. 2. The method according to claim 1, wherein the raw milk has a protein content of 3 to 10% by weight based on the total weight of the raw milk.

4. 2. The method according to claim 1, wherein the raw milk is treated with 10 to 500 ppm of protein glutaminase based on the total weight of the raw milk.

5. 2. The method according to claim 1, wherein the raw milk is treated with protein glutaminase in an amount of 0.01 to 5 U per gram of protein in the raw milk.

6. 2. The method according to claim 1, wherein the raw milk is treated with protein glutaminase at a treatment temperature of up to 50°C.

7. 2. The method according to claim 1, wherein the raw milk is treated with protein glutaminase for 15 minutes to 24 hours.

8. 2. The method of claim 1, further comprising heat sterilizing the modified milk after treating the raw milk with protein glutaminase and before fermenting the modified milk.

9. The method according to claim 8, wherein the heat sterilization is carried out at a temperature of 70 to 95°C for 1 to 30 minutes.

10. 10. The method of claim 1, further comprising adding a stabilizer to the raw milk before treating the raw milk with protein glutaminase.

11. 2. The method of claim 1, further comprising treating the raw milk with a reducing agent before or simultaneously with treating the raw milk with protein glutaminase.

12. 12. The method according to claim 11, wherein the raw milk is treated simultaneously with a reducing agent and protein glutaminase.

13. The method according to claim 11, wherein the raw milk is treated with 10 to 500 ppm of the reducing agent based on the total weight of the raw milk.

14. 12. The method of claim 11, wherein the reducing agent is yeast extract.

15. 2. The method of claim 1, wherein the modified milk is fermented with 0.0001 to 1% by weight of a starter culture relative to the total weight of the modified milk.

16. The method of claim 1 , wherein no thickener is used.

17. The raw milk is treated with protein glutaminase and transglutaminase at the same time.

10. The method of claim 1, further comprising treating with an enzyme.

18. 2. The method of claim 1, wherein protein glutaminase is the only enzyme used that acts on glutamine residues of milk proteins contained in the raw milk.

19. 10. The method of claim 1, wherein the stirred yogurt has a viscosity of 134,000 cP to 250,000 cP as measured on a Brookfield viscometer at 4.0 rpm and 40°F (4.4°C).

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