EA045075B1 - FERMENTED PRODUCT BASED ON MAMMAL MILK OR VEGETABLE MILK BASE AND ITS PREPARATION USING PHOSPHOLIPASE - Google Patents

Description

Field of invention

The present invention essentially relates to methods for producing fermented milk products.

Prior Art

Dairy products, such as fermented milk products, are known in the art. For dairy products, texture is a very important quality parameter. Many consumers desire a product that has a soft consistency and gel strength that provides a pleasant mouthfeel.

A standard technique for improving the texture of fermented milk products is the addition of protein, typically skim milk powder or whey protein. Thickeners or other texture agents such as modified starch, cornstarch, pectin, gelatin, or agar are often used. However, added proteins or structure-providing agents can be expensive. Thus, it would be advantageous to develop a process for producing a fermented milk product in which the addition of such agents can be reduced or eliminated. This is also desirable due to the fact that there is a growing demand in the market for fermented milk products that meet the clean label definition, i.e. no added stabilizing or texturing agents.

Another common technique for imparting good texture to fermented milk products is homogenization. It is carried out before fermentation to crush the milk fat into smaller sizes. The smaller fat globules produced by this process can be easily suspended in solution and are therefore not present as a separate layer in the milk. Homogenization can be accomplished, for example, by forcing milk through a fine filter or restriction valve at high pressures, resulting in an emulsion with a reduced particle size. The homogenization pressure typically used in the dairy industry is around 150-250 bar depending on the product. However, homogenization is expensive because it requires significant energy expenditure. Thus, it is advantageous to develop a process that reduces the need for homogenization, thereby reducing the operating costs of the manufacturer.

Attempts have been made to solve this problem. Many of these relate to the development of new strains of lactic acid bacteria, which can result in better texture. For example, WO 2007/095958A1 (Chr. Hansen) discloses the use of certain starter cultures that produce extracellular polysaccharides to improve the texture of fermented milk products. There is a constant need in the art to improve the organoleptic properties of fermented milk products, in particular the taste impression and coating formation in the oral cavity.

Summary of the invention

The present invention is based in part on the unexpected discovery that phospholipases have a positive effect on the organoleptic quality of fermented milk products. By using phospholipase(s) in the preparation method, the use of proteins, texture agents or stabilizing agents in fermented milk products can be reduced or even eliminated. It is also possible to reduce the cost of homogenization required during the production process.

The present invention provides a new use of phospholipase in a method for preparing a fermented milk product. A fermented milk product, such as yogurt, can be prepared from a homogenized and heat-treated milk base with standardized fat and protein content. The milk is then inoculated with a starter culture and fermented.

It was observed that phospholipase can actively participate in the fermentation process under the influence of lactic acid bacteria, resulting in an increase in the viscosity of the finished fermentation product. The present invention therefore provides a method for producing a fermented milk product in which a phospholipase-containing milk base is subjected to fermentation.

This application provides a method comprising adding a starter culture to a milk base, fermenting the milk base for a period of time to achieve a target pH at which at least one phospholipase is added to the milk base. Phospholipase can be added before, at the beginning or during fermentation. Preferably, the phospholipase is added before or at the beginning of fermentation. Once the target pH is reached, a fermented milk product can be obtained.

This application proposes a method for producing a fermented milk product, including the stages:

a) adding to the milk base a starter culture containing at least one strain of lactic acid bacteria,

b) fermenting the milk base for such a period of time as to achieve the target pH, and

c) adding at least one phospholipase to the milk base before, at the beginning or during the fermentation period.

Phospholipase that can be used for the present application includes phospholipase A such as phospholipase A1 (EC 3.1.1.32) and phospholipase A2 (EC 3.1.1.4), and phospholipase B (EC 3.1.1.5), phospholipase C (EC 3.1.4.3 ) and phospholipase D (EC 3.1.4.4).

This application includes a fermented milk product obtained by the methods disclosed in this document. In another aspect, the present invention provides a fermented milk product containing phospholipase.

In a further aspect, the present invention provides a kit comprising a starter culture and at least one phospholipase useful for making fermented milk products.

Other features and advantages of the invention will become apparent upon reading the following description in conjunction with the accompanying drawings.

Brief description of graphic materials

In fig. 1 depicts a hysteresis curve for fermented milk products prepared in Example 1, showing the dependence of shear stress on shear rates.

In fig. Figure 2 shows the influence of factors on the response variable (viscosity at 60 1/s) in fermented milk products prepared in example 2.

In fig. Figure 3 shows the influence of factors on the response variable (viscosity at 300 1/s) in fermented milk products prepared in example 2.

In fig. 4 shows a hysteresis curve for fermented milk products prepared in example 3, showing the dependence of shear stress on shear rates.

Detailed Description of the Invention

This invention relates to fermented milk products obtained by fermenting milk with selected microorganisms. Different types of fermented milk can be characterized by the specific starter cultures used in fermentation. For example, symbiotic cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is used as a starter culture for yogurt, while Lactobacillus acidophilus is used to produce acidophilus milk. To produce quark or low-fat soft cheese, other mesophilic lactic acid bacteria are used.

To prepare the products according to the present application, first of all, a milk base is provided as a starting material. Dairy base is used broadly herein to refer to a milk-based composition or milk components that can be used as a medium for the growth and fermentation of a starter culture. Milk in general refers to the secretion of milk obtained by milking any mammal such as cows, sheep, goats, buffaloes or camels. The milk base can be obtained from any raw and/or processed milk material or from reconstituted milk powder. The milk base may also be a vegetable base, i.e. may be made from plant material such as soy milk.

Useful milk bases include, but are not limited to, solutions/suspensions of any protein-containing dairy or milk-like products, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, evaporated milk, powdered milk.

In the methods and products of the present invention, the milk base is preferably prepared from cow's milk or milk components.

Depending on consumer needs, the milk base may also have a reduced lactose content. Lactose-reduced milk can be produced by any method known in the art, including hydrolysis of lactose to glucose and galactose by the enzyme lactase, or methods of nanofiltration, electrodialysis, ion exchange chromatography and centrifugation.

Pasteurization

Before fermentation, the milk base is preferably pasteurized according to methods known in the art. As used herein, pasteurization means treating a milk base to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a given temperature for a given period of time. The set temperature is usually achieved by heating. Those of ordinary skill in the art are able to select temperatures and durations to kill or inactivate specific microorganisms. This may be followed by a rapid cooling step.

Homogenization

Homogenization refers to the process of homogenizing a mixture to produce a homogeneous liquid composition of immiscible components. During the homogenization process, milk fat is broken down into smaller particles so that it can no longer be separated from the milk. This can be done by forcing milk through small holes under high pressure. In the dairy industry, homogenization is usually used after or simultaneously with pasteurization. As will be apparent from the following description, the present invention provides methods in which the need for homogenization is reduced. Thus, in some embodiments of the invention, the milk base is homogenized, albeit at a lower pressure than typically required, and in other embodiments of the invention, the milk base is not homogenized.

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Fermentation

A starter culture is added to ferment the milk base. When used in the present context, the term starter or starter culture refers to a culture of one or more food grade microorganisms, in particular lactic acid bacteria, responsible for acidifying the milk base. Starter cultures can be fresh, frozen or freeze-dried. Determining the starter culture to be used and its quantities is within the competence of ordinary practitioners.

Fermentation in general means the conversion of carbohydrates into alcohols or acids by the action of a microorganism. In the present invention, fermentation in the methods of the invention refers to the conversion of lactose into lactic acid.

Starter culture

In accordance with the present invention, the starter culture contains at least one strain of lactic acid bacteria. Lactic acid bacteria are widely used to produce fermented foods and their use is well known in the art. As used herein, the term lactic acid bacteria or LAB is used to refer to food-grade bacteria that produce lactic acid as a product of the fermentation of carbohydrates as the main metabolic end product. These bacteria are related in their general metabolic and physiological characteristics and are typically Gram-positive, acid-stable, non-sporulating, non-respiring rod-shaped bacilli or cocci with low DNA GC content. During the fermentation stage, consumption of lactose by these bacteria causes the formation of lactic acid, a decrease in pH and leads to the formation of protein coagulum.

A starter culture useful for the present invention may have a composition of strains of any conventional lactic acid bacteria starter culture, including a single strain culture or mixed cultures depending on the particular type of fermented milk product. In addition to the starter culture, other beneficial bacteria can also be included in the fermentation, including the probiotic bacteria Bifidobacterium spp.

In one embodiment of the invention, the starter culture contains thermophilic bacteria to produce a thermophilic fermented milk product. The term thermophilic fermented milk product refers to fermented milk products prepared by thermophilic fermentation of a thermophilic starter culture, examples of which include fermented milk products such as thermostatic yogurt, tank yogurt, and drinking yogurt such as Yakult. On an industrial scale, the most beneficial thermophilic bacteria (thermophiles) include Streptococcus spp. and Lactobacillus spp. The term thermophilic fermentation as used herein refers to fermentation at a temperature higher than about 35°C, for example from about 35 to about 45°C. Thermophiles in general are microorganisms that grow and develop best at temperatures above 35°C.

In another embodiment of the invention, the starter culture contains mesophilic bacteria to produce a mesophilic fermented milk product.

The term mesophilic fermented milk product refers to fermented milk products prepared by mesophilic fermentation of a mesophilic starter culture, examples of which include fermented milk products such as buttermilk, sour milk, fermented milk, sour cream, sour heavy cream, kefir and young cheese such as quark, cottage cheese and cream cheese. Beneficial mesophilic bacteria (mesophiles) include Lactococcus spp. and Leuconostoc spp. The term mesophilic fermentation as used herein refers to fermentation at a temperature of from about 22 to about 37°C. The term mesophiles generally refers to microorganisms that grow and develop best at moderate temperatures (15-35°C).

Lactic acid bacteria useful for making fermented milk product include, but are not limited to, bacteria belonging to the genus Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus rhamnosus, Bifidobacterium breve and Leuconostoc spp . The choice of specific strains in the starter culture will depend on the specific type of dairy product being produced.

In a preferred embodiment of the invention, the starter culture contains at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus.

In a preferred embodiment of the invention, the lactic acid bacteria are selected from the group consisting of bacteria of the genera Lactobacillus, Streptococcus, Lactococcus and Leuconostoc.

In a specific embodiment of the invention, the fermented milk product is a product obtained using one or more strains of lactic acid bacteria selected from the group consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

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Fermentation method

After adding the starter culture and bringing the milk culture to suitable conditions, the fermentation process is started and continued for a period of time. One of ordinary skill in the art will know how to select appropriate process conditions, such as temperature, oxygen supply, carbohydrate addition, amount and characteristics of microorganism(s), and process time. This process may take three, four, five, six hours or longer.

These conditions include setting the temperature appropriate for the specific starter culture strains. For example, if the starter culture contains mesophilic lactic acid bacteria, the temperature may be set at about 30°C, and if the culture contains thermophilic strains of lactic acid bacteria, the temperature is maintained in the range of about 35 to 50°C, such as 40 to 45°C. The fermentation temperature setting also depends on the enzyme(s) added to the fermentation, as can be easily determined by one of ordinary skill in the art. In a specific embodiment of the invention, the fermentation temperature is from 35 to 45°C, preferably from 37 to 43°C and more preferably from 40 to 43°C.

Fermentation can be stopped by any means known in the art. Typically, depending on various process parameters, fermentation can be stopped, rendering the milk base unsuitable for the growth of the starter culture strain(s). For example, stopping can be accomplished by rapidly cooling the fermented milk product when the target pH is reached. It is known that during fermentation, acidification occurs, which leads to the formation of a three-dimensional network consisting of clusters and chains of caseins. The term target pH means the pH at which the fermentation step ends. The target pH depends on the fermented milk product to be produced and can be easily determined by one of ordinary skill in the art.

In specific embodiments of the invention, fermentation is carried out until a pH of 5.0 is reached, including a pH of 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8 or 3.7. Preferably, fermentation is carried out until a target pH of 4.0 to 5.0, and more preferably 4.0 to 4.6, is achieved. In a preferred embodiment of the invention, fermentation is carried out until the target pH is below 4.6.

Phospholipase

Milk contains phospholipids. Phospholipids are associated with milk fat due to their nonpolar lipophilic properties. Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in the outer (sn-1) and middle (sn-2) positions and esterified with phosphoric acid in the third position. Phosphoric acid, in turn, can be esterified to an amino alcohol. Phospholipids can be hydrolyzed by phospholipase to lysophospholipid, which in turn can be hydrolyzed by lysophospholipase.

Phospholipases are major enzymes that play a critical role in living organisms in general and in the metabolism and biosynthesis of phospholipids in particular. These enzymes are involved in the hydrolysis of phospholipids, and several types of phospholipase activity can be distinguished. Phospholipase A can be further classified as phospholipase A1 (EC 3.1.1.32) or A2 (EC 3.1.1.4.), which hydrolyze one fatty acyl group (at the sn-1 and sn-2 positions, respectively) to form a lysophospholipid. Phospholipase B (EC 3.1.1.5) hydrolyzes the remaining fatty acyl group in the lysophospholipid. Other phospholipases are phospholipase C (EC 3.1.4.3) and phospholipase D (EC 3.1.4.4). The phospholipase for use in the method of the invention can be any phospholipase or any combination of phospholipases.

In a preferred embodiment of the invention, the phospholipase(s) useful for the present application is phospholipase A. More preferably, the phospholipase is phospholipase A1 (EC 3.1.1.32) and/or phospholipase A2 (EC 3.1.1.4). In other embodiments of the invention, the phospholipase is phospholipase B (EC 3.1.1.5) or C (EC 3.1.4.3).

According to the standard classification of enzymes, phospholipase A1 is defined as EC 3.1.1.32.

Official name: Phospholipase A1

Reaction catalyzed: phosphatidylcholine + water <=> 2-acylglycerophosphatidylcholine + fatty acid anion

Note: has significantly broader specificity than EU 3.1.1.4.

According to the standard classification of enzymes, phospholipase A2 is defined as EC 3.1.1.4

Official name: Phospholipase A2

Alternative names: phosphatidylcholine-2-acylhydrolase, lecithinase a; phosphatidase or phosphatidolipase.

Catalyzed reaction: phosphatidylcholine + water <=> i-acylglycerophosphocholine + fatty anion

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Note: Also acts on phosphatidylethanolamine, choline plasmalogen and phosphatides by removing the fatty acid attached at the 2-position.

The phospholipase can be of any origin, for example animal origin (such as, for example, from mammals), for example, from the pancreas (for example, the pancreas of cattle or pigs), or from snake or bee venom. Alternatively, the phospholipase may be derived from microorganisms, for example filamentous fungi, yeasts or bacteria, for example from the genus or species of Aspergillus, for example A. niger; Dictyostelium, for example D. discoideum; mucor, for example M. javanicus, M. mucedo, M. subtilissimus; Neurospora, eg N. crassa; Rhizomucor, for example R. pusillus; Rhizopus, for example R. arrhizus, R. japonicus, R. stolonifer; Sclerotinia, for example S. libertiana; Trichophyton, for example T. rubrum; Whetzelinia, for example W. sclerotiorum; Bacillus, for example B. megaterium, B. subtilis; Citrobacter, for example C. freundii; Enterobacter, for example E. aerogenes, E. cloacae; Edwardsiella, E. tarda; Erwinia, for example E. herbicola; Escherichia, for example E. coli; Klebsiella, for example K. pneumoniae; Proteus, for example P. vulgaris; Providencia, eg P. stuartii; Salmonella, for example S. typhimurium; Serratia, for example S. liquefasciens, S. marcescens; Shigella, for example S. flexneri; Streptomyces, for example S. violeceoruber; Yersinia, for example Y. enterocolitica. The phospholipase may be of fungal origin, for example the class Pyrenomycetes, such as the genus Fusarium, for example a strain of F. culmorum, F. heterosporum, F. solani, F. venenatum or a strain of F. oxysporum. The phospholipase may also be derived from a strain of filamentous fungi within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae. A preferred phospholipase is obtained from a strain of the genus Fusarium, in particular F. venenatum or F. oxysporum, for example from strain DSM 2672, as disclosed in WO 98/26057, in particular as disclosed in claim 36 of WO 98/26057. In further embodiments of the invention, the phospholipase is the phospholipase disclosed in WO 00/32758 (Novozymes A/S, Denmark). Another preferred phospholipase A is phospholipase A2 from streptomycetes, such as PLA2 from S. violaceoruber.

Phospholipases are commercially available. Phospholipase A1 from Thermomyces lanuginosus/Fusarium oxysporum, expressed in Aspergillus oryzae, is commercially available under the trade name Lecitase®Ultra (Novozymes A/S, Denmark). Another preferred phospholipase A1 is commercially available under the trade name YieldMAX® or YieldMAX®PL (Chr. Hansen A/S, Denmark), which is obtained by deep fermentation of a strain of Aspergillus oryzae.

A particularly preferred phospholipase A is phospholipase A1 from Fusarium spp.

Phospholipase A2 can be found under different trade names. For example, phospholipase A2 of animal origin (from porcine pancreas), developed for degumming vegetable oils, is commercially available under the name Lecitase®10L (Novozymes A/S, Denmark). Maxapal® A2 (DSM Food Specialties, The Netherlands) is produced by microbial fermentation of a specific strain of Aspergillus niger, designed to improve the emulsifying properties of chicken eggs and egg yolks. CakeZyme® and BakeZyme® are also phospholipase A2 from DSM Food Specialties (Netherlands). Additional examples include microbial phospholipase A2 Rohalase®MPL (AB Enzymes, Germany) and LysoMax® (Genencor, USA).

In a preferred embodiment of the invention, the phospholipase is YieldMAX® or YieldMAX®PL (Chr. Hansen A/S, Denmark). Other phospholipases suitable for use in the food industry are well known to those skilled in the art and can be found, for example, in the work of Casado, Victor, et al. Phospholipases in food industry: a review. Lipases and Phospholipases: Methods and Protocols (2012): 495-523.

In some embodiments of the invention, the phospholipase(s) used in the method of the invention are or can be obtained from any source specified herein. In this context, the term obtained means that the phospholipase can be isolated from an organism where it is present in its native form, i.e., the amino acid sequence of this enzyme is identical to the amino acid sequence of the native enzyme. As used herein, the term "derived" also means that the enzymes can be produced recombinantly in a host organism, wherein the recombinantly produced enzyme has an identical or modified amino acid sequence to the native enzyme, for example having a deletion, insertion and/or substitution of one or more amino acids, i.e. That is, the enzyme obtained by the recombinant method is a mutant and/or fragment of the native amino acid sequence. Natural variants are included in the meaning of native enzyme. In addition, the term obtained includes enzymes obtained synthetically, for example by peptide synthesis. The term derived also covers enzymes that have been modified, for example by glycosylation, phosphorylation, etc., either in vivo or in vitro. As used herein, the term produced means that the enzyme has an amino acid sequence identical to that of the native enzyme. The term covers an enzyme that has been isolated from an organism where it is present in its native form, or expressed recombinantly in the same or a different type of organism, or enzymes produced synthetically, such as by peptide synthesis. In relation to a recombinantly produced enzyme, the terms produced and obtained refer to the identity of the enzyme, but do not refer to the identity of the host organism in which it is produced recombinantly.

Accordingly, the phospholipase can be obtained from the microorganism by using any suitable method. For example, a phospholipase enzyme preparation can be prepared by fermenting a suitable microorganism and then isolating the phospholipase from the resulting liquid fermentation medium or microorganism by methods known in the art. Phospholipase can also be produced using recombinant DNA methods. Such a method typically involves culturing a host cell transformed with a recombinant DNA vector containing a DNA sequence encoding the phospholipase of interest and a DNA sequence operably linked to an appropriate expression signal such that the cell is capable of expressing the phospholipase in a culture medium under conditions permissive for expression of the enzyme and recovering the enzyme from the culture. The DNA sequence may also be inserted into the genome of a host cell. The DNA sequence may be of genomic, cDNA, or synthetic origin, or any combination thereof, and may be isolated or synthesized according to methods known in the art. Such phospholipase may be purified. As used herein, the term purified includes the phospholipase enzyme free of components of the organism from which it is derived. As used herein, the term purified also includes a phospholipase enzyme free of components of the organism from which it is derived; it is also referred to as essentially pure phospholipase, and may be particularly suitable for phospholipases that occur naturally and have not been genetically modified, for example, by deletion, substitution or insertion of one or more amino acid residues. Purification can be accomplished, for example, by filtration, precipitation or chromatography.

The phospholipases used herein may be in any suitable form, such as a dry powder or granulate, a dust-free granule, a liquid, a stabilized liquid, or a protected enzyme. The granules can be prepared, for example, as disclosed in US Pat. No. 4,106,991 and US Pat. No. 4,661,452, and can optionally be coated by methods known in the art. Liquid enzyme preparations can be stabilized according to well-established methods, for example by adding stabilizers such as sugar, sugar alcohol or other polyol, lactic acid or other organic acid. Protected enzymes can be obtained in accordance with the method disclosed in European Patent No. 238216.

Phospholipase activity can be defined as the rate of consumption of sodium hydroxide during neutralization of a fatty acid. Such activity can be expressed in lecitase units (LEU) relative to a standard lecitase (phospholipase) sample. These techniques are known in the art. For example, phospholipase A1 activity can be measured relative to a phospholipase standard using lecithin as a substrate. Phospholipase A1 catalyzes the hydrolysis of lecithin to lysolecithin and free fatty acid. The released fatty acid is titrated with 0.1 N. sodium hydroxide solution under standard conditions (pH 8.0; 40° ± 0.5). 1 LEU is defined as the amount of enzyme that, under standard conditions (pH 8.0; 40° ± 0.5), results in the same rate of sodium hydroxide consumption (in microeq/min) as a standard lecitase sample diluted to a nominal activity of 1 LEU/g. This technique can be performed using either an automated system or standard laboratory equipment for performing titration experiments.

Addition of phospholipase

Phospholipase can be added to the milk base before, at the beginning of, or during the fermentation period. The expression at the beginning of fermentation means a short time before, simultaneously with, or a short time after the addition of the starter culture to the milk base. In this case, the term short time means less than 30 minutes. The expression during the fermentation period means any time during the fermentation period after the start of fermentation and before the end of fermentation.

In one embodiment of the present invention, phospholipase is added to the milk base prior to the fermentation step. In another embodiment of the present invention, phospholipase is added to the milk base at the beginning of the fermentation step. In another embodiment of the present invention, phospholipase is added to the milk base during the fermentation period. The fermentation step can be stopped by a cooling treatment.

The amount of phospholipase to add to the milk base depends on a number of parameters, including the phospholipase activity and the composition of the milk base, e.g. fat content, etc. This amount can be determined by standard experiments.

Suitable conditions for performing phospholipase treatment can be determined by one skilled in the art using techniques well known in the art to optimize enzymatic reactions. One skilled in the art will know how to adjust parameters such as pH, temperature and amount of lipase to achieve the desired results based on the desired properties of the fermented milk product. The amount of phospholipase to be used in the method of the invention may depend on the activity of the particular phospholipase on the phospholipids present under the particular processing conditions. For example, when using phospholipase A as shown in the example, the amount of phospholipase added may be from 0.1 to 50 LEU per gram of fat, for example from 0.5 to 25 or 1 to 10 LEU per gram of fat.

Dairy products

The term fermented milk product is a term substantially defined in accordance with current official regulations and standards well known in the art. The expression fermented milk product means a food or feed product where the preparation of this food or feed product involves fermentation of the milk base by lactic acid bacteria. As used herein, fermented milk product includes, but is not limited to, products such as thermophilic fermented milk products (e.g., yogurt) and mesophilic fermented milk products (e.g., sour heavy cream and buttermilk, as well as soured whey, quark, and low fat soft cheese). Unlike cheese products, fermented milk products generally have a lower target pH at the end of fermentation.

More specifically, the method disclosed herein can be used to produce thermophilic fermented milk products such as yogurt and, in particular, thermostatic yogurt, and mesophilic fermented milk products such as quark.

In one embodiment of the invention, the fermented milk product is a yoghurt product. The term yogurt has its usual meaning and is essentially defined in accordance with current official regulations and standards well known in the art. The yogurt product may be selected from the group consisting of a tank product, a thermostat product, or a drink product.

A reservoir product is a fermented milk product that can withstand mechanical processing after fermentation. Mechanical processing is typically, but not exclusively, achieved by mixing, pumping, filtering or homogenizing the gel or by mixing with other ingredients. A thermostatic product is a milk-based product that is inoculated with a starter culture, packaged, and fermented in the package. The term drinkable product includes beverages such as drinking yoghurt, diluted drinking yoghurt and the like, which may be a dairy product produced by fermentation with a combination of thermophiles from various species of lactobacilli and streptococci.

In a preferred embodiment of the invention, the fermented milk product is selected from the group consisting of quark, cream cheese, low fat soft cheese, Greek yogurt, soy yogurt, skyr, labneh, buttermilk, sour milk, fermented milk, kefir, lassi, ayran, pressed cottage cheese, tan , sour cream, yakult and dahi. More preferably, the fermented milk product is sour milk, fermented milk, kefir, lassi, ayran, tan, yakult or dahi.

Protein content

Fermented milk products produced by the method disclosed herein may contain protein in a concentration of from 0.5% by weight to 10% by weight. The fermented milk product may also be a low protein product in which the protein concentration ranges from 1% by weight to 4.0% by weight. Alternatively, the fermented milk product may be a high protein product in which the protein concentration is greater than 3.5% by weight.

In a specific embodiment of the invention, the milk base is characterized by a protein content (w/w) of less than 0.5%, less than 0.7%, less than 0.9%, less than 1.0%, less than 1.3%, less than 1.5 %, less than 2.0%, less than 2.5%, less than 3.0%, less than 3.5%, less than 4.0%, less than 4.5%, less than 5.0%, less than 5.5%, less than 6.0%, less than 6.5%, less than 7.0%, less than 7.5%, less than 8.0%, less than 8.5%, less than 9.0%, less than 9.5%, less than 10 ,0%, less than 10.5%, less than 11.0%, less than 11.5%, less than 12.0%, less than 12.5%, less than 13.0%, less than 13.5%, less than 14.0 %, less than 14.5% or less than 15.0%.

Fat content

The authors of the present application have discovered the positive effects of phospholipases on the texture of fermented milk products. They observed that their effects were greater the higher the fat content of the milk base, making them particularly suitable for use in fermentation products containing a high concentration of fat.

In specific embodiments of the invention, the milk base is characterized by a fat content (w/w) of at least 0.5%, for example at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.3%, at least 1.5%, at least 2.0%, at least 2.5%, at least 3.0%, at least 3.5%, at least 4.0%, at least 4.5%, at least 5.0%, at least 5, 5%, at least 6.0%, at least 6.5%, at least 7.0%, at least 7.5%, at least 8.0%, at least 8.5% , at least 9.0%, at least 9.5%, at least 10.0%, at least 10.5%, at least 11.0%, at least 11.5%, according to

- 7 045075 at least 12.0%, at least 12.5%, at least 13.0%, at least 13.5%, at least 14.0%, at least 14.5% or at least 15.0%. In some embodiments of the invention, the fat content is at least 16.0%, at least 17.0%, at least 18.0%, at least 19.0%, at least 20.0%, at least 21.0% at least

22.0%, at least 23.0%, at least 24.0%, at least 25.0%, at least

26.0%, at least 27.0%, at least 28.0%, at least 29.0%, at least

30.0%, at least 35.0%, at least 40.0%, at least 45.0%, at least

50.0% or more. One skilled in the art will be aware of adjusting the fat content of the milk base, for example, by adding cream to the milk base.

In preferred embodiments of the invention, the milk base has a fat content of from 0.5 to 10.0% (w/w), preferably from 1.0 to 9.0%, preferably from 3.0 to 7.0%, preferably from 4.0 to 6.0% and more preferably 4.5 to 5.5%. It was found that the higher the fat content, the greater the effect of phospholipase(s) on texture.

Homogenization

By following the teachings of the present invention, it is possible to provide fermented milk products where the level of homogenization typically required for such products can be reduced or even eliminated, thereby reducing the operating costs of the manufacturer. Thus, in one embodiment of the invention, the milk base used to prepare such a product is not homogenized. However, in some preferred embodiment of the invention, depending on the types of fermented milk products, the milk base can still be homogenized. The homogenization level is preferably below 250 bar, including less than 240 bar, less than 220 bar, less than 200 bar, less than 190 bar, less than 180 bar, less than 170 bar, less than 160 bar, less than 150 bar, less than 140 bar, less than 130 bar , less than 120 bar, less than 110 bar, less than 100 bar, less than 90 bar, less than 80 bar, less than 70 bar, less than 60 bar, less than 50 bar. In the context of the present application, if homogenization is carried out in several stages, the level of homogenization refers to the total pressure applied in all stages. For example, if homogenization is carried out in two stages, the first at 120 bar and the second at 60 bar, the level of homogenization of the milk base is 180 bar.

Chymosin

The present inventors have also discovered that additional addition of chymosin (EC 3.4.23.4) may be beneficial for improving the texture of the fermented milk product. Chymosin is an aspartic protease belonging to the broad class of peptidases and is often used in cheese production as a milk-clotting enzyme.

Bovine chymosin, particularly veal chymosin, is commercially available both as gastric enzyme extracts (rennet; contains natively produced chymosin) and as recombinantly produced chymosin (which is typically expressed in bacterial, yeast or fungal cells - owners) (see, for example, WO 95/29999). Non-bovine chymosins, such as Camelus dromedarius, or variants thereof are disclosed in WO 2002036752, WO 2013174840 and WO 2015/128417. Preferred chymosins include commercially available chymosins such as CHY-MAX®, CHY-MAX® M and CHY-MAX® Plus (Chr. Hansen A/S, Denmark).

In a preferred embodiment of the invention, the method further includes the step of adding chymosin to the milk base before, at the beginning or during the fermentation period. The amount of chymosin to add to the milk base depends on a number of parameters including the activity of the chymosin, the composition of the milk base, eg protein content such as casein content, etc. Suitable conditions can be selected by one skilled in the art and in accordance with methods known in the art for optimizing enzymatic reactions. Assays for determining the activity of chymosin are known in the art. It can be measured according to a standardized method and expressed in International Milk Clotting Units (IMCU)/g.

In one preferred embodiment of the invention, a method for producing a fermented milk product includes adding a starter culture with at least one strain of lactic acid bacteria to a milk base, fermenting the milk base for a period of time to achieve a target pH, wherein phospholipase and chymosin are added to the milk base simultaneously or alternately at the beginning of the fermentation period.

In one preferred embodiment of the invention, a method for producing a thermophilic fermented milk product includes adding a starter culture with at least one strain of lactic acid bacteria to a milk base, fermenting the milk base for a period of time to achieve a target pH, wherein phospholipase and chymosin are added to the milk base separately prior to fermentation period.

In yet another preferred embodiment of the invention, a method for producing a yogurt product includes adding a starter culture with at least one strain of lactic acid bacteria to a milk base, fermenting the milk base for a period of time to achieve a target pH, wherein phospholipase and chymosin are added to the milk base simultaneously for a period of time. - 8 045075 fermentation period.

Storage

The fermented milk product is preferably stored for at least two days, for example at least 3 days, at least 4 days, more preferably at least 5 days, at least 6 days, at least 7 days, at least 8 days , at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days and at least 14 days. In fresh dairy production, products typically reach the consumer within about 3-5 days and usually no later than 7 days after production.

As used herein, the term storage or stored refers to the holding of products under suitable conditions before they are shipped to consumers for consumption. Such conditions are known in the industry and can be determined by the practitioner.

Kit

In another aspect, the present invention provides a kit containing a starter culture and one or more phospholipases. The starter culture contains one or more lactic acid bacteria useful for producing a fermented milk product as mentioned herein. The lactic acid bacteria contained in the kit may include, for example, Lactobacillus spp. and Lactococcus spp. They can be provided in the form of frozen or freeze-dried cultures for propagation of production starters or so-called Direct Vat Sets (DVS), intended for direct seeding of a fermentation vessel or tank for the production of a fermented milk product. The phospholipase included in the kit may be phospholipase A, phospholipase A1, phospholipase A2, phospholipase B or phospholipase C, or a combination thereof. The kit may further contain one or more chymosins.

Texture of fermented milk products

The present invention also provides a new use of phospholipase to improve the texture of fermented milk products. Texture can be characterized by rheological properties by methods well known in the art, including measuring the shear stress (viscosity) or gel strength of the product. The SI unit of shear stress and gel strength is Pascal (Pa). The inventors observed that both gel strength and viscosity could be improved.

In specific embodiments of the invention, the use increases the viscosity of a fermented milk product, such as yogurt, measured at 13°C at a shear rate of 60 1/s after 7 days of storage.

The term shear stress defines viscosity. Viscosity (unit is Pa in s) is defined as shear stress (Pa)/shear rate (1/s). The magnitude of shear stress can be recorded at various points. Experiments to determine sensory properties have shown that the best correlations between rheological parameters and sensory properties are found when measuring viscosity at a shear rate of 60 1/s (high correlation with first taste impression) and at a shear rate of 300 1/s (correlation of cohesiveness with difficulty in swallowing ).

The gel strength of fermented milk products can be measured by the so-called frequency swing, measuring the total modulus (G*) of the gel as a function of the oscillation frequency. The G* value at 1.52 Hz can be used as the gel strength of the product. The viscosity of a product can be measured by measuring the so-called rate constant, which determines the shear stress of the product as a function of the shear rate.

When producing a fermented milk product in accordance with the provisions of the present invention, it is possible to achieve an increase in its viscosity or gel strength, measured at a shear rate of 60 1/s or 300 1/s, compared to a control product, which is a fermented milk product obtained in the same way, but without the use of phospholipase.

In one embodiment, the use of phospholipase creates a fermented milk product viscosity, measured at a shear rate of 60 1/s (Pa), that is at least about 5% higher, at least about 10% higher, at least about 15% higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 50% higher than the viscosity created in the product obtained without the use of phospholipase.

In another embodiment, the use of phospholipase creates a fermented milk product viscosity, measured at a shear rate of 300 1/s (Pa), that is at least about 5% higher, at least about 10% higher, at least about 15% higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least about 50% higher than the viscosity created in the product obtained without the use of phospholipase.

In another embodiment, the use of phospholipase creates a gel strength (G* at 1.52 Hz) of a fermented milk product that is at least about 5% higher, at least about 10% higher, at least about 15% higher, at least about 20% higher, at least about 25%

- 9 045075 higher, at least about 30% higher, than the gel strength created in the product obtained without the use of phospholipase.

In the context of the description of the invention (especially in the context of the claims), the use of singular terms and similar references should be understood to include both the singular and the plural, unless otherwise indicated herein and the context clearly conflicts with them. The terms including, having, including and containing are to be understood in their open sense (i.e., including without limitation) unless otherwise noted. In this document, the indication of ranges of quantities is intended solely as a means of abbreviated individual identification of each individual quantity within that range, unless otherwise specified herein, and each individual quantity is included in the description as if it were individually stated herein. All methods disclosed herein can be performed in any order unless otherwise indicated herein and unless the context clearly conflicts with them. Unless otherwise indicated, all exact values given herein are representative of the corresponding approximate values (e.g., all exact illustrative values given with respect to a particular factor or indicator may also be considered to indicate the corresponding approximate figure, modified as appropriate by the term about). The use of any and all examples or illustrative language (such as the following) provided herein is intended solely to better illuminate the invention and is not intended to limit the scope of the invention unless otherwise stated in the claims. Nothing in the specification should be construed as indicating any element not claimed as essential to the practice of the invention.

Examples

The invention described and claimed herein should not be limited in scope by the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be included within the scope of this invention. Indeed, based on the above description and the following examples, various modifications of the invention in addition to those shown and disclosed herein will become apparent to one skilled in the art. Such modifications are also intended to be included within the scope of the appended claims. In the event of a conflict, the definitions of this specification will control.

Example 1.

Yogurt products containing 1.5 or 5.0% fat were prepared. On the 7th and 30th days, the effect of phospholipase on the rheological properties was assessed.

Preparing the milk base

Skim milk powder and whole milk powder (Fonterra Co-operative Group Limited, New Zealand) were used to prepare the milk base for the examples below.

Table 1. Composition of milk base (w/w)

Sample Skimmed milk powder Whole milk powder Water 1 8.60% 5.30% 86.10% 2 8.60% 5.30% 86.10% 3 8.10% 9.00% 82.90% 4 8.10% 9.00% 82.90%

The ingredients were mixed with water using a Silverson mixer at 45-50°C. The hydration time was 20 minutes. The milk base was homogenized using a GEA Lab homogenizer in two stages (first stage 150 bar and second stage 50 bar; total: 200 bar) and pasteurized at 85°C for 30 min and cooled.

Starter culture:

The starter culture consisted of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.. bulgaricus.

Phospholipase:

Phospholipase A1 (YieldMAX®PL from Chr. Hansen A/S, Denmark; average activity 2600 LEU/ml, dosage 0.021650% at 5% fat) was added at the beginning of fermentation. Fat and protein content was determined using a MilkoScan analyzer.

Fermentation:

Fermentation was carried out at 43°C for 4 h and 50 min, and a final pH of 4.55 ± 0.05 was achieved. The fermented product was cooled after treatment by means of a plate heat exchanger to 23-25° C. and brought to homogeneity by means of a check valve at a pressure of 1 bar at the outlet of the cooler.

The samples were stored in a cold room at a temperature of 5-7°C.

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Table 2. Composition and description of fermentation

Sample Protein content Fat content Phospholipase Final pH Fermentation time 1 4.00% 1.50% - 4.51 4 hours 50 min 2 4.00% 1.50% 0.00345% 4.49 4 hours 50 min 3 4.00% 5.00% - 4.51 4 hours 50 min 4 4.00% 5.00% 0.01265% 4.49 4 hours 50 min

On the 7th and 30th days of storage, samples were tested for viscosity and gel strength.

Measuring shear stress and gel strength

The rheological properties of the sample were assessed using a rheometer (Anton Paar Modular Compact Rheometer MCR 302, Anton Paar® GmbH, Austria). During the measurements, the rheometer was set to a constant temperature of 13 °C. We used the Stirred_oscillation+Up_DownFlow program.

Shear stress at 60 1/s and at 300 1/s were chosen for analysis because they correlated with oral texture sensation (first taste impression) and cohesiveness (when swallowing a fermented milk product).

The gel strength (total modulus G* at 1.52) was assessed using a rheometer (Anton Paar Modular Compact Rheometer MCR 302, Anton Paar® GmbH, Austria).

The results are presented below and the hysteresis curve is shown in Fig. 1.

Table 3. Rheological assessment of the sample on day 7

Sample First taste impression (viscosity at 60 1/s) Cohesiveness Difficulty swallowing (viscosity at 300 1/s) 1 422.71 119.44 2 475.66 128.50 3 907.30 196.31 4 1092.9 212.45

Table 4. Rheological assessment of the sample on day 30

Sample First taste impression (viscosity at 60 1/s) Cohesiveness Difficulty swallowing (viscosity at 300 1/s) 1 412.25 110.38 2 495.85 128.72 3 966.16 202.41 4 1078.80 207.96

As shown in table. 3 and 4, the viscosity of samples treated with phospholipase (samples 2 and 4) at 60 1/s (first taste impression) and at 300 1/s (cohesiveness) was improved compared to control samples (samples 1 and 3). The effects were stronger in the higher fat sample (sample 4).

Example 2.

1. Experimental model

A fractional-replicate factorial design was chosen to test the 5 factors described below in 16 trials. The goal is to confirm the effect of phospholipases on texture in a larger design and to test for statistical significance, interaction with homogenization pressure level and interaction with the enzyme chymosin, as well as interaction with starter cultures.

Table 5. Experimental model: factors

Factors were coded orthogonally:

Factors -1 +1 1 - Homogenization pressure 150 bar 300 bar 2 – Starter culture 1 2 3 - Protein content 2.50% 4.50% 4 - Chymosin No There is 5 - Phospholipase No There is

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Table 6. Experimental model: design

1 2345 2 1345 3 1245 4 1235 12 345 13 245 14 235 23 145 24 135 34 125 123 45 124 35 134 25 234 15 1234 5

The fifth factor is examined by third-order interaction (5 = 1234). Thus, all first-order interactions can be calculated with reasonable accuracy, taking into account the high probability of very minor or negligible second-order interactions.

2. Methodology

Yogurt products containing 5% fat were prepared with different parameters.

Preparation of milk base:

Skimmed milk powder and cream powder (Fonterra Co-operative Group Limited, New Zealand) were used to prepare the milk base described below.

Table 8. Composition of milk base 1 (protein = 2.50%, fat = 5.00%)

Ingredient Dose (%) Dry low fat milk 3.39 Dry cream 9.07 Water 87.54

- 12 045075

Table 9. Composition of milk base 2 (protein = 4.50%, fat = 5.00%)

Ingredients Dose (%) Skimmed milk powder 9.68 Dry cream 8.98 Water 81.34

Skim milk powder, cream powder and water were mixed using a Silverson mixer at 8-10° C. for at least 2 hours. The milk base was homogenized using a GEA Lab homogenizer. Depending on the experimental model, homogenization was carried out at 150 bar (first stage 120 bar and second stage 30 bar) or at 300 bar (first stage 240 bar and second stage 60 bar). The homogenized milk base was then pasteurized at 85°C for 30 min and cooled.

Pasteurization and fermentation

Depending on the experimental model, phospholipase and/or chymosin were added at the beginning of fermentation along with the starter culture.

Starter culture

The starter culture from Example 1 was used (starter culture 1, level -1). For comparison, a second starter culture (starter culture 2, level +1) with different fermentation kinetics was included. Both starter cultures consisted of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.. bulgaricus.

Phospholipase and chymosin

Phospholipase A1 (YieldMAX®PL manufactured by Chr. Hansen A/S, Denmark; average activity 2600 LEU/ml, dosage 0.021650% at 5% fat)

Bovine chymosin (CHY-MAX® Plus from Chr. Hansen A/S, Denmark; average activity 200 IMCU/ml, dosage 0.00026% for 2% casein). Fat and protein content was determined using a MilkoScan analyzer.

Table 10. Composition and description of fermentation of samples 1-16

Trial Homogenization (bar) Starter culture Protein Chymosin (%) Phospholip per (%) Farm time. Final pH 1 150 1 2.50% 0 0.01265 3 hours 55 minutes 4.47 2 300 1 2.50% 0.00026 0.01265 3 hours 55 minutes 4.47 3 150 2 2.50% 0.00026 0.01265 4 hours 35 minutes 4.52 4 300 2 2.50% 0 0.01265 4 hours 35 minutes 4.54 5 150 1 4.50% 0.00046 0.01265 5 hours 30 minutes 4.52 6 300 1 4.50% 0 0.01265 5 h 00 min 4.51 7 150 2 4.50% 0 0.01265 6 hours 25 minutes 4.56 8 300 2 4.50% 0.00046 0.01265 6 hours 25 minutes 4.52 9 150 1 2.50% 0.00026 0 4 h 10 min 4.52 10 300 1 2.50% 0 0 4 h 10 min 4.52 eleven 150 2 2.50% 0 0 4 hours 20 minutes 4.54 12 300 2 2.50% 0.00026 0 4 hours 20 minutes 4.55 13 150 1 4.50% 0 0 5 h 00 min 4.54 14 300 1 4.50% 0.00046 0 5 h 00 min 4.53 15 150 2 4.50% 0.00046 0 6 hours 35 minutes 4.54 16 300 2 4.50% 0 0 6 hours 35 minutes 4.53

Fermentation was carried out at 43 °C until pH reached 4.55 ± 0.05. The fermentation time was recorded. The fermented product was cooled after treatment by means of a plate heat exchanger to 23-25° C. and brought to homogeneity by means of a check valve at a pressure of 1 bar at the outlet of the cooler.

The samples were stored in a cold room at a temperature of 5-7°C.

Samples after storage on day 7 were tested for viscosity and gel strength as in Example 1. The results are presented below.

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Table 11. Rheological assessment of samples on day 7

Homogenization -tion (bar) Starter culture Protein Chymosin (%) Phospholipase (%) Gel strength (G*) Viscosity at 60 1/s Viscosity at 300 1/s 1 150 1 2.50% 0 0.01265 129.22 425.4 97.0 2 300 1 2.50% 0.00026 0.01265 230.46 578.0 110.9 3 150 2 2.50% 0.00026 0.01265 198.86 715.3 150.6 4 300 2 2.50% 0 0.01265 156.17 633.7 168.3 5 150 1 4.50% 0.00046 0.01265 725.84 1559.6 261.2 6 300 1 4.50% 0 0.01265 531.91 1378.3 275.3 7 150 2 4.50% 0 0.01265 527.07 1745.6 375.4 8 300 2 4.50% 0.00046 0.01265 814.83 2255.2 416.2 9 150 1 2.50% 0.00026 0 173.49 441.4 88.1 10 300 1 2.50% 0 0 156.15 454.4 100.1 AND 150 2 2.50% 0 0 128.64 538.7 139.1 12 300 2 2.50% 0.00026 0 218.96 726.7 154.6 13 150 1 4.50% 0 0 387.86 1066.2 237.4 14 300 1 4.50% 0.00046 0 779.04 1694.5 292.9 15 150 2 4.50% 0.00046 0 655.44 1816.1 354.5 16 300 2 4.50% 0 0 561.71 1889.3 387.7

Rheological responses were analyzed using stepwise regression analysis with a multilinear model including main effects and first-order interactions:

Answer = cste + a.F1 + b.F2 + c.F3 + d.F4 + e.F5 + f.F1 .F2 + g F1 .F3... All effects where the risk of error was 5% (P value more 0.05) were excluded.

Viscosity at 60 1/s

Model summary:

S R-sq R-sq (corr.) R-sq (prev.)

46.9055 99.78% 99.44% 98.41%

This model explains more than 98% of the experimental variation.

Odds:

Index Coef. SE coefficient T value P value Constant 1119.9 I,7 95.50 0.000 Homogeneous. 81.4 I,7 6.94 0.000 Culture 170.2 I,7 14.51 0.000 Protein 555.7 I,7 47.39 0.000 Chymosin 103.5 I,7 8.82 0.000 Phospholipase 41.5 I,7 3.54 0.012 Homog.*Protein 47.4 I,7 4.04 0.007 Homog.*Phosph. -31.5 I,7 -2.68 0.036 Cult. *Protein 80.8 I,7 6.89 0.000 Protein*Chem. 52.3 I,7 4.46 0.004

The effects of various factors are presented graphically in FIG. 2.

Viscosity at 300 1/s

Model summary:

S R-sq R-sq (corr.) R-sq (prev.)

8.81291 99.64% 99.41% 98.87%

This model explains more than 98% of the experimental variation.

Odds:

Index Coef. SE coefficient T value P value Constant 225.57 2.20 102.38 0.000 Homogeneous. 12.68 2.20 5.75 0.000 Culture 42.71 2.20 19.39 0.000 Protein 99.49 2.20 45.16 0.000 Phosph. 6.28 2.20 2.85 0.019 Hom.*Protein 5.29 2.20 2.40 0.040 Cult. *Protein 15.67 2.20 7,I 0.000

The effects of various factors are presented graphically in FIG. 3.

Gel strength on day 7 (G*)

Model summary:

S R-sq R-sq (corr.) R-sq (prev.)

9.66033 99.96% 99.86% 99.38%

This model explains more than 99% of the experimental variation.

- 14 045075

Odds:

Index Coef. SE coefficient T value P value Constant 398.48 2.42 165.00 0.000 Homogeneous. 32.68 2.42 13.53 0.000 Culture 9.23 2.42 3.82 0.019 Protein 224.48 2.42 92.95 0.000 Chymosin 76.14 2.42 31.53 0.000 Phofsolipase 15.82 2.42 6.55 0.003 Homog.*Protein 16.23 2.42 6.72 0.003 Homog.*Phosph. -13.63 2.42 -5.64 0.005 Cult. *Protein 7.57 2.42 3.13 0.035 Cult. *Chem. -11.82 2.42 -4.90 0.008 Protein*Chem. 44.69 2.42 18.50 0.000 Protein*Phosph. 11.13 2.42 4.61 0.010

As evident from the above data, the effect of phospholipase measured at both viscosity measurement points was confirmed at a high level of significance (p value = 0.012 and 0.019), indicating its positive effect on taste impression and cohesiveness. The effect of phospholipase was greater at lower levels of homogenization (significant negative interaction, p value = 0.036). The maximum viscosity increase is +145 mPa s at a homogenization level of 150 bar. No interaction with culture was observed; phospholipase appears to act in the same way regardless of fermentation kinetics. The absence of a negative interaction between phospholipase and chymosin indicates that their combined effect is purely additive, and the maximum increase can reach +457 mPa-s. This confirms that the addition of chymosin can further improve texture.

Regarding the gel strength response, similar strong effects can be observed and the same conclusions can be drawn.

Example 3.

Soy yogurt containing 2.03% fat and 4.05% protein was prepared. On the 7th day, the effect of phospholipase on rheological properties was assessed.

Table 12. Composition used to prepare soy yogurt (w/w)

Ingredient Specification Dose (%) Chinese soybean powder Redman 22.50 Sugar KSL, refined sugar 5.00 Water City Water, PUB 72.50

Table 13. Samples of Example 3

Sample Culture Dose Enzyme S1 YE-L DA01 200 Units/MT - S2 YF-L DA01 200 Units/MT Yieldmax® PL 0.0046%

Pasteurization and fermentation

Depending on the experimental model, phospholipase was added before, at the beginning, or during the fermentation period.

Example 3 used the method described below. The composition presented in table. 12, mixed using a Silverson mixer. Mixing temperature/hydration time was 4550°C/20 min. Homogenization was carried out using a GEA Lab homogenizer heated to 65-70°C, and the pressure was 150 bar for the 1st stage and 50 bar for the 2nd stage for a total of 200 bar. The process was carried out in a Scandinox water bath under pasteurization conditions of 85°C/30 min and at a cooling temperature of less than 10°C. Fermentation was carried out in a Scandinox water bath at a temperature of 43 °C and a final pH of 4.55 ± 0.05.

Subsequent processing of soy yogurt consisted of the following: a pilot process unit (PTU) with a back pressure of 1.0 bar and a cooling temperature of 23-25°C was used.

Rheological studies of the samples obtained in example 3 were carried out as described below. Rheological studies were carried out using a modular compact rheometer Anton Paar MCR 302. The measurement temperature was 13°C, and the program used was the Stirred oscillation+Up_DownFlow program.

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Claims (10)

Table 14. Description of fermentation Recipe S1 S2 Final pH 4.60 4.58 Fermentation time 9 hours 10 minutes 9 hours 10 minutes pH at D+7 4.52 4.53 Table 15. Oscillation and hysteresis curve after storage period D+7 days Oscillation Hysteresis curve Shelf life Sample G* at 1.52 Hz (Pa) Vis pt 1 Vis pt 5 Vis pt 21 Square D+7 S1 452.43 74453 1178.8 295.65 6396.17 S2 537.42 86539 1342.8 317.83 7145.82 Vis pt 1: Viscosity (mPa-s) at 0.271 1/s (initial state, resistance when spooning yoghurt) Vis pt 5: Viscosity at 60 1/s (well correlated with mouthfeel) Vis pt 21: Viscosity at 60 1/s (cohesiveness (difficulty in swallowing)/thickness in the mouth) Hysteresis area: correlates with shear strength. G* oscillation at 1.52 Hz: correlates with gel strength As is evident from the above data for soy yogurt, the addition of phospholipase (YieldMAX®PL) has a positive effect on the texture of soy yogurt, with Vis pt 5 of sample S2 being 13.9% higher compared to S1. Organoleptic evaluation of soy yogurt showed that S2 had a thicker, creamier texture and less pungent flavor compared to S1. Thus, the addition of phospholipase (YieldMAX®PL) improves the texture and taste of soy yogurt. CLAIM 1. Use of phospholipase A to increase the viscosity of a product selected from a mammalian milk-based fermented product and a plant milk-based fermented product. 2. Application according to claim 1, including a process for obtaining a product, including the stages: a) adding to a milk base characterized by a fat content of at least 1.5% (w/w) a starter culture containing at least one strain of lactic acid bacteria, b) fermenting the milk base for a period of time until the target pH is reached, where the target pH is 5.0 or lower, and c) adding at least one phospholipase A to the milk base at the beginning of the fermentation period, where the milk base is a mammalian milk base or a plant milk base. 3. Use of phospholipase A and chymosin (EC 3.4.23.4) to increase the viscosity of a product selected from a mammalian milk-based fermented product and a plant milk-based fermented product. 4. Application according to claim 3, including a process for obtaining a product, including the stages: a) adding to a milk base characterized by a fat content of at least 1.5% (w/w) a starter culture containing at least one strain of lactic acid bacteria, b) fermenting the milk base for a period of time until the target pH is reached, where the target pH is 5.0 or lower, and c) adding at least one phospholipase A to the milk base at the beginning of the fermentation period, and further comprising adding chymosin (EC 3.4.23.4) to the milk base before, at the beginning of, or during the fermentation period, where the milk base is a mammalian milk base or plant-based milk base. 5. Use according to any one of claims 1 to 4, wherein the phospholipase is phospholipase A1 (EC 3.1.1.32). 6. Use according to any one of claims 1 to 4, wherein the phospholipase is phospholipase A2 (EC 3.1.1.4). 7. Use according to any one of claims 2 and 4-6, where the target pH is 4.6 or lower. 8. Use according to any one of claims 2 and 4-7, wherein the milk base is characterized by a fat content of at least 2.0% (w/w). 9. Use according to any one of claims 2 and 4-8, where the lactic acid bacteria are selected from the group consisting of bacteria of the genera Lactobacillus, Streptococcus, Lactococcus and Leuconostoc. 10. Use according to claim 2 or 4, where the starter culture contains at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus. -