Method for producing fermented dairy products for storage at room temperature

Abstract

The present invention is in the field of dairy technology. It relates to a method for producing a fermented milk product containing live bacteria, which has no or low / reduced post-acidification when the product is stored at ambient temperature. Furthermore, the present invention relates to the fermented milk product produced by the method.

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to a method for producing a fermented milk product, in particular a fermented milk product containing live bacteria for shelf-stable use with reduced post-acidification. [Background technology]

[0002] 2. Background of the Invention Fermented milk products and methods for their production are known in the art. Generally, fermented milk products require storage and distribution at cool temperatures to prevent further fermentation and acidification by the strains contained in the starter culture after production is complete, i.e., post-acidification, and to reduce the activity of any undesirable microorganisms that would cause spoilage of the product. In regions where cold storage and distribution are difficult or non-existent, desirable methods have been developed to produce fermented milk products that are more or less stable at ambient temperatures. In particular, methods for controlling post-acidification have been addressed.

[0003] Methods have been described that include steps to inactivate starter culture strains and undesirable microorganisms, and heat treatment is traditionally applied as the inactivation step, however heat can affect product attributes such as appearance, texture, flavor, etc.

[0004] In some countries, the presence of live bacteria in a product is required by law in order to be labeled as "yogurt". Furthermore, the presence of certain live bacteria, e.g., probiotic bacteria, in a product may provide producers with a desirable option for claiming health benefits. However, the presence of live bacteria poses challenges, especially at ambient temperatures, particularly due to post-acidification.

[0005] The use of lactose-deficient strains has been applied in several ways, for example, to reduce post-acidification in the produced products: US10072310 (Ling) relates to a process for preparing a fermented milk beverage that retains a high viable cell count at ambient temperature, involving the use of the lactose-deficient strain ATCC 53103. WO2005 / 089560 (Nestec SA) describes shelf-stable dairy products containing live microorganisms that cannot use lactose for nutrition, and methods for producing such products, however only one-step fermentation with a single strain, CNCM I-2116, was demonstrated. WO 2019 / 092064 (Tetra Laval Holdings & Finance SA) describes a method for producing a package containing a fermented milk product for ambient distribution, which contains a live dedicated culture that is free of bacteria that cannot consume lactose. WO2019 / 206754 (Chr. Hansen) describes a process for producing a dairy product by fermenting a milk base with a lactose-deficient strain and subsequently adding a probiotic strain.

[0006] However, there remains a need for improved methods for producing fermented milk products containing live bacteria which have no or only low / reduced post-acidification when stored at ambient temperature. Summary of the Invention

[0007] The present invention provides a method for preparing a fermented dairy product. In a first aspect, the present invention relates to a method for producing a fermented milk product using live bacteria, the method comprising the steps of: (a) providing a milk base comprising at least one carbohydrate and a first culture comprising one or more lactic acid bacteria strains capable of metabolizing the carbohydrate and producing at least one monosaccharide; (b) fermenting the milk base at a first temperature of less than or equal to 45° C. for a period of time until a first target pH of less than or equal to pH 4.7 is reached to obtain a first fermented milk base comprising at least one monosaccharide; (c) treating the first fermented milk base, thereby inactivating or removing the bacterial strains contained in the first culture; (d) adding to the treated first fermented milk base a second culture comprising one or more lactic acid bacteria strains, wherein said strains are lactose deficient and capable of metabolizing at least one monosaccharide produced during the first fermentation; (e) fermenting the treated first fermented milk base at a second temperature of 45° C. or less for a period of time until a second target pH of 4.4 or less is reached, wherein the second target pH is determined by depletion of at least one monosaccharide, and wherein the second target pH is lower than the first target pH; The method further comprises:

[0008] In a second aspect, the present invention provides a fermented milk product produced by the method.

[0009] definition Before describing the invention in more detail, a set of terms and general conventions are defined: The term "milk" should be understood as the lacteal secretion obtained by milking any mammal, including but not limited to cows, sheep, goats, buffalo, camels, llamas, mares, deer, etc. In a preferred embodiment, the milk is cow's milk.

[0010] "Homogenization" as used herein means vigorous mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, it can be performed to disperse the milk fat into smaller sizes so that it does not separate from the milk. This can be accomplished by forcing the milk through small orifices at high pressure.

[0011] "Pasteurization" as used herein refers to the treatment of a milk base to reduce or eliminate the presence of viable organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specific temperature for a specific time. Typically, the specific temperature is achieved by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful germs. This may be followed by a rapid cooling step.

[0012] In the present context, the term "starter culture" refers to a culture that is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to help initiate the fermentation process in the preparation of fermented products such as various food, feed and beverage products. In the present context, a "yoghurt starter culture" is a bacterial culture comprising one or more strains of Lactobacillus bulgaricus and one or more strains of Streptococcus thermophilus. According to this specification, "yoghurt" refers to a fermented milk product obtained by inoculating a milk base with a composition comprising strains of Lactobacillus bulgaricus and Streptococcus thermophilus and fermenting it.

[0013] The term "normal temperature" or "room temperature" in the context of this specification means a temperature above 10°C; 15°C; 20°C; 25°C, or a temperature between 10-50°C; 10-40°C; 10-30°C; 15-45°C; 15-35°C; 15-25°C; 20-40°C; 20-30°C.

[0014] With respect to lactic acid bacteria strains, the term "CFU" or "cfu" refers to colony forming units as determined by growth (colony formation) on MRS agar plates incubated for 3 days at 37° C. under anaerobic conditions. For details as used in the context of the present invention, see the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention Fermented dairy products such as yogurt can be produced from a milk base that is standardized with respect to lipid and protein content, homogenized, and pasteurized. In a typical process, the milk base is inoculated with a starter culture including selected microorganisms such as lactic acid bacteria (LAB). Fermentation is carried out under specified conditions (time, temperature, oxygen, etc.) until a desired target pH is reached, and then the fermentation is typically terminated by lowering the temperature. The fermented dairy product is preferably stored at cool temperatures, often at 4°C. Fermented dairy products are characterized by the particular starter culture used for fermentation.

[0016] In the absence of cold transport and / or cold storage of the fermented milk product or when the cold chain is incomplete, undesirable acidification continues after fermentation, resulting in a low pH, i.e. post-acidification of the milk product. Post-acidification can result in undesirable changes in product properties such as appearance, taste and texture, among others. Furthermore, depending on the degree of post-acidification, the viability of any bacteria present in the product may be affected, leading to low viability or even bacterial death.

[0017] The post-acidification caused by the starter culture may be prevented by inactivating the microorganisms contained in the starter culture. However, the desire to produce products with live bacteria, e.g. probiotic bacteria, requires the addition of live bacteria after the inactivation step. The added live bacteria may continue the acidification of the dairy product at room or ambient temperature, resulting in a change in said dairy product.

[0018] The present invention relates to a two-step fermentation process for producing fermented milk products suitable for storage at ambient temperature. Current methods for preparing fermented milk products, such as post-pasturization yogurt (PPY), struggle to reduce post-acidification caused by live bacteria present in the final product. Surprisingly, as demonstrated by the present invention, acidification by live bacteria added after post-pasteurization can be advantageously utilized in the fermentation process to produce dairy products.

[0019] Thus, in one embodiment, the present invention relates to a method for producing a fermented milk product using live bacteria, comprising the following steps: (a) providing a milk base comprising at least one carbohydrate and a first culture comprising one or more lactic acid bacteria strains capable of metabolizing the carbohydrate and producing at least one monosaccharide; (b) fermenting the milk base at a first temperature of less than or equal to 45° C. for a period of time until a first target pH of less than or equal to pH 4.7 is reached to obtain a first fermented milk base comprising at least one monosaccharide; (c) treating the first fermented milk base, thereby inactivating or removing the bacterial strains contained in the first culture; (d) adding to the treated first fermented milk base a second culture comprising one or more lactic acid bacteria strains, wherein said strains are lactose deficient and capable of metabolizing at least one monosaccharide produced during the first fermentation; (e) fermenting the treated first fermented milk base at a second temperature of 45° C. or less for a period of time until a second target pH of 4.4 or less is reached, wherein the second target pH is determined by depletion of at least one monosaccharide, and wherein the second target pH is lower than the first target pH; The present invention relates to a method comprising the steps of:

[0020] The milk base can be any raw milk and / or processed milk-derived raw material or other material that can be fermented according to the method of the present invention. Useful milk bases therefore include, but are not limited to, any dairy or milk-like product solution or suspension that contains proteins, such as whole milk, full fat milk, nonfat milk, low fat milk, skim milk, buttermilk, low lactose milk, concentrated milk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream. Obviously, the milk base can be derived from any mammal, for example, substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least a portion of the proteins in the milk base are proteins naturally occurring in mammalian milk, such as casein or whey protein.

[0021] In one embodiment, the present invention relates to a method, wherein the milk base is derived from an animal, such as a mammal, hi one embodiment, the present invention relates to a method, wherein the mammal is selected from the group consisting of cows, sheep, goats, buffaloes, camels, llamas, mares, and deer, hi a preferred embodiment, the mammal is a cow.

[0022] Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art.

[0023] Mammalian-derived milk bases contain lactose as the main carbohydrate, which is hydrolyzed by lactic acid bacteria during fermentation into the monosaccharides glucose and galactose. If the lactic acid bacteria cannot metabolize lactose, i.e. are lactose deficient, it may be necessary to add a suitable carbohydrate to the milk base in order to have at least one carbohydrate available for the production of at least one monosaccharide available to the lactic acid bacteria of the second fermentation.

[0024] The term "lactose deficient" is used in the context of the present invention to characterize lactic acid bacteria that have partially or completely lost the ability to use lactose as a source to maintain cell viability or cell growth. Lactose deficient bacteria are able to metabolize one or more carbohydrates selected from sucrose, galactose, glucose, and / or other fermentable carbohydrates. As these carbohydrates are not naturally present in milk in sufficient amounts to support fermentation by lactose deficient bacteria, they need to be added to the milk base.

[0025] The carbohydrate suitable for addition is determined by the lactic acid bacteria or bacteria of the first culture and the monosaccharides that need to be available to the lactic acid bacteria or bacteria of the second culture. Examples of suitable carbohydrates are lactose, sucrose, maltose, or trehalose. Sucrose is hydrolyzed to the monosaccharides glucose and fructose, maltose to glucose, and trehalose to glucose. In one embodiment, the present invention relates to a method, wherein at least one carbohydrate in the milk base is selected from lactose, sucrose, maltose, or trehalose. In one embodiment, the present invention relates to a method, wherein at least one carbohydrate in the milk base is added to the milk base.

[0026] The amount of carbohydrates present in the milk base must be sufficient for a first fermentation to occur producing at least one monosaccharide that must be available for the second fermentation. However, the amount of carbohydrates does not need to be excessive if the lactic acid bacteria or bacteria of the second culture are able to metabolize the carbohydrates. In the situation where the lactic acid bacteria of both the first and second cultures are able to metabolize the carbohydrates, the amount of carbohydrates must be selected, i.e., limiting the carbohydrates that must be used up after the first fermentation or, alternatively, that must be used up when the second fermentation reaches the second target pH. In one embodiment, the invention relates to a method, in which the amount of at least one carbohydrate is used up during the first fermentation. In one embodiment, the invention relates to a method, in which the amount of at least one carbohydrate is used up when the second fermentation reaches the second target pH.

[0027] The first fermentation begins when a first culture is added to the milk base. The first culture may be any starter culture, such as, for example, a yogurt starter culture. The term "starter" or "starter culture" as used in the context of this specification refers to a culture of one or more food-grade microorganisms, in particular lactic acid bacteria, which are involved in the acidification of a milk base. The starter culture may be fresh, frozen or freeze-dried. In one embodiment, the present invention relates to a method, wherein the one or more lactic acid bacteria strains of the first culture are selected from the genus Streptococcus, such as S. thermophilus, or the genus Lactobacillus, such as L. delbrueckii subsp. bulgaricus.

[0028] The one or more lactic acid bacteria strains of the first culture may be lactose deficient. Examples of suitable lactose deficient strains can also be found in WO2015 / 193459. In one embodiment, the present invention relates to a method, wherein the one or more lactic acid bacteria strains of the first culture are lactose deficient. In one embodiment, the present invention relates to a method, wherein the lactose deficient strain of the first culture is selected from the group consisting of: DSM28952, DSM28953, DSM28910, DSM32600, DSM32599.

[0029] The first fermentation is terminated when a sufficient amount of at least one monosaccharide is produced. The first fermentation is terminated when a first target pH is reached. The first target pH should be higher than the second target pH and should be selected to provide a pH range that allows for further acidification during the second fermentation. In one embodiment, the invention relates to a method, wherein the first target pH is less than or equal to pH 4.70:4.65;4.60;4.55;4.50;4.45;4.40; or is within the range of pH 4.70-4.00;4.70-4.10;4.70-4.20;4.70-4.30;4.70-4.40;4.70-4.45;4.65-4.50;4.60-4.55; or is about 4.70:4.65;4.60,4.55;4.50;4.45; or 4.40.

[0030] Since the temperature affects the fermentation speed, it should preferably be kept stable or constant at a defined temperature during the first fermentation. In one embodiment, the present invention relates to a method, wherein the first temperature is less than 25; 30; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45 °C; or is within the range of 20-45; 25-45; 30-45; 40-45; 25-40; 30-40; 35-40 °C; or is about 20; 25; 30; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45 °C.

[0031] To terminate and prevent further fermentation by the first culture, it needs to be inactivated or removed. The first fermented milk base can be inactivated in several ways. In one embodiment, the present invention relates to a method, wherein the treatment of the first fermented milk base is a treatment with heat, ultrasound, radiation, such as UV irradiation, centrifugal sterilization, or microfiltration. Treatment with heat or heat treatment can also be called post-pasteurization. In one embodiment, the invention relates to a method, wherein the heat treatment is carried out in the range of 65-75°C for at least 1-30 minutes, at least 60;65;70; or 75°C for 1;5;10;15;20;25; or 30 minutes; or in the range of 70-90 for 10-50 seconds; or at least 70;75;80;85; or 90°C for at least 10;15;20;25;30;35;40;45; or 50 seconds; or in the range of 65-90°C; 70-85°C; 75-80°C for 10-50 seconds; or at 75°C for 25 seconds; or at 75°C for 50 seconds.

[0032] In one embodiment, the present invention relates to a method, wherein the first fermentation is terminated by a cooling step. Preferably, the temperature used for the cooling step is about 4°C, such as 2°C, 3°C, 4°C, 5°C, or 6°C.

[0033] Preferably, the second culture is added under sterile conditions, i.e. without or with minimal introduction of any microorganisms other than the lactic acid bacteria or bacteria of the second culture. The second culture may be added as one or more bulks or as a continuous feed to the production line. The second culture may be added in the form of a liquid culture, a frozen culture or a lyophilized culture. Preferably, the culture is a concentrated culture.

[0034] The one or more lactic acid bacteria strains of the second culture are lactose deficient and metabolize at least one monosaccharide produced during the first fermentation. Thus, the second fermentation is terminated when at least one monosaccharide is depleted from the first fermented milk base and a second target pH is reached. In one embodiment, the invention provides a method for fermenting milk having a second target pH of less than or equal to pH 4.5; 4.4; 4.3; 4.2; 4.1; 4.0; 3.9; 3.8; 3.7; 3.6; 3.5; or a pH of 4.50-3.50; 4.50-4.05; 4.45-4.10; 4.45-4.15; 4.40-4.20; 4.40-4.25; 4.35-4.30; 4. 3.75-3.60; or about pH 4.40; 4.35; 4.30; 4.25; 4.20; 4.15; 4.10; 4.05; 4.00; 3.90; 3.80; 3.70; 3.60; 3.50.

[0035] In one embodiment, the present invention relates to a method for producing a lactobacillus strain or strains of a lactobacillus strain, comprising the step of: culturing one or more lactobacillus strains of the second culture; ... reuteri, and Lactobacillus johnsonii; as well as the genus Bifidobacterium, such as Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp.lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, and Bifidobacterium infantis; or Streptococcus spp., such as S. thermophilus.

[0036] Consumption of probiotic bacteria is believed to be beneficial for the health of an individual. Therefore, a certain amount of live probiotic bacteria in the fermented milk product is desirable. In one embodiment, the present invention relates to a method, wherein the one or more lactic acid bacteria of the second culture are probiotic bacteria.

[0037] The one or more lactic acid bacteria strains of the second culture may be lactose deficient and selected from the group consisting of: ATCC 53103, CNCM I-2116, and DSM 16572. In one embodiment, the invention relates to a method, wherein the bacteria is ATCC 53103, CNCM I-2116, and / or DSM 16572.

[0038] It has been shown that the method of the invention results in a fermented milk product with no or low / reduced acidification. In the examples, this is shown as a change in acidification during storage from time t1 onwards. This can result in a change in pH measured in the product. The change in pH can be an increase in pH or a decrease in pH. Post-acidification is always shown as a decrease in pH.

[0039] In one embodiment the invention relates to a method, wherein the pH of the fermented milk product changes by less than 0.80; 0.70; 0.60; 0.50; 0.40; 0.35; 0.30, 0.25, 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 pH units after storage at 25° C. for 6 months. In one embodiment the invention relates to a method, wherein the pH of the fermented milk product changes by less than 0.80; 0.70; 0.60; 0.50; 0.40; 0.35; 0.30, 0.25, 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 pH units after storage for 2 months at 37° C. or after storage for 1 month at 42° C.

[0040] In one embodiment the invention relates to a method, wherein the fermented milk product comprises at least 1.0E+03;1.0E+04;1.0E+05;1.0E+06;1.0E+07;1.0E+08;1.0E+09;1.0E+10 cfu / g live bacteria after 6 months of storage at 25° C. In one embodiment the invention relates to a method, wherein the fermented milk product comprises at least 1.0E+06 cfu / g live bacteria after 2 months of storage at 37° C. or after 1 month of storage at 42° C. In one embodiment the invention relates to a method, wherein the fermented milk product comprises at least 1.0E+07 cfu / g live bacteria after 2, 3, 4 or 5 weeks of storage at 45° C. In one embodiment the invention relates to a method, wherein the live bacteria comprises or contain probiotic bacteria.

[0041] In one embodiment, the invention relates to a method, wherein the one or more lactic acid bacteria of the second culture are capable of growing cells and increasing cell number during or during and after the second fermentation, hi one embodiment, the invention relates to a method, wherein the cell number is enhanced by 0.5; 1.0; 1.5; 2.0; 2.5; or 3.0 log.

[0042] In one embodiment, the present invention relates to a method further comprising the addition of flavorings, thickeners, emulsifiers, and / or stabilizers, such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginates, agar, guar gum, and the like. In one embodiment, the present invention relates to a method further comprising the addition of a sweetener, such as a chemical / artificial sweetener (such as sucralose, isomaltulose, acesulfame potassium), a sugar alcohol (such as maltitol or isomaltitol (12 carbons), erythritol (4 carbons), xylitol (5 carbons), sorbitol (6 carbons), etc. Sugar alcohols vary in number of carbons, so in one embodiment, the present invention relates to a method, wherein the sugar alcohol is selected from the group consisting of 4-, 5-, 6-, or 12-carbon sugar alcohols. In one embodiment, the present invention relates to a method further comprising the sugar alcohol erythritol and / or maltitol. In one embodiment, the mass ratio of erythritol:maltitol is (0.5-4.0):(0-4.0). The sugar alcohol may be used in a total amount of 0.5-8.0, 2.5-6.0, or 4.5 w / w% based on the total weight of the fermented milk product.

[0043] In one embodiment, the present invention relates to a fermented milk product produced by the method. The term "fermented milk product" as used herein refers to a food or feed product, the preparation of which involves fermentation of a milk base with lactic acid bacteria according to the present invention. "Fermented milk product" as used herein includes, but is not limited to, dairy products such as yoghurt. In one embodiment, the present invention relates to a fermented milk product that is a food or feed product. In one embodiment, the present invention relates to a fermented milk product that is a dairy product, such as yoghurt (set or stirred); Greek yoghurt; yoghurt-based products such as fruit yoghurt, yoghurt-based beverages; buttermilk; kefir; Labneh, Quark, etc. Preferably, the fermented milk product is yoghurt.

[0044] In one embodiment, the present invention relates to a fermented milk product, wherein said product is an ambient-stable fermented milk product. The term "ambient-stable fermented milk product" refers to a fermented milk product, which is suitable for ambient storage over a period of time. The storage period may be between 1 and 12 months, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

[0045] Fermented milk products typically contain protein at a level of 2.0-3.5% w / w. The fermented milk product may be a low protein product with a protein level of 1.0-2.0% w / w. Alternatively, the fermented milk product may be a high protein product with a protein level of more than 3.5 or 5.1% w / w, for example 3.5-5.1%, 3.5-10.5% or 5.1-10.5% w / w. The protein may be derived from milk, for example whey or casein.

[0046] It is recommended to consume a certain amount of probiotic bacteria to obtain health benefits. It is therefore desirable for the fermented milk product to contain a certain level of probiotic bacteria. In one embodiment, the present invention relates to a fermented milk product, said product comprising at least 1.0E+05;1.0E+06;1.0E+07;1.0E+08;1.0E+09;1.0E+10;1.0E+11;1.0E+12 cfu / serving of probiotic bacteria.

[0047] Taxonomy It is understood that the taxonomy of the Lactobacillus genus has been updated in 2020. The new taxonomy is disclosed in Zheng et al. 2020 Int. J. Syst. Evol. Microbiol. DOI 10.1099 / ijsem.0.004107 and is summarized herein unless otherwise indicated. For the purposes of the present invention, the following table shows a list of the old and new names of some Lactobacillus species relevant to the present invention.

[0048] Table 1. Old and new names of Lactobacillus species relevant to this invention [Table 1]

[0049] Table 2. Strains [Table 2] EXAMPLES

[0050] Example 1 - YF-L904 & LGG A defined milk base for two-step fermented ambient yoghurt with live bacteria is prepared. The defined milk base is suitable for using a normal yoghurt culture as the first fermentation culture. In this defined milk base fermentation, the normal yoghurt culture uses lactose to produce lactic acid while galactose is left in the yoghurt. The amount of galactose is different when the fermentation is stopped at different pH. When a heat treatment is applied to inactivate the first culture and a second lactose negative culture LGG is aseptically injected into the heat treated yoghurt, LGG continues to lower the pH using the galactose left over from the first fermentation, and when the galactose is used up, the pH of the final yoghurt enters a plateau during which LGG grows to a high cell number and remains stable at ambient storage.

[0051] material: Table 3. Dairy based [Table 3]

[0052] culture F-DVS YF-L904 (Batch 3551191Chr Hansen) F-DVS LGG® (Batch 3584797Chr Hansen)

[0053] method: The milk base was prepared according to the table above and pasteurized at 134°C for 4 seconds. First fermentation - Inoculate milk base with 100 u / T (units / ton) of YF-L904 and ferment at 43°C until a first target pH of 4.7, 4.6, or 4.5 is reached. The curd is crushed and the yogurt bases of three different pH values ​​are heat treated at 75°C for 25 seconds.

[0054] Second fermentation - F-DVS LGG is aseptically inoculated into pasteurized yogurt of three different pH values, at a dose of 100u / T (time t0). The second fermentation is carried out at 25°C, 30°C or 35°C until a stable pH is reached. Store at room temperature -25℃. The cell number of L. rhamnosus LGG® was determined by using Difco MRS agar, pour plate method with anaerobic incubation at 37° C. for 3 days.

[0055] result: Table 4. Post acidification for the second fermentation at 35°C [Table 4]

[0056] Table 5. Post acidification for the second fermentation at 30°C [Table 5]

[0057] Table 6. Post acidification for the second fermentation at 25°C [Table 6]

[0058] Table 7. LGG number [Table 7]

[0059] YF-L904 hydrolyzes lactose, which is naturally present in milk, into glucose and galactose. The glucose is metabolized and the galactose is left in the milk base. LGG can metabolize the galactose.

[0060] Different first target pH leads to different second target pH. At higher pH, first fermentation is terminated and at higher pH, second fermentation reaches stable pH. Thus, first target pH should be determined according to the requirements of the final product. LGG reaches stable pH faster at high heat during second fermentation compared to lower temperature (35°C vs. 30°C and 25°C). LGG reaches cell count levels >1.0E+08 cfu / g at all second fermentation temperatures.

[0061] Example 2 - ACIDIFIX & LGG, 2. Effect of temperature during fermentation A milk base with limited amount of sugar (0.75%) was prepared in this experiment. The first fermentation was carried out with Acidifix 1.0 until a stable pH. The second fermentation was carried out with LGG. The second fermentation culture metabolizes the monosaccharides produced during the first fermentation until a stable pH is reached. The second fermentation was carried out at 25°C, 33°C, and 40°C until a stable pH. The product was then stored at 25°C and checked for cell count and post acidification at various intervals.

[0062] material: Table 8. Dairy-based content [Table 8]

[0063] Culture: F-DVS YoFlex® Acidifix™ 1.0 (Batch 3586293, Chr. Hansen A / S) F-DVS LGG® (Batch 3584797, Chr. Hansen A / S)

[0064] method: The milk base was prepared according to the table above and pasteurized at 134°C for 4 seconds. First fermentation - A milk base containing a limited amount of sucrose, i.e. 0.75 w / w%, was fermented with Acidifix 1.0 100u / t milk base at 43°C until sugars were exhausted and a stable pH was reached, i.e. a first target pH of 4.50. Heat treatment - 75°C for 49.2 seconds.

[0065] Second fermentation - Lactose-deficient LGG at a concentration of 3.5E+06 cfu / g LGG(1) or 9.0E+06 cfu / g LGG(2) was added to the fermented milk base containing the monosaccharide fructose produced during the first fermentation (time t0) and fermented at 25°C, 30°C, or 40°C until a stable pH was reached (time t1) at a second target pH of about pH 4.3. Store at room temperature -25℃.

[0066] result: Table 9. pH over time for methods according to the invention where the second fermentation is carried out at temperatures of 25°C, 33°C or 40°C [Table 9]

[0067] Table 10. Cell count over time for methods according to the invention where the second fermentation is carried out at temperatures of 25°C, 33°C or 40°C [Table 10]

[0068] Fermentation / acidification with a second culture is part of the method of the invention. LGG ferments / acidifies the heat treated fermented milk base as evident from the pH difference between pH t0 and pH t1. No further fermentation (post acidification) takes place during storage as can be observed by comparing pH t1 with pH week 7. A stable pH was observed over all temperatures tested. A stable pH is important to maintain the taste of the product during storage.

[0069] The LGG grown at both LGG inoculum doses was tested during the second fermentation as can be observed by comparing the cell counts at t0 and t1. Storage at room temperature (25°C) for 7 weeks shows that the cell count of the product remains stable. The stable cell count provides an adequate amount of live bacteria to the end consumer.

[0070] Example 3 - Storage stability at room temperature (25°C) material: Table 11. Dairy based [Table 11]

[0071] culture F-DVS YoFlex® Acidifix™ 1.0 (Batch 3586293Chr Hansen A / S) F-DVS LGG® (Batch 3584797Chr Hansen A / S) method: The milk base was prepared according to the table above and pasteurized at 134°C for 4 seconds. First fermentation - Inoculate the milk base with Acidifix 100u / T (units / tonne) and ferment at 43°C until a first target pH of 4.50 is reached. The curd is broken and the yogurt base is heat treated at 75°C for 25 seconds.

[0072] Second Fermentation - F-DVS LGG is aseptically inoculated in two doses: dose (1) 100u / T, 7.0E+06 cfu / g; dose (2) 200u / T, 1.2E+07 cfu / g was added to the heat treated yogurt base prepared by the first fermentation (time t0). The second fermentation is carried out at 25°C, 33°C, 40°C until a stable pH is reached (time t1) at a second target pH of about pH 4.3. Store at room temperature -25℃.

[0073] result: The same cell counts were obtained for the three temperatures tested, therefore storage stability data from one temperature (25° C.) is presented in the table below.

[0074] Table 12. pH and cell count during storage at room temperature (25°C) [Table 12]

[0075] A stable pH was achieved at all temperatures tested. Both LGG inoculum doses achieved higher cell counts (>1.0E+8 cfu / g) at t1 than the inoculum dose. After 2 months of ambient storage (25°C), the pH and cell count of the product remained stable.

[0076] Example 4 - Shelf life of six different fermented dairy products The first fermentation was carried out with two different types of cultures (Acidifix1.0 and YF-L904). The second fermentation was carried out with LGG or Fresh Q2. The second fermentation culture metabolizes the monosaccharides produced during the first fermentation until a stable pH is reached. The second fermentation was carried out at 25° C. for 3 days or until a stable pH. The product was then stored at 25° C. and checked for cell count and post-acidification at various intervals.

[0077] material: Three different milk bases with different amounts of sucrose were used and prepared according to the following table: no sucrose, limited amount of sucrose (0.75%) and excess amount of sucrose (7.00%).

[0078] Table 13. Dairy base 1 [Table 13]

[0079] Table 14. Dairy base 2 [Table 14]

[0080] Table 15. Dairy base 3 [Table 15]

[0081] The following cultures were used in the fermentation and were added at a concentration of 100 u / t: F-DVS YF-L904 (Batch 3551191, Chr. Hansen A / S) F-DVS YoFlex® Acidifix™ 1.0 (Batch 3586293, Chr. Hansen A / S) F-DVS LGG® (Batch 3584797, Chr. Hansen A / S) F-DVS FQ®2 (Batch 3589655, Chr. Hansen A / S)

[0082] method: The milk base prepared according to the above table was pasteurized at 134°C for 4 seconds. Fermentation was set up according to the following table with no sucrose (S1), limited amounts of sucrose (S2-S3) or excess amounts of sucrose (S4-S6). YF-L904 and FQ2 hydrolyze the lactose present in the milk base to glucose and galactose, where glucose is consumed and galactose is left in the milk base. Acidifix and LGG are lactose deficient and therefore hydrolyze the sucrose added to the milk base to glucose and fructose, where glucose is consumed and fructose is left in the milk base. LGG could grow on fructose but was very slow on galactose.

[0083] First fermentation - 100 units / ton of 1. culture was inoculated into milk base according to the table below. Fermentation was carried out at 43°C until a first target pH of 4.50 was reached. The curd was broken and the yogurt base was heat treated at 75°C for 49.2 seconds.

[0084] Second fermentation - 5.0E+06 cfu / g of 2. culture was inoculated into the heat treated yogurt base prepared by the first fermentation (time t0). The second fermentation was carried out at 25°C until a stable pH was reached (time t1) at a second target pH of about pH 4.3. Store at room temperature -25℃.

[0085] Table 16. Fermentation settings [Table 16]

[0086] result: Table 17. pH and cell count for fermentation settings 1-6 [Table 17]

[0087] Table 18a. pH during storage at 42°C [Table 18]

[0088] Table 18b. Cell counts during storage at 42°C [Table 19]

[0089] Table 19a. pH during storage at 37°C [Table 20]

[0090] Table 19b. Cell counts during storage at 37°C [Table 21]

[0091] Table 20a. pH during storage at 25°C [Table 22]

[0092] Table 20b. Cell counts during storage at 25°C [Table 23]

[0093] The method according to the invention is illustrated in S1 and S2, where post-acidification during storage was very low or non-existent. Controls S3 and S5 show that post-acidification occurs when a lactose fermentation culture is used as the second culture. In controls S4, S5, and S6, sucrose is added in excess, so that it is available for the second culture for post-acidification.

[0094] Example 5 - Post-acidification in the presence of sweeteners Products were prepared according to the invention in the presence of various sweeteners and stored at 25° C. and acidification was measured at various time points during the storage period. material: Three different dairy bases, each containing a different sweetener (maltitol, isomaltulose, and erythritol), were prepared according to the table below.

[0095] Table 21. Milk base with maltitol (MB-M) [Table 24]

[0096] Table 22. Isomaltulose-based milk base (MB-I) [Table 25]

[0097] Table 23. Milk-based Erythritol (MB-E) [Table 26]

[0098] The following cultures were used in the fermentation at a concentration of 100u / t: F-DVS YoFlex® Acidifix™ 1.0 (Batch 3586293, Chr. Hansen A / S) F-DVS LGG® (Batch 3584797, Chr. Hansen A / S)

[0099] method: The milk base was prepared according to the table above and pasteurized at 134°C for 4 seconds. For the first fermentation, 100 units / ton of YoFlex® Acidifix® culture was inoculated into the milk base and fermentation was carried out at 43° C. until a first target pH of 4.50 was reached. The curd was broken and the yogurt base was heat treated at 75°C for 49.2 seconds.

[0100] A second fermentation was carried out with an inoculum of 5.0E+06 cfu / g of LGG® in the heat-treated yogurt base prepared by the first fermentation. The second fermentation was carried out at 25° C. and was terminated after 72 hours (day 0). The products were stored at temperatures of 25°C, 37°C and 42°C, and the titratable acidity, TA(°T), was measured during storage according to the Chinese National Standardization Method (GB5009.239-236 China Food Safety Standard, Determination of Acidity in Food).

[0101] result: Table 24. TA during storage at room temperature and 25℃ [Table 27]

[0102] Table 25. TA during storage at 37°C [Table 28]

[0103] Table 26. TA during storage at 42°C [Table 29]

[0104] The products made by the two-step fermentation process resulted in different post-acidification with the various sweeteners used.

[0105] Example 6 - Use of an alternative lactose-deficient strain for the second fermentation The method of the present invention was carried out using the lactose deficient strain L. casei 02 for the second fermentation.

[0106] material: Two different milk bases with different amounts of sucrose were prepared according to Table 3 in Example 1 (MB1 without sucrose) and Table 11 in Example 3 (MB2 with 0.75% sucrose). The following cultures were used in the fermentation at a concentration of 100u / t: F-DVS YoFlex® Acidifix™ 1.0 (Batch 3586293, Chr Hansen A / S) F-DVS YF-L904 (Batch 3551191, Chr Hansen A / S) F-DVS L. casei 02 (Batch 3565988, Chr Hansen A / S)

[0107] Table 27. Fermentation settings [Table 30]

[0108] method: The milk base was prepared according to the table and pasteurized at 134°C for 4 seconds. First fermentation - Inoculate the milk base with YF-L904 100u / T (units / ton) in MB1 and Acidifix 100u / T (units / ton) in MB2 and ferment at 43°C until a first target pH of 4.50 is reached. The curd is broken and the yogurt base is heat treated at 75°C for 25 seconds.

[0109] Second fermentation - F-DVS L. casei 02 is aseptically inoculated into the heat-treated yogurt base prepared by the first fermentation (time t0) at a dosage of 0.00663%, corresponding to 5.5E+6 cfu / g. The second fermentation is carried out at 25°C until a stable pH is reached (time t1) at a second target pH of about pH 4.2-4.3. They were stored at temperatures of 25°C, 37°C, and 42°C. Cell numbers were determined as described in Example 1.

[0110] result: Table 28. Acidification after measurement as pH for samples stored at 25°C [Table 31]

[0111] Table 29. Cell counts in samples stored at 25°C [Table 32]

[0112] Table 30. Acidification after measurement as pH for samples stored at 37°C [Table 33]

[0113] Table 31. Cell counts in samples stored at 37°C [Table 34]

[0114] Table 3. Acidification after pH measurement for samples stored at 42°C [Table 35]

[0115] Table 33. Cell counts in samples stored at 42°C [Table 36]

[0116] Both S7 and S8 reached a stable level of pH during storage at 25°C, 37°C and 42°C. However, S7 took longer to reach such a level of pH and the pH level was lower than that of S8. This is an indication that the type and amount of monosaccharides left by the starter culture in the first fermentation is different in S7 compared to S8. L. casei 02 shows a similar effect to that observed with LGG®. In both S7 and S8, L. casei 02 grew to cell numbers higher than the inoculum level, however, the stability varied at different temperatures. The most stable cell numbers were observed for S8 at 25°C, where the cell numbers remained above 1.0E+8 cfu / g for 3 months.

Claims

1. 1. A method for producing a fermented milk product using live bacteria, comprising the steps of: (a) providing a milk base comprising at least one carbohydrate and a first culture comprising one or more lactic acid bacteria strains capable of metabolizing the carbohydrate and producing at least one monosaccharide; (b) fermenting the milk base at a first temperature of less than or equal to 45°C for a period of time until a first target pH of less than or equal to 4.7 is reached to obtain a first fermented milk base comprising at least one monosaccharide; (c) treating the first fermented milk base, thereby inactivating or removing the bacterial strains contained in the first culture; (d) adding to the treated first fermented milk base a second culture comprising one or more strains of lactic acid bacteria, wherein said strains are lactose deficient and capable of metabolizing at least one monosaccharide produced during the first fermentation; (e) fermenting the treated first fermented milk base at a second temperature of not more than 45°C for a period of time to reach a second target pH of not more than pH 4.4 to obtain a fermented milk product, wherein the second target pH is determined by depletion of at least one monosaccharide, and wherein the second target pH is lower than the first target pH; The method comprising:

2. 10. The method of claim 1, wherein the at least one carbohydrate in the milk base is selected from lactose, sucrose, maltose, or trehalose.

3. 10. The method of claim 1, wherein at least one carbohydrate in the dairy base is added to the dairy base.

4. 4. The method according to claim 1, wherein the amount of the at least one carbohydrate is depleted during a first fermentation.

5. 4. The method according to any one of claims 1 to 3, wherein the one or more strains of lactic acid bacteria of the first culture are selected from the genus Streptococcus, such as S. thermophilus, or the genus Lactobacillus, such as L. delbrueckii subsp. bulgaricus.

6. The method according to any one of claims 1 to 3, wherein the one or more lactic acid bacteria strains of the first culture are lactose deficient.

7. 7. The method of claim 6, wherein the lactose-deficient strain of the first culture is selected from the group consisting of: DSM28952, DSM28953, DSM28910, DSM32600, DSM32599.

8. 4. The method of any one of claims 1 to 3, wherein the first target pH is equal to or less than pH 4.70: 4.65; 4.60; 4.55; 4.50; 4.45; 4.40; or is within the range of pH 4.70 to 4.00; 4.70 to 4.10; 4.70 to 4.20; 4.70 to 4.30; 4.70 to 4.40; 4.70 to 4.45; 4.65 to 4.50; 4.60 to 4.55; or is about 4.70: 4.65; 4.60, 4.55; 4.50; 4.45; or 4.

40.

9. 4. The method of claim 1, wherein the first temperature is less than 25; 30; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45°C; or is in the range of 20 to 45; 25 to 45; 30 to 45; 40 to 45; 25 to 40; 30 to 40; 35 to 40°C; or is about 20; 25; 30; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45°C.

10. 4. The method according to any one of claims 1 to 3, wherein the treatment of the first fermented milk base is a treatment using heat, wherein the heat treatment is carried out in the range of 65-75°C for at least 1 to 30 minutes, at least 60; 65; 70; or 75°C for 1; 5; 10; 15; 20; 25; or 30 minutes; or in the range of 70-90 for 10 to 50 seconds; or at least 70; 75; 80; 85; or 90°C for at least 10; 15; 20; 25; 30; 35; 40; 45; or 50 seconds; or in the range of 65-90°C; 70-85°C; 75-80 for 10 to 50 seconds; or at 75°C for 25 seconds; or at 75°C for 50 seconds.

11. The second target pH is equal to or less than pH 4.5; 4.4; 4.3; 4.2; 4.1; 4.0; 3.9; 3.8; 3.7; 3.6; 3.5; or pH 4.50 to 3.50; 4.50 to 4.05; 4.45 to 4.10; 4.45 to 4.15; 4.40 to 4.20; 4.40 to 4.25; 4.35 to 4.30; 4.00 to 3.50; 3.95 4. The method of any one of claims 1 to 3, wherein the pH is within the range of 3.0 to 3.50; 3.90 to 3.55; 3.85 to 3.60; 3.80 to 3.65; 3.75 to 3.60; or about pH 4.40; 4.35; 4.30; 4.25; 4.20; 4.15; 4.10; 4.05; 4.00; 3.90; 3.80; 3.70; 3.60; 3.

50.

12. The one or more lactic acid bacteria of the second culture may be bacteria of the genus Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactiprantibacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, and Lactobacillus johnsonii; and bacteria of the genus Bifidobacterium, such as Bifidobacterium longum, Bifidobacterium adrenergica, Bifidobacterium oxysporum ...

4. The method of claim 1, wherein the bacterial strain is selected from the group consisting of Bifidobacterium suscentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angratum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, and Bifidobacterium infantis; or Streptococcus, such as S. thermophilus.

13. The method according to any one of claims 1 to 3, wherein the one or more lactic acid bacteria of the second culture are probiotic bacteria.

14. The method according to any one of claims 1 to 3, wherein the bacterium is ATCC53103, CNCM I-2116, and / or DSM16572.

15. 4. The method according to any one of claims 1 to 3, wherein the pH of the fermented milk product changes by less than 0.80; 0.70; 0.60; 0.50; 0.40; 0.35; 0.30, 0.25, 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01 pH units after storage at 25°C for 6 months.

16. 4. The method of claim 1, wherein the fermented milk product comprises at least 1.0E+03; 1.0E+04; 1.0E+05; 1.0E+06; 1.0E+07; 1.0E+08; 1.0E+09; or at least 1.0E+10 cfu / g of viable bacteria after storage at 25°C for 6 months.

17. 4. The method according to claim 1, wherein the one or more lactic acid bacteria of the second culture are capable of growing cells and increasing cell number during, or during and after, the second fermentation.

18. 18. The method of claim 17, wherein the cell number is enhanced by 0.5; 1.0; 1.5; 2.0; 2.5; or 3.0 logs.

19. A fermented milk product produced by the method according to any one of claims 1 to 3.

20. 20. The product of claim 19, wherein the fermented milk product comprises at least 1.0E+05; 1.0E+06; 1.0E+07; 1.0E+08; 1.0E+09; 1.0E+10; 1.0E+11; 1.0E+12 cfu / serving of probiotic bacteria.