Method for producing fermented milk products with improved stability

Abstract

The present disclosure is in the dairy product arts and relates to methods for producing acidified or fermented dairy products with improved stability. The present disclosure also provides acidified or fermented dairy products produced by the methods.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of acidified or fermented milk, and in particular to methods for producing acidified or fermented milk products with improved stability, and to acidified or fermented milk products produced thereby. [Background technology]

[0002] Flavor and gas development are important characteristics that affect customer acceptability of acidified or fermented dairy products. Insufficient cooling or temperature changes during transportation and storage can alter the flavor and / or gas development of fermented dairy products. In addition to changes in the product's taste and texture in the mouth, gas development can lead to pressure buildup, expansion, and even rupture of the final product package.

[0003] Thus, there is a need for improved methods for producing acidified or fermented dairy products in which the final product remains stable during storage, particularly where a cold chain is not possible or there is a risk of the cold chain being disrupted or partially disrupted. Summary of the Invention

[0004] In a first aspect, the present disclosure provides a method for producing an acidified or fermented milk product, the method comprising the steps of: (a) adding one or more sweeteners to a milk base or a pre-acidified milk and acidifying the milk base to a first target pH of 4.7 or less to obtain a pre-acidified milk; (b) optionally heat treating the pre-acidified milk; (c) inoculating the pre-acidified milk with lactose-deficient lactic acid bacteria until a second target pH of 4.6 or less is reached to obtain a milk product; and The conversion of citric acid to acetic acid in the dairy product is delayed during storage compared to a dairy product produced without the addition of one or more sweeteners.

[0005] In a second embodiment, the present disclosure provides an acidified or fermented milk product obtainable by the method.

[0006] As described below, it has surprisingly been found that the development of off-flavors and gases during the storage period of acidified or fermented milk products can be suppressed by slowing the conversion of citric acid to acetic acid. The present disclosure provides a solution in which said conversion is suppressed by adding certain sweeteners, as described in more detail above.

[0007] The present disclosure relates to a method for producing an acidified or fermented milk product, comprising the steps of: (a) adding one or more sweeteners to a milk base or a pre-acidified milk and acidifying the milk base to a first target pH of 4.7 or less to obtain a pre-acidified milk; (b) optionally heat treating the pre-acidified milk; (c) inoculating the pre-acidified milk with lactose-deficient lactic acid bacteria until a second target pH of 4.6 or less is reached to obtain an acidified milk product; and The conversion of citric acid to acetic acid in the dairy product is delayed during storage compared to an acidified dairy product made without the addition of one or more sweeteners.

[0008] In one embodiment, the present disclosure relates to a method wherein the lactose-deficient lactic acid bacteria in step (c) is a strain of the genus Lacticaseibacillus.

[0009] In one embodiment, the present disclosure relates to a method wherein the strain of Bacillus lactis is selected from the species rhamnosus, casei, and paracasei.

[0010] In one embodiment, the present disclosure relates to a method, wherein the lactose-deficient lactic acid bacteria strain in step (c) is selected from one or more of ATCC53103, CNCM I-2116, and DSM16572.

[0011] In one embodiment, the present disclosure relates to a method wherein lactose-deficient lactic acid bacteria in step (c) ferment the pre-acidified milk to obtain a fermented milk product.

[0012] The term "milk" is intended to mean the lacteal secretion obtained by milking any mammalian animal, including but not limited to cows, sheep, goats, buffalo, camels, llamas, mares, deer, etc. In a preferred embodiment, the milk is cow's milk.

[0013] A "dairy base" can be any raw and / or processed milk component or other milk-derived material that can be acidified according to the methods of the present invention. Useful milk bases therefore include, but are not limited to, any solution or suspension of milk or milk-like product containing protein, such as whole milk, full-fat milk, nonfat milk, low-fat milk, skim milk, buttermilk, lactose-reduced milk, concentrated milk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, lactose crystallization mother liquor, whey protein concentrate, or cream. Of course, the milk base can be derived from any mammal, e.g., substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least a portion of the protein in the milk base is a protein naturally occurring in mammalian milk, such as casein or whey protein.

[0014] In one embodiment, the present invention relates to a method wherein the milk base is of animal origin, 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, buffalo, camels, llamas, mares, and deer, hi a preferred embodiment, the mammal is a cow.

[0015] Prior to acidification, the milk base can be homogenized and pasteurized according to methods known in the art. As used herein, "homogenization" refers to high-speed agitation and mixing to obtain a soluble suspension or emulsion. Homogenization prior to fermentation can disperse milk fat into smaller particles to prevent separation from the milk. This can be achieved by forcing the milk through small orifices at high pressure. As used herein, "pasteurization" refers to treating the milk base to reduce or remove the presence of living organisms, such as microorganisms. Pasteurization is preferably achieved by maintaining a specific temperature for a specific period of time. The specified temperature is usually achieved by heating. The temperature and time may be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0016] Mammalian-derived milk bases contain lactose as the primary carbohydrate, which is hydrolyzed to the monosaccharides glucose and galactose by lactose-metabolizing (lactose-fermenting) lactic acid bacteria. If the lactic acid bacteria are unable to metabolize lactose, i.e., lactose-deficient, or if the pre-acidification of the milk base is chemical, it is necessary to add a suitable carbohydrate to the milk base or pre-acidified milk to make at least one carbohydrate and / or monosaccharide available for the lactic acid bacteria to ferment up to the second target pH.

[0017] The term "lactose-deficient" is used in the context of the present invention to describe lactic acid bacteria that have partially or completely lost the ability to utilize lactose as a source to maintain cell viability or cell growth. Lactose-deficient bacteria are capable of metabolizing one or more carbohydrates selected from sucrose, galactose, glucose, and / or other fermentable carbohydrates. These carbohydrates are not naturally present in milk in sufficient amounts to support fermentation by lactose-deficient bacteria and therefore must be added to the milk base or pre-acidified milk.

[0018] Pre-acidification is terminated upon reaching a first target pH, which must be the same as or higher than the second target pH and can be selected to be in a pH range that allows for the possibility of further acidification to the second target pH. In one embodiment, the present disclosure relates to a method 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 in 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 approximately 4.70; 4.65; 4.60; 4.55; 4.50; 4.45; or 4.40.

[0019] In one embodiment, the present disclosure provides a method for treating a maltodextrin-containing ... For fermentation the values ​​are 4.25; 4.20; 4.15; 4.10; 4.05; 4.00; 3.90; 3.80; 3.70; 3.60; 3.50.

[0020] Temperature affects the rate of acidification and fermentation, and it is preferred to keep the temperature stable or constant during acidification. In one embodiment, the present disclosure relates to a method wherein the acidification and / or fermentation temperature is 25°C or less, 30°C or less, 35°C or less, 36°C or less, 37°C or less, 38°C or less, 39°C or less, 40°C or less, 41°C or less, 42°C or less, 43°C or less, 44°C or less, 45°C or less, or in the range of 20-45°C, 25-45°C, 30-45°C, 40-45°C, 25-40°C, 30-40°C, 35-40°C, or approximately 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C or less, 45°C.

[0021] In one embodiment, the present disclosure relates to a method wherein the one or more sweeteners are sugars and / or sugar alcohols.

[0022] In one embodiment, the disclosure relates to a method wherein the sugar is selected from fructose, galactose, glucose, and sucrose. In one embodiment, the disclosure relates to a method wherein the sugar concentration is 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, or 0.50%, or in the ranges of 0.05-0.50%, 0.10-0.40%, or 0.15-0.30%. Unless otherwise specified, percentages (%) in percentage disclosures are expressed as weight / volume (w / v), i.e., % w / v.

[0023] In one embodiment, the disclosure relates to a method wherein the sugar is fructose at a concentration ranging from 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment, the disclosure relates to a method wherein the sugar is glucose at a concentration ranging from 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment, the present disclosure relates to a method wherein the sugar is a combination of fructose and glucose, and the concentration of fructose is in the range of 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%, and the concentration of glucose is in the range of 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment, the present disclosure relates to a method wherein the sugars are 0.10% fructose and 0.10% glucose, 0.15% fructose and 0.15% glucose, 0.20% fructose and 0.20% glucose, 0.10% fructose and 0.15% glucose, 0.10% fructose and 0.20% glucose, 0.15% fructose and 0.10% glucose, or 0.20% fructose and 0.10% glucose.

[0024] In one embodiment, the present disclosure provides a sweetener comprising one or more C4 sugar alcohols [C4H 10 O4] and C5 sugar alcohols [C5H 12 O5].

[0025] In one embodiment, the disclosure relates to a method wherein the C4 sugar alcohol is erythritol, D-threitol, or L-threitol and the C5 sugar alcohol is xylitol, ribitol, D-arabitol, L-arabitol, D-lyxitol, or L-lyxitol, hi one embodiment, the disclosure relates to a method wherein the sugar alcohol concentration is 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%, or in the range of 0.5-5.0%, 1.0-4.5%, 1.5-4.0%, 2.0-3.5%, or 2.5-3.0%.

[0026] In one embodiment, the disclosure relates to a method wherein the sugar alcohol is xylitol at a concentration selected from 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%, or at a concentration in the range of 0.5-5.0%, 1.0-4.5%, 1.5-4.0%, 2.0-3.5%, or 2.5-3.0%. In one embodiment, the disclosure relates to a method wherein the sugar alcohol is xylitol at a concentration of 2.0%.

[0027] The sugar alcohol may be added as a single sugar alcohol or as a mixture of two or more sugar alcohols. In one embodiment, the present invention relates to a combination of at least one C4 sugar alcohol and at least one C5 sugar alcohol. In one embodiment, the at least one C4 sugar alcohol comprises erythritol. In one embodiment, the at least one C5 sugar alcohol comprises xylitol. In one embodiment, the present invention relates to a combination comprising or present erythritol and xylitol.

[0028] The ratio of C4:C5 sugar alcohols may vary. In one embodiment, the C4:C5 ratio is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1. In one embodiment, the ratio of erythritol to xylitol (Ery:Xyl) is selected from 1:99, 10:90, 20:80, 30:70, 40:60, 45:55, 50:50, 55:45, 60:40, 70:30, 80:20, 90:10, or 99:1.

[0029] In one embodiment, the disclosure relates to a method wherein the one or more sweeteners are a combination of a sugar alcohol and a sugar. In one embodiment, the disclosure relates to a method wherein the one or more sweeteners include a combination of xylitol and fructose and / or glucose. In one embodiment, the disclosure relates to a method wherein the one or more sweeteners include a combination of 2% xylitol and 0.1-0.2% fructose and / or 0.1-0.2% glucose.

[0030] In one embodiment, the disclosure relates to a method where one or more sweeteners are added to a milk base. In one embodiment, the disclosure relates to a method where one or more sweeteners are added to a previously acidified milk. In one embodiment, the disclosure relates to a method where one or more sweeteners are added to a heat-treated previously acidified milk.

[0031] It is clear from the present disclosure that by carefully selecting the type, concentration and combination of one or more sweeteners, the conversion of citric acid to acetic acid can be controlled, thereby providing a method for producing dairy products with improved stability in terms of flavor, gas generation, taste and mouthfeel.In one embodiment, the present disclosure relates to using one or more sweeteners to control the conversion of citric acid to acetic acid.The specific combinations disclosed in the present method also apply to the use of one or more sweeteners to control the conversion, as in the examples.

[0032] Acidification can be achieved by chemical acidification by adding an acid or a composition containing an acid. Examples of acids include, but are not limited to, acetic acid (commonly found in vinegar), adipic acid, citric acid (commonly found in lemon juice), fumaric acid, glucono-delta-lactone, hydrochloric acid, lactic acid, malic acid, phosphoric acid, succinic acid, and tartaric acid. In one embodiment, the present disclosure relates to a method in which the chemical acidification is achieved by adding an acid selected from acetic acid, adipic acid, citric acid, fumaric acid, glucono-delta-lactone, hydrochloric acid, lactic acid, malic acid, phosphoric acid, succinic acid, and tartaric acid.

[0033] When added to a milk base or previously acidified milk containing one or more substrates fermentable by lactic acid bacteria, acidification can also be the result of fermentation by lactic acid bacteria.

[0034] Acidification can also occur by chemical acidification in combination with lactic acid fermentation. Chemical acidification and lactic acid fermentation can occur in the same step or in separate steps. In one embodiment, the present invention relates to a method in which acidification is the result of both chemical acidification and lactic acid fermentation.

[0035] In one embodiment, the present invention relates to a method wherein the prior acidification of the milk base in step (a) is chemical acidification and / or fermentation with one or more lactic acid bacteria.

[0036] In one embodiment, the present invention relates to a method wherein the prior acidification of the milk base in step (a) is by fermentation with one or more lactic acid bacteria and the prior acidified milk is heat treated.

[0037] In one embodiment, the present invention relates to a method wherein the one or more lactic acid bacteria are of the genus Streptococcus, such as Streptococcus thermophilus, or of the genus Lactobacillus, such as Lactobacillus delbrueckii subsp. bulgaricus.

[0038] The one or more lactic acid bacteria used to acidify the milk base to obtain the pre-acidified milk may be lactose-deficient. Examples of suitable lactose-deficient strains may be found in WO2015 / 193459. In one embodiment, the present invention relates to a method wherein the one or more lactic acid bacteria are lactose-deficient. In one embodiment, the present invention relates to a method wherein the lactose-deficient strain is selected from the group consisting of DSM28952, DSM28953, DSM28910, DSM32600, and DSM32599.

[0039] In a second aspect, the present disclosure relates to an acidified or fermented dairy product obtained by the method described herein. Accordingly, any disclosure regarding the method may also be applied to the resulting dairy product. As used herein, the term "acidified dairy product" or "fermented dairy product" refers to a food or feed product whose production includes acidifying and / or fermenting a milk base (see above). The fermentation is carried out by lactic acid bacteria according to the present disclosure. As used herein, "acidified dairy product" and "fermented dairy product" include, but are not limited to, dairy products such as yogurt. In one embodiment, the present disclosure relates to an acidified or fermented dairy product that is a food or feed product. In one embodiment, the present disclosure relates to an acidified or fermented dairy product that is a dairy product such as yogurt (set or stirred), Greek yogurt, fruit yogurt, and yogurt-based beverages such as drinking yogurt, buttermilk, kefir, labneh, or quark. Preferably, the acidified or fermented dairy product is yogurt.

[0040] In this context, the term "starter" refers to a culture that is a preparation (composition) of one or more bacterial strains (e.g., lactic acid bacteria strains) to help initiate the fermentation process in the production of various food, feed, beverage, and other fermented products. In this context, "yogurt starter" refers to a bacterial culture containing one or more Lactobacillus bulgaricus strains and one or more Streptococcus thermophilus strains. As used herein, "yogurt" refers to a fermented dairy product obtained by inoculating a milk base with a composition containing Lactobacillus bulgaricus and Streptococcus thermophilus strains and fermenting the mixture.

[0041] In one embodiment, the disclosure relates to an acidified or fermented milk product that is a shelf-stable product. The term "shelf-stable product" or "shelf-stable yogurt" refers to a product that is suitable for storage at room temperature for a period of time. The shelf life is 1 to 12 months, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The term "ambient temperature" or "room temperature" in this context refers to a temperature above 10°C, 15°C, 20°C, or 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, or 20-30°C.

[0042] In one embodiment, the present disclosure relates to an acidified or fermented milk product, which is a post-pasteurized product, such as, for example, post-pasteurized yogurt (PPY). The term "post-pasteurized product" or "post-pasteurized yogurt" refers to a product that has been heat-treated (pasteurized) after the acidification / fermentation step.

[0043] Acidified or fermented dairy products typically contain protein levels of 2.0-3.5% w / w. Acidified or fermented dairy products may be low-protein products with protein levels of 1.0-2.0% w / w. Alternatively, acidified or fermented dairy products may be high-protein products with protein levels greater than 3.5% w / w to 5.1% w / w, e.g., 3.5-5.1%, 3.5-10.5%, or 5.1-10.5% w / w. The protein may be derived from milk, such as whey or casein.

[0044] The product obtained by the method of the present disclosure may contain additional ingredients. For example, if the sweetness is not sufficiently high, additional sweeteners may be added, such as artificial sweeteners such as aspartame, acesulfame K (Ace-K), sucralose, neotame, advantame, saccharin, or sweet mixtures such as preparations containing fruit. In one embodiment of the present disclosure, the product further contains an additional sweetening component. In one embodiment, the sweetening component is an artificial sweetener or a sweetening mixture. In one embodiment, the sweetening component is selected from aspartame, acesulfame K (Ace-K), sucralose, neotame, advantame, and saccharin. In one embodiment, the sweetening mixture is a preparation containing fruit.

[0045] The method for producing a fermented product described in this disclosure is suitable for producing a product containing probiotic lactic acid bacteria.

[0046] In one embodiment, the present disclosure provides a method for preparing a lactic acid bacteria product comprising the step of culturing one or more lactic acid bacteria of the second culture, the step of culturing one or more lactic acid bacteria of the genus Lactobacillus, such as Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Lactobacillus johnsonii. johnsonii, and the genus Bifidobacterium, such as Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum, Bifidobacterium infantis, or S.The method relates to a method in which the bacterium is selected from the group consisting of Streptococcus spp., such as B. thermophilus.

[0047] The intake of probiotic bacteria is believed to be beneficial to an individual's health. Therefore, it is desirable that a certain amount of live probiotic bacteria is contained in the fermented dairy product. In one embodiment, the present invention relates to a method in which one or more species of lactic acid bacteria in the second culture are probiotic bacteria.

[0048] In particular, probiotic lactic acid bacteria are lactose-deficient (lactose negative). The effectiveness of probiotic lactic acid bacteria depends on the number of bacteria present in the fermented product, i.e., the number of bacteria, expressed as colony-forming units per gram of fermented product, i.e., cfu / g. In one embodiment of the present disclosure, the amount of probiotic lactic acid bacteria after storage of the fermented product at room temperature for 3 months is at least 1E+05 cfu / g, at least 5E+05 cfu / g, at least 1E+06 cfu / g, at least 5E+06 cfu / g, at least 1E+07 cfu / g, at least 5E+07 cfu / g, at least 1E+08 cfu / g, at least 5E+08 cfu / g, at least 1E+09 cfu / g, at least 5E+09 cfu / g, at least 1E+10 cfu / g, at least 5E+10 cfu / g, at least 1E+11 cfu / g, at least 5E+11 cfu / g, at least 1E+12 cfu / g, or at least 5E+12 cfu / g. In one embodiment, the ambient temperature is 25°C. In another embodiment, the ambient temperature is 37°C. In a preferred embodiment, the lactic acid bacteria count is 5E+06 cfu / g or more after 6 months of storage at 25° C. In another preferred embodiment, the lactic acid bacteria count is 5E+06 cfu / g or more after 4 months of storage at 37° C. [Example]

[0049] Example 1 Citric acid consumption and acetic acid production during the shelf life of post-pasteurized yogurt containing LGG®.

[0050] The conversion of citric acid to acetic acid by a Lactobacillus rhamnoces strain (F-DVS LGG®, Chr. Hansen) in post-pasteurized yogurt (PPY) during its shelf life was investigated.

[0051] Briefly, PPY was produced as follows: a milk base containing 3.2% fat, 3.1% protein, 0.78% sucrose, 1.5% modified starch (Clearam CJ 5025, Roquette), 0.1% pectin (106-AS YA, CP Kelco), and 3.5% maltitol (SweetPearl® P200, Roquette) and 0.1% erythritol (Zerosodium EDTA) as sugar alcohols. TM The milk base, containing 1% erythritol 16952 (Cargill), was heat-treated at 134°C for 4 seconds and then cooled to 5°C. The milk base was fermented with F-DVS Acidifix 1.0 (Chr. Hansen) at 43°C to an initial target pH of 4.45. The fermented milk was heat-treated at 75°C for 20 seconds, aseptically filled into cups, and refrigerated at 6°C. For the second fermentation, F-DVS LGG® (Chr. Hansen) was diluted with B-milk (skim milk powder reconstituted with 9.5% MSNF and heat-treated at 99°C for 30 minutes) and inoculated at 1 mL / 100 mL cup to a final concentration of 5.3E+06 CFU / g yogurt. The LGG® fermentation was carried out at 25°C for 72 hours to reach a second target pH of ≤4.41. No citric acid consumption was observed during the second fermentation. Samples were stored at 25° C. or 37° C. The concentrations of citric acid and acetic acid were tracked over time (see table below).

[0052] [Table 1]

[0053] Example 2 Inhibitory Effects of Erythritol and Maltitol on the Conversion of Citric Acid by LGG®

[0054] The effect of erythritol (ERY) and / or maltitol (MAL) on the conversion of citric acid to acetic acid in pre-acidified milk supplemented with different concentrations of fructose (FRU), galactose (GAL), and / or sucrose (SUC) was investigated.

[0055] To avoid interactions with the lactic acid concentration produced by LGG® during growth, pre-acidified milk was prepared at pH 4.5 by adding 3 mL of 5 M hydrochloric acid to 210 mL of milk. LGG® was incubated in 2 mL of each milk in 96 deep-well plates at 37°C for 3 days after analyzing the concentrations of citric acid (data not shown) and acetic acid (see table below) in the dairy products as shown in the table below.

[0056] [Table 2]

[0057] Example 3 The decrease in CO2 production correlates with the decrease in acetate production.

[0058] The correlation between CO2 bubble formation, citric acid and acetic acid concentrations, and the presence of different concentrations of galactose and sucrose in pre-acidified milk fermented with LGG® was investigated.

[0059] Pre-acidified milk at pH 4.5 was prepared by adding 3 mL of 5 M hydrochloric acid to 210 mL of milk. After analyzing the citric acid and acetic acid concentrations in the milk, LGG® dispensed 2 mL of each milk into a 96-deep-well plate and incubated it in replicates for 40 hours at 37°C. Additionally, the bottom of the plate was visually inspected four times to quantify bubble (CO2) formation.

[0060] [Table 3A]

[0061] [Table 3B]

[0062] Example 4 Effect of available sugars and pH on citrate utilization in LGG®

[0063] The effect of adding different sugars to milk on citric acid consumption by LGG® was investigated. Sugars such as glucose (GLC), galactose (GAL), or fructose (FRU) were added to milk and pre-acidified milk. Pre-acidified milk at pH 4.5 was prepared by adding 3 mL of 5 M hydrochloric acid to 210 mL of milk. LGG® was administered to 2 mL of each milk in a 96-deep-well plate and incubated at 37°C for 40 hours. The plates were stored at room temperature, and citric acid concentrations were measured by HPLC after 0, 16, 23, 40, and 500 hours. The detection limit was 0.04 g / L.

[0064] LGG consumed all of the citric acid in the samples after 40 hours in milk (Table 4A). However, in pre-acidified milk, LGG consumed very little or no citric acid in the presence of low concentrations of glucose or fructose (0.1-0.2%) (Table 4B). In pre-acidified milk, citric acid was completely consumed without added sugars or at low galactose concentrations (0.1%). At high galactose concentrations (≥0.5%), citric acid consumption decreased in pre-acidified milk (Table 4B). This data suggests that adequate sugar availability, along with pH, ​​plays a major role in citric acid consumption by LGG® in milk. Citric acid consumption resulted in the production of acetic acid in all samples (Tables 4C and 4D).

[0065] [Table 4A]

[0066] [Table 4B]

[0067] [Table 4C]

[0068] [Table 4D]

[0069] Example 5 Effect of sugar alcohols on citric acid consumption in LGG®

[0070] LGG® was incubated at 37°C using pre-acidified milk supplemented with different sugars and / or sugar alcohols at the concentrations shown in the table below. The sugars were glucose (GLC), galactose (GAL), or fructose (FRU), and the sugar alcohols were erythritol (ERY) or xylitol (XYL). The fermented milk products were stored at 37°C and the citric acid concentration was measured after 4 days and 1 month of storage.

[0071] Surprisingly, both erythritol and xylitol were found to affect citric acid consumption after 4 days of incubation at 37°C. After 1 month of storage at 37°C, only the combination of xylitol and 0.2% fructose resulted in zero citric acid consumption. This result demonstrates that sweeteners can be used to control citric acid consumption, i.e., by selecting the type and amount of sugar and / or sugar alcohol.

[0072] [Table 5]

[0073] Example 6 Effect of sweeteners on lactose-negative strains of different species

[0074] The effects of different sweeteners on the conversion of citric acid to acetic acid by two lactose-negative strains of Bacillus lacticasei were investigated.

[0075] Pre-acidified milk (pH 4.5) containing 210 mL of milk supplemented with different concentrations of fructose and 3 mL of 5 M hydrochloric acid was inoculated with L. rhamnosus (LGG) or L. paracasei (DSM16572). Each strain was cultured in 2 mL of each milk in a 96-deep-well plate at 37°C for 3 days. The milk was analyzed for citric acid (data not shown) and acetic acid (Table 6). The detection limit was 0.15 g / L.

[0076] [Table 6]

[0077] Example 7 Effect of additional sugars added after primary fermentation

[0078] The results of Example 6 demonstrated that the addition of additional sugar during secondary fermentation delayed the conversion of citric acid to acetic acid. Furthermore, to investigate the effect of adding additional sugar after primary fermentation, the following process was employed: Milk bases (MB1-MB4) were prepared and pasteurized at 134°C for 4 seconds. The milk bases were inoculated with 100 U / 1000 L of F-DVS YoFlex® Acidifix® 1.0 (Chr. Hansen A / S) and fermented at 43°C until the initial approximate target pH of 4.45 was reached. The fermented milk was heat-treated at 75°C for 25 seconds. Samples were stored refrigerated in sterile cups / bottles until use. Samples were pre-equilibrated to 25°C, and each cup was aseptically spiked with 0.009% F-DVS nu-trish® LGG® AI (Chr. Hansen A / S) + / - 0.1% fructose. The samples were thoroughly shaken and mixed (using a sterile spoon or mixer for bottles) to remove air and until homogenous. Secondary fermentation was carried out at 25°C until the pH reached 4.3 or less. At the end of secondary fermentation (day 1), samples were stored at 25°C or 37°C and then analyzed.

[0079] Milk bases 1 to 4 were produced by adding sugar alcohols according to the following table to the following composition: 92.1% milk, 1.4% H2O, 0.9% sucrose, 1.5% modified starch, and 0.1% pectin.

[0080] [Table 7-1]

[0081] [Table 7-2]

[0082] Cell counts, measured as colony forming units (cfu) per gram, were followed over time for samples stored at either 25°C or 37°C.

[0083] [Table 8a]

[0084] [Table 8b]

[0085] The post-acidification of the samples was followed over time. The combination of LGG with fermentable sugars can result in undesirable post-acidification. The effect of fructose addition was dependent on the milk base and, therefore, the presence of sugar alcohols. From the table below, it can be seen that the post-acidification when fructose was added with LGG was lowest for MB3 (Ery+Xyl) and MB4 (Ery+Mal) measured at both 25°C and 37°C.

[0086] [Table 9a]

[0087] [Table 9b]

[0088] The evolution of CO2 concentration in the samples was tracked over time. Briefly, samples were placed in glass bottles with rubber stoppers. The caps were pierced with a needle, and CO2 in the headspace of the bottles was measured using a portable headspace gas analyzer, Dansensor® CheckPoint3.

[0089] The results in the table below show that adding fructose along with LGG can delay the evolution of CO2, this delay depends on the milk base and therefore the presence of sugar alcohols.

[0090] [Table 10a]

[0091] [Table 10b]

[0092] The conversion of citric acid to acetic acid in the samples was followed over time.

[0093] The results in the table below show that adding fructose along with LGG can delay the conversion of citric acid to acetic acid at day 28. This delay depends on the storage temperature, the milk base and the presence of sugar alcohols.

[0094] [Table 11a]

[0095] [Table 11b]

[0096] [Table 11c]

[0097] [Table 11d]

[0098] Example 8 Effect of pH on the efficacy of sugar alcohols

[0099] The effect of pH on the conversion of citric acid to acetic acid by Bacillus lacticasei strains in the presence and absence of sweeteners was investigated. Pre-acidified milk was prepared at four pH levels (4.5, 5.0, 5.5, and 6.0) by adding HCl to milk supplemented with 0.1% glucose (GLC) and + / - 2% xylitol (XYL). The pre-acidified milk was inoculated with Bacillus lacticasei rhamnosus (F-DVS LGG®, Chr. Hansen) or Bacillus lacticasei paracasei (DSM16572). Two mL of each milk was cultured at 37°C for 3 days in a 96-deep-well plate, and the samples were then stored at 30°C. The citric acid and acetic acid contents (g / L) in the milk were analyzed by HPLC after 3, 14, and 28 days.

[0100] The results in the table below are consistent with those in Example 4, showing that citric acid consumption in unacidified milk is faster than that in pre-acidified milk. Furthermore, the effect of sweeteners was observed after 28 days at pH 4.5, but only after 3 days at pH 5. Above pH 5.0, no effect of sweeteners was observed.

[0101] [Table 12a]

[0102] [Table 12b]

[0103] The present invention has been described with reference to various embodiments, aspects, examples, etc. These elements should not be construed independently of one another. Thus, the present disclosure contemplates combining two or more of the embodiments, aspects, examples, etc. All embodiments described herein are intended to be included within the scope of the disclosed invention. All embodiments described herein are intended to be included within the scope of the disclosed invention. These and other embodiments of the present invention will be readily apparent to those skilled in the art from the following detailed description of the preferred embodiments with reference to the entire specification, but the present invention is not limited to the specific preferred embodiments disclosed.

Claims

1. 1. A method for producing a fermented milk product, comprising the steps of: a) adding one or more sweeteners to a milk base or a previously acidified milk and acidifying the milk base to reach a first target pH of 4.7 or less to obtain a previously acidified milk; b) optionally heat treating the pre-acidified milk; c) inoculating the previously acidified milk with lactose-deficient lactic acid bacteria until a second target pH of 4.6 or less is reached to obtain a milk product, wherein the first target pH is equal to or higher than the second target pH; and wherein the conversion of citric acid to acetic acid in the dairy product is delayed during storage compared to a dairy product produced without the one or more sweeteners.

2. 2. The method according to claim 1, wherein the lactose-deficient lactic acid bacteria in step (c) are strains of the genus Lacticaseibacillus.

3. 3. The method of claim 2, wherein the strain of Bacillus lacticasei is selected from the group consisting of Rhamnosus sp., Casei sp., and Paracasei sp.

4. 4. The method according to claim 1, wherein the lactose-deficient lactic acid bacteria in step (c) ferment the pre-acidified milk to obtain a fermented milk product.

5. The method according to any one of claims 1 to 4, wherein the one or more sweeteners are sugars and / or sugar alcohols.

6. 6. The method of claim 5, wherein the sugar is selected from fructose, galactose, glucose, and sucrose.

7. 7. The method of claim 5, wherein the sugar concentration is 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, or 0.50%, or in the range of 0.05-0.50%, 0.10-0.40%, or 0.15-0.30%.

8. 6. The method of claim 5, wherein the one or more sweeteners are C4 sugar alcohols [C ４ H １０ O ４ ] and C5 sugar alcohol [C ５ H １２ O ５ The sugar alcohol is selected from the group consisting of:

9. 9. The method of claim 8, wherein the C4 sugar alcohol is erythritol, D-threitol, or L-threitol, and the C5 sugar alcohol is xylitol, ribitol, D-arabitol, L-arabitol, D-lyxitol, or L-lyxitol.

10. 10. The method of any one of claims 5, 8 and 9, wherein the concentration of the sugar alcohol is 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%, or in the range of 0.5-5.0%, 1.0-4.5%, 1.5-4.0%, 2.0-3.5%, or 2.5-3.0%.

11. 11. The method according to any one of claims 1 to 10, wherein the prior acidification of the milk base in step (a) is chemical acidification or by fermentation with one or more lactic acid bacteria.

12. 12. The method of claim 11, wherein the pre-acidification of the milk base in step (a) is by fermentation with one or more lactic acid bacteria, and the pre-acidified milk is heat treated.

13. 13. The method according to claim 11 or 12, wherein the one or more lactic acid bacteria are of the genus Streptococcus, such as S. thermophilus, or of the genus Lactobacillus, such as L. delbrueckii subsp. bulgaricus.

14. 14. The method according to claim 11 or 13, wherein the one or more lactic acid bacteria are lactose deficient.

15. 15. The method of claim 14, wherein the lactose-deficient strain is selected from the group consisting of DSM28952, DSM28953, DSM28910, DSM32600, and DSM32599.

16. An acidified or fermented milk product obtainable by the method according to any one of claims 1 to 15.

17. 17. The dairy product according to any one of claims 1 to 16, wherein the product is a dairy product such as yogurt (set or stirred), a yogurt-based product such as Greek yogurt, fruit yogurt, and a yogurt-based drink such as drinking yogurt, buttermilk, kefir, labneh, or quark.

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