EP4672965A1

EP4672965A1 - Method for producing a fermented milk product with improved stability

Info

Publication number: EP4672965A1
Application number: EP24706772.1A
Authority: EP
Inventors: Solvej PRECHT; Mette OEHRSTROEM; Hui Han; Victoria PREBNER
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-02-28
Filing date: 2024-02-27
Publication date: 2026-01-07
Also published as: MX2025009558A; JP2026505994A; WO2024180057A1; CN120475902A

Abstract

The present disclosure is in the field of dairy technology. It relates to a method for producing acidified or fermented milk products with improved stability. The disclosure also provides an acidified or fermented milk product produced therefrom.

Description

METHOD FOR PRODUCING A FERMENTED MILK PRODUCT WITH IMPROVED STABILITY

FIELD

The present disclosure generally relates to the field of acidified or fermented milk. In particular to a method for producing an acidified or fermented milk product with improved stability, and acidified or fermented milk products made by said method.

BACKGROUND

Flavor and gas formation are important attributes affecting customer acceptance of acidified or fermented milk products. Due to lack of cooling or temperature variation during transport and storage the flavor and/or gas formation of fermented milk products may change. In addition to change in the taste and mouth feel of the product, gas formation may also result in increased pressure, swelling and potentially blowing of the final product package.

Thus, there is a need for improved methods for producing acidified or fermented milk products where the final product remains stable during storage. In particular when a cold chain is not possible or is at risk of being broken or partially disrupted.

SUMMARY

In a first aspect the present disclosure provides a method for producing an acidified or fermented milk product comprising the steps of:

(a) adding one or more sweetener to a milk base or a pre-acidified milk wherein the milk base is acidified until a first target pH of no more than 4.7 is reached to obtain the pre-acidified milk;

(b) optionally heat treating the pre-acidified milk;

(c) inoculate the pre-acidified milk with a lactose-deficient lactic acid bacteria, until a second target pH of no more than 4.6 is reached to obtain the milk product; and wherein conversion of citric acid to acetic acid in the milk product is delayed during storage as compared to a milk product produced without the one or more sweetener.

In a second embodiment the disclosure provides an acidified or fermented milk product obtained by the method. DETAILED DESCRIPTION OF THE INVENTION

As described infra it has surprisingly been found that off-flavor and gas formation during shelf life of an acidified or fermented milk product can be controlled by delaying the conversion of citric acid to acetic acid. The disclosure provides a solution where said conversion is controlled by addition of specific sweetener as described more in detail supra.

This disclosure relates to a method for producing an acidified or fermented milk product comprising the steps of:

(b) optionally heat treating the pre-acidified milk;

(c) inoculate the pre-acidified milk with a lactose-deficient lactic acid bacteria, until a second target pH of no more than 4.6 is reached to obtain the acidified milk product; and wherein conversion of citric acid to acetic acid in the milk product is delayed during storage as compared to an acidified milk product produced without the one or more sweetener.

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

In one embodiment the disclosure relates to the method, wherein the strain of the genus lacticaseibacillus is selected from the species rhamnosus, casei, and paracasei.

In one embodiment the disclosure relates to the method, wherein the lactose-deficient lactic acid bacteria strain in step (c) is one or more selected from ATCC53103, CNCM 1-2116, and DSM16572.

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

The term "milk" is to be understood as the lacteal secretion obtained by milking an animal such as any mammal including but not limited to cow, sheep, goat, buffalo, camel, lama, mare, and deer. In a preferred embodiment, the milk is cow's milk.

A "milk base" may be any raw and/or processed milk ingredient or other material derived from milk that can be subjected to acidification according to the method of the invention. Thus, useful milk bases include, but are not limited to, solutions or suspensions of any milk or milk like products comprising protein, such as whole milk, full fat milk, fat-free milk, lowfat milk, skim milk, buttermilk, lactose-reduced milk, concentrated milk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquid from crystallization of lactose, whey protein concentrate, or cream. Obviously, the milk base may originate from any mammal, e.g. being substantially pure mammalian milk, or reconstituted milk powder. Preferably, at least part of the protein in the milk base are proteins naturally occurring in mammalian milk, such as e.g. casein or whey protein.

In one embodiment the disclosure relates to the method, wherein the milk base is derived from an animal such as e.g. a mammal. In one embodiment the disclosure relates to the method, wherein the mammal is selected from the group consisting of cow, sheep, goat, buffalo, camel, lama, mare, and deer. In a preferred embodiment, the mammal is a cow.

Prior to acidification, the milk base may be homogenized and pasteurized according to methods known in the art. "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices. "Pasteurizing" as used herein means treatment of the milk base to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

The milk base derived from mammals comprises lactose as the main carbohydrate. The lactose is hydrolyzed by lactose-metabolizing (lactose-positive) lactic acid bacteria into the monosaccharides glucose and galactose. If the lactic acid bacteria is not able to metabolize lactose i.e. is lactose- deficient or if pre-acidification of the milk base is a chemical acidification it may be necessary to add a suitable carbohydrate to the milk base or the pre-acidified milk to obtain at least one carbohydrate and/or monosaccharide available for the lactic acid bacteria for the fermentation to the second target pH.

The term "lactose-deficient" are used in the context of the present invention to characterize lactic acid bacteria which partly or completely have lost the ability to use lactose as a source for maintaining 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. Since these carbohydrates are not naturally present in sufficient amounts in milk to support fermentation by lactose-deficient bacteria they must be added to the milk base or the preacidified milk. The pre-acidification is terminated when the first target pH has been reached. The first target pH must be identical or higher than the second target pH and may be selected to provide a pH range allowing for a potential further acidification to a second target pH. In one embodiment the disclosure relates to the method wherein the first target pH is no more 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 is about 4.70: 4.65; 4.60; 4.55; 4.50; 4.45; or 4.40.

In one embodiment the disclosure relates a fermentation wherein the second target pH is no more 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 is in the range of pH 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; 4.00-3.50; 3.95-3.50; 3.90-3.55; 3.85-3.60; 3.80-3.65; 3.75-3.60; or is 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.

Temperature affects the speed of acidification and fermentation and should preferably be kept stable or constant at a defined temperature during the acidification. In one embodiment the disclosure relates to the method, wherein the acidification and/or fermentation temperature is no more than 25; 30; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45°C; or in 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.

In one embodiment the disclosure relates to the method, wherein the one or more sweetener is a sugar and/or a sugar alcohol.

In one embodiment the disclosure relates to the method, wherein the sugar is selected from fructose, galactose, glucose, and sucrose. In one embodiment the disclosure relates to the method, wherein the concentration of sugar 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 range of 0.05-0.50%, 0.10-0.40%, or 0.15-0.30%. Unless otherwise indicated percentages (%) in the precent disclosure are given in weight/volume (w/v), i.e. %\N/\

In one embodiment the disclosure relates to the method, wherein the sugar is fructose in a concentration of 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or in the range of 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment the disclosure relates to the method, wherein the sugar is glucose in a concentration of 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or in the range of 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment the disclosure relates to the method, wherein the sugar is a combination of fructose and glucose wherein the concentration of fructose is 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or in the range of 0.001%-0.10%, 0.10-0.25%, 0.15-0.20% and the concentration of glucose is 0.001%, 0.005%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, or in the range of 0.001%-0.10%, 0.10-0.25%, 0.15-0.20%. In one embodiment the disclosure relates to the method, wherein the sugar is 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.

In one embodiment the disclosure relates to the method, wherein the one or more sweetener is a sugar alcohol selected from a C4 sugar alcohol [C4H10O4] and a C5 sugar alcohol [C5H12O5].

In one embodiment the disclosure relates to the 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. In one embodiment the disclosure relates to the method, wherein the concentration of sugar alcohol 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%.

In one embodiment the disclosure relates to the method, wherein the sugar alcohol is xylitol in 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 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 the method, wherein the sugar alcohol is xylitol in a concentration of 2.0%.

Sugar alcohol may be added as a single sugar alcohol or added as a mixture of two or more sugar alcohols. In one embodiment the disclosure 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 disclosure relates to a combination comprising or existing of Erythritol and Xylitol.

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 Erythritol to Xylitol (Ery:Xyl) 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 disclosure relates to the method, wherein the one or more sweetener is a combination of a sugar alcohol and a sugar. In one embodiment the disclosure relates to the method, wherein the one or more sweetener comprise xylitol in combination with fructose and/or glucose. In one embodiment the disclosure relates to the method, wherein the one or more sweetener comprise 2% xylitol in combination with 0.1-0.2% fructose and/or 0.1-0.2% glucose.

In one embodiment the disclosure relates to the method wherein the one or more sweetener is added to the milk base. In one embodiment the disclosure relates to the method wherein the one or more sweetener is added to the pre-acidified milk. In one embodiment the disclosure relates to the method wherein the one or more sweetener is added to the heat treated and pre-acidified milk.

From the present disclosure it is apparent that by carefully selecting the type, concentration and combination of one or more sweetener it is possible to control conversion of citric acid to acetic acid thereby providing the method for producing a milk product with improved stability with respect to flavor, gas formation, taste, and mouth feel. In one embodiment the disclosure relates to use of one or more sweetener to control conversion of citric acid to acetic acid. The specific combinations disclosed for the method as well as in the examples also applies to the use of one or more sweetener for controlling said conversion.

Acidification may be brought about by chemical acidification by adding an acid or compositions comprising 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 disclosure relates to the method, wherein chemical acidification is due to addition of 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.

Acidification may also be a result of fermentation by lactic acid bacteria(s) when added to a milk base or a pre-acidified milk comprising one or more substrate(s) fermentable by the lactic acid bacteria(s).

Acidification may also be a result of chemical acidification in combination with fermentation by lactic acid bacteria. Chemical acidification and fermentation by lactic acid bacteria may be conducted in the same step or in separate steps. In one embodiment the disclosure relates to the method wherein the acidification is a result of both chemical acidification and fermentation by lactic acid bacteria.

In one embodiment the disclosure relates to the method, wherein pre-acidification of the milk base in step (a) is a chemical acidification and/or is due to fermentation by one or more lactic acid bacteria.

In one embodiment the disclosure relates to the method, wherein pre-acidification of the milk base in step (a) is due to fermentation by one or more lactic acid bacteria and wherein the pre-acidified milk is heat treated.

In one embodiment the disclosure relates to the method, wherein the one or more lactic acid bacteria is of the genus Streptococcus such as S. thermophilus, or the genus Lactobacillus such as L. delbrueckii subsp. bulgaricus. The one or more lactic acid bacteria used for acidifying the milk base to obtain an pre-acidified milk may be lactose-deficient. Examples of suitable lactose-deficient strains may be found in WO2015/193459. In one embodiment the disclosure relates to the method wherein the one or more lactic acid bacteria is lactose-deficient. In one embodiment the invention relates to the method wherein the lactose-deficient strain is selected from the group consisting of: DSM28952, DSM28953, DSM28910, DSM32600, and DSM32599.

In a second aspect the disclosure relates to an acidified or fermented milk product obtained by the method described herein. Thus any disclosure made for the method may also be applied to the milk product obtained. The term "acidified milk product" or "fermented milk product" as used herein refers to a food or feed product wherein the preparation of the food or feed product involves acidification and/or fermentation of a milk base (c.f. supra). Fermentation is conducted by lactic acid bacteria according to the disclosure. "Acidified milk product" and "fermented milk product" as used herein includes but is not limited to dairy products such as yogurt. In one embodiment the disclosure relates to an acidified or fermented milk product which is a food or feed product. In one embodiment the disclosure relates to an acidified or fermented milk product which is a dairy product such as Yogurt (set or stirred); Greek yogurt; Yogurt based products such as fruit yogurt, and yogurt based beverages such as drinking yogurt; Buttermilk; Kefir; Labneh, Quark. Preferably, the acidified or fermented milk product is a yogurt.

In the present context the term "starter culture" is a culture which is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to assist the beginning of the fermentation process in preparation of fermented products such as various foods, feeds and beverages. In the present context, a "yoghurt starter culture" is a bacterial culture which comprises one or more Lactobacillus bulgaricus strains and one or more Streptococcus thermophilus strains. In accordance herewith, a "yoghurt" refers to a fermented milk product obtainable by inoculating and fermenting a milk base with a composition comprising a Lactobacillus bulgaricus and a Streptoccocus thermophilus strain.

In one embodiment the disclosure relates to the acidified or fermented milk product wherein said product is an ambient storage product. The term "ambient storage product" or "ambient storage yogurt" means a product, which is suitable for storage at ambient temperature for a period of time. Storage period may be between 1-12 month, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 month. The term "ambient temperature" or alternatively "room temperature" in the present context means a temperature above 10°C; 15°C; 20°C; 25°C; or 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. In one embodiment the disclosure relates to the acidified or fermented milk product wherein said product is a post-pasteurized product, such as a post-pasteurized yogurt (PPY). The term "postpasteurized product" or "post-pasteurized yogurt" means a product which has been heat treated (pasteurized) after an acidification/fermentation step.

The acidified or fermented milk product typically contains protein in a level of between 2.0-3.5% w/w. The acidified or fermented milk product may be a low protein product with a protein level of between 1.0-2.0% w/w. Alternatively, the acidified or fermented milk product may be a high protein product with a protein level of above 3.5, or 5.1% w/w e.g between 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.

The product obtained by the method in the present disclosure may comprise further ingredients. For example if the sweetness is not sufficiently high then further sweetener such as an artificial sweetener such as e.g. Aspartame, Acesulfame potassium (Ace-K), Sucralose, Neotame, Advantame, and Saccharin, or sweet mixtures such as preparations comprising fruit may be added. In one embodiment of the present disclosure the product further comprises additional sweetening ingredients. In one embodiment the sweetening ingredient is an artificial sweetener or a sweet mixture. In one embodiment the sweetening ingredient is selected from Aspartame, Acesulfame potassium (Ace-K), Sucralose, Neotame, Advantame, and Saccharin. In one embodiment the sweet mixture is a preparation comprising fruit.

The method of producing fermented products described in the present disclosure is suitable for producing products comprising probiotic lactic acid bacteria.

In one embodiment the disclosure relates to the method wherein the one or more lactic acid bacteria of the second culture is selected from the group consisting of 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; 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 the genus Streptococcus such as S. thermophilus.

Consumption of probiotic bacteria is considered beneficial for the health of an individual. Thus a certain amount of live probiotic bacteria in the fermented milk product is desirable. In one embodiment the invention relates to the method wherein the one or more lactic acid bacteria of the second culture is a probiotic bacteria.

In particular, the probiotic lactic acid bacteria is lactose deficient (lactose negative). The effect of the probiotic lactic acid bacteria is dependent on the amount i.e. cell count present in the fermented product. The cell count is a number provided as colony forming unit per gram of fermented product, i.e. cfu/g. In one embodiment of the present disclosure the amount of probiotic lactic acid bacteria is more than 1E+05 cfu/g, more than 5E+05 cfu/g, more than 1E+06 cfu/g, more than 5E+06 cfu/g, more than 1E+07 cfu/g, more than 5E+07 cfu/g, more than 1E+08 cfu/g, more than 5E+08 cfu/g, more than 1E+09 cfu/g, more than 5E+09 cfu/g, more than 1E+10 cfu/g, more than 5E+10 cfu/g, more than 1E+11 cfu/g, more than 5E+11 cfu/g, more than 1E+12 cfu/g, more than 5E+12 cfu/g after storage of the fermented product at ambient temperature for 3 month. In one embodiment the ambient temperature is 25°C. In another embodiment the ambient temperature is 37°C. In a preferred embodiment the amount of lactic acid bacteria is more than 5E+06 cfu/g after storage at 25°C for 6 month. In another preferred embodiment the amount of lactic acid bacteria is more than 5E+06 cfu/g after storage at 37°C for 4 month.

EXAMPLES

Example 1 - Citric acid consumption and acetic acid production during shelf life of postpasteurized yogurt comprising LGG*.

Conversion of citric acid to acetic acid by a strain Lacticaseibacillus rhamnosus (F-DVS LGG®, Chr. Hansen) in post-pasteurized yogurt (PPY) during shelf life was studied.

In brief, production of PPY was as follows. Milk base with 3.2% fat, 3.1% protein, 0.78% sucrose, 1.5% modified starch (Clearam CJ 5025, Roquette) and 0.1% pectin (106-AS YA, CP Kelco), comprising 3.5% of the sugar alcohols maltitol (SweetPearl* P200, Roquette) and 1% of erythitol (Zerose™ erythritol 16952, Cargill) was heat treated at 134°C for 4 seconds and subsequently cooled to 5°C. The milk base was fermented with F-DVS Acidifix 1.0 (Chr. Hansen) at 43°C to a first target pH 4.45. Fermented milk was heat treated at 75°C for 20 seconds and filled aseptically in cups and stored cold at 6°C . For the second fermentation F-DVS LGG* (Chr. Hansen) was diluted in B-milk (reconstituted skimmed milk with 9.5% MSNF, heat treated at 99°C for 30 min) and ImL/lOOmL cup was inoculated to give a final concentration of 5.3E+06 CFU/g yogurt. LGG* fermentation took place at 25°C for 72 hours to a second target pH <4.41. No citric acid consumption was observed during the second fermentation. Samples were stored at 25°C or 37°C. The concentration of citric acid and acetic acid were followed over time (see table below). Table 1: Concentration of Citric acid and Acetic acid (g/L) at different storage temperature and time.

Example 2 - Inhibitory effect of erythritol and maltitol on citrate conversion by LGG®.

The contribution of erythritol (ERY) and/or maltitol (MAL) on citric acid conversion to acetic acid in pre-acidified milk supplemented with different concentrations of fructose (FRU), galactose (GAL) and/or sucrose (SUC) were studied.

Pre-acidified milk pH 4.5 were prepared by adding 3mLof 5M HCI to 210mL milk to avoid interaction with the lactate concentrations produced by LGG® during growth. LGG® was grown in 2mL of the different milks as indicated in the table below in 96 deep-well plates at 37°C for three days after which the concentration of citric acid (data not shown) and acetic acid (see table below) in the milk product were analyzed.

Table 2: Erythritol and not maltitol had an impact on acetic acid concentration (g/L).

Example 3 - Reduced CO2 formation correlate with decrease in acetic acid production. We studied the correlation of CO2 bubble formation, concentration of citric acid and acetic acid, and the presence of different concentration of galactose and sucrose in pre-acidified milk fermented with LGG®.

Pre-acidified milk pH 4.5 were prepared by adding 3mL of 5M HCI to 210mL milk. LGG® was grown in 2mL of the different milks in 96 deep-well plates at 37°C for 40 hours in duplicate after which the concentration of citric acid and acetic acid content in the milk were analyzed. The bottom of the plates were furthermore inspected visually in quadruplicates to quantify air (CO2) bubble formation.

Table 3A: Correlation of CO₂ bubble formation with concentration of citric acid and acetic acid in the presence of different concentrations of galactose.

Table 3B: Correlation of CO₂ bubble formation with concentration of citric acid and acetic acid in the presence of different concentrations of fructose.

Example 4 - Influence of available sugar and pH on citric acid utilization in LGG®.

We studied the influence of adding different sugars in milk on citric acid consumption of LGG*. Milk and pre-acidified milk were supplemented with the sugars: glucose (GLC), galactose (GAL) or fructose (FRU). Pre-acidified milk pH 4.5 were prepared by adding 3mL of 5M HCI to 210mL milk. LGG* was inoculated and grown in 2mL of the different milks in 96 deep-well plates at 37°C for 40 hours. The plates were stored at room temperature and citric acid concentrations were measured at 0, 16, 23, 40, and 500 hours by HPLC. The detection limit was 0.04 g/L.

LGG consumed all citric acid in the samples after 40 hours in milk (Table 4A), whereas LGG in preacidified milk lead to no or much lower citric acid consumption in the presence of low concentrations of glucose or fructose (0.1-0.2%) (Table 4B). No addition of sugars and low concentrations of galactose (0.1%) resulted also in full consumption of citric acid in pre-acidified milk. High galactose concentrations (>0.5%) resulted in reduced consumption of citric acid in preacidified milk (Table 4B). This data suggests that the pH together with the right sugar availability play a major role in the consumption of citric acid by LGG* in milk. Citric acid consumption resulted in acetic acid formation in all samples (Table 4C, 4D).

Table 4A: Concentration of citric acid (g/L) measured during storage of milk. Table 4B: Concentration of citric acid (g/L) measured during storage of pre-acidified milk.

Table 4C: Concentration of acetic acid (g/L) measured during storage of milk.

Table 4D: Concentration of acetic acid (g/L) measured during storage of pre-acidified milk.

Example 5 - Influence of sugar alcohols on citric acid consumption on LGG®.

LGG* was grown in pre-acidified milk supplemented with different sugars and/or different sugar alcohols at 37°C at the concentrations indicated 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 concentration of citric acid was measured at 4 days and at 1 month of storage.

Surprisingly, we could identify both erythritol and xylitol to have an effect on citric acid consumption after 4 days of incubation at 37°C. After 1 month of storage at 37°C, only xylitol together with 0.2% fructose resulted in no citric acid consumption. This finding showed that we could use the sweetener to control the citric acid consumption, i.e. by selecting the type and amount of sugar and/or sugar alcohol.

Table 5: Citric acid concentrations measured during storage at 37°C. Example 6 - Influence of sweetener on lactose negative strains from different species. We studied the effect of different sweetener on the conversion of citric acid to acetic acid by lactose negative strains of two species of Lacticaseibacillus.

Pre-acidified milk pH 4.5 prepared by adding 3mL of 5M HCI to 210mL milk supplemented with different concentration of fructose were inoculated with either a Lacticaseibacillus rhamnosus strain (LGG) or a Lacticaseibacillus paracasei strain (DSM16572). Each of the strains were grown in 2mL of the different milks in 96 deep-well plates at 37°C for 3 days after which the content of both citric (data not shown) and acetic acid (Table 6) in the milk were analyzed. Detection limit was 0.15 g/L-

Table 6: Concentrations of acetic acid (g/L) in milk inoculated with different lactose-negative strains

Example 7 - Influence of additional sugar added after the first fermentation.

The result in example 6 showed that addition of additional sugar during the second fermentation delayed the conversion of citric acid to acetic acid. To further study the effect of adding additional sugar after the first fermentation the following process were used. Milk bases (MB1 to MB4) were prepared and sterilized at 134°C for 4 seconds. The Milk bases were inoculated with 100U/1000L F- DVS YoFlex® Acidifix® 1.0 (Chr. Hansen A/S) and fermented at 43°C until first approx, target pH 4.45. The fermented milks were heat treated at 75°C for 25 seconds. Samples were stored cold in sterile cups/bottles until use. Samples were equilibrated to 25°C before aseptically dosing 0.009% F-DVS nu-trish® LGG® Al (Chr. Hansen A/S) +/- 0.1% fructose into each cup. Samples were shaken thoroughly to homogeneity without introducing air (a sterile spoon or mixer were used for bottles). The second fermentation were conducted at 25°C until pH 4.3 or just below. At the end of the second fermentation (Day 1) the samples were stored at 25°C or 37°C and analyzed during the subsequent period.

Milk bases 1 to 4 were produced by adding sugar alcohol(s) according to the table below to the following composition: 92.1% Milk, 1.4% H2O, 0.9% Sucrose, 1.5% Modified starch, and 0.1% pectin.

Table 7: Sample set up.

Cell count measured as colony forming unit (cfu) pr gram (g) sample stored at 25°C or 37°C were followed over time. Table 8a: Cell count in samples stored at 25°C.

* TNTC = too numerous to count

Table 8b: Cell count in samples stored at 37°C.

* TNTC = too numerous to count

Post-acidification of the samples were followed over time. Addition of a fermentable sugar together with LGG could potentially result in undesired post-acidification. The effect of additional fructose were dependent on the milk base and thus the presence of sugar alcohol(s). From the tables below it seems that post-acidification when adding fructose together with LGG were lowest in MB3 (Ery+Xyl) and MB4 (Ery+Mal) measured at both 25°C and 37°C.

Table 9a: pH in the samples stored at 25°C.

Table 9b: pH in the samples stored at 37°C.

Development of CO2 in the samples were followed over time. In short, samples were placed in glass bottles with a rubber stopper lid. The lid was penetrated with a needle and %CC>2 in the head space of the bottle was measured using the portable Headspace Gaz Analyzer Dansensor® Checkpoints. The results in the tables below show that by adding fructose together with LGG development of CO2 may be delayed. This delay is dependent on the milk base and thus the presence of sugar alcohol(s).

Table 10a: % CO₂ in samples stored at 25°C. Table 10b: % CO₂ in samples stored at 37°C.

Citric acid conversion to acetic acid in the samples were followed over time.

The results in the tables below show that by adding fructose together with LGG conversion of citric acid to acetic acid at day 28 may be delayed. This delay is dependent on storage temperature, the milk base and thus the presence of sugar alcohol(s). It

Table Ila: Citric acid consumption in samples stored at 25°C.

Table 11b: Acetic acid production in samples stored at 25°C.

Table 11c: Citric acid consumption in samples stored at 37°C.

Table lid: Acetic acid production in samples stored at 37°C.

Example 8 - Influence of pH on the effect of sugar alcohols.

We studied the influence of pH of pre-acidified milk on citric acid to acetic acid conversion by strains of the species Lacticaseibacillus in the presence or absence of sweetener. Milk pre-acidified to four different pH (4.5; 5.0; 5.5; 6.0) were prepared by adding HCI to milk supplemented with 0.1% glucose (GLC) and +/- 2% xylitol (XYL). The pre-acidified milks were inoculated with either a Lacticaseibacillus rhamnosus strain (F-DVS LGG®, Chr. Hansen) or a Lacticaseibacillus paracasei strain (DSM 16572). Each of the strains were grown in 2mL of the different milks in 96 deep-well plates at 37°C for 3 days after which the samples were stored at 30°C. The content (g/L) of both citric acid and acetic acid in the milk were analyzed after 3, 14, and 28 days by HPLC.

The results in the tables below are in alignment with the results in Example 4 showing that citrate consumption is faster in non-acidified milk as compared to citrate consumption in pre-acidified milk. Furthermore, the effect of the sweetener could be observed after 28 days at pH 4.5, but only after 3 days at pH 5. Above pH 5.0 no effect of the sweetener was seen. Table 12a: Effect of pH on the concentration of citrate and acetate (g/L) in Milk +0.1% GLC +/- 2% XYL inoculated with LGG.

Table 12b: Effect of pH on the concentration of citrate and acetate (g/L) in Milk +0.1% GLC +/- XYL inoculated with DSM 16573.

The present invention has been described with reference to various embodiments, aspects, examples, or the like. It is not intended that these elements be read in isolation from one another.

Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like. All embodiments described herein are intended to be within the scope of the invention disclosed. These and other embodiments of the present invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the whole description, the invention not being limited to any particular preferred embodiment(s) disclosed.

Claims

1. A method for producing a fermented milk product comprising the steps of: a) adding one or more sweetener to a milk base or a pre-acidified milk wherein the milk base is acidified until a first target pH of no more than 4.7 is reached to obtain the pre-acidified milk; b) optionally heat treating the pre-acidified milk; c) inoculate the pre-acidified milk with a lactose-deficient lactic acid bacteria, until a second target pH of no more than 4.6 is reached to obtain the milk product, wherein the first target pH is identical or higher than the second target pH; and wherein conversion of citric acid to acetic acid in the milk product is delayed during storage as compared to a milk product produced without the one or more sweetener.

2. The method according to claim 1, wherein the lactose-deficient lactic acid bacteria in step (c) is a strain of the genus lacticaseibacillus.

3. The method according to claim 2, wherein the strain of the genus lacticaseibacillus is selected from the species rhamnosus, casei, and paracasei.

4. The method according to any of claims 1-3, wherein the lactose-deficient lactic acid bacteria in step (c) is fermenting the pre-acidified milk to obtain a fermented milk product.

5. The method according to any of claims 1-4, wherein the one or more sweetener is a sugar and/or a sugar alcohol.

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

7. The method according to any of claims 5-6, wherein the concentration of sugar 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. The method of claim 5, wherein the one or more sweetener is a sugar alcohol selected from a C4 sugar alcohol [C4H10O4] and a C5 sugar alcohol [C5H12O5].

9. The method according to claim 8, wherein the C4 sugar alcohol is 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. The method according to any of claims 5, 8-9, wherein the concentration of 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. The method according to any of the preceding claims, wherein pre-acidification of the milk base in step (a) is a chemical acidification or is due to fermentation by one or more lactic acid bacteria.

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

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

14. The method according to any of claims 11-13, wherein the one or more lactic acid bacteria is lactose-deficient.

15. The method according to claim 14, wherein the lactose-deficient strain is selected from the group consisting of: DSM 28952, DSM 28953, DSM 28910, DSM 32600, and DSM 32599.

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

17. The milk product according to any of claims 1-16, wherein the product is a dairy product such as Yogurt (set or stirred); Greek yogurt; Yogurt based products such as fruit yogurt, and yogurt based beverages such as drinking yogurt; Buttermilk; Kefir; Labneh, Quark.