Fermented milk ingredient

Technical field of the invention

The present invention relates to a method of preparing a fermented milk ingredient and the fermented milk material obtained by said method. In

particular, the present invention relates to a fermented milk ingredient prepared by hydrolysis of lactose and fermentation of a liquid milk material comprising a high amount of lactose and a low amount of protein. In addition, the present invention relates to a beverage comprising the fermented milk ingredient, in particular a carbonated beverage.

Background of the invention

Sweetened and flavoured beverage consumption has highly increased over the past years with carbonated beverages. For example, the consumption of carbonated soft drinks by children, teenagers and young adults has increased over the past 10 years.

In the preparation of commonly known carbonated beverages, acids are added. Acids are added both to assist in giving the carbonated beverages a distinct taste, but acids is also added in order to reduce growth of bacteria and fungi and thus to improve the shelf life. A low pH of the carbonated beverage is necessary in order to obtain a shelf life of 6 months or more. For example, phosphoric acid is often added to cola drinks, while citric acid is added to citrus drinks and other drinks to provide a tangy taste and as a preservative. Malic acid occurs naturally in apples, pears, and cherries, and is added to many noncarbonated beverages such as fruit drinks, fortified juices, sports drinks, and iced teas because it enhances the intrinsic flavour. Malic acid is also added to artificially sweetened carbonated beverages to intensify taste and reduce the amount of other added flavourings.

However, while adding acids to the beverages, the beverages becomes very sour and it is therefore also necessary to add sugars or other sweeteners in high amounts. These high amounts of sugar have disadvantaged effects, since the sugar results in a high calorie content and cause increased risk of carries to the teeth.

Hence, a new ingredient which can be added to beverages in order to decrease the pH of the beverages, but where the sourness of the beverages are not decreased, would be advantageous. In addition, a more natural and healthy alternative having a refreshing taste and a low pH (below 4.5) obtained from natural fermentation would be advantageous. Furthermore, a beverage made with no or a low amounts of organic acids and other additives, but comprises naturally occurring metabolites from a fermentation process would be advantageous.

Summary of the invention

Thus, an object of the present invention relates to providing a fermented milk ingredient and a method of preparing said fermented milk ingredient which can be used in the preparation of clear beverages. In addition, an object of the present invention is to provide a fermented milk ingredient that that solves the above mentioned problems of the prior art. In particular, it is an object of the present invention to provide a fermented milk ingredient and a method of preparing said fermented milk ingredient to be used in the preparation of clear carbonated beverages based on milk. In addition, it is an object to provide a clear beverage which has a low pH and has a unique fermented and refreshing taste, while not being as sour as compared to conventional unsweetened beverages where pH has been decreased by addition of acids. Furthermore, it is an object to provide a beverage which is heathy and is a natural alternative to commonly known soft drinks, i.e. reduced addition of additives. Furthermore, it is an object to prepare a beverage comprising the fermented milk ingredient that comprises flavor and aroma compounds made during the fermentation process, such as ketones, aldehydes, acids, alcohols, esters and sulphuric components.

Thus, one aspect of the invention relates to a method of preparing a fermented milk ingredient for use in the preparation of beverages comprising the following steps: a) providing a liquid milk material comprising lactose in an amount of at least 4% by weight and protein in an amount of 1% by weight or less of the liquid milk material;

b) adding lactase to said liquid milk material to provide hydrolysis of lactose in the liquid milk material;

c) adding one or more microorganisms and fermenting the liquid milk material to obtain a fermented milk ingredient;

wherein hydrolysis in step b) is before, after or simultaneously with fermentation in step c).

A further aspect of the present invention relates to a fermented milk ingredient obtainable by the method according to the invention.

Another aspect of the invention is to provide a fermented milk ingredient for use in the preparation of beverages obtainable by the following steps:

a) providing a liquid milk material comprising lactose in an amount of at least 4% by weight and protein in an amount of 1% by weight or less of the liquid milk material;

b) adding lactase to said liquid milk material to provide hydrolysis of lactose in the liquid milk material;

c) adding one or more microorganisms and fermenting the liquid milk material to obtain a fermented milk ingredient;

wherein hydrolysis in step b) is before, after or simultaneously with fermentation in step c).

Yet another aspect of the present invention is to provide a beverage comprising the fermented milk ingredient according to the invention in an amount of 5-99% by weight, a flavouring agent in an amount of 1-50% by weight, water in an amount of 0-90% by weight and optionally a pH regulating agent.

Still another aspect of the present invention is to provide a method of preparing a carbonated beverage comprising the steps of

i) mixing 5-99% by weight of the fermented milk ingredient according to the invention with 1-50% by weight of a flavouring agent, 0-90% by weight water and optionally a pH regulating agent; ii) carbonize the mixture of step i)

ii) pasteurize the carbonized mixture.

Brief description of the figures

Figure 1 shows a flow diagram of the method of preparing the fermented milk ingredient according to the present invention where hydrolysis and fermentation is in two steps.

Figure 2 shows a flow diagram of the method of preparing the fermented milk ingredient according to the present invention where hydrolysis and fermentation is in one step.

Figure 3 shows a flow diagram of the method of preparing a carbonated beverage according to the present invention.

Figure 4 shows the effect on acidification by adding a malt extract

Figure 5 shows the effect acidification by adding yeast extract and malt extract Figure 6 shows the effect of fermentation temperature on acidification

Figure 7 shows the correlation between fermentation temperature and

fermentation time to reach pH 4.5 Figure 8 shows samples of fermented milk ingredient before pasteurization prepared from liquid milk material with different amounts of protein.

Figure 9 shows samples of fermented milk ingredient after pasteurization prepared from liquid milk material with different amounts of protein.

Figure 10 shows the acidification profile of the milk ingredient by various microbial cultures.

The present invention will now be described in more detail in the following. Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

In the context of the present invention, mentioned percentages are weight/weight percentages unless otherwise states.

The term "and/or" used in the context of "X and/or Y" should be interpreted as "X", or "Y", or "X and Y".

Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 4 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the present invention shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary by the context in which the reference is made.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Method of preparing a fermented milk ingredient

One aspect of the invention relates to a method of preparing a fermented milk ingredient for use in the preparation of beverages comprising the following steps: a) providing a liquid milk material comprising lactose in an amount of at least 4% by weight and protein in an amount of 1% or less by weight of the liquid milk material;

b) adding lactase to said liquid milk material to provide hydrolysis of lactose in the liquid milk material; c) adding one or more microorganisms and fermenting the liquid milk material to obtain a fermented milk ingredient;

wherein hydrolysis in step b) is before, after or simultaneously with fermentation in step c).

In the context of the present invention, the term "liquid" in "liquid milk material" refers to a material obtained from a milk product that is in a liquid state. The solid content of the liquid may vary and the liquid may have a higher solid content than the original milk. However, the milk material is not in the form of a powder or granulate when used for hydrolysis and fermentation. The milk material may however be a powder reconstituted in water.

The term "milk material" refers in the context of the present invention to a milk material obtained from a milk product, and the milk material may for example be a fraction of milk obtained by membrane filtration of milk.

In skim milk from cows, the amount of fat is approximately 0.05% by weight, the protein content is approximately 3.6% by weight and lactose content is

approximately 4.55% by weight (total carbohydrate content is approximately 4.9% by weight). In whole milk, the fat content is about 3.4% by weight, the protein content is about 3.5% by weight, the lactose content is about 4.8% by weight, however the amounts of protein and fat in milk products may vary. The protein content in milk products may be within the range of 3.1 to 3.8% by weight and the lactose content is about 4.2 to 5.5% by weight.

The liquid milk material used in the process according to the present invention should comprise lactose in an amount of at least 4% by weight and protein in an amount of 1% by weight or less.

It is important that the protein content in the liquid milk material (measured as Nx6.38) is 1% by weight or less. Of the total protein in milk, about 80% by weight is casein and 20% by weight is whey protein. Casein, if present in high amounts, will coagulate during the fermentation process, since casein coagulates at acidic pH. The whey proteins in the milk product will not coagulate at acidic pH. However, it is still desired to have the total protein content below 1%, since some of the whey proteins may denature during heat treatment and precipitate. Thus, the total protein content of the liquid milk material should be 1% by weight or less in order to avoid coagulation and precipitation. If a milk material has a protein content above 1%, for example 3.2-3.6% as in whole milk or skim milk, the milk will coaguale and hence form clumps when the pH is decreased to below 4.5 during the fermentation step.

In a preferred embodiment of the invention, the liquid milk material comprises protein in an amount of 0.7% by weight or less based on the total weight of the liquid milk material, more preferably the liquid milk material comprises protein in an amount of 0.5% by weight or less, such as 0.4% by weight or less.

If the proteins in the liquid milk material are a combination of whey proteins and casein, it is preferred that the amount of casein is not more than 0.5% by weight of the liquid milk material, such as not more than 0.4% by weight of the liquid milk material, preferably not more than 0.3% by weight of the liquid milk material, even more preferably not more than 0.25% by weight of the liquid milk material.

In another preferred embodiment, the ratio between the casein and whey protein in the liquid milk material is 80: 20 or less, such as 70:30 or less, preferably,

60:40 or less, even more preferably 50: 50 or less.

In an embodiment of the invention, the liquid milk material is i) an ultrafiltration permeate obtained by ultrafiltration of a milk product, or ii) a nanofiltration retentate obtained by nanofiltration of said ultrafiltration permeate, or iii) a de- caseinated milk material. In a preferred embodiment, the liquid milk material is i) an ultrafiltration permeate obtained by ultrafiltration of a milk product, or ii) a nanofiltration retentate obtained by nanofiltration of said ultrafiltration permeate. In an even further preferred embodiment of the invention, the liquid milk material is a nanofiltration retentate obtained by nanofiltration of an ultrafiltration permeate. If skim milk, whole milk or another milk product is used for

ultrafiltration and nanofiltration, the ratio between casein and whey protein in the liquid milk material is typically 80: 20. The ratio of casein to whey may however be lower if the milk product is de-caseinated before ultrafiltration and nanofiltration. Ultrafiltration is preferably prepared with an ultrafiltration membrane having a cutoff in the range from 5 to 20 kDa, such as from 8 to 15 kDa, preferably about 10 kDa. The ultrafiltration membrane used may for example be a membrane from Koch Dairy Pro 10K or from Alfa Laval GR73PE. Nanofiltration is preferably prepared with an nanofiltration membrane having a cutoff in the range from 100 to 700 Da, such as from 200 to 500 Da, preferably about 300 Da. The

nanofiltration membrane used may for example be from DOW Filmtec NF or from Hydranautics Dairy NF.

In an embodiment, it is preferred that the liquid milk material comprises protein in an amount of 0.5% or less.

Protein is in the context of the present invention measured by measuring the nitrogen (N) content and measure protein as Nx6.38.

For example, the protein content can be determined by:

1) Determining the total nitrogen of the sample following ISO 8968-1/2IIDF

020-1/2-Milk - Determination of nitrogen content - Part 172:

Determination of nitrogen content using Kjeldahl method

2) Calculating the total amount of protein as: Nx6.38

In an embodiment of the invention, the liquid milk material is a de-caseinated milk. The de-caseinated milk can be obtained by microfiltration and diafiltration of milk (such as whole milk, low-fat milk, reduced fat milk, semi-skimmed milk, skim milk and butter milk) with a microfiltration membrane, such that casein is retained by the membrane and present in the retentate, while whey proteins, lactose and minerals is flushed through the membrane and collected in the permeate. When using a de-caseinated milk product as the liquid milk material, the amount of casein is less than 5% by weight of the total protein content, preferably less than 2% by weight of the total protein content. Furthermore, the casein to whey protein in de-caseinated milk as the liquid milk material is 30:70 or less, such as 30: 70 or less, preferably, 20:80 or less, even more preferably 10:90 or less. The microfiltration membrane used for making de-caseinated milk should have a cut- off in the range of from 0.05 to 03 pm, such as about 0.1 pm. In an embodiment of the invention, the protein in the liquid milk material is free of casein. By the term "free of" is meant that the liquid milk material comprise casein in an amount of 5% by weight or less based on the total protein content in the liquid milk material, such as casein in an amount of 4% by weight or less, preferably casein in an amount of 3% by weight or less, even more preferably casein in an amount of 2% by weight or less, most preferred an amount of 1% by weight or less based on the total protein content in the liquid milk material.

In addition, it is important to have a lactose content in the liquid milk material of 4% by weight or more. For the fermentation step, it is important with a substrate for the microorganisms. Either the substrate may be the lactose in the liquid milk material or more preferably, the substrate for the microorganisms is glucose obtained after hydrolysis of lactose. It is important to have a certain amount of substrate for the microorganisms in order to obtain an efficient fermentation.

In an embodiment of the invention, the amount of lactose in the liquid milk material is 6% by weight or more, such as 8% by weight or more, preferably 10% by weight or more, such as 13% by weight or more, more preferably 15% by weight or more. In a most preferred embodiment of the invention, the lactose content in the liquid milk material is 18% by weight or more.

The liquid milk material comprising lactose in an amount of at least 4% by weight and protein in an amount of 1% by weight or less can be obtained from treating a milk product such that a milk material with the given amounts of lactose and protein is obtained. The liquid milk material may for example be obtained by membrane filtration of a milk product.

In the context of the present invention, the term "milk product" refers to any product normally considered as milk, i.e. whole milk, low-fat milk, reduced fat milk, semi-skimmed milk, skim milk, butter milk, reconstituted milk powder, heat treated milk (e.g. pasteurized milk, sterilized milk, condensed milk, evaporated milk and UHT milk), raw unfiltered milk, de-caseinated milk and homogenized milk.

Furthermore, the milk product used for obtaining the liquid milk material according to the present invention may be based on milk from mammals such as cows, buffalos, goats, sheep, yaks, pigs, camels, horses, ewes, mares or mixtures thereof. In a preferred embodiment of the present invention, the milk product is based on milk from cows.

The terms "milk product" and "liquid milk material" do not comprise whey. Whey is the liquid remaining after milk has been curdled and strained in connection with the production of cheese and therefore whey is a by-product of the manufacture of cheese. In the production of cheese, rennet or an edible acid is added to heated milk. This will decrease pH to below 4.6 and the milk will coagulate or curdle. Afterwards the milk solids (curds) can be separated from the liquid whey. Sweet whey is the by-product of rennet-coagulated cheese, and acid whey (also called sour whey) is the by-product of acid-coagulated cheese.

Whey will have another composition of ingredients than milk. Furthermore, the pH of whey will be reduced to about pH 4.6 during the coagulation process.

Therefore, the taste of whey will be different from the taste of milk. In the context of the present invention it is not desired to use whey, and in an embodiment of the invention, the liquid milk material does not comprise whey and the "milk product" is not whey.

In an embodiment of the present invention, the liquid milk material is selected from the group of:

i) an ultrafiltration permeate obtained by ultrafiltration of a milk product, or ii) a nanofiltration retentate obtained by nanofiltration of the ultrafiltration permeate in i),

iii) de-caseinated milk

or a combination of one or more of i), ii) and iii).

In a further embodiment of the present invention, the liquid milk material is selected from the group of:

i) an ultrafiltration permeate obtained by ultrafiltration of a milk product, or ii) a nanofiltration retentate obtained by nanofiltration of the ultrafiltration permeate in i),

or a combination of one or more of i), and ii). The liquid milk material according to i) or ii) is obtained by membrane filtration and without addition of any additional compounds. Thus, the pH of the

ultrafiltration permeate of i) and the nanofiltration retentate of ii) is similar to the starting milk.

In an embodiment of the invention, the liquid milk material is obtained by membrane filtration according to i) ultrafiltration and/or ii) nanofiltration of a milk product, where the milk product is de-caseinated milk.

Hydrolysis

In the method according to the present invention, lactase is added to the liquid milk material to provide hydrolysis of the lactose present in the liquid milk material.

With hydrolysis of lactose in the liquid milk material, lactose is split to the monosaccharides glucose and galactose. Glucose and galactose is much sweeter than lactose. With sucrose as a reference (sweetness of 1.0), lactose has a sweetness of about 0.16 while glucose has a sweetness of about 0.74-0.8 and galactose has a sweetness of about 0.60. Thus, the fermented and hydrolysed milk ingredient obtained according to the present invention has a sweet taste as compared to non-hydrolysed fermented milk ingredients.

In a preferred embodiment of the invention, hydrolysis is conducted before or simultaneously with the fermentation step and the resulting monosaccharides glucose and galactose are used as substrate for the microorganisms during fermentation. Most microorganisms metabolise monosaccharides (glucose and galactose) faster than disaccharides (lactose).

Thus, with the present invention, it is possible to provide a fermented milk ingredient where lactose from milk is split into monosaccharides, and the monosaccharides are used as substrate during the fermentation and as a sweetener.

In an embodiment of the invention, hydrolysis is carried out until lactose is reduced to 1.0 g lactose per 100 g liquid milk material or less. Hydrolysis is preferably carried out until lactose is reduced to 0.8 g lactose per 100 g liquid milk material or less, such as until lactose is reduced to 0.5 g lactose per 100 g liquid milk ingredient or less, more preferably until lactose is reduced to 0.3 g lactose per 100 g liquid milk ingredient.

Lactose content can be measured using Ion Chromatography with a suitable column (Dionex CarboPac PA210).

Lactose may also be hydrolysed in order to obtain a lactose-free product. Then hydrolysis is carried out until lactose is reduced to 0.1 g lactose per 100 g liquid milk material or less, and preferably, until lactose is reduced to 0.05 g lactose per 100 g liquid milk material or less, and even more preferably until lactose is reduced to 0.01 g lactose per 100 g liquid milk material or less. A beverage prepared by the use of a lactose-free fermented milk ingredient is suitable for people suffering from lactose intolerance and lactose allergy.

Fermentation

The present invention for preparing a fermented milk ingredient also comprises addition of one or more microorganisms to the liquid milk material. By inoculating the liquid milk material with one or more microorganisms, the substrate (lactose, glucose, galactose) is subsequently fermented. Inoculation with microorganisms and adding microorganism will in the context of the present invention be understood as the same and may be used interchangeably.

The fermentation involves fermenting with one or more predetermined

microorganisms to produce organic acids, alcohols and flavour compounds in various ratios under fermentation conditions described later on. The

microorganisms primarily uses either lactose, or glucose and/or galactose as substrate. The organic acid produced is depending on the bacteria used. The organic acid produced may for example be lactic acid, gluconic acid, acetic acid, and succinic acid.

After fermentation, and thus production of an organic acid, the acidity of the fermented milk ingredient is decreased. The decrease in acidity is depending on the amount of organic acid produced which is determined by substrate availability and fermentation efficiency. Thus, the acidity can be determined by measuring the pH of the fermented milk ingredient.

The one or more microorganisms used for the fermentation step may be selected from the group consisting of lactic acid bacteria, acetic acid bacteria and yeast. Preferably, the microorganisms used for the fermentation step is selected from the lactic acid bacteria and acetic acid bacteria.

Examples of multi-species cultures include, but is not limited to, kombucha culture and kefir culture.

Lactic acid bacteria

In the context of the present invention, any lactic acid bacteria may be used for fermentation. However, in an embodiment of the invention the lactic acid bacteria is one or more selected from the genus Lactobacillus, the genus Streptococcus, the genus Bifidobacterium , the genus Lactococcus, the genus Propionibacterium , the genus Pediococcus and the genus Leuconostoc.

Examples of lactobacillus strains that can be used for fermentation in the present invention is Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei ("preferably Lactobacillus paracasei subsp. paracasei F-19), Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus delbrueckii, (preferably

Lactobacillus delbrueckii subsp. bulgaricus ), and Lactobacillus fermentum. Other strains of Lactobacillus may also be used. The strains can be used alone or in combination.

Preferably, the lactic acid bacteria is one or more of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei , Lactobacillus delbrueckii subspecies bulgaricus and Lactobacillus fermentum, and more preferably Lactobacillus plantarum, Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus casei, and Lactobacillus paracasei, and even more preferably Lactobacillus plantarum and Lactobacillus delbrueckii subspecies bulgaricus.

An example of a Streptococcus strain that can be used in the fermentation according to the present invention is Streptococcus thermophilus, The one or more microorganism used in the present invention may also be a Lactococcus. An example of a Lactococcus strain is Lactococcus lactis, preferably Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and

Lactococcus lactis subspecies lactis biovar diacetylactis.

Examples of Bifidobacterium strains that can be used in the fermentation in the present invention include Bifidobacterium animalis, preferably subsp. lactis. Examples of Leuconostoc strains that can be used in the fermentation in the present invention includes Leuconostoc mesenteroides, preferably Leuconostoc mesenteroides subsp. cremoris.

Examples of Propionibacterium strains that can be used in the fermentation of the present invention include Propionibacterium freudenreichii, P. jensenii, P.

acidipropionici, and P. thoenii

The one or more microorganisms used in the fermentation step in the present invention may be one or more bacteria selected from the group consisting of Lactobacillus, Streptococcus, Lactococcus, Bifidobacterium and Leuconostoc strains.

Acetic acid bacteria

In an embodiment of the invention, the acetic acid bacteria is one or more selected from the group of the genus Gluconobacter and the genus Acetobacter.

Examples of Gluconobacter strains used in the present invention include

Gluconobacter oxydans. Examples of Acetobacter strains used in the present invention include Acetobacter pasteurianus.

Yeast: In an embodiment of the invention, the yeast is one or more selected from the group of the genus Candida, the genus Pichia, the genus Saccharomyces, the genus Kluveromyces, the genus Torulaspora and the genus Wickerhamomyces.

Preferably, the yeast is a strain selected from the group of Pichia kluveri,

Saccharomyces cerevisiae, Saccharomyces italicus, Candida kefyr, Kiuyveromyces marxianus, and Kiuyveromyces lactis. More preferably, the yeast is selected from the group of Pichia kluveri, Sacchraromyces cerevisiae, Saccharomyces italicus and Kiuyveromyces lactis.

The amount of microorganism used for the fermentation is dependent on the microorganism used and should therefore not be seen as a limitation. However, as a guidance, the microorganism is added in an amount of from 0.02 to 0.2% by weight. Preferably, the microorganism is added in an amount of from 0.03 to 0.15% by weight, most preferably from 0.04% to 0.1% by weight, such as about 0.05% by weight.

Fermentation conditions:

Conditions for the fermentation are for example specific temperature, time and oxygen content. The fermentation is carried until a desired acidity or pH is reached. The fermentation may be aerobic or anaerobic depending on the microorganism.

In an embodiment of the invention, the fermentation is performed at a

temperature of 10°C to 50°C for 1 to 75 hours. The temperature and time used for fermentation is dependent on the microorganisms used for the fermentation. For example, the fermentation may be performed at a temperature of 15°C to 45°C, such as 25°C to 45°C for 3 to 50 hours, such as 25°C to 40°C for 10 to 48 hours.

For fermentation with lactic acid bacteria, the conditions may for example be 25°C to 50°C for 5 to 60 hours, preferably 30°C to 45°C for 5 to 60 hours, more preferably 37°C to 43°C for 5 to 60 hours, even more preferably 37°C to 43°C for 30 to 50 hours. In other embodiments of the invention, the fermentation temperature is 15°C to 40°C, preferably 20°C to 37°C, and even more preferably 25°C to 33°C when a lactic acid bacteria is used for the fermentation with a fermentation time of 10 to 48 hours.

For fermentation with yeast, the conditions for the fermentation may be 10°C to 30°C for 1 to 75 hours, preferably 15°C to 25°C for 12 to 60 hours, more preferably 18°C to 23°C for 24 to 48 hours.

For fermentation with acetic acid bacteria, the fermentation could for example be carried out at 20°C to 45°C for 10 to 100 hour, preferably 20°C to 45°C for 20 to 90 hours such as for 30 to 85 hours, even more preferably for 40 to 80 hours.

Fermentation with a combination of lactic acid bacteria, acetic acid bacteria and yeast, the conditions for the fermentation could for example be 20°C to 35°C for 5 to 60 hours, preferably 23°C to 32°C for 15 to 50 hours, more preferably 25°C to 30°C for 20 to 50 hours.

Thus, the temperature and time is dependent on the microorganism used for the fermentation. In general, the fermentation is performed at a temperature of 10°C to 50°C for 1 to 75 hours, preferably 22°C to 48°C for 1 to 75 hours, such as 24°C to 45°C for 1 to 75 hours, more preferably 25°C to 42°C for 1 to 75 hours, even more preferably 27°C to 40°C for 1 to 75 hours.

The fermentation is carried out until the desired pH is reached. After the fermentation and hydrolysis, the fermented and hydrolysed liquid milk material is considered the fermented milk ingredient which can be used as an ingredient in the preparation of beverages.

The pH of the liquid milk ingredient is during fermentation decreased due to formation of organic acids. The pH of the fermented milk ingredient will be in the range of 3.0 to 6.7, preferably the pH will be lowered during fermentation to 3.3 to 6.7, more preferably to 3.5 to 6.5, such as 3.6 to 6.0, preferably 3.6 to 5.5, and even more preferably to 3.8 to 5.0, even more preferably to 4.0 to 4.5.

The taste of the fermented milk ingredient is dependent on the choice of the microorganism used in the fermentation and the metabolites produced. In the method of preparing the fermented milk ingredient according to the present invention, the hydrolysis step may be before, after or simultaneously with the fermentation step.

In an embodiment of the invention, the hydrolysis step b) is before or

simultaneously with the fermentation step c). By having hydrolysis before or simultaneously with the fermentation, the monosaccharides (glucose and galactose) may be used as substrate in the fermentation. The monosaccharides is much easier consumed by the bacteria during fermentation than lactose.

The fermented milk ingredient obtained by the method of the present invention has a strong refreshing taste. It has a low pH, from about 3.0 to 7.0, such that beverages prepared based on the fermented milk ingredient can be stored for more than 6 months. In addition, the taste is sweet because lactose has been split to glucose and galactose. Therefore, there is no need for addition of additional sweeteners to a beverage prepared based on the fermented milk ingredient.

Additional sweeteners, such as sugar, may however be added, but in lower amounts than conventional soft drinks.

After the desired acidity is obtained in the fermentation step, the fermentation is preferably stopped by either a pasteurization step to inactivate the

microorganisms or a microfiltration step to remove the microorganisms. If a pasteurization step is used, the pasteurization will preferably be at 65°C to 90°C for 5 seconds to 5 minutes, preferably 70°C to 80°C for 5 seconds to 5 minutes, such as at 70°C to 80°C for 10 seconds to 30 seconds.

If the fermentation is stopped by removal of the microorganisms by use of microfiltration, a suitable membrane pore size should be used, such as about from 0.2 to 2.0pm, such as from 0.4 to 1.8 pm, preferably from 0.8 to 1.4 pm.

Addition of acidification accelerating additives:

In an embodiment of the invention, an acidification accelerating additive can be added. The acidification accelerating additive can be added before, after or simultaneously with addition of the microorganism in step c). The acidification accelerating additive is in an embodiment of the present invention a malt extract. The acidification accelerating agent, such as malt extract, may be added in an amount of from 0.5% to 10% by weight, such as from 0.7% to 5% by weight, even more preferably from 1% to 3% by weight.

Heat treatment and/or filtration step:

In an embodiment of the present invention, the liquid milk material provided in step a) is subjected to heat treatment and/or filtration before hydrolysis and fermentation in steps b) and c) and wherein said heat treatment is performed at 70°C to 145°C for 4 seconds to 15 minutes. The heat treatment may for example be at 90°C to 100°C for 2 to 12 minutes, preferably at 92°C to 97°C for 3 to 7 minutes, more preferably at 95°C for 5 minutes.

The heat treatment step may also be referred to as a pasteurization step of the liquid milk material. The terms "heat treatment" and "pasteurization" may in the context of the present invention be used interchangeably. Pasteurization is the process of heating to a specific temperature to slow microbial growth in the food. Sterilization on the contrary refers to the process of eliminating all forms of bacteria from any product. The heat treatment of the liquid milk material before hydrolysis and fermentation may not sterilize the liquid milk material, but pasteurize it.

Alternative to heat treatment, the liquid milk ingredient may be subjected to a filtration step in order to remove contaminants. The filtration step is preferably a microfiltration step with a suitable membrane pore size, such as from 0.2 to 1.4pm.

Figure 1 shows an example of preparing a liquid milk ingredient according to the present invention where a nanofiltration retentate comprising 15.3% lactose and having a pH of 6.6 is used as a liquid milk material. The nanofiltration retentate is obtained by nanofiltration of an ultrafiltration permeate from ultrafiltration of skim milk. First, the nanofiltration retentate is subjected to pasteurization at 80°C for 90 seconds. After cooling to about 10°C, the lactose is hydrolysed by addition of 0.2% lactase (Maxilact from DSM). Afterwards, the hydrolysed mixture is heated to 30 °C and inoculated with Lactobacillus plantarum. The fermentation is stopped after 48 hours (pH below 4.5) by microfiltration of the mixture using a membrane having a 0.2pm pore size. Hereby, a fermented milk ingredient is prepared which is ready to be used in the preparation of beverages.

Figure 2 shows an example of preparing a liquid milk ingredient according to the present invention where a nanofiltration retentate comprising 15-20% lactose and having a pH of 6.7 is used as a liquid milk material. The nanofiltration retentate is obtained by nanofiltration of an ultrafiltration permeate from ultrafiltration of skim milk. The nanofiltration retentate is subjected to pasteurization at 80°C for 90 seconds. After cooling to about 30°C, the pasteurized nanofiltration retentate is subjected to a combined step of hydrolysing lactose with lactase (0.1% Maxilact from DSM) and fermentation with Lactobacillus plantarum. The fermentation is stopped after 48 hours (pH below 4.5) by heat treatment of microfiltration.

Hereby, is prepared a fermented milk ingredient ready to be used in the

preparation of beverages.

Fermented milk ingredient

An aspect of the present invention is to provide a fermented milk ingredient obtainable by the method according to the invention.

Another aspect of the invention is to provide a fermented milk ingredient for use in the preparation of beverages obtainable by the following steps:

a) providing a liquid milk material comprising lactose in an amount of at least 4% by weight and protein in an amount of 1% or less by weight of the liquid milk material;

b) adding lactase to said liquid milk material to provide hydrolysis of lactose in the liquid milk material;

c) adding one or more microorganisms and fermenting the liquid milk material to obtain a fermented milk ingredient;

wherein hydrolysis in step b) is before, after or simultaneously with fermentation in step c).

The fermented milk ingredient according to the present invention comprises a lactose content of 1.0% by weight or less and a protein content of 1% by weight or less, and has a pH in the range of 3.0 to 7.0. The fermented milk ingredient according to the present invention preferably has a lactose content of 0.5% by weight or less, such as a lactose content of 0.3% by weight or less, more preferably 0.1% by weight or less. In an embodiment, the fermented milk ingredient according to the present invention is lactose-free and therefore comprises a lactose content of 0.05% by weight or less, preferably a lactose content of 0.03% by weight or less, and even more preferably a lactose content of 0.01% by weight or less. The protein content in the fermented milk ingredient according to the present invention comprises a protein content of 1% by weight or less. Preferably, the protein content is lower than 1% by weight because a content of protein of 1% by weight may cause small precipitates when the fermented milk ingredient is used in the preparation of a beverage. Therefore, the amount of protein in the fermented milk ingredient is preferably 0.8% by weight or less, such as 0.7% by weight or less, preferably 0.5% by weight or less.

The fermented milk ingredient will be acidic or slightly acidic and have a pH in the range of 3.0 to 6.7. Preferably, the pH will be lowered during the fermentation step such that the pH of the fermented milk ingredient obtained is in the range of about 3.5 to 6.5, more preferably to about 3.8 to 6.0, such as 4.0 to 4.5.

The fermented milk ingredient also comprises one or more organic acids. The organic acids may be one or more from the group of lactic acid, acetic acid, gluconic acid and succinic acid. The organic acids are for example present in the fermented milk ingredient in an amount of from 0.6 to 1.2% by weight.

Beverage

An aspect of the present invention is also to provide a beverage comprising the fermented milk ingredient according to the present invention. The beverage comprises the fermented milk ingredient in an amount of from 5 to 99% by weight, a flavouring agent in an amount of 1 to 50% by weight, water in an amount of 0-90% by weight and optionally a pH regulating agent. The flavouring agent may be any kind of compound, composition or agent which may provide flavour to a liquid drink. The flavouring agent is soluble in water. The flavouring agent may be any of a concentrate, a powder, a granulate or emulsion, but it has to be able to solubilise in an aqueous solution, because it is wished to obtain a clear beverage without any undissolved solids or precipitates in it.

The flavouring agent may for example be one or more selected from the group of syrups, malt extracts, juices, juice concentrates, kombucha, and aroma

compounds. Kombucha may be added both as a flavouring agent, but also as a culture during the fermentation step.

Syrups may both be a fruit syrup, but it may also be a syrup of sugar, i.e. a honey syrup or a of monosaccharides or disaccharides, such as a glucose syrup or sucrose syrup and fructose syrup.

In a preferred embodiment the flavouring agent used is a fruit syrup, fruit juice or fruit concentrate. The fruit syrup, fruit juice or fruit concentrate may be based on any kind of fruit and the beverage according to the present invention is not limited to the syrup used. Different fruit syrups may give different taste. The fruit syrup may for example be a syrup of apricot, blueberry, blackberry, cherry, cranberry, coconut, grape, grapefruit, kiwi, lemon, lime, lychee, mango, melon, nectarine, orange, papaya, passionfruit, peach, pear, plum, pineapple, pomelo, raspberry, redcurrant and strawberry.

In another preferred embodiment, the flavouring agent may be kombucha.

Kombucha which may also be referred to as a variety of fermented effervescent sweetened black or green tea. Kombucha tea is supposed to have health benefits.

In still another embodiment of the present invention, the flavouring agent is one or more malt extracts.

The beverage according to the present invention may comprise the flavouring agent in an amount of 1 to 50% by weight of the beverage. In a preferred embodiment, the beverage comprises the flavouring agent in an amount of 2 to 40% by weight, such as 3 to 30% by weight, even more preferably in an amount of 4 to 20% by weight.

The beverage comprises the fermented milk ingredient according to the invention in an amount of 5 to 99% by weight of the beverage. In a preferred embodiment of the invention, the beverage comprises the fermented milk ingredient in an amount of 10 to 90% by weight, more preferably in an amount of 15 to 85% by weight.

In the embodiments of the invention where the fermented milk ingredient is prepared with a nanofiltration retentate as the liquid milk ingredient, the beverage preferably comprises the fermented milk ingredient according to the invention in an amount of 5 to 30% by weight of the beverage. In a preferred embodiment of the invention, the beverage comprises the fermented milk ingredient in an amount of 10 to 27% by weight, more preferably in an amount of 12 to 25% by weight of the beverage.

In the embodiments of the invention where the fermented milk ingredient is prepared with an ultrafiltration permeate as the liquid milk material, the beverage may comprise a high concentration of the fermented milk ingredient and low amounts of water. In such embodiments, the beverage preferably comprises the fermented milk ingredient according to the invention in an amount of 50 to 99% by weight of the beverage. In a preferred embodiment of the invention, the beverage comprises the fermented milk ingredient in an amount of 60 to 95% by weight, more preferably in an amount of 70 to 90% by weight, even more preferably 75 to 85% by weight.

Furthermore, the beverage may comprise water, preferably degassed water.

Degassed water is commonly known by the skilled person in the production of carbonated beverages.

The beverage according to the present invention may optionally comprise a pH regulating agent added to reach a pH below 4.5. The pH regulating agent may be any edible organic acid, such as acetic acid, citric acid, lactic acid and malic acid, or a naturally acidic fruit juice such as lomon or lime juice. If a pH regulating agent is added it is in amounts much lower than when for example conventional soft drinks are prepared. This is due to the fermented milk ingredient used to prepare the beverage is also acidic and will reduce the pH of the beverage to some extend.

A beverage based on the fermented milk ingredient according to the present invention is besides from having a refreshing taste and a low lactose content, also low in calories and contains some of the minerals and vitamins originating from milk. No additional sugar needs to be added. The product is a clear product low in alcohol and can be stored for 6 months or longer due to its acidity (low pH). The refreshing taste mainly comes from the fermented milk ingredient and the flavouring agent is added in order to give the beverage for example a fruity taste.

In a preferred embodiment, the beverage according to the present invention is a carbonated beverage. The carbonated beverage comprises carbon dioxide.

Method of preparing a carbonated beverage

An aspect of the invention relates to providing a method of preparing a

carbonated beverage comprising the steps of

i) mixing 5-99% by weight of a fermented milk ingredient according to the invention with 1-50% by weight of a flavouring agent, 0-90% by weight water and optionally a pH regulating agent;

ii) carbonize the mixture of step i)

iii) pasteurize the carbonized mixture.

In an embodiment of the invention, the carbonized mixture according to step ii) may be placed in bottles before pasteurization in step iii). The bottles may vary in size but could be in the sizes of 330 ml, 500 ml, 1000 ml or 1500 ml.

The pasteurization in step iii) may for example be at 75°C to 95°C for 3 to 15 minutes, such as 77°C to 90°C for 5 to 13 minutes, preferably at 80°C for 10 minutes.

Carbonation of the beverage means in the present context addition of bubbles to the beverage. The carbonation process is well known for the skilled person and relates to a process where carbon dioxide gas under pressure has been dissolved. By the carbonation process, the carbonated liquid becomes effervescent. The present invention is not limited to a specific method of carbonation and any method available in the art may be used.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1 - Composition of a nanofiltration retentate versus skim milk

The liquid milk material used to prepare a fermented milk ingredient according to the present invention may be a nanofiltration retentate obtained by:

i) ultrafiltration of 100 L skim milk (3.6% w/w protein and 4.6% w/w lactose) with a 10 kDa cutoff ultrafiltration membrane (CF=2) from Koch Dairy Pro 10K to obtain a ultrafiltration retentate (50 L with 7.1% w/w protein and 4.7% w/w lactose) and ultrafiltration permeate (50L with 0.2% w/w protein and 4.3% w/w lactose) and subsequently perform nanofiltration of said ultrafiltration permeate with a nanofiltration membrane having a 300 Da cutoff from DOW Filmtect NF (CF=4) to obtain a nanofiltration retentate (12.5L with 0.3% w/w protein and 20.4% w/w lactose) and a nanofltration permeate (37.5 L with 0.1% w/w protein and 0.4% w/w lactose . The ultrafiltration retentate will comprise most proteins from the milk while the ultrafiltration permeate will comprise a low amount of proteins. The lactose content in the milk is split into the retentate and permeate with about 47% of the lactose from milk in the retentate and about 43% of the lactose in the permeate. Below in table 1 is shown the composition of a nanofiltration retentate (NFR), and ultrafiltration permeate (UFP) and of the skim milk used for preparing the ultrafiltration permeate and nanofiltration retentate. Table 1:

Example 2 - Preparation of fermented milk ingredient in two steps

1 kg of a nanofiltration retentate as shown in example 1 was used as the liquid milk material. The nanofiltration retentate having a pH of 6.6 and a Brix of 15.3 was subjected to pasteurization by heat treatment to 80°C for 90 seconds. After heat treatment the nanofiltration retentate was cooled to 10°C. After cooling, the nanofiltration retentate was subjected to hydrolysis of lactose by adding 2 g lactase (Maxilact LGi 5000 (DSM)). Hydrolysis was performed at 10°C for 16 hours. The hydrolysed nanofiltration retentate was then subjected to a filtration with a microfiltration membrane having a pore size of 0.2 pm. This filtration step is to remove possible contaminants. The filtration step is optional in the method of the present invention and may also be replaced by a heat treatment step.

Lactobacillus plantarum was added in an amount of approximately 10 ⁶ cells/ml and fermentation was performed for 48 hours at 30°C to obtain a hydrolysed and fermented nanofiltration retentate, herein also called a fermented milk ingredient.

In figure 1 is a flow diagram shown of the method of preparing a fermented milk ingredient described in example 2.

Example 3 - Preparation of fermented milk ingredient in one step

1 kg of a nanofiltration retentate as shown in example 1 was used as the liquid milk material. The nanofiltration retentate having a pH of 6.6 and a Brix of 15.3 was subjected to pasteurization by heat treatment to 80°C for 90 seconds. After heat treatment the nanofiltration retentate was cooled to 20-30°C and subjected to hydrolysation and fermentation by adding 2 g lactase (Maxilact LGi 5000 (DSM)) and Lactobacillus plantarum (10 ⁶ cells/ml)

Hydrolysis and fermentation was performed for 72 hours at 30°C and a

hydrolysed and fermented nanofiltration retentate was obtained (fermented milk ingredient), which can be used in the preparation of beverages with a refreshing taste.

In figure 2 is a flow diagram shown of the method of preparing a fermented milk ingredient described in example 2

Example 4 - A beverage comprising the fermented milk ingredient In table 2 below is shown an example of a refreshing beverage prepared by using a fermented milk ingredient as prepared in example 2.

Table 2:

The beverages shown in table 2 had a refreshing, slightly acidic taste. The beverages had a fruity, caramel-cerealie like taste and the beverages did not have a milky taste. The beverages was clear and dark brown in appearance and was similar in appearance to a cola soft drink.

Example 5 - A method of preparing a carbonated beverage comprising a fermented milk ingredient

12,5 kg of a flavouring composition from Dohler was added to 25,0 kg of fermented milk ingredient as prepared in example 2 and 212.5 kg de-gassed water. 1 kg of a multi fruit base was added and the ingredients was mixed.

The mixture was carbonated by adding carbon dioxide under pressure.

The carbonated beverage was the added to 330 ml bottles and pasteurized at 80°C for 10 minutes.

Example 6 - Preparation of different beverages, different microorganism

An ultrafiltration permeate comprising 5.5% lactose obtained by ultrafiltration of skim milk was pasteurization by heat treatment to 95°C for 5 minutes. The pasteurized ultrafiltration permeate was divided in three tanks and hydrolysed with lactase (from Novozymes Lactozym Pure 6500 L) simultaneous with fermentation with the culture described below as code 1-3. The day after (24-30 hours later), a sample from each tank was taken out and diluted in water (1 part sample and 8 parts water). Fruit bases was added to prepare a drink.

Code 1 : Microorganism was Probat 505 FRO 500 DCU from Danisco which was a combination of Lactococcus lactis subsp. lactis, lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar. diacetulactis and leuconostoc

mesenteroides subsp. cremoris.

Code 2: Microorganism was CHOOZIT™ Kefir DT LYO 1000 I from Danisco comprising Lactococcus lactis subsp., Leuconostoc sp., Lactobacillus sp., and Streptococcus thermophilus.

Code 3 : Microorganism was YO-MIX™ 321 FRO 500 DCU from Danisco comprising Streptococcus thermophilus and lactobacillus delbrueckii subsp. bulgaricus.

The pH of the fermented nanofiltration retentates were before mixing the following :

Code 1 : pH 5.47 after 7 hours fermentation and pH 5.38 after 10.5 hour

Code 2: pH 5.75 after 7 hours fermentation and pH 5.67 after 10.5 hour

Code 3 : pH 5.32 after 7 hours fermentation and pH 4.76 after 10.5 hour

The taste, flavour and other parameters of the three different beverages (code 1-

3) observed were:

Code 1 : Good taste, balanced acidity, fresh flavor, good mouthfeel

Code 2: Sparkling mouthfeel, slight astringency, slightly un-clean flavor

Code 3 : Sharp acidity, sour, strong yoghurt smell, astringent

Example 7 - Preparation of fermented milk ingredient by use of

Lactobacillus plantarum with yeast extract and malt extract

The object of the present example was to determine the effect of a yeast extract and the effect of a malt extract in the method of preparing a fermented milk ingredient according to the invention. The lactose hydrolysis and fermentation was made in two steps with the use of a ultrafiltration permeate comprising 14% lactose as the liquid ingredient material. The ultrafiltration permeate was subjected to pasteurization by heat treatment to 80°C for 90 seconds. After heat treatment the ultrafiltration permeate was cooled to 10°C. After cooling, the ultrafiltration permeate was subjected to hydrolysis of lactose by adding lactase (Maxilact LGi 5000 (DSM)) in a concentration of 0.2% by weight. Hydrolysis was performed at 10°C for 16 hours. The hydrolysed ultrafiltration permeate was then subjected to a filtration with a microfiltration membrane having a pore size of 0.2 pm. This filtration step is to remove possible contaminants. The filtration step is optional in the method of the present invention and may also be replaced by a heat treatment step.

Lactobacillus plantarum (Harvest LB-1) was added in an amount of 0.05 g/L. The fermentations were also performed with 0.02 g/L Lactobacillus plantarum (Harvest LB-1). Furthermore, either a yeast extract (Bactivaid from Christian Hansen) or a malt extract (from maltbazaren.dk; Spraymalt extra light, No. 84113) were added.

The yeast extract was added in different doses; 0 g/L (reference), 0.01 g/L (extremely low concentration), 0.05 g/L, 0.15 g/L (recommended), 0.5 g/L, and 2 g/L (extremely high concentration). The concentration 0.15 g/L is the

recommended dosage in most wine production and therefore it was considered that this concentration also would be good for fermentation of the ultrafiltration permeates.

1 g/L (1%) of the malt extract was used corresponding to 0.01-0.05% yeast extract. The malt extract was added in different doses: 0 g/L (reference), 5 g/L (extremely low), 20 g/L(average) and 50 g/L (extremely high).

The fermentation was performed for 48 hours at 30°C to obtain a hydrolysed and fermented ultrafiltration permeate, herein also called a fermented milk ingredient.

The pH of the fermented ultrafiltration permeates were measured at different time periods after start of the fermentation and evaluated as being an indication of the effect of the the yeast extract and malt extract on the fermentation. It was observed that using 0.05 g/L Lactobacillus plantarum as compared to using 0.02 g/L resulted in an increased acidification rate.

In addition, it was observed that the yeast extract did not result in faster acidification. However, addition of the malt extract did result in increased acidification.

Figure 4 shows the effect of the malt extract on the acidification, where

BH1 is the reference with 0 g/L malt extract added

M10 is atlso a reference with 0 g/L malt extract added

Mil is the fermentation liquid after 5 g/L malt extract is added

M12 is the fermentation liquid after 20 g/L malt extract is added

M13 is the fermentation liquid after 50 g/L malt extract is added.

The x-axis represent the time in hours of fermentation while the y-axis represents the pH of the fermentation liquid. Figure 4 shows that with malt extract added, the acidification is increased. The acidification increases the more malt extract added.

A second experiment was performed repeating the experiment described above, but using the following concentrations of added yeast extract and malt extract.

FI - fermentation 1 with no yeast extract or malt extract added (reference),

F2 - fermentation with 0.15 g/L yeast extract added (recommended),

F3 - fermentation with 15 g/L yeast extract added (100 fold-overdosis).

F4 - fermentation with 5 g/L malt extract (0.5% malt extract)

F5 - fermentation with 10 g/L malt extract (1.0% malt extract)

The pH of the fermented permeates were measured at different time period after start of the fermentation. The result is shown in figure 5. The x-axis represent the time in hours of fermentation while the y-axis represents the pH of the fermentation liquid.

Figure 5 shows that reference fermentation and the fermentation with the recommended amount of yeast extract (0.15 g/L) acidify in a similar manner. Thus, the yeast extract does not have added value at this dosage. The fermentation with 1.5% yeast extract (100 fold the recommended dosis) showed an improved effect on the acidification (down to pH 4,8 within 24 h), but does not go all the way to pH below 4.5. Besides, using 15 g/L of the yeast extract would not be recommended, because it is very concentrated, have a yeasty taste and would be expensive to prepare with such a high concentration.

Figure 5 also shows that the use of 0.5% malt extract (5 g/L) gives similar acidification as the fermentation with 100 fold-overdose of yeast extract. The use of 0.5% malt extract gives an acidification down to pH 4.8 with 24 hours. The fermentation with 1% added malt extract (10 g/L) showed a proper acidification, i.e. down to pH 4.3 within 24 hours.

Hence, it seems like the lowest pH the ferementation with lactobacillus plantarum in a concentration of 0.05% can reach with no additives is about 5.2-5.5.

However, if for example a malt extract is added in concentrations about 0.5-1.0%, the pH can be lowered to 4.3-4.8 within 24 hours. If higher concentrations of malt extract is used, the acidification is faster and the pH can become lower. For example, the use of 5% malt extract can give a pH of 3.9 within 24 hours.

Example 8 - Preparation of fermented milk ingredient at different fermentation temperature

An experiment was made to evaluate the effect of the fermentation temperature on the acidification.

The lactose hydrolysis and fermentation was made in two steps with the use of a ultrafiltration permeate comprising 14% lactose as the liquid ingredient material.

The ultrafiltration permeate was subjected to pasteurization by heat treatment to 80°C for 90 seconds. After heat treatment the ultrafiltration permeate was cooled to 10°C. After cooling, the ultrafiltration permeate was subjected to hydrolysis of lactose by adding lactase (Maxilact LGi 5000 (DSM) in a concentration of 0.2%. Hydrolysis was performed at 10°C for 16 hours. The hydrolysed ultrafiltration permeate was then subjected to a filtration with a microfiltration membrane having a pore size of 0.2 pm. This filtration step is to remove possible contaminants. The filtration step is optional in the method of the present invention and may also be replaced by a heat treatment step.

0.05 g/L Lactobacillus plantarum (Harvest LB-1) and 10 g/L malt extract (from maltbazaren.dk; Spraymalt extra light, No. 84113) were added.

The fermentation was performed for at different temperatures; 10°C, 15°C, 20°C, 25°C, 30°C and 37. The pH was measured at different times and the result is shown in figure 6.

In figure 6, the following refers to:

Tl : temperature of 10°C was used

T2: temperature of 15°C was used

T3: temperature of 20°C was used

T4: temperature of 25°C was used

T5: temperature of 30°C was used

T6: temperature of 37°C was used

Figure 6 shows that the higher the temperature is, the faste the fermentation liquid reaches a low pH (such as pH 4.5). Hence, there is an proportional-like relationship between the fermentation temperature and the fermentation time to reach pH 4,5. From figure 6, it can be concluded that temperatures below 15°C and above 40°C is not recommended. Increasing the concentrationof malt extract could however, increase the fermentation rate.

Figure 6 shows the correlation between fermentation temperature and

fermentation time for reaching pH 4.5.

Example 9 - Preparation fermented milk ingredient with different amount of protein in the liquid milk material

The object of the example is to analyse the effect protein present in the starting material (i.e. the liquid milk material) on the turbidity and formation of

precipitation upon acidification and pasteurization. A nanofiltration retentate as shown in Table 1 in example 1 and skim milk is mixed to reach samples of liquid milk material having different protein

concentrations. The protein concentrations are 0% (reference), 0.3% by weight, 0.5% by weight, 0.6% by weight, 0.8% by weight and 1% by weight.

The samples are acidified to pH 4.2 by addition of lactic acid (80%) before pasteurization at 90°C for 5 min. Pictures were taken of the samples before and after pasteurization showing the turbidity and precipitate formation dependent on the protein concentration. Figure 8 shows pictures of samples before

pasteurization and Figure 9 shows pictures of the samples after pasteurization.

The samples shown in Figure 8 and 9 show from left to right the reference, the sample with 0.3% w/w protein, the sample with 0.5% w/w protein, the sample with 0.6% w/w protein, the sample with 0.8% w/w protein and the sample with 1.0% w/w protein.

Figure 8 shows that the turbidity and precipitation formation before pasteutization is increased when the protein concentration is increased. In the reference, there is clearly no precipitation, while more than 50% of the sample with 1% w/w protein is turbid.

Figure 9 shows that the turbidity and precipitation after pasteurization also is increased when the protein concentration is increased, but less than the unpasteurized samples.

Example 10 - Sensory evaluation of fermented milk ingredient with different microorganisms

A nanofiltration retentate as shown in Table 1 in example 1 is mixed with 1% malt extract and pasteurized at 90°C for 5 min. After cooling to 10°C, the nanofiltration retentate was subjected to hydrolysis of lactose by adding lactase (Maxilact LGi 5000 (DSM)) in a concentration of 0.2% by weight. Hydrolysis was performed at 10°C for 16 hours. The hydrolysed nanofiltration permeate was then subjected to a filtration with a microfiltration membrane having a pore size of 0.2 pm before fermentation at 35°C for 23 hours. The fermentation was performed with different microorganisms as mentioned below. The fermentation liquid was cooled to 5°C before sensory evaluation.

Fermentation 1 : Lactobacillus plantarum from Christian Hansen (Harvest LB-1) was used as the microorganism. Fermentation was performed 23 hours to pH 4.60.

Fermentation 2: A Preparation comprising Lactococcus lactis/cremoris and Lactococcus diacetylactis and Leuconostoc cremoris from Dupont (Probat 505) was used as the microorganism. Fermentation was performed 23 hours to pH 4.64.

Fermentation 3: A preparation of Streptococcus thermophiles and Lactobacillus bulgaricus from Dupont (VEGE033) was used as the microorganism. Fermentation was performed 23 hours to pH 4.46.

Fermentation 4: A preparation comprising Streptococcus thermophiles,

Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus

casei/paracasei from Dupont (VEGE068) was used as the microorganism.

Fermentation was performed 23 hours to pH 4.35.

Fermentation 5: A preparation comprising Lactobacillus plantarum and

Pediococcus from Dupont (VEGE081) was used as the microorganism.

Fermentation was performed 23 hours to pH 4.60.

Fermentation 6: A preparation comprising Saccharomyces cerevisiae from Bulldog Brews (B5 American West ale yeast) was used as the microorganism.

Fermentation was performed for 14 days to pH 4.23.

Figure 10 shows the pH of the fermentation liquid dependent on the fermentation time. Figure 10 shows that using yeast as the microorganism does not provide an effective acidification. After 23 hours of fermentation, the fermented liquid made with Saccharomyces cerevisiae had a pH of abput 5.4.

On the contrary, the samples of fermented liquid made with the use of

Lactobacillus, Leuconostoc, Streptococcus or Pediococcus all provided acidification to a pH below 4.7 within 23 hours.

In table 3 below is the sensory evaluation of the samples of fermented liquid given (fermentation 1-6) shown

In conclusion, the microorganisms from the genus Lactobacillus, Leuconostoc Streptococcus and Pediococcus all provided fermented liquid with an acceptable taste and smell. On the contrary. The fermented liquid made in fermentation 6 with the use of Saccharomyces cerevisiae from Bulldog Brews resulted in an unpleasant taste and smell. However, other strains or sources of yeast could result in a more pleasant taste and smell.