Method for producing a fermented milk product

Abstract

The present invention is in the field of dairy technology. It relates a method for producing fermented milk products comprising the steps of: (a) Exposing a lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners to obtain a pre-treated strain; (b) Providing a milk base comprising one or more second sweeteners; (c) Fermenting the milk base with the pre-treated strain to produce the fermented milk product; and wherein the fermented milk product after days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain. The invention also provides a fermented product produced therefrom.

Description

[0001] METHOD FOR PRODUCING A FERMENTED MILK PRODUCT

[0002] FIELD

[0003] The present disclosure is in the field of Dairy. In general it relates to fermented milk products, their use and methods of producing them. In particular, the disclosure relates to stability of fermented products.

[0004] BACKGROUND

[0005] Flavor and gas formation are important attributes affecting customer acceptance of fermented products. Changes in various conditions during fermentation, transport and storage of fermented products may affect conversion of citrate thereby leading to unwanted acetate and CO2. Due to lack of cooling or temperature variations during transport and storage the flavor and / or gas formation of fermented products may change. In addition to changes in the organoleptic and / or sensory characteristics of fermented products, gas formation may also potentially lead to increased pressure, swelling and blowing of the product package.

[0006] Thus, there is a need for improved methods for producing fermented milk products where the final product remains stable during production, transport and storage. In particular when a cold chain is not possible or is at risk of being broken or partially disrupted.

[0007] SUMMARY

[0008] Off-flavor and gas formation during shelf life of a fermented milk product can be wholly or partly controlled by delaying the conversion of citric acid to acetic acid. The present disclosure provides a solution wherein said conversion is controlled by pre-treating the fermenting lactic acid bacteria as described more in detail supra.

[0009] In a first aspect the present disclosure relates to a method for producing a fermented milk product comprising the steps of:

[0010] (a) Exposing a lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners to obtain a pre-treated strain;

[0011] (b) Providing a Milk base comprising one or more second sweeteners;

[0012] (c) Fermenting the Milk base with the pre-treated strain to produce the fermented milk product; wherein the fermented milk product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

[0013] In a second aspect the present disclosure relates to fermented milk products made by said method

[0014] In a third aspect the present disclosure relates to a method for obtaining a pre-treated strain comprising the steps of:

[0015] (a) Providing a lactic acid bacteria strain;

[0016] (b) Exposing the lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners to obtain a pre-treated strain; and wherein the pre-treated strain when used to ferment a milk base comprising one or more second sweeteners produce a fermented milk product which product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

[0017] The method of the present disclosure relates to lactic acid bacteria and fermented milk products made by said lactic acid bacteria with reduced citrate conversion. While not wishing to be bound by theory, it is believed that citrate conversion results in unwanted flavor and gas-formation.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] The citric acid metabolism in lactic acid bacteria goes through several steps involving the enzymes: citrate lyase; malate dehydrogenase; fumarate hydratase; succinate dehydrogenase; oxaloacetate decarboxylase; malolactic enzyme; lactate dehydrogenase; acetolactate synthase; acetolactate decarboxylase; pyruvate formate lyase; acetaldehyde dehydrogenase; alcohol dehydrogenase; phosphotransacetylase; and acetate kinase. Citric acid is thereby converted into the substances succinate, lactate, acetate, and ethanol or acetylacetone through the intermediate metabolite oxaloacetate. The citric acid metabolism starts when extracellular citric acid is transported to the cell through a membrane-associated permease. Inside the cell citrate is converted into acetate and oxaloacetate under the catalysis of citric acid lyase complex. The oxaloacetate is decarboxylated by oxaloacetate decarboxylase to produce pyruvate and carbon dioxide.

[0020] The citric acid metabolism is together with the amino acid metabolism the main source for the biosynthesis of flavor substances provided by lactic acid bacteria. However, due to the variety of lactic acid bacteria, fermentation substrates, and flavors substances produced, the flavor of the final product is difficult to control. Furthermore, the stability of the strain is easily affected by many factors during fermentation and may thus confer a change in the flavor substances produced.

[0021] Thus, citrate consumption may result in often unwanted substances such as acetate and diacetyl under the formation of CO2. For that reason it is important to identify when this carbon source is utilized during milk fermentations and know how to control it.

[0022] The present disclosure relates to a method for producing a fermented milk product comprising the steps of:

[0023] (a) Exposing a lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners to obtain a pre-treated strain;

[0024] (b) Providing a Milk base comprising one or more second sweeteners;

[0025] (c) Fermenting the Milk base with the pre-treated strain to produce the fermented milk product; wherein the fermented milk product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

[0026] In one embodiment the disclosure relates to the method, wherein the strain has been exposed to the pre-fermentation media for at least 1 hour. The strain may have been exposed to the pre-fermentation media for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0027] 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.

[0028] A "milk base" may be any raw and / or processed milk or 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, low fat 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.

[0029] 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.

[0030] To ferment a milk base a starter culture is added. The term "starter" or "starter culture" as used in the present context refers to a composition or culture comprising one or more food-grade microorganisms in particular lactic acid bacteria (LAB), which are responsible for the fermentation and acidification of the milk base. The starter culture may be a yogurt starter culture. Starter cultures may be fresh, frozen or freeze-dried. It is within the skills of ordinary practitioners to determine the starter culture and amounts to be used. The pre-treated strain of the present disclosure may be comprised in or constitute the starter culture.

[0031] 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.

[0032] In one embodiment the disclosure relates to the method, wherein the one or more first sweetener and the one or more second sweetener is a sugar or a sugar alcohol.

[0033] In one embodiment the disclosure relates to the method, wherein the sugar is Lactose, Glucose, Galactose, Sucrose, or Fructose. In one embodiment the disclosure relates to the method, 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%. Unless otherwise indicated percentages (%) in the precent disclosure are given in weight / volume (w / v), i.e. %w / v. In one embodiment the disclosure relates to the method, wherein the sugar alcohol is Erythritol, Xylitol, Allulose, Tagatose, Maltitol, Lactitol, Mannitol, Sorbitol, Pa latinitol, or Isomaltulose. In one embodiment the disclosure relates to the method, wherein the one or more sweetener is a sugar alcohol selected from a 04 sugar alcohol [C4H10O4] and a C5 sugar alcohol [C5H12O5]. In one embodiment the disclosure relates to the method, wherein the 04 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 0.5%, 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%.

[0034] In one embodiment the disclosure relates to the method, wherein one sweetener of the one or more first sweetener is identical to one sweetener of the one or more second sweetener. In one embodiment the disclosure relates to the method, wherein at least one sweetener of the one or more first sweetener is identical to at least one sweetener of the one or more second sweetener. In one embodiment the disclosure relates to the method, wherein all of the one or more first sweetener are identical to all of the one or more second sweetener.

[0035] In one embodiment the disclosure relates to the method, wherein the one or more first sweetener is a sugar and the one or more second sweetener is a sugar and / or a sugar alcohol.

[0036] In one embodiment the disclosure relates to the method, wherein the one or more first sweetener is Lactose or Glucose and the one or more second sweetener is Lactose supplemented with any of Glucose, Galactose, Sucrose or Fructose.

[0037] In one embodiment the disclosure relates to the method, wherein the one or more second sweetener is further comprising a sugar alcohol.

[0038] In one embodiment the disclosure relates to the method, wherein the sugar alcohol is Erythritol and / or Xylithol. In one embodiment the disclosure relates to the method, wherein the sugar alcohol is xylitol in a concentration selected from 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%.

[0039] The strain of the present disclosure must be able to metabolize at least one sugar. The sugar may be one or more selected from lactose, glucose, galactose, sucrose and fructose. The term "lactose-positive" is to be understood that a strain is capable of growing in a media where lactose is the sole carbon source. The same understanding should be applied to other terms of the form "sugar-positive". In one embodiment the disclosure relates to the method, wherein the strain is lactose-positive, glucose-positive, galactose-positive, and / or sucrose-positive.

[0040] In one embodiment the disclosure relates to the method, wherein the strain is of the genus Lacticaseibacillus. In one embodiment the disclosure relates to the method, wherein the strain is of the species L. rhamnosus, L. paracasei, L. zeae, L. casei, or L. chiayiensis.

[0041] In one embodiment the disclosure relates to the method, wherein the fermentable base is milk wholly or partly of mammalian origin. 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 one embodiment the disclosure relates to the method, wherein the milk of mammalian origin is selected from cow, sheep, goat, buffalo, camel, lama, mare, and deer. In a preferred embodiment, the mammal is a cow.

[0042] The present disclosure furthermore relates to a fermented milk product obtainable by the method of the invention. In one embodiment the present disclosure relates to a fermented milk product comprising the pre-treated strain, wherein the product has a reduced citrate conversion as compared to a fermented milk product without the pretreated strain.

[0043] The term "fermented milk product" means a product wherein the preparation of the product involves fermentation of a milk base with lactic acid bacteria or a starter culture. "Fermented milk product" as used herein includes but is not limited to products such as thermophilic fermented milk products, e.g. yoghurt, mesophilic fermented milk products, e.g. sour cream and buttermilk, as well as fermented whey.

[0044] In one embodiment of the invention, the fermented milk product is selected form the group consisting of yogurt, fresh cheese, cream cheese, tvarog, quark, sour milk, sour cream, buttermilk, creme fraiche, fromage frais, skyr, fermented whey, cultured milk, smetana, kefir, drinking yogurt, and Yakult. Preferably, the yogurt is selected from set yogurt, stirred yogurt and drinking yogurt. Preferably the cheese is selected from quark, tvarog and cream cheese.

[0045] The fermented milk product typically contains protein in a level of between 2.0-3.5% w / w. The fermented milk product may be a low protein product with a protein level of between 1.0-2.0% w / w. Alternatively, the 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. In one embodiment the disclosure relates to the 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 1-6 month or 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.

[0046] In one embodiment the disclosure relates to the fermented milk product wherein said product is a post-pasteurized product, such as a post-pasteurized yogurt (PPY). The term "post-pasteurized product" or "post-pasteurized yogurt" means a product which has been heat treated (pasteurized) after an acidification / fermentation step.

[0047] In one embodiment the disclosure relates to a method for obtaining a pre-treated strain comprising the steps of:

[0048] (a) Providing a lactic acid bacteria strain;

[0049] (b) Exposing the lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners for at least 1 hour to obtain a pre-treated strain; and wherein the pre-treated strain when used to ferment a milk base comprising one or more second sweeteners produce a fermented milk product which product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

[0050] In one embodiment the disclosure relates to the method, wherein the strain is lactosepositive, glucose-positive and / or galactose-positive. In one embodiment the disclosure relates to the method, wherein the strain is of the genus Lacticaseibacillus. In one embodiment the disclosure relates to the method, wherein the strain is of the species L. rhamnosus, L. paracasei, L. zeae, L. casei, or L. chiayiensis.

[0051] EXAMPLES

[0052] Example 1 - Strains with preference for different carbon sources.

[0053] The Lacticaseibacillus paracasei strain DSM 23035 has been described previously in WO2013 / 153074 and DSM 32092 has been described in W02017 / 037046. The sugars fermentable by the strain was investigated and both of the strains were able metabolize lactose, glucose, galactose, whereas only DSM 23035 was able to metabolize sucrose. X-gal is a lactose analog that can be hydrolyzed by beta-galactosidase, which cleaves it into galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter compound dimerizes spontaneously and gets oxidized into a blue color. In this way, X-gal can be used as a readout for expression and activity of beta-galactosidase. In our experiment it was used as an estimate, when the strains are expressing the necessary genes to utilize lactose. For this we grew the strain over night at 37°C in MRS-IM with different carbon sources in decreasing concentrations in the presence of X-gal. The growth of the culture was determined by absorbance measurements in a plate reader at 600 nm, while the blue color formation was observed by eye.

[0054] Example 2 - Effect of pre-fermentation on citrate consumption and acetate production.

[0055] Pre-treatment - Strains were inoculated into MRS-IM (MRS that does not contain beef extract or a carbon source) containing 2% glucose or 2% lactose overnight at 37°C in 9 mL tubes.

[0056] Acidification in milk - Next day 10 pl of the overnight culture were inoculated into 2 mL heat sterilized milk supplemented with different carbon sources at different concentrations. The plate was incubated at 37°C for 2 days and subsequently stored at room temperature. To follow the acidification a pH indicator was added to the milk and the color change was measured by a flatbed scanner and converted by a calibration curve to pH values as described in Kuzina et al.

[0057] The strains were pre-treated with glucose or lactose and used to ferment milk + / - different concentrations of glucose, galactose or fructose according to the process as described above.

[0058] Pre-treatment of the strain DSM 23035 in media comprising glucose as only carbon source delayed citrate consumption in the presence of only certain sugars whereas pretreatment in media comprising lactose as only carbon source delayed citrate consumption for all sugars tested measured at 500 hours.

[0059] Table la: Consumption of citric acid (g / L) during fermentation of Milk supplemented with the indicated sugar by the strain DSM 23035 pre-treated with MRS-IM + Glucose.

[0060] Table lb: Consumption of citric acid (g / L) during fermentation of Milk supplemented with the indicated sugar by DSM 23035 pre-treated with MRS-IM + Lactose.

[0061] Example 3 - Effect of pre-fermentation on citrate consumption and acetate production.

[0062] The strains were pre-treated with glucose or lactose and used to ferment milk + / - different concentrations of glucose, galactose or fructose according to the process as described in example 2.

[0063] Pre-treatment of the strain DSM 32092 in media comprising glucose as only carbon source delayed citrate consumption in the presence of only certain sugars whereas pretreatment in media comprising lactose as only carbon source delayed citrate consumption for all sugars tested measured at 500 hours. Table 2a: Consumption of citric acid (g / L) during fermentation of Milk supplemented with the indicated sugar by the strain DSM 32092 pre-treated with MRS-IM + Glucose.

[0064] Table 2b: Consumption of citric acid (g / L) during fermentation of Milk supplemented with the indicated sugar by DSM 32092 pre-treated with MRS-IM + Lactose.

[0065] Example 4 - Effect of pre-treatment on acidification in milk + / - added sugar and / or sugar alcohol.

[0066] The strains were pre-treated with glucose or lactose and used to ferment milk + / - different concentrations of glucose or fructose in the presence of + / - 2% erythritol or 2% xylitol according to the process as described in example 2.

[0067] Dependent on pre-treatment, sugar and sugar alcohol a delay in acidification to pH 4 could be observed.

[0068] Table 3: Acidification time (minutes) to pH 4.0

[0069] Example 5 - Effect of pre-treatment on citrate consumption after acidification in milk + / - added sugar and / or sugar alcohol. The strains were pre-treated with glucose or lactose and used to ferment milk + / - different concentrations of glucose or fructose in the presence of + / - 2% erythritol or 2% xylitol according to the process as described in example 2. Citrate consumption were measured after 80 hours from the start of fermentation.

[0070] Even after the end of fermentation an effect of pre-treatment in lactose could be observed.

[0071] Table 4: Concentration of citric acid (g / L) after fermentation in Milk supplemented with sugar and / or sugar alcohol. 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

CLAIMS1. A method for producing a fermented milk product comprising the steps of: a) Exposing a lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners to obtain a pre-treated strain; b) Providing a milk base comprising one or more second sweeteners; c) Fermenting the milk base with the pre-treated strain to produce the fermented milk product; wherein the fermented milk product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

2. The method according to claim 1, wherein the strain has been exposed to the pre-fermentation media for at least 1 hour.

3. The method according to any of the preceding claims, wherein the one or more first sweetener and the one or more second sweetener is a sugar or a sugar alcohol.

4. The method according to claim 3, wherein the sugar is Lactose, Glucose, Galactose, Sucrose, or Fructose.

5. The method according to claim 3, wherein the sugar alcohol is Erythritol, Xylitol, Allulose, Tagatose, Maltitol, Lactitol, Mannitol, Sorbitol, Palatinitol, or Isomaltulose.

6. The method according to any of the preceding claims, wherein one sweetener of the one or more first sweetener is identical to one sweetener of the one or more second sweetener.

7. The method according to any of the preceding claims, wherein the one or more first sweetener is a sugar and the one or more second sweetener is a sugar and / or a sugar alcohol.

8. The method according to claim 7, wherein the one or more first sweetener is Lactose or Glucose and the one or more second sweetener is Lactose supplemented with any of Glucose, Galactose, Sucrose or Fructose.

9. The method according to claim 8, wherein the one or more second sweetener is further comprising a sugar alcohol.

10. The method according to claim 9, wherein the sugar alcohol is Erythritol and / or Xylithol.

11. The method according to any one of the preceding claims, wherein the strain is lactose-positive, glucose-positive, galactose-positive, and / or sucrose-positive.

12. The method according to any one of claims 1-11, wherein the strain is of the genus Lacticaseibacillus.

13. The method according to claim 12, wherein the strain is of the species L. rhamnosus, L. paracasei, L. zeae, L. casei, or L. chiayiensis.

14. The method according to any one of the preceding claims wherein the fermentable base is milk wholly or partly of mammalian origin.

15. A method for obtaining a pre-treated lactic acid bacteria strain comprising the steps of: a) Providing a lactic acid bacteria strain; b) Exposing the lactic acid bacteria strain to a pre-treatment media comprising one or more first sweeteners for at least 1 hour to obtain a pre-treated strain; wherein the pre-treated strain when used to ferment a milk base comprising one or more second sweeteners produce a fermented milk product which product after 20 days of storage at room temperature has reduced citrate conversion as compared to a fermented milk product produced without the pre-treated strain.

Images