Process for producing cottage cheese

Abstract

The present disclosure relates to processes for the production of cottage cheese using thermophilic lactic acid bacteria and lactose-deficient mesophilic lactic acid bacteria. The disclosure further relates to bacterial compositions suitable for use in these processes and for cottage cheese produced by the processes of the disclosure.

Description

[0001] PROCESS FOR PRODUCING COTTAGE CHEESE

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to the field of fresh cheese, and in particular the production of cottage cheese via the use of lactose deficient lactic acid bacteria.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] A general concern of the Fresh Dairy industry is the short shelf-life of the products leading to logistic constraints and a large quantity of products ending in the waste as they have to be removed from the shelf. The shelf-life limitation is mostly due to the fact that dairy products include living organisms, which continue to be active in the product and degrade the product or seriously affect the taste of the product, because of acidification originating from continuing lactose conversion into lactic acid during storage.

[0006] Currently, for example, the shelf-life of green-labelled cottage cheese varies from 2 to 3 weeks (or about 16 to 21 days, depending on the respect of the cold chain), after which post-acidification occurs and the cheese is rejected by consumers as too acidic. Within this short life-time, after manufacturing, the product is stored (2-3 days) to allow for quality control to happen before being shipped to the merchant or retailer to be made available to consumers. The shipping takes another 2- 3 days. These different steps further reduce the shelf-life of the product once in the hands of the retailer or consumer.

[0007] As an attempt to solve the post-acidification problem, it has been disclosed to introduce lactase enzymes in the starting milk to consume most lactose upstream of the product manufacturing process in order to avoid remaining lactose in the final product. For example, WO2022 / 258817A1 discloses a process for producing a fermented milk product, e.g., cottage cheese, having a relatively stable pH value after the end of fermentation. A lactase is added to the milk before the milk seeding step to convert lactose in the milk into glucose and galactose. Then, a starter culture composed of lactose-deficient lactic acid bacteria, capable of metabolizing the produced glucose and galactose is used. This technique makes it possible to produce an adequate and limited quantity of glucose / galactose to limit the activity of the bacteria and thus control the growth of the culture to reach a precise pH of interest in order to limit post-acidification. However, it is not desirable, and not always allowed from a regulatory perspective, to have remaining enzymes in dairy food products.

[0008] W02019 / 043115A1 discloses a process for producing mesophilic fermented milk products by using lactose-deficient lactic acid bacteria (LAB) with the objective of reducing post-acidification. Starter cultures composed of at least one mesophilic lactose-deficient strain of Lactococcus lactis and at least one thermophilic lactose-deficient strain of Streptococcus thermophilus, both able to metabolize non-lactose carbohydrates, along with a defined amount of a non-lactose carbohydrate are added to milk. Fermentation occurs up to a defined target pH reached once all sugar is consumed. No further acidification is expected. This process appears suitable for fermented dairy products such as buttermilk, sour milk, cultured milk, smetana, sour cream, kefir, and fresh cheese such as quark and cream cheese. However, as shown in the present disclosure, this approach is not suitable for manufacturing cottage cheese.

[0009] Indeed, the cottage cheese manufacturing process comprises steps that are not needed for fermented dairy products such as buttermilk, sour milk, cultured milk, smetana, sour cream, kefir and fresh cheese such as quark and cream cheese. For cottage cheese, a curd having a specific texture has to be obtained and separated from the whey. The whey is drained using pumps. The curd is further mixed with cream, thereby reintroducing an amount of lactose in the process. The applicant has tested the starter cultures disclosed in W02019 / 043115A1 to produce cottage cheese, but were not successful. The desired curd texture was never obtained: the grains were soft and breakable, desired firmness before cutting was obtained at pH much higher than expected (about 4.85 instead of around 4.65) and the moisture retention was too high. In addition, fermentation time had to be increased to over 16 hours and the whey was too viscous to be drained efficiently.

[0010] The inventors have therefore developed an improved process to produce cottage cheese with an improved shelf life.

[0011] SUMMARY OF THE DISCLOSURE

[0012] In a first main aspect, the disclosure relates to a process for producing a cottage cheese comprising steps of:

[0013] (a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition comprises less than 5% hyper-texturizing bacteria;

[0014] (b) Fermenting the milk base until a gel is obtained;

[0015] (c) Cutting the gel;

[0016] (d) Stirring and scalding to obtain a curd and a whey;

[0017] (e) Removing the whey;

[0018] (f) Processing the curd further into cottage cheese. In a further main aspect, the disclosure relates to a process for producing a cottage cheese comprising steps of:

[0019] (a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889;

[0020] (b) Fermenting the milk base until a gel is obtained;

[0021] (c) Cutting the gel;

[0022] (d) Stirring and scalding to obtain a curd and a whey;

[0023] (e) Removing the whey;

[0024] (f) Processing the curd further into cottage cheese.

[0025] In a further main aspect, the disclosure relates to a process for producing a cottage cheese comprising steps of:

[0026] (a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and glucose-deficient thermophilic lactic acid bacteria;

[0027] (b) Fermenting the milk base until a gel is obtained, wherein preferably the firmness of the gel that is obtained is between about 100 to 270 Pa;

[0028] (c) Cutting the gel;

[0029] (d) Stirring and scalding to obtain a curd and a whey;

[0030] (e) Removing the whey;

[0031] (f) Processing the curd further into cottage cheese.

[0032] In one embodiment of this aspect, the bacterial composition comprises, or further comprises, a glucose-positive mesophilic strain and a glucose-producing thermophilic strain.

[0033] In a further main aspect, the disclosure relates to cottage cheese obtained or obtainable by the process of the disclosure as described herein.

[0034] In an even further main aspect, the disclosure relates to a bacterial composition suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria and thermophilic lactic acid bacteria, wherein the bacterial composition comprises less than 5% hyper-texturizing bacteria.

[0035] In an even further main aspect, the disclosure relates to a bacterial composition suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889.

[0036] In an even further main aspect, the disclosure relates to a bacterial composition suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and glucose- deficient thermophilic lactic acid bacteria, wherein preferably the bacterial composition comprises, or further comprises, a glucose-positive mesophilic strain and a glucose-producing thermophilic strain.

[0037] DETAILED DESCRIPTION OF THE DISCLOSURE

[0038] Definitions

[0039] The process of the disclosure uses a milk base. The term "milk base" refers to animal milk, preferably cow milk or goat milk.

[0040] As used herein, the terms "lactic acid bacteria" designate gram-positive, microaerophilic or anaerobic bacteria, which ferment sugar with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as frozen or freeze- dried compositions for bulk starter propagation or as so-called "Direct Vat Set" (DVS) compositions, intended for direct inoculation into a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product. Such compositions are in the art also referred to as "starter cultures" or "starters".

[0041] The term "mesophilic" herein refers to microorganisms that thrive best at moderate temperatures (15°C-35°C). The industrially most useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "thermophilic" herein refers to microorganisms that thrive best at temperatures above 35°C. The industrially most useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp.

[0042] The terms "lactose-deficient" and "lactose-negative" in short "Lac(-)" are used in the context of the present disclosure to characterize lactic acid bacteria which either partially or completely lost the ability to use lactose as a source for cell growth or maintaining cell viability. Such lactic acid bacteria are capable of metabolizing one or several carbohydrates selected from sucrose, galactose and / or glucose or another fermentable carbohydrate. Since these carbohydrates are not naturally present in milk in sufficient amounts to support fermentation by lactose deficient mutants, it is necessary to add these carbohydrates to the milk. Lactose deficient and partially deficient lactic acid bacteria can be characterized as white colonies on a medium containing lactose and X-Gal.

[0043] Similarly, "glucose-deficient" and "glucose-negative" in short "Glu(-)" are used in the context of the present disclosure to characterize lactic acid bacteria which either partially or completely lost the ability to use glucose as a source for cell growth or maintaining cell viability. Such lactic acid bacteria are capable of metabolizing one or several carbohydrates selected from sucrose, galactose and / or lactose or another fermentable carbohydrate.

[0044] The terms "lactose-positive" and "lactose-metabolizing" in short "Lac(+)" are used to refer to strains capable of using lactose as a source for cell growth or maintaining cell viability. Similarly, the terms "glucose-positive" and "glucose-metabolizing" in short "Glu(+)" are used to refer to strains capable of using glucose as a source for cell growth or maintaining cell viability and the terms "sucrosepositive" and "sucrose-metabolizing" in short "Suc(+)" refer to strains capable of using sucrose as a source for cell growth or maintaining cell viability.

[0045] In the context of bacterial compositions and in the context of carbohydrates added, percentages (%) refer to weight percentages.

[0046] The term "hyper-texturizing" herein refers to a thermophilic strain, such as a Streptococcus thermophilus strain, which, under the conditions described in Example 5, generates fermented milk gels with firmness index greater than 16 at pH 4.60.

[0047] Further aspects and embodiment of the disclosure

[0048] The inventors made the surprising observation that when lactose-deficient bacterial compositions as described in W02019 / 043115A1 were used for the production of cottage cheese (with the objective of avoiding post-acidification of the product during storage) the gel firmness at cutting was compromised (too high at acceptable pH range) and the whey became viscous, compromising its removal by pumping. It was found that these problems could be avoided by reduction of high texturizing components in the bacterial culture.

[0049] Thus, in a first main aspect, the disclosure relates to a process for preparing cottage cheese comprising the steps of:

[0050] (a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition comprises less than 5% hyper-texturizing bacteria;

[0051] (b) Fermenting the milk base until a gel is obtained;

[0052] (c) Cutting the gel;

[0053] (d) Stirring and scalding to obtain a curd and a whey;

[0054] (e) Removing the whey;

[0055] (f) Processing the curd further into cottage cheese.

[0056] Cottage cheese is a widely known and consumed food product. It may be seen as a cheese curd product with a mild flavor. It is typically drained, but not pressed so some whey may remain and the individual curds may remain loose. Different styles of cottage cheese are made from milks with different protein levels and in small curd or large curd preparations.

[0057] The process of the disclosure aims at controlling (reducing) post-acidification and this is in part achieved by the use of bacterial compositions, starter cultures, that contain mesophilic lactic acid bacteria that are lactose-deficient and metabolize an added non-lactose carbohydrate during fermentation. Production of cottage cheese typically involves adding lactose-containing cream to the final product, but the lactose-deficient mesophilic lactic acid bacteria will not be able to ferment the lactose, thus avoiding post-acidification. Thermophilic lactic acid bacteria are included, inter alia, to reduce fermentation time.

[0058] Preferably, there is inoculated at least 104cfu / ml of bacteria to the milk, such as inoculated from 104to IO10cfu / ml, for example inoculated from 105to 106cfu / ml.

[0059] In one embodiment, the non-lactose carbohydrate that is added in step (a) of the process of the disclosure is sucrose, glucose, galactose and / or fructose. In one embodiment, the non-lactose carbohydrate is sucrose. In one embodiment, the non-lactose carbohydrate is sucrose and the bacterial composition comprises sucrose-positive mesophilic lactic acid bacteria and / or sucrosepositive thermophilic lactic acid bacteria. Further embodiments of the bacterial composition are described herein below. In one embodiment, the added non-lactose carbohydrate, for example sucrose, represents at least 0.4% by weight of the milk base. In one embodiment, the added nonlactose carbohydrate represents between 0.1% and 1%, preferably between 0.3% and 0.7%, for example 0.4%, 0.5% or 0.6% by weight of the milk base.

[0060] In one embodiment, the milk base is animal milk, such as cow milk or goat milk. In one embodiment, wherein the milk base comprises a protein level between 3.0% and 3.8%, preferably a protein level ranging from 3.5% to 3.7%.

[0061] In step (b), the milk base is fermented until a gel (also termed "coagulum") is obtained. In one embodiment, the milk base is fermented for a period of time until the gel has a pH between 4.55 and 4.80, preferably between 4.65 and 4.75. Fermentation is typically is carried out at temperatures between 31°C and 35°C. Optionally, a milk coagulating enzyme may be added, e.g. rennet or chymosin, before, during or after step (b).

[0062] In step (c), the gel is cut. In one embodiment, at cutting, the gel has a firmness of between 100 and 270 Pa. In one embodiment, at cutting, the gel has a firmness index of between 10 and 20 when tested using a Chymograph as described in Example 6.

[0063] Step (d) comprises stirring and scalding to obtain a curd and a whey. Scalding is not carried out in the manufacture of the fresh dairy products, such as disclosed in W02019 / 043115A1. The scalding step can also be called cooking step and, in cottage cheese-making, it aims to drain the curd and to allow the development of a coating layer around the grains. During this step the whey may be separated from the curd, and removed, allowing the curd to be "drained" and to achieve suitable moisture. Later in this step, a fast temperature increase is typically carried out leading to the development of a coating layer around the grains, which will give suitable texture (crunchy and not breakable grains characteristic of cottage cheese). Thus, step (d) is achieved by applying a suitable heating and stirring process (stirring helps to remove the whey; to increase temperature by favoring heat exchange; and to keep the temperature homogeneous in the cheese vats).

[0064] The following parameters of the scalding step are preferred:

[0065] • Duration: suitable from 60 to 180 min, preferably from 90 to 150 min, most preferably 120 min

[0066] • Temperature increase: progressive increase temperature from fermentation to final scalding temperature in increasing rates varying from 0.1 to 2°C per minute (increasing rate is managed by regulating heating / stirring profile)

[0067] • Final scalding temperature from 55°C to 65°C , for example from 58°C to 63°C. In step (e), the whey is removed, for example by draining. In one embodiment, the whey has a viscosity below 0.9 Pa, where viscosity is measured by shear stress, for example using rheometer as described in Example 6 herein.

[0068] Step (f) comprises processing the curd further into cottage cheese. This step may comprise: (fl) Washing the curd, and (f2) Mixing the curd with cream to obtain cottage cheese, optionally including the addition of salt. Step (fl) may be performed more than once, for example, two, three or more times. The curd is typically washed in cold water. Washing steps remove non-used lactose from curd, which may avoid taste issues as cottage cheese curd is not sweet. Preferably, the curd comprises at least 16% of dry matter, such as at least 18% of dry matter.

[0069] A draining step is typically performed after step (fl), before proceeding with step (f2). Addition of salt during step (f2) may further inhibit fermentation upon storage.

[0070] The process of the disclosure is advantageously devoid of (added) lactase.

[0071] The final product typically has a pH of about 5.0 to 5.2 (when non-fermented dressing is used) and is ideally stored at cold temperatures from 4°C to 6°C.

[0072] As described above, the process of the disclosure involves the use of a bacterial composition comprising lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria. The phrase "lactose-deficient mesophilic lactic acid bacteria" indicates that at least one strain of lactose-deficient mesophilic lactic acid bacteria is present in the culture, i.e. the composition may contain one or more than one strain of lactose-deficient mesophilic lactic acid bacteria, such as two, three, four or more strains. Similarly "thermophilic lactic acid bacteria" indicates that at least one strain of thermophilic lactic acid bacteria is present in the culture, i.e. the composition may contain one or more than one strain of thermophilic lactic acid bacteria, such as two, three, four or more strains. Furthermore, the term "comprising" has its usual meaning, i.e. it does not exclude the presence of additional components.

[0073] In one embodiment, the lactose-deficient mesophilic lactic acid bacteria comprise, or consist of, a Lactococcus lactis strain. In one embodiment, the lactose-deficient mesophilic lactic acid bacteria comprise, or consist of, a Lactococcus lactis strain and the Lactococcus lactis strain is of a subspecies selected from lactis, lactis biovar diacetylactis, and / or cremoris. In one embodiment, the lactose- deficient mesophilic lactic acid bacteria comprise two or more strains of lactose-deficient mesophilic lactic acid bacteria, such as a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain. In one embodiment, the mesophilic lactic acid bacteria do not comprise lactose-metabolizing mesophilic lactic acid bacteria. In one embodiment, the lactose-deficient mesophilic lactic acid bacteria comprise, or consist of, a lactose-deficient, sucrose-metabolizing Lactococcus lactis subsp. lactis strain and a lactose-deficient, sucrose-deficient, glucose-metabolizing Lactococcus lactis subsp. cremoris strain.

[0074] In one embodiment, the thermophilic lactic acid bacteria comprise lactose-deficient thermophilic lactic acid bacteria. In another embodiment, the thermophilic lactic acid bacteria comprise lactose-positive thermophilic lactic acid bacteria. In one embodiment, the thermophilic lactic acid bacteria comprise both lactose-deficient thermophilic lactic acid bacteria and lactosepositive thermophilic lactic acid bacteria. In one embodiment, the thermophilic lactic acid bacteria comprise, or consist of, one or more Streptococcus thermophilus strains, such as one or two Streptococcus thermophilus strains. In one embodiment, the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria, such as a glucose-deficient Streptococcus thermophilus strain. In one embodiment, the thermophilic lactic acid bacteria comprise glucose- deficient thermophilic lactic acid bacteria, such as a glucose-deficient Streptococcus thermophilus strain. In one embodiment, the thermophilic lactic acid bacteria comprise lactose-positive, sucrosepositive, glucose-deficient thermophilic lactic acid bacteria, such as a lactose-positive, sucrosepositive, glucose-deficient Streptococcus thermophilus strain. In one embodiment, the thermophilic lactic acid bacteria comprise, or consist of, a lactose-deficient, sucrose-positive Streptococcus thermophilus strain and a lactose-positive, sucrose-positive, glucose-deficient Streptococcus thermophilus strain.

[0075] In one embodiment, the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain and wherein the thermophilic lactic acid bacteria comprise one or more Streptococcus thermophilus strains.

[0076] In one embodiment, the lactic acid bacteria comprise at least one strain of glucose- metabolizing mesophilic lactic acid bacteria, such as a strain of glucose-metabolizing Lactococcus lactis subsp. cremoris and at least one strain of glucose-producing thermophilic lactic acid bacteria, such as a strain of glucose-producing Streptococcus thermophilus.

[0077] Various ratios of lactose-deficient mesophilic lactic acid bacteria to thermophilic lactic acid bacteria in the bacterial composition to be used in the process of the disclosure may be used. In one embodiment of the process, the bacterial composition comprises: 65% to 95% of lactose-deficient mesophilic lactic acid bacteria, and 5% to 35% of thermophilic lactic acid bacteria. In one embodiment of the process, the bacterial composition comprises: 70% to 95% of lactose-deficient mesophilic lactic acid bacteria, and 5% to 30% of thermophilic lactic acid bacteria. In one embodiment of the process, the bacterial composition comprises: 70% to 75% of lactose-deficient mesophilic lactic acid bacteria, and 25% to 30% of thermophilic lactic acid bacteria.

[0078] As described above, in one aspect, the bacterial composition used in the process of the disclosure comprises less than 5%, for example less than 4%, such as less than 3%, for example less than 2%, such as less than 1%, for example less than 0.5% hyper-texturizing bacteria, such as no hypertexturizing bacteria. Hyper-texturizing bacteria are bacteria that produce a relatively large amount of exopolysaccharides. Exopolysaccharide production may for example be determined as described in Mizuno et al. 2020 Front. Microbiol., Sec. Food Microbiology, Volume 11 - article 894.

[0079] In one embodiment, the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides, than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889, for example when tested as described by Mizuno et al. 2020, for example after an incubation time of at least 10 hours.

[0080] A number of suitable strains for use in the process of the disclosure have been described in the art.

[0081] Lactose-deficient Lactococcus lactis bacteria suitable for use in the process of the disclosure may include one or more of the strains deposited under the following numbers: DSM32398; DSM18882; DSM18893; DSM32601; DSM32602; DSM32603; DSM32604; DSM32605; DSM32829; DSM32830; DSM32832.

[0082] The thermophilic Streptococcus thermophilus bacteria suitable for use in the process of the disclosure may include one or more of the strains deposited under the following numbers: DSM 25850; DSM25851; DSM26722; DSM28889; DSM32227; DSM33719; DSM33720; DSM28952, DSM28953, DSM32559, DSM32560 and DSM25012.

[0083] In one embodiment of the process of the disclosure, the bacterial composition comprises or consists of 35-45% of strain DSM32832, 35-40% of strain DSM18893 and 5-25% of strain DSM28889.

[0084] In a further main aspect, the disclosure relates to cottage cheese obtained or obtainable by the process of the disclosure as described herein.

[0085] Bacterial compositions

[0086] As describes above, in a further main aspect, the disclosure relates to compositions suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria and thermophilic lactic acid bacteria, wherein the composition comprises less than 5% hyper-texturizing bacteria. In one embodiment of the composition, the lactose-deficient mesophilic lactic acid bacteria comprise, or consist of, a Lactococcus lactis strain. In one embodiment of the composition, the lactose- deficient mesophilic lactic acid bacteria comprise, or consist of, a Lactococcus lactis strain and the Lactococcus lactis strain is of a subspecies selected from lactis, lactis biovar diacetylactis, and / or cremoris. In one embodiment of the composition, the bacterial composition comprises two or more strains of lactose-deficient mesophilic lactic acid bacteria, such as a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain. In one embodiment of the composition, the bacterial composition does not comprise lactose-metabolizing mesophilic lactic acid bacteria. In one embodiment of the composition, the lactose-deficient mesophilic lactic acid bacteria comprise, or consist of, a lactose-deficient, sucrose-metabolizing Lactococcus lactis subsp. lactis strain and a lactose-deficient, sucrose-deficient, glucose-metabolizing Lactococcus lactis subsp. cremoris strain.

[0087] In one embodiment of the composition, the thermophilic lactic acid bacteria comprise lactose- deficient thermophilic lactic acid bacteria. In another embodiment of the composition, the thermophilic lactic acid bacteria comprise lactose-positive thermophilic lactic acid bacteria. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise both lactose-deficient thermophilic lactic acid bacteria and lactose-positive thermophilic lactic acid bacteria. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise, or consist of, one or more Streptococcus thermophilus strains, such as one or two Streptococcus thermophilus strains. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria, such as a glucose-deficient Streptococcus thermophilus strain. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria, such as a glucose-deficient Streptococcus thermophilus strain. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise lactose-positive, sucrose-positive, glucose-deficient thermophilic lactic acid bacteria, such as a lactose-positive, sucrose-positive, glucose-deficient Streptococcus thermophilus strain. In one embodiment of the composition, the thermophilic lactic acid bacteria comprise, or consist of, a lactose-deficient, sucrosepositive Streptococcus thermophilus strain and a lactose-positive, sucrose-positive, glucose-deficient Streptococcus thermophilus strain.

[0088] In one embodiment of the composition, the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain and wherein the thermophilic lactic acid bacteria comprise one or more Streptococcus thermophilus strains. In one embodiment of the composition, the bacterial composition comprises at least one strain of glucose-metabolizing mesophilic lactic acid bacteria, such as a strain of glucose-metabolizing Lactococcus lactis subsp. cremoris and at least one strain of glucose-producing thermophilic lactic acid bacteria, such as a strain of glucose-producing Streptococcus thermophilus.

[0089] Lactose-deficient Lactococcus lactis bacteria suitable for use in the composition of the disclosure may include one or more of the strains deposited under the following numbers: DSM32398; DSM18882; DSM18893; DSM32601; DSM32602; DSM32603; DSM32604; DSM32605; DSM32829; DSM32830; DSM32832.

[0090] The thermophilic Streptococcus thermophilus bacteria suitable for use in the composition of the disclosure may include one or more of the strains deposited under the following numbers: DSM25850; DSM25851; DSM26722; DSM28889; DSM32227; DSM33719; DSM33720; DSM28952, DSM28953, DSM32559, DSM32560 and DSM25012.

[0091] Various ratios of lactose-deficient mesophilic lactic acid bacteria to thermophilic lactic acid bacteria in the bacterial composition of the disclosure may be used. In one embodiment of the composition, the bacterial composition comprises: 65% to 95% of lactose-deficient mesophilic lactic acid bacteria, and 5% to 35% of thermophilic lactic acid bacteria. In one embodiment of the composition, the bacterial composition comprises: 70% to 95% of lactose-deficient mesophilic lactic acid bacteria, and 5% to 30% of thermophilic lactic acid bacteria. In one embodiment of the composition, the bacterial composition comprises: 70% to 75% of lactose-deficient mesophilic lactic acid bacteria, and 25% to 30% of thermophilic lactic acid bacteria.

[0092] As described above, in one aspect, the composition comprises less than 5%, for example less than 4%, such as less than 3%, for example less than 2%, such as less than 1%, for example less than 0.5% hypertexturizing bacteria, such as no hyper-texturizing bacteria.

[0093] In one embodiment, the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides, than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889, for example when tested as described by Mizuno et al. 2020, for example after an incubation time of at least 10 hours.

[0094] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. EMBODIMENTS

[0095] 1. A process for preparing cottage cheese comprising the steps of:

[0096] (a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition comprises less than 5% hyper-texturizing bacteria;

[0097] (b) Fermenting the milk base until a gel is obtained;

[0098] (c) Cutting the gel;

[0099] (d) Stirring and scalding to obtain a curd and a whey;

[0100] (e) Removing the whey;

[0101] (f) Processing the curd further into cottage cheese.

[0102] 2. The process according to embodiment 1, wherein the non-lactose carbohydrate is sucrose, glucose, galactose and / or fructose.

[0103] 3. The process according to embodiments 1 or 2, wherein the non-lactose carbohydrate is sucrose.

[0104] 4. The process according to any of the preceding embodiments, wherein the added non-lactose carbohydrate represents between 0.1% and 1%, preferably between 0.3% and 0.7%, by weight of the milk base.

[0105] 5. The process according to any one of the preceding embodiments, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain.

[0106] 6. The process according to any one of the preceding embodiments, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain and wherein the Lactococcus lactis strain is of a subspecies selected from lactis, lactis biovar diacetylactis, and / or cremoris.

[0107] 7. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises two or more strains of mesophilic lactic acid bacteria, such as a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain.

[0108] 8. The process according to any one of the preceding embodiments, wherein the thermophilic lactic acid bacteria comprise lactose-deficient thermophilic lactic acid bacteria.

[0109] 9. The process according to any one of the preceding embodiments, wherein the thermophilic lactic acid bacteria comprise lactose-positive thermophilic lactic acid bacteria.

[0110] 10. The process according to any one of the preceding embodiments, wherein the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria.

[0111] 11. The process according to any one of the preceding embodiments, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain and wherein the thermophilic lactic acid bacteria comprise a Streptococcus thermophilus strain.

[0112] 12. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises at least one strain of glucose-metabolizing mesophilic lactic acid bacteria, such as a strain of glucose-metabolizing Lactococcus lactis subsp. cremoris and at least one strain of glucose-producing thermophilic lactic acid bacteria, such as a strain of glucose-producing Streptococcus thermophilus.

[0113] 13. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises one or more lactose-deficient Lactococcus lactis bacteria selected from the group consisting of: DSM32398; DSM18882; DSM18893; DSM32601; DSM32602; DSM32603; DSM32604; DSM32605; DSM32829; DSM32830 and DSM32832.

[0114] 14. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises one or more thermophilic Streptococcus thermophilus bacteria selected from the group consisting of: DSM25850; DSM25851; DSM26722; DSM28889; DSM32227; DSM33719; DSM33720; DSM28952, DSM28953, DSM32559, DSM32560 and DSM25012.

[0115] 15. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises:

[0116] • 65% to 95% of lactose-deficient mesophilic lactic acid bacteria, and

[0117] • 5% to 35% of thermophilic lactic acid bacteria.

[0118] 16. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises:

[0119] • 70% to 95% of lactose-deficient mesophilic lactic acid bacteria, and

[0120] • 5% to 30% of thermophilic lactic acid bacteria.

[0121] 17. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises:

[0122] • 70% to 75% of lactose-deficient mesophilic lactic acid bacteria, and

[0123] • 25% to 30% of thermophilic lactic acid bacteria.

[0124] 18. The process according to any one of the preceding embodiments, wherein the bacterial composition comprises less than 4%, such as less than 3%, for example less than 2%, such as less than 1%, for example less than 0.5% hyper-texturizing bacteria, such as no hyper-texturizing bacteria.

[0125] 19. The process according to any one of the preceding embodiments, wherein the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889.

[0126] 20. The process according to any one of the preceding embodiments, wherein the milk base is fermented for a period of time until the gel has a pH between 4.55 and 4.80, preferably between 4.65 and 4.75.

[0127] 21. The process according to any one of the preceding embodiments, wherein the gel at cutting has a firmness of between 100 and 270 Pa.

[0128] 22. The process according to any one of the preceding embodiments, wherein the whey has a viscosity below 0.9 Pa.

[0129] 23. The process according to any one of the preceding embodiments, wherein curd comprises at least 16% of dry matter, such as at least 18% of dry matter.

[0130] 24. The process according to any one of the preceding embodiments, wherein the milk base comprises a protein level between 3.0 and 3.8%, preferably a protein level ranging from 3.5% to 3.7%.

[0131] 25. The process according to any one of the preceding embodiments, wherein step (f) comprises additional steps of:

[0132] (fl) Washing the curd, and

[0133] (f2) Mixing the curd with cream to obtain cottage cheese, optionally including the addition of salt.

[0134] 26. Cottage cheese obtained or obtainable by the process according to one of the preceding embodiments.

[0135] 27. A bacterial composition suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria and thermophilic lactic acid bacteria, wherein the composition comprises less than 5% hyper-texturizing bacteria.

[0136] 28. The bacterial composition according to embodiment 27, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain.

[0137] 29. The bacterial composition according to embodiments 27 or 28, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain and wherein the Lactococcus lactis strain is of a subspecies selected from lactis, lactis biovar diacetylactis, and / or cremoris.

[0138] 30. The bacterial composition according to any one of embodiments 27 to 29, wherein the bacterial composition comprises two or more strains of mesophilic lactic acid bacteria, such as a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain.

[0139] 31. The bacterial composition according to any one of embodiments 27 to 30, wherein the thermophilic lactic acid bacteria comprise lactose-deficient thermophilic lactic acid bacteria.

[0140] 32. The bacterial composition according to any one of embodiments 27 to 31, wherein the thermophilic lactic acid bacteria comprise lactose-positive thermophilic lactic acid bacteria. 33. The bacterial composition according to any one of embodiments 27 to 32, wherein the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria.

[0141] 34. The bacterial composition according to any one of embodiments 27 to 33, wherein the lactose- deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis subsp. lactis strain and a Lactococcus lactis subsp. cremoris strain and wherein the thermophilic lactic acid bacteria comprise a Streptococcus thermophilus strain.

[0142] 35. The bacterial composition according to any one of embodiments 27 to 34, wherein the bacterial composition comprises at least one strain of glucose-metabolizing mesophilic lactic acid bacteria, such as a strain of glucose-metabolizing Lactococcus lactis subsp. cremoris and at least one strain of glucose-producing thermophilic lactic acid bacteria, such as a strain of glucose-producing Streptococcus thermophilus.

[0143] 36. The bacterial composition according to any one of embodiments 27 to 35, comprising at least one glucose-deficient Streptococcus thermophilus strain capable of metabolizing lactose.

[0144] 37. The bacterial composition according to any one of embodiments 27 to 36, wherein the bacterial composition comprises one or more lactose-deficient Lactococcus lactis bacteria selected from the group consisting of: DSM32398; DSM18882; DSM18893; DSM32601; DSM32602; DSM32603; DSM32604; DSM32605; DSM32829; DSM32830 and DSM32832.

[0145] 38. The bacterial composition according to any one of embodiments 27 to 37, wherein the bacterial composition comprises one or more thermophilic Streptococcus thermophilus bacteria selected from the group consisting of: DSM25850; DSM25851; DSM26722; DSM28889; DSM32227; DSM33719; DSM33720; DSM28952, DSM28953, DSM32559, DSM32560 and DSM25012.

[0146] 39. The bacterial composition according to any one of embodiments 27 to 38, wherein the bacterial composition comprises:

[0147] • 65% to 95% of lactose-deficient mesophilic lactic acid bacteria, and

[0148] • 5% to 35% of thermophilic lactic acid bacteria.

[0149] 40. The bacterial composition according to any one of embodiments 27 to 39, wherein the bacterial composition comprises:

[0150] • 70% to 95% of lactose-deficient mesophilic lactic acid bacteria, and

[0151] • 5% to 30% of thermophilic lactic acid bacteria.

[0152] 41. The bacterial composition according to any one of embodiments 27 to 40, wherein the bacterial composition comprises:

[0153] • 70% to 75% of lactose-deficient mesophilic lactic acid bacteria, and

[0154] • 25% to 30% of thermophilic lactic acid bacteria. 42. The bacterial composition according to any one of embodiments 27 to 41, wherein the bacterial composition comprises less than 4%, such as less than 3%, for example less than 2%, such as less than 1%, for example less than 0.5% hyper-texturizing bacteria, such as no hyper-texturizing bacteria. 43. The bacterial composition according to any one of embodiments 27 to 42, wherein the bacterial composition produces lower levels, such as two or more fold lower levels, of exopolysaccharides than a reference composition comprising: 39% DSM32832, 39% DSM18893, 17% DSM28952, and 5% DSM28889.

[0155] EXAMPLES

[0156] Material

[0157] F-DVS® FRESCO® 3000-80 (Catalogue no. 721603, Chr. Hansen A / S) in short "FRESCO®" a commercially available Lac(+) starter culture for Cottage cheese production was used as reference.

[0158] NATUREN® Premium 145 NB (Catalogue no. 113504, Chr. Hansen A / S).

[0159] Example 1 - Post-acidification in cottage cheese samples produced with lactose-deficient cultures (known cultures)

[0160] To evaluate the potential of application of lactose-deficient starters to control post-acidification in cottage cheese, an assay was carried out to investigate pH evolution of cottage cheese samples produced with a lactose-deficient (Lac(-)) culture as described in W02019 / 043115, in comparison with Lac(+) FRESCO® produced at the same day.

[0161] Samples of cottage cheese were produced with organic skim milk containing 3.25% protein; pasteurized at 73°C / 20 sec; with addition of 7.7 IMCU / 100L of NATUREN® Premium 145 NB (Catalogue no. 113504, Chr. Hansen A / S); with 0.53% of sucrose added. Fermentation was carried out at 33°C, using a dosage of 10U / 100L of a Lac(-) experimental culture as described in W02019 / 043115. Since target pH for gel cutting (4.65) was never reached in these conditions, gel was cut after 16 hours of fermentation, at pH 4.80, according to its firmness (empirically determined by touching and based on cheese-maker experience). The other process steps were carried out according to traditional cottage recipe (cooking for 2 hours, reaching final temperature of 57°C; whey removal; curd washing in 2 steps with cold water at 4°C to cool down the curd and reach °Brix < 1 in final washing water; draining in static perforated belt for 15 min). The obtained curd was mixed with cream in a ratio of 50:50 (curd:cream) and stored at 4°C up to 30 days. This first Lac(-) culture evaluated was not suitable for cottage cheese production, because it did not decrease pH to its target for gel cutting, delayed fermentation time to 16 hours (usually fermentation for cottage cheese production takes 5-6 hours) and negatively affected the whey separation, leading to a soft and breakable curd, different in texture from commercially available cottage cheese. However we use the obtained product to perform a first comparison between the pH evolution along shelf-life of a product produced with Lac(-) culture and a product from the market, produced with Lac(+) culture. Samples of cottage cheese commercially available in the Polish market, with same manufacturing date as that of our pilot product and produced with FRESCO® were collected for comparison. All cheeses were stored at 4°C for up to 30 days. Samples were collected after 10, 20 and 30 days of storage for measurement of pH. pH measurements were carried out at the same temperature for all samples (about 7-8°C).

[0162] Table 1. Evolution of pH of cottage cheese samples stored at 4°C. "Commercial" refers to a standard commercially available Lac(+) starter culture.

[0163] These first results indicated no post-acidification for samples produced with Lac(-) cultures. pH remained stable during at least 30 days. After that, samples started to show signs of spoilage by yeasts and other contaminant microbiota and the measurements were discontinued. For the commercial samples, a pronounced reduction in pH was observed at 30 days of storage. In the field, postacidification of cottage cheese produced without preservatives is expected to happen before (about 16 to 21 days of storage), but in our laboratorial trials the control of cold temperature was more strict than in the field, where cold chain breaks boost the appearance of post-acidification. These results suggested that the use of Lac(-) strains in cottage cheese fermentation could be a solution to control post-acidification in this type of cheese. However, the data also showed that adjustment of the bacterial culture and / or the process is needed, because the target pH was not reached, fermentation time was delayed and the whey separation was negatively affected, leading to a soft and breakable curd.

[0164] Example 2 - Testing other Lac(-) experimental cultures to evaluate the most suitable for cottage cheese production

[0165] To try and find a Lac(-) culture suitable for cottage cheese technology we tested the acidification performance of 4 different Lac(-) experimental cultures (composed of different strains and / or ratios).

[0166] Table 2. Composition of Lac(-) cultures used in the trial for selecting the most suitable candidate for cottage cheese production.

[0167] The trials were conducted with organic skim milk (pasteurized at 73°C / 20 sec) containing 3.5% protein and added of 1% sucrose. The amount of sucrose added was increased (from 0.53% to 1%) to ensure that all tested cultures would have enough substrate to decrease pH to the target value of 4.65. Experimental cultures were inoculated in a dosage of 33U / 100L. Fermentation was carried out in water bath at 32°C, in 200 mL scale glass bottles, and the pH was continuously measured by Axone System. FRESCO® was used in the same dosage of experimental cultures for comparison of the acidification performance.

[0168] Table 3. Time to reach target pH 4.65 of 4 different lactose deficient cultures compared with a lactose positive reference culture

[0169] Results showed that all 4 lactose deficient cultures were slower in acidification compared with the lactose-positive reference culture (FRESCO®). Blend 10 was the closest to reference, but still presented a large delay in time to reach target pH 4.65 (tpH 4.65), when compared with reference Lac(+) FRESCO® (8.9 and 5.9 hours, respectively).

[0170] We also evaluated the capability of these Lac(-) cultures to ferment lactose. For this experiment we tested the 4 different Lac(-) experimental cultures added in milk without sucrose addition. We used a high culture dosage (50U / 100L) and fermented at 32°C. As controls we added a "Blank" (milk without culture inoculation) and FRESCO®. The results (not shown) demonstrated that, even at higher dosages (50U / 100L), the Lac(-) cultures were not able to ferment milk without sucrose addition (same behavior as sample of milk without inoculation - blank), confirming that these cultures do not ferment lactose.

[0171] Even though experimental Lac(-) Blend 10 was 3 hours longer to reach tpH 4.65, compared to reference, this culture was chosen for further trials as it was the closest to Lac(+) FRESCO® reference.

[0172] Example 3 - Cottage cheese-making in pilot scale using selected experimental Lac(-) culture

[0173] To try and evaluate the performance of experimental Lac(-) culture Blend 10 in cottage cheese-making we ran a trial in pilot vat (25L scale) using organic skim milk (pasteurized at 73°C / 20 sec; with 3.5% protein) added of 1% sucrose. Experimental culture was inoculated in a dosage of 33U / 100L and fermentation was carried out at 32°C, up to gel cutting. Trials were run in 2 consecutive days in which we tested different targets for gel cutting. In both days one vat of experimental Lac(-) culture Blend 10 was produced in parallel with one vat of Lac(+) reference culture FRESCO®, also inoculated at dosage of 33U / 100L. The downstream process steps were carried out according to traditional cottage recipe (cooking for 2 hours, reaching final temperature of 57°C; whey removal; curd washing in 2 steps with cold water at 4°C to cool down the curd and reach °Brix < 1 in final washing water; draining in static perforated belt for 15 min). The obtained curd was mixed with cream in a ratio of 60:40 (curd:cream) and stored at 4°C.

[0174] In the first day of trials we cut the gel obtained with either FRESCO® or Blend 10 cultures at the same firmness (131 Pa, firmness of reference culture at pH 4.65). In the second day of trials the gels in both vats were cut at the same target pH (4.65).

[0175] Table 4. Results from cheese-making trial comparing Lac(-) Blend 10 with FRESCO® culture.

[0176] Regardless of the target for gel cutting (set at pH or gel firmness), in both days of trials experimental Lac(-) culture Blend 10 was longer in fermentation time (from 2 hours to 2h40 min) than reference FRESCO® culture. The curd obtained with this culture was also higher in moisture when compared with reference (about 3% to 5% higher), this fact led to a soft curd with breakable texture, not suitable for cottage cheese. In addition, in sensory evaluation, the curd obtained with Lac(-) culture was sweeter than that obtained with reference FRESCO®. This observation was confirmed by a sugar analysis of both curd samples which showed that curd obtained with Blend 10 had higher residual lactose when compared with curd obtained with reference culture (results are presented in Table 5).

[0177] Another relevant observation during cheese-making was that fermentation with Lac(-) Blend

[0178] 10 led to viscosity in the whey and it is not a desired feature during cottage cheese-making.

[0179] Table 5. Sugar contents of curd obtained with Lac(-) Blend 10 or Lac(+) FRESCO® culture

[0180] Example 4 - Optimization of Lac(-) concept in cottage cheese application

[0181] In this experiment, the Lac(-) culture was modified in order to try to optimize the culture for use in cottage cheese technology by increasing the ratio of mesophilic versus thermophilic bacteria. The Pilot blend tested was composed of 22% of thermophilic components and 78% of mesophilic components.

[0182] Table 6. Composition of Blend P.

[0183] Blend P was tested versus FRESCO®, a reference Lac(+) cottage cheese culture. The fermentation conditions were the same used in the previous pilot trial, i.e., organic skim milk (pasteurized at 73°C / 20 sec; with 3.5% protein and added of 1% sucrose), culture dosage of 33U / 100L, temperature of 32°C and gel cut determined by target pH 4.65. The scalding step was adapted to improve whey draining and curd firmness (heating at higher temperature of about 61-63°C and for longer time, about lh). The curd was washed three times to better remove remaining sucrose and non-utilized lactose and more water was used at each washing step (100% of initial volume of milk) to further help to remove remaining sugars.

[0184] Table 7. Comparison of Lac(-) Blend P with reference Lac(+) FRESCO® culture.

[0185] The fermentation time for the optimized Lac(-) culture was still more than 2.5h longer than reference (7h45 min vs 5h). In addition, the longer scalding at higher temperature did not help to better drain the curd produced with the Lac(-) blend, which presented higher moisture than reference, leading to a softer and breakable texture. Reducing the ratio of thermophilic components in the culture did not solve the issue of whey viscosity.

[0186] Example 5 - Further optimization of Lac(-) bacterial cultures for cottage cheese production and characterization of the texturizing properties of S. thermophilus (ST) strains

[0187] Aiming at solving the issue of whey viscosity and soft breakable curd we carried out a small scale trial with different Lac(-) cultures varying in the ratios of mesophilic / thermophilic bacteria. It was hypothesized that the suboptimal results were due to high production of exopolysaccharides (EPS) by certain strains, in particular Streptococcus thermophilus strain DSM28952. The strategy was to test different formulations in 200mL pots and simulate fermentation and cut steps to observe how syneresis happens.

[0188] The criteria for selecting the best blend option for the further pilot trial were:

[0189] • Fermentation time no longer than 9 hours (540 min)

[0190] • Good firmness at cutting: arbitrary scale from 1 (soft) to 4 (very firm) established by comparison with references (FRESCO® and a Lac(-) culture according to W02019 / 043115)

[0191] • Low or no viscosity in whey: arbitrary scale from 0 (no viscosity) to 3 (high viscosity) established by comparison with references (FRESCO® and a Lac(-) culture according to W02019 / 043115)

[0192] • Good syneresis after cutting - indicating capability to drain the way along cooking step (measurement of whey released after healing, by weight)

[0193] Trial design was as described below:

[0194] • 8 different lab-made blends (Pilot Blend P, as above, and Blends A, B, C, D, E, F and G) with different ratios of components were tested. Bacterial components are indicated in Table 8 as percentages.

[0195] • References: FRESCO® and a Lac(-) culture according to W02019 / 043115 (Lac(-) ref)

[0196] • Scale: 200 mL pots heated in water bath and gel cut with a knife

[0197] • Milk: skim milk (73°C / 20s) to which 1% sucrose was added for Lac(-) cultures

[0198] • Culture dosage: 33U / 100L • Fermentation T°C: 35°C

[0199] • Target for gel cutting: pH 4.65

[0200] Table 8: Blends tested in small scale pre-pilot trial and results

[0201] Blends with low percentages of Streptococcus thermophilus strain DSM28952 (Blend A) and blends without this strain (Blends E, F & G) were found to result in lower whey viscosity and more suitable firmness at cutting. Amongst these four experimental blends (A, E, F & G), blend G presented the shortest fermentation time (490 min) as compared to the others (505, 550 and 510 min for blends A, E & F, respectively). For this reason Blend G was selected for further trials.

[0202] These results supported the hypothesis that high firmness and whey viscosity issues were related to the high production of EPS by the ST strain DSM28952. Therefore, in the context of this invention Streptococcus thermophilus DSM28952 was considered as hyper-texturizing.

[0203] To better define the texturizing properties of ST strains used in cottage cheese cultures we run a trial comparing three ST strains:

[0204] • DSM28952, present in the blends which gave, in the small scale trial, higher gel firmness and whey viscosity.

[0205] • DSM28889, present in blends E, F & G (without DSM28952) which gave similar firmness to reference FRESCO® culture and did not generate viscosity in the whey.

[0206] • DSM25012, which is the ST strain present in FRESCO®, which gives reference firmness for cottage cheese make and no whey viscosity. To characterize the texturizing properties of these three strains we evaluated their capability to give texture to milk along fermentation. Each strain was inoculated in skim milk (3.3% protein, heat treatment at 85°C / 30 s, added of 0.6% sucrose) in a dosage of 33U / 100L and fermented at 35°C. Fermentation (acidification profile) was followed by continuous pH measurement with Axone system (ARDPI, FR) and texture was assessed by the evolution of gel firmness, measured with ChymoGraph® equipment (FR2991771B1, Chr-Hansen A / S). The characterization of the texturizing properties of these three strains was assessed by the gel firmness obtained at pH 4.60. The choice of pH 4.60 was based on the fact that it is within the range of target pH values for cutting the gel for cottage cheese make and it gives a good discrimination of strains with different texturizing properties.

[0207] Table 9. Time to reach pH 4.60 and gel firmness at pH 4.60 (followed by Axone and ChymoGraph®, respectively) of skim milk fermented with three Streptococcus thermophilus strains.

[0208] We observed that the three strains gave different gel firmness values at pH 4.60 (16.6, 15.5 and 13.5 for DSM28952, DSM28889 and DSM25012, respectively) allowing their discrimination as hypertexturizing or not hyper-texturizing, wherein hyper-texturizing strains are strain giving a Firmness Index at pH 4.60 greater than 16 and not hyper-texturizing strains are giving a Firmness Index at pH 4.60 lower than 16.

[0209] Example 6 - Evaluation of whey viscosity and gel firmness at cutting during cottage cheese make with optimized blend

[0210] To confirm that Blend G would be suitable for cottage cheese make, giving appropriate gel firmness at cutting and no viscosity in whey, we compared the performance of this culture with that of two references:

[0211] • FRESCO®, positive reference for cottage cheese make, with appropriate gel firmness at cutting and no viscosity in whey

[0212] • Lac(-) reference culture from W02019 / 043115, negative reference for cottage cheese make, with too much firmness at gel cutting and unacceptable whey viscosity.

[0213] Two trials were run: • Evaluation of gel firmness at cutting obtained after milk fermentation with each of the three cultures and assessed by ChymoGraph® (Chr-Hansen A / S)

[0214] • Evaluation of the viscosity in the whey obtained after milk fermentation with each of the three cultures, assessed by rheometer.

[0215] Milk fermentation step was the same for both analysis, skim milk (3.5% protein, heat treatment at 73°C / 20s, added of 0.6% sucrose for Lac(-) cultures) was inoculated with 33U / 100L of each culture and fermented at 35°C up to pH 4.65. Acidification profile (pH evolution along time) was measured by Axone System and used to detected when pH 4.65 was reached indicating end of fermentation and the cutting time. Gel firmness at cutting was evaluated on a ChymoGraph® (Chr-Hansen A / S), considering firmness at pH 4.65 as target for cutting.

[0216] Table 10. Gel firmness at cutting (pH 4.65) for milk samples fermented with different cultures.

[0217] We observed that positive reference Fresco® culture, containing a non-texturizing ST strain, gave a gel with a firmness index of about 15, while the presence of the texturizing ST strain DSM28889 in Blend G contributed to the higher gel firmness at cutting observed for this culture (firmness index of about 18.5), but this value is still acceptable for cottage cheese make and allows a proper cutting of the gel without breaking it. The firmness index observed for the Lac(-) reference (negative reference for cottage cheese make) was the highest one (about 20.5). This firmness level may compromise the quality of the curd and was considered too high for cottage cheese make. According to the these results we considered that a suitable firmness index for gel cutting in cottage cheese make, measured by ChymoGraph® (Chr-Hansen A / S), should be between 12 and 19.

[0218] Whey viscosity was assessed on a rheometer (Anton Paar Physica Rheometer with ASC, Automatic Sample Changer, Anton Paar® GmbH, Austria), by using a bob-cup. Samples were prepared as follow: after milk fermentation (as mentioned above), gel was cut with a knife and left to heal for 15 min (without stirring, to allow whey expulsion). After healing, whey was collected (separated from the curd) with the help of a sieve and cooled down to 13°C previous to being loaded on the rheometer for the analysis. Setting parameters from rheometer were the following: constant temperature of 13°C; holding time of 5 min (without any physical stress); oscillation step with constant strain = 0.3 %, frequency (f) = [0.5...8] Hz, 6 measuring points over 60 s; rotation step (to measure shear stress at 300 1 / s) with shear rate = [0.3-300] 1 / s, 21 measuring points over 210 s. Viscosity in whey was assessed as shear stress (in Pa) observed with a shear rate of 300 1 / s.

[0219] Table 11. Shear stress of whey samples obtained from skim milk fermented with three different cultures and evaluated by rheometer at shear rate of 300 1 / s.

[0220] Data from rheometer showed that the whey samples obtained with Blend G and FRESCO® cultures presented similar shear stress (about 0.85 Pa). Both whey samples were considered as non-viscous and suitable for cottage cheese make. On the other hand, the whey sample obtained with Lac(-) reference culture (containing DSM28952 strain) presented a much higher shear stress of about 1.24 Pa. This whey was considered as viscous and not suitable for cottage cheese make. According to these data we considered that whey with shear stress (at shear rate of 300 / s) lower than 0.90 can be considered as non-viscous and suitable for cottage cheese make.

[0221] Example 7 - Evaluation of post-acidification of cottage cheese produced using optimized blend

[0222] Blend G from the small scale experiment described in Example 5 was tested at larger scale to test the potential of this culture to limit post-acidification in cottage cheese products relative to cottage cheese produced using FRESCO®. Besides the cheese-making, the results from this trial were also used to confirm in a pilot-scale (25L) that Blend G would be suitable for cottage cheese-making, i.e., with fermentation time lower than 9 hours, no viscosity in the whey and proper curd draining leading to acceptable texture (not breakable curd similar to reference).

[0223] The following conditions were used:

[0224] Gel firmness at cutting was measured by CoaguSens® Flex (Rheolution, France) at pH of cutting for each culture and whey viscosity was evaluated by Rheometer (Anton Paar®, Austria) as described in Example 5. Curd moisture was evaluated by FoodScan (Foss®, Denmark).

[0225] For post-acidification assessment, the final product was stored at 8°C and the pH of samples was measured on day (D) D+l, D7 and from D13 onwards every 2 days to identify the beginning of post-acidification. When post-acidification was detected (i.e. a pH reduction of more than 0.2 pH units), this was considered to be the end of self-life.

[0226] Cheese-making results confirmed that Blend G was suitable for making cottage cheese with acceptable fermentation time and gel firmness at cutting, without viscosity in the whey and allowing good draining of the curd resulting in good moisture and no breakable curd.

[0227] Table 12. Cheese-making of cottage cheese using Lac(-) Blend G or Lac(+) FRESCO® culture.

[0228] In this cottage cheese trial gel firmness was measured with CoaguSens® Flex (Rheolution, France) and we observed that suitable gel firmness for cottage cheese gels would vary from about 100 to 270 Pa.

[0229] Table 13. Post-acidification at 8°C of samples produced with Lac(-) or Lac(+) cultures.

[0230] *n / a: not analysed. We observed that Blend G delayed post-acidification in cottage cheese samples for at least 10 days at 8°C. For samples made with reference FRESCO® culture, begin of post-acidification (reduction of 0.18 pH units) was detected after 15 days of incubation and after 19 days at 8°C a strong post-acidification was observed (reduction of 0.38 pH units). Analysis of FRESCO® samples was interrupted due to the end of shelf-life being reached. For samples made with Blend G, no post-acidification was observed for up to 21 days. Only after 28 days of storage at 8°C a beginning of post-acidification was detected (reduction of 0.16 pH units).

Claims

CLAIMS1. A process for preparing cottage cheese comprising the steps of:(a) Adding a non-lactose carbohydrate and a bacterial composition to a milk base, wherein the bacterial composition comprises lactose-deficient mesophilic lactic acid bacteria capable of metabolizing the non-lactose carbohydrate and thermophilic lactic acid bacteria, wherein the bacterial composition comprises less than 5% hyper-texturizing bacteria;(b) Fermenting the milk base until a gel is obtained;(c) Cutting the gel;(d) Stirring and scalding to obtain a curd and a whey;(e) Removing the whey;(f) Processing the curd further into cottage cheese.

2. The process according to claim 1, wherein the non-lactose carbohydrate is sucrose, glucose, galactose and / or fructose.

3. The process according to any one of the preceding claims, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain.

4. The process according to any one of the preceding claims, wherein the lactose-deficient mesophilic lactic acid bacteria comprise a Lactococcus lactis strain and wherein the Lactococcus lactis strain is of a subspecies selected from lactis, lactis biovar diacetylactis, and / or cremoris.

5. The process according to any one of the preceding claims, wherein the thermophilic lactic acid bacteria comprise lactose-deficient thermophilic lactic acid bacteria.

6. The process according to any one of the preceding claims, wherein the thermophilic lactic acid bacteria comprise lactose-positive thermophilic lactic acid bacteria.

7. The process according to any one of the preceding claims, wherein the thermophilic lactic acid bacteria comprise glucose-deficient thermophilic lactic acid bacteria.

8. The process according to any one of the preceding claims, wherein the bacterial composition comprises:• 65% to 95% of lactose-deficient mesophilic lactic acid bacteria, and• 5% to 35% of thermophilic lactic acid bacteria.

9. The process according to any one of the preceding claims, wherein the bacterial composition comprises less than 4%, such as less than 3%, for example less than 2%, such as less than 1%, for example less than 0.5% hyper-texturizing bacteria, such as no hyper-texturizing bacteria.

10. The process according to any one of the preceding claims, wherein the milk base is fermented for a period of time until the gel has a pH between 4.55 and 4.80, preferably between 4.65 and 4.75.

11. The process according to any one of the preceding claims, wherein the gel at cutting has a firmness of between 100 and 270 Pa.

12. The process according to any one of the preceding claims, wherein the whey has a viscosity below 0.9 Pa.

13. The process according to any one of the preceding claims, wherein step (f) comprises additional steps of:(fl) Washing the curd, and(f2) Mixing the curd with cream to obtain cottage cheese, optionally including the addition of salt.

14. Cottage cheese obtained or obtainable by the process according to one of the preceding claims.

15. A bacterial composition suitable for use in the production of cottage cheese, comprising, either as a mixture or as a kit-of-parts, lactose-deficient mesophilic lactic acid bacteria and thermophilic lactic acid bacteria, wherein the composition comprises less than 5% hyper-texturizing bacteria.

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