WO2022238329A1

WO2022238329A1 - Compositions and methods for producing fermented milk products

Info

Publication number: WO2022238329A1
Application number: PCT/EP2022/062487
Authority: WO
Inventors: Mette WINTHER BØRSTING; Andrea Budde-Niekiel; Antje-Kristin MOMMSEN; Dirk KUCKELSBERG
Original assignee: Dupont Nutrition Biosciences Aps
Priority date: 2021-05-11
Filing date: 2022-05-09
Publication date: 2022-11-17

Abstract

Provided herein are compositions, e.g., starter cultures, containing Lactococcus laudensis and methods of using the same to produce fermented milk products.

Description

COMPOSITIONS AND METHODS FOR PRODUCING FERMENTED MILK PRODUCTS

FIELD OF THE INVENTION

Provided herein are compositions, e.g., starter cultures, containing Lactococcus laudensis and methods of using the same to produce fermented milk products. BACKGROUND TO THE INVENTION

Starter cultures are used extensively in the food industry to manufacture fermented products including milk products, such as cheese and yoghurt. The use of starter cultures in the manufacture of such fermented foods can reduce production heterogeneity, thus providing consistent and predictable products. There remains a need for starter cultures capable of producing consistent and predictable fermented products, e.g., cheeses, yoghurts, with desirable organoleptic profiles. The compositions and methods provided herein address such needs.

SUMMARY OF THE INVENTION

Provided herein are starter cultures containing Lactococcus laudensis. In some embodiments, the starter cultures further contain species from the genera Lactococcus, Lactobacillus,

Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, Enterococcus or Bifidobacterium, or any combination of species from the foregoing.

Also provided herein are methods for producing a fermented milk product, the methods including fermenting a milk substrate as described herein with a starter culture as described herein to produce a fermented milk product. In some embodiments, the milk substrate is dairy milk.

Use of a starter culture according to any of the embodiments described herein for preparing a fermented milk product is also provided.

In some embodiments, the fermented milk product is a cheese. In some embodiments, the cheese is a semi-hard cheese.

Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect. DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions, such as starter cultures, and methods for producing fermented milk products, such as cheese. In some embodiments, the compositions include species Lactococcus laudensis. In some embodiments, the composition is a starter culture. In some embodiments, the milk product is a cheese. In some embodiments, the cheese is a semi-hard cheese. In some embodiments, the methods including the compositions, e.g., starter cultures, described herein produce a consistent and predictable fermented milk product.

A. General definitions

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

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

I. Compositions and Methods for Producing a Fermented Milk Product

The compositions and methods described herein are useful in the production of fermented milk products. In some embodiments, the production of the fermented milk product is facilitated by using compositions, e.g., starter cultures, including bacteria of the species Lactococcus laudensis (L laudensis). In some embodiments, the method for producing a fermented milk includes inoculating a milk substrate, for example as described in Section l-A below, with bacteria or compositions thereof, for example as described in Section l-B below, and fermenting the inoculated milk substrate, for example as described in Section l-C below.

The methods and compositions provided herein are useful for producing a consistent fermented milk product. Exemplary fermented milk products are described in Section l-D.

A. Milk Substrate

In some embodiments, the fermented milk is produced by inoculating and fermenting a milk substrate. It is contemplated herein that a milk substrate for producing the fermented milk may be any raw and/or processed milk material that can be subjected to fermentation, e.g., mesophilic or thermophilic fermentation, according to the methods provided herein. Thus, useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk-like products comprising protein, such as full fat or reduced fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, whey protein concentrate, or cream. In some embodiments, the milk substrate may originate from any mammal, e.g., being substantially pure mammalian milk, or reconstituted milk powder. The milk may be in the native state, reconstituted milk, ora skimmed milk. In some embodiments, the milk is supplemented with compounds necessary for the growth of bacteria or for the subsequent processing of fermented milk, such as fat, proteins of a yeast extract, peptone and/or a surfactant, for example.

In some embodiments, milk is a lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes, zebras, horses, donkeys, camels, and the like. In some embodiments, the milk substrate is dairy milk. In some embodiments, the milk is cow milk.

In some embodiments, the milk substrate is a full fat milk. For example, in some cases, the full fat milk is milk that has not had fat, e.g., milk fat, content removed. In some embodiments, the milk substrate is a reduced fat milk. For example, in some cases, the reduced fat milk is milk that has had a portion of its fat, e.g., milk fat, content removed. In some embodiments, the portion removed is, is about, or is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fat content, e.g., by weight. In some embodiments, the milk substrate is non-fat milk, also referred to as skim milk. For example, in some cases, the non-fat milk is milk that has had about 100% of its fat, e.g., milk fat, content removed. In some embodiments, the non-fat milk is or is about 0% fat by weight. In some embodiments, the percentage of fat by weight in a non-fat milk is negligible, e.g., at or below a level of detection or quantification. Suitable methods for measuring fat in milk, e.g., milk fat, include, but are not limited to, the Rose-Gottlieb reference method, the Gerber butyrometric method, the Mojonnier method, spectrophotometry, e.g., UV, and spectroscopy, e.g., infrared. In some embodiment, the full fat, reduced fat, low fat, and/or non-fat milk referred to herein is a dairy milk, optionally cow milk.

In some embodiments, the milk substrate is commercial UHT milk.

In some cases, the milk substrate may be homogenized and/ortreated with heat, i.e. , pasteurized.

In some embodiments, the milk substrate is mesophilically fermented. In some embodiments, the milk substrate is thermophilically fermented. Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

In some cases, homogenization is or includes intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed to break up fat in the milk substrate, e.g., 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.

In some embodiments, pasteurization includes treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. In some embodiments, pasteurization is attained by maintaining a specified temperature for a specified period of time. In some embodiments, the specific temperature is attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. In some embodiments, the milk substrate is heated to a temperature between or between about 62 to 65 °C. In some embodiments, the milk substrate is heated to a temperature between or between about 72 to 75 °C. In some embodiments, the milk substrate is heated to a temperature between or between about 85 to 127 °C. In some embodiments, the milk substrate is heated for or for about 30 to 32 minutes. For example, when the milk substrate is heated in the range of about 62 to 65 °C, the duration of heating is in the range of about 30 to 32 minutes. In some embodiments, the milk substrate is heated for or for about 15 to 30 seconds. For example, when the milk substrate is heated in the range of about 72 to 75 °C or about 85 to 127 °C, the duration of heating is in the range of about 15 to 30 seconds. The type of pasteurization process can be selected depending on the species of bacteria, e.g., harmful or undesirable bacteria, that are to be reduced, eliminated, or inactivated in the milk substrate.

In some embodiments, the milk substrate may undergo thermisation. In some cases, thermisation is useful for preserving the properties of the milk substrate while significantly reducing, eliminating, or inactivating unwanted or harmful bacteria. In some embodiments, thermisation includes heating the milk substrate to a temperature of or of about 57 °C. In some embodiments, thermisation includes heating the milk substrate to a temperature in the range of or of about 64 to 69 °C. In some embodiments, the milk substrate is heated for at least 15 seconds. For example, when the milk substrate is heated to a temperature of or of about 57 °C, the duration of heating is for at least 15 seconds. In some embodiments, the milk substrate is heated for or for about 5 to 40 seconds. For example, when the milk substrate is heated to a temperature of or of about 64 to 69 °C, the duration of heating is or is about 5 to 40 seconds. Thermisation can be employed depending on milk quality and cheese making technology to be used.

In some embodiments, after heating, the milk substrate is cooled. In some embodiments, the milk substrate is cooled to a temperature between or between about 30 to about 50°C, inclusive. In some embodiments, the cooling is rapid cooling. In some embodiments, the milk substrate is cooled for less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute. In some embodiments, the milk substrate is cooled for less than 10 minutes. In some embodiments, the milk substrate is cooled for less than 5 minutes. In some embodiments, the milk substrate is cooled for or for about 10 minutes. In some embodiments, the milk substrate is cooled for or for about 5 minutes. In some embodiments, the milk substrate is inoculated with a bacterial composition, e.g., starter culture as described in Section l-B, to facilitate fermentation.

B. Bacteria and Compositions Thereof

The milk substrate, e.g., as described in Section l-A, may be inoculated with one or more bacterial strains to facilitate fermentation. In some embodiments, the one or more bacterial strains are contained in a bacterial composition, such as a starter culture. In some embodiments, the one or more bacterial strains are contained in a starter culture. In some embodiments, the starter culture is a preparation of living bacteria able to assist in fermentation, e.g., mesophilic or thermophilic fermentation. In some embodiments, the milk substrate is inoculated with a starter culture. The terms inoculating and adding may be used interchangeably to refer to contacting a milk substrate with with one or more bacteria, for example as contained in a bacterial composition, e.g., starter culture.

In some embodiments, the milk substrate is inoculated with one or more bacterial strains separately. For example, the strains are not mixed together prior to being added to the milk substrate. In some embodiments, the strains are mixed together prior to being added to the milk substrate. Regardless of how a strain is added to the milk substrate, the strain or mixtures of strains used for inoculation may be referred to as or included in a starter culture. In some embodiments, the starter culture is a mesophilic starter culture, and the composition of the starter culture is suitable for mesophilic fermentation as described in Section l-C. In some embodiments, the starter culture is a thermophilic starter culture, and the composition of the starter culture is suitable for thermophilic fermentation as described in Section l-C. In some embodiments, the starter culture includes a combination of bacteria suitable for mesophilic and/or thermophilic fermentation.

In some embodiments, the starter culture is a pure culture, i.e., comprises or consists of a single bacterial strain. In some embodiments, the starter culture is a mixed culture, i.e. comprises or consists of at least one bacterial strain of the invention as described herein and at least one other bacterial strain. For example, at least 1 or more, and in particular 1 , 2, 3, 4 or 5, other bacterial strains are included in the starter culture. In some embodiments, the starter culture contains about 10 to about 40 bacterial strains, e.g., 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 bacterial strains. In some embodiments, the starter culture contains at least 10 bacterial strains. In some embodiments, the starter culture contains at most 40 bacterial strains. In some embodiments, the starter culture contains at least 10 and at most 40 bacterial strains. In some embodiments, the starter culture contains one or more lactic acid bacteria. As it is normal in lactic acid bacterial fermentation processes to apply a mixed culture as a starter culture, the composition may in some embodiments include a multiplicity of strains either belonging to the same species or belonging to different species.

In some embodiments, the starter culture includes at least bacteria of the genera Lactococcus. In some embodiments, the starter culture includes or consists of bacteria of the genera Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, Enterococcus, Bifidobacterium, Paralactobacillus, Acetilactobacillus, Agrilactobacillus, Amylolactobacillus, Apilactobacillus, Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus, Fioizapfeiia, Lacticaseibacillus, Lactiplantibacillus, Lapidilactobacillus, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacilus, Paucilactobacillus, Schleiferilactobacillus, Secundilactobacillus, or any combination thereof. In some embodiments, the starter culture includes or consists of bacteria of the genera Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, Enterococcus or Bifidobacterium, or any combination thereof. In some embodiments, the starter culture includes or consists of bacteria of the genera Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, or Bifidobacterium, or any combination thereof.

In some embodiments, the starter culture includes at least one Lactococcus lactis strain. In some embodiments, the starter culture includes of consists of Lactococcus lactis, Lactococcus laudensis, and Leuconostoc pseudomesenteroides.

In some embodiments, the starter culture is or includes one or more L laudensis strains.

In some embodiments the presence of one or more strain of L laudensis in the starter culture may improve or support diacetyl production, acetaldehyde conversion and breakdown, the viscosity, gel stiffness, mouth coating, flavor, post acidification, and/or acidification speed in the fermented milk.

In some embodiments, the starter culture, either as a pure or mixed culture as defined above, is in frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder. In some embodiments, the starter culture is in a frozen format or in the form of pellets or frozen pellets, in particular contained in one or more box or sachet. In some embodiments, the starter cultures as defined herein are in a powder form, such as a dried or freeze-dried powder, in particular contained in one or more box or sachet. In some embodiments, the liquid format is a bulk starter, such as a bacterial strain or mixture of bacterial strains previously propagated in a growth medium to obtain the required concentration for inoculation.

Commercial non-concentrated cultures of bacteria are referred to in industry as 'mother cultures', and are propagated at the production site, for example a dairy, before being added to an edible starting material, such as milk substrate, for fermentation.

In some embodiments, the starter culture, either as a pure culture or mixed culture as defined above, and whatever the format (frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder) includes the bacterial strain(s) described herein in a concentration in the range of or of about 10⁵ to 10¹³ cfu (colony forming units) per gram of the starter culture. In some embodiments, the concentration of the bacterial strain(s) described herein within the starter culture of the invention is in the range of or of about 10⁵ to 10¹³ cfu per gram of the starter culture, and in particular at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹ CFU/g of the starter culture. In some embodiments, when in the form of frozen or dried concentrate, the concentration of bacterial strain(s) described herein - as pure culture or as a mixed culture - within the starter culture is in the range of or of about 10⁵ to 10¹³ cfu/g of frozen concentrate or dried concentrate, and more preferably at least 10⁶, least 10⁷, least 10^s, at least 10⁹, at least 10¹⁰, at least 10¹¹ , at least 10¹², or at least 10¹³cfu/g of frozen concentrate or dried concentrate.

In some embodiments, the L laudensis is at least 0.05%, e.g., 0.1 , 0.5, 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, of the total starter culture. In some embodiments, L laudensis is or is about 0.05 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 5 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 10 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 20 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 30 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 40 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 50 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 60 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 70 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 80 to about 90% of the total starter culture. In some embodiments, L laudensis is or is about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% of the total starter culture. In some embodiments, the starter culture consists of L laudensis. Starter cultures may be prepared by techniques well known in the art such as those disclosed in

US 4,621 ,058. By way of example, starter cultures may be prepared by the introduction of an inoculum, for example a bacterium, to a growth medium to produce an inoculated medium and ripening the inoculated medium to produce a starter culture. Dried starter cultures may be prepared by techniques well known in the art, such as those discussed in US 4,423,079 and US 4,140,800. In some embodiments, the starter culture is a bulk starter culture. In some embodiments, the starter culture is in the form of concentrated frozen pellets. In some embodiments, the addition of the starter culture to the milk substrate is direct, e.g., as a direct vat inoculate, direct vat starter, direct vat set culture.

In some embodiments, the milk substrate may be inoculated with the starter culture or bacteria of the starter culture prior to initiating fermentation. In some embodiments, the milk substrate is inoculated with the starter culture or bacteria of the starter culture following, e.g., at a time point after, fermentation is initiated. In some embodiments, the milk substrate is inoculated with the starter culture or bacteria of the starter culture during fermentation.

Further microorganisms and/or additives may be added to the milk substrate before, during or after fermentation, e.g., mesophilic or thermophilic fermentation, of the milk substrate. For example, boosters, such as yeast extracts and/or amino acid containing compositions may be added to the milk substrate in addition to a starter culture, to support or promote microorganism function and facilitate successful production of the fermented milk product. Microorganisms that may be added to the milk substrate include those that will contribute in an advantageous manner to the properties of the fermented milk product. For example, added microorganisms, yeast extracts, or amino acid compositions may improve or support diacetyl production, acetaldehyde conversion and breakdown, the viscosity, gel stiffness, mouth coating, flavor, post acidification, and/or acidification speed in the fermented milk. Optionally, other ingredients may be added to the milk substrate, such as colors, stabilizers, e.g., pectin, starch, modified starch, CMC, etc.; or polyunsaturated fatty acids, e.g. omega-3 fatty acids. Such ingredients may be added at any point during the production process, e.g. before or after fermentation.

Components such as cryoprotectants and/or conventional additives including nutrients such as yeast extracts, sugars and vitamins, e.g., vitamin A, C, D, K or vitamins of the vitamin B family may also be added to the milk substrate either with or separate from the starter culture or other additives. Suitable cryoprotectants that may be added include components that improve the cold tolerance of the microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose and fructose. Other additives may include carbohydrates, flavors, minerals, enzymes (e.g., rennet, lactase and/or phospholipase).

In some embodiments, additives, microorganisms, or other components as described in the preceding paragraphs are added simultaneously or nearly simultaneously with the starter culture to the milk substrate, e.g., at the same time as inoculation of the milk substrate with the starter culture. In some embodiments, additives, microorganisms, or other components as described in the preceding paragraphs are added to the milk substrate before, e.g., temporally prior to, inoculation of the milk substrate with the starter culture. In some embodiments, additives, microorganisms, or other components as described in the preceding paragraphs are added after, e.g., temporally following, inoculation of the milk substrate with the starter culture. In some embodiments, additives, microorganisms, or other components as described in the preceding paragraphs may be added to the milk substrate before, during, or after fermentation, for example independent of the inoculation with the starter culture.

In some embodiments, the milk substrate is inoculated with the starter culture, and optionally additives, microorganisms, or other components as described in the preceding paragraphs above, by any suitable method. For example, the milk substrate may be inoculated by direct inoculation into a fermentation vessel.

C. Fermentation

The methods for producing a fermented milk product include mesophilic or thermophilic fermentation of a milk substrate, for example a milk substrate as described in Section l-A, inoculated with a starter culture, and optionally other additives, microorganisms, and components, as described in Section l-B.

Fermentation according to the methods provided herein facilitates the conversion of carbohydrates into alcohols or acids through the action of a microorganism, e.g., bacteria present, for example, in a starter culture. In some embodiments, fermentation according to the methods provided herein includes conversion of lactose to lactic acid.

Mesophilic and thermophilic fermentation processes may be selected to optimize parameters such as temperature, oxygen, and process time.

In some embodiments, the inoculated milk substrate is fermented at a temperature of or of about 12°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 50°C, 60°C, 70°C, 75°C, or 80 °C. In some embodiments, the inoculated milk substrate is fermented at a temperature of between or between about 15 to 80°C, 20 to 80°C, 30 to 80°C, 40 to 80°C, 50 to 80°C, 60 to 80°C, or 70 to 80°C. In some embodiments, more than one temperature or range of temperatures may be used for fermentation. For example, as known in the art, different steps of a cheese making process may take place at the same or different temperatures.

In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 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. In some embodiments, mesophilic fermentation of the inoculated milk substrate is carried out for between or between about 2 to 24, 2 to 23, 2 to 22, 2 to 21, 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 4 to 5 hours, or 2 to 3 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for at least 2 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 3 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 4 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 5 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 10 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 15 hours. In some embodiments, fermentation of the inoculated milk substrate is carried out for or for about 20 hours.

In some embodiments, fermentation of the inoculated milk substrate is carried out for an amount of time needed for the milk substrate to reach a target pH. In some embodiments, the target pH is in a range of or of about 3.5 to 6. In some embodiments, the target pH is in a range of or of about 4 to 6. In some embodiments, the target pH is in a range of or of about 5 to 6.

In some embodiments, the fermented milk product may be stored. In some embodiments, the storage is refrigerated (e.g., 6°C). In some embodiments, the fermented milk product is stored at a temperature of between or between about 5 to 15 °C. In some embodiments, the fermented milk product is stored at a temperature of between or between about 22 to 23 °C. In some embodiments, the fermented milk product is stored at a higher temperature, e.g., about 22 to 23 °C, for a first duration and then stored at a lower temperature, e.g., 5 to 15 °C, for a second duration. In some embodiments, the fermented milk product is stored for 1 , 2, 3, 4, 5, or 6 days. In some embodiments, the fermented milk product is stored for 1 , 2, 3, or 4 weeks. In some embodiments, the fermented milk product is stored for 1 , 2, 3, 4, 5, 6, or more months. In some embodiments, the fermented milk is stored for a duration, e.g., first duration, of between or between about 10 to 30 days. In some embodiments, the fermented milk is stored for a duration, e.g., second duration, of between or between about 10 to 30 days. The temperatures and duration, e.g., first and second durations, of storage may be selected depending on the type of cheese making technology used.

D. Fermented Milk Products

Also provided herein are fermented milk products obtained by any of the methods or compositions provided herein. In some aspects, the fermented milk product contains L laudensis.

Suitable fermented milk products include, but are not limited to, a food product, which include, but are not limited to, dairy foods and dairy food-derived products.

The term "food" is used in a broad sense and includes feeds, foodstuffs, food ingredients, food supplements, and functional foods. Here, the term "food" is used in a broad sense - and covers food for humans as well as food for animals (i.e. a feed). In a preferred aspect, the food is for human consumption.

In some embodiments, the fermented milk produced according to the methods described herein is a functional food. As used herein, the term "functional food" means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a further beneficial effect to consumer. Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that there are foods marketed as having specific health effects.

In some embodiments, the bacterial strains or bacterial compositions, e.g., of the starter culture, described herein may be - or may be added to - a food ingredient, a food supplement, or a functional food.

In some embodiments, the food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration.

In some embodiments, the bacterial strains or bacterial compositions, e.g., starter cultures, described herein can be used in the preparation of food products such as one or more of fermented milk products.

In some embodiments, the fermented milk product is cheese. In some embodiments, the fermented milk product is a Cheddar cheese. In some embodiments, the fermented milk product is a cottage cheese. In some embodiments, the fermented milk product is a cream cheese. In some embodiments, the fermented milk product is a hard cheese. In some embodiments, the fermented milk product is a pasta filata cheese. In some embodiments, the fermented milk product is a white cheese. In some embodiments, the fermented milk product is a blue cheese. In some embodiments, the fermented milk product is tvorog. In some embodiments, the fermented milk product is a traditional soft cheese. In some embodiments, the fermented milk product is a stabilized soft cheese. In some embodiments, the fermented milk product is a semi-hard cheese. In some embodiments, the fermented milk product is a processed cheese. In some embodiments, the fermented milk product is a butter, e.g., fermented cream butter or cultured butter. In some embodiments, the fermented milk product is butter milk. In some embodiments, the fermented milk product is sour cream.

EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 : L laudensis in Cultures

The presence and percentage of L laudensis in starter cultures, including complex cultures and bulk starter cultures, for cheese production was assessed by droplet digital polymerase chain reaction (ddPCR). A recA-based PCR assay specific to L laudensis was used to determine absolute genomic copy numbers in cultures. A bacteria-specific PCR assay was used as a PCR positive control. Results are shown in Tables E1-E3 below.

Table E1 : Absolute genomic copy (AGC) numbers and detection of L laudensis in cultures. 0 - no detection of L laudensis ; 1 - L laudensis detect; nt - not tested. Calculations are based on the total plate count numbers and AGC/ml of L laudensis.

Table E2: Absolute and viable copy numbers (samples treated with 5 mM PMAxx) of L laudensis in frozen cultures.

* VGC - Viable Genomic Copies (PMAxx 5mM)

** the proportion of L laudensis (%) was calculated based on vddPCR data for L. laudensis and plate count data for the total cell count. Due to the circumstance that vddPCR values are by trend higher than plate count values, the real proportion may be lower than demonstrated Table E3: Viable genomic copy numbers (samples treated with 5 μM PMAxx) of L laudensis in bulk starters.

The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Although the invention may be described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A starter culturing comprising L laudensis.

2. The starter culture of claim 1 , further comprising one or more species from the genera Lactococcus, Lactobacillus, Streptococcus, Lacticaseibacillus, Leuconostoc, Pediococcus, Enterococcus or Bifidobacterium, or any combination of species from the foregoing.

3. The starter culture of claims 1 or 2, which culture is comprising or consisting of L laudensis and 1 , 2, 3, 4 or 5, 10 to about 40 different bacterial strains, such as 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 different bacterial strains.

4. The starter culture of claims 1-3, wherein L laudensis is at least 0.05%, such as at least

0.1 , 0.5, 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, of the total colony forming units of the starter culture.

5. The starter culture of claims 1-4, wherein L laudensis is present in an amount of at least about 10⁵ to 10¹³ colony forming units per gram of the starter culture, such as at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹ , or at least 10¹² colony forming units per gram of the starter culture.

6. A method for producing a fermented milk product, the method comprising fermenting a milk substrate inoculated with a starter culture of any one of claims 1-5 to produce a fermented milk product.

7. The method of claim 6, wherein the milk substrate is dairy milk.

8. Use of a starter culture of claims 1-5 for preparing a fermented milk product.

9. The method of claim 6 or claim 7 or the use of claim 8, wherein the fermented milk product is a cheese.

10. The method or use of claim 9, wherein the cheese is a semi-hard cheese.