EP4705431A1

EP4705431A1 - Novel lactobacillus strains

Info

Publication number: EP4705431A1
Application number: EP24727652.0A
Authority: EP
Inventors: Paul Klaassen; Jeroen SCHOEVERS; Thomas Hendrik ECKHARDT; Daniel Avraham SCHWARTZ
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-05-04
Filing date: 2024-05-06
Publication date: 2026-03-11
Also published as: WO2024227959A1; CN121311578A

Abstract

The present invention relates to novel Lactobacillus strains with extended shelf- life properties, i.e., capability to extend the shelf-life at ambient temperature of a food product, such as e.g., a fermented milk product, when added to such products, without heavily diminishing the antimicrobial, antifungal and/or anti- yeast effect of said strains. According to another aspect, the present invention relates to an antimicrobial, antifungal or anti-yeast composition. According to yet another aspect, the present invention relates to a food product. According to another aspect, the invention relates to a process for manufacturing a food product. According to another aspect, the present invention relates to the use of a Lactobacillus strain for providing an antifungal, antimicrobial and/or anti-yeast effect, for example in a fermented milk product.

Description

Novel Lactobacillus strains

The present invention relates to novel Lactobacillus strains with extended shelflife properties, i.e., capability to extend the shelf-life at ambient temperature of a food product, such as e.g., a fermented milk product, when added to such products, without heavily diminishing the antimicrobial, antifungal and/or antiyeast effect of said strains. According to another aspect, the present invention relates to an antimicrobial, antifungal or anti-yeast composition. According to yet another aspect, the present invention relates to a food product. According to another aspect, the invention relates to a process for manufacturing a food product. According to another aspect, the present invention relates to the use of a Lactobacillus strain for providing an antifungal, antimicrobial and/or antiyeast effect, for example in a fermented milk product.

Lactic acid bacteria (LAB) are known for their role in the preparation of fermented foods, such as for instance cheese, yoghurt, meat, and vegetablebased products.

Fermented foods, such as fermented food products, can be susceptible to the growth of undesired microorganisms, such as yeast(s), mould(s) and (pathogenic) bacteria. Fungi, such as moulds and yeasts, could grow abundantly in fermented food products, even under cold conditions.

Food grade chemical preservatives such as potassium sorbate and benzoate are traditionally adequate measures to prevent the undesired growth of yeasts, moulds, and bacteria. However, a drawback of these food grade chemical preservatives is that they are non-natural products and thus the products preserved with such a food grade chemical preservative do not have a clean label. This is undesirable in view of the increasing demand for natural products.

As an alternative, LAB with protective characteristics can be used. Fermented milk products often comprise LAB. During fermentation, lactic acid and other organic compounds are being produced by the LAB, thereby reducing the pH of the food product, and consequently making it unfavorable to the growth of undesired microorganisms, such as yeast(s), mould(s) and (pathogenic) bacteria. The use of LAB strains to prevent said undesired growth of contaminants, such as e.g., yeast, mould or pathogenic bacteria, is also known as bioprotection.

The bioprotective effect of LAB strains contributing to the production of a milk- derived bioactive peptide, which shows growth-inhibitory activity against Debaromyces hansenii, has been described by McNair et al. (FEMS Yeast Research, 2018, Vol. 18, No. 8).

In EP3279312 a single Lactobacillus rhamnosus species CBS 141584 is described having antimicrobial, antifungal and/or anti-yeast properties.

The effect on bioprotection using a combination of strains from Lactobacillus rhamnosus strain together with strains from Lactobacillus paracasei has been shown in the past, see e.g., WO2013/153074 or WO2012/136830.

An example of commercial use of a combination of Lactobacillus rhamnosus and Lactobacillus paracasei is FreshQ®, which is a (bio)protective culture for fermented milk products available from Chr. Hansen, Denmark.

Although the presence of LAB in fermented milk products is desired, particularly with regards to extension of shelf-life and enhanced safety of food, more particularly in view of an increasing demand in the market for products that are suitable for transport, distribution and/or storage at ambient temperatures, it is a challenge to use them to reduce the undesired, non-controlled growth of contaminant yeasts, moulds and bacteria in fermented milk products.

The use of bioprotective cultures consisting of combinations of different species may increase the risk that the additional species introduce flavor to a food product, such as a fermented milk product. This is a disadvantage because it limits the applications of the bioprotective culture. Further developments were made to prepare LAB strains with improved characteristics on this point.

A further well-known problem in the production of fermented milk products is post-acidification. Post-acidification or post-fermentation acidification is an undesired process in fermented milk products whereby continued acidification beyond the optimal range occurs during shelf-life of the fermented milk product. The post-acidification predominantly takes place during shelf-life of the fermented milk product. It may for example take 5 to 12 days before a customer opens a package. If during this period the product acidifies further this can affect taste in a detrimental manner, even if from a pH perspective the further acidification may be very limited.

PCT patent application no. PCT/EP2022/080223 describes a Lactobacillus casei strain deposited as CBS 148322 or mutants derived therefrom, wherein said mutants have the same or improved antimicrobial, antifungal and/or anti yeast properties as strain CBS 148322. The strain is described to have a reduced contribution to post-acidification.

PCT patent application no. PCT/EP2022/080233 describes a Lactobacillus rhamnosus strain deposited as CBS 148323 or mutants derived therefrom, wherein said mutants have the same or improved antimicrobial, antifungal and/or anti yeast properties as strain CBS 148323. The strain is described to have a reduced contribution to post-acidification.

Thus, there is still a need in dairy industry to improve bioprotection and shelflife of products, particularly fermented milk products, more particularly extension of the shelf-life of such products at ambient temperature. Thus, it is an object of the present invention to provide novel strains of lactic acid bacteria with high efficacy as bioprotective agents, but improved characteristics with regards to post-acidification, especially post-acidification at ambient temperature (20° C).

Surprisingly, we now identified novel LAB strains showing reduced or low postacidification at ambient temperature as well as maintained bioprotective properties as well as, compared to known LAB strains. Such improved strains can be identified via adaptive evolution.

Thus, the present invention is directed to novel LAB strains, preferably bioprotective Lactobacillus bacterial strains, said new strains being capable of reducing post-acidification in a fermented milk product such as e.g., yogurt, particularly at ambient temperature, in comparison to known LAB strains.

The novel LAB strains, such as strains with bioprotective properties, identified herein might be generated via an enrichment process, preferably via application of adaptive laboratory evolution (ALE), resulting in strains of Lactobacillus with low or reduced post-acidification at ambient temparature.

Preferably, such improved strains are selected from Lactobacillus strains CBS 149825, CBS 149826 or CBS 151609 and mutants or derivatives thereof. Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

The articles "a" and "an" are used herein to refer to one or more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element. When referring to a noun (e.g., a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g., a "strain", this means "at least one" of that strain, e.g., "at least one strain", unless specified otherwise.

When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

As used herein, the term "lactic acid bacteria" (LAB) or "lactic bacteria" refers to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low- GC, acid tolerant, non-sporulating, non-respiring, rod-shaped bacilli, or cocci. During the fermentation stage, the consumption of sugars by these bacteria causes the formation of lactic acid and reduces the pH. These bacteria are thus responsible for the acidification and in some cases (e.g., in case of milk fermentations) for the texture of the fermented product. As used herein, the term "LAB" or "lactic bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbruekii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus lactis, Bifidobacterium animalis, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus acidophilus and Bifidobacterium breve.

The term "milk" is intended to encompass milks from mammals, from plant sources or recombinant produced milk. Preferably, the milk is from a mammal source. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, llama, mare and deer. In a preferred embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, llama, mare and deer, and combinations thereof. Plant sources of milk include, but are not limited to, milk extracted from soybean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed, sunflower seed, and/or mixtures thereof. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof.

As used in the present specification, the term "fermented milk product" refers to a product that has been fermented with LAB. Examples of LAB are Streptococcus thermophilus and Lactobacillus delbruekii subsp. bulgaricus, but also, optionally, other microorganisms such as for instance Lactobacillus delbruekii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus casei, or any microorganism derived therefrom. The fermentation process increases the shelf-life of the product while enhancing and improving the digestibility of milk. Many different types of fermented milk products can be found in the world today. Examples are soured milk (e.g., buttermilk), sour cream and yogurt.

The term "starter culture" (also referred to as "starter") as used herein refers to a composition comprising one or more LAB strains, which are responsible for the acidification of the milk or milk base. Starter cultures compositions may be fresh (liquid), frozen or freeze-dried. Freeze dried cultures need to be regenerated before use. For the production of a fermented milk product, the starter cultures composition is usually added in an amount from 0.01 to 3%, preferably from 0.01 and 0.02 % by weight of the total amount of milk or milk base.

The term "novel strain" or "inventive strain" should be understood as a strain having the characteristics as described herein, i.e., (bio)protective activity combined with improved post-acidification at ambient temperature, such as e.g. a strain derived from a so called "mother strain", "parent strain" or "ancestral strain", obtained by means of e.g., chemical mutagenesis, radiation, genetic engineering or even enrichment, or even arise in a population that is not actively treated to obtain derivatives or mutants, such as by means of errors during DNA replication (so-called "spontaneous" mutations). The terms "inventive strain" and "derivative" are sometimes used interchangeably herein and also include "mutant strains". Many methods are known in the art for obtaining said inventive strains and particular methods for selecting said strains including derivatives or mutants with desired properties are well known. A particularly useful method for generation of inventive strains, particularly mutant strains as described herein, is the adaptive laboratory evolution (ALE) technique, whereby mutant strains are generated that are functionally equivalent to the original or mother strain, such as e.g., having more or less the same antifungal or antimicrobial properties but improved post-acidification performance especially at a given temperature, such as e.g., at ambient temperature. The inventive strains in the scope of the present invention might be also generated in an enrichment process, resulting in the selection of strains with more or less the same antifungal or antimicrobial activities but improved post-acidification performance especially at ambient temperature and as further described herein.

The term "mutant" should be understood as a strain derived from a strain of the invention, also called mother strain or ancestral strain, obtained by means of e.g., chemical mutagenesis, radiation, or genetic engineering. Mutants may even arise in a population that is not actively treated to obtain mutants, by means of errors during DNA replication (so-called "spontaneous" mutations) and are also included herein. Many methods are known in the art for obtaining mutants and methods for selecting mutants with desired properties are well known. A particularly useful method for generation of mutant strains in the scope of the present invention is the adaptive laboratory evolution (ALE) technique further described herein. Preferably, the mutant is functionally equivalent to the original or mother strain, in the sense that the mutant has the same or preferably improved properties, such as e.g., the ability to produce antifungal or antimicrobial compounds. Preferably, any mutant as described herein has a nucleic acid sequence with at least about 70% identity, more preferably with at least about 80, 90, 95, 99 or 99.9% identity with the nucleic acid sequence of the ancestral strain from which it is derived. Preferably, any mutant as described herein has an improved post-acidification property at ambient temperature as compared to the parent strain.

The term "mesophile" or "mesophilic" herein refers to LAB that thrive best at temperatures lower than about 41°C, such as e.g., between about 15°C and about 40°C. Examples of mesophilic lactic acid bacteria with industrial relevance include for instance Lactococcus ssp. and Leuconostoc ssp. Hence, a "mesophilic fermentation" herein refers to a fermentation that is being done at a temperature between about 20°C and 36°C, such as e.g., at about 28°C.

The term "fermentation" herein refers to a metabolic process wherein sugar(s) are being converted into acids, gases, or alcohol. Fermentation occurs in many different cell types, such as e.g., yeasts and bacteria. Preferably, fermentation comprises the conversion of lactose into lactic acid.

"Undesired microorganisms", "undesired contaminants" and "contaminants" herein refer to the occurrence of microorganisms, such as bacteria, yeasts, moulds, or a combination thereof, which bring about a negative perception of the food. The contaminant may be pathogenic, may have the ability to deteriorate food products or, give rise to an unpleasant smell, taste, or appearance of the food product. The strain of the invention is providing a solution in the prevention of the appearance and/or growth of such contaminants by inhibiting and/or preventing their growth upon entry in the fermented milk matrix, such as e.g., a yogurt or a sour cream product. The prevention of the growth of contaminants due to the action or the presence of Lactobacillus bacterial strain can be expressed by e.g., a lower number of contaminant cell counts in a fermented milk product prepared with the present Lactobacillus bacterial strain, compared to a similar product, without the presence of said Lactobacillus bacterial strain.

As used herein, the term antimicrobial, an antifungal or an anti-yeast composition means a composition suitable for providing an antimicrobial, an antifungal or an anti-yeast efficacy. The Lactobacillus bacterial strains as used herein preferably have antimicrobial, antifungal and/or anti yeast effect, particularly with regards to contaminations used in the dairy industry and are thus used/added to dairy products as bioprotective adjunct strains.

The terms "bioprotective" or "protective" strains in relation to LAB strains are used interchangeably herein and refer to the properties of said strains to control, particularly inhibit or delay, the growth of undesired microorganism such as e.g., yeast, moulds and other (pathogenic) bacteria. Said process is also referred to as "bioprotection" or "biopreservation".

In one aspect, the present invention provides a Lactobacillus bacterial strain, having antimicrobial, antifungal and/or anti yeast properties and having a viability, as expressed in the number of colony forming units per gram (cfu/g) on MRS-agar after 7 days, wherein the viability at about 48° C, such as e.g. at about 49 or 50° C, is at least about 50%, such as e.g. at least about 70, 80 or 90%, of the viability at 37° C.

According to the present invention, the Lactobacillus bacterial strain to be used as parent strain might be selected from a Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei or a Lactobacillus casei strain, said strains being known for their bioprotective properties.

A particularly useful Lactobacillus bacterial strain is selected from strain CBS 149825 and CBS 149826 deposited on 8 March 2023 at the Westerdijk Fungal Biodiversity Institute (CBS, formally named the Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3508 AD Utrecht, The Netherlands under the provisions of the Budapest Treaty.

A furthermore particularly useful Lactobacillus bacterial strain si selected from CBS 151609 deposited on 02 May 2024 at the Westerdijk Fungal Biodiversity Institute (CBS, formally named the Centraalbureau voor Schimmelcultures), Uppsalalaan 8, 3508 AD Utrecht, The Netherlands under the provisions of the Budapest Treaty.

The particular useful strains might be obtainable from a parent strain via mutagenesis, thus as e.g., described herein.

The inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, preferably have a limited growth rate, particularly a growth rate, measured as the number of colonies of at least about 1 mm diameter, such as e.g. of at least about 2, 3, 4, 5 diameter, after 7 days on MRS- agar, wherein the growth rate at 48°C, preferably at 49° C, and more preferably at 50°C, is equal to or less than 90%, such as e.g. equal or less than 80, 70 or 50% of the growth rate at 37°C.

In one embodiment, the inventive Lactobacillus bacterial strain, particularly strains CBS 149825, CBS 149826 or CBS 151609, have a growth rate, measured as the number of colonies of at least about 1 mm diameter, such as e.g. of at least about 2, 3, 4, 5 diameter, after 7 days on MRS-agar, wherein the growth rate at 37° C, preferably at 40°C, is equal to or less than 90%, such as e.g. equal or less than 80, 70 or 50% of the growth rate at 48° C.

In one embodiment, the inventive Lactobacillus bacterial strain, such as e.g., particularly strains CBS 149825, CBS 149826 or CBS 151609, have an average colony diameter, measured after 7 days of growth on MRS-agar, wherein the average diameter at 37° C, preferably at 40°C, is equal to or less than 90%, such as e.g. equal or more than 80, 70 or 50%, of the average colony diameter at 48° C.

In another embodiment, the inventive Lactobacillus bacterial strains, such as e.g., Lactobacillus strains CBS 149825, CBS 149826 or CBS 151609, have a mean colony diameter, measured after 7 days of growth on MRS-agar, wherein the mean diameter at 37° C, preferably at 40° C, is equal to or less than 90%, such as e.g. equal or less than 80, 70, or 50%, of the mean colony diameter at 48°C.

In a further embodiment, the inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, have a growth rate, measured as the amount of biomass Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar, wherein the amount of biomass at 37° C, preferably at 40°C, is equal to or less than 90%, such as e.g. equal or less than 80, 70 or 50%, of the amount of biomass at 46°C, preferably at 48°C.

In a further embodiment, the inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, has an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar at a certain temperature, wherein the optimum growth rate occurs at a temperature of at least 43°C, such as e.g. of at least 44, 45, 46 or 48° C.

In some embodiments, the inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, have an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS- agar, wherein the optimum growth rate lies outside the temperature range of 37° to 43°C, preferably outside of 35° to 45°C.

The above-mentioned embodiments can be cross-combined and can be combined with the preferences for the viability as mentioned-above.

Preferably, the inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, have the ability to grow one or more colonies of at least 1 mm diameter at a temperature of at least 48° within 7 days. More preferably, the Lactobacillus bacterial strain has the ability to grow one or more colonies of at least 1 mm diameter at a temperature of at least 49°C within 7 days. Most preferably, the Lactobacillus bacterial strain has the ability to grow one or more colonies of at least 1 mm diameter at a temperature of at least 50°C within 7 days.

In some particular embodiments, in 12 wt% reconstituted skim milk (RSM) and at an inoculation rate of 10⁷ CFU/g, when combined with a starter culture comprising or consisting of Streptococcus thermophilus and Lactobacillus delb rue kii subsp. bulgaricus, the inventive Lactobacillus bacterial strains, particularly strains such as CBS 149825 or CBS 149826, have an acidification profile exhibiting a Time To Reach ("TTR") pH 4.6 of 225 minutes or less, which is about 10 to 15 minutes less than a respective sample comprising the starter culture only but no further Lactobacillus bacterial strains as of the present invention added as bioprotective adjunct culture.

Preferably, in 12 wt% reconstituted skim milk (RSM) and at an inoculation rate of 10⁷ CFU/g, when combined with a starter culture comprising or consisting of Streptococcus thermophilus and Lactobacillus delb ruekii subsp. bulgaricus, the inventive Lactobacillus bacterial strain, particularly strains CBS 149825, CBS 149826 or mutants thereof added as (bio)protective adjunct has an acidification profile exhibiting a Time To Reach ("TTR") pH 4.6 in the range of about 240 or less, preferably in the range of 230, 220, 200 minutes or less.

Preferably, the inventive Lactobacillus bacterial strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, increase the pH of a fermented milk product, comprising said Lactobacillus bacterial strain, during storage after fermentation in comparison to a fermented milk product without the addition of such an inventive strain, or even as compared to fermented milk products comprising a known (bio)protective strain such as e.g., strain CBS 148322 or CBS 141584. The increase in pH is at least by a value of 0.1, such as e.g. at least by a value of 0.2, 0.3, 0.4 or more and wherein the increase in pH is determined after storage of the fermented milk product, fermented with a starter culture, such starter culture preferably comprising or consisting of Streptococcus thermophilus and Lactobacillus delbruekii subsp. bulgaricus, and optionally the known (bio)protective Lactobacillus strains CBS141584 or CBS 148322, in a concentration of at least 10⁷ CFU/g over about 14 days at 20°C.

The inventive Lactobacillus bacterial strains as defined herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, have bioprotective properties, such as e.g., being capable of limiting the fungal growth of known contaminants in dairy industry, including but not limited to strains of Aspergillus, Fusarium, Penicillium, Toluraspora, Debaryomyces, Pichia, Zagosaccharomyces, Yarrowia, Kluyveromyces, Saccharomyces or other known moulds and yeast.

Preferably, the Lactobacillus bacterial strains according to the invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, have a greater antimicrobial effect in a food product than potassium sorbate, wherein the potassium sorbate is dosed in an amount of 0.05% (w/w) of the food product and wherein the inventive Lactobacillus bacterial strain is dosed in an amount of 0.001 %(w/w) to 0.1%(w/w) of a medium or substrate, such as for example milk.

Particularly, the inventive strains, particularly strains CBS 149825, CBS 149826 or CBS 151609, exhibit at least about 80%, such as e.g., at least about 90, 95, 100% of the antimicrobial, antifungal, and/or anti-yeast effect compared with the strain CBS 141584 or strain CBS 148322 if compared under equal conditions.

Preferably, the inventive strains provide said antifungal, anti-yeast and/or antimould effect without introducing a flavor effect.

Preferably the Lactobacillus bacterial strains disclosed in the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, are preferably produced, supplied or otherwise present in frozen, dried, or freeze-dried form.

Preferably, the Lactobacillus bacterial strains disclosed in the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, are dosed, each individually, in an amount of 0.001 %(w/w) to 0.1%(w/w) of a medium or substrate, such as for example milk.

In one aspect, this invention provides an antimicrobial and/or antifungal and/or anti-yeast composition comprising a Lactobacillus bacterial strain as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, comprised in such composition, particularly a composition comprising a Lactobacillus strain selected CBS 151609, CBS 149825 or CBS 149826, wherein preferably said derivative has the same or improved antimicrobial, antifungal and/or anti-yeast properties as the strain originating from, such as ancestral strain CBS 148322.

Preferably, the amount of the inventive Lactobacillus bacterial strain in the antimicrobial, antifungal and/or anti-yeast composition is sufficient to provide an antimicrobial, an antifungal or an anti-yeast effect. This enables the antimicrobial, antifungal or anti-yeast composition suitable to provide an antimicrobial, an antifungal or an anti-yeast effect.

More preferably, the antimicrobial and/or antifungal and/or anti-yeast composition comprises both (i) a Lactobacillus bacterial strain as described above, particularly strains CBS 149825, CBS 149826 or CBS 151609, and (ii) an, optionally additional, preferably bioprotective, Lactobacillus casei strain, Lactobacillus paracasei strain, Lactobacillus plantarum strain and/or a Lactobacillus rhamnosus strain, optionally further supplemented by (iii) one or more other lactic acid strains, such as for example one or more Streptococcus thermophilus strains and/or one or more Lactobacillus delb ruekii subsp. bulgaricus strains.

In a preferred embodiment, the present antimicrobial, antifungal and/or antiyeast composition comprises:

(i) a Lactobacillus bacterial strain, particularly selected from strains CBS 149825, CBS 149826 or CBS 151609, wherein preferably said strain has the same or improved antimicrobial, antifungal and/or anti-yeast properties as e.g., strain CBS 148322 or CBS 141584; and

(ii) optionally additional, preferably bioprotective, Lactobacillus casei strain, Lactobacillus paracasei strain, Lactobacillus plantarum strain and/or a Lactobacillus rhamnosus strain; and

(iii) optionally one or more other lactic acid strains, such as one or more Streptococcus thermophilus strains and/or one or more Lactobacillus delbrue kii subsp. bulgaricus strains.

Preferably, the amount of the Lactobacillus bacterial strain(s) according to the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, and as described herein comprised in the antimicrobial, antifungal and/or anti-yeast composition is sufficient to provide an antimicrobial, an antifungal or an antiyeast effect. This enables the antimicrobial, antifungal or anti-yeast composition suitable to provide an antimicrobial, an antifungal or an anti-yeast effect.

The present antimicrobial, antifungal and/or anti-yeast composition preferably comprises the Lactobacillus bacterial strain(s) as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, and according to all aspects of the present invention in a concentrated form including frozen, dried or freeze-dried concentrates.

The present antimicrobial, antifungal and/or anti-yeast composition preferably comprises the Lactobacillus bacterial strain(s) according to all aspects of the invention and as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, in a concentration of viable cells, which is, each individually, preferably in the range of 10⁴ to 10¹³ cfu (colony forming units) per gram of the composition including at least about 10⁴ cfu per gram of the composition, such as at least about 10⁵ cfu/g, e.g., at least about 10⁵ cfu/g, such as at least about 10⁷ cfu/g, e.g., at least about 10⁸ cfu/g, such as at least about 10⁹ cfu/g, e.g., at least about 10¹⁰ cfu/g, such as at least about 10¹¹ cfu/g. Thus, the antimicrobial, antifungal or anti-yeast composition of the invention is preferably present in a frozen, dried, or freeze-dried form, e.g., as a Direct Vat Culture (DVC). However, as used herein the composition may also be a liquid that is obtained after suspension of the frozen, dried, or freeze-dried cell concentrates in a liquid medium such as water, milk, or PBS buffer. Where the composition of the invention is a suspension, the concentration of viable cells is in the range of 10⁴ to 10¹² cfu (colony forming units) per ml of the composition including at least about 10⁴ cfu per gram of the composition, such as at least about 10⁵ cfu/ml, e.g., at least about 10⁵ cfu/ml, such as at least about 10⁷ cfu/ml, e.g., at least about 10⁸ cfu/ml, such as at least about 10⁹ cfu/ml, e.g., at least about 10¹⁰ cfu/ml, such as at least about 10¹¹ cfu/ml.

The present antimicrobial, antifungal or anti-yeast composition may further comprise 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. Suitable cryoprotectants that may be added to the composition of the invention are components that improve the cold tolerance of the microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, sucrose, and/or fructose. Other additives may include, e.g., carbohydrates, flavors, minerals, and/or enzymes (e.g., rennet, lactase and/or (phospho)lipase).

More preferably, the present antimicrobial, antifungal or anti-yeast composition is packed. Preferably, in a package which is suitable for shipment and/or storage of the present antimicrobial, antifungal or anti-yeast composition for at least about 1 month, such as at least about 3 months. Preferably, the amount of antimicrobial, antifungal or anti-yeast composition in the package is at least about 50, 100 or 500 grams, such as, e.g. at least about 50 to 500 grams, such as e.g. at least about 100 to 500 grams.

In a further aspect, the present invention relates to a food product comprising the Lactobacillus bacterial strain(s) as described herein and according to all aspects of the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, and optionally other LAB strains, or the present antimicrobial and/or antifungal composition.

The present invention relates to a food product comprising an amount of the Lactobacillus bacterial strain(s) according to the present invention and as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, which is effective for imparting antimicrobial properties to the food product. More preferably, wherein the presence of the Lactobacillus bacterial strain(s) as described herein does not introduce a flavor to the food product.

In a further preferred embodiment, the present food product comprises the Lactobacillus bacterial strain(s) according to the present invention and as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, in an amount, each individually, which is in the range of 10⁴ to 10¹² cfu (colony forming units) per gram of the food product including at least about 10⁴ cfu per gram of the food product, such as at least about 10⁵ cfu/g, e.g., at least about 10⁵ cfu/g, such as at least about 10⁷ cfu/g, e.g., at least about 10⁸ cfu/g, such as at least about 10⁹ cfu/g, e.g., at least about 10¹⁰ cfu/g, such as at least about 10¹¹ cfu/g of the food product. More preferably, the present food product comprises the Lactobacillus bacterial strain(s) according to the present invention and as described herein in an amount, each individually, which is in the range of 10⁴ to 10¹² cfu (colony forming units) per cm² surface of the food product including at least about 10⁴ cfu/cm² of the food product, such as at least about 10⁵ cfu/cm², e.g., at least about 10⁵ cfu/cm², such as at least about 10⁷ cfu/cm², e.g., at least about 10⁸ cfu/cm², such as at least about 10⁹ cfu/cm², e.g., at least about 10¹⁰ cfu/cm², such as at least about 10¹¹ cfu/cm² surface of the food product.

In a further preferred embodiment, the present food product comprises further any (optionally additional) Lactobacillus casei strain, Lactobacillus paracasei strain, Lactobacillus plantarum strain and/or Lactobacillus rhamnosus strain, if present, each individually, in an amount which is in the range of 10⁴ to 10¹² cfu (colony forming units) per gram of the food product including at least about 10⁴ cfu per gram of the food product, such as at least about 10⁵ cfu/g, e.g., at least about 10⁵ cfu/g, such as at least about 10⁷ cfu/g, e.g., at least about 10⁸ cfu/g, such as at least about 10⁹ cfu/g, e.g., at least about 10¹⁰ cfu/g, such as at least about 10¹¹ cfu/g of the food product. More preferably, the present food product comprises the second Lactobacillus strain in an amount which is in the range of 10⁴ to 10¹² cfu (colony forming units) per cm² surface of the food product including at least about 10⁴ cfu per cm² of the food product, such as at least about 10⁵ cfu/cm², e.g., at least about 10⁵ cfu/cm², such as at least about 10⁷ cfu/cm², e.g., at least about 10⁸ cfu/cm², such as at least about 10⁹ cfu/cm², e.g., at least about 10¹⁰ cfu/cm², such as at least about 10¹¹ cfu/cm² surface of the food product. Preferably, the present food product has a flavor profile which is comparable or indistinguishable from the food product, or from the same food product, which does not comprise the present Lactobacillus bacterial strain, particularly strains CBS 149825, CBS 149826 or CBS 151609. In other words, the inventive Lactobacillus bacterial strain and optionally any further additional strain does not introduce a flavor to a food product to which the Lactobacillus bacterial strain and optionally the further strain is added.

The Lactobacillus bacterial strains according to the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, might be used for providing an antimicrobial and/or antifungal and/or anti-yeast effect in a food product, preferably in a fermented milk product.

Preferably, the present food product as described herein comprising the Lactobacillus bacterial strains according to the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, are a fermented milk product. More preferably a mesophilic or thermophilic fermented milk product. Most preferably, the fermented milk product is a yogurt. Examples of a fermented milk product include regular yoghurt, plain yogurt, low fat yoghurt, nonfat yoghurt, tvarog, kefir, dahi, ymer, buttermilk, butter, sour cream, and sour whipped cream. Another preferred example of a fermented milk product is cheese. For example, fresh cheeses, un-ripened cheeses, or curd cheeses. Alternatively, the fermented milk product is a ripened cheese. The food product can also be a milk, whey, milk powder or whey powder.

According to yet another aspect, the present invention relates to a method for manufacturing a food product comprising adding at least one Lactobacillus bacterial strain as described herein, particularly strains CBS 149825, CBS 149826 or CBS 151609, or the present antimicrobial composition, antifungal composition, or anti-bacterial composition, during manufacture of the food product. Preferably, the Lactobacillus bacterial strain according to the the present invention and as described herein is added to the milk together with LAB used for fermentation of the milk. More preferably, the present method comprises a step of fermenting milk with LAB. The present inventors found that a high efficacy against yeast and moulds can be obtained if the Lactobacillus bacterial strain as described herein and according to the present invention is present during fermentation of the food product.

Thus, in a preferred embodiment, the method comprises one or more fermentation steps. Preferably, the method comprises fermenting a milk substrate with a starter culture comprising at least one strain of the genera selected from Lactobacillus, Streptococcus, Lactococcus and/or Leuconostoc. The present step of fermenting a milk substrate can be fermenting under mesophilic or under thermophilic conditions. More preferably, the present method is a method for manufacturing yogurt comprising adding at least one Lactobacillus bacterial strain according to the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, or the present antimicrobial composition, antifungal composition, or anti-bacterial composition, during fermentation of milk with a starter culture comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Alternatively, the present method is a method for manufacturing sour cream comprising adding at least one Lactobacillus bacterial strain according to the the present invention, or the present antimicrobial composition, antifungal composition, or antibacterial composition, during fermentation of milk with a starter culture comprising Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis.

According to another aspect, the present invention relates to the use of the present Lactobacillus bacterial strain, particularly strains CBS 149825, CBS 149826 or CBS 151609, for providing an antimicrobial effect in food products, preferably fermented milk products, preferably for providing an antifungal effect and/or an anti-yeast effect in food products, preferably fermented milk products. More preferably wherein the food product is a fermented milk product. Even more preferably a fermented milk product such as various types of regular yoghurt, low fat yoghurt, nonfat yoghurt, kefir, dahi, ymer, buttermilk, butter, sour cream, and sour whipped cream. Other examples of a fermented milk product wherein the present Lactobacillus bacterial strain can be used is cheese. For example, fresh cheeses, un-ripened cheeses, or curd cheeses. Alternatively, the fermented milk product is a ripened cheese.

In a preferred embodiment, the present invention relates to the use of the present Lactobacillus bacterial strain, particularly strains CBS 149825, CBS 149826 or CBS 151609, for providing an antimicrobial effect in silage, preferably for providing an antifungal effect and/or an anti-yeast effect in silage.

As explained below and illustrated by the examples, the invention advantageously allows for a bioprotective solution which not only affords a favorable shelf-life stability but also has reduced post-acidification compared to prior art compositions. It is especially advantageous that such further reduced post-acidification can be obtained even at ambient temperature, such as e.g., at about 20°C.

According to one aspect of the present invention, the mutant Lactobacillus bacterial strains as described herein are generated from a parent strain by means of Adaptive Laboratory Evolution (ALE). ALE can be described as a laboratory method in which microbial cells are harnessing biology to selfoptimize by natural (or induced) mutations through selection. Adaptation of a microbial culture to a new environment necessarily involves the enhancement of certain traits, leading to improved function and an increase in fitness under the applied conditions. Detailed guidance on experimental evolution of microbes can be found in Bachmann et al. (FEMS Microbiology Reviews, 2017, Vol. 41, No. Supp 1) using Escherichia coli, concluding that, while lots of progress on genome dynamics and cellular resource allocation may be made in recent years, there are still many open questions on how different constraints during the evolution influence such trade-offs and eventually the fitness of an organism in a particular environment.

The present invention conveniently provides a method for obtaining a mutant LAB, more preferably a Lactobacillus bacterial strain, preferably capable of reducing post-acidification in a fermented milk product in comparison to the respective ancestral Lactobacillus bacterial strain, wherein the method comprises subjecting the parent LAB, respectively Lactobacillus bacterial strain, to ALE, said ALE comprising a series of evolutionary changes wherein each next descendent strain of a preceding strain is subjected to, and preferably grown at, a higher environmental temperature than the preceding strain. Different forms of ALE exist and have been described (see e.g., Dragosits and Mattanovich, 2013, Microb Cell Fact 12, 64. [https://doi.org/10.1186/1475-2859-12-64]; Sandberg et al., 2019, Metabolic Engineering 56, 1-16.

[https://www.sciencedirect.com/science/article/pii/S1096717619301533]; W02019/043055; Bennett and Lenski, 2007, PNAS, vol. 104, suppl. 1, 8649-8654. [www.pnas.orgcgidoi10.1073pnas.0702117104]).

More preferably, the invention provides a method for obtaining a mutant LAB strain, preferably capable of reducing post-acidification in a fermented milk product in comparison to the respective ancestral LAB strain, wherein the method comprises subjecting an LAB strain to ALE, particularly wherein the LAB strain has protective properties, more preferably wherein the LAB strain is selected from a Lactobacillus strain, even more preferably wherein the LAB strain is selected from strain CBS 148322 or CBS 141584, said ALE comprising a series of evolutionary changes wherein each next generation descendent of a preceding parent strain is subjected to, and preferably grown at, an environmental or incubation temperature that is at least 1°C higher than the environmental or incubation temperature at which the preceding parent strain was grown, wherein the LAB strain preferably is a Lactobacillus bacterial strain. ALE consists of several rounds of mutations or adaptions as described herein, preferably with 1 to 10 rounds, such as e.g., 1 to 5, 1 to 7, 1 to 8 rounds, with characterization of the obtained mutants after the final round.

Thus, the present invention includes a Lactobacillus bacterial strain as described herein, particularly a mutant strain derived from strain CBS 148322 or 141584, said strain being obtained, obtainable or produced by the above method using ALE.

It was surprisingly found that said Lactobacillus bacterial strain CBS 149825, CBS 149826, CBS 151609 provides for an improved post-acidification, especially at ambient temperature (20°C), compared to the ancestral strain CBS 148322 or CBS 141584 and also as compared to further mutant strains derivable from several rounds of ALE (see Examples).

The Lactobacillus bacterial strains according to the present invention, particularly strains CBS 149825, CBS 149826 or CBS 151609, such as strains obtainable via ALE, preferably have antimicrobial, antifungal and/or anti-yeast effect, wherein through the application of ALE the shelf-life with regards to postacidification is improved, particularly if said mutant strain is added as bioprotective to a dairy product such as e.g. a yogurt.

Mutants as used in the above context mean Lactobacillus bacterial strains which are derived/obtained from e.g., CBS 148322 or CBS 141584, having mutation(s) in comparison with the parent Lactobacillus bacterial strain, wherein the mutation(s) do not alter the bioprotective phenotype of the derived Lactobacillus bacterial strain. Preferably, the mutant strain has the same or improved antimicrobial antifungal and/or anti-yeast properties as the mother strain. Preferably, the derived Lactobacillus bacterial strain is suitable for providing an antifungal effect and/or an anti-bacterial effect, as found for or like the mother strain, e.g., CBS 148322 or even better. The mutant derived from the mother strains, such as e.g., CBS 148222, might have at least about 80%, such as e.g., about 90, 95, 100% or more of the antimicrobial, antifungal, and/or antiyeast effect compared with the mother strain, such as e.g., strain CBS 148322, if compared under equal conditions. In a particularly preferred embodiment, the present bioprotective Lactobacillus bacterial strain is a strain selected from Lactobacillus strain CBS 148322, further most preferably the mutant strain with improved post-acidification properties as described herein is selected from strain CBS 151609 but furthermore includes other mutant strains derived from strain CBS 148322 as described herein.

Particularly, the inventive strains established throughout the present invention, are mutant strains generated via ALE, are Lactobacillus bacterial strains, preferably mutants of strain CBS 148322, such as e.g. strain CBS 151609 deposited on 02 May 2024 at the Westerdijk Fungal Biodiversity Institute (The Netherlands).

Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined and the described preferences for one of the above aspects of the invention also apply to the other aspects of the invention.

The present invention is directed to the following particular embodiments (1) to (17):

(1) A Lactobacillus bacterial strain, having antimicrobial, antifungal and/or anti yeast properties and having a viability, as expressed in the number of colony forming units per gram (cfu/g) on MRS-agar after 7 days, wherein the viability at 48°C is equal to or more than 50% of the viability at 37° C.

(2) A Lactobacillus bacterial strain, having antimicrobial, antifungal and/or anti yeast properties and having an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar, wherein the optimum growth rate lies outside the temperature range of 37° to 43°C, preferably outside the range of 35° to 45°C.

(3) A Lactobacillus bacterial strain as of embodiments (1) or (2), having an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar, wherein the optimum growth rate occurs at a temperature of equal to or more than 43°C, preferably equal to or more than 45° C.

(4) A Lactobacillus bacterial strain as of embodiments (1), (2) and/or (3), wherein the Lactobacillus bacterial strain is a Lactobacillus rhamnosus strain, a Lactobacillus plantarum strain, a Lactobacillus paracasei strain, or a Lactobacillus casei strain.

(5) A Lactobacillus bacterial strain as of embodiments (1), (2, (3) and/or (4), wherein (a) the Lactobacillus bacterial strain increases the pH of a fermented milk product, comprising the Lactobacillus bacterial strain, during storage after fermentation in comparison to a fermented milk product, comprising a Lactobacillus bacterial strain deposited as CBS 148322, wherein the increase in pH is at least by a value of 0.1, and wherein the increase in pH is determined after storing the fermented milk product, fermented with a starter culture and the Lactobacillus bacterial strain, respectively the Lactobacillus bacterial strain deposited as CBS 148322, in a concentration of at least 10⁷ CFU/g over 14 days at 20°C; and (b) the Lactobacillus bacterial strain is preferably a Lactobacillus casei strain.

(6) A Lactobacillus bacterial strain deposited as CBS 149825 or CBS149826 or mutants derived therefrom, wherein said mutants have the same or improved antimicrobial, antifungal and/or anti yeast properties as strain CBS 149825 or CBS149826.

(7) A Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6) in frozen, dried, or freeze-dried form.

(8) An antimicrobial and/or antifungal and/or anti yeast composition comprising a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6).

(9) A food product comprising a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6) or an antimicrobial and/or antifungal and/or anti yeast composition as of embodiment (8).

(10) A food product as of embodiment (9), wherein the food product is a fermented milk product, preferably cheese, sour cream, or yoghurt.

(11) A process for manufacturing a food product comprising adding a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6), or the antimicrobial and/or antifungal and/or anti yeast composition as of embodiment (8), is added after a step of fermenting the food product.

(12) The process of embodiment (11), wherein a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6), or the antimicrobial and/or antifungal and/or anti yeast composition as of embodiment (8), is added before or during a step of fermenting the food product. (13) The process of embodiment (11), wherein a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6), or the antimicrobial and/or antifungal and/or anti yeast composition as of embodiment (8), is added after a step of fermenting the food product.

(14) The process of embodiment (11), (12), and/or (13), wherein the food product is a fermented milk product, preferably cheese, sour cream, or yoghurt.

(15) Use of a Lactobacillus bacterial strain as of embodiments (1), (2, (3), (4), (5) and/or (6) for providing an antimicrobial and/or antifungal and/or anti yeast effect in a food product, preferably in a fermented milk product.

(16) Method for obtaining a mutant lactic acid bacterial strain, preferably Lactobacillus bacterial strain, preferably capable of reducing post-acidification in a fermented milk product in comparison with a parent Lactobacillus bacterial strain, wherein the method comprises subjecting the parent lactic acid bacterial strain to an adaptive evolution, which adaptive evolution comprises a series of evolutionary changes wherein each next descendent strain of a preceding strain is subjected to a higher environmental temperature than the preceding strain.

(17) A Lactobacillus bacterial strain obtained or obtainable by a method as of embodiment (16).

Figures

Figure 1: impact of incubation temperature on the number of viable cells of prior art strain CBS 148322.

Figure 2: post-acidification (PA) measured in days for strain CBS 149826 (i.e. "#1R") compared to strain CBS 141584 or a culture without any bioprotective strain ("None") in dependence of the pH (Fig. 2A); post-acidification (PA) measured in days for strain CBS 149825 (i.e. "#2C") compared to strain CBS 148322 or a yogurt sample without any bioprotective strain ("None") in dependence of the pH (Fig. 2B).

Figure 3: challenge test, day 6, 20°C incubation temperature. NC = no inoculation with a mold, PR = Penicillium roqueforti ATCC 34905, PB = Penicillium brevicompactum CBS 110070, None = yogurt sample without any bioprotective strain. Fig. 3A shows the results for strain CBS 149826 (i.e. "#1R") compared to strain CBS 141584. Fig. 3B shows the results for strain 149825 (i.e. "#2C") compared to strain CBS 148322. The following examples are illustrative only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications, cited throughout this application are hereby incorporated by reference, in particular EP3279312, WO2013/153074, WO2012/136830, PCT/EP2022/080223, and W02019/043055.

Examples

Example 1: General methods and materials

All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989) or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

Strains. Bioprotective strains, all deposited at Westerdijk Fungal Biodiversity Institute (the Netherlands) under the provisions of the Budapest Treaty, as well as contaminants and starter cultures are shown in Table 1.

Table 1: list of (deposited) strains including bioprotective adjuncts ("BP"), starter cultures ("Starter": mix of S. thermophilus and L. delbrueckii bulgaricus commercially available as Delvo®Fresh YS-141 form DSM Delft, NL) or fungal contaminants (moulds and yeast). "Name" means the naming or abbreviation used througout this invention. For more details, see text/ Exam pies.

Strains used are available from ATCC (USA) CBS (Westerdijk Fungal Biodiversity Institute, The Netherlands) or DSM DSM Food and Beverages, Delft, The Netherlands).

Adaptive Laboratory Evolution (ALE). For generation of strain CBS 151609 (mutant strain #3) and further mutant strains, including mutant #1, #2, #4, ALE is performed with strain CBS 148322 as mother strain, with generation of an initial population with or without genotypic diversification (e.g., by mutagenesis), followed by evolution under selected growth conditions, for a desired time, or until the population exhibits a desired phenotypical change.

After several rounds of ALE, in which the population has evolved such that a changed phenotype has become apparent, populations and/or isolates are analyzed for beneficial mutations. The resulting isolates can be used directly as they are or serve as input for (a) next round(s) of ALE.

If desired, mutations are reimplemented in a clean strain (reverse engineering) to validate the contribution of the individual mutations to the phenotype, allowing to assess which mutation(s) are directly implicated with the phenotype, but also to eliminate the effect of coincidental mutations which do not, or negatively impact the desired phenotype.

Normally, colonies for ALE are selected and resuspended in 1mL or MRS-broth. An equal volume of 20% glycerol is added for conservation purposes (glycerol stock), at -80°C. A loopful of material from the glycerol stocks is streaked on MRS agar plates and incubated at a starting temperature, e.g., 47° C. This procedure is repeated for several rounds, with selection of colonies and resuspension in broth and glycerol.

Through temperature increase by 1°C per round, novel mutations are allowed to be introduced into the genome, leading to a higher permissive growth temperature. From the same sample, an agar-plate is prepared and incubated at 37°C to check for normal growth under standard growth conditions.

As the incubation temperature increased, the incubation time to obtain colonies of a certain size increases as well. Usually, it takes cells to grow to colonies of approximately 1-4 mm diameter in 2 days at 37° C. With increasing incubation temperature, the time required to obtain such colonies needs to be extended as well, ultimately to 7 days at the incubation temperature of 50°C.

Milk fermentation. 12% (w/v) RSM (reconstituted skim milk) was pasteurized by heating in a water bath for 20 minutes at 100°C. After pasteurization, the milk was quickly cooled in ice-water and kept at 4°C.

The pasteurized milk was inoculated with the bioprotective cultures at an inoculation rate of 10⁷ CFU/mL. In addition, 1U/1000L of a yogurt starter culture Delvo®Fresh YS-141 (comprising Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus, commercially obtainable from DSM, Delft, Netherlands) were added. The inoculated milk was incubated at 42°C. The pH was continuously monitored using a CINAC apparatus (Ysebaert, France). Alternatively, acidification was performed in microplates using fluorescent pH probes for measuring the acidification. Several methods have been published and are known to those skilled in the art.

Simultaneously, for the purpose of additional measurements, samples have been prepared in an identical manner. For instance, in cups (e.g., 150 ml volume, diameter 5.5 cm), in tubes (e.g., 50 mL) or in microplates.

Samples for additional analysis (for instance: post-acidification, texture analysis, and/or challenge tests for bioprotective activity) were harvested when the pH of the fermented milk reached pH 4.6. The fermented milks thus obtained were cooled and stored at 4°C until further analysis (yogurt samples).

Post-acidification (PA) measurement. The degree of post-acidification was determined by measurement of the pH of the yogurt samples in time. To this end, the yogurt samples were incubated at different temperatures to mimic storage of the product, at for instance 7°C and 20°C (see Example 3). For each time point, at each temperature, a separate small sample (1 - 10 mL) is being prepared, which was discarded after the pH measurement.

Challenge test. The fermented milk products were subjected to a challenge test, to show the bioprotective activity of the bioprotective adjunct strains as specified in the Examples. Each sample was divided: one part was not contaminated; one part was contaminated with mould or yeast spores of strains indicated in Table 1. The contaminants were added to the fermented milk products at about 50 spores per spot, contaminants are well-known in the dairy industry.

The fungal spores were pipetted on top of the yogurt samples that were previously mixed with an equal volume of agar melted in water, and that were allowed to cool and solidify in plates (about 50 g per plate).

The cups, tubes or microplates were closed with appropriate lids and stored at the desired temperature (for instance, 20°C; see Example 3). Cups were inspected and photographed at regular intervals (days, weeks) to look for the occurrence of mould/yeast growth on the surface of the products, up to 21 days.

Example 2: Milk fermentations with of new strains as bioprotective adjuncts

Strains CBS 149825 (= strain #1) as well as CBS 149825 (= strain #2) were tested for temperature robustness at a range of 46 to 51°C, whereby the formation of colonies at the tested temperarture was compared to the number of colonies upon incubation of the same population of cells at 37°C. Precultures of strains were plated on several MRS-agar plates, at several different dilutions and incbated at different temperatures. Both strains were able to form colonies at 46°C, 47° C, 48°C, 49° C, and 50°C. No colonies were observed at 51°C (not shown).

Yogurts were made using 1U/1000L of a starter culture (Delvo®Fresh YS-141, commercially available from DSM, Delft, The Netherlands) comprising Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus, supplemented with or without a bioprotective culture (bioprotective adjunct), i.e., strain CBS 148322 or CBS 141584 as known strains or the newly identified strains according to Table 1. Milk fermentation was performed as described in Ex. 1, with inoculation of overnight cultures of the bioprotective cultures at an inoculation rate of 10⁷ cfu/ml. As soon as the pH reached the value of 4.6, the fermentation was stopped by cooling the fermented milk for 15 min in ice water. The results are shown in Table 2. Table 2A: growth performance ("TTR pH 4.6") of yogurts comprising starter culture YS-141 and strain #1 or CBS 141584 ("BP adjunct) at 42°C. "None" means the starter culture only without any BP adjunct. The results are the average of 2 biological replicates. For more details, see text.

Table 2A: growth performance ("TTR pH 4.6") of yogurts comprising starter culture YS-141 and strain #2 or CBS 148322 ("BP adjunct) at 42°C. "None" means the starter culture only without any BP adjunct. The results are the average of 2 biological replicates. For more details, see text.

Table 2C: growth performance ("TTR pH 4.6") of yogurts comprising starter culture YS-141 and mutant #3 or CBS 148322 ("BP adjunct) at 42°C. "None" means the starter culture only without any BP adjunct. The results are given as average ± standard deviation of 3 biological replicates. For more details, see text. As showns in Tab. 2A, addition of strain #1 reduces the TTR pH 4.6 by about 10 - 15 minutes.

As shown in Table 2, the speed of yogurt fermentation as indicated by TTR pH 4.6 is similar for the new strains compared to CBS 148322 or CBS 141584 when added as BP adjuncts. Performance of strain #1 was slightly different, i.e., it reduces the TTR pH 4.6 by about 10 - 15 minutes when compared to known strain CBS 141584.

Example 3: Long-term PA-performance of mutant strains

Shelf-life of mutant strains (see Table 1) were tested at 20°C (see Table 3A, 3B) for strain #1 and strain #2 as well as both 7°C and 20°C (see Table 4A, 4B) for mutant #3 in a yogurt with probes taken at several time points (0 to 14 days) as indicated in the tables below. The experimental set-up was as described above. The results are shown below.

Table 3A: PA at 20°C using strain CBS 141584 or strain #1 as BP adjunct. A yoghurt comprising starter culture YS-141 only (without any BP adjunct) is taken as reference ("none"). The results are given as average of 2 biological replicates. For more details, see text.

Table 3B: PA at 20°C using strain CBS 148322 or strain #2 as BP adjunct. A yoghurt comprising starter culture YS-141 only (without any BP adjunct) is taken as reference ("none"). The results are given as average of 2 biological replicates. For more details, see text.

As shown above for strain #1 (see Tab. 3A), a decreased contribution to PA, i.e. improved PA-profile) at 20°C by 0.2 pH units compared to known strain CBS 141584 could be shown after 14 days. Similarly, strain #2 shows improved PA- profile compared to known strain CBS 148322 by 0.1 pH units after 14 days at 20°C.

Table 4A: PA at 20°C using strain CBS 148322 or different mutant #3 as BP adjunct. A yoghurt comprising starter culture YS-141 only (without any BP adjunct) is taken as reference ("none"). The results are given as average of 3 biological replicates. For more details, see text.

Table 4B: PA at 7°C using strain CBS 148322 or mutant strain #3 as BP adjunct. A yoghurt comprising starter culture YS-141 only (without any BP adjunct) is taken as reference ("none"). The results are given as average of 3 biological replicates. For more details, see text.

As shown for mutant #3 at 20°C, the addition of bioprotective mutant #3 performed better than the addition of its ancestral strain, i.e. the known bioprotective strain CBS 148322. When compared to a yogurt comprising the starter culture only and no BP-adjunct, addition of mutant #3 (i.e., strain CBS 151609) resulted in a pH decrease of only 0.2 after 13 days at 20°C as compared to a BP-free yogurt (see "none" in Tab. 4A). Under the same conditions, addition of strain CBS 148322 into a yogurt leads to a pH reduction of 0.4 compared to the pH in a yogurt comprising the starter culture only and no bioprotective adjunct (see "none" in Tab. 4A).

Comparing the shelf-life of yogurt at 7°C as shown in Tab. 4B, the performance of samples comprising a bioprotective adjunct was slightly better than the performance of a yogurt comprising a starter culture only without addition of further bioprotective strains. Whereas the pH dropped in the yogurt comprising the starter culture only by 0.3 after 11 days, the pH reduction in a yogurt supplemented with a bioprotective strain (mutant #3) was in the range of 0.1 to 0.2 after 11 days.

Example 4: Inhibition of fungal growth by mutant strains

A challenge test was performed to test the bioprotective activity of the mutant strains independently of the decreased contribution to the PA (see Ex. 1) using various yeast or mould as contaminants. Strains #1 and #2 were tested as bioprotectives against Penicillium roqueforti (PR) or Penicillium brevicompactum (PB), both at 7°C and 20°C (see Table 5 and 6).

Mutant strains #1, #2, #3, and #4 were tested against further fungal contaminants (see Tab. 7) products. For complete list of strains see Table 1 (Ex. 1).

Table 5A: bioprotection against known contaminants as indicated and measured after incubation of a yogurt sample at 7°C for max. 21 days, wherein the performance of strain #1 is compared to CBS 141584 added as bioprotective adjunct or to a yogurt comprising starting culture YS-141 and without any bioprotective added ("none"). Scores are given from 1 to 5 as follows: "5" means no mould growth observed; "4" means first sign of growth /yogurt surface has changed; "3" means visible mold colonies; "2" means advanced mold growth, but yogurt surface not fully covered; "1" means surface completely covered with sporulated mould. For more details, see text. Table 5B: bioprotection against known contaminants as indicated and measured after incubation of a yogurt sample at 20°C for max. 21 days, wherein the performance of strain #1 is compared to CBS 141584 added as bioprotective adjunct or to a yogurt comprising starting culture YS-141 and without any bioprotective added ("none"). For more details, see text or Fig. 5A.

As apparent from Table 5, mould growth is progressing faster at 20°C compared to 7°C. A picture was taken at day 6 after inoculation of the mold spores and incubation at 20°C (see Fig. 2A). As shown, the sample without bioprotectant ("none") is completely overgrown by the moulds. The mould growth is partially inhibited by the bioprotective adjunct strains #1 and CBS141584.

Table 6A: bioprotection against known contaminants as indicated and measured after incubation of a yogurt sample at 7°C for max. 21 days, wherein the performance of strain #2 is compared to CBS 148322 added as bioprotective adjunct or to a yogurt comprising starting culture YS-141 and without any bioprotective added ("none"). For more details, see text or Fig. 4A.

Table 5B: bioprotection against known contaminants as indicated and measured after incubation of a yogurt sample at 20°C for max. 21 days, wherein the performance of strain #2 is compared to CBS 148322 added as bioprotective adjunct or to a yogurt comprising starting culture YS-141 and without any bioprotective added ("none"). For more details, see text or Fig. 5A. As apparent from Table 6, mould growth is progressing faster at 20°C compared to 7°C. Under the more challenging conditions of 20°C, new strain #2 outperforms prior art strain CBS148322. That is, at 20°C strain #2 provides more adequate bioprotection besides better post-acidification performance as shown in Ex.3.

A picture was taken at day 6 after inoculation of the mold spores and incubation at 20°C (see Fig. 2A). As shown, the sample without bioprotectant ("none") is completely overgrown by the moulds. The bioprotective activity (i.e., mould growth inhibition) is even better in strain #2 compared to the activity of the known strain CBS 148322.

The bioprotective activity of mutant #1, #2, #3, #4 compared to the activity of strain CBS 148322 was also tested at 20°C incubation. The results are shown in Tab. 6.

Table 6: bioprotection against selected mould and yeast strains as indicated ("contaminant"; see also Table 1) and measured after incubation of a yogurt sample at 20°C for 11 days, wherein the performance of the ancestor strain CBS 148322 ("ancestor") is compared to mutant strains #1, 2, 3 or 4 (see Ex. 1; mutant #1 has been deposited as strain CBS 151609) added as bioprotective adjunct to a yogurt comprising starting culture YS-141. means no inhibition, "+" means weak inhibition, "++" means strong inhibition and "+++" means complete inhibition of growth of contaminants as indicated in the table. For more details, see text.

The bioprotective activity of the mutant strains #1, 2, 3 and 4 was more or less in line with the ancestral strain CBS 148322, in addition to the fact that particularly mutant strain #3 (i.e., strain CBS 151609) has better post-acidification properties.

Claims

1. A bioprotective Lactobacillus bacterial strain capable of reducing postacidification in a fermented milk product at ambient temperature measured via increase in pH by at least a value of 0.1 as compared to a Lactobacillus bacterial strain deposited under CBS 148322.

2. The Lactobacillus bacterial strain according to claim 1, having antimicrobial, antifungal and/or anti yeast properties and having a viability, as expressed in the number of colony forming units per gram (cfu/g) on MRS-agar after 7 days, wherein the viability at 48°C is equal to or more than 50% of the viability at 37° C.

3. The Lactobacillus bacterial strain according to claim 1 or 2, having an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar, wherein the optimum growth rate lies outside the temperature range of 37° to 43°C, preferably outside the range of 35° to 45° C.

4. The Lactobacillus bacterial strain according to any one of claims 1 to 3, having an optimum growth rate, as measured by the amount of biomass of the Lactobacillus bacterial strain in grams after, in order of preference, 1, 2, 3, 4, 5, 6 or 7 days of growth on MRS-agar, wherein the optimum growth rate occurs at a temperature of equal to or more than 43°C, preferably equal to or more than 45° C.

5. The Lactobacillus bacterial strain according to any one of the preceding claims, wherein the Lactobacillus bacterial strain is a Lactobacillus rhamnosus strain, a Lactobacillus plantarum strain, a Lactobacillus paracasei strain, or a Lactobacillus casei strain.

6. The Lactobacillus bacterial strain according to any one of claims 1 to 5, being selected from a strain deposited under CBS 149825, CBS 149826 or CBS 151609.

7. The Lactobacillus bacterial strain according to any one of the preceding claims, wherein:

(a) the Lactobacillus bacterial strain increases the pH of a fermented milk product, comprising the Lactobacillus bacterial strain, during storage after fermentation in comparison to a fermented milk product, comprising a Lactobacillus bacterial strain deposited as CBS 148322, wherein the increase in pH is at least by a value of 0.1, and wherein the increase in pH is determined after storing the fermented milk product, fermented with a starter culture and the Lactobacillus bacterial strain, respectively the Lactobacillus bacterial strain deposited as CBS 148322, in a concentration of at least 10⁷ CFU/g over 14 days at 20°C; and

(b) the Lactobacillus bacterial strain is preferably a Lactobacillus casei strain.

8. An antimicrobial and/or antifungal and/or anti yeast composition comprising a Lactobacillus bacterial strain according to any one of claims 1 to 7.

9. A food product comprising a Lactobacillus bacterial strain according to any one of claims 1 to 7 or an antimicrobial and/or antifungal and/or anti yeast composition according to claim 8.

10. A food product according to claim 8, wherein the food product is a fermented milk product, preferably cheese, sour cream, or yogurt.

11. A process for manufacturing a food product comprising adding a Lactobacillus bacterial strain according to any one of claims 1 to 7, or an antimicrobial and/or antifungal and/or anti yeast composition according to claim 8, during manufacture of the food product.

12. The process according to claim 11, wherein Lactobacillus bacterial strain according to any one of claims 1 to 7, or the antimicrobial and/or antifungal and/or anti yeast composition according to claim 8, is added before or during a step of fermenting the food product.

13. The process according to claim 11, wherein the Lactobacillus bacterial strain according to any one of claims 1 to 7, or the antimicrobial and/or antifungal and/or anti yeast composition according to claim 8, is added after a step of fermenting the food product.

14. The process according to any one of claims 12 to 13, wherein the food product is a fermented milk product, preferably cheese, sour cream, or yogurt.

15. Use of a Lactobacillus bacterial strain according to any one of claims 1 to 7 for providing an antimicrobial and/or antifungal and/or anti yeast effect in a food product, preferably in a fermented milk product.