WO2023073183A1

WO2023073183A1 - Bioprotection of dairy products

Info

Publication number: WO2023073183A1
Application number: PCT/EP2022/080223
Authority: WO
Inventors: Paul Klaassen; Sofia DASHKO; Paul-Johan DE VOOGD; Margaretha Adriana DIRVEN-NEELE
Original assignee: Dsm Ip Assets B.V.
Priority date: 2021-10-28
Filing date: 2022-10-28
Publication date: 2023-05-04

Abstract

The present invention relates to a novel Lactobacillus easel strain which is suitable for providing an antimicrobial effect in dairy and in dairy based products. 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 method for manufacturing a food product. According to another aspect, the present invention relates to the use of a Lactobacillus easel strain for providing an antifungal effect in silage or in dairy products.

Description

BIOPROTECTION OF DAIRY PRODUCTS Field of the invention The present invention relates to a novel Lactobacillus casei strain which is suitable for providing an antimicrobial effect in dairy and in dairy based products. 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 method for manufacturing a food product. According to another aspect, the present invention relates to the use of a Lactobacillus casei strain for providing an antifungal effect in silage or in dairy products. Background of the invention Lactic acid bacteria are known for thousands of years for their role in the preparation of fermented foods, such as for instance dairy, meat, and vegetable-based products. During fermentation, lactic acid and other organic compounds are being produced by the lactic acid bacteria, thereby reducing the pH of the food product, and consequently making it unfavorable to the growth of undesired micro-organisms, such as yeast(s), mould(s) and (pathogenic) bacteria. Without wishing to be bound by theory, examples of compounds that are being produced by lactic acid bacteria during fermentation have been described by Ananou et al. (“Biopreservation, an ecological approach to improve the safety and shelf-life of foods” in Mendez-Vilas A. (Ed.) Communicating Current Research and Educational Topics and trends in Applied Microbiology, Formatex (2007) 475-486), Delavenne et al. (Int. J. Food Microbiol. 155 (2012) 185-190) and Woraprayote et al. (Meat Science 120 (2016) 118-132). Bioprotection, or biopreservation, is defined as the extension of shelf life and enhanced safety of foods using natural or controlled microbiota and/or antimicrobial compounds. To this end, certain microbial ingredients, such as e.g., lactic acid bacteria, are being used to control the growth of undesired microorganisms such as yeasts, moulds, and bacteria. Besides fungi, lactic acid bacteria are of particular interest for bioprotection since some may have antagonistic properties which make them useful as a bioprotectant. Dairy products generally comprise living microorganisms such as lactic acid bacteria. However, fungi such as moulds and yeasts could grow abundantly in dairy products, even under cold conditions. Although the presence of lactic acid bacteria in dairy products is desired, there is a challenge to reduce the undesired, non-controlled growth of contaminant yeasts and moulds. Food grade chemical preservatives such as potassium sorbate and benzoate are common adequate measures to prevent the undesired growth of yeasts, moulds, and bacteria. 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. WO 2013/153074 describes a Lactobacillus rhamnosus strain CHCC5366 and Lactobacillus paracasei strains CHCC12777 and CHCC14676. It is disclosed that the combination of the Lactobacillus rhamnosus strain with the Lactobacillus paracasei strain provides a synergistic effect in comparison with the strains alone. WO 2012/136830 describes an antimicrobial composition comprising at least one Lactobacillus rhamnosus strain and at least one Lactobacillus paracasei strain. A significant synergistic antimicrobial effect if both strains are combined is reported. The commercial use of a combination of Lactobacillus rhamnosus and Lactobacillus paracasei is FreshQ^®, which is a protective culture for fermented milk products available from Chr. Hansen, Denmark. The use of protective cultures consisting of combinations of different species may increase the risk that the additional species introduce flavor to the food product. This is a disadvantage because it limits the applications of the protective culture. In EP 3279312 an antimicrobial composition is described which is effective with a single Lactobacillus rhamnosus species CBS141584. Still, the use of protective Lactobacillus cultures requires further improvement. There are several problems associated with the production of fermented dairy products. Firstly, there is the problem of reduction of bioprotective activity of the Lactobacillus cultures in cases where heat treatment is involved in the production process. The latter is, for example, the case in the manufacturing of pasta filata cheeses. Secondly, there is the problem of undesired production of gas. This is a phenomenon that regularly occurs, particularly in dairy products that comprise fruit or other sources of gas-producing compounds such as malic acid. Thirdly, there is the problem of loss of color in certain fermented dairy products. Discoloration may occur because of the undesired conversion or degradation of dyes, for example of azo dyes. Finally, there is the problem of post-acidification, which is an undesired process in fermented dairy products whereby continued acidification beyond the optimal range occurs. The gas production, dye discoloration and post-acidification predominantly take place during shelf-life of the fermented dairy 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. The development of gas may lead to a billowing or even bursting package. Gas development or discoloration make the product less appealing to a customer. Therefore, there is a need to develop a bioprotective solution which not only affords a favorable shelf-life stability per se, but also resolves any or all the problems mentioned above. Legend to the Figures Figure 1: Contribution of bioprotective cultures CBS141584 and CBS148322 to post- acidification of yogurts during storage at 21°C. X axis shows days of storage. Y axis shows the values of delta pH with respect to day 0. Change of pH in yogurts without a bioprotective culture (Blank) are marked as a black cross, yogurts with CBS141584 are marked with black circles and yogurts made with CBS 148322 are marked with black diamonds. Figure 2: Contribution of bioprotective cultures CBS141584 and CBS148322 to post- acidification of yogurts during storage at 30°C. X axis shows days of storage. Y axis shows the values of delta pH with respect to day 0. Change of pH in yogurts without a bioprotective culture (Blank) are marked as a black cross, yogurts with CBS141584 are marked with black circles and yogurts made with CBS 148322 are marked with black diamonds. Figure 3: Contribution of bioprotective cultures CBS141584 and CBS148322 and a blend with CBS148322 to post-acidification of yogurts during storage at 21°C. X axis shows days of storage. Y axis shows the pH values in yogurts. pH of yogurts without a bioprotective culture (Blank) are marked as a black cross, yogurts with CBS141584 are marked with black circles and yogurts made with CBS 148322 are marked with black diamonds. The blend containing CBS 148322 and CBS 148323 is marked in open diamond and dashed line. Figure 4: Contribution of bioprotective cultures CBS141584 and CBS148322 and a blend with CBS148322 to post-acidification of yogurts during storage at 30°C. X axis shows days of storage. Y axis shows the pH values in yogurts. pH of yogurts without a bioprotective culture (Blank) are marked as a black cross, yogurts with CBS141584 are marked with black circles and yogurts made with CBS 148322 are marked with black diamonds. The blend containing CBS 148322 and CBS 148323 is marked in open diamond and dashed line. Figure 5: Challenge test with two mould contaminants using YOA plates from yogurt samples prepared with the bioprotective culture CBS148322, the bioprotective culture CBS141584, the bioprotective blend CBS148322 + CBS148323 and the commercial blend product FreshQ^®. Y axis shows the size of the contaminant colony (cm), X axis shows tested bioprotective cultures or blends and a reference yogurt without a bioprotective culture (Blank). Trellis on the right indicates the day when the contaminant growth was recorded (3, 5, 6, 10, 13 and 17 days). Grey bars show the growth of Penicillium brevicompactum, black bars show the growth of Aspergillus niger. Figure 6: Challenge test with two yeast contaminants using YOA plates from yogurt samples prepared with the bioprotective culture CBS148322, the bioprotective culture CBS141584, the bioprotective blend CBS148322 + CBS148323 and the commercial blend product FreshQ^®. Y axis shows the size of the contaminant colony (cm), X axis shows tested bioprotective cultures or blends and a reference yogurt without a bioprotective culture (Blank). Trellis on the right indicates the day when the contaminant growth was recorded (3, 5, 6, 10, 13 and 17 days). Grey bars show the growth of Debaryomyces hansenii, black bars show the growth of Torulaspora delbrueckii. Figure 7: Gas development in yogurts without added L-malic acid. X axis shows gas tube reading (in ml). Y axis shows the number of days from yogurt make. Yogurts without a bioprotective culture are shown as black, yogurts made with CBS148322 are shown in grey, yogurts with CBS116412 are marked in check pattern and yogurts with CBS 141584 are marked with stripe line pattern. Figure 8: Gas development in yogurts with 0.15% ^L-malic acid. X axis shows gas tube reading (in ml). Y axis shows the number of days from yogurt make. Yogurts without a bioprotective culture are shown as black, yogurts made with CBS148322 are shown in grey, yogurts with CBS116412 are marked in check pattern and yogurts with CBS141584 are marked with stripe line pattern. Figure 9: Color stability of yogurts recorded at 7°C and 21°C storage temperatures. Y axis shows color difference expressed as the Euclidean distance between L, a and b values (ΔE). X axis shows days of yogurt storage. Top trellis indicates the type of colorant added to yogurt samples (Allura and Annato). Right trellis shows storage temperature applied to yogurts (7 and 21°C). Yogurts prepared without a bioprotective cultures are marked as black squares. Yogurts with CBS148322 are marked with stars, yogurts with CBS 141584 are marked with crosses. Line on Y axis indicates the threshold at which color difference are significantly visible. Figure 10: Challenge test with Penicillium brevicompactum using YOA plates made from yogurt samples subjected to 30 min of the heat exposure at 60°C (bottom row) or kept at 21°C (top row). Photos of the plates taken on day 6 of incubation at 25°C. Plates on the left contain yogurts made with the addition of CBS 141584, plates in the middle are made with CBS 148322, plates on the right are made without added bioprotective culture. Summary of the invention The objective of the present invention is the provision of novel strains of lactic acid bacteria with high efficacy as bioprotective agents, but no effect or an improved effect on attributes such as flavor, post-acidification, gas production in the presence of fruit, color, and the like. This objective, amongst other objectives, is met by providing a Lactobacillus casei strain according to the appended claim 1. More specifically, this objective, amongst other objectives, is met by providing a Lactobacillus casei strain as found in deposit CBS148322, or mutants derived therefrom. According to another aspect, the present invention relates to an antimicrobial composition comprising the present Lactobacillus casei strain. According to another aspect, the present invention relates to an antifungal composition comprising the present Lactobacillus casei strain. According to yet another aspect, the present invention relates to a food product comprising the present Lactobacillus casei strain, the present antimicrobial composition, or the present antifungal composition. According to yet another aspect, the present invention relates to a method for manufacturing a food product comprising adding the present Lactobacillus casei strain, the present antimicrobial composition, or the present antifungal during manufacture of the food product. According to yet another aspect, the present invention relates to the use of the present Lactobacillus casei for providing an antifungal effect in dairy products or in silage. Detailed description 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 "lactic acid bacteria" 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 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, Ilama, mare and deer. In an embodiment, the milk is from a mammal selected from the group consisting of cow, sheep, goat, buffalo, camel, Ilama, 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 and sunflower seed. 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 lactic acid bacteria. Examples of lactic acid bacteria 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 lactic acid bacteria, 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 "mutant" should be understood as a strain derived from a strain of the invention, 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. 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 that has equal to or more than 70% identity, more preferably equal to or more than 80% identity, even more preferably equal to or more than 90% identity, still even more preferably equal to or more than 95% identity, yet even more preferably equal to or more than 99% identity and most preferably equal to or more than 99.9% identity with the nucleic acid sequence of the parent strain from which it is derived (such as for example the Lactobacillus casei strain deposited as CBS148322, respectively the Lactobacillus rhamnosus strain deposited as CBS148323) . The term "thermophile" or "thermophilic" herein refers to lactic acid bacteria that thrive well at temperatures above 41°C. The most useful thermophilic lactic acid bacteria include Lactobacillus spp. and Streptococcus ssp. Hence, a "thermophilic fermentation" herein refers to a fermentation that is being executed at a temperature above about 35°C, for example between about 35°C and about 45°C, such as e.g., at 42°C. The term "mesophile" or "mesophilic" herein refers to lactic acid bacteria that thrive best at temperatures lower than 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° and 36°C, such as e.g., at 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 micro-organisms", "undesired contaminants" and "contaminants" herein refer to the occurrence of micro-organisms, 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 dairy matrix, such as e.g., a yogurt or a sour cream product. The prevention of the growth of contaminants due to the action the present Lactobacillus casei strain can be expressed by e.g., a lower number of contaminant cell counts in a dairy product prepared with the present Lactobacillus casei, compared to a similar product, without the present Lactobacillus casei 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. In a first aspect, the inventors have identified a novel Lactobacillus casei strain suitable for providing an antimicrobial effect. It was surprisingly found that this Lactobacillus casei strain provides an equal or even improved antimicrobial effect in view of the commonly used chemical preservative potassium sorbate. Therefore, in a preferred embodiment, the present Lactobacillus casei strain has an improved 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. Preferably, wherein the Lactobacillus casei 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. Further, the inventors found that the present Lactobacillus casei strain can match or even improve the antimicrobial effect of a combination of another Lactobacillus casei strain and Lactobacillus paracasei strain such as FreshQ^®. Furthermore, the present inventors found that the present Lactobacillus casei strain does not introduce a flavor effect to the food product. This enables the present Lactobacillus casei strain to be used in a broad range of food applications without altering the flavor profile of the food item. Therefore, in a preferred embodiment, the present Lactobacillus casei strain is suitable as bioprotectant without introducing a flavor effect to the food product the strain is added to. The present Lactobacillus casei strain is as found in deposit CBS148322 or mutants derived therefrom. Strain CBS148322 is deposited on 03/09/2021 at the Centraalbureau voor Schimmelcultures (Fungal Biodiversity Centre), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands under the provisions of the Budapest Treaty. Mutants derived therefrom as used in the present context means Lactobacillus casei strains which are derived from CBS148322, or obtained from CBS148322, and may have mutations in comparison with Lactobacillus casei CBS148322 wherein the mutations do not alter the bioprotective phenotype of the derived Lactobacillus casei strain. Preferably, the mutant strain has the same or improved antimicrobial antifungal and / or anti-yeast properties as the mother strain CBS148322. Preferably, the derived Lactobacillus casei strain is suitable for providing an antifungal effect and/or an anti-bacterial effect, as found for or like strain CBS148322. Preferably, the mutant derived from CBS148322 has at least 80% or more, preferably at least 90% or more, more preferably at least 95% or more or even up to 100% or more than 100% of the antimicrobial, antifungal, and / or anti-yeast effect compared with strain CBS148322 if compared under equal conditions. In a more preferred embodiment, the present Lactobacillus casei strain is strain CBS148322. Therefore, in a preferred embodiment, the present Lactobacillus casei strain is suitable for providing an antifungal effect and/or an anti-yeast effect, more preferably without introducing a flavor effect. According to another aspect, the present invention relates to an antimicrobial, an antifungal and/or an anti-yeast composition comprising the present Lactobacillus casei strain. Preferably, the amount of the present Lactobacillus casei 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. In a preferred embodiment, the present antimicrobial, antifungal and/or anti-yeast composition further comprises a Lactobacillus rhamnosus strain, preferably a Lactobacillus rhamnosus strain as found in deposit CBS148323 or mutants derived therefrom. The present invention therefore advantageously also provides an antimicrobial, antifungal and/or an anti-yeast composition comprising: a) a Lactobacillus casei strain deposited as CBS148322 or mutants derived therefrom, wherein preferably said mutants have the same or improved antimicrobial, antifungal and/or anti-yeast properties as strain CBS148322; and b) a Lactobacillus rhamnosus strain deposited as CBS148323 or mutants derived therefrom, wherein preferably said mutants have the same or improved antimicrobial, antifungal and/or anti-yeast properties as strain CBS148323. As illustrated in the examples, such antimicrobial, antifungal and/or an anti-yeast composition comprising both a Lactobacillus casei strain deposited as CBS148322 or mutants derived therefrom and a Lactobacillus rhamnosus strain deposited as CBS148323 or mutants derived therefrom advantageously may have improved bioprotection characteristics compared to market product FreshQ^®. From a bioprotection perspective such a composition may match the performance of bioprotective culture CBS141584 of EP3279312 that also outperformed market product FreshQ^®. However, compared to previous culture CBS141584 of EP3279312, such antimicrobial, antifungal and/or an anti-yeast composition comprising both a Lactobacillus casei strain deposited as CBS148322 or mutants derived therefrom and a Lactobacillus rhamnosus strain deposited as CBS148323 or mutants derived therefrom may have improved post- acidification characteristics. Preferences for such antimicrobial, antifungal and/or an anti-yeast composition are the same as described for the single strain as described herein above and hereinbelow. By the Lactobacillus rhamnosus strain CBS148323 is suitably understood the strain as found in deposit CBS148323 deposited on 03/09/2021 at the Centraalbureau voor Schimmelcultures (Fungal Biodiversity Centre), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands under the provisions of the Budapest Treaty. Mutants derived therefrom as used in the present context are suitably understood to be Lactobacillus rhamnosus strains which are derived from CBS148323, or obtained from CBS148323, and may have mutations in comparison with Lactobacillus rhamnosus CBS148323 wherein the mutations do not alter the bioprotective phenotype of the derived Lactobacillus rhamnosus strain. Preferably, the mutant strain has the same or improved antimicrobial antifungal and / or anti-yeast properties as the parent strain CBS148323. Preferably, the derived Lactobacillus rhamnosus strain is suitable for providing an antifungal effect and/or an anti-bacterial effect, as found for or like strain CBS148323. Preferably, the mutant derived from CBS148323 has at least 80% or more, preferably at least 90% or more, more preferably at least 95% or more or even up to 100% or more than 100% of the antimicrobial, antifungal, and / or anti-yeast effect compared with strain CBS148323 if compared under equal conditions. In a more preferred embodiment, the Lactobacillus rhamnosus strain is strain CBS148323. The present antimicrobial, antifungal and/or anti-yeast composition preferably comprises the present Lactobacillus casei strain, such as for example the Lactobacillus casei strain deposited as CBS148322 or one or more mutants derived therefrom, in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells, which is preferably in the range of 10⁴ to 10¹³ cfu (colony forming units) per gram of the composition including at least 10⁴ cfu per gram of the composition, such as at least 10⁵ cfu/g, e.g., preferably at least 10⁶ cfu/g, such as at least 10⁷ cfu/g, e.g., more preferably at least 10⁸ cfu/g, such as at least 10⁹ cfu/g, e.g., most preferably at least 10¹⁰ cfu/g, such as at least 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 preferably in the range of 10⁴ to 10¹² cfu (colony forming units) per ml of the composition including at least 10⁴ cfu per gram of the composition, such as at least 10⁵ cfu/ml, e.g., at least 10⁶ cfu/ml, such as at least 10⁷ cfu/ml, e.g., at least 10⁸ cfu/ml, such as at least 10⁹ cfu/ml, e.g., at least 10¹⁰ cfu/ml, such as at least 10¹¹ cfu/ml. If present, the antimicrobial, antifungal and/or anti-yeast composition preferably comprises the Lactobacillus rhamnosus strain deposited as CBS148323 or one or more mutants derived therefrom in a concentrated form including frozen, dried or freeze-dried concentrates typically having a concentration of viable cells, which is preferably in the range of 10⁴ to 10¹³ cfu (colony forming units) per gram of the composition including at least 10⁴ cfu per gram of the composition, such as at least 10⁵ cfu/g, e.g., preferably at least 10⁶ cfu/g, such as at least 10⁷ cfu/g, e.g., more preferably at least 10⁸ cfu/g, such as at least 10⁹ cfu/g, e.g., most preferably at least 10¹⁰ cfu/g, such as at least 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 preferably in the range of 10⁴ to 10¹² cfu (colony forming units) per ml of the composition including at least 10⁴ cfu per gram of the composition, such as at least 10⁵ cfu/ml, e.g., at least 10⁶ cfu/ml, such as at least 10⁷ cfu/ml, e.g., at least 10⁸ cfu/ml, such as at least 10⁹ cfu/ml, e.g., at least 10¹⁰ cfu/ml, such as at least 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 fructose. Other additives may include, e.g., carbohydrates, flavors, minerals, 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 1 month, such as at least 3 months. Preferably the amount of antimicrobial, antifungal or anti-yeast composition in the package is at least 50 grams, such as at least 100 grams or 500 grams. In a further aspect, the present invention relates to a food product comprising the present Lactobacillus casei strain and optionally the presence of a second Lactobacillus strain, or the present antimicrobial and/or antifungal composition. The present invention relates to a food product comprising an amount of the present Lactobacillus casei strain and optionally an amount of the second Lactobacillus rhamnosus strain which is effective for imparting antimicrobial properties to the food product. More preferably, wherein the presence of the present Lactobacillus casei strain and optionally an amount of the second Lactobacillus rhamnosus strain does not introduce a flavor to the food product. In a further preferred embodiment, the present food product comprises the present Lactobacillus casei strain in an amount which is preferably in the range of 10⁴ to 10¹² cfu (colony forming units) per gram of the food product including at least 10⁴ cfu per gram of the food product, such as at least 10⁵ cfu/g, e.g., preferably at least 10⁶ cfu/g, such as at least 10⁷ cfu/g, e.g., more preferably at least 10⁸ cfu/g, such as at least 10⁹ cfu/g, e.g., most preferably at least 10¹⁰ cfu/g, such as at least 10¹¹ cfu/g of the food product. More preferably, the present food product comprises the present Lactobacillus casei 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 10⁴ cfu/cm² of the food product, such as at least 10⁵ cfu/cm², e.g., at least 10⁵ cfu/cm², such as at least 10⁷ cfu/cm², e.g., more preferably at least 10⁸ cfu/cm², such as at least 10⁹ cfu/cm², e.g., most preferably at least 10¹⁰ cfu/cm², such as at least 10¹¹ cfu/cm² surface of the food product. In a further preferred embodiment, the present food product comprises the second Lactobacillus rhamnosus strain in an amount which is preferably in the range of 10⁴ to 10¹² cfu (colony forming units) per gram of the food product including at least 10⁴ cfu per gram of the food product, such as at least 10⁵ cfu/g, e.g., more preferably at least 10⁶ cfu/g, such as at least 10⁷ cfu/g, e.g., even more preferable at least 10⁸ cfu/g, such as at least 10⁹ cfu/g, e.g., most preferably at least 10¹⁰ cfu/g, such as at least 10¹¹ cfu/g of the food product. More preferably, the present food product comprises the second Lactobacillus rhamnosus 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 10⁴ cfu per cm² of the food product, such as preferably at least 10⁵ cfu/cm², e.g., at least 10⁵ cfu/cm², such as at least 10⁷ cfu/cm², e.g., more preferably at least 10⁸ cfu/cm², such as at least 10⁹ cfu/cm², e.g., most preferably at least 10¹⁰ cfu/cm², such as at least 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 casei strain and optionally the second Lactobacillus rhamnosus strain. In other words, the present Lactobacillus casei strain and optionally the second Lactobacillus rhamnosus strain do not introduce a flavor to a food product to which the Lactobacillus casei strain and optionally the second Lactobacillus rhamnosus strain is added. Preferably, the present food product in all aspects of the present invention is a dairy product. More preferably a fermented milk product, such as a mesophilic or thermophilic fermented milk product. Most preferably the present food product is sour cream or yogurt. Examples of a fermented dairy product of the present invention are various types of regular yoghurt, plain yogurt, low fat yoghurt, nonfat yoghurt, tvarog, kefir, dahi, ymer, buttermilk, butter, sour cream, and sour whipped cream. Other examples of fermented dairy product are cheese. For example, fresh cheeses, un-ripened cheeses, or curd cheeses. Alternatively, the fermented dairy product is a ripened cheese. Alternatively, the present food product is a dairy product, more preferably 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 casei strain and optionally a Lactobacillus rhamnosus strain as described herein, or the present antimicrobial composition, antifungal composition, or anti-bacterial composition, during manufacture of the food product. Preferably, the present method comprises addition of the present Lactobacillus casei strain and optionally a Lactobacillus rhamnosus strain and/or the present antimicrobial, antifungal or anti-yeast composition to milk before fermentation of the milk. More preferably, the present Lactobacillus casei strain and optionally a Lactobacillus rhamnosus strain and/or the present antimicrobial, antifungal or anti-yeast composition is added to the milk together with lactic acid bacteria used for fermentation of the milk. More preferably, the present method comprises a step of fermenting milk with lactic acid bacteria. Even more preferably, the present method comprises a step of fermenting milk with lactic acid bacteria in the presence of the present Lactobacillus casei strain and optionally a Lactobacillus rhamnosus strain and/or in the presence of the present antimicrobial, antifungal or anti-yeast composition. The present inventors found that a high efficacy against yeast and moulds can be obtained if the present Lactobacillus casei strain 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 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 casei strain and optionally a Lactobacillus rhamnosus strain as described herein, 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 casei strain and optionally a Lactobacillus rhamnosus strain as described herein, or the present antimicrobial composition, antifungal composition, or anti-bacterial 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 casei strain for providing an antimicrobial effect in dairy products, preferably for providing an antifungal effect and/or an anti-yeast effect in dairy products. More preferably wherein the dairy product is a fermented dairy product. More preferably a fermented dairy 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 dairy product wherein the present Lactobacillus casei strain can be used is cheese. For example, fresh cheeses, un-ripened cheeses, or curd cheeses. Alternatively, the fermented dairy product is a ripened cheese. In a preferred embodiment, the present invention relates to the use of the present Lactobacillus casei strain for providing an antimicrobial effect in silage, preferably for providing an antifungal effect and/or an anti-yeast effect in silage. The invention is further illustrating using the following non limiting examples.

EXAMPLES Materials and methods Strains and products Table 1. List of all the lactic acid bacteria cultures and fungal contaminants used in the Examples

Yogurt fermentation Yogurts for Examples 1, 2, 4, 5 and 6 were prepared with the starter culture Delvo^®Fresh YS-141 (dosed as recommended) and addition of the bioprotective cultures (details can be found in the Tables for each Example). In case of Example 3, Delvo^®Tam FVV-122 was applied as a starter culture, according to the supplier’s recommendations. For Examples 1, 3, 5 and 6 the bioprotective cultures were pre-grown in MRS (37°C) overnight. In Examples 2 and 4, frozen pellets with an average cell count of 10¹¹ cfu/g were applied. Regular full-fat milk, 3.4% fat, 3.4% protein, was heat-treated at 85°C for 30 min and cooled at 4°C overnight prior usage for Example 1. For Examples 2 and 3 semi-skimmed milk (1.5% fat, 4.2% protein) was applied. For Example 4 semi skimmed milk with 3% protein was used. Yogurts were prepared at 42°C. When the pH of the milk fermentation reached 4.6, yogurt bottles were immediately cooled in ice water and processed for further analysis. Assessment of post-acidification of yogurts pH measurements with a pH meter (post-acidification) were performed on the yogurts stored at 7°C, 21°C and 30°C. The samples were measured at regular intervals for a period of up to 8 weeks. One separate tube per measurement (time point) was used and discarded after the pH measurement. The pH meter was calibrated before each use. Yogurts used for measurements were prepared as described in “Yogurt fermentation”. Challenge tests with fungal contaminants Yogurts used for the challenge tests were prepared as described in materials and method section. Yogurts were mechanically smoothened and mixed with 2% sterile agar solution in the ratio 1:1. After swift mixing, the content was poured into sterile Petri dishes and left to solidify. Three so- called YOA-plates were prepared per variation (variations are listed in the Examples, different combinations of starter culture and bioprotective cultures). One plate was not contaminated, one plate was contaminated with mould spores (7 µl containing approx.200 spores) spotted and one plate was contaminated with yeast cells (7 µl containing approx.200 cells). Mould contaminants were Aspergillus niger and Penicillium brevicompactum (Table 1). The yeast contaminants were Debaryomyces hansenii and Torulaspora delbrueckii (Table 1). Species were chosen because of their relevance in dairy matrices. The above-listed contaminants were spotted on the plate surface. YOA plates were incubated at 25°C. The colony size of the contaminants was measured at regular intervals, for up to about 4 weeks. The data was visualized by plotting the size of contaminant colonies recorded on a day of observation. Detection of gas production in yogurts Gas production was monitored in the yogurt samples. Each yogurt sample was split into two sample series. One sample series were kept as such. The second series included the addition of sterile solution of L-malic acid (0.15% final concentration in yogurt) to mimic the effect of added fruit. The detection protocol was based on a method in which rubber stoppers, with inserted serological pipette containing an agar plug (0.75% agar), are used to close glass Schott bottles containing the two series of yogurt samples. The samples were incubated at room temperature (20-21°C). If gas production took place in time, this gas production caused a displacement of the agar plug. The displacement of the agar plug was recorded as the height of the agar plug in the serological pipettes. Monitoring discoloration in yogurts supplemented with food colorants An effect of a bioprotective culture on food colorant stability in yogurt was evaluated by measuring the color stability in yogurt using a flatbed scanner. To this end, one synthetic colorant Allura Red (E129, at 0.03% w/v final concentration) and one natural colorant Annato (E160b, dosed at 0.01% w/v final concentration) were added to the yogurt prepared as described in Materials and Methods section. Amounts of the colorants added are specified in brackets. Yoghurt with the colorants added was pipetted into a 24 wells cell-culture plate to enable measurement with a flatbed scanner. Samples were added to the plate in triplicate (technical replicates). The plates were stored at three temperatures: 7°C, 21°C and 30°C. For 20 days, at regular time points, the color of each well with yoghurt with colorant was measured. Output from the L A B channels was applied to calculate ΔE in order to detect a possible color change. Formula and criteria for a color change decision are listed below. Difference in color was calculated as the Euclidean distance between L a b values (Jain, Anil K. (1989) Fundamentals of Digital Image Processing. New Jersey, United States of America: Prentice Hall. pp.68, 71, 73. ISBN 0-13-336165-9.).

L measures perceptual lightness, a indicates the position of the color between red and green, and b indicates its position between yellow and blue. ΔE*ab value is used to determine the extent of color change: 0.0 … 0.5 no color difference 0.5 … 1.0 difference only perceivable for experienced observers 1.0 … 2.0 minimal color difference 2.0 … 4.0 perceivable color difference 4.0 … 5.0 significant color difference > 5 different color Heat challenge test with fungal contaminants Heat resistance of the bioprotective candidate CBS148322 was evaluated. A heat test was performed on the yogurt samples, prepared as described in Materials and Methods and exposed to the heat treatment in a water bath, preheated to 60°C. Three samples per yogurt variant were prepared (for t0, t10 min and t30 min treatments).10 minutes of preheating time was applied to all the samples except for t0. After the preheating step, samples were kept in the water bath for 10 minutes or 30 minutes for t10 min and t30 min treatments respectively. Afterwards, the samples were taken out of the water bath, and were placed on ice to cool down. After cooling for 15 minutes, one part of each yogurt sample was sampled for the viable cell count analysis. The second part of the sample was mixed with agar and used in the challenge test with fungal contaminant Penicillium brevicompactum CBS110070 as described in the Materials and Methods above. Example 1 Contribution of single strain CBS148322 to post-acidification in yogurts Table 2 List of yogurt samples for the Example 1

Yogurts were produced as described in Materials and Methods section. Subsequently, the samples were divided and stored at 21°C and 30°C. At regular intervals, the pH was measured of one of the tubes. The results are shown in Figures 1 (21°C storage) and 2 (30°C storage). At 21°C already after 5 days of storage CBS148322 showed substantially reduced contribution to post-acidification when compared to CBS141584. At 30°C the differences between the two cultures were even more prominent. These results show that the new strain CBS148322 has a reduced contribution to post-acidification of the final fermented milk product when compared to the existing bioprotective solutions, like CBS141584. Example 2 Contribution of blends with CBS148322 to post-acidification in yogurts Table 3a List of yogurt samples for the Example 2

Yogurts were produced as described in the Materials and Methods section. Subsequently, the samples were divided in subsamples and stored at 21°C and 30°C. At regular time intervals, the pH was measured. The results are shown in Figures 3 (21°C storage) and Figure 4 (30°C storage) and summarized in Table 3b below. At both temperatures, yogurt containing the bioprotective blend with CBS148322 (black diamonds) showed substantially reduced contribution to post-acidification when compared to strain CBS141584 (black circles). These results show that the blends containing CBS148322 showed reduced post-acidification of the final product when compared to existing bioprotective solutions, like CBS141584. Also the blend containing CBS 148322 and CBS 148323 (open diamonds) showed a reduced contribution to post-acidification when compared to strain CBS141584 (black circles). Table 3b

Example 3 Yogurt challenge test Yogurts were produced as described in the Materials and Methods section. The yogurt samples are the same as mentioned in Example 2.. In addition, the commercial blend product FreshQ^® was tested. The challenge test with fungal contaminants was performed as described above in the Materials and Methods section. During storage at 25°C, mould contaminants were completely inhibited in yogurts where CBS148322 and the blends with CBS148322 were added (Figure 5). As illustrated in Figure 5, the bioprotective culture CBS148322 and the bioprotective blend CBS148322 + CBS148323 matched the performance of the bioprotective culture CBS141584 of EP3279312 and the commercial blend product FreshQ^® Yeast contaminant T. delbrueckii was inhibited only partially in yogurts with CBS148322 but remained completely inhibited when the CBS148322 + CBS148323 blend was added (Figure 6). D. hansenii was inhibited effectively in yogurts with CBS148322 up to day 6. The CBS148322 + CBS148323 blend was more effective in inhibiting D. hansenii. As illustrated in Figure 6, in respect of T. delbrueckii, the bioprotective blend CBS148322 + CBS148323 matched the performance of the bioprotective culture CBS141584 of EP3279312 and showed an improvement in antimicrobial effect over the commercial blend product FreshQ^®. These results show that CBS148322, being a single strain of Lactobacillus casei provides an excellent degree of biopreservation against mould contaminants comparable to the existing market solutions and prevents outgrowth of fungal contaminants in yogurt matrix when used as a single strain or as part of the bioprotective blend. Inhibition of yeast contaminants is, in general, less effective, but can be improved when blending with another bioprotective strain, as shown in this Example by the combination CBS148322 + CBS148323. Moreover, the example showed that the bioprotective blend CBS148322 + CBS148323 matched the performance of the bioprotective culture CBS141584 of EP3279312 and outperforms market product FreshQ^®. Example 4 Solving the problem of gas production from fruit preparations Table 4 List of yogurt samples for the Example 4

Gas development in yogurts was calculated by the movement of the agar plug in the serological pipette, inserted in the sealed bottles. The yogurts for the test were prepared as described in the Materials and Methods section and kept at room temperature (20-21°C) for the whole time of the gas production experiment. Figure 7 shows the results of gas development in yogurts without added L-malic acid. Figure 8 contains the results with 0.15% added L-malic acid. Some minor noise was introduced due to temperature fluctuations but overall, little to no gas was detected without ^L-malic acid in all the samples tested (Figure 7). When L-malic acid was added to yogurts commercially available bioprotective culture CBS141584 as well as CBS116412 produced substantial amounts of gas (Figure 8). In contrast, CBS148322 did not show any gas formation and was comparable to the reference yogurts without a bioprotective culture or combination of bioprotective cultures. This result showed that CBS148322 is a suitable bioprotective culture that can be applied in yogurts with L-malic acid present in a form of a fruit puree. Example 5 Problem of coloration Table 5 List of yogurt samples for the Example 5

The stability of 2 colorants (Allura Red and Annato) was evaluated in yogurts with the bioprotective cultures tested (Table 5). Color stability of yogurts was recorded at 2 storage temperatures, 7°C and 21°C. No discoloration was observed at 7°C in any of the samples (Figure 9), as the (delta)E value remained below 5 in all cases. At 21°C, the color stability of Annato also remained comparable in yogurts with and without the bioprotective cultures (Figure 9). In contrast, the yogurts containing the Allura-dye showed substantial discoloration in case CBS141584 or CBS148323 were added, when stored at 21°C. Surprisingly, CBS148322 did not cause any visible discoloration of yogurt in any of the tested conditions. This demonstrates that CBS148322 is suitable bioprotective culture in dairy applications where azo-dyes like Allura Red are applied. Example 6 Solving the problem of loss of bioprotective activity in products involving heat treatment Table 6 List of yogurt samples for the Example 6

To determine whether the bioprotective culture CBS148322 is suitable for applications where a heat treatment above 59°C is involved, a heat-challenge test at 60°C was performed. The yogurt samples with and without the bioprotective cultures listed in Table 6 were subjected to a heat challenge test as described in the Materials and Methods. Samples from t0, t10 min and t30 min treatments were used to tests the bioprotective activity against the fungal contaminant, P. brevicompactum (Table 1). After 30 minutes of the heat treatment, yogurts with CBS141584 were less resistant to the mould contaminant (Figure 10). In contrast, the yogurts prepared with CBS148322 still inhibited the mould contaminant growth. This result demonstrates that CBS148322 is a bioprotective strain suitable for applications where heat treatment is applied.

Claims

1. A Lactobacillus easel strain deposited as CBS148322 or mutants derived therefrom, wherein said mutants have the same or improved antimicrobial, antifungal and/or anti-yeast properties as strain CBS148322.

2. Lactobacillus easel strain according to claim 1 in frozen, dried, or freeze-dried form.

3. An antimicrobial and/or antifungal composition comprising the Lactobacillus easel strain as defined in any of the preceding claims, optionally further comprising a facultative heterofermentative Lactobacillus strain.

4. Composition according to claim 3 wherein said facultative heterofermentative Lactobacillus strain is a Lactobacillus rhamnosus strain, preferably wherein said Lactobacillus rhamnosus strain is deposited as CBS148323.

5. A food product comprising a Lactobacillus easel strain as defined in any of claims 1 to 2 or an antimicrobial and/or antifungal composition according to any of claims 3 to 4.

6. Food product according to claim 5 comprising an amount of Lactobacillus easel strain and/or an amount of Lactobacillus rhamnosus strain, which amounts are effective for imparting antimicrobial and/or antifungal properties to the food product.

7. Food product according to claims 5 to 6, wherein the food product is a dairy product, preferably cheese, sour cream, or yoghurt.

8. A method for manufacturing a food product comprising adding the Lactobacillus easel strain as defined in any of claims 1 to 2, or an antimicrobial and/or antifungal composition according to any of claims 3 to 4 during manufacture of the food product.

9. Method according to claim 8, wherein the Lactobacillus easel strain is added before or during a step of fermenting the food product.

10. Method according to claim 9, wherein the method comprises fermenting milk with a starter culture comprising at least one strain of the genera selected from Lactobacillus, Streptococcus, Lactococcus and Leuconostoc.

11. Use of a Lactobacillus easel strain deposited as CBS148322 for providing an antifungal effect in a dairy product, preferably further providing reduction of post-acidification.

12. Use according to claim 11 further providing reduction of gas production in said dairy product.

13. Use according to claim 12 wherein said dairy product comprise L-malic acid.

14. Use according to any of claims 11 to 13 further providing prevention of discoloration of said dairy product.

15. Use according to any of claims 11 to 14, wherein the dairy product is cheese, sour cream, or yogurt.