Strains of lactic acid bacteria and uses thereof in packing water for improving mozzarella flavoring

Abstract

The present invention belongs to the field of food products, in particular fresh mozzarella, and provides packing water suitable for directly acidified fresh mozzarella comprising at least one lactic acid bacterium (LAB), preferably wherein the at least one LAB strain is selected from a Lactobacillus strain, a Lactiplantibacillus strain, a Lacticaseibacillus strain or a Lactococcus strain. The invention further provides LAB suitable for being comprised in the packing water of the present invention and methods for producing packaged fresh mozzarella using the packing water and LAB of the present invention.

Description

[0001] STRAINS AND USES THEREOF FOR IMPROVING MOZZARELLA FLAVORING

[0002] FIELD OF THE INVENTION

[0003] The present invention belongs to the field of food products, in particular packaged directly acidified (or "citric") fresh mozzarella and provides means for improving the flavor of directly acidified citric fresh mozzarella, including lactic acid bacteria strains and their use therefor.

[0004] BACKGROUND OF THE INVENTION

[0005] Mozzarella is an unripened and smooth, elastic cheese with a long stranded parallel- orientated fibrous protein structure without evidence of curd granules. The cheese is rindless and may be formed into various shapes. Mozzarella is made by "pasta filata" (stretched curd) processing, which consists of heating curd of a suitable pH value kneading and stretching until the curd is smooth and free from lumps. It may be manufactured from buffalo or cow milk. Two types of mozzarella exist: low moisture, mostly used for dressing pizza, and high moisture, used as a table cheese (McMahon, Oberg, & McManus, 1993). Mozzarella with a high moisture content (moisture equal or higher than 52%, such as between 52% and 60%, or 62%, or 65%, or more), also referred to as "fresh mozzarella", is a soft cheese with overlying layers that may form pockets containing liquid of milky appearance. It may be packed with or without the liquid, also known as packing water. The cheese has a near white color. It is usually consumed fresh as table cheese.

[0006] Fresh mozzarella cheese can be manufactured by direct acidification with organic acids (e.g., citric, lactic, acetic, etc.), also known as "directly acidified" or "citric" fresh mozzarella, to demineralize the milk protein as opposed to addition of acidifying bacteria such as, e.g., lactic acid bacteria, also known as "fermented fresh mozzarella". Direct acidification by addition of acids such as citric acid to milk is very popular in the dairy industry because it is cheaper and allows better standardization than starter fermentation, see, e.g., M. Faccia, et al., "Short communication: Relationships between milk quality and acidification in the production of table Mozzarella without starters", Journal of Dairy Science, 2009, 92(9): 4211-4217. This process eliminates reliance on biological acid production and accelerated coagulation and curd-firming processes, see, e.g., R. Micketts, N.F. Olson, "Manufacture of mozzarella cheese by direct acidification with reduced amounts of rennet and pepsin, Journal of Dairy Science, 1974, 57(3): 273-279. This unfermented Mozzarella better maintains its textural integrity at relatively higher pH and has a longer shelf-life (e.g., 25 - 30 days, although in some cases it can go up to 35-40 days) but usually appears less flavorful and faint than fermented mozzarella. Fresh mozzarella is usually packed and stored with a covering or preserving liquid that is water or a diluted brine containing mono and / or divalent salts and calcium (e.g., NaCI and CaCl2), known as "packing water" or "preservative liquid". Mozzarella cheese is often sold immersed in this packing water and is exposed to it until reaching the consumer.

[0007] The flavor of fresh mozzarella, in particular citric fresh mozzarella, as perceived visually and orally by consumer is an important attribute of mozzarella cheese during the entire shelf-life.

[0008] Hence, there is a need for a technology to enable a desirable flavor profile over the shelf-life of the fresh mozzarella, in particular of citric fresh mozzarella.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have found that by adding at least one lactic acid bacterium (LAB) strain to the packaging water, sensory attributes of flavor are improved while still allowing for an easy and industry-friendly process.

[0011] Hence, the present invention relates to a packing water suitable for fresh mozzarella comprising at least one lactic acid bacterium (LAB) strain, preferably wherein the the packing water comprises water, NaCI, and calcium, and wherein the pH of the packing water is between 4.5 and 6.

[0012] The at least one LAB strain is preferably selected from a Lactobacillus strain, a Lactiplantibacillus strain, a Lacticaseibacillus strain or a Lactococcus strain, more preferably from a Lactobacillus rhamnosus strain, a Lactococcus lactis subsp. lactis biovar. diacetylactis strain, a Lacticaseibacillus paracasei strain, a Lactobacillus paracasei strain, a Lactobacillus para case / subsp. paracasei strain, a Lactobacillus acidophilus strain, a Lactobacillus helveticus strain, a Lactiplantibacillus plantarum strain or a Lactococcus lactis subsp. lactis strain.

[0013] The present invention also relates to lactic acid bacteria suitable for being comprised in the packing wated of the present invention, and to cultures or composition comprising the same. In particular, the present invention provides a LAB selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666 and DSM 23035. Strains with deposit number DSM 34775, DSM 34776, DSM 34777, DSM 34778, DSM 34779, DSM 34780 and DSM 34781 were deposited on October4, 2023 (2023-10-04). Strain with deposit number DSM 24616 was deposited on March 1, 2011 (2011-03-01). Strain with deposit number DSM 18875 was deposited on December 19, 2006 (2006-12-19). Strain with deposit number DSM 32389 was deposited on November 2, 2016 (2016-11-02). Strain with deposit number DSM 32666 was deposited on October 17, 2017 (2017-10-17). Strain with deposit number DSM 23035 was deposited on October 14, 2009 (2009-10-14).

[0014] Further, the invention also relates to a fresh mozzarella product comprising a packing water as defined in the present invention, and to the use of the packing water of the present invention to produce a directly acidified fresh mozzarella product.

[0015] The present invention also relates to compositions and starter cultures comprising the strains of the invention strains as well as to methods of using the packing water and strains of the invention for fresh mozzarella production and flavor improvement.

[0016] BRIEF DESCRIPTION OF FIGURES

[0017] Figure 1: Acid score of the fresh mozzarella samples.

[0018] Figure 2: Milky score of the fresh mozzarella samples.

[0019] Figure 3: Fresh score of the fresh mozzarella samples.

[0020] Figure 4: Diacetyl score of the fresh mozzarella samples. Figure 5: Bitter score of the fresh mozzarella samples.

[0021] Figure 6: Off flavor score of the fresh mozzarella samples.

[0022] Figure 7: Creamy score of the fresh mozzarella samples.

[0023] Figure 8: pH evolution of the packing water during the shelf-life.

[0024] Figure 9: pH evolution of each packed citric fresh mozzarella cheese over time.

[0025] Figure 10: Citric acid concentration in the fresh mozzarella cheeses over time.

[0026] Figure 11: Lactic acid concentration in the fresh mozzarella cheeses over time.

[0027] Figure 12: Relative abondance of diacetyl (S / N, signal to noise) in the fresh mozzarella cheeses over time.

[0028] Figure 13: Relative abondance of acetoin (S / N, signal to noise) in the fresh mozzarella cheeses over time.

[0029] Figure 14: Relative abondance of ethanol (S / N, signal to noise) in the fresh mozzarella cheeses over time.

[0030] Figure 15: Total free amino acids (mmol / kg) present in the fresh mozzarella cheeses overtime.

[0031] DETAILED DESCRIPTION

[0032] Definitions

[0033] All definitions of herein relevant terms are in accordance with what would be understood by the skilled person in relation to the herein relevant technical context.

[0034] In the context of the present application, the term "milk" is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals (e.g., cows, sheep, goats, buffaloes, camel, etc.) or produced using plant bases. Preferably, the milk in the context of the present invention is buffalo's milk (also referred to as "buffalo water"), cow's milk or goat's milk. The term "milk" may also encompass plant-source milk. 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.

[0035] The term "milk substrate" may be any raw and / or processed milk material that can be subjected to transformation according to the method for preparing fresh mozzarella described in the present invention. Thus, useful milk substrates include, but are not limited to, solutions / suspensions of any milk or milk like products comprising protein, such as whole or low-fat milk, skim milk, reconstituted milk powder, etc. Obviously, the milk substrate may originate from any mammal, e.g., being substantially pure mammalian milk, or reconstituted milk powder.

[0036] Prior to the cheese making process, the milk substrate may be homogenized and pasteurized according to methods known in the art. The milk is preferably pasteurized before treatment with the organic acids and rennet (coagulant).

[0037] "Homogenizing" as used in the context of the present invention in any of its embodiments, means intensive mixing to obtain a soluble suspension or emulsion.

[0038] "Pasteurizing" as used in the context of the present invention in any of its embodiments, means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

[0039] "Fermentation" in the context of the present invention in any of its embodiments means the conversion of carbohydrates into acids or alcohols or a mixture of both -through the action of microorganisms (e.g., lactic acid bacteria (LAB).

[0040] In the context of the present invention in any of its embodiments, the expression "lactic acid bacteria" or "lactic acid bacterium" ("LAB") designates 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 Gram positive, low-GC, acid tolerant, non-sporulating, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. In the present context the term "starter culture" is a culture or composition which comprises a mixture of bacterial strains (such as lactic acid bacteria strains).

[0041] "Flavor" is an important quality factor for directly acidified fresh mozzarella, and consumer acceptance is often closely linked to their flavor profiles. The flavor of directly acidified fresh Mozzarella cheese is dependent on various aspects of taste, including, but not limited to, e.g., the acidity (pH of the cheese and / or packing water), the freshness, the bitterness, the creaminess (assessed by expert panellists), and different levels of organic compounds present in the cheese or packing water, including, e.g., the levels of organic acids such as citric acid, free amino acids and / or volatile compounds such as diacetyl.

[0042] "Texture" is also an important quality factor for fermented milk products such as fresh mozzarella cheese, and consumer acceptance is often closely linked to texture properties. In the context of the present invention, the texture of the fresh mozzarella cheese can be inferred from the turbidity of the packing water. During the storage of the samples and due to the action of the pH of the packing water, the surface of the cheese can start to be described as slimy. The solubilisation of the cheese surface will lead to an unpleasant perception of the sample, as well as an increase in the turbidity of the packing water.

[0043] In the context of the present invention, "directly acidified fresh mozzarella" refers to high moisture mozzarella cheese ("fresh") (i.e., mozzarella cheese with a moisture content of at least 52%, such as a moisture content of 52% to 60%, or with a moisture content of 52% to 65%, or more) which has been prepared by "direct acidification", i.e., without involvement of acidifying starter cultures to generate the curd. This mozzarella is also referred to as "citric fresh mozzarella" or "unfermented mozzarella". Directly acidified fresh mozzarella cheese is made by direct acidification of the milk with an acid such as citric acid, lactic acid, acetic acid, etc. to lower the pH of the solution. The milk is subsequently coagulated using, e.g., rennet or purified chymosin. The curd is then stretched, and the fresh mozzarella cheese is produced.

[0044] "Packing water" in the context of the present invention refers to the liquid with which fresh mozzarella is generally packaged. As described above, fresh mozzarella is usually packed and stored with a covering or preserving liquid. This covering or preserving liquid is the "packing water" in the context of the present invention. It generally consists of water or a diluted brine (saline solution) comprising salts and calcium. For instance, the packing water may comprise mono and / or divalent salts such as NaCI, CaCb or calcium lactate (CeHwCaOe). The packing water may comprise other components and is critical for the shelf life of the product.

[0045] In the context of present invention, the term "directly acidified fresh mozzarella product" (or synonyms as "citric fresh mozzarella product" or "unfermented mozzarella product" refers to a product comprising both the directly acidified fresh mozzarella and the packing water of the present invention (e.g., a product comprising the directly acidified fresh mozzarella packed in the packing water of the present invention). Hence, the directly acidified fresh mozzarella can be stored in the packing water of the present invention, and the packing water of the present invention can be used to store a directly acidified fresh mozzarella.

[0046] In the context of the present invention, "shelf life" of a food product, e.g., fresh mozzarella cheese, refers to the period for which it can be stored without becoming unfit for use, consumption or sale. For instance, the shelf-life of fresh unfermented mozzarella may be about 28 to 40 days if kept in the unopened package at about 4°C.

[0047] As used herein, the term "about" (or "around") means the indicated value ± 1 % of its value, or the term "about" means the indicated value ± 2 % of its value, or the term "about" means the indicated value ± 5 % of its value, the term "about" means the indicated value ± 10 % of its value, or the term "about" means the indicated value ± 20 % of its value, or the term "about" means the indicated value ± 30 % of its value; preferably the term "about" means exactly the indicated value (± 0 %).

[0048] Throughout the description and claims the word "comprise" and variations of the word (e.g., "comprising", "having", "including", "containing") typically is not limiting and thus does not exclude other features, which may be for example technical features, additives, components, or steps. However, whenever the word "comprise" is used herein, this also includes a special embodiment in which this word is understood as limiting; in this particular embodiment the word "comprise" has the meaning of the term "consist of". The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0049] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0050] The packing water of the present invention

[0051] In a first aspect, the present invention provides a packing water suitable for being used with mozzarella cheese, in particular with fresh mozzarella produced by direct acidification, as described above. The packing water of the present invention comprises at least one lactic acid bacterium (LAB). The LAB belongs to a certain strain, so in the context of the present invention it is stated that the packing water of the present invention comprises at least one LAB strain.

[0052] In the context of the present invention the terms "packing water" and "brine" are used interchangeably herein. In the context of the present invention in any of its embodiments, the expression "packing water" means a solution comprising water and salts that is used for the preservation of the cheese and / or the improvement of flavor of the cheese as described in the embodiments of the invention.

[0053] As described above, fresh mozzarella is generally sold packaged in packing water. The inventors have found that, when at least one LAB strain is added to the packing water, the flavor of the fresh mozzarella is improved as compared with the flavor of fresh mozzarella with no LAB in the packing water, as shown in the examples. The packing water of the present invention comprises water. It preferably also comprises salts, such as sodium chloride (NaCI), and calcium, such as calcium chloride (CaCh) or calcium lactate (CeHwCaOe). In a preferred embodiment, the pH of the packing water is between about 4.5 and about 6, preferably between about 5 and about 6. It is preferred that the pH of the packing water is not significantly reduced over the shelf-life of the cheese. For instance, the pH of the packing water is not reduced more than the reduction experimented by the same packing water without comprising at least one LAB under the same conditions and for the same time (reference packing water). Hence, in a preferred embodiment, the pH of the packing water is kept in the range of between about 4.5 and about 6 over the shelf-life of the cheese, preferably in the range of between about 4.5 and 5.5, even more preferably at a pH of about 5.

[0054] In a preferred embodiment, the packing water of the present invention comprises calcium at a concentration of between about 0.5 and about l g / L, preferably at a concentration of between about 0.6 and about 0.8 g / L, even more preferably at a concentration of about 0.8 g / L.

[0055] The amount of NaCI in the packing water, if present, is preferably the same % as the NaCI% comprised in the fresh mozzarella. For instance, this can be between about 0.3% (w / w) and about 0.6% (w / w), such as between about 0.42% (w / w) and about 0.48% (w / w), preferably about 0.46% (w / w).

[0056] Hence, as described above, the packing water of the present invention comprises at least one LAB strain. Of course, this at least one LAB strain is a strain which is not involved in the manufacturing of the mozzarella, i.e., it is not a strain which was used for the manufacturing of the curd in the process of generating the cheese.

[0057] "High-moisture mozzarella" (HMM) (also referred to as "fresh mozzarella" in the present invention) is a fresh pasta filata cheese that originated in Italy in the 17th century. It has a moisture content equal to or higher than 52%, such as between 52% and 60%, or 62%, or 65%, or more. The production technology is based on acidification of the curd that determines casein demineralization and makes it stretchable in hot water. Curd acidification can be obtained by starter fermentation (traditional method) or by direct acidification of milk (industrial method) with organic acids (e.g., citric, lactic, acetic, etc.). Figure 1 of Natrella, M. et al. ("Short communication: Sensory characteristics and volatile organic compound profile of high-moisture mozzarella made by traditional and direct acidification technology", Journal of Dairy Science, 2020, 103(3):2089-2097) shows an example of a direct acidification mozzarella manufacturing protocol commonly used in Southern Italy. The starting milk can be raw milk, as indicated in Figure 1 of Natrella, M. et al. or can be pasteurized. The skilled person is aware of methods for manufacturing fresh mozzarella cheese by direct acidification from raw or pasteurized milk, preferably from pasteurized milk, e.g., as described in Figure 1 of Natrella, M. et al. The fresh mozzarella cheese of the present invention is not produced by fermentation. It is produced by direct acidification with organic acids, e.g., as described above. In a preferred embodiment, the acid used in the direct acidification method to manufacture fresh mozzarella cheese is citric acid. As the skilled person would recognize, any suitable coagulant (rennet) may be used in the production of directly acidified fresh mozzarella. For instance, the rennet (coagulant) used in the direct acidification method to manufacture fresh mozzarella cheese may be chymosin (EC 3.4.23.4), for instance a chymosin recombinantly produced, e.g., by fermentation on a vegetable substrate with Aspergillus niger var. awamori. Chymosin is an aspartic protease which hydrolyzes the linkage between Phel05 and Metl06 in the K-casein molecule, resulting in the destabilization of casein micelles and causing flocculation. The final product of this flocculation process is the generation of milk curds. For instance, the chymosin used in the direct acidification method of the present invention may be Chymax or Chymax M from Chr. Hansen A / S (Hprsholm, Denmark). In other preferred embodiment, the rennet (coagulant) used in the direct acidification method to manufacture fresh mozzarella cheese is a microbial coagulant such as mucorpepsin, which can be produced by submerged fermentation on a vegetable substrate with a select strain of the fungus Rhizomucor miehei. For instance, the rennet (coagulant) used it the direct acidification method to manufacture fresh mozzarella cheese is MICROLANT® Supreme, from Chr. Hansen (a mucorpepsin). Hence, the fresh mozzarella cheese according to the present invention is an "unfermented mozzarella" because it has not been produced using acidifying bacteria such as, e.g., lactic acid bacteria.

[0058] In a preferred embodiment, the amount of lactic acid in the fresh mozzarella cheese packaged with the packing water of the present invention is not increased over the shelf life of the fresh mozzarella. Lactose is converted to lactic acid through fermentation and ripening of cheese using starter cultures. Therefore, in general, fresh mozzarella cheeses in which manufacturing process acidifying starter cultures to generate the curd are involved show very high amounts of lactic acid. However, in directly acidified fresh mozzarella, since there is no involvement of acidifying starter cultures to generate the curd, the lactic acid levels in the cheese remain much lower, (e.g., 2 mg / g or lower). Therefore, in the context of the present invention, the amount of lactic acid in the fresh mozzarella cheese packaged with the packing water of the present invention remains low over the shelf life of the fresh mozzarella, e.g., lower than 15 mg / g, such as lower than 10 mg / g, or lower than 8 mg / g, or lower than 7 mg / g, or lower than 5 mg / g, or 2 mg / g or lower than. The amount of lactic acid in the cheese can be quantified using HPLC by known methods.

[0059] As described above, the packing water of the present invention comprises at least one LAB. Hence, in another embodiment, the present invention provides at least one lactic acid bacteria that is suitable of being comprised in the packing water of the present invention.

[0060] The at least one LAB strain of the present invention may be an isolated strain, e.g., isolated from a naturally occurring source, or may be a non-naturally occurring strain, e.g., obtained recombinantly. Recombinant strains will differ from naturally occurring strains by at least the presence of the nucleic acid construct(s) used to transform or transfect the mother strain. The strains of the present invention may also be strains derived from those covered by the invention.

[0061] In the present context, the terms "strains derived from", "derived strain" or "mutant" should be understood as a strain derived from a strain of the invention by means of, e.g., genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. It is preferred that the derived strain is a functionally equivalent mutant, e.g., a strain that has substantially the same, or improved, properties with respect to water holding capacity as the mother strain. Such a derived strain is a part of the present invention. Especially, the term "derived strain" or "mutant" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenizing treatment including treatment with a chemical mutagen, such as ethane methane sulphonate (EMS) or / V-methyl- / V'-nitro- / \ / -nitroguanidine (NTG), UV light or to a spontaneously occurring mutant.

[0062] A mutant may have been subjected to several mutagenizing treatments (a single treatment should be understood as one mutagenizing step followed by a screening / selection step), but it is presently preferred that no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred derived strain, less than 1%, or less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion, or a combination thereof) compared to the mother strain.

[0063] As stated above, the packing water of the present invention comprises at least one LAB strain. But the packing water of the present invention may comprise more than one LAB strain, such as 2, 3, 4 or 5 LAB strains, or more. For instance, the packing water of the present invention may comprise two LAB strains. For instance, the packing water of the present invention may comprise three LAB strains. For instance, the packing water of the present invention may comprise four LAB strains. For instance, the packing water of the present invention may comprise five LAB strains.

[0064] Preferably, the at least one LAB comprised in the packing water of the present invention is a citrate-positive strain. In the context of the present invention, a "citrate-positive strain" refers to a LAB strain that is able to ferment citrate. The skilled person is aware of means for ascertaining whether a certain LAB strain is a citrate-positive strain. For instance, the LAB strains can be seeded on Simmons Citrate agar, dispensed into inclined tubes, and then incubated at 30°C for 48 hours. The change of the medium colour to blue indicates a positive reaction. In the opposite, the medium remains greenish and the test is denoted negative, see, e.g., Rhaiem N. et al., "Identification and characterization of lactic acid bacteria isolated from cow milk and olives brine", J. Mater. Environ. Sei., 20167, 5: 1504-1509. Another way of ascertaining whether a LAB strain is citrate positive or not is described in Aditi FY et al., " study on the microbiological status of mineral drinking water", Open Microbiol J., 2017, 11:31- 44. Briefly, Simmons citrate agar slants of 2 ml in each vial are prepared by autoclaving at 15 psi 121°C. Using sterile technique, small amount of the bacteria is inoculated into the vials by means of a streak inoculation method with an inoculating needle and the vials are incubated for 48 hours at 37°C. Following incubation, citrate positive cultures are recognized by the presence of growth on the surface of the slant of Simmons citrate agar and deep Prussian blue coloration of the medium. Another way of ascertaining whether a LAB strain is citrate positive or not involves the use of a plate count media. Modified leesment agar with calcium citrate added (based on ISO 17792 | IDF 180:2006) is used to enumerate and to detect and quantify mesophilic citrate-positive bacteria. The media is incubated aerobically at 25°C ± 1°C for 5 days. If cells are citrate positive, they will generate a clean area around the colonies. The mesophilic citrate-positive bacteria will - due to the fermentation of the citrate - make clear zones.

[0065] In another preferred embodiment, the at least one LAB strain comprised in the packing water of the present invention is capable of producing diacetyl. Diacetyl is an aroma compound which can be produced as a product resulting from citric acid fermentation. In LAB, citrate metabolism, like glycolysis, leads to the formation of pyruvate. The synthesis of pyruvate from citrate requires three steps: citrate transport by a permease, its conversion into oxaloacetate by citrate lyase (CL), and further conversion of oxaloacetate into pyruvate and CO2 by the oxaloacetate decarboxylase (OAD). Metabolism of pyruvate can yield in LAB different endproducts such as lactate, formate, acetate, ethanol and the aroma compounds of four carbons (C4 compounds) diacetyl, acetoin and butanediol, see, e.g., Garcia Quintans N. et al., "Citrate metabolism and aroma compound production in lactic acid bacteria", Molecular Aspects of Lactic Acid Bacteria for Traditional and New Applications, 2008: ISBN: 978-81-308-0250-3 Editors: Baltasar Mayo, Paloma Lopez and Gaspar Perez-Martinez. The skilled person is aware of means for ascertaining whether a certain LAB strain is capable of producing diacetyl. For instance, as described in Natrella, M. et al. (cited above), diacetyl (such as other volatile compounds) can be detected and quantified by headspace solid-phase microextraction coupled to GC-MS or by gas chromatography with mass spectrometric detection. In one preferred embodiment, the at least one LAB comprised in the packing water of the present invention belongs to Lactobacillus, Lactiplantibacillus, Lacticaseibacillus or Lactococcus. Hence, in a preferred embodiment, the at least one LAB strain comprised in the packing water of the present invention is selected from a Lactobacillus strain, a Lactiplantibacillus strain, a Lacticaseibacillus strain or a Lactococcus strain. Preferably, the LAB is selected from Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis biovar. diacetylactis, Lacticaseibacillus paracasei, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum or Lactococcus lactis subsp. lactis. Hence, in a preferred embodiment the at least one LAB strain comprised in the packing water of the present invention is selected from a Lactobacillus rhamnosus strain, a Lactococcus lactis subsp. lactis biovar. diacetylactis strain, a Lacticaseibacillus paracasei strain, a Lactobacillus paracasei strain, a Lactobacillus paracasei subsp. paracasei strain, a Lactobacillus acidophilus strain, a Lactobacillus helveticus strain, a Lactiplantibacillus plantarum strain or a Lactococcus lactis subsp. lactis strain. In a more preferred embodiment, the at least one LAB strain is a Lactobacillus rhamnosus strain. Hence, in a more preferred embodiment, the LAB comprised in the packing water of the present invention belongs to the species Lactobacillus rhamnosus.

[0066] In a preferred embodiment, the packing water of the present invention comprises lactic acid bacteria belonging at least to: a. Lactococcus lactis subsp. lactis biovar. diacetylactis; b. Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis; c. Lacticaseibacillus paracasei, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei and Lactococcus lactis subsp. lactis; or d. Lactobacillus rhamnosus.

[0067] More preferably, the packing water of the present invention comprises at least one LAB strain selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, underthe accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666 and DSM 23035.

[0068] In another embedment of the present invention, the packing water comprises LAB from the following strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the following accession numbers: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035,

[0069] In a more preferred embodiment, the packing water comprises LAB from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 32666 and / or under the accession number DSM 23035. In an even more preferred embodiment, the the packing water comprises LAB from the strain deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 23035.

[0070] Preferably, the LAB comprised in the packing water of the present invention are present in a concentration of more than about 5xl08CFU / L, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably in a concentration of about 5 xlO10CFU / L.

[0071] For instance, the packing water of the present invention may comprise the following LAB at the following concentrations: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779 at a total concentration of LAB (CFU) in the packing water of more than about 5xl08CFU / L of packing water, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably about 5 xlO10CFU / L; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777 at a total concentration of LAB (CFU) in the packing water of more than about 5xl08CFU / L of packing water, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably about 5 xlO10CFU / L; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874, at a total concentration of LAB (CFU) in the packing water of more than about 5xl08CFU / L of packing water, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably about 5 xlO10CFU / L; d. DSM 32666 at a total concentration of LAB (CFU) in the packing water of more than about 5xl08CFU / L of packing water, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably about 5 xlO10CFU / L; and / or e. DSM 23035 at a total concentration of LAB (CFU) in the packing water of more than about 5xl08CFU / L of packing water, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably about 5 xlO10CFU / L.

[0072] The lactic acid bacteria and cultures of the present invention

[0073] In another embodiment, the present invention provides a lactic acid bacterium suitable for being comprised in the packing wated of the present invention, and cultures or compositions comprising the same. In particular, the present invention provides a LAB selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and / or DSM 23035, preferably from DSM 32666 and DSM 23035. Strains with deposit number DSM 34775, DSM 34776, DSM 34777, DSM 34778, DSM 34779, DSM 34780 and DSM 34781 were deposited on October 4, 2023 (2023-10-04). Strain with deposit number DSM 24616 was deposited on March 1, 2011 (2011-03-01). Strain with deposit number DSM 18875 was deposited on December 19, 2006 (2006-12-19). Strain with deposit number DSM 32389 was deposited on November 2, 2016 (2016-11-02). Strain with deposit number DSM 32666 was deposited on October 17, 2017 (2017-10-17). Strain with deposit number DSM 23035 was deposited on October 14, 2009 (2009-10-14).

[0074] In a particular embodiment, the present invention provides a lactic acid bacterium with deposit number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 34777 and / or DSM 34775.

[0075] The present invention provides a culture (also referred to as "composition" in the present invention) comprising or consisting of at least one, such as one, or at least two, or at least three, or at least four, or at least five LAB selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666 and DSM 23035.

[0076] Hence, the culture (composition) of the present invention comprises or consists of LAB belonging at least to: a. Lactococcus lactis subsp. lactis biovar. diacetylactis; b. Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis; c. Lacticaseibacillus paracasei, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei and Lactococcus lactis subsp. lactis; or d. Lactobacillus rhamnosus.

[0077] In a preferred embodiment, the culture comprises or consists of LAB from the following strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH underthe following accession numbers: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035.

[0078] Preferably, the culture comprises or consists of LAB from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 32666 and / or under the accession number DSM 23035, even more preferably under the accession number DSM 23035.

[0079] LAB, including bacteria of the present invention, are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set cultures", intended for inoculation in the dairy products, such as in the packing water of fresh mozzarella produced by direct acidification, as described in the present invention. Accordingly, the composition (or culture) of the present invention may be frozen or freeze- dried. In addition, the composition (or culture) of the present invention may be provided in liquid form. Thus, in one embodiment, the composition (or culture) is in frozen, dried, freeze- dried or liquid form.

[0080] The compositions or cultures of the present invention may also additionally comprise cryoprotectants, antioxidants, nutrients, fillers or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, land / or antioxidants and most preferably cryoprotectants. Use of protectants such as cryoprotectants are known to a skilled person in the art. Suitable cryoprotectants include mono-, di-, tri- and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tri polyphosphate). In one embodiment, the compositions or cultures according to the present invention may comprise one or more cryoprotective agent(s) selected from the group consisting of inosine- 5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'- monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0081] In one embodiment the compositions or starter cultures of the invention contains or comprises from 0.2 % to 20 % of the cryoprotective agent or mixture of agents measured as % w / w of the material. It is, however, preferable to add the cryoprotective agent or mixture of agents at an amount which is in the range from 0.2 % to 15 %, from 0.2 % to 10 %, from 0.5 % to 7 %, and from 1 % to 6 % by weight, including within the range from 2 % to 5 % of the cryoprotective agent or mixture of agents measured as % w / w of the frozen material by weight. In a preferred embodiment the culture comprises approximately 3 % of the cryoprotective agent or mixture of agents measured as % w / w of the material by weight. The amount of approximately 3 % of the cryoprotective agent corresponds to concentrations in the 100 mM range. It should be recognized that for each aspect of embodiment of the invention the ranges may be increments of the described ranges.

[0082] The fresh mozzarella product of the present invention

[0083] The present invention also relates to a fresh mozzarella product comprising a packing water as defined in the present invention. As described above in this description, the fresh mozzarella of the present invention is a fresh or high moisture mozzarella, i.e., it has a moisture content equal to or higher than about 52%, such as between about 52% and about 65%, such as between about 55% and about 65%, or about 62%, or about 64%, or about 65%, or more. For instance, the fresh mozzarella of the present invention has a moisture content of about 64,5%. In addition, the mozzarella of the present invention is an unfermented mozzarella, i.e., it has not been produced using acidifying bacteria such as, e.g., lactic acid bacteria. The mozzarella of the present invention has been produced by direct acidification with organic acids, preferably with citric acid, e.g., as described above. As the skilled person would recognize, any suitable organic acid may be used in the production of directly acidified fresh mozzarella. In a preferred embodiment, the acid used in the direct acidification method to manufacture fresh mozzarella cheese is citric acid. As the skilled person would recognize, any suitable coagulant (rennet) may be used in the production of directly acidified fresh mozzarella. For instance, the rennet (coagulant) used in the direct acidification method to manufacture fresh mozzarella cheese may be chymosin (EC 3.4.23.4), for instance a chymosin recombinantly produced, e.g., by fermentation on a vegetable substrate with Aspergillus niger var. awamori (e.g., Chymax or Chymax M from Chr. Hansen A / S (Hprsholm, Denmark)). In other preferred embodiment, the rennet (coagulant) used in the direct acidification method to manufacture fresh mozzarella cheese is a microbial coagulant such as mucorpepsin, which can be produced by submerged fermentation on a vegetable substrate with a select strain of the fungus Rhizomucor miehei. For instance, the rennet (coagulant) used it the direct acidification method to manufacture fresh mozzarella cheese is MICROLANT® Supreme, from Chr. Hansen (a mucorpepsin). In one preferred embodiment, the mozzarella of the present invention has been produced by direct acidification of pasteurized milk with citric acid, and the rennet (coagulant) used is a microbial coagulant such as mucorpepsin, preferably MICROLANT® Supreme, from Chr. Hansen.

[0084] Once the mozzarella has been produced, it is packaged with the packing water of the present invention, to conform the fresh mozzarella product of the present invention, and can be stored, preferably at about 4°C.

[0085] Methods and uses of the present invention

[0086] The present invention further relates to uses and methods for producing fresh mozzarella as described herein. Hence, in one embodiment, the present invention relates to the use of the packing water of the present invention to produce a fresh mozzarella (the fresh mozzarella, as described above, has been produced by direct acidification and can be referred to as "directly acidified fresh mozzarella" or "citric fresh mozzarella", as described above).

[0087] In addition, the present invention provides the use of at least one lactic acid bacterium (LAB) (or LAB strain, also used in the present description) as defined in the present invention for producing a packing water suitable for directly acidified fresh mozzarella. As shown in the examples, the use of at least one LAB as defined herein in the packing water of fresh mozzarella leads to an improved flavor of the cheese, as compared with fresh mozzarella packed in a packing water without the at least one LAB. In particular, the acid score, milky score, fresh score, the diacetyl score, bitter score and the off-flavor score of fresh mozzarella packed with the packing water of the present invention are better than the acid score, milky score, fresh score, the diacetyl score, the bitter score and / or the off-flavor score of fresh mozzarella packed with a packing water not comprising the at least one LAB. Hence, the present invention relates to the use of at least one LAB for improving the flavor of directly acidified fresh mozzarella, preferably wherein the LAB is a LAB as described in the present invention.

[0088] The present invention further provides a method for producing directly acidified fresh mozzarella, wherein the method comprises a step in which the packing water of the present invention is used.

[0089] For instance, the method of the present invention comprises the following steps: a. Providing a fresh mozzarella (i.e., a high moisture content mozzarella, as described herein), wherein the fresh mozzarella has been produced by direct acidification, preferably comprising the use of citric acid; b. Packing the fresh mozzarella in a packing water comprising at least one LAB, as defined herein. c. Screening for directly acidified fresh mozzarella with improved flavor, as compared with directly acidified fresh mozzarella comprising the same packing water without the at least one LAB. The flavor improvement can be evaluated by an expert panel, during different storage time of the samples, see the Examples section for further details. Briefly, the expert panellists (panellists expert in testing this kind of products) test each sample of the fresh mozzarella cheese (packaged in packing water comprising LAB) and quantifiy each sensory attribute as compared with a reference fresh mozzarella (packaged in packing water without LAB).

[0090] Step c. (screening step) can be performed up to 40 days after the fresh mozzarella has been packaged in the packing water of the present invention (step b.).

[0091] The evaluation of the flavor of the packaged fresh mozzarella of the present invention can be performed as described in the examples. For instance, the evaluation of the flavor can be performed 4, 10, 20 31 or 40 days of storage time (e.g., 4, 10, 20, 31 or 40 days after the fresh mozzarella has been packaged with the packing water of the present invention), kept at a temperature of about 4°C. The evaluation of the flavor can be performed by analysing the following sensory attributes: "Fresh score", the "Diacetyl score", the "Bitter score" the "Off flavors" score, the "Acid score" and / or the "Milky score" of the packaged mozzarella, preferably all of the scores, using the methodology as described above and in the examples.

[0092] For instance, the expert panellists (experts in testing this kind of product) may test each sample of the fresh mozzarella cheese in order to evaluate the flavor, by analysing the following sensory attributes: "Fresh score", the "Diacetyl score", the "Creamy score", the "Bitter score" the "Off flavors" score, the "Acid score" and / or the "Milky score". Each session can start by tasting the reference sample (fresh mozzarella packed in packing water without addition of LAB cultures) and align the levels of the different descriptors (sensory attributes) for the reference sample. The panellists may subsequently test the different samples (fresh mozzarella packaged in packing water comprising each of the LAB cultures as defined herein), and each expert (panellist) can evaluate each descriptor (sensory attribute) in comparison to the same sensory attribute in the reference cheese.

[0093] It is preferred that the texture of the fresh mozzarella (as measured, e.g., by studying the turbidity of the packing water) is not worsened during the storage time. For instance, the increase in turbidity of a certain packing water as compared with the same packing water lacking the at least one LAB may be an indication that the texture of the fresh mozzarella packaged with that packing water is getting worse. In addition, the expert panellists may note changes in the surface of the products. This changes in the surface of the products, if any, can then be correlated with the colour of the packing water. A transparent packing water may be indicative of a good structure of the cheese, as it may indicate that the surface of it is not losing its integrity. A less transparent and / or whitish packing water may be indicative of a loss of texture (e.g., a slimier surface of the mozzarella cheese).

[0094] Any combination of the above-described elements, aspects and embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Embodiments of the present invention are described below, by way of examples only.

[0095] CULTURES

[0096] The present invention further provides the following cultures or compositions:

[0097] CULTURE 1: Comprises four different strains of Lactococcus lactis subsp. lactis biovar. Diacetylactis (Accession Nos. DSM 34778, DSM 34779, DSM 34780, and DSM 34781).

[0098] CULTURE 2: Comprises four different strains of Lactobacillus acidophilus (Accession No. DSM 34776), Lactobacillus helveticus (Accession No. DSM 19499), Lactiplantibacillus plantarum (Accession No. DSM 34777), and Lactococcus lactis subsp. Lactis (Accession No. DSM 18874).

[0099] CULTURE 3: Comprises five different strains of Lacticaseibacillus paracasei (Accession No. DSM 34775), Lactobacillus rhamnosus (Accession No. DSM 24616), Lactobacillus paracasei (Accession No. DSM 32389), Lactobacillus paracasei subsp. Paracasei (Accession No. DSM 18875), and Lactococcus lactis subsp. Lactis (Accession No. DSM 18874).

[0100] CULTURE 4: Comprises Lactobacillus rhamnosus (Accession No. DSM 32666).

[0101] CULTURE 5: Comprises Lactobacillus rhamnosus (Accession No. DSM 23035). EXAMPLES

[0102] Evaluation of the flavor profile of Mozzarella using packing water inoculated with LAB strains.

[0103] Materials and methods

[0104] In order to select the flavoring non-gas producing LAB that will be injected in the governing liquid (packing water) for driving the flavor differentiation in the fresh mozzarella cheese, 8oz fresh mozzarella samples produced by direct acidification ("citric" fresh mozzarella), as described in the art (high moisture mozzarella, with a moisture content of 64,5%) are repackaged in a brine (packing water) inoculated with LAB cultures (see below) and incubated at 4°C (previously, a compositional analysis is performed on the cheese for preparing the packing water according to the average cheese composition and pH). The amount of LAB inoculated in the packing water ranged from about 5xl08CFU / L of packing water to about 5 xlO11CFU / L. For instance, packing water comprising Cultures 4 and 5 had an amount of LAB of about 5 xlO10CFU / L.

[0105] The following packing waters (also referred to as "brine" in these examples) were evaluated. In all cases the packing water comprises sterilized water, CaCl2 at a concentration of about 0.8 g of Ca / L and NaCI at a concentration of 0.46% (w / w) (same % (w / w) as the amount of NaCI of the cheese), at a pH of about 6 (adjusted with lactic acid 85%). The packing water is divided in equal volumes, each one being inoculated with the corresponding culture (with the exception of the reference packing water, which does not comprise any LAB).

[0106] Packing water 1: Comprising Culture 1 (strains DSM 34778, DSM 34779, DSM 34780, and DSM 34781, 40U / 100L of packing water, e.g., about 5xl08CFU / L of packing water to about 5 xlO11CFU / L of packing water);

[0107] Packing water 2: Comprising Culture 2 (strains DSM 34776, DSM 19499, DSM 34777 and DSM 18874, 50U / 100L of packing water, e.g., about 5xl08CFU / L of packing water to about 5 xlO11CFU / L of packing water);

[0108] Packing water 3: Comprising Culture 3 (strains DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874, 50U / 100L of packing water, e.g., about 5xl08CFU / L of packing water to about 5 xlO11CFU / L of packing water); Packing water 4: Comprising Culture 4 (strain DSM 32666, 5OU / 1OOL of packing water, e.g., about 5 xlO10CFU / L of packing water);

[0109] Packing water 5: Comprising Culture 5 (strain DSM 23035, 50U / 100L of packing water, e.g., about 5 xlO10CFU / L of packing water);

[0110] Packing water 6: Control; packing water comprising a blend of defined yeast strains, 1 pouch (l,2xlO10CFU) in 250 L of packing water) Packing water 7: Reference (packing water without LAB).

[0111] The ratio of cheese:packing water was 1:1. Once the citric fresh mozzarellas were packed with the individual packing waters, the containers (packaged mozzarellas) were stored at 4°C during the entire time. To evaluate the flavor evolution during time, samples were collected after 4, 10, 20, 31 and 40 days. Hence, the shelf life or storage time (at 4°C) of each packaged fresh mozzarella was 40 days, and samples were collected after approximately 4, 10, 20, 30 and 40 days of storage at 4°C. In some cases, samples were collected at day 11 instead of day 10, or at day 21 instead of day 20, or at day 32 instead of day 30.

[0112] At each sampling point, the following analysis were performed:

[0113] • pH of the packing water

[0114] • Visual evaluation of the packing water

[0115] • pH of the citric fresh mozzarella samples

[0116] • Tasting evaluation, with expert panelist

[0117] • Organic acids (citric and lactic acid)

[0118] • Free amino acids

[0119] • Volatile compounds (diacetyl, acetoin and ethanol).

[0120] Tasting sessions were organized for day (approximately) 10, day 20, day 30, and day 40 of incubation to compare flavor development between samples. One reference sample (no culture in the packing water), one control sample (no LAB in the packing water, but yeast) and five trial samples (comprising cultures 1-5, as described above).

[0121] The main steps used to conduct the trials were: 1) Measure the composition (Foodscan), as well as salt (Titration) and pH of 3 samples of citric fresh mozzarella balls;

[0122] 2) Prepare a brine containing the same % of NaCI of the cheese balls (calculate the average of the 3 samples), 0,46% in this case;

[0123] 3) Add 80 mg of Ca / lOOg of packing water and adjust pH to pH of the sample with lactic acid 85% (use sterilized water), in this case, pH 6,0.

[0124] 4) Split the packing water in 7, keep 1 volume for the control and add the cultures to each packing water, as described above.

[0125] Results

[0126] Tasting evaluation, with expert panelist

[0127] The cheese sensory characteristics (flavor) were evaluated by a panel composed of 4 trained panellists (experts on this type or product). The expert panellists tested each sample of the fresh mozzarella cheese in order to evaluate the flavor, by analysing the following sensory attributes: "Fresh score", the "Diacetyl score", the "Creamy score", the "Bitter score" the "Off flavor" score, the "Acid score" and / or the "Milky score". Each session started by tasting the reference sample (fresh mozzarella packed in packing water without addition of LAB cultures) and aligning the levels of the different descriptors (sensory attributes) for the reference sample. The panellists tested subsequently the different samples (fresh mozzarella packaged in packing water comprising each of the LAB cultures as defined herein), and each expert (panellist) evaluated each descriptor (sensory attribute) in comparison to the same sensory attribute in the reference cheese.

[0128] The following sensory attributes were evaluated on each cheese by each panelist: fresh score, diacetyl score, bitter score, milky score, acid score, creamy score and off-flavor score. Figures 1 to 7 show the results of the panellists' evaluation of the different fresh mozzarella samples. As can be seen from these figures, the presence of the LAB in the packing water resulted in better sensory attributes than cheese packaged with packing water without the LAB (reference). In particular, all cultures resulted in a higher acid score than the reference. Regarding milky score, cultures 1, 2, 4 and 5 perform similarly as the reference, in particular cultures 2 and 5 achieve a very similar milky score than the reference after 40 days storage. Culture 5 had a superior fresh score as compared with the reference for the whole shelf life, and cultures 1, 2 and 4 performed also better than the reference after 40 days storage. The diacetyl score was also higher than the reference for all evaluated cultures, at least after 4, 10, and 40 days of storage. Of note, cultures 1, 4 and 5 showed a much higher diacetyl score as compared with the reference during the whole storage time. All cultures also showed a lower bitter score as compared with the reference at 40 storage days. Of note, cultures 4 and 5 showed a very low bitter score, lower than the reference at almost all time points. With the exception of culture 3, all other cultures showed similar or better off-flavor score than the reference. Finally, the creamy score of cultures 1, 4 and 5 was higher than the reference in the first days of storage. The creamy score of culture 5 was higher than the reference at all timepoints.

[0129] These results thus show that the presence of at least one LAB in the packing water, in particularthe ones comprised in cultures 1 to 5 as described herein, leads to improved sensory attributes (improved flavor) in the packed fresh mozzarella, as compared with fresh mozzarella packed with packing water without LAB. Particularly good performing cultures are cultures 1, 2, 4 and 5, in particular cultures 4 and 5. pH of the packing water

[0130] The pH of the packing water of each packaged citric fresh mozzarella was measured at the different storage times. Figure 8 shows the pH evolution of the packing water of each packaged citric fresh mozzarella during the shelf-life of the product. As shown in this figure, the pH of the packing water comprising cultures 4 and 5 was not significantly decreased over the entire storage time (up to 40 days) of the packaged citric direct mozzarella. pH of the citric fresh mozzarella samples The pH of each packaged citric fresh mozzarella was measured at the different storage times. Figure 9 shows the pH evolution of each packaged citric fresh mozzarella during the shelf-life of the product. As shown in this figure, the pH of the fresh mozzarella packed with packing water comprising cultures 4 and 5 was not significantly decreased over the entire storage time (up to 40 days). Absence or low acidification of the fresh mozzarella is good for keeping the texture of the product. These results correlate with the observation by the expert panellists that the packing water was kept transparent during the storage time (up to 40 days, data not shown), similar to the reference packing water (without LAB).

[0131] Citric acid consumption

[0132] The consumption of citric acid by the LAB comprised in the different packing waters was also evaluated. For this, the concentration of citric acid (mg of citric acid per g of cheese) in each cheese sample at different time points (0, 4, 11, 21, 32 and 40 days of storage at 4°C) was measured. As shown in Figure 10, no citrate was detected in any cheese samples at 32 or 40 days (with the exception of the packing water comprising yeast). A faster citrate consumption was shown in packing water comprising culture 1 (absence already after 21 days). These results correlate with a faster production of diacetyl as compared with the reference packing water (without LAB), see Figure 12.

[0133] Lactic acid production

[0134] The production of lactic acid by the LAB comprised in the different packing waters was also evaluated. For this, the concentration of lactic acid (mg of lactic acid per g of cheese sample) in each cheese sample at different time points (0, 4, 11, 21, 32 and 40 days of storage at 4°C) was measured. As shown in Figure 11, the concentration of lactic acid overtime was relatively low in all samples at lower storage times.

[0135] Production of volatile compounds (diacetyl, acetoin, ethanol)

[0136] The production of volatile compounds (diacetyl, acetoin, ethanol) by the LAB comprised in the different packing waters was also evaluated. For this, the relative abondance of diacetyl, acetoin and ethanol in each cheese sample at different time points (0, 4, 11, 21, 32 and 40 days of storage at 4°C) was measured by gas chromatography with mass spectrometric detection. The results are shown in figures 12-14, respectively. As shown in Figure 12, Culture 5 was the highest diacetyl-producing culture, followed by Culture 1 and cultures 3 and 4. All cultures produced higher relative abondance of diacetyl than the reference and the control culture (yeast). Culture 1 was the highest acetoin-producing culture, as shown in Figure 13. Finally, Culture 1 (which comprises different strains of Lactococcus lactis subsp. lactis biovar. Diacetylactis, as well as the yeast-comprising culture, are producing ethanol, see Figure 14.

[0137] Concentration of free amino acids

[0138] The concentration of free amino acids in the different cheeses (mmol / kg) was also measured at different storage times. The presence of free amino acids in the packaged fresh mozzarella cheeses was evaluated by methods known in the art (e.g., liquid chromatography with mass spectrometric detection). The results are shown on Figure 15. As shown in this figure, the total amount of free amino acids in the cheeses is very low, in agreement with their young age as well as with their storage conditions (4°C).

[0139] Hence, the inventors conclude that by using the technology of present invention, it is possible to increase the freshness and improve the flavor of non-fermented fresh mozzarella by inoculating the packaging water with at least one LAB, preferably with the cultures described in the present application.

[0140] DEPOSITS

[0141] The strain DSM 34775 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34775 on 2023-10-04.

[0142] The strain DSM 34776 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34776 on 2023-10-04.

[0143] The strain DSM 34777 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34777 on 2023-10-04. The strain DSM 34778 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34778 on 2023-10-04.

[0144] The strain DSM 34779 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34779 on 2023-10-04.

[0145] The strain DSM 34780 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34780 on 2023-10-04.

[0146] The strain DSM 34781 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 34781 on 2023-10-04.

[0147] The strain DSM 24616 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 24616 on 2011-03-01.

[0148] The strain DSM 18875 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 18875 on 2006-12-19.

[0149] The strain DSM 32389 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 32389 on 2016-11-02.

[0150] The strain DSM 32666 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 32666 on 2017-10-17. The strain DSM 23035 has been deposited by Chr. Hansen A / S at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbFI, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM 23035 on 2009-10-14.

[0151] The deposits have been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0152] The Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.

[0153] ITEMS OF THE PRESENT INVENTION

[0154] 1. A packing water suitable for directly acidified fresh mozzarella comprising at least one lactic acid bacterium (LAB).

[0155] 2. The packing water according to claim 1, wherein the packing water comprises water, NaCI, and calcium, and wherein the pH of the packing water is between 4.5 and 6, preferably wherein the pH of the packing water is not significantly reduced over the shelf-life of the directly acidified fresh mozzarella.

[0156] 3. The packing water according to any one of claims 1 or 2, wherein the pH of the packing water is between 5 and 6.

[0157] 4. The packing water according to any one of claims 1 to 3, wherein the packing water comprises calcium at a concentration of between 0.5 and 1 g / L, preferably at a concentration of between 0.6 and 0.8 g / L, even more preferably at a concentration of about 0.8 g / L, and optionally wherein the concentration of NaCI in the packing water is essentially the same as the NaCI concentration in the fresh mozzarella cheese to be packaged with the packing water, such as between about 0,30% (w / w) and about 0,60% (w / w), such as between about 0,42% (w / w) and about 0,48% (w / w), preferably about 0,46% (w / w). 6. The packing water according to any one of claims 1 to 5, wherein the amount of lactic acid in the packing water is not significantly increased over the shelf life of the directly acidified fresh mozzarella.

[0158] 7. The packing water according to any one of claims 1 to 6, wherein the at least one LAB strain comprised in the packing water:

[0159] (i) is a citrate-positive strain; and / or

[0160] (ii) is capable of producing diacetyl.

[0161] 8. The packing water according to any one of claims 1 to 7, wherein the at least one LAB strain is selected from a Lactobacillus strain, a Lactiplantibacillus strain, a Lacticaseibacillus strain or a Lactococcus strain, preferably from a Lactobacillus rhamnosus strain, a Lactococcus lactis subsp. lactis biovar. diacetylactis strain, a Lacticaseibacillus paracasei strain, a Lactobacillus paracasei strain, a Lactobacillus paracasei subsp. paracasei strain, a Lactobacillus acidophilus strain, a Lactobacillus helveticus strain, a Lactiplantibacillus plantarum strain or a Lactococcus lactis subsp. lactis strain, preferably wherein the at least one LAB strain is a Lactobacillus rhamnosus strain.

[0162] 9. The packing water according to claim 8, wherein the packing water comprises LAB belonging at least to: a. Lactococcus lactis subsp. lactis biovar. diacetylactis; b. Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis; c. Lacticaseibacillus paracasei, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei and Lactococcus lactis subsp. lactis; or d. Lactobacillus rhamnosus.

[0163] 10. The packing water according to any one of claims 1 to 10, wherein the at least one LAB strain is selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666, and DSM 23035.

[0164] 11. The packing water according to any one of claims 1 to 10, wherein the packing water comprises LAB from the following strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035, preferably wherein the packing water comprises LAB from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 32666 and / or under the accession number DSM 23035.

[0165] 12. The packing water according to any one of claims 1 to 11, wherein the packing water comprises at least 20 U LAB / 100 L of water, preferably at least 40 U LAB / 100 L of water, more preferably 50 U LAB / 100 L of water, preferably wherein the packing water comprises LAB in a concentration of more than about 5xl08CFU / L, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably in a concentration of about 5 xlO10CFU / L.

[0166] 13. A directly acidified fresh mozzarella product comprising a packing water as defined in any one of claims 1 to 12.

[0167] 14. Use of the packing water as defined in any one of claims 1 to 12 to produce a directly acidified fresh mozzarella product.

[0168] 15. A LAB selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666 and DSM 23035, wherein strains with deposit number DSM 34775, DSM 34776, DSM 34777, DSM 34778, DSM 34779, DSM 34780 and DSM 34781 were deposited on October 4, 2023 (2023-10-04), strain with deposit number DSM 24616 was deposited on March 1, 2011 (2011-03-01), strain with deposit number DSM 18875 was deposited on December 19, 2006 (2006-12-19), strain with deposit number DSM 32389 was deposited on November 2, 2016 (2016-11-02), strain with deposit number DSM 32666 was deposited on October 17, 2017 (2017-10-17) and strain with deposit number DSM 23035 was deposited on October 14, 2009 (2009-10-14).

[0169] 16. A composition comprising at least one LAB as defined in claim 15, preferably wherein the composition comprises the following LAB strains, deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the following accession numbers: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035.

[0170] 17. The composition according to claim 16, wherein the composition further at least one cryoprotectant, one antioxidant, nutrients, fillers and / or mixtures thereof.

[0171] 18. Use of at least one lactic acid bacteria (LAB) strain as defined in any one of claims 5 to 12 or 15 for producing a packing water suitable for directly acidified fresh mozzarella.

[0172] 19. Use of at least one lactic acid bacteria (LAB) strain for improving the flavor of directly acidified fresh mozzarella preferably wherein the LAB strain is a LAB strain as defined in any one of claims 5 to 12 or 15.

[0173] 20. A method for producing directly acidified fresh mozzarella, wherein the method comprises a step in which the packing water as defined in any one of claims 1 to 12 is used. method according to claim 20, wherein the method comprises the following steps: a. Providing a fresh mozzarella (i.e., a high moisture content mozzarella, as described herein), wherein the fresh mozzarella has been produced by direct acidification, preferably comprising the use of citric acid; b. Packing the fresh mozzarella in a packing water as defined in any one of claims 1 to 12. c. Screening for a fresh mozzarella with improved flavor, as compared with the same fresh mozzarella comprising the same packing water but without the at least one LAB strain, wherein the evaluation of the fresh mozzarella flavor is performed up to 40 days of storage at a temperature of about 4°C-, and wherein the evaluation of the fresh mozzarella flavor is performed by the analysis of the following sensory attributes: "Fresh score", the "Diacetyl score", the "Bitter score" the "Off flavors" score, the "Acid score" and / or the "Milky score" of the packaged mozzarella by expert panellists, as compared with a reference fresh mozzarella sample (packaged in packing water without LAB).

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[0183] FOR RECEIVING OFFICE USE ONLY

[0184] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A packing water suitable for directly acidified fresh mozzarella comprising at least one lactic acid bacterium (LAB), wherein the packing water preferably comprises water, NaCI, and calcium, and wherein the pH of the packing water is between 4.5 and 6, preferably wherein the pH of the packing water is not significantly reduced over the shelf-life of the directly acidified fresh mozzarella, and the amount of NaCI if present is preferably the same as the % NaCI% comprised in the fresh mozzarella.

2. The packing water according to claim 1, wherein the packing water comprises calcium at a concentration of between 0.5 and 1 g / L, preferably at a concentration of between 0.6 and 0.8 g / L, even more preferably at a concentration of about 0.8 g / L.

3. The packing water according to any one of claims 1 to 2, wherein the at least one LAB strain comprised in the packing water:(i) is a citrate-positive strain; and / or(ii) is capable of producing diacetyl.

4. The packing water according to any one of claims 1 to 3, wherein the at least one LAB strain is selected from a Lactobacillus strain, a Lactiplantibacillus strain, a Lacticaseibacillus strain or a Lactococcus strain, preferably from a Lactobacillus rhamnosus strain, a Lactococcus lactis subsp. lactis biovar. diacetylactis strain, a Lacticaseibacillus paracasei strain, a Lactobacillus paracasei strain, a Lactobacillus paracasei subsp. paracasei strain, a Lactobacillus acidophilus strain, a Lactobacillus helveticus strain, a Lactiplantibacillus plantarum strain or a Lactococcus lactis subsp. lactis strain, preferably wherein the at least one LAB strain is a Lactobacillus rhamnosus strain.

5. The packing water according to claim 4, wherein the packing water comprises LAB belonging at least to: a. Lactococcus lactis subsp. lactis biovar. diacetylactis; b. Lactobacillus acidophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis;36c. Lacticaseibacillus paracasei, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei and Lactococcus lactis subsp. lactis; or d. Lactobacillus rhamnosus.

6. The packing water according to any one of claims 1 to 5, wherein the at least one LAB strain is selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 24616, DSM 18875, DSM 32389, DSM 32666 and DSM 23035, preferably from DSM 32666, and DSM 23035.

7. The packing water according to any one of claims 1 to 6, wherein the packing water comprises LAB from the following strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035, preferably wherein the packing water comprises LAB from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the accession number DSM 32666 and / or under the accession number DSM 23035.

8. The packing water according to any one of claims 1 to 7, wherein the packing water comprises LAB in a concentration of more than about 5xl08CFU / L, such as for instance from about 5xl08CFU / L to about 5 xlO12CFU / L, such as from about 5xl08CFU / L to about 5 xlO11CFU / L, preferably from about 5xl09CFU / L to about 5 xlO11CFU / L, even more preferably in a concentration of about 5 xlO10CFU / L.

9. A directly acidified fresh mozzarella product comprising a packing water as defined in any one of claims 1 to 8.

10. Use of the packing water as defined in any one of claims 1 to 8 to produce a directly acidified fresh mozzarella product.

11. A LAB selected from the strains deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, by Chr. Hansen A / S, under the accession number DSM 34781, DSM 34780, DSM 34778, DSM 34779, DSM 34776, DSM 19499, DSM 18874, DSM 34777, DSM 34775, DSM 18875, and DSM 32389, wherein strains with deposit number DSM 34775, DSM 34776, DSM 34777, DSM 34778, DSM 34779, DSM 34780 and DSM 34781 were deposited on October 4, 2023 (2023-10-04), strain with deposit number DSM 18875 was deposited on December 19, 2006 (2006-12-19), and strain with deposit number DSM 32389 was deposited on November 2, 2016 (2016-11-02).

12. A composition comprising the following LAB strains, deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under the following accession numbers: a. DSM 34781, DSM 34780, DSM 34778 and DSM 34779; b. DSM 34776, DSM 19499, DSM 18874 and DSM 34777; c. DSM 34775, DSM 24616, DSM 18875, DSM 32389 and DSM 18874; d. DSM 32666; and / or e. DSM 23035, and preferably, wherein the composition further at least one cryoprotectant, one antioxidant, nutrients, fillers and / or mixtures thereof.

13. Use of at least one lactic acid bacteria (LAB) strain as defined in any one of claims 1 to 8 or 11, for producing a packing water suitable for directly acidified fresh mozzarella.

14. A method for producing directly acidified fresh mozzarella, wherein the method comprises a step in which the packing water as defined in any one of claims 1 to 8 is used.

15. The method according to claim 14, wherein the method comprises the following steps:a. Providing a fresh mozzarella (i.e., a high moisture content mozzarella, as described herein), wherein the fresh mozzarella has been produced by direct acidification, preferably comprising the use of citric acid; b. Packing the fresh mozzarella in a packing water as defined in any one of claims 1 to 8. c. Screening for a fresh mozzarella with improved flavor, as compared with the same fresh mozzarella comprising the same packing water but without the at least one LAB strain, wherein the evaluation of the fresh mozzarella flavor is performed up to 40 days of storage at a temperature of about 4°C, and wherein the evaluation of the fresh mozzarella flavor is performed by the analysis of the following sensory attributes: "Fresh score", the "Diacetyl score", the "Bitter score" the "Off flavors" score, the "Acid score" and / or the "Milky score" of the packaged mozzarella by expert panellists, as compared with a reference fresh mozzarella sample (packaged in packing water without LAB).

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