Lacticaseibacillus rhamnosus strain and use thereof for preserving casein quality and stability in milk products

Abstract

The present invention relates to a novel lactic acid bacterium (LAB), namely a novel Lacticaseibacillus rhamnosus strain or its mixture with one or more further LAB strains and their uses in preserving casein quality and stability in milk products. Furthermore, methods for producing milk products are also disclosed.

Description

[0001] P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0002] “LACTICASEIBACILLUS RHAMNOSUS STRAIN AND USE THEREOF FOR PRESERVING CASEIN QUALITY AND STABILITY IN MILK PRODUCTS”

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a novel lactic acid bacterium (LAB), namely a novel Lacticaseibacillus rhamnosus strain or its mixture with one or more further LAB strains and their uses in preserving casein quality and stability in milk products.

[0005] Furthermore, methods for producing milk products are also disclosed.

[0006] STATE OF THE ART

[0007] Raw milk is a non-sterile substrate, as milking procedures are not conducted under sterile conditions. As a result, raw milk naturally contains microorganisms, which are typically eliminated through pasteurization or other thermal treatments. Prior to processing, raw milk is generally stored in refrigerated tanks, with storage duration varying based on production needs, ranging from a few hours to up to 72 hours. Despite refrigeration at temperatures below 10°C, microbial growth can still occur. Specifically, psychrophilic and psychrotrophic bacteria, capable of proliferating at low temperatures, constitute a significant portion of the microflora present in raw milk. These bacteria encompass various genera and species, contributing to milk spoilage.

[0008] During cold storage, psychrotrophic bacterial populations dominate, producing extracellular enzymes such as proteases and lipases that degrade milk components and negatively impact dairy product quality. The composition and enzymatic activity of psychrotrophic bacteria in raw milk are influenced by factors such as farm-specific conditions and seasonal variations. Novel psychrotrophic species are frequently isolated from raw milk. The dominant psychrotrophic bacterial genera include Gammaproteobacteria, Bacilli, and Actinobacteria, while minor classes comprise Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria. In particular, Pseudomonas and Acinetobacter (Gammaproteobacteria') predominantly exhibit lipolytic activity, Micro bacterium (Actinobacteria) demonstrates both lipolytic and proteolytic activity. The composition of psychrotrophic bacterial flora in raw milk is thus a critical determinant of milk quality.

[0009] Psychrotrophic bacteria produce extracellular heat-resistant enzymes that remain active even after thermal processing, impacting dairy product characteristics. A highP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0010] psychrotrophic bacterial load in raw milk correlates with thermoresistant enzymes that alter cheese texture, induce sensory defects, and / or reduce industrial yield. These bacteria influence as1- and p-casein fractions, while cheese maturation time affects as1- and y-casein fractions. Bacterial proteases, primarily metalloproteinases, cleave peptide bonds within protein chains, facilitating microbial assimilation. The partial degradation of - and K-caseins leads to reduced cheese yield, the formation of low molecular weight peptides results in a bitter taste, and proteolytic activity contributes to slicing difficulties and loss of elasticity.

[0011] Psychrotrophic bacterial populations reaching densities of 6 to 7 log colony-forming units (CFU) per milliliter synthesize these enzymes at concentrations sufficient to compromise milk stability. Such enzymatic activity can lead to technological issues, including alterations in cheese texture and the gelation of ultra-high temperature (UHT) milk. Among psychrotrophic bacteria, Pseudomonas spp. represent a predominant group of Gramnegative bacteria found in raw milk, particularly at milking sites. These bacteria are strongly associated with quality issues in refrigerated milk, as they rapidly adapt to low temperatures and exhibit short generation times under refrigeration conditions.

[0012] The pre- maturation treatment of refrigerated raw milk is a technique used to improve the physicochemical and microbiological quality of milk, with the aim of increasing yield in cheese making. Here are some key points about this process.

[0013] The benefits of pre-maturation are different. The first one to be mentioned is the caseinpreservation: pre-maturation helps in preserving the casein structure. This point is crucial to maintaining proper clotting times and clot structure during cheese-making. Following, the increase of yield: the preservation of caseins in an intact form leads to an increase in cheese yield, as the proteins are better utilized during the clotting process. Finally, the improvement of milk quality: the addition of cultures with protective effects inhibits or reduces the growth of undesired microorganisms, either by nutrient competition / sequestering or by the production of antimicrobial compounds, leading to milk with improved microbiological quality.

[0014] Cheese production involves the clotting ("curdling") and acidification of milk to produce curd by adding starter cultures to the milk. The curd is then processed, for example, by pressing to remove excess liquid (whey), before the ripening process begins, during which flavors and texture properties develop.

[0015] Microorganisms such as lactic acid bacteria (LAB) are used in cheese production, includingP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0016] hard cheeses, smear cheeses, and soft cheeses. The food industry utilizes mostly LAB, not only to improve the taste and texture of foods but also to extend their shelf life. In fact, LAB are extensively used in the dairy industry as cultures with protective effect.

[0017] A better and more efficient preservation of milk as the main ingredient in dairy preparation allows for the production of dairy products with improved quality and texture. Furthermore, there is an increasing need in the dairy industry to reduce costs and improve yields in production processes.

[0018] The technical problem to be addressed is the lack of an effective method for preserving casein quality avoiding its degradation due to heat-stable proteolytic and lipolytic enzymes released by psychrotrophic bacteria, which negatively impact milk quality and yield of the derived dairy products.

[0019] SUMMARY OF THE INVENTION

[0020] The inventors of the present invention have surprisingly identified a Lacticaseibacillus rhamnosus strain with surprising qualities, that when used alone or in combination with one or more other lactic acid bacteria strains, allows to improve the quality of raw milk, allowing to obtain a milk with higher protein integrity / stability, and also having positive repercussions on the yields of the dairy products produced therefrom, for example cheese.

[0021] The invention therefore relates to a Lacticaseibacillus rhamnosus strain, wherein said strain is called LRH05 and is deposited with accession number LMG P- 20354 and its identification reference is LR12 at the Belgian Coordinated Collection of Microorganisms BCCM / LMG under the Budapest Treaty on 31 January, 2001.

[0022] In a second aspect the present invention relates to a composition comprising the Lacticaseibacillus rhamnosus strain LMG P-20354 deposited at the BCCM, according to the invention.

[0023] In a third aspect the present invention relates to the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or of the composition comprising the same, for the preparation of a pre-maturation culture.

[0024] In a fourth aspect the present invention relates to the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or of the composition comprising the same, for the preparation of a food product.

[0025] In a fifth aspect the present invention relates to a method for preserving casein quality and stability in milk products, said method comprising the step of:

[0026] a) adding a Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0027] 20354 to raw milk, obtaining a pre-maturation mixture;

[0028] b) incubating the pre-maturation mixture of step a) at a temperature in the range from 4°C to 15°C, preferably at a temperature in the range from 4°C to 7°C; and

[0029] c) thermal treating (e.g. pasteurizing) the mixture of step b) by heating to a temperature in the range from 60 to 140 °C for a period of time from 1 second to 30 minutes to obtain a milk product with at least the same protein quality of the raw milk, wherein the protein quality is measured by the Ramsdell assay.

[0030] In a sixth aspect the present invention relates to a milk product obtainable by a method for preserving casein quality and stability in milk products using Lacticaseibacillus rhamnosus strain LMG P-20354.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Examples given for illustrative and non-limiting purposes, and from the annexed Figures 1-8, wherein:

[0033] Figure 1 : Experimental flow for 24 - 48 - 72 hours milk pre-maturation.

[0034] Figure 2: Results achieved with 24 hours of pre-maturation of raw milk contaminated with psychrophilics cocktail at a concentration of 103CFU / mL. Pre-maturation started 3 hours (A) and 12 hours (B) after milking.

[0035] Figure 3: Results achieved with 24 hours of pre-maturation of raw milk contaminated with psychrophilics cocktail at a concentration of 105CFU / mL. Pre-maturation started 3 hours (A) or 12 hours (B) after milking.

[0036] Figure 4: Results achieved with 24 - 48 - 72 hours of pre-maturation of raw milk contaminated with psychrophilics cocktail at a concentration of 103CFU / mL. Pre-maturation was done using strain LRH05 (A) or a mixture of strain LRH05 with another LAB strain (B). Figure 5: Results achieved with 24 - 48 - 72 hours of pre-maturation of raw milk contaminated with psychrophilics cocktail at a concentration of 105CFU / mL. Pre-maturation was done using strain LRH05 (A) or a mixture of strain LRH05 with another LAB strain (B). Figure 6: Microbiological quality of the pre-maturated milk with the strain LRH05 ora mixture of strain LRH05 with a LAB strain, with respect to the untreated raw milk. Raw milk contaminated with psychrophilic cocktail at a concentration of 103CFU / mL. Pre-maturation was started 12 hours after milking.

[0037] Figure 7: Pre-maturation trials done with the strains LRH05, LRH08, LRH01 or by using EDTA (positive control). (A) Psychrophilics counts achieved at different length of pre-P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0038] maturation; (B) Ramsdell score achieved in the sample pre-maturated with different L. rhamnosus strains / EDTA 5 hours after milking and for 24 hours. Scores achieved in milk contaminated with a psychrophilic cocktail at a concentration of 103CFU / mL, treated 3 hours (C), 24 hours (D) and 72 hours (E) after milking and pre-matured for 24 hours. Figure 8: Thermal inactivation of the culture of LRH05. (A) Experimental set up of the inactivation trial; (B) Microbial cell counts of LRH05 achieved before and after the thermal treatment.

[0039] Figure 9: HPLC assessment of the milk protein quality. (A) HPLC profiles of milk contaminated with the psychrophilic cocktail at the concentration of 103CFU / mL stored for 72 hours with or without the strain LRH05. (B) Specific details of the HPLC profile. Specifically in boxes A, B and C are shown the peaks kept with higher area when LRH05 strain is added.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to a novel L. rhamnosus strain with exceptional properties. This strain, either used alone or in combination with one or more lactic acid bacteria, significantly enhances the quality of raw milk. Its application results in milk with superior protein integrity and stability, which is crucial for maintaining and producing high-quality dairy products, with high yield.

[0042] Furthermore, using this novel strain contributes to a reduced microbiological load in the milk, ensuring safer and more hygienic raw ingredients for dairy production. The positive effects of this strain extend beyond milk quality, also leading to improved yields in various dairy products, such as cheese, which benefit from better consistency, flavor, and overall production efficiency. This discovery opens new possibilities for optimizing dairy processes, enhancing product quality, and improving the overall sustainability of dairy production. The invention therefore relates to a Lacticaseibacillus rhamnosus strain, wherein said strain is called LRH05.

[0043] As used herein, the term “LRH05” or “LR12” or “LMG P-20354” is a Lacticaseibacillus rhamnosus or Lactobacillus rhamnosus or L. rhamnosus strain deposited with accession number LMG P-20354 and its identification reference is LR12 at the Belgian Coordinated Collection of Microorganisms BCCM / LMG under the Budapest Treaty on January 31st, 2001.

[0044] As used herein, the term “lactic acid bacterium” or “LAB” designates a gram-positive microaerotollerant bacterium, which ferments sugars with the production of organic acidsP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0045] including lactic acid - as the predominantly produced acid - acetic acid and propionic acid. The industrially most useful bacteria are found within the order “Lactobacillales" which includes, for example, Carnobacterium sp., Lactobacillus delbrueckii, Lactobacillus acidophilus group, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Lactiplantibacillus plantarum group, Lacticaseibacillus paracasei and Lacticaseibacillus casei and other bacteria such as Streptococcus thermophilus. These bacteria are normally supplied to the dairy industry either as frozen or freeze-dried cultures for bulk-set or Direct-Vat Set (DVS) application, for the production of a dairy product, such as a fermented milk product.

[0046] As used herein, the term “milk” refers to the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein / fat solutions made of plant materials, e.g. soy milk, almond milk, oat milk and coconut milk.

[0047] As used herein, the term "dairy products" refers to raw and processed or manufactured milk and milk-derived products. Non-limiting exemplary dairy products include plain yoghurt, flavoured yoghurt, fruit-yoghurt, flavoured milk drinks, fermented milk drinks with or without fruit preparation or flavouring, hard cheese or soft cheese, ice cream, cream or creambased desserts.

[0048] As used herein, the term “thermally treating” or “thermal treatment” is meant to involve heating milk to a specific temperature for a set period of time, in particular the milk is heated to a temperature in the range from 60 to 140 °C for a period of time from 1 second to 30 minutes. Treatment at higher temperatures such as 140°C (e.g. with flowing steam) typically lasts 1-5 seconds. Pasteurization typically heats milk to around 72°C for 15 seconds (high-temperature short-time method) or to 63°C for 30 minutes (low-temperature long-time method).

[0049] As used herein, the term “protein quality” is measured by the Ramsdell assay, a commonly used method in dairy plants to easily assess the protein quality of milk, especially if it has to be treated through UHT. Specifically, the assay evaluates milk coagulation following treatment with a 0.1 N KH2PO4solution and assigns a corresponding score to the sample, as described in detail in Example 1.

[0050] Advantageously, the inventors have found that the L. rhamnosus strain LRH05 and its combination with one or more LAB strains, surprisingly has the capacity to contain the psychrotrophic bacteria, naturally present in raw milk, even if stocked at low temperature (e.g. 5 °C). In fact, the quality of the resulting milk inoculated (pre-maturation) with the strainP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0051] LRH05 or strain LRH05 in combination with one or more LAB strains, was seen to be higher, since the caseins were kept intact during the cold storage, with notable advantages in the subsequent technological processes.

[0052] As can be seen from the results in Example 1, the addition of the strain LRH05 according to the invention or the mixture as herein described resulted in the containment of the psychrotrophic bacteria that were used for the milk contamination, and it had a significant impact on the milk quality preservation. In fact, after the Ramsdell test, the pre-matured milk with LRH05 or with its mixture with one or more LAB strains gave an higher quality score with respect to the control.

[0053] In a second aspect the present invention relates to a composition comprising the Lacticaseibacillus rhamnosus strain LMG P-20354 deposited at the BCCM, according to the invention.

[0054] In a preferred aspect the comprising the Lacticaseibacillus rhamnosus LMG P-20354 strain further comprises one or more further LAB strains selected from the group consisting of Carnobacterium sp., Lactobacillus delbrueckii, Lactobacillus acidophilus group, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Lactiplantibacillus plantarum group, Lacticaseibacillus paracasei and Lacticaseibacillus casei. In a still more preferred aspect the further LAB strain belongs to the species Carnobacterium divergens.

[0055] The composition according to the present invention can preferably be freeze-dried, tyndallized, frozen, for example under liquid nitrogen or in the form of a liquid suspension. Preferably in the composition as herein described, the Lacticaseibacillus rhamnosus strain LMG P-20354 is in an amount in the range from 1X108CFU / L to 1X109CFU / L and the one or more further LAB strains is in an amount in the range from 1X108CFU / L to 1X109CFU / L. In a third aspect the present invention relates to the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or of the composition comprising the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 as specified above, for the preparation of a pre-maturation culture.

[0056] In a fourth aspect the present invention relates to the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or of the composition comprising the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 as specified above, for the preparation of a food product.

[0057] In a preferred aspect, the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or the composition the same or further comprising one orP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0058] more LAB strains, wherein the food product is selected from the group consisting of milk, cheese, butter, yogurt, fresh cream, UHT or pasteurised cream, ice cream, cottage cheese, ricotta cheese, milk powder, condensed milk, kefir, fermented milk, cream cheese, frozen yogurt, clotted cream, buttermilk, fromage blanc, paneer and ghee.

[0059] In a preferred aspect, the use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 or the composition the same or further comprising one or more LAB strains, wherein said milk is selected from the group consisting of cow milk, sheep milk, goat milk, buffalo milk, camel milk or vegetable milk.

[0060] "Vegetable milk" is also known as "plant-based milk" or "plant milk", and can be selected from the group comprising almond milk, oat milk, coconut milk and soy milk).

[0061] In a fifth aspect the present invention relates to a method for preserving casein quality and stability in milk products, said method comprising the step of:

[0062] a) adding the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 to raw milk, obtaining a pre-maturation mixture;

[0063] b) incubating the pre-maturation mixture of step a) at a temperature in the range from 4°C to 15°C, preferably 4-7°C; and

[0064] c) thermally treating (e.g. pasteurizing) the mixture of step b) by heating to a temperature in the range from 60 to 140 °C for a period of time from 1 second to 30 minutes to obtain a milk product with at least the same protein quality of the raw milk, wherein the protein quality is measured by the Ramsdell assay.

[0065] In a preferred aspect, the thermal treatment (thermally treating of step c)) is a step of heating the mixture of step b) at a temperature in the range from 60 to 140°C, for a time in the range from 1 second to 30 minutes.

[0066] Preferably in the method according to the invention, the one or more further LAB strains are selected from the group consisting of Carnobacterium sp., Lacticaseibacillus rhamnosus, Latilactobacillus sakei ssp. sakei, Lactiplantibacillus plantarum ssp. plantarum, Lacticaseibacillus paracasei ssp. paracasei and Schleiferilactobacillus harbinensis, is added to step a) to obtain the pre-maturation mixture.

[0067] Still more preferably the Lacticaseibacillus rhamnosus strain LMG P-20354 is in an amount in the range from 1X108CFU / L to 1X109CFU / L and the one or more further lactic acid bacteria (LAB) strains is in an amount in the range from 1X108CFU / L to 1X109CFU / L. In a preferred aspect of the method:

[0068] - step b) is carried out for at least 24 hours, and for not more than 96 hours; andP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0069] - the raw milk in step a) is stored at a temperature in the range from 4°C to 15°C, preferably in the range from 4 to 7°C.

[0070] In a sixth aspect the present invention relates to a milk product obtainable by a method for preserving casein quality and stability in milk products using Lacticaseibacillus rhamnosus strain LMG P-20354.

[0071] Preferably the milk product is selected from the group consisting of milk (whole, skim, 2%, etc.), cheese (Cheddar, mozzarella, gouda, brie, feta, cream cheese, etc.), butter (unsalted, salted), yogurt (regular, Greek, flavored, non-fat, low-fat), cream (heavy cream, whipping cream, sour cream, clotted cream, fresh cream, UHT or pasteurized cream), ice cream (regular, low-fat, non-dairy alternatives), cottage cheese, ricotta cheese, milk powder (whole, skim), kefir, fermented milk, cream cheese, whey protein (used in supplements and bars), evaporated milk, condensed milk, powder milk, frozen yogurt, clotted cream, buttermilk (traditional, cultured), fromage blanc, paneer (Indian-style cheese), and ghee (clarified butter). These products come in various forms, catering to different tastes, diets, and culinary needs.

[0072] The pre-maturation of milk with the strain described in the present invention or the mixture of the same with another LAB strain(s), has repercussions in technological applications as shown in Example 3. Specifically, experimental trials were set up in a dairy plant producing pizza cheese. The pre-maturation of the milk with 1X108CFLI / L of the strain LRH05 resulted in higher yields of cheese production, with the consequent reduction of the liters of milk needed to produce the same amount of cheese. The pre-maturation kept a higher milk protein quality, with consequent higher kilos of cheese produced with the same amounts of milk.

[0073] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0074] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

[0075] Example 1. Laboratory experiments in raw milk contaminated with psychrophilic bacteria: 24 hours milk pre-maturation

[0076] Preliminary tests in the laboratory were set up to evaluate capability of the selected strains in the reduction of the psychrophilic bacteria. A cocktail of twenty different psychrophilic bacterial strains isolated from dairy plants and products was created.P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0077] The resulting psychrophilic bacterial mix, called psychrophilics cocktail, contained 20 strains each belonging to one of the following bacterial species:

[0078] Pseudomonas putida

[0079] Pseudomonas chlororaphis

[0080] Pseudomonas aeruginosa

[0081] Pseudomonas fluorescens

[0082] Pseudomonas jessenii

[0083] Pseudomonas koreensis

[0084] Pseudomonas luteola

[0085] Pseudomonas lactis

[0086] Corynebacterium callunae

[0087] Micro bacterium foliorum

[0088] Acinetobacter baumanii

[0089] Rouxiella aceris / yersinia kristensenii / yersinia ruckeri

[0090] Enterobacter spp

[0091] Bacillus infantis

[0092] Escherichia coli

[0093] Macrococcus caseoliticus

[0094] Serratia marcesciens

[0095] Paenibacillus odorifer

[0096] Aeromonas bestiarum

[0097] All psychrophilic strains were mixed in the same amount to reach the final bacterial count needed (e.g., 103CFU / mL, 105CFU / mL).

[0098] Fresh raw milk batches were collected from a cowshed and immediately stored at 4°C. The psychrophilics cocktail was used to further contaminate the raw milk at 0.5 hours after milking. The amount of psychrophilics contaminants used resembles the values often reported in dairy plants.

[0099] The contaminated raw milk was stored at 4°C and at different times from the milking (3 and 12 hours) the pre-maturation strain of Lacticaseibacillus rhamnosus LRH05, deposited with accession number LMG P-20354 at the BCCM or a mixture of strain LRH05 with another LAB strain.

[0100] In the examples the results obtained with a strain of Carnobacterium divergens are shown, but similar results can be achieved with other LAB strains selected from the group consistingP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0101] of Carnobacterium sp., Lactobacillus delbrueckii, Lactobacillus acidophilus group, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Lactiplantibacillus plantarum group, Lacticaseibacillus paracasei and Lacticaseibacillus casei.

[0102] The final concentrations used for the pre- maturation step are 5X108CFLI / L (dose 1) or 1X109CFLI / L (dose 2). The resulting samples were kept at 4°C, as normally done in industrial storage tanks.

[0103] Twenty-four hours after the addition of the pre-maturation culture / s (LRH05 alone or in a mixture with another LAB), the Ramsdell assay (Ramsdell et al., 1931) was used to assess the milk protein quality and the consequent heat stability.

[0104] The specific experimental flow is reported in Fig. 1.

[0105] Ramsdell method consists in the addition of different volumes of a 0,1 N monobasic potassium phosphate (KH2PO4) solution to a milk sample: KH2PO4 boosts the milk coagulation, if broken caseins are present in the samples. If milk caseins are native and in a good quality, KH2PO4 will not affect the coagulation of the milk.

[0106] Ramsdell method is commonly used in dairy plants to easily assess the protein quality of milk, especially if it has to be treated through UHT. When coagulation is observed with 0,1 N KH2PO4 solution, the milk is corrected accordingly by adding specific concentration of disodium phosphate in order to help in increasing the milk protein quality for the thermal treatment.

[0107] In detail, the Ramsdell method has the following protocol:

[0108] 10 mL of milk dispensed in 8 different tubes

[0109] - Addition of increasing volumes of 0,1 N KH2PO4, starting from tube 1 to 8:

[0110]

[0111] Thermal treatment at 97°C for 10 minutes

[0112] Cooling down to 4°C and holding for 10 minutes

[0113] Coagulation level assessment

[0114] Scoring:

[0115] o 1 = no coagulation high protein quality

[0116] o 0,5 = partial coagulation partial reduction in the protein quality of the mil o 0 = full coagulation high protein degradation

[0117] The higher the volume of the buffer needed to induce coagulation, the higher the quality ofP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0118] the milk.

[0119] The milk contaminated with the psychrophilics cocktail at the final concentration of 103CFU / mL without the pre-maturation step showed coagulation already after the first two or maximum three volumes added in the Ramsdell method after 24 hours storage at 4°C (Fig.

[0120] 2 and 3).

[0121] This confirms that the psychrophilic bacteria contained in the cocktail (in addition to those already present in the raw milk) effectively reduced the milk quality, possibly acting on the milk caseins.

[0122] Notably, the pre-maturation of the milk in the presence of the strain LRH05 or the mixture of LRH05 with another LAB prevented premature coagulation of the milk (Fig. 2 and 3). Surprisingly, higher volume of the monobasic potassium phosphate solution had to be added before achieving coagulation (Fig. 2 and 3).

[0123] The milk quality is preserved when the pre-maturation strains are added 3 hours after milking (Fig. 2A), or 12 hours (Fig. 2B) after milking.

[0124] These results reproduce what often happens in the dairy chain: after milking, the milk may not be immediately stocked at cold temperatures and often some hours are needed before it will be properly stocked.

[0125] The addition of a pre-maturation culture thus helps in preserving the milk quality a lot, while raw milk is transported from the place of milking to the stock tank.

[0126] The start of the pre-maturation in the range of 3 to 12 hours after milking, is effective in keeping the milk quality higher, if pre-maturation is extended until 24 hours.

[0127] When the psychrophilic cocktail contamination is increased until 105CFU / mL, the contribution of the pre-maturation culture / s (LRH05 alone or in a mixture with a LAB) in keeping a higher milk quality is still visible (Fig. 3).

[0128] If not pre-matured with strain LRH05 or a combination of strain LRH05 and another LAB, all samples analyzed 24 hours after contamination time showed poor quality: the two lower quantities of KH2PO4 solution are enough to determine the premature coagulation of the milk (Fig. 3).

[0129] The addition of the pre-maturation strain / s LRH05 alone or in a mixture with another LAB increased the milk quality: in particular, significantly higher amounts of KH2PO4 solution are needed in the Ramsdell test to achieve milk coagulation.

[0130] Pre-maturation starting as quickly as possible is preferable, but addition of the pre-maturation strains even 12 hours after milking is anyway improving the milk quality, both inP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0131] conditions where the psychrophilic contamination is 103CFU / mL (Fig. 2B) or 105CFU / mL (Fig. 3B).

[0132] Both LRH05 alone or in a mixture with another LAB performed well in the pre-maturation treatments.

[0133] Example 2. Laboratory experiments of raw milk contaminated with psychrophilic bacteria: milk pre-maturation extension until 72 hours.

[0134] At this point, further experiments were carried out after 48 or 72 hours of pre-maturation at 4°C.

[0135] As previously done, the raw milk was contaminated with the psychrophilics cocktail at the two final concentrations of 103or 105CFU / mL. The pre-maturation strain LRH05 alone or in a mixture with another LAB were added 3 or 12 hours after milking.

[0136] The reported data (Fig. 4 and 5) are the average of the milk quality assessed with a specific pre-maturation treatment, independently from the time after milking the culture / s (LRH05 alone or in a mixture with a LAB) were added (the Ramsdell scores and the microbial cells counts are expressed as average of the conditions 3 and 12 hours after milking). The following doses were used:

[0137] - LRH05 at 5X108CFU / L (dose 1);

[0138] - LRH05 + LAB together at 1X109CFU / L (dose 2).

[0139] All trials were done in triplicate, and the average values are reported.

[0140] LRH05 or its mixture with another LAB were also counted during storage conditions: their cell count is kept stable during the pre-maturation step. The final cell counts of the strains LRH05 alone or in a mixture with another LAB do not negatively impact the overall milk quality. High casein quality of the raw milk is kept independently from the count of LRH05 alone or its mixture with another LAB during storage.

[0141] Independently from the time of addition of LRH05 alone or its mixture with another LAB, it is evident that the pre-maturation treatment keeps a higher Ramsdell score of the raw milk, both when the psychrophilic contamination is reaching a count of 103or 105CFU / mL (Fig. 4 and 5).

[0142] A pre-maturation extended to 48 hours seems optimal in keeping higher casein quality, giving higher Ramsdell scores. Pre-maturation step with the strain L. rhamnosus LRH05, deposited with accession number LMG P-20354 at the BCCM, alone or in a mixture with another LAB for a period of 24, 48 or 72 hours results in milk with higher quality (higher Ramsdell score) (Fig. 4 and 5).P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0143] When the psychrophilics cocktail is inoculated at 103CFU / mL (Fig. 6), the pre- maturation with the strain LRH05 alone or in a mixture with another LAB, results in a substantial reduction of its growth, with respect to the raw milk untreated with pre-maturation LAB. Starting the pre-maturation 12 hours after milking significantly reduced the psychrophilics growth, especially when maturation was extended for 24 - 48 hours (Fig. 6).

[0144] In general, a greater reduction in the growth of the contaminants is achieved when using the strain LRH05 in a mixture with another LAB (Fig. 6).

[0145] Due to the heterogeneity of the species composing the psychrophilics cocktail and to their different growth kinetics, the data regarding the spoilage growth reduction may vary in time. The main evidence of the beneficial effect of the pre-maturation treatment is the keeping of high quality in the proteins present in the raw milk.

[0146] Example 3: Pre-maturation impact on cheese-making.

[0147] Technical trials in dairy plants were set up with the aim of analyzing the structural gains of casein after the pre-maturation treatment of raw milk refrigerated in “silos”, using L. rhamnosus LRH05.

[0148] The main focus of the work was the evaluation of the yield in cheese making, due to the preservation of casein during cold milk storage achieved with pre-maturation trials.

[0149] The L. rhamnosus LRH05, deposited with accession number LMG P-20354 was used for the pre-maturation of refrigerated raw milk, before pasteurization. The pre-maturation treatment was carried out while the milk was stored in the refrigerated silos.

[0150] The pre-maturation treatment of refrigerated raw milk is overall useful to improve the physicochemical quality of milk, with the final gain of increasing yield in cheese making. In detail, the following pre-maturation steps were applied:

[0151] Forty-eight hours after milking, the milk was received in the dairy plant through refrigerated trucks. The dairy plant recorded the receiving temperature, acidity (°D), cryoscopy, reductase, antibiotics / inhibitors and levels of fat and protein.

[0152] The milk was released for collection, unloaded from the trucks and stored in refrigerated tanks at temperature from 4 to 7°C. The strain L. rhamnosus LRH05 was added to reach the cell count of 1X108CFU / L of raw milk. The strain was previously diluted in 1 liter of milk and added to the trucks, immediately before the transfer of the milk to the storage silos.

[0153] - As negative control, milk without addition of LRH05 for pre-maturation was stored in a silos.P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0154] Reductase, lacto-fermentation (incubation of the milk at 37°C) and physicochemical patterns were evaluated after 24 and 48 hours of cold storage at 4-7 °C.

[0155] - After 48 hours of pre-maturation (or just cold storage for the negative control), the milk was pasteurized and transformed following the standard production procedures for pizza-cheese applied in the dairy plant.

[0156] The main goal of this experiment was the evaluation of the structural gains of casein and the impact on the cheese-making yield, as well as to identify the microbiological problems associated with the prolonged storage of refrigerated raw milk.

[0157] At different sampling times the following analyses were done:

[0158] • Casein structural analysis: using spectroscopy techniques to evaluate the integrity and structure of casein.

[0159] • Microbiological evaluation: microbial cell count in Enterobacteriaceae (EBCD) and lacto-fermentation tests. The lacto-fermentation test was set-up to evaluate the microbiological quality of the milk as a function of the activity of the various types of microorganisms present in the sample. It is based on the incubation of 10 mL of milk at 37°C for 24 hours. After incubation, milk is analyzed and scored according to the odor and the clot obtained. It is an important qualitative test for screening raw milk and pasteurized milk and it is a quick test that is useful to understand if an undesired fermentation occurred.

[0160] • Yield in cheese-making: measurement of the yield of cheeses produced from milk pre-maturated with L. rhamnosus LRH05 compared to untreated milk. Empirical methods were used in the perception of yield (liters of milk / kilos of cheese); yield with adjusted moisture and yield calculated by Van Slyke formula, adapted to pizzacheese production.

[0161] Van Slyke formula for forecasting manufacturing yields is:

[0162] >

[0163]

[0164] The elements of the formula are:

[0165] - Yield: refers to the percentage yield, that is, the forecast of how many kg of cheese wouldP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0166] be obtained from 100 liters of milk.

[0167] - Milk fat: milk fat content.

[0168] - 0.93%: Van Slyke assumed that in Cheddar cheese about 93% of the milk fat is transferred to the curds.

[0169] - Milk casein: casein content of milk.

[0170] - 0.1: Van Slyke assumed that in Cheddar cheese about 96% of the casein in the milk is transferred to the curd. For better understanding: if in 100 liters of milk containing 2.5 kg of casein, 0.1 kg of protein is subtracted, by rule of three it is concluded that in 100 kg this subtraction corresponds to a loss of 4 kg of casein (4%).

[0171] - 1.09: fixed factor to represent the solids (apart from fat and casein) retained in the whey of the cheese matrix, for example, mineral salts, ashes, lactic acid, lactose and eventually sodium chloride (since Cheddar is salted in the dough). 1.09 applies to Cheddar, a cheese with about 37-38% moisture (fresh) and when it comes to cheeses with higher moisture content (such as pizza-cheese) it will certainly increase.

[0172] - T arget cheese moisture refers to the desired or anticipated moisture content in the cheese. It can be the final moisture content, found in the finished cheese.

[0173] This formula was used for more than a century by American and other cheese-makers (data not shown) to predict yield in the manufacture of Cheddar cheese and it is considered quite reliable, although exact results are not obtained, due to the natural variability observed in cheese manufacturing.

[0174] Barbano's formula is, obviously, the same as Van Slyke's, but he considered a fat use of only 85% (considering losses in whey and losses during the stretching process).

[0175]

[0176] Taking into account the higher moisture content of pizza-cheese, Barbano changed the fixed factor to 1.13, relative to soluble solids (apart from fat and casein) retained in the whey of the cheese matrix, such as mineral salts, ashes, lactic acid, lactose.

[0177] The pre- maturation treatment with L. rhamnosus LRH05 resulted effective in preserving the casein structure. Chromatographic analyses indicated a lower degradation of caseinP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0178] micelles compared to untreated milk. Spectroscopy revealed a higher composition of quantified caseins in the milk compared, which contributes to a better yield in cheesemaking.

[0179] Concerning the cheese making, a significant increase in the yield of cheeses produced from milk pre-treated with L. rhamnosus LRH05 was recorded (Table 1).

[0180] The preservation of casein during cold storage resulted in a higher efficiency in coagulation and clot formation.

[0181] Table 1: Cheese yields achieved in the dairy plant.

[0182] <

[0183]

[0184] Globally, 69100 liters of milk were used in these trials, using at least three different silos for the cold stock of the milk (see vat names, Table 1). Overall, when the milk is pre-treated with the strain LRH05, 2.7% more pieces of pizza-cheese were produced. Furthermore, theP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0185] pizza-cheese produced using the milk pre-maturated with the strain LRH05, kept a 3.6% higher weight after packaging, than the ones produced with the untreated milk. Overall, 6908 kg of cheeses were produced when the milk was pre-treated with the strain LRH05, while just 6668 kg when using untreated milk. Globally, 240 kg of extra cheese were obtained using milk pre-matured with the strain LRH05.

[0186] Further analyses were done to assess the microbiological quality of milk. The improvement in the quality of the pre-matured milk was assessed through microbiological analysis, lactofermentation tests and milk reductase assay (Table 2). Differences in pH and acidity between the pre-matured milk with LRH05 and the untreated milk were not detected, while significant differences were found in the final pasteurized milk solid contents (data not shown). In fact, the pasteurized milk resulted in a significant lower solids contents when pre-matured with LRH05.

[0187] Table 2: Microbiological quality of the milk (CCS: count of somatic cells).

[0188]

[0189]

[0190] P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0191] cheese preparation, yields increased between the 0.8 and the 2.8 % were always recorded when using milk pre-treated with LRH05.

[0192] In external application trials, a dairy plant working 80-10000 liters of milk per day, applied LRH05 for pre-maturation in the milk silos during storage, with different lengths (24-72 hours) according to practical needs for one month. In parallel, the same amount of milk was stored at refrigerated temperatures without the addition of LRH05. Both the milk prematurated with LRH05 and the milk without addition of LRH05 were later used for the production of different cheeses. Following we report the average increase in yields achieved during 1 month of manufacturing for different cheese types when using milk stored with the addition of the strain LRH05 (increase in yield when compared to the same cheese produced with milk without addition of LRH05).

[0193] Danbo cheese: + 2,00% yield increase;

[0194] Mozzarella cheese: +2,54% yield increase;

[0195] Danbo cheese Top quality: +1,7% yield increase.

[0196] Other important achievements in terms of yield increase were noted by measuring the yield increased on Mondays, i.e. , when the milk was used after 72 hours of cold storage in the presence of LRH05:

[0197] Danbo cheese: +1,76% yield increase;

[0198] Mozzarella cheese: +3,2% yield increase;

[0199] Danbo cheese Top quality: +4,02% increase.

[0200] Example 4: Pseudomonas cocktail containment and milk quality by using L. rhamnosus strains different from LRH05 (LRH08 and LRH01) and EDTA addition

[0201] An experiment similar to the one set up in Example 1 was carried out with the aim to test two strains of L. rhamnosus different from LRH05.

[0202] Pre-maturation trials were performed using L. rhamnosus strain LRH05, L. rhamnosus strain LRH08, or L rhamnosus strain LRH01 at two levels, e.g. 5X108CFLI / L (dose 1) or 1X109CFU / L (dose 2).

[0203] L. rhamnosus strain LRH08, is deposited with accession number DSM 21690 at the Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures under the Budapest Treaty on 25 July, 2008.

[0204] The psychrophilic cocktail was used at a concentration of 103CFU / mL or 105CFU / mL. Also, a further test has been set up: a raw milk sample was added with 14 mg / L of EDTA to understand if the chelating capability (one of the possible mechanisms of LRH05 to keep aP024209WO-02 Notarbartolo & Gervasi S.p.A.

[0205] higher milk protein quality) may be the mechanism responsible for preserving the milk caseins quality. The capability of chelating ions (possibly co-factors of the proteases secreted by psychrophilic bacteria) is known among LAB and considered crucial in the containment of spoilage microorganisms. In previous studies and inventions is indeed reported the capability of few LAB strains in up-taking ions such as manganese. In this experiment EDTA was used in order to assess if the chelation of cations could limit the negative effects of the psychrophilic bacteria on the milk quality.

[0206] Ramsdell method and total psychrophilic cell counts were measured in order to assess the quality of the treated milk.

[0207] The pre-maturation started 3,5 or 12 hours after milking and lasted for 24,48 or 72 hours. Compared to strain LRH08, strain LRH05 was clearly better in containing the development of psychrophilic bacteria (Fig. 7A) and keeping a higher milk quality as assessed using the Ramsdell method. As an example, the score achieved in milk treated 5 hours after milking and pre-matured for 24 hours is reported for the strain LRH08 (Fig. 7B). Similar differences in milk quality among the samples were recorded also when pre-maturation started 3 hours after milking and when pre-maturation was prolonged to 48 hours.

[0208] Also, in comparison with the strain LRH01 , LRH05 resulted clearly better in keeping a higher milk quality as assessed using the Ramsdell method. In Fig. 7C we present the scores achieved in milk contaminated with a psychrophilic cocktail at a concentration of 103CFU / mL, treated 3 hours after milking and pre-matured for 24 hours. Fig. 7D and 7E clearly show the efficacy of LRH05 in keeping a higher milk quality in contamination conditions by a psychrophilic cocktail (105CFU / mL), even when the addition of the strain with protective effect has been done 12 hours after milking. LRH05 is better than LRH01 both in 24 hours pre-maturation (Fig. 7D) and 72 hours of pre-maturation time (Fig. 7E). Strain LRH05 clearly maintains a higher milk quality than strain LRH08 and LRH01.

[0209] EDTA is still effective but not as much as the LRH05 strain according to the present invention. The ions chelation may be one of the mechanisms of the milk quality preservation since higher scores are recorded with respect to the un-matured milk, but the highest scores are always registered for the samples with LRH05, indicating that the strain effect relies on a complex mechanism of action, that goes beyond the ions chelating capacity.

[0210] Example 5: Thermal treatment effects on L. rhamnosus strain LRH05

[0211] The pre-maturation of raw milk implies the addition of the strain LRH05 in the cold-stored raw milk. After storage, the raw milk is thermally treated before being processed further.P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0212] The set-up experiment had the aim to demonstrate that the levels of the strain applied in the pre-maturation is significantly reduced after the thermal treatment, and consequently the strain will not impact the following dairy processes: this aspect ensures no negative repercussions on the starter cultures fermentation profile neither in the aroma of the final products. An experiment was set up, pasteurizing the pre-maturated milk containing the strain LRH05: the thermal treatment effectively reduced the load of LRH05 below the experimental detection limit, as expected normally by this treatment and in compliance with the quality standards of a dairy company (Fig. 8).

[0213] This demonstrates that the use of strain LRH05 in the pre-maturation is safe for the following dairy processes: the culture used for the pre-maturation is removed through the thermal treatment and therefore the following dairy steps will be not influenced by the culture previously used for the pre-maturation.

[0214] Example 6: HPLC analysis of the milk proteins quality preservation by the strain LRH05 HPLC was applied to confirm that the strain LRH05 used for the milk pre-maturation during cold storage reduced the milk protein fragmentation caused by enzymatic activities of contaminating psychotropic bacteria. The peptide profiles of milk samples collected after 72 hours of storage at cold temperature were obtained. The non-protein fractions of the milk samples were extracted using 12% trichloroacetic acidic solution in milliQ water (1:4 ratio) and incubated overnight at 4 C. Supernatant was separated by centrifugation (12000 ref, 3 min, 4 C) collected and filtered using 0.45-pm syringe cellulose filters (15 mm diameter, Sartorius Minisart RC hydrophilic, Germany) and 50 pL were injected onto the column to obtain the profiles.

[0215] The separation of milk peptides was performed in reversed-phase mode using an Agilent Zorbax 300SB C8 column (4.6 mm i.d. x 150 mm, Agilent Technologies, USA). A step gradient of water TFA 0.1% and Acetonitrile TFA 0.1%. From 0 to 5 min 100% water 0.1% TFA; from 5 to 60 min linear increase to 50% AON TFA 0.1%; from 60 to 65 linear increase to 60% AON TFA 0.1%; from 65 to 70min 95% ACN 0.1% TFA; from 75-80 min 100% water 0.1% TFA was applied. Flow was kept constant to 0.36 ml / min throughout the separation. The HPLC equipment used was a SHIMADZU LC2020 (SHIMADZU, JAPAN)) with a quaternary pump and a multi wavelength detector SPD-M30A (SHIMADZU, JAPAN). Chromatograms were registered wavelength at 214 nm and data analysis was performed after background subtraction.P024209WO-02 Notarbartolo & Gervasi S.p.A.

[0216] HPLC analysis was used to compare the profiles of milk contaminated with the psychrophilic bacteria and the same contaminated milk where also LRH05 was added. As example, Fig.

[0217] 9A reports the HPLC profiles of the milk contaminated with the psychrophilic cocktail at the concentration of 103CFU / mL stored for 72 hours and the milk contaminated as well but added of the strain LRH05 used for a pre-maturation of 72 hours at 5°C. Fig. 9B shows specific details of the HPLC profile. Specifically, boxes A and B of Fig. 9B show the peaks kept with smaller area when LRH05 strain was added. Interestingly, the addition of LRH05 can be identified by a signature peptide appearing around the 31 ml of elution, visible in all the profiles where LRH05 was added (Fig. 9B, box C).

[0218] These results confirm that the strain LRH05 is effective in keeping higher milk protein quality, counteracting the protein hydrolysis caused by the psychrophilic bacteria; the peaks of the milk protein / peptides change when LRH05 is added, thus suggesting a reduced protein hydrolysis.

[0219] From the above description and the above-noted examples, the advantage attained by the strains described and obtained according to the present invention are apparent.(Original in Electronic Form)

[0220]

[0221]

[0222] (Original in Electronic Form)

[0223] FOR RECEIVING OFFICE USE ONLY

[0224]

[0225] FOR INTERNATIONAL BUREAU USE ONLY

[0226]

Claims

P024209WO-02 Notarbartolo & Gervasi S.p.A.CLAIMS1. Use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 at the Belgian Coordinated Collections of Microorganisms (BCCM) or the composition comprising the Lacticaseibacillus rhamnosus strain LMG P-20354 deposited at the BCCM, for the preparation of a pre- maturation culture.

2. Use of the Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 according to claim 1 or the composition comprising the Lacticaseibacillus rhamnosus strain LMG P-20354 deposited at the BCCM, for the preparation of a food product.

3. The use according to claims 1 or 2, wherein said composition comprises one or more further lactic acid bacteria (LAB) strains selected from the group consisting of Carnobacterium sp., Lactobacillus delbrueckii, Lactobacillus acidophilus group, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Lactiplantibacillus plantarum group, Lacticaseibacillus paracasei and Lacticaseibacillus casei.

4. The use according to any one of claims 1-3, wherein the further LAB strain is Carnobacterium divergens.

5. The use according to any one of claims 1 to 4, wherein the composition is freeze-dried, tyndallized, frozen using liquid nitrogen or in the form of a suspension.

6. The use according to any one of claims 1 to 5, wherein the Lacticaseibacillus rhamnosus strain LMG P-20354 is in an amount in the range from 1X108CFU / L to 1X109CFU / L.

7. The use according to any one of claims 3 to 6, wherein the one or more further lactic acid bacteria (LAB) strains is in an amount in the range from 1X108CFU / L to 1X109CFU / L.

8. The use according to any one of claims 2-7, wherein the food product is selected from the group consisting of milk, cheese, butter, yogurt, cream, ice cream, cottage cheese, ricotta cheese, milk powder, condensed milk, kefir, fermented milk, cream cheese, fresh cream, UHT or pasteurised cream cheese, frozen yogurt, clotted cream, buttermilk, fromage24P024209WO-02 Notarbartolo & Gervasi S.p.A.blanc, paneer and ghee.

9. The use according to claim 8, wherein said milk is selected from the group consisting of cow milk, sheep milk, goat milk, buffalo milk, camel milk or plant-based milk.

10. A method for preserving casein quality and stability in milk products, said method comprising the step of:a) adding a Lacticaseibacillus rhamnosus strain deposited with accession number LMG P-20354 to raw milk, obtaining a pre-maturation mixture;b) incubating the pre-maturation mixture of step a) at a temperature in the range from 4°C to 15°C; andc) thermally treating the mixture of step b) by heating to a temperature in the range from 60 to 140 °C for a period of time from 1 second to 30 minutes to obtain a milk product with at least the same protein quality of the raw milk, wherein the protein quality is measured by the Ramsdell assay.

11. The method according to claim 10, wherein one or more further lactic acid bacteria (LAB) strains selected from the group consisting of Carnobacterium sp., Lactobacillus delbrueckii, Lactobacillus acidophilus group, Lacticaseibacillus rhamnosus, Latilactobacillus sakei, Lactiplantibacillus plantarum group, Lacticaseibacillus paracasei and Lacticaseibacillus casei is added to step a) to obtain the pre-maturation mixture.

12. The method according to any one of claims 10 or 11, wherein the Lacticaseibacillus rhamnosus strain LMG P-20354 is in an amount in the range from 1X108CFU / L to 1X109CFU / L.

13. The method according to any one of claims 10 to 12, wherein the one or more further lactic acid bacteria (LAB) strains is in an amount in the range from 1X108CFU / L to 1X109CFU / L.

14. The method according to any one of claims 10 to 13, wherein step b) is carried out for at least 24 hours, and for not more than 96 hours.P024209WO-02 Notarbartolo & Gervasi S.p.A.

15. The method according to any one of claims 10 to 14, wherein the raw milk in step a) is stored at a temperature in the range from 4 to 15°C.

16. The method according to any one of claims 10 to 15, wherein the raw milk in step a) is stored at a temperature in the range from 4 to 7°C.

17. A milk product with a preserved casein quality and stability obtainable by the method using Lacticaseibacillus rhamnosus strain LMG P-20354 according to any one of claims 10 to 16.

18. The milk product according to claim 17, wherein the milk product is selected from the group consisting of milk, cheese, butter, yogurt, cream, ice cream, cottage cheese, ricotta cheese, milk powder, condensed milk, kefir, fermented milk, cream cheese, fresh cream, UHT or pasteurised cream, cheese, frozen yogurt, clotted cream, buttermilk, fromage blanc, paneer and ghee.

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