Strain of lactiplantibacillus plantarum and its use for cheese protection

Abstract

The present invention concerns a new strain of an anti-Clostridium Lactiplantibacillus plantarum and its use for hard cheese protection as an antimicrobial agent.

Description

[0001] STRAIN OF LACTIPLANTIBACILLUS PLANTARUM AND ITS USE FOR CHEESE PROTECTION

[0002] The present invention concerns a strain of Lactiplantibacillus plantarum and its use for cheese protection. In particular, the invention refers to a new strain of an anti-Clostridium Lactiplantibacillus plantarum and its use for hard cheese protection as an antimicrobial agent.

[0003] It is known that the occurrence of late blowing defect (LBD) in hard and semi-hard cheeses is a significant problem in cheese manufacturing. Butyric acid fermentation by butyric Clostridia remains as one of the main causes of spoilage in semi-hard and hard cheeses (Brandie et al. 2018). In particular, germination of the Clostridia spores, specifically of Clostridium tyrobutyricum, leads to the late blowing defect during ripening with the appearance of irregular eyes, slits and off-flavours (Brasca et al. 2022).

[0004] Conventional protective methods are not fully satisfactory and are do not meet to market needs.

[0005] To date, lysozyme is still the most widely applied and most effective tool for limiting the occurrence of the LBD in hard cheeses. However, the increasing focus on clean-label products and the legal obligation to indicate lysozyme as a potential allergen, urges the industry to find alternative solutions (Gomez -Torres et al. 2014; Favaro et al. 2015; Trejo-Gonzalez et al. 2022).

[0006] To prevent LBD, various methods have been investigated and developed, ranging from the addition of antimicrobial agents, physical methods such as microfiltration or bactofugation, to the use of microbial cultures or lytic phages with anti-clostridia activity (Avila et al. 2023; Bintsis and Papademas 2024).

[0007] Although a variety of approaches have been investigated, the use of bacteriocin-producing strains to prevent late blowing in cheese seems to be the most feasible alternative to the addition of lysozyme.

[0008] Among the bactericides, nisin, a polypeptide consisting of 34 amino acids produced by Lactococcus lactis spp lactis, is certainly the one most investigated and offers the most information. This specific bacteriocin inhibits vegetative cells of C. tyrobutyricum and is a food additive authorised in the European Union under Annex II of Regulation (EC) 1333 / 2008 for use in several food categories, including dairy products (EFSA, 2017).In addition to nisin, strains producing other bacteriocins have been studied and proposed for their action against Clostridia. Lactin 3147, produced by L lactis subsp. lactis DPC3147, has been shown to inhibit C. tyrobutyricum spores in milk. It has also been demonstratedthat the in situ activity of lactin 3147, in a model curd system, significantly reduced the number of Clostridium spores after 13 days, compared to a non-bacteriocin-producing control (Carmen Martinez-Cuesta et al., 2010). In addition, a bacteriocin produced by Streptococcus thermophilus showed the ability to inhibit C. tyrobutyricum in a ripening curd model for up to 14 days (Mathot et al., 2003).

[0009] However, it must be emphasized that despite the myriads of basic and applied researches on the identification, characterisation and development of lactic acid-producing bacteriocins, very few formulations have reached the dairy market and are still struggling to find wide application. In fact, bacteriocin-producing cultures have severallimitations, such as low production levels, difficult replicability in the food system, antagonism with other useful microorganisms and interaction between the bacteriocin and the food matrix.

[0010] In the light of the above, it is therefore apparent the need to provide new and effective tools to counteract the occurrence of late blowing defect (LBD) in cheese caused in particular by Clostridium tyrobutyricum.

[0011] According to the present invention a new lactic acid bacteria strain (Lactiplantibacillus plantarum LPI25) is provided as a protective tool in dairy production. The strain, according to the invention, by inhibiting Clostridium tyrobutyricum, is useful in the prevention of late blowing defects in hard cheeses. The present invention represents an alternative biopreservation tool to conventional additives, such as lysozyme, and is more effective than commercially available protective cultures. The advantages of the strain according to the present invention lie in its mechanism of action, which is not based on the production of secondary metabolites such as bacteriocins. Instead, the antimicrobial activity of the strain of the invention can be attributed to various primary metabolites, such as organic acids and short-chain fatty acids, the production of which is more repeatable in food models and cheese during ripening.

[0012] This strain according to the present invention is useful for preserving hard cheese in order to avoid late swelling during ripening caused by butyric Clostridia and in particular by Clostridium tyrobutyricum. The safety of the strain of theinvention and its anti-clostridium efficacy have been demonstrated in vitro and validated in the food system as shown in the Example below. Therefore, the present invention is a viable alternative to conventional protective tools. As shown below, the efficacy of the strain is superimposable on that produced by lysozyme (a conventionally used as additive) and appears clearly superior to that expressed by bacteriocin-producing protective microbial cultures.

[0013] Moreover, as shown in the Example below, the use of the protective culture LPI25 has demonstrated comprehensive efficacy both in vitro and in situ during ripening of hard cheeses. These results are particularly interesting considering that there is often a discrepancy in efficacy between in vitro and in situ conditions.

[0014] On the basis of the above, the present invention constitutes an important building block for the development of food products that correspond to the health needs expressed by the market and the public, as the bearers of interests that are to be found in the broad concept of one health.

[0015] It is therefore specific object of the present invention the Lactiplantibacillus plantarum LPI25 strain deposited in the Spanish Type Culture Collection with accession number 31123.

[0016] In particular, the Lactiplantibacillus plantarum strain of the present invention identified with the depositor identification reference LPI25 was deposited by Mediterranea Biotecnologie srl, in accordance with the Budapest Treaty, at the Spanish Type Culture Collection (Coleccion Espanola De Cultivos Tipo - CECT, Edificio 3 CUE, Parc Cientific Universitat de Valencia, Catedratico Agustin Escardino, 9 46980 Paterna, Valencia, Espana) on 10 September 2024 with accession number 31123 or CECT 31123.

[0017] A further object of the present invention is a composition, such as a starter culture (i.e. a composition or preparation of living microorganisms), comprising the Lactiplantibacillus plantarum LPI25 strain as defined in claim 1. According to a preferred embodiment of the present invention, said composition comprises vital cells of said strain.

[0018] According to the present invention, said composition can further comprise one, more than one or all the following specific compounds from its metabolism: propionic acid, isobutyric acid, phenyl-lactic acid, valeric acid, caproic acid, isovaleric acid. Moreover, according to the invention, said composition can further comprise co-formulants capable to promote LPI25 reactivation.In addition, according to a preferred embodiment of the invention, said composition can further comprise a carrier material.

[0019] According to the present invention, said composition can be in lyophilized, liquid, or frozen form.

[0020] The present invention also concerns the use of the Lactiplantibacillus plantarum LPI25 as defined above or of a composition as defined above as an antimicrobial agent in a dairy product or in dairy production or in a dairy production process.

[0021] In particular, the dairy product can be cheese, preferably hard cheese or semi-hard cheese, for example Grana type cheese.

[0022] According to the present invention, said Lactiplantibacillus plantarum LPI25 or composition can be used as an anti-Clostridium agent, in particular as an anti-Clostridium tyrobutyricum agent. In particular, the Lactiplantibacillus plantarum LPI25 or composition according to the present invention are able to inhibit Clostridium bacteria, in particular Clostridium tyrobutyricum, also in the form of spores.

[0023] It is a further object of the present invention a method for producing a dairy product, preferably cheese, more preferably hard cheese or semi-hard cheese, for example Grana type cheese, said method comprising the step of a) adding Lactiplantibacillus plantarum LPI25 as defined in claim 1 or a composition as defined in any one of claims 2-5 in a tank containing milk, preferably pasteurized milk, and subsequently b) inducing coaugulation of the milk obtained in step a).

[0024] According to the present invention, step b) can be carried out by adding a coagulant. Preferably, step a) is carried out about 30-50 minutes before step b).

[0025] According to the present invention, the method can also comprise a sub-step of hydrating the strain before adding it to the milk.

[0026] According to the method of the invention, said Lactiplantibacillus plantarum LPI25 strain can be added in combination with other conventional starter cultures strains.

[0027] In fact, the strain LPI25 according to the invention can be advantageously used together with other starter culture strains, since it does not have any antagonistic action towards said strains.

[0028] The present invention also concerns a dairy product, preferably cheese, more preferably hard cheese or semi-hard cheese, for example Grana type cheese,comprising Lactiplantibacillus plantarum LPI25 as defined above. More in detail, the diary product according to the invention can comprise vital and / or dead cells of Lactiplantibacillus plantarum LPI25 and / or genetic material belonging to Lactiplantibacillus plantarum LPI25 strain.

[0029] The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings, wherein:

[0030] - Figure 1 shows box and whiskers showing the intensity of antimicrobial activity (expressed as size of halo of inhibition) against Clostridium tyrobutyricum of Lactiplantibacillus plantarum LPI25 strain and of the known strains NIS_T_pc, BAC_T_pc and BAC_C_pc;

[0031] - Figure 2 shows a scatter dot plot showing mean, standard deviation and minimum and maximum values of the antimicrobial activity expressed by the post-biotics of the four investigated cultures (LPI25, NIS_T_pc, BAC_T_pc and BAC_C_pc);

[0032] - Figure 3 shows the antimicrobial activity expressed by the post-biotics of different microbial cultures (LPI25, NIS_T_pc, BAC_T_pc and BAC_C_pc) after being treated at different time-temperature combinations;

[0033] - Figure 4 shows a scatter plot (showing average value) of the level of the different metabolites detected in post-biotics from four anti-Clostridium strains (LPI25, NIS_T_pc, BAC_T_pc and BAC_C_pc);

[0034] - Figure 5 shows a radar graph representing the effect of strain LPI25 on sensory attributes;

[0035] - Figure 6 shows box and whiskers showing the compatibility values of strain LPI25 with three different conventional starter cultures commonly used in cheesemaking processes. In all cases, the strain shows values above 0.1, denoting excellent compatibility;

[0036] - Figure 7 shows a line plot showing Clostridia trend during the ripening of hard cheeses intentionally inoculated with Clostridium spores and without any protective agents (Control) or plus lysozyme (Lysozyme), BAC_C_pc, BAC_T_pcor LPI25 as protective agent; and

[0037] - Figure 8 shows the centre section of cheeses obtained at different ripening months from the batches (lysozyme, LPI25, BACT_T_pc and BAC_C_pc).EXAMPLE 1: Study of the anti-Clostridium activity, technological suitability, safety and action on hard cheese of the strain Lactiplantibacillus plantarum LPI25 according to the present invention.

[0038] Microbial Strains

[0039] Microbial cultures with potential protective activity were evaluated for their ability to inhibit Clostridium tyrobutyricum, which is responsible for the late blowing defect.

[0040] Anti-Clostridium tyrobutyricum strains

[0041] Lactiplantibacillus plantarum LPI25 strain, previously isolated from naturally fermented dairy products and selected for its antagonistic activities against undesirable micro-organisms, was evaluated for its ability to inhibit the etiological agent of late blowing defect in cheeses.

[0042] Three protective strains, detailed below, were used as control strains:

[0043] - NIS_T_pc: nisin-producing Lactococcus lactis strains from a commercial formulation;

[0044] - BAC_T_pc: a Lactococcus lactis strain from a commercial formulation producing bacteriocins other than nisin;

[0045] - BAC_C_pc: Lactiplantibacillus plantarum K46 strain, belonging to the collection of the AAA Department of the University of Molise and producing bacteriocin-like antimicrobial metabolites.

[0046] Indicator strain

[0047] Clostridium tyrobutyricum DSM 2637 was used as indicator (target) strain causing late blowing defect. The strain was cultivated in Reinforced Clostridial Medium (RCM, Oxoid - Italy) at 37 °C for 48 h in anaerobic conditions (jars with an H2 plus CO2 generating kit -AnaeroGen, Oxoid, Basingstoke, UK). Bacteria were stored at -80°C as stock cultures in their corresponding culture media supplemented with 5% glycerol, and sub-cultured twice before their use. To give rise to LBD in cheese, a stock of C. tyrobutyricum spores in Skim Milk (Oxoid) was prepared.

[0048] Detection and characterisation of anti -Clostridium activity

[0049] Growing cells activityThe antimicrobial activity expressed by growing cells of producers against C. tyrobutyricum was evaluated in plates using the spot-on-the-lawn technique. For this purpose, overnight pre-cultures in de Man, Rogosa and Sharp medium (MRS, BD Difco, Detroit, USA) of each producer strain containing ca. 10E8 CFU / mL were spotted (10pL) in plates containing 10 mL of nutrient agar (Oxoid) and incubated at 28 °C for 24 hours under anaerobic conditions. After incubation, the plates were covered with 10 mL of Reinforced Clostridial Medium (RCM, BD Difco, Detroit, USA) whit 10g / L of the agar (Oxoid Ltd., UK), previously inoculated with the indicator strain to approximately 10E7 CFU / mL. Antimicrobial activity was determined by evaluating halo size formed around the spots using Adobe Photoshop CS4 Extended software. Plate images were acquired using a calibrated GS-800 densitometer (Biorad).

[0050] As shown in Figure 1, all the strains investigated showed good inhibitory activity against the Clostridium tyrobutyricum strain. It is quite evident that no differences in anti-Clostridium tyrobutyricum activity were found between the various strains.

[0051] Dose-related anti-clostridium activity

[0052] The antimicrobial activity of the producer strains utilized in five different doses (8, 7, 6, 5 and 4 log CFU / mL) was tested against the indicator strain which was used in the concentration of 4, 5 and 6 log CFU / mL. Therefore, 15 different combinations were assayed for each producer strain. The antimicrobial activity was evaluated in plates by the spot-on-the-lawn technique as reported above. The results of the dosedependent anti-Clostridium activity are shown in Table 1. In particular, Table 1 shows the intensity of inhibitory activity against Clostridium tyrobutyricum as a dosedependent function of the producer and indicator strains.

[0053] Table 1

[0054] STRAINS indicator Clostridium tyrobutyricum concentrations

[0055] producers 6 5 4

[0056] (log CFU / mL)

[0057] 8 0,32 0,4 1,01 7 0,23 0,34 1 LPI2560,2 0,3 0,65

[0058] 5 0,05 0,22 0,3 4 0,01 0,1 0,23

[0059]

[0060] 8 0,18 0,25 0,34

[0061] 7 0,1 0,2 0,25 NIS_T_pc 6 0,1 0,15 0,21

[0062] 5 0,1 0,12 0,2 4 0,08 0,12 0,16 8 0,3 0,39 0,82 7 0,18 0,35 0,58 BAC_T_pc 6 0,1 0,28 0,41

[0063] 5 0,05 0,25 0,38 4 0,2 0,13 0,25 8 0,27 0,35 0,9 7 0,2 0,35 0,9 BAC_C_pc 6 0,12 0,3 0,48

[0064] 5 0,07 0,15 0,26 4 0,02 0,09 0,21

[0065]

[0066] The particular experimental design adopted highlighted differences between the various strains and allowed to identify for each strain the charge levels useful in exhibiting significant an -Clostridium tyrobutyricum activity. The most notable differences emerged when the Clostridium tyrobutyricum strain was used in doses of 6 log CFU. In this case, the NIS_T_pc culture showed no significant inhibitory activity, whereas the LPI25, BAC_C_pc and BAC_T_pc coltures produced a significant inhibition when used at concentrations of 10E6, 10E7 and 10E8, respectively.

[0067] Postbiotics Activity

[0068] The antimicrobial activity of the post-biotics (cell free supernatant), broth cultures of cell-free test strains, was tested.

[0069] 5 mL of the overnight broth culture of each strain was centrifuged for 4 minutes at 4 °C at 10000 rpm and the supernatant obtained was microfiltered through a sterile 0.22 pm filter.

[0070] 1 mL of C. tyrobutyricum overnight culture was collected and transferred to a plate containing 19 mL RCM agar. When the medium had solidified, four small holes were punched and 50 pL of the supernatant from the Lpb. plantarum strain wasadded. The plates were incubated anaerobically at 37 °C for 24 h in anaerobic conditions. At the end of the incubation period, any inhibition halos were measured using using Adobe Photoshop CS4 Extended software.

[0071] The results (figure 2) show that all post-biotics exhibit anti-Clostridium tyrobutyricum activity with an intensity comparable to that previously exhibited by the growing cells of the producing strains.

[0072] Thus, it is possible to assume that the inhibitory activity is not due to a simple competition between cells but to the production of extracellular metabolites.

[0073] Metabolic profile of post-biotic

[0074] To identify bioactive metabolites produced by the two novel strains, postbiotics from Lpb. plantarum LPI25 and Lpb. plantarum K46 strains were collected by bacterial cultures centrifugation (8000 x g, 10 min, 4 °C) and filter-sterilized using 0.2pm syringe filters (Sartorius Ministart Syringe hydrophilic filter). Each postbiotics(CFS) was characterized as follows:

[0075] - temperature test. Specifically, 1 ml of CFS was incubated at temperature from 4 °C to 80 °C, for 1 h; 90 °C, and 100 °C for 30 minutes, and 121 °C for 15 minutes, and the antimicrobial activity against C. tyrobutyricum was evaluated according to Sorrentino et al. (2018).

[0076] - bioactive molecule size determination. The analysis of the size of the hypothetical bacteriocin produced was performed using two different columns with filtration membranes of different sizes. The ultrafiltration devices were used for the concentration and purification of all supernatants.

[0077] 12 ml of supernatant was added to the column Sartorius Vivaspin 20 100 KDa to 3KDa and centrifugated at 4500xg for 30 minutes at 4°C. A second filtration was performed using Amicon Ultra Centrifugal Filter Ultracel -3K Sigma Aldrich and centrifugated at 4500xg for 30 minutes at 4°C.

[0078] - analysis of Organic acids. Liquid chromatography (HPLC) coupled with quadrupole-time-of-flight mass spectrometry was applied. Compounds were separated at 30 °C on a Zorbax Extend C18 column (201 x 150 mm, 5pm) equipped with a pre-column (2.1 x 12.5mm, 5pm, Agilent Technologies), using a binary solvent gradient (A: water with 10 mM ammonium formate and 0.1% formic acid; B: acetonitrile with 0.1% formic acid). The mass spectrometer was operated in negative electrospray ionization mode.Compounds were identified and quantified using a standard mixture of the standard organic acids at 0, 1, 5, 10, 30 and 50 ppm.

[0079] - analysis of short-chain fatty acid (SCFAs). Each supernatant was analyzed by Gas Chromatography (GC) to detect Short-chain fatty acids (SCFAs). FID (Flame Ionization Detector) (LISTA GCFID2 EQ-292-CCE) with column DB- WAXtr (100%polietilenglicol, 60m, 0.325x0.25) was used for the SCFAs analysis in the different CFS samples.

[0080] The overall results of these investigations show that the LPI25 strain differs from all other protective cultures in terms of the metabolites produced involved in antimicrobial activity.

[0081] The results shown in Figure 3 indicate that the anti-Clostridium action expressed by strain LPI25 is attributable, unlike that expressed by all the other strains considered, to thermostable metabolites.

[0082] In fact, on one hand, the anti-Clostridium action of post-biotics from commercially available protective culture strains (NIS_T_pc and BAC_T_pc) or from collections known for their anti-Clostridium action (BAC_C_pc) was impaired when the relevant CFS were treated at 90 °C or higher temperatures; on the other hand, the post-biotic from strain LPI25 showed good antimicrobial activity even when treated with the most severe time-temperature combinations.

[0083] Based on the results shown in Figure 3, it appears crucial to understand the difference between the different strains in their post-biotics by investigating the presence of other organic compounds such as organic acids, short-chain fatty acids etc.

[0084] The results shown in Figure 4 show that the post-biotic strain LPI25 differs from the others in the higher presence of propionic, isobutyric, valeric, isovaleric, caproic and phenyl-lactic acid.

[0085] The particular combination propionic: isobutyric:phenyl-lactic:valeric:caproic: isovaleric acid in the ratio 4:2:2: 1.5:1:1 is responsible for the effective anti-clostridium activity. Indeed, antimicrobial plate tests have shown that when the concentration of one of the metabolites changes (decreases), the antagonistic action is significantly impaired.

[0086] Evaluation of Lpb. plantarum LPI25 technological suitability Acidifying activity and effect on sensorial attributesThe acidifying effect of the selected Lpb. plantarum strain was evaluated in a milk model. For this purpose, a substrate containing 10% Skim Milk power (Biolife) was prepared and inoculated with 1% of the bacterial culture. Periodically, at 0, 1, 2, 3, 4, 5, 6, 7, 8 h and after 24 h of incubation at 30 °C, the pH was measured with a pH meter (FiveEasyplus, Mettler Toledo) and a panel test was performed to evaluate the sensory characteristics.

[0087] To assess the different sensory characteristics of the sample at the end of the acidification process, a sensory analysis was carried out by 25 trained tasters. A tasting sheet was created to measure the intensity of each selected descriptor using an unstructured intensity scale presented on a wheel. Three replicates of the sample were sensory analysed. The sensory attributes assessed as olfactory descriptors were fresh cheese, herbaceous, floral, animal, barnyard / sheep and buttery. The scores used ranged from 0 (no perception) to 9 (highest perception). Strain LPI25 exhibited moderate acidifying activity fully compatible with that expressed by the starter culture conventionally used in cheese-making processes.

[0088] Furthermore, its action has no negative effect on the sensory profile.

[0089] The results shown in Figure 5 highlight how the strain positively influenced the most sought-after sensory attributes without causing any undesired effect Detection of Interactions between Lactoplantibacillus plantarum LPI25 Strains and Conventional Starter

[0090] The effect of Lpb. plantarum strain on the growth of conventional starter cultures was assessed in skin milk inoculated with growing cells of indicator strains combined with:

[0091] - cell-free supernatants of producer strain;

[0092] - growing cells of producer’s strain.

[0093] The microbial growth of conventional starter culture (thermophilic lactic acid bacteria strains) after incubation in skin milk at 35 °C for 30 h was ascertained by plate counts performed in MRS agar and in M17 agar. Based on these values, an arbitrary index was estimated and calculated as follows:

[0094] >.,... ^endjexp ~ ^0_ic

[0095] Compatiabihty = — - - -

[0096]

[0097] ‘end tc ' 0 ic

[0098] where Yend c and Yendjexp represents the count levels of indicators cells at the end of incubation alone or in combination with Lpb. plantarum CFS, respectively, and Yo_ic represents the count levels of indicator cells in the batches at the beginningof the incubation (tO) without Lpb. plantarum CFS. Values lower than 0 indicate inhibitory action against the indicators, whereas positive values correspond to a stimulation effect of producers on indicator strains. Values close to zero correspond to a neutral interaction. Specifically, considering previous results, the following breakpoints were defined:

[0099] - high compatibility: Indicator compatibility ≥ 0.1;

[0100] - moderate compatibility: -0.1 < Indicator compatibility < 0.1;

[0101] - low compatibility: Indicator compatibility ≤ −0.1.

[0102] Values showing the compatibility of the Lpl25 strain with cultures conventionally used in the cheesemaking of hard cheeses are shown in Figure 6.

[0103] The LPI25 strain showed excellent compatibility with the starter cultures without experiencing any competition. Even a slight growth stimulation of the strains that make up the conventionally used starter cultures was appreciated.

[0104] Evaluation of Lpb. plantarum LPI25 Safety

[0105] Antibiotic Sensitivity

[0106] Sensitivity to antibiotics was assessed by the E-test method (Epsilometer test) (Biomerieux, Marcy-l'Etoile, France), a quantitative method based on a plastic strip with a predetermined antibiotic gradient, fixed on one side, and MIC interpretation scale printed on the other. The method is based on the diffusion of antibiotics from a plastic strip into an agar medium, thus providing quantitative and repeatable MICs results for the isolates tested.

[0107] The results (not shown) highlighted that the strain was sensitive to all antibiotics tested. Thus, LPI25 does not manifest antibiotic resistance Biogenic Amines

[0108] The ability of the strains to produce biogenic amines was evaluated by qualitative analysis in a specific culture medium prepared as described below according to the protocol of Bover-Cid et al., (1999). The medium contained the amine precursor amino acid and an indicator (bromocresol purple), so any positive result was evidenced by the turning of the culture medium from yellow to purple; specifically, the production of the biogenic amines results in an alkalization of the medium. Only in the specific case of tyrosine, which shows low solubility, and the culture medium is opaque, is the positive result evidenced by the appearance of a clarifying halo. Culture medium without the precursor amino acids was used as anegative control. Results (data not shown) showed that strain LPI25 does not produce biogenic amines, therefore confirming the safety of the strain.

[0109] Formulation of Anti -Clostridium tyrobutyricum

[0110] The protective formulation was prepared with the lyophilised culture of LPI25 so that after hydration for 30 minutes it is able to live and viable and produce (among the different metabolites) propionic, isobutyric, phenyl-lactic, valeric, caproic and isovaleric acids in a 4:2:2: 1.5:1 ratio.

[0111] Evaluation Anti-Clostridium tyrobutyricum action in hard cheese To investigate the inhibition of C. tyrobutyricum by the Lpb. plantarum formulation LPI25, a specific challenge test on pilot scale was conducted involving the intentional use of Clostridium botulinum in spore form. The cheesemaking was carried out with pasteurised cow's milk (72 C° for 20 seconds), with rennet and starter culture (7 log CFU / mL) added, and divided into five independent batches, each with 100 L of pasteurised milk, as follows:

[0112] 1. control batch: the Clostridium tyrobutyricum 2637 spore suspension (1000spore / L) was added without the use of any preservative;

[0113] 2. lysozyme batch: C. tyrobutyricum spores suspension and lysozyme (2.5 g / L milk) were added;

[0114] 3. LPI25: C. tyrobutyricum spores and LPI25 protective culture (7 log CFU / mL) were added,

[0115] 4. BAC_T_pc batch: C. tyrobutyricum and commercial protective culture BAC_T_pc were added;

[0116] 5. BAC_C_pc: C. tyrobutyricum spores suspension and protective culture BAC_C_pc were added.

[0117] The production technology of the hard cheese included scalding the curd at a temperature of 50 °C, pressing, forming and salting in brine (22%). The obtained cheeses were subjected to microbiological analysis of spore count at 6 hours after formation, after salting at 1 day and throughout the aging time. Ripening was conducted at 12 °C and 85% relative humidity for 180 days.

[0118] Figure 7 shows the development of Clostridium tyrobutyricum, intentionally inoculated in the form of spores in milk, in cheeses prepared without protective agents (bach control) or with the use of protective agents such as lysozyme (lysozyme batch), or the protective cultures LPI25 (LPI15 batch), BAC_T_pc (BAC_T_pc) or BAC_C_pc).Significant differences (p < 0.05) were found as function of time and batch. In particular, the control batch showed an increase in Clostridium levels over three logarithmic cycles during the observation period. A significantly different trend (p > 0.01 ) from that characterizing the above-mentioned batches was shown by samples from the Lysozyme and LPI25 batches. The latter batches showed a containment of Clostridium loads and then a significant decrease in load levels. In particular, a decrease in charge levels was observed in both batches, which was not detectable at the end of the observed maturation period. The inhibition of Clostridium tyrobutyricum prevented the occurrence of the late blowing defect (Image 1). Interestingly, the late blowing defect was also found in the samples from the batches with the bacteriocin (BAC_T_pc) or bacteriocin-like (BAC_C_pc) producing protective cultures.

[0119] It is therefore clear that the use of a protective culture capable of producing primary metabolites with antimicrobial activity is a bio-protective strategy of extraordinary industrial interest.

[0120] References

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[0129] 10. Akbal, S., & Oner, Z. Inhibition of Clostridium species responsible for late-blowing defect using different antimicrobial agents. Pakistan Journal of Agricultural Sciences, 2024, 67(1).

Claims

CLAIMS1) Lactiplantibacillus plantarum LPI25 strain deposited in the Spanish Type Culture Collection with accession number 31123.2) Composition, such as a starter culture comprising the Lactiplantibacillus plantarum LPI25 strain as defined in claim 1.3) Composition according to claim 2 further comprising one, more than one or all the following compounds: propionic acid, isobutyric acid, phenyl-lactic acid, valeric acid, caproic acid, isovaleric acid.4) Composition according to any one of claims 2-3, said composition further comprising a carrier material.5) Composition according to any one of claims 2-4, said composition being in lyophilized, liquid, or frozen form.6) Use of the Lactiplantibacillus plantarum LPI25 as defined in claim 1 or of a composition as defined in any one of claims 2-5 as an antimicrobial agent in a dairy product or in dairy production.7) Use according to claim 6, wherein the dairy product is cheese, preferably hard cheese or semi-hard cheese, for example Grana type cheese.8) Use according to any one of claims 6-7, wherein said Lactiplantibacillus plantarum LPI25 or composition are used as an anti-Clostridium agent, in particular as an anti-Clostridium tyrobutyricum agent.9) Method for producing a dairy product, preferably cheese, more preferably hard cheese or semi-hard cheese, for example Grana type cheese, said method comprising the step of a) adding Lactiplantibacillus plantarum LPI25 as defined in claim 1 or a composition as defined in any one of claims 2-5 in a tank containing milk, preferably pasteurized milk, and subsequently b) inducing coaugulation.10) Method according to the preceding claim, wherein said Lactiplantibacillus plantarum LPI25 strain is added in combination with other starter cultures strains.11) Dairy product, preferably cheese, more preferably hard cheese or semi-hard cheese, for example Grana type cheese, comprising Lactiplantibacillus plantarum LPI25 as defined in claim 1.

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