Advantages and disadvantages of autochthonous enterococci strains for their potential use in cheese ripening: a preliminary study

Summary The enzymatic activity exhibited by native enterococci could, in turn, result in a reduction in the cheese rip-ening time. However, they may also carry virulence factors or resistance to antibiotics that prevent their use in food. A total of 69 enterococci isolates were obtained from a Spanish traditional raw goat’s milk cheese and identiﬁed by Maldi-Tof/MS. Diﬀerent enzymatic activities of technological interest were evaluated and the safety of those with greater technological suitability was studied. The strains were identiﬁed as E. malodoratus , E. italicus , E. gilvus , E. faecium , E. durans , E. casseliﬂavus and E. faecalis . Some strains had very high acidifying and proteolytic activities. Most strains had medium or low amino and carboxy-peptidase activities and high dipeptidase activities, while esterase activities were medium. A total of 10 strains were selected based on their enzymatic activities. Of these, ﬁve showed b -haemolysin activity, eight showed gelatinase activity and eight produced tyramine. These activities represent virulence factors that could potentially aﬀect food safety. Finally, all selected strains were susceptible to the antibiotics tested.


Introduction
The genus Enterococcus is the third largest genus of lactic acid bacteria (LAB) with about 50 species.Enterococci are Gram-positive catalase-negative cocci that are usually present in large numbers in vegetables and foods, especially those of animal origin, such as raw dairy products (Oladipo et al., 2015;Ben Bra€ ıek & Smaoui, 2019).
It is well established that enterococci are among the microorganisms that gain access to milk and cheese with contaminated water, air, manure or processing equipment during the production of these foods.However, it should be noted that enterococci in food are not always due to faecal contamination and there is currently no limit for their presence in foods (Commission Regulation, 2007).It is possible for enterococci to be present in large numbers in raw milk and dairy products (up to 10 8 cfu g À1 ).They are ubiquitous in cheeses made from raw milk, cow, sheep and goat milk.They are characteristic of Mediterranean raw milk cheeses and possess an exceptional ability to survive in adverse conditions.
Enterococci contribute to the development of cheese flavour and texture through their proteolytic and esterolytic activities and play an important role in modulating the cheese microbiota (Ben Bra€ ıek & Smaoui, 2019;Câmara et al., 2020).Enterococci exhibit acidifying, proteolytic and lipolytic activity and undoubtedly contribute to the development of the sensory characteristics of the cheeses in which they are present as part of the natural microbiota.Depending on their concentration and enzymatic activity, they can contribute positively to flavour or negatively, contributing to the development of bitter flavours or generating biogenic amines.On the other hand, several strains of enterococci producing broad-spectrum bacteriocins (enterocins) with antimicrobial activity against pathogenic and spoilage microorganisms have been described (Oladipo et al., 2015;Khalkhali & Mojgani, 2017).Moreover, enterococci isolated from dairy products could survive the conditions of the gastrointestinal tract, demonstrating probiotic properties (Terzi c-Vidojevi c et al., 2021).Despite their desirable technological characteristics in cheeses, the selection of Enterococcus spp.strains for use as starter cultures remains a matter of debate, due to the uncertainty on their safety (Ramos et al., 2020).Additionally, they carry virulence factors and can be opportunistic pathogens.These virulence factors are present in virtually all enterococci, and the current question is to what extent these virulence factors can be expressed in cheese and compromise its safety (Dapkevicius et al., 2021).The genus Enterococcus includes strains that are opportunistic pathogens causing several diseases in humans as bacteremia, endocarditis and other infections.Thus, Enterococcus genus does not have Qualified Presumption of Safety (QPS) status in the European Union and are not Generally Regarded as Safe (GRAS) in the United States of America (Russo et al., 2018;de Sousa et al., 2020).They can be reservoirs of antibiotic resistance and virulence genes and are intrinsically resistant to several antibiotics (de Sousa et al., 2020;Terzi c-Vidojevi c et al., 2021).Moreover, they can easily accumulate mutations and exogenous genes that confer additional resistances (Russo et al., 2018).In recent decades, one of the major resistance implications that have emerged are vancomycin-resistant strains of enterococci, causing major problems in infection control (Khalkhali & Mojgani, 2017).On the other hand, the enterococci decarboxylase systems may lead to the accumulation of hazardous biogenic amines (BA) and the consumption of food products with high concentrations of BA may result in toxicological effect.Numerous studies have reported that enterococci have often been identified as one of the main BA producers in cheese (histamine, tyramine, 2-phenylethylamine, cadaverine and putrescine) (Park et al., 2020;Dapkevicius et al., 2021).Despite the safety implications of enterococci, these microorganisms are part of the microbiota present in raw milk cheeses, contributing to their ripening through their enzymatic potential (Terzi c-Vidojevi c et al., 2021).Some authors advocate their use in the dairy industry with caution, considering that the desirable or undesirable aspects are strain-dependent characteristics and studies indicate that some strains may be safe for use as starter cultures or probiotics as they do not carry virulence factors and antibiotic resistance genes (Terzi c-Vidojevi c et al., 2021).Moreover, there is currently a growing interest in incorporating native starter and non-starter strains in cheese cultures.It is important to consider that they may have enzymatic activities that have an impact on cheese ripening.In relation to this, the technological role of enterococci has been suggested.However, as they are not recognised as safe, their use as secondary adjunct cultures for cheeses is compromised.Nevertheless, they could be selected so that they do not contain virulence factors or produce biogenic amines that could compromise the safety of the product, depending on the concentrations in which they are present.
A preliminary study is necessary to ascertain the characteristics of enterococci strains that may or may not permit their inclusion as culture starters and to subject them to cheese-making trials.Therefore, this study aimed to evaluate the enzymatic activities of several autochthonous strains of Enterococcus and select those with the best technological properties to decide whether they can be used as ripening agents, considering the presence or absence of virulence factors.

Indigenous enterococci strains
The present study included 69 isolates of enterococci from Spanish raw goat milk cheeses (Armada cheese), previously isolated and identified by classical methods (Tornadijo et al., 1995).The manufacturing process of this traditional cheese includes a predominantly lactic coagulation and involves draining the curd in sheets and then salting and soaking the curd until it was moulded.It is manufactured only by artisanal methods in the eastern mountains of the province of Le on, in north-western Spain.The strains were isolated during the production and ripening of an artisanal goat's cheese (Armada cheese).The strains were isolated on different culture media (M17, MSE and KAA) from the spring-summer and autumn-winter batches and at different stages of cheese manufacture and ripening.The origin is indicated in the modified Table 1.time-of-flight mass spectrometry).Mass spectra were acquired using a Microflex TM mass spectrometer (Bruker Daltonik, Germany) and compared with the reference database (Bruker Daltonik, Germany).

Technological characterisation of enterococci strains
Growth ability for different temperatures and NaCl concentrations Growth in Tryptone Soya Broth (TSB) (Oxoid, Unipath Ltd., Basingstoke, UK) at different temperatures (10 °C, 40 °C and 45 °C for 7 days) was determined following the method described by Albayrak & Duran (2021).The analysis of salt tolerance was carried out as described De Almeida J unior et al. (2015).In both cases, growth was determined by observing the appearance of turbidity.

Acidifying activity
To assess acidifying activity, 0.5 mL of activated cultures in MRS broth (Oxoid, Unipath Ltd., Basingstoke, UK) were inoculated into tubes with 50 mL of sterile skim milk (reconstituted to a 10%).9 mL of the milk culture was sampled at 4, 8 and 24 h of incubation at 37 °C and the pH and titratable acidity analysed.

Proteolytic activity
Extracellular quantitative proteolytic activity was determined as described Church et al. (1983) using o-pthaldialdehyde (OPA).The absorbance results at 340 nm were quantified as mmol of Glycine L À1 of milk using a calibration curve performed with glycine.

Intracellular proteolytic and esterolytic activities
Cell-free extracts (CFE) of the enterococci isolates were obtained as described Abarquero et al. (2022) and protein concentration of the CFE were analysed according to Lowry et al. (1951).
The aminopeptidase (AP), carboxypeptidase (CP) and dipeptidase (DP) activities were analysed by the methodology described by Abarquero et al. (2022).One unit of AP activity (U) was defined as the increase in absorbance of 0.001 units at 410 nm in 1 min.One CP activity unit (U) corresponded to a 0.01 units increase in absorbance at 570 nm for 1 min.Finally, one DP activity unit (U) to a 0.1 units increase in absorbance at 507 nm for 1 min.AP, CP and DP were expressed as number of units (U) per mg of protein in cell-free extracts.

Isolates selection, molecular identification and PCR fingerprinting
After characterisation, best isolates (at least one of each species) were selected on the basis of the results of each of the enzyme activities studied and were identified at the molecular level by 16S rRNA gene sequencing and random amplification of polymorphic DNA-PCR (RAPD-PCR) was used for genetic typing.For typing, BoxA2R, M13 and OPA18 primers were used.The obtained banding patterns were subjected to clustering.The analysis of similarity in patterns was carried out using GeneTools software version 4.03 (SynGene, Cambridge, UK).The primers used for 16S rRNA gene sequencing and RAPD-PCR are listed in Table 2.

International Journal of Food Science and Technology 2024
Safety evaluation of the selected enterococci strains

Phenotypical assessment of virulence factors
Evaluation of haemolytic activity was carried out by culturing selected enterococci strains on blood agar plates (Condalab, Madrid, Spain) incubated at 37 °C for 48 h according to the fabricant recommendations.Haemolytic reaction was evaluated as follows: a-haemolysis: characterised by the formation of a green zone as a result of partial hydrolysis of red blood cells; bhaemolysis: formation of a clear zone around the bacterial colony by complete hydrolysis of the red blood cells; c-haemolysis: no changes in reaction.
DNase activity was detected by the strains culture on DNase agar (Condalab) for 48 h at 37 °C as described by Câmara et al. (2020).After application of a 1 mol L À1 HCl solution, the reaction was considered positive if pink areas appeared around the colonies.
Finally, the methodology described by Câmara et al. (2020) was used for the study of gelatinase activity.Gelatinase production was demonstrated by the formation of transparent halos around the colonies after flooding with saturated ammonium sulfate.

Resistance/susceptibility to antibiotics
The minimum inhibitory concentrations (MIC), defined as lowest concentration (lg mL À1 ) at which no visible growth was observed, of six antibiotics (penicillin-G, clindamycin, cefotaxime, tetracycline, gentamycin and vancomycin) in the selected enterococci strains were analysed by the MICEvaluator (MICE) system (Oxoid).Briefly, individual colonies were suspended in sterile saline (NaCl 0.9%) to McFarland's standard 1 and incubated for 48 h at 37 °C in MRS agar plates with a strip of the required antibiotic.The MIC was determined as the first concentration at which the inhibition halo contacted the antibiotic strip.

Biogenic amines production
BAs study was assayed in MRS broth supplemented with 1 mmol L À1 of Tyr, His, Lys or Orn (Sigma-Aldrich).After incubation for 48 h at 37 °C, the cultures were centrifuged (15 000 9 g for 10 min).Supernatants were filtered through a 0.22 lm cellulose membrane (Millipore, Burlington, USA) and derivatised with DEEMM (Ethoxymethylenemalonic acid diethyl ester; Sigma-Aldrich).BAs were identified and quantified by ultra-high performance liquid chromatography (UHPLC) according to Redruello et al. (2013).

Statistical analysis
All technological characterisation tests were carried out in triplicate and those for biogenic amines were carried out in duplicate.SPSS v.25 software (SPSS, Chicago, USA) was used for statistical analysis.Kruskal-Wallis test was applied to identify statistical differences (P < 0.05) between isolates of the same species, and a post hoc Mann-Whitney to determine differences between groups.The AP, DP and esterase activities were performed by factor analysis with principal component extraction (FAEPC).

Identification of enterococci strains
The 69 native enterococci, identified at the species level by Maldi-Tof/MS (Table 3), were assigned to the following species: E. malodoratus (1), E. italicus (1), E. gilvus (24), E. faecium (1), E. durans (6), E. casseliflavus (1) and E. faecalis (35).These species represent some of the most isolated enterococci found in raw milk cheeses (Fuka et al., 2017;Terzi c-Vidojevi c et al., 2020).However, a higher number of E. gilvus strains were found.All E. gilvus strains of included in this study were isolated on MSE agar at different times of ripening and from different batches of these artisanal goat cheeses (Table 3).This species was described for the first time in 2002 (Tyrrell et al., 2002) and then, isolated from meat and cheese products by Mart ın et al. (2009) and Zago et al. (2009).Other authors also isolated E. gilvus in cheeses made from raw milk, with levels between 0.1% and 10% of the total enterococci counted in KAA in the cheeses studied (Margalho et al., 2020).The presence of E. gilvus in cheeses may have a positive impact from a technological and nutritional point of view, as it is attributed to the ability to produce carotenoids, which may act as free radical chelators in the oxidation process (Zago et al., 2009).In relation to the production of carotenoid compounds in cheeses, smear ripened cheeses are a source of carotenoids from the microbial biomass of the cheese.As mentioned above, E. gilvus also has this capacity and can be a source of carotenoids of technological interest as a source of colorants, food supplements or pharmaceuticals (Yeluri Jonnala et al., 2021).Indeed, there is a wide range of benefits associated with carotenoids, some of which act as precursors of vitamin A, which in turn is positively involved in the functioning of the immune system, vision and regulation of adipose tissue, while other carotenoids, non-vitamin A, also play beneficial roles in the body, such as improving cognitive function, reducing the risk of macular degeneration and protecting against various types of cancer (Yeluri Jonnala et al., 2021).On the other hand, its involvement in infections in humans is unclear.E. gilvus can ferment lactose with production of lactic acid, which may have an impact on lactic fermentation in cheeses.However, little information is known about the role or technological characteristics of E. gilvus in cheese or its safety implications (Zago et al., 2009).Figure 1 illustrates the dendrogram generated from the comparison of the mass spectra of the enterococci isolates included in this study, using MALDI Biotyper software.The proximity between the different isolates reflects the similarity between them, suggesting that some of them could be clones of the same strain.However, this technique is not yet fully reliable for typing strains of the same species.Although, under experimental conditions, the comparison of mass spectra obtained by this technique has been used to type methicillin-resistant Staphyococcus aureus strains, for example, the information obtained is not sufficient, which means that genetic techniques are still necessary to perform typing (Singhal et al., 2015;Gantzias et al., 2020).

Technological characterisation of Enterococcus isolates
The results of growth at different temperatures and salt concentrations are included in Fig. 1.Enterococci have generally been described as microorganisms with the ability to grow in the presence of adverse conditions such as 6.5% NaCl and temperatures of 45 °C.In view of the results, all isolates grew at both 10 °C and 40 °C.However, at 45 °C, E. gilvus isolates were not able to grow, as well as 10 isolates of E. faecalis isolates and the E. malodoratus and E. italicus isolates.The 12 strains identified as E. gilvus were not ascribed by classical methodology to this species, since their previous identification dates to before 2002, when this species was described (Tyrrell et al., 2002), and their particularity with respect to the rest of enterococci was that they did not grow at 45 °C or at 40 g L À1 NaCl, as well as the isolate of E. malodoratus.
Finally, growth on medium supplemented with 65 g L À1 NaCl was not recorded in seven isolates of E. faecalis, 15 isolates of E. gilvus and the isolate of E. italicus.In summary, E. gilvus, E. malodoratus TAUL1318 and E. italicus TAUL250 were not able to survive in the most extreme culture conditions (45 °C and 65 g L À1 NaCl).These results suggest that enterococci are highly adaptable microorganisms that are able to resist harsh environmental conditions (Russo et al., 2018).However, some differences were observed among the six species studied.
The LAB proteolytic activity is one of the most outstanding enzymatic properties of strains intended for cheese production, since casein hydrolysis plays a key role in the sensory characteristics of cheese (Frau et al., 2016).Regarding this matter, it can be stated that the main involvement of enterococci is in the proteolysis of cheese and, consequently, in the texture and volatile components.From this point of view, they could intervene by reducing the ripening time.
Proteolytic activity (Table 5) showed significant differences (P < 0.001) among the species studied.In general, low proteolytic activity (<1 mmol Gly L À1 milk) predominated.However, nine E. faecalis isolates (TAUL42, TAUL44, TAUL46, TAUL49, TAUL50, TAUL207, TAUL261, TAUL807 and TAUL1400) showed very high proteolysis values (above 4 mmol Gly L À1 milk), being E. faecalis TAUL50 the one reporting the highest activity value (9.14 AE 0.59 mmol Gly L À1 milk).The results are consistent with those of other studies that have reported an overall poor proteolytic activity in enterococci isolates, except for some isolates of E. faecalis.(Albayrak & Duran, 2021).Similarly, differences between strains of the same species were also observed in other studies (Yerlikaya & Akbulut, 2020).Notably, in our study the most proteolytic E. faecalis isolates were also the most acidifying.However, no clear relationship between proteolytic and acidifying activities was observed as previously reported by Giraffa (Giraffa, 2003).
The presence of intracellular proteolytic activities is also important during the hydrolysis of peptides generated from the hydrolysis of caseins, although it is an atypical activity in LAB (Abarquero et al., 2022).The peptidase activities of LAB have been extensively investigated.However, there are few studies specific to these  International Journal of Food Science and Technology 2024 activities in enterococci (Serio et al., 2010).The specific AP, CP and DP activities of enterococci isolates were significantly different (P < 0.001) in all activities between isolates of different species.Likewise, differences were also observed between isolates of the same species (P < 0.05) for each of the substrates tested in the three proteolytic activities.These results indicate that endopeptidase activities are strain-dependent.They highlight the interest in the selection of autochthonous enterococci strains.Aminopeptidase activity allows the release of flavour compounds and precursors (small peptides and amino acids), which also help to reduce the bitterness of cheese by hydrolysing proline-rich oligopeptides derived from casein proteolysis (Terzi c-Vidojevi c et al., 2021).The AP activities ( ).Finally, E. faecium TAUL656 showed the highest Phe-Ala activity with a value of 563.48 AE 6.65 U mg À1 .To observe the DP activities of the isolates, a FAEPC was performed (Fig. 3).It can be seen how several E. gilvus isolates were separated from the rest of the isolates because they recorded high activity values for almost all the substrates tested.These strains may be of interest for inclusion in non-starter cultures with the objective of intensifying the ripening of cheeses and thus shortening the ripening time.Unlike what was observed in other LAB genera, the results observed in enterococci isolates, isolated from traditional cheeses, showed high endopeptidase activities, especially high for CP and DP (Câmara et al., 2019;Abarquero et al., 2022).The auxotrophy of enterococci for several amino acids would explain that these microorganisms have endopeptidases to release essential amino acids for their growth in dairy products (Dapkevicius et al., 2021).
Esterase and lipase activities are important in the development of cheese aroma, especially in long-ripened cheeses (Câmara et al., 2020).Table 7 shows results of   Table 6 Dipeptidase a -and carboxypeptidase b -specific activities of crude cell-free extract of 69 enterococci isolates from raw milk cheeses

Dipeptidase activity Carboxypeptidase
Ala-Ala Leu-Gly Leu-Leu Phe-Ala Tyr-Leu N-CBZ-Leu <50 50-200 a Dipeptidase-specific activity expressed as units of enzyme activity (U) mg À1 protein.One unit of dipeptidase activity was defined as the increase in absorbance of 0.01 units at 505 nm in 1 min.b Carboxypeptidase activity expressed as units of enzyme activity (U) mg À1 protein.One unit of carboxypeptidase activity was defined as the increase in absorbance of 0.01 units at 570 nm in 1 min.International Journal of Food Science and Technology 2024 carried out to study globally the esterase activities of the isolates.Some E. gilvus isolates showed different esterase activity from other enterococci: TAUL40t, TAUL1351t and TAUL1276, as well as, E. faecalis TAUL232.In general, LAB exhibit limited enzymatic metabolism of fats (Bintsis, 2018).However, the enterococci esterolytic system is particularly more effective and complex compared to other LAB genera, with E. faecalis and E. faecium standing out as the main esterolytic species within the group of enterococci (Serio et al., 2010).

Isolates selection, molecular identification and PCR fingerprinting
From a technological standpoint, the enterococci strains were selected.For isolate selection, each enterococci isolate included in this study was given a total score (TS), Table 7 Esterase a -specific activities of crude cell-free extract of 69 enterococci isolates from raw milk cheeses obtained by adding the points of each of the activities tested (acidifying, proteolytic, aminopeptidase, carboxypeptidase, dipeptidase and esterase) according to the parameters listed in Table 8.The 13 highest scoring isolates were selected (Table 9) by comparison of total scores between isolates of the same species.
The 13 selected isolates were submitted to reidentification by sequencing the 16S rRNA gene, confirming the detection obtained by Maldi-TOF/MS, and typed to determine if any of the selected isolates were clones of the same species.After typing, 10 different RAPD profiles were obtained, taking into account the 84% identity threshold.Thus, it was established that each of these 10 profiles corresponded to different types of strains: E. malodoratus (1), E. italicus (1), E. gilvus (2), E. faecium (1), E. durans (1), E. casseliflavus (1) and E. faecalis (3).Typing revealed that E. faecalis isolates TAUL49 and TAUL261 were probably the same strain.Also, E. gilvus isolates TAUL35 and TAUL1210, as well as isolates TAUL267 and TAUL1238, could be the same strain, showing identical RAPD profile.

Safety evaluation of the selected enterococci strains
The existence of virulence factors in the enterococci strains selected in the previous section was evaluated phenotypically and the results are shown in Table 10.None of the enterococci strains produced DNase.The production of DNase allows cells to escape host defences and spread through their tissues by degrading extracellular DNA (Câmara et al., 2020).However, the presence of this activity in enterococci strains has not been described in previous works (Tsanasidou et al., 2021).On the other hand, after evaluation of haemolytic activity, a-haemolysis was not observed in any of them.Fortunately, five strains did not show haemolytic activity.However, b-haemolysis was detected in the remaining five strains: E. gilvus TAUL267 and TAUL1210, E. faecium TAUL656, E. durans TAUL210 and E. casseliflavus strain TAUL610.The presence of b-haemolysis in enterococci isolated from food has also been described in other studies and, in view of the results, these five strains with b--haemolysis activity could not be used in food production (Oruc et al., 2021).Finally, 8 of the 10 strains selected produced gelatinase.The gelatinase contributes to the virulence of the strains, as it facilitates tissue penetration by hydrolysis of collagen and gelatin (Câmara et al., 2020).
Enterococci are remarkable for their ability to exchange genes with other bacteria, thus favouring their adaptation to seemingly hostile conditions International Journal of Food Science and Technology 2024 (Dapkevicius et al., 2021).This ability to adapt, coupled with the extensive use of antibiotics in recent years in both animals and humans, has led to a widespread incidence of antibiotic resistance in food-borne isolates (Câmara et al., 2020).The susceptibility/resistance results to six clinically used antibiotics are shown in Table 10.All enterococci were sensitive to vancomycin (MIC < 30 lg), tetracycline (MIC < 30 lg), penicillin (MIC <10 lg) and cefotaxime (MIC < 30 lg).However, all enterococci strains showed resistance to gentamycin (MIC > 10 lg) and in six strains were proved resistance to clindamycin (MIC >2 lg): E. malodoratus TAUL1318, E. italicus TAUL250, E. gilvus TAUL1210, E. faecium TAUL656 and E. faecalis TAUL49 and TAUL791.Vancomycin resistance has become a major concern over the decades, causing major problems in infection control (Khalkhali & Mojgani, 2017).Enterococci are intrinsically resistant to both low levels of aminoglycosides, such as gentamycin, and low levels of clindamycin, and are of no clinical concern (Dapkevicius et al., 2021).Therefore, resistance to gentamycin and clindamycin in the enterococci strains could be classified as of low importance.
Finally, the production of biogenic amines in the selected Enterococcus strains was investigated (Table 10).Hazardous biogenic amines present in food are known to have harmful effects on human health and it is highly recommended to select strains that do not produce BA (Khalkhali & Mojgani, 2017).No histamine, putrescine, cadaverine or spermidine production was detected by any of the 10 strains selected.However, tyramine production was observed in eight of the 10 enterococci strains.Tyramine levels in the tyramine-producing strains ranging from 300.56 to 402.35 mg L À1 , with no significant differences (P > 0.05) between strains.However, problematic levels of tyramine are around two to three times higher than these levels (Paulsen et al., 2012).Tyramine biosynthesis seems to be a characteristic feature at the species level for E. faecium, E. faecalis and E. durans (Ladero et al., 2012).The results obtained in our study are consistent with this statement, and the BAs production levels observed were similar to those  previously reported by other authors (Park et al., 2020;Tsanasidou et al., 2021).

Conclusions
Enterococci strains isolated from raw milk cheeses exhibited strain-to-strain variation in their enzyme activities.For this reason, the selection of native enterococci strains for their enzymatic activity could find interesting technological applications in cheese ripening, for example, accelerating the ripening of cheeses.Some of these indigenous Enterococcus strains could be of technological and economic benefit to the pressed cheese industry, which usually has long ripening times.Their proteolytic activity, such as that exhibited by some strains, could help to reduce the ripening time, since the main effect of a higher intensity of proteolysis is on texture and the formation of flavour compounds.In this study, the selected enterococci strains were negative for DNase production, none of them showed resistance to the tested antibiotics, and five also exhibited no haemolytic activity or histamine production.In contrast, they exhibited gelatinase activity and generated tyramine in vitro.The findings of this preliminary study indicate the potential for further investigation into the influence of these strains on the cheese ripening process.However, the identification of a virulence factor in the strains may restrict their utilisation.It is important to note that an ecosystem is created within the cheese matrix, where a multitude of factors can interact and influence the evolution of microbial populations and the development of chemical and sensory characteristics.International Journal of Food Science and Technology 2024 Identification of the isolates was confirmed by Maldi-Tof/MS (matrix-assisted laser desorption/ionisation International Journal of Food Science and Technology 2024 Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).

ÓFigure 1
Figure 1 Main spectrum (MSP) dendrogram of matrix-assisted laser desorption ionisationtime-of-flight (MALDI-TOF) mass spectral profiles generated by the MALDI Biotyper.Distance is displayed in relative units.

Figure 2
Figure 2 Factor analysis with principal component extraction (FAEPC) of the aminopeptidase activities (Ala, Arg, Leu, Lys and Proaminopeptidase) by the 69 enterococci isolates from raw milk cheeses.

Ó
2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).
specific activity expressed as lmoles of b-naphthol released per min mg À1 protein of cell-free extract.b NI: number of isolates.International Journal of Food Science and Technology 2024 Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).

Table 1
Native enterococci strains, isolation medium, batch and sampling point of 69 enterococci isolated from raw milk cheeses Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).

Table 2
Primers used for the identification and PCR fingerprinting of selected enterococci isolates from raw milk cheeses International Journal of Food Science and Technology 2024 Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).

Table 3
Species, number and identification by Maldi-Tof/MS of 69 enterococci isolates from raw milk cheeses

Table 4
Acidifying activity a of 69 enterococci isolates from raw milk cheeses

Table 5
protein) and high (>200 U mg À1 protein) for most of the isolates (Table6).Particularly it was high for most of the tested substrates in the E. gilvus isolates.The highest activity for Ala-Ala and Leu-Leu substrates was recorded by E. gilvus isolate TAUL1250 (704.88AE 65.88 U mg À1 and 521.75 AE 23.52 U mg À1 , respectively).For the Leu-Gly substrate, E. gilvus TAUL1208t recorded the highest activity with 543.69 AE 5.07 U mg À1 , while for the Tyr-Leu substrate, E. gilvus TAUL1208o showed the highest activity (163.10AE 6.08 U mg À1

Table 5
Proteolytic activity a and aminopeptidase b specific activity of crude cell-free extract of 69 enterococci isolates from raw milk cheeses Proteolytic activity (mmol Gly L À1 of milk) measured using the O-phthaldialdehyde (OPA) spectrophotometric assay.b Aminopeptidase activity expressed as units of enzyme activity (U) mg À1 protein.One unit of aminopeptidase activity was defined as the increase in absorbance of 0.001 at 410 nm in 1 min.
a c NI: number of isolates.International Journal of Food Science and Technology 2024 Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).(69.90 AE 2.83 lmol b-naphthol).Finally, E. faecalis TAUL117 recorded the highest C18 activity (13.30AE 0.04 lmol b-naphthol).FAEPC analysis (Fig. 4) was

Table 8
Scoring parameters for each enzyme activity included in this study Acidifying activity measured by monitoring pH and total acidity (g lactic acid 100 mL À1 ) after 8 and 24 h of incubation.bProteolyticactivity(mmol Gly L À1 of milk).Intracellular peptidase activities expressed as units of enzyme activity (U) mg À1 protein.dEsterase-specificactivity expressed as lmoles of b-naphthol released min À1 mg À1 protein.
a c

Table 9
Enterococci isolates from raw milk cheeses, selected through the designed scoring system Total Score calculated by summing S pH + S TA + S Prot + S AP + S CP + S DP + S ES .b S pH : Acidifying activity score calculated by summing S pH8 + S pH24 .: Acidifying activity score calculated by summing S TA8 + S TA24 .S AP : Aminopeptidase activity score calculated by summing S Ala-AP + S Arg-AP + S Leu-AP + S Lys-AP + S Pro-AP .f S CP : Carboxypeptidase activity score calculated from S Leu-CP .g S DP : Aminopeptidase activity score calculated by summing S Ala-Ala + S Leu-Gly + S Leu-Leu + S Phe-Ala + S Tyr-Leu .h S ES : Esterase activity score calculated by summing S C4 + S C8 + S C14 + a TS: c S TA d S Prot : Proteolytic activity score.e International Journal of Food Science and Technology 2024 Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).
a Ó 2024 The Author(s).International Journal of Food Science & Technology published by John Wiley & Sons Ltd on behalf of Institute of Food Science & Technology (IFST).