Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening

ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 25 (2008) 366–377 www.elsevier.com/locate/fm

Influence of selected lab cultures on the evolution of free amino acids, free fatty acids and Fiore Sardo cheese microflora during the ripening Nicoletta P. Mangia, Marco A. Murgia, Giovanni Garau, Maria G. Sanna, Pietrino Deiana Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agro-alimentari, Universita` degli Studi di Sassari, V. le Italia 39, 07100 Sassari, Italy Received 31 July 2007; received in revised form 20 September 2007; accepted 30 September 2007 Available online 12 October 2007

Abstract Fiore Sardo Protected Denomination of Origin is a traditional Sardinian (Italy) hard cheese produced exclusively from whole raw ovine milk and coagulated with lamb rennet paste. Currently, Fiore Sardo is still produced by shepherds at the farmhouse level without the addition of any starter culture and the cheese-making process is characterized by significant waste. The first objective of the present work was to investigate the autochthonous microflora present in milk and Fiore Sardo cheese in order to select lactic acid bacterial (LAB) cultures with suitable cheese-making attributes and, possibly reduce the production waste. Secondly, the ability of selected cultures to guarantee cheese healthiness and quality was tested in experimental cheese-making trials. In this study, we show that the typical lactic microflora of raw ewe’s milk and Fiore Sardo cheese is mostly composed of mesophilic LAB such as Lactococcus lactis subsp. lactis, Lactobacillus plantarum and Lactobacillus casei subsp. casei. Moreover, strains belonging to the species were selected for cheese-making attributes and used in experimental cheese-making trials carried out in different farms producing Fiore Sardo. The evolution of the cheese microflora, free amino acids and free fatty acids during the ripening showed that the experimental cheeses were characterized by a balanced ratio of the chemical constituents, by a reduced number of spoilage microorganisms and, remarkably, by the absence of production waste that were significant for the control cheeses. r 2007 Elsevier Ltd. All rights reserved. Keywords: Fiore Sardo cheese; Autochthonous microflora; Starter cultures; Free amino acids (FAAs); Free fatty acids (FFAs)

1. Introduction Fiore Sardo Protected Denomination of Origin (PDO; Gazzetta Ufficiale della Comunita` Europea, 1996) is a hard cheese produced exclusively from whole, raw ovine milk coagulated with lamb rennet paste. Currently this traditional Sardinian (Italy) cheese is still produced by shepherds at the farmhouse level. Usually, bulk milk from the evening and the morning milking are mixed together and, traditionally, lactic acid bacteria (LAB) are not used for Fiore Sardo production. Remarkably, the manufacturing of Fiore Sardo cheese has always been accompanied by a significant production waste likely due to the difficulties faced by producers during the cheesemaking process. This latter, as already mentioned, is characterized by the use of raw milk and, quite often, is Corresponding author. Tel.: +39 079 229288; fax: +39 079 229370.

E-mail address: [email protected] (P. Deiana). 0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2007.09.009

carried out without the addition of LAB cultures. Recently, the majority of the Fiore Sardo cheese producers have improved and optimized several technological phases and, according to the new EU Hygiene Directive 853/2004, the overall hygienic quality of milk. According to this EU Directive this cheese should be produced from ewe’s raw milk containing less than 500,000 bacterial cfu ml1. In several cases, this poor microbial content is not sufficient to carry out a suitable acidification and a proper ripening process. As a consequence, a constant guarantee of the organoleptic and sensory features of Fiore Sardo cheese is currently lacking. Some recent findings showed that autochthonous LAB can improve, to some extent, the typical sensory characteristics of Fiore Sardo cheese (Pisano et al., 2007). Indeed, this suggests that the use of autochthonous LAB cultures, that is allowed by the manufacturing procedures specified under the PDO (Gazzetta Ufficiale della Comunita` Europea, 1996), could be helpful to achieve a better

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management of the process, decrease the production waste and maintain the Fiore Sardo ‘‘typicality’’ as previously showed for similar cheeses (Macedo et al., 2004). In this context, the selection of autochthonous lactic species must be necessarily based on the knowledge of their presence in the raw milk and in the traditional cheese. Moreover, the evaluation of different LAB strains for selected cheesemaking attributes, or technological features, can be crucial in order to make up a starter culture able to influence or maintain the chemical and organoleptic cheese qualities. This approach revealed successful for the selection of an appropriate autochthonous starter culture for Pecorino Sardo PDO, a half-cooked paste cheese made from thermized ewe’s milk (Madrau et al., 2006). In this study, we show the suitability of this strategy for the selection of an autochthonous starter culture for a raw ewe’s milk cheese such as Fiore Sardo. In particular, in this work, we first provided a global picture of the lactic microflora of Fiore Sardo cheese and secondly, we select an autochthonous starter (with suitable cheese-making attributes) made up by the most representative LAB species that were identified in the traditional product. The influence of the selected LAB cultures on the microbiological and physicochemical characteristics of the cheese has been finally evaluated in experimental cheese-making trials. In particular, the evolution of free amino acids (FAAs), free fatty acids (FFAs) and cheese microflora have been followed in experimental and control Fiore Sardo cheeses during the ripening process.

2. Materials and methods 2.1. Isolation and identification of autochthonous LAB from milk and Fiore Sardo cheese Ewes’ raw milk and Fiore Sardo cheese samples (made from this milk) were collected from five producers of Fiore Sardo cheese in Sardinia (Italy). Curd and cheese samples were collected at 5, 30, 90, 150 and 210 days of ripening. LAB strains were isolated from these samples as previously described (IDF, 1988b; Caridi, 2003) and tested for Gram stain, catalase production and shape morphology (phase contrast microscopy; Zeiss, Gottingen, Germany). Gram positive, coccal-shaped and catalase negative LAB were identified according to Bridge and Sneath (1983) and Schleifer et al. (1985). Gram positive, catalase negative rods were identified according to Kandler and Weiss (1986). LAB identification was confirmed by carbohydrate fermentation patterns determined using API 50 CH and API 20 STREP test galleries (API System bioMerieux, Marcy l’Etoile, France). All the strains were stored at 80 1C in MRS (rods) or M17 (cocci) broth with 30% (v/v) glycerol. The evolution of lactobacilli and lactococci was also recorded as described below.

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2.2. Evaluation of cheese-making attributes of LAB strains and preparation of the experimental starter All the LAB strains belonging to the species Lactobacillus casei subsp. casei, Lactobacillus plantarum and Lactococcus lactis subsp. lactis were tested for growth kinetic, acidifying and proteolytic activities in sterile whole ewes’ milk. Selected mixed cultures were only tested for growth kinetics and acidifying activity on the same substrate. Growth kinetics were determined by inoculating each LAB into sterile whole ewes’ milk at 106 cells ml1 and viable counts carried out at 0, 2, 4, 6, 8, 10 and 24 h of incubation at 30 1C in anaerobic conditions (Gas-Pack; Oxoid, Milan, Italy). For cocci and rods enumeration, the counting procedure as well as the media utilized have been previously described (Madrau et al., 2006). The acidifying activity of each strain was determined by quantifying the amount of lactic acid produced (%) at each time-point in 10 ml of sterile whole ewes’ milk after titration with 10 M NaOH (phenolphthalein was used as indicator). The proteolytic activity of the different strains was determined in ewe’s raw milk using the spectrophotometric method of Church et al. (1983) and using o-phthaldialdehyde (OPA) as derivatizing agent and N,N0 -dimethyl-2-mercaptoethylammonium chloride as thiol reagent. This assay detects released a-amino groups which results from the proteolysis of milk proteins. The absorbance of the samples (at 340 nm), measured after 24 h of incubation, was used to quantify the proteolysis. Selected strains that showed suitable acidifying, growth and proteolytic activities were used to make up the experimental starter culture. This latter was prepared in the laboratory by mixing selected strains of L. lactis subsp. lactis, Lb. casei subsp. casei and Lb. plantarum in a final ratio of 3:0.5:0.5, respectively. These ratios were based on the relative abundance of the LAB species during the fermentative phase of Fiore Sardo and on the need to obtain a not too acidic curd as this can result in texture defects (crumbliness) of Fiore Sardo. LAB strains were grown separately in sterile ewes’ milk at 30 1C for 12 h before their employment. 2.3. Experimental cheese-making trials Experimental cheese-making trials were carried out at five traditional dairies in Sardinia (Italy) using 500 l of ewes’ milk in every batch. Two batches were prepared at each dairy: control batches in which Fiore Sardo cheese was manufactured without the addition of the starter culture and experimental batches in which the selected autochthonous starter was added before the addition of the lamb rennet (Caglificio Manca, Thiesi, Italy; strength 1:10,000). The starter was added at a level of 1%. The manufacturing procedure, employed in all the cheese-making trials, was in accordance with the specifications indicated in the standard of production (Fig. 1).

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Raw ewes’ milk

Heating of milk to 35°C Only in experimental cheese Addition of LAB cultures Addition of lamb rennet paste 35–40 g h−1 (strength 1:10,000) Coagulation of milk after ca. 30 min

Cutting of coagulum (size of the curd after cutting: ca. 0.3 cm)

Pressing of the curd at the bottom of the vat

Removal of the curd that is poured into plastic or stainless steel moulds with a cut-down cone shape that confers to the cheese the typical “mule’s back” form

Salting by immersion in brine for ca. 48 h Smoked for ca. 10 days using Pistacia lentiscus L. wood or brush Ripening at ca. 13–17°C for 180–360 days Fig. 1. Cheese-making technology of Fiore Sardo cheese.

2.4. Microbiological and physicochemical features of the experimental and control cheeses In each dairy, milk and cheese samples (from three different cheeses) were collected for each batch (experimental and control) at different sampling times. Milk samples were collected from the vat, while in the case of the experimental batches, milk samples were collected before the addition of the starter culture. Curd samples were collected at moulding while cheese samples were collected at 1, 5, 30, 90, 150 and 210 days of ripening. Cheese sampling was carried out using the standard methods (IDF, 1995). At each time point microbiological and physicochemical analyses were carried out for experimental and control cheeses. For microbiological analysis milk (10 ml), curd and cheese (10 g) samples were homogenized in 90 ml sterile Ringer’s solution for 2 min in a Stomacher Lab Blender 80 (PBI, Milan, Italy). Aliquots (1 ml) were 10-fold diluted in Ringer’s solution and plated/inoculated on the specific media used to quantify different microbial groups. In particular microbiological analysis targeted the presence of the following microbial groups: aerobic mesophilic bacteria; lactococci and lactobacilli, heterofermentative LAB, staphylococci, yeasts, total and faecal coliforms and spores of sulphite-reducing clostridia. The following media and incubation conditions were used for the enumeration of the

different microbial groups: aerobic mesophilic bacteria, Plate Count Agar (Oxoid), 30 1C for 48 h; lactococci, M17 (Terzaghi and Sandine, 1975) 22 1C for 72 h in anaerobic conditions (Gas-Pack; Oxoid); lactobacilli, MRS agar (Oxoid) acidified to pH 5.4, 22 1C for 72 h in anaerobic conditions (Gas-Pack; Oxoid); heterofermentative LAB, MRS broth with Durham tubes, 37 1C for 24 h (MPN method); staphylococci, Baird Parker Agar (Oxoid) supplemented with Egg Yolk Tellurite Emulsion (Oxoid), 37 1C for 48 h; yeasts, YPDA (1% w/v yeast extract, 2% w/v dextrose, 2% w/v peptone, 1.5% w/v agar, pH 4.5) 25 1C for 48 h; total and faecal coliforms, Brilliant Green Bile Broth (Oxoid), 37 and 44 1C, respectively (MPN method) for 48 h; spores of sulphite-reducing clostridia, DRCM broth (Oxoid) after heat treatment (80 1C for 10 min) of the samples, inoculation and incubation at 37 1C for 48 h in anaerobic conditions. Physicochemical parameters followed during the ripening were: total solids (TS) (IDF, 1982), ash (IDF, 1964), fat (IDF, 1986), NaCl (IDF, 1988a) and pH (Balestrieri and Marini, 1996). Water activity (aw), lactose and lactic acid were determined as previously described (St-Gelais et al., 1991; Madrau et al., 2006). Total (TN) and non-protein (NPN) nitrogen were determined by Kjeldahl according to Bu¨tikofer et al. (1993). FAAs were extracted from cheeses using the procedure described by Aristoy and Toldra´ (1991). The identification

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and quantification of FAAs were achieved using an HP 1050 HPLC system with an HP 1046A fluorescence detector and the HP Chemstation Rev. A.06.03 software (Hewlett-Packard Co., Wilmington, USA). A Hypersil C18 AA column (200  2.1 mm, 5 mm) with a guard column (Agilent Technologies Company, Palo Alto, USA) was used. All of the instrumental analytic conditions were as described by Gratzfeld-Hu¨esgen (1999). FFAs were extracted from cheeses and analysed by gas chromatography using the procedures described by de Jong and Badings (1990), with some minor modifications that have been fully described in Madrau et al. (2006). Briefly, FFAs (C4–C18:3) were separated using a Nukol capillary column (15 m, 0.53 mm i.d., 0.50 mm Df; Sigma-Aldrich Co., St. Louis, USA) using an HP 5890 series II gas chromatograph (Hewlett-Packard Co.) with an autosampler and a flame ionization detector. Data acquisition was carried out using the HP Chemstation Rev. A.06.03 software (Hewlett-Packard Co.). 2.5. Statistical analysis All the chemical and microbiological determinations were carried out in triplicate on each sample. For each time point significant differences between means (experimental vs. control) were evaluated by one-way analysis of variance (one-way ANOVA) followed by a Student’s t-test. Differences were considered significant at Po0.05. 3. Results and discussion 3.1. Autochthonous microflora of Fiore Sardo cheese A total of 492 LAB strains were isolated from ewe’s raw milk and Fiore Sardo cheese manufactured with the traditional technology and without the addition of any starter culture (Table 1). Moreover, the evolution of lactococci and lactobacilli during the ripening was also recorded (Table 2). Overall, the LAB recovered at different ripening times were all identified as mesophilic species as previously Table 1 LAB species and number of strains isolated from raw milk, curd and Fiore Sardo cheese at different ripening times Isolated species

Milk Curd Ripening time (days)

Lactococcus lactis subsp. lactis 28 Lactobacillus plantarum 2 Lactobacillus casei subsp. casei 1 Lactobacillus pentosus 0 Lactobacillus brevis 2 Lactobacillus spp. 3 Enterococcus faecium 6 Enterococcus faecalis 6 Enterococcus avium 6

26 4 3 1 4 4 9 9 6

5

30

90

150

210

34 10 7 1 1 2 12 10 2

24 14 8 0 5 7 10 8 2

18 16 9 0 6 4 10 8 4

8 19 12 2 9 4 11 6 0

6 8 14 7 14 4 12 4 0

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shown for different raw ewe’s milk cheeses (Macedo et al., 2004). Lactococci dominated the fermentative phase while lactobacilli were prevailing after 5 days (Table 2). This can likely be explained with a reduced sensitivity of lactobacilli to the pH decrease (Macedo et al., 2004). In particular, L. lactis subsp. lactis was the main responsible of the fermentative phase of Fiore Sardo cheese (Table 1) confirming previous observations (Bottazzi et al., 1978; Ledda et al., 1994). Moreover, the important role of this LAB species in the fermentation of cheeses made from ewe’s raw milk, such as Pecorino Sardo and Serra da Estrela (Madrau et al., 2006; Macedo et al., 2004) seems confirmed by our results. Facultative heterofermentative lactobacilli (FHL) such as Lb. plantarum, Lb. casei and Lb. pentosus were dominating the ripening phase of Fiore Sardo (Table 1). In addition to these latter, several other FHL species were previously isolated from traditional Fiore Sardo cheese (Mannu et al., 2000). This discrepancy in the composition and evolution of FHL can be due to the different microbial composition of raw milk and/or to the different microbial environment of the dairy farm as previously pointed out (Mannu et al., 2000). Among lactobacilli, Lb. plantarum could be considered the predominant species in the first 150 days (Table 1) while subsequently its number declined in favour of Lb. brevis and Lb. casei that were prevalent at the end of the ripening. Interestingly, this behaviour was also observed by Sa`nchez et al. (2006) studying the dynamics of Lactobacillus community in artisanal Manchego cheese (a Spanish cheese made from ewe’s raw milk). Enterococci were abundantly recovered in milk and cheese during both the fermentative and the ripening phases. A significant presence of Enterococcus faecium and Enterococcus faecalis in milk and cheese suggested a substantial role of these microrganisms in cheese evolution. On the contrary, Enterococcus avium was abundant during the fermentative phase and progressively disappeared during the ripening (Table 1). The recovery of Enterococcus species in cheeses made from raw ovine milk is well documented and can be considered as common (Ledda et al., 1994; Arizcun et al., 1997; Medina et al., 2001; Caridi, 2003; Madrau et al., 2006). Their proteolytic and esterolytic activities, as well as their ability to metabolize citrate, may contribute to cheese ripening and flavour development (Morandi et al., 2006; Giraffa, 2003) even though in some cases they can represent a spoilage problem (Giraffa et al., 1997). 3.2. Cheese-making attributes of LAB strains All the LAB strains belonging to the lactic species predominant in milk and cheese were characterized from a technological point of view (or for cheese-making attributes). In particular, all the strains belonging to the species L. lactis subsp. lactis (n ¼ 144), Lb. casei subsp. casei (n ¼ 54) and Lb. plantarum (n ¼ 73) (Table 1) were

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Table 2 Mesophilic lactobacilli and cocci evolution in Fiore Sardo cheese manufactured with the traditional technology (mean values of five cheese-making trials) Microbial groups

Milk (cfu ml1) Curd (cfu g1) Ripening times 5 days (cfu g1) 30 days (cfu g1) 90 days (cfu g1) 150 days (cfu g1) 210 days (cfu g1)

Mesophilic lactobacilli 1.00E+04 Mesophilic cocci 8.00E+04

7.00E+04 3.20E+06

7.00E+07 2.00E+10

separately tested for growth kinetic, acidifying and proteolytic activity. Despite Enterococcus strains were abundantly recovered in milk and cheese they were not evaluated for cheese-making attributes since their use can raise some concerns (Giraffa et al., 1997). Overall, lactobacilli demonstrated a lower acidifying activity compared to lactococci and, interestingly, this finding is conflicting with previous reports (Bottazzi, 1993). In our study, most of the strains of L. lactis (75%) and only a low number of Lb. casei subsp. casei (35%) showed a significant acidifying activity at 24 h while most of the Lb. plantarum showed a poor or intermediate acidifying ability (Fig. 2). Remarkably, strains of L. lactis subsp. lactis, Lb. plantarum and Lb. casei subsp. casei isolated from Armada cheese (made from raw goat milk), showed acidifying features at 24 h very similar to those reported here (Herreros et al., 2003). The proteolytic activity of the different strains was quantified by the OPA spectrophotometric assay (Fig. 3). Most of the strains of L. lactis subsp. lactis showed a medium to weak proteolytic activity similar to that observed for strains of this species isolated from Pecorino Sardo cheese (Madrau et al., 2006). Strains of Lb. casei subsp. casei showed a variable proteolytic activity. Most of them exhibited a very low proteolytic activity whilst approximately the 20% of the strains examined was highly proteolytic, especially if compared with what reported in other studies (Madrau et al., 2006). Despite strains of Lb. plantarum, isolated from goats’ milk cheese, have previously shown a low proteolytic activity (Sa`nchez et al., 2005), most of the strains characterized in this study displayed a medium to high activity. The values of proteolytic activity (OPA spectrophotometric assay) for the LAB strains used in the experimental starter culture were: 0.1590 for L. lactis subsp. lactis strain CFM7, 0.1742 for Lb. casei subsp. casei strain Lc101 and 0.3345 for Lb. plantarum Lp17. Growth abilities were determined for all the L. lactis subsp. lactis (n ¼ 144), Lb. casei subsp. casei (n ¼ 54) and Lb. plantarum (n ¼ 73) grown as single cultures. The growth abilities of selected strains (L. lactis subsp. lactis strain CFM7, Lb. casei subsp. casei Lc101 and Lb. plantarum Lp17) are shown in Tables 3 and 4 along with their acidifying capacities. Growth and acidifying activities of these strains were also evaluated in mixed culture (Tables 3 and 4) since they showed the suitable cheesemaking attributes as single culture.

3.20E+08 2.00E+07

8.00E+06 1.90E+05

8.00E+06 2.70E+05

6.50E+04 1.00E+05

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

L. lactis subsp. lactis

Lb. casei subsp. casei

0.28–0.38%;

0.39–0.55%;

Lb. plantarum 0.56–0.85%

Fig. 2. Lactic acid produced at 24 h (&: 0.28–0.38%; : 0.39–0.55%; ’: 0.56–0.85%) by L. lactis subsp. lactis, Lb. casei subsp. casei and Lb. plantarum strains. In the Y-axis is indicated the percentage of strains.

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

L. lactis subsp. lactis 0.0583–0.0972;

Lb. casei subsp. casei 0.0973–0.1840;

Lb. plantarum

0.1841–0.3057;

0.3058–0.5690

Fig. 3. Proteolytic activity (OPA method; higher optical density values denote higher proteolysis) at 24 h (&: 0.0583–0.0972; : 0.0973–0.1840; ’: 0.1841–0.3057; : 0.3058–0.5690) by L. lactis subsp. lactis, Lb. casei subsp. casei and Lb. plantarum strains. In the Y-axis is indicated the percentage of strains.

Overall, mixed cultures showed improved cheese-making attributes such as growth rate and acidifying activity with respect to single cultures (Tables 3 and 4). This synergistic effect between mesophilic lactococci and lactobacilli appeared particularly evident for the growth rates. All the strains in mixed cultures reached, in the first 4 h of

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Table 3 Growth kinetics (average of three batches) of single and mixed strains of L. lactis subsp. lactis (CFM 7), Lb. casei subsp. casei (Lc 101) and L. plantarum (Lp 17) Incubation time (h)

0 2 4 6 8 10 24

Lc 101 (cfu ml1)

CFM 7 (cfu ml1)

1.25E+06 5.80E+06 1.72E+07 4.71E+07 1.26E+08 7.38E+08 9.10E+08

Lp 17 (cfu ml1)

2.31E+06 8.51E+06 2.91E+07 5.30E+07 8.50E+07 6.60E+07 9.10E+07

1.79E+06 4.76E+06 1.00E+07 8.38E+07 3.44E+08 4.22E+08 3.68E+08

CFM7+Lc 101 (cfu ml1)

CFM7+Lp 17 (cfu ml1)

CFM7+Lc 101+Lp 17 (cfu ml1)

CFM7

Lc 101

CFM7

Lp 17

Cocci

Lactobacilli

3.42E+06 6.18E+07 3.36E+08 7.20E+08 3.48E+08 4.00E+08 4.80E+08

4.00E+06 2.00E+07 4.50E+07 2.80E+08 2.00E+08 2.40E+08 2.80E+08

8.00E+06 1.50E+07 1.00E+08 1.00E+08 2.40E+08 3.00E+08 3.60E+08

4.16E+06 8.80E+07 4.08E+08 4.10E+08 8.00E+08 1.36E+09 1.72E+09

2.75E+06 2.08E+08 3.24E+08 3.45E+08 5.68E+08 1.54E+09 5.62E+08

9.20E+06 1.20E+08 1.46E+08 2.40E+08 7.00E+08 2.00E+09 2.50E+09

Table 4 Acidifying activity (pH and % lactic acid) of single and mixed strains (average of three batches) of L. lactis subsp. lactis (CFM 7), Lb. casei subsp. casei (Lc 101) and Lb. plantarum (Lp 17) Incubation time (h)

0 2 4 6 8 10 24

CFM 7

Lc 101

Lp 17

pH

% Lactic acid

pH

% Lactic acid

pH

6.62 6.57 6.17 5.85 5.12 4.9 4.42

0.17 0.19 0.20 0.25 0.36 0.51 0.72

6.69 6.41 6.33 6.1 5.66 5.78 4.86

0.17 0.19 0.20 0.23 0.29 0.31 0.56

6.5 6.41 6.26 5.93 5.94 5.67 5.38

CFM 7+Lp 17

CFM 7+Lc 101

CFM7+Lc 101+Lp 17

% Lactic acid

pH

% Lactic acid

pH

% Lactic acid

pH

% Lactic acid

0.18 0.18 0.23 0.26 0.28 0.31 0.47

6.35 5.86 5.29 4.80 4.59 4.49 4.20

0.20 0.23 0.35 0.51 0.55 0.63 0.78

6.52 6.27 5.90 5.43 5.09 4.87 4.28

0.18 0.21 0.26 0.36 0.45 0.57 0.85

6.41 6.27 5.59 4.98 4.76 4.51 4.17

0.19 0.21 0.29 0.44 0.57 0.63 0.89

incubation, 108 cfu ml1; the only exception was Lc101 mixed with CFM7. The synergistic effect seemed to be more relevant for rods and for Lb. plantarum in particular. In mixed culture, this latter, showed improved growth ability with respect to Lb. casei. This behaviour is likely due to a better adaptability of Lb. plantarum to complex substrates such as raw ewe’s milk. Moreover, this should not be surprising since strains of this species have been recovered from very different food matrices (De Vuyst and Neysens, 2005; Ercolini et al., 2006; Mangia et al., 2007). The same synergic effect between lactococci and mesophilic lactobacilli was also observed in Roncal-type cheeses made from ewe’s milk (Ortigosa et al., 2006). 3.3. Microflora evolution in experimental and control cheeses The control cheeses made without the addition of the starter culture showed a poor quality in two out of five dairies (Fig. 4). In particular, in one of the farm, the production waste for the control cheese was very high and around 40%. For these reasons, the analytical results (microbiological and physicochemical) from these two dairies (but only referring to the control cheeses) were not taken into account. Indeed, all the results presented for the control cheeses are average values from three dairies. Microbiological analyses showed that the addition of the

autochthonous starter induced a marked increase in the mesophilic lactic microflora both in the curd and after 24 h of ripening compared to the control (Table 5). Remarkably, these high values were maintained throughout the ripening. The lactococci developed rapidly, particularly in the experimental cheeses, often reaching very high numbers and representing therefore the dominant fermentative microflora (Table 5). The evolution of the mesophilic lactobacilli followed a trend that is typical for this group (Mannu et al., 2000); indeed, their initial growth was slow while after 5 days of ripening they reached their highest number (Table 5). We could not observe any substantial increase of spoilage microorganisms, such as coliforms, in the experimental cheeses. In general, these latter presented a more homogeneous structure characterized by the absence or a rare occurrence of cavities (Fig. 3). In some cases, to the contrary, the fermentative activity operated by coliforms in the control cheeses (Fig. 3) resulted in early swelling and favored the development of Phiophila casei (Fig. 4). Different factors could have contributed to the decline of spoilage microflora during the ripening of the experimental cheeses; these include a rapid production of lactic acid, a fast decrease of pH and the inhibition of spoilage bacteria by LAB (Psoni et al., 2003). The heterofermentative LAB were recovered in low number in both experimental and control cheeses (Table 5). Staphylococci that were present in high number in the milk were

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et al., 2000). Yeasts, that were present in both milk and cheeses, showed a significant increase during the first 5 days of ripening. A similar yeast count was previously observed by Fadda et al. (2004) in ripened Fiore Sardo. This likely highlights the relevancy of these microorganisms for taste and flavour development in this cheese.

3.4. Physicochemical features of the experimental and control cheeses

Fig. 4. Examples of control (a) and experimental (b) Fiore Sardo cheeses at 5 days of ripening. To be noticed the massive presence of cavities in the control cheese (a) compared to the experimental one (b). The excessive presence of cavities in the control cheese is most likely due to the significant number of total and faecal coliforms at this time point (see Table 5) and to their fermentative activity. Control cheese at 150 days of ripening infested by Phiophila casei larvae (c).

also recovered in the cheeses. In the control cheese, in particular, their population reached a significant size. It should also be mentioned that they were all coagulasenegative staphylococci and, therefore, they could have been played a substantial role in the ripening process (Ingham

Physicochemical analyses of control and experimental batches have been carried out for curds and cheeses at 1, 5, 30, 90, 150 and 210 days of ripening. For brevity, we only report in Table 6 the physicochemical features of Fiore Sardo curds and cheeses at 210 days of ripening (the complete set of data is available upon request). At 210 days of ripening there were no significant differences (P40.05) between the experimental and control cheeses. The total solids (TS) increased progressively during the ripening reaching mean values of 76.770.2% for the experimental cheeses and of 78.970.8% for the control. The ashes increased over time, particularly after the salting, until they reached a mean value of 6.2% at 7 months. Overall, the NaCl concentration remained constant during the last 6 months of ripening. The levels of total fats increased progressively due to the decrease in humidity up to the end of the ripening (30%). The lactose that was present in the curds at concentrations of 2.48–3.38%, was metabolized during the first days of ripening (not shown); in contrast, lactic acid increased from 0.11% and 0.15% (curds) up to 1.38% and 1.47% after 1 day of ripening for the control and experimental cheeses, respectively. This is an indication of a good lactic fermentation that occurred quickly especially in the experimental cheeses. Moreover, this behaviour is likely due to the metabolic activity of the starter microflora which induced a decrease in the pH over the first day, from 6.6570.02 to 5.2170.20 for the experimental samples, and from 6.6970.02 to 5.3970.22 for the controls. During the ripening, the pH showed a slight increase to 5.5370.07 for the experimental samples, and to 5.4170.04 for the controls. In agreement with the increase in the TS, the water activity (0.98870.003 for the experimental curds and 0.98970.003 for the controls) decreased slowly to 0.84970.004 and 0.83870.001 for experimental and control samples at 210 days of ripening. A significant increase of WSN and NPN was observed during the ripening while the ripening coefficient (WSN  100/TN) reached after 7 months a mean value of 27.0% and 23.6% for the experimental and controls cheeses, respectively. A slightly higher ripening coefficient in the experimental cheeses can be likely due to the proteolytic activity of the mesophilic lactobacilli present in the starter culture. In particular, Lb. plantarum that preferentially metabolizes b-casein (Khalid and Marth, 1990) can be responsible for an ‘‘accelerated’’ cheese ripening.

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Table 5 Microflora evolution in (a) experimental Fiore Sardo cheese manufactured with the autochthonous starter (mean values of five cheese-making trials) and (b) control Fiore Sardo cheese manufactured without the addition of the starter (mean values of three cheese-making trials) Microbial groups

Ripening times Milk (cfu ml1)

Curd (cfu g1)

1 day (cfu g1)

5 days (cfu g1)

(a) Experimental Fiore Sardo cheese manufactured with the autochthonous starter Colony count 2.5E+05 6.8E+08 4.3E+10 3.6E+10 210 210 240 11 Total coliformsa Fecal coliformsa 210 210 240 11 o3 o3 o3 o3 Clostridial sporesa Lactic o3 o3 o3 o3 heterofermentativea Mesophilic lactobacilli 3.8E+04 1.5E+06 1.1E+10 5.3E+10 Mesophilic cocci 2.1E+05 3.9E+07 7.1E+10 7.2E+10 Staphylococci 5.3E+04 1.2E+05 2.6E+05 8.3E+04 Yeasts 4.2E+02 7.8E+02 1.9E+03 3.5E+05 (b) Control Fiore Sardo cheese manufactured without Colony count 1.8E+05 5.0E+05 Total coliformsa 210 210 Fecal coliformsa 210 210 Clostridial sporesa o3 o3 o3 o3 Lactic heterofermentativea Mesophilic lactobacilli 3.9E+04 1.4E+05 Mesophilic cocci 4.4E+04 2.3E+06 Staphylococci 5.3E+04 1.8E+05 Yeasts 5.0E+02 2.2E+03 a

the addition of the starter 8.0E+10 2.5E+10 2400 2400 2400 2400 o3 o3 o3 o3 2.0E+08 1.2E+09 3.1E+06 5.5E+01

3.5E+09 3.9E+10 5.8E+06 1.9E+05

30 days (cfu g1)

90 days (cfu g1)

150 days (cfu g1)

210 days (cfu g1)

2.1E+10 7 7 o3 460

2.3E+08 o3 o3 o3 210

1.3E+07 o3 o3 o3 210

2.9E+07 o3 o3 o3 460

9.0E+09 1.8E+10 7.4E+04 1.0E+01

1.7E+08 1.5E+08 8.0E+04 1.0E+01

3.5E+06 1.0E+06 5.0E+02 1.0E+01

1.5E+06 4.1E+06 1.1E+03 1.0E+01

1.1E+10 150 150 o3 460

1.7E+07 o3 o3 o3 21

1.5E+07 o3 o3 o3 21

1.4E+06 o3 o3 o3 150

3.3E+09 4.5E+09 2.9E+06 3.2E+05

1.2E+07 4.1E+06 1.4E+04 1.0E+01

1.0E+06 1.7E+06 1.5E+02 1.0E+01

2.5E+06 3.5E+05 1.0E+01 4.5E+01

MPN method, MPN ml1 or g1.

Table 6 Compositional analysis of curd, experimental (E; n ¼ 5 batches) and control (C; n ¼ 3 batches) Fiore Sardo cheeses at 210 days of the ripening (mean values7S.D.) Time (days)

NaCla

TSa

Ashesa

Fata

Proteinsb

SNa

NPNa

Lactosea

Lactic acida

pH

aW

Curd

E C

0.270.01 0.170.01

41.671.7 39.071.1

2.270.1 2.170.1

19.070.3 17.771.7

17.970.6 17.270.3

0.4670.08 0.3170.01

0.1570.06 0.1570.06

2.4870.78 3.3870.39

0.1570.04 0.1170.02

6.6570.02 6.6970.02

0.98870.003 0.98970.003

210 days

E C

2.670.4 2.470.1

76.770.2 78.970.8

6.470.1 6.170.2

28.870.5 31.570.3

31.970.7 33.271.0

1.3570.06 1.2370.05

0.9270.04 0.9070.12

0.0270.02 0.0570.02

2.0670.04 2.1470.06

5.5370.07 5.4170.04

0.84970.004 0.83870.001

TS, total solids; SN, soluble nitrogen; NPN, non-protein nitrogen. a Expressed as g 100 g1 of cheese. b Total nitrogen  6.38.

3.5. Evolution of free amino acid (FAAs) and free fatty acid (FFAs) in experimental and control cheeses Tables 7 and 8 show the evolution of FAAs and FFAs for the experimental and control cheeses during the ripening. The results presented are significant since, to our knowledge, this is the first study presenting the content and the evolution of FAAs and FFAs in Fiore Sardo cheese. At the end of the ripening (210 days) leucine, valine, isoleucine and phenylalanine were the more abundant amino acids detected. These latter constituted 63% of the total FAAs recovered in the experimental samples and 61% of the control ones. Interestingly, lysine was present at very low levels (approximately 28 mg 100 g1) with

respect to the Pecorino Sardo cheese (approximately 300 mg 100 g1) that, differently from Fiore Sardo, was manufactured with heat-treated ewes’ milk and with the addition of mesophilic and thermophilic LAB (Madrau et al., 2006). In experimental and control cheeses, the glutamic acid reached the highest content at 90 days of ripening while in the following period its content decreased remarkably. This aminoacid could be utilized by the mesophilic lactobacilli during the last part of the ripening when their number is substantially high (Table 4). It should be mentioned that the content of glutamic acid in Fiore Sardo is contrasting with those found in different cheeses (made from ewe’s milk) at the end of the ripening (Poveda et al., 2004; Madrau et al., 2006; Barcina et al., 1995). Also

374

Table 7 Evolution of free amino acids (mg 100 g1 of cheese) during the ripening of experimental (E; n ¼ 5 batches) and control (C; n ¼ 3 batches) Fiore Sardo cheese (mean values7S.D.) Ripening time (days) Curd

1 day

a a a a a a a a a a a a a a a a a a a a

0.1970.06 2.8870.11 N.D. 0.2570.13 N.D. 0.3870.05 0.5170.19 N.D. 0.4970.13 N.D. 1.7970.58 1.9070.28 1.2170.03 2.0270.48 N.D. 0.2770.05 0.2570.19 N.D. 1.1370.01 0.2870.07 0.2370.01 13.7772.16 b

E a a a a a a a a b a b a b a b

0.6470.31 4.4771.54 2.1970.13 0.6970.11 2.6171.07 2.0070.14 0.7670.27 1.0670.36 2.4770.16 N.D. 3.6570.36 1.8570.03 3.7370.48 2.4770.70 0.7770.21 3.2070.31 0.7170.25 1.8070.26 4.3670.32 4.7470.44 7.4770.48 51.6272.40 a

C a a a a a a a a a a a a a a a a a a a a

0.7370.31 4.2870.17 0.3570.07 0.9070.24 0.8870.38 1.3570.37 0.3470.07 0.6270.02 2.2670.20 N.D. 1.8070.10 N.D. 2.7070.13 2.2970.09 0.1270.02 1.5970.36 0.9170.18 0.9170.05 3.0170.11 1.9970.15 3.7071.18 30.7071.64 b

E a a b a b b b a a b b a b b a b b b b

1.5970.54 3.2770.50 5.4470.45 0.9570.17 6.7971.27 2.3070.29 0.9870.09 2.0170.36 5.3070.96 N.D. 4.8172.10 N.D. 14.1771.60 5.6471.09 0.9270.10 10.0571.19 4.1471.06 6.8570.46 18.3473.00 12.8771.45 7.7070.66

C a a a a a a a a a a a a a a a a a a a

1.8570.24 6.1470.31 1.0070.23 0.9470.39 4.6270.28 1.4770.33 0.5870.16 1.4970.65 5.5870.45 1.3470.13 2.2570.54 N.D. 9.6472.58 4.2070.97 0.3170.01 5.7871.74 3.1270.76 3.1472.24 11.4273.01 6.2572.77 5.2171.77

90 days

E a b b a b b b a a a a b a b b a b b b b

2.1470.33 19.2875.12 8.5971.83 1.9170.38 13.5170.17 1.8070.07 2.9470.27 4.4270.32 10.7571.82 1.4270.05 8.1773.08 1.3570.15 39.8673.50 11.7570.82 1.1170.50 32.8871.47 15.8170.62 13.3870.23 68.0270.92 29.8973.19 12.8671.55

114.09715.82 76.31718.03 301.8175.23 a b a

C a a a a a a a a a a a a a a a a a a a a a

3.5070.21 35.7471.38 3.9471.75 2.2970.24 9.8972.89 1.1270.36 4.1970.09 5.0270.07 15.8471.18 2.2470.06 8.5371.72 2.3370.26 52.7970.95 16.6070.41 1.5370.49 41.7971.23 18.5370.27 21.2570.82 78.5271.05 41.0471.41 16.8571.10 383.5074.64 b

E

150 days C

b 8.1072.08 a 7.0271.91 a b 75.7878.48 a 71.2578.53 a a 31.0876.13 a 21.7679.08 a a 12.1672.32 a 14.9772.39 a a 16.1372.01 a 15.7571.74 a a 1.4670.27 a 2.6870.86 b a 11.6870.54 a 10.9670.99 a a 15.5771.63 a 14.1871.26 a b 45.2272.25 a 45.6773.39 a a 4.1370.20 a 3.8170.17 a b 21.4575.75 a 22.5072.80 a a 6.6370.25 a 7.6271.72 a b 110.6876.25 a 105.8675.96 a a 56.0475.09 a 54.9472.45 a b 5.2570.78 a 5.1571.01 a b 95.7874.36 a 93.8576.24 a b 85.2274.93 a 81.2074.80 a a 24.7073.44 a 26.3078.93 a b 181.3675.00 a 181.8079.96 a b 71.2473.11 a 69.4975.85 a a 60.2972.27 a 57.0472.76 a

E 1.9970.69 a 31.5579.06 a 24.3175.61 a 11.1371.83 a 16.4670.97 a 2.7770.23 a 10.2770.42 a 16.0972.64 a 44.4675.02 a 3.2370.20 a 21.9171.98 a 8.4870.52 a 119.07731.68 a 66.87713.84 a 7.2170.62 a 115.50728.14 a 111.95730.40 a 11.4675.68 a 296.46741.86 a 37.0277.86 a 78.33712.93 a

210 days C 2.7571.42 a 32.0674.38 a 23.8276.18 a 15.5271.11 b 14.9273.76 a 3.8570.32 b 10.3770.37 a 14.6770.33 a 47.1371.51 a 3.2570.55 a 23.3471.52 a 8.2971.18 a 113.46734.93 a 66.78719.09 a 7.0372.75 a 111.73731.06 a 104.06733.71 a 12.6371.49 a 290.57738.02 a 37.27710.26 a 72.61711.15 a

E 2.4070.34 a 24.5874.33 a 16.6171.74 a 12.5572.59 a 9.6572.02 a 3.5170.21 a 10.9770.25 a 15.5371.89 a 46.0671.16 a N.D. 28.3676.79 a 9.4070.78 a 127.6173.81 a 73.4875.13 a 7.9571.63 a 119.96715.78 a 126.2977.94 a 7.2171.06 a 276.61720.11 a 28.9071.95 a 92.8772.76 a

939.92725.89 913.76745.37 1036.497133.29 1016.107163.63 1040.46751.84 a a a a a

Different letters (a, b) on the same row and only at the same ripening time indicate significant differences (Po0.05). N.D., not detectable; FAA, free amino acids.

C 1.6370.39 b 28.7177.33 a 15.9576.41 a 16.8172.11 b 7.7672.30 a 4.1470.67 a 10.9371.73 a 14.5771.34 a 47.8372.46 a N.D. 27.3471.40 a 9.3170.49 a 125.7979.50 a 71.5774.72 a 7.4672.41 a 109.35711.27 a 122.54711.07 a 9.3673.05 a 271.77733.59 a 27.5974.20 a 91.7876.62 a 1022.18797.63 a

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ASP 0.4670.11 GLU 2.9170.65 ASN 0.6670.55 SER 0.4270.20 GLN 0.7870.04 HIS 0.7570.34 GLY 0.7470.18 THR 0.6470.24 ALA 1.2170.66 ARG+GABA N.D. TYR 2.6071.01 CYS–CYS 1.8870.18 VAL 3.8171.28 MET 2.1670.49 TRP 0.3170.01 PHE 2.6071.36 ILE 0.5870.26 ORN 0.7070.02 LEU 3.4571.04 LYS 0.2970.06 PRO 4.8671.59 P FAA 31.7878.72 a

C

30 days

N.P. Mangia et al. / Food Microbiology 25 (2008) 366–377

E

5 days

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Different letters (a, b) on the same row and only at the same ripening time indicate significant differences (Po0.05). N.D., not detectable; FFA, free fatty acids.

FFA 74.54716.65 a 55.82713.04 a 104.9573.71 a 92.5876.33 b 229.17735.74 a 226.2678.48 a 815.96730.28 a 887.57752.27 a 1921.307194.14 a 1904.987123.58 a 2067.06769.95 a 2346.977142.92 b 2859.337159.36 a 2786.557208.88 a P

254.97749.41 a 124.35714.13 a 79.8075.26 a 228.74715.06 a 123.42710.61 a 318.88730.95 a 760.90746.65 a 243.31719.53 a 490.16755.32 a 135.87720.71 a 26.1574.46 a 230.11714.32 a 109.2975.71 a 71.9477.24 a 212.48716.10 a 120.9976.88 a 315.88716.96 a 805.64741.41 a 271.59738.34 a 537.29778.10 a 157.18736.69 a 26.9672.60 a 219.50712.65 b 101.2072.35 b 66.7178.87 b 187.68726.54 a 105.51714.07 b 259.02720.97 b 638.48745.65 b 220.74715.47 b 407.83744.50 a 115.3779.23 a 24.9475.55 a 192.5879.66 a 81.5677.26 a 49.6673.82 a 152.00716.18 a 83.57710.52 a 208.15714.86 a 554.41723.87 a 186.0178.29 a 417.1973.77 a 115.5575.55 a 26.3972.85 a 184.62724.01 a 82.0379.49 a 48.0374.40 a 142.68716.12 a 78.07712.92 a 187.28731.58 a 505.63722.76 a 175.96717.67 a 362.01720.18 a 115.58714.51 a 23.0970.56 a 182.76724.92 a 79.0875.52 a 46.7473.90 a 139.86711.29 a 78.5476.23 a 196.55718.32 a 522.30760.50 a 170.90719.24 a 362.10746.62 a 116.13712.69 a 26.3573.66 a 104.2774.74 b 41.4673.16 b 21.3070.93 b 71.1173.16 a 40.8471.87 b 90.2575.41 b 247.22714.72 b 74.9077.46 a 147.00716.04 a 35.4476.23 a 13.7970.91 a a 92.4571.15 a a 35.9170.52 a a 18.0970.76 a b 62.3477.90 a a 34.6171.40 a a 74.1774.60 a a 225.5977.19 a a 74.1575.45 a a 150.6575.95 a a 34.3672.06 a b 13.6570.65 a 24.1073.55 9.5270.56 3.9870.95 17.9671.90 13.2972.12 19.0973.12 48.5077.95 26.8974.16 48.1076.92 10.5874.21 7.1870.11 a 9.9471.96 b a 3.2370.16 a a 0.5070.17 b a 4.6370.23 b a 7.3671.16 a a 7.5272.19 a a 17.4273.18 a a 15.6471.06 b a 20.6473.47 a a 5.7270.33 a N.D. 12.9271.20 3.4370.33 0.9970.22 7.1470.92 7.4170.38 8.2670.89 19.0070.69 18.3170.47 20.5070.56 6.9970.81 N.D. 3.4071.22 2.0570.63 0.3370.09 5.0071.47 5.4370.83 3.8171.04 8.8071.56 8.7671.39 13.5073.09 4.7670.54 N.D. C4 C6 C8 C10 C12 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3

4.7571.40 2.2170.59 0.5070.18 5.4771.17 6.8170.97 5.0171.70 13.4672.34 14.1972.79 16.9273.15 5.2470.83 N.D.

a a a a a a a a a a

C

a a a a a a b b a a

E E

C

E

a a a a a a a a a a a

26.1871.59 8.8370.80 3.3270.70 15.0570.73 12.5270.55 17.1770.36 47.1671.41 26.0970.89 51.2175.13 11.0671.68 7.6970.15

E C

E

C

E

C

E

C

210 days 150 days 90 days 30 days 5 days 1 day Curd

Ripening time (days)

Table 8 Evolution of free fatty acids (mg 100 g1 of cheese) during the ripening of experimental (E; n ¼ 5 batches) and control (C; n ¼ 3 batches) Fiore Sardo cheese (mean values7S.D.)

C

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375

proline was present at important levels and its content increased progressively during the ripening. This can likely suggest a substantial idrolysis of the b-casein during the maturation process (Poveda et al., 2004; Frau et al., 1997). In Table 8, we report the evolution of FFAs in experimental and control Fiore Sardo cheeses during the ripening. Overall, the FFAs content of the experimental and control cheeses increased progressively during the ripening and was very similar across the two batches. At the end of the ripening, the average content of FFAs of Fiore Sardo (approximately 2800 mg 100 g1) was considerably greater than that reported for Pecorino Sardo (approximately 700 mg 100 g1) (Madrau et al., 2006), as already mentioned was manufactured using a different technology and a different starter culture. Similarly to Pecorino Sardo, the most representative FFAs at 210 days were palmitic, oleic, myristic and stearic acids (Madrau et al., 2006). Remarkably a high content of short-chain FFAs, above all butyric acid, was also found at the end of the ripening. A high content of this FFA was previously observed in similar cheeses made from ewe’s raw milk such as Idiazabal (Hernandez et al., 2005). This can be due to the specific activity of the lipases present in the lamb rennet (Virto et al., 2003). Interestingly, butyric acid was found as a major FFA even in cheeses made from raw cow’s milk (Prieto et al., 2002). In this case, the high content of butyric acid has been attributed to the lipolytic activity of the lactic microflora mainly composed by Lactobacillus and Lactococcus species. In general, the accumulation of FFAs seems more influenced by the use of raw milk, which retains its total content of constitutive lipases. Moreover, this is particularly evident for cheeses that do not undergo heat treatment like Fiore Sardo. Substantial amounts of linoleic acid C18:2 were found at the end of the ripening (approximately 145 mg 100 g1) in comparison with Pecorino Sardo cheese (approximately 25 mg 100 g1) (Madrau et al., 2006). Despite its origin can be attributed to the constitutive milk lipase and to the rennet lipase, linoleic acid can also be originated by the lipolytic activity of the mesophilic lactic microflora. Specific lipase from Lactobacillus casei (Yu, 1986), a LAB species highly recovered from Fiore Sardo and utilized in the experimental starter, could have been contributed to the accumulation of this long chain fatty acid. 4. Conclusions Globally, this study provides a general picture of the lactic microflora associated to Fiore Sardo cheese and highlights the importance of mesophilic lactococci and lactobacilli in cheese-making processes that utilize raw ewe’s milk. In particular, mesophilic lactococci and lactobacilli seemed responsible for a balanced lipolytic, proteolytic and fermentative activity that conferred desired attributes to the experimental cheese and, remarkably, reduced significantly the production waste. Moreover, the experimental cheese-making trials, carried out following

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