Reduction of 25% salt in Prato cheese does not affect proteolysis and sensory acceptance

International Dairy Journal 75 (2017) 101e110

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Reduction of 25% salt in Prato cheese does not affect proteolysis and sensory acceptance
bora Parra Baptista a, *, Francisca Diana da Silva Araújo b, Marcos Nogueira Eberlin b, De Mirna Lúcia Gigante a a b

Department of Food Technology, Faculty of Food Engineering, University of Campinas, UNICAMP, 13083-862 Campinas, SP, Brazil ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas, UNICAMP, POB 6154, 13083-970 Campinas, SP, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 March 2017 Received in revised form 28 July 2017 Accepted 1 August 2017 Available online 10 August 2017

The effect of salt reduction (25% and 50%) on proteolysis, firmness, and sensory acceptance of Prato cheese was investigated throughout 60 days of ripening. Proteolysis was assessed by nitrogen fractionation, degradation of caseins by capillary electrophoresis (CE), and analysis of peptides by matrixassisted laser desorption/ionisation mass spectrometry (MALDI-TOF-MS). The salt reduction did not affect proteolysis, as determined by the nitrogen fractionation, and hydrolysis profile of caseins by CE. In addition, the salt reduction did not increase the relative intensity of known bitter-tasting peptides in cheeses, as assessed by MALDI-TOF-MS. Cheeses with 50% salt reduction were less firm and less sensory acceptable than the control cheese and the cheese with 25% salt reduction. However, the reduction of 25% salt content resulted in cheeses with similar peptide profile, firmness, and sensory acceptance when compared with the control cheese. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Sodium is an essential nutrient with important functions in the regulation of extracellular fluid volume and transport of molecules through cell membranes. However, excessive sodium intake is associated with the prevalence of hypertension, prehypertension (Doyle & Glass, 2010) and other adverse health effects, which justifies the recommendation of the World Health Organisation of a daily intake of less than 2 g sodium per day (5 g salt per day; WHO, 2012). Salt reduction in cheese is considered a major technological challenge due to the importance of salt to the characteristics of this product. In addition to providing flavour, salt affects microbial growth, enzyme activity, and syneresis, which in turn affects the cheese composition (Guinee & Fox, 2004). The cheese composition and microbial and enzymatic activities are closely related to the ripening, providing the typical flavour and texture of each variety. Proteolysis is the most complex biochemical change during ripening, being characterised by a sequence of steps that include initial hydrolysis of casein to long-chain peptides by the action of

* Corresponding author. Tel.: þ55 19 35213993. E-mail address: [email protected] (D.P. Baptista). http://dx.doi.org/10.1016/j.idairyj.2017.08.001 0958-6946/© 2017 Elsevier Ltd. All rights reserved.

the residual coagulant and plasmin, hydrolysis of long-chain peptides to the medium and short chain peptides by the action of lactic acid bacteria and fungi, with subsequent hydrolysis to dipeptides, tripeptides, and free amino acids (Farkye, 2004; Schornsteiner, Mann, Bereuter, Wagner, & Schmitz-Esser, 2014). The effect of salt on the proteolysis in cheese is due to its ability to stimulate or inhibit microbial and enzymatic activities (Guinee & Fox, 2004). In general, sensory defects including the development of bitterness and reduction of firmness have been associated with salt reduction and the related changes in the proteolysis behaviour (Mistry & Kasperson, 1998; Rulikowska et al., 2013). Bitterness in cheese is commonly associated with the accumulation of hydrophobic peptides formed through the hydrolysis of aS1- and b-caseins (aS1and b-CNs), such as the fragment b-CN f193e209 and some peptides formed by the hydrolysis of the fragment aS1-CN f1e23 (Broadbent, Strickland, Weimer, Johnson, & Steele, 1998; Soeryapranata, Powers, & Ünlü, 2008; Soeryapranata et al., 2002; Visser, 1993). Although different authors have associated bitterness to a greater proteolysis due to salt reduction (Mistry & Kasperson, 1998; Rulikowska et al., 2013), the peptide profiles during ripening of Prato cheeses with different salt contents has not been reported. € (2012) studied the effect of salt Møller, Rattray, Høier, and Ardo reduction on the peptide profile of Cheddar cheese with equal

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moisture content and verified an inverse dependence on the salt concentration of the majority of the identified peptides. The evaluation of proteolysis is directly related to the sensitivity of the methodology used. Traditionally, studies on proteolysis use non-specific methodologies, such as the monitoring of soluble nitrogen in buffer solutions at pH 4.6 and trichloroacetic acid, without identification of the compounds formed. The use of electrophoresis, chromatography, and mass spectrometry techniques allow greater elucidation of the mechanisms involved in the proteolysis through the identification of the peptides formed (Fox, Guinee, Cogan, & McSweeney, 2000; Fox, McSweeney, & Singh, 1995; Sousa, Ardo, & McSweeney, 2001), thus increasing the possibility of correlating the changes between cheese characteristics and development of proteolysis. Different strategies for reducing salt levels in cheeses have been found in the literature, ranging from a simple reduction (Ganesan, Brown, Irish, Brothersen, & McMahon, 2014; Mistry & Kasperson, 1998; Rulikowska et al., 2013), partial or total replacement of NaCl by other salts (e.g., KCl, CaCl2 and MgCl2) (Grummer, Karalus, Zhang, Vickers, & Shoenfuss, 2012; Thibaudeau, Roy, & St-Gelais, 2015) and salt reductions in combination with flavour enhancers such as hydrolysed vegetable protein, yeast extract, disodium inosinate, and disodium guanylate (Grummer, Bobowski, Karalus, & Vickers, 2013). According to Guinee and Sutherland (2011), there is an ideal salt-in-moisture ratio for each cheese variety, and the sensory defects can be perceived only below that range, possibly due to the growth of undesirable bacteria and/or uncontrolled enzymatic activity. Thus, the simple reduction of salt to levels that do not compromise the physicochemical and sensory quality of the product is a promising approach, as reported for mozzarella and Cheddar cheeses (Ganesan et al., 2014; Mistry & Kasperson, 1998; Rulikowska et al., 2013). The objective of this study was to evaluate the impact of salt reduction on the proteolysis, firmness, and sensory acceptance of Prato cheese during ripening (60 days). The strategy was 25% and 50% reduction of salt levels, and the proteolysis was monitored by both non-specific and specific methods to evaluate the hydrolysis of the casein fractions by CE and the peptide profile by matrixassisted laser desorption/ionisation mass spectrometry (MALDITOF-MS). The effects of sodium reduction on other functions of salt, such as food safety or reduction of spoilage by undesired bacteria were not investigated in the study. 2. Materials and methods 2.1. Preparation of cultures Lyophilised lactic mesophilic cultures of Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris (R 704; Chr. Hansen, Valinhos, Brazil) were reactivated in 10% (w/v) sterile reconstituted skim milk, and incubated at 30  C for 8 h. The activated culture was subsequently reactivated in 10% (w/v) sterile reconstituted skim milk, and incubated at 30  C for 8 h, and used in cheese manufacture. 2.2. Prato cheese manufacture and sampling The cheese was produced by a traditional manufacturing method as described by Mazal, Vianna, Santos, and Gigante (2007) with modifications, using 100 L of heat-treated whole milk (68  C for 2 min). Milk was cooled to 35  C, and the lactic mesophilic cultures Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris (R 704; Chr. Hansen), were added (1%, v/v). Calcium chloride (250 ppm), annatto dye (80 ppm) and fermentation-produced chymosin (Ha

La 1175, Chr. Hansen) sufficient to coagulate the milk within 35 min were added. The curd was cut into 1 cm cubes and submitted to slow continuous mixing for 15 min, which was followed by indirect heating to 42  C, increasing the temperature by 1  C every 3 min. This temperature was maintained for 40 min. After cooking, all the whey was drained off and curd was placed in rectangular plastic molds (13 cm  8 cm  7 cm) and pressed at room temperature with successive turns (0.1 MPa for 15 min; 0.1 MPa for 15 min; 0.24 MPa for 30 min, and 0.31 MPa for 90 min). Cheeses (~0.5 kg) were fermented for 5 h at room temperature (~25  C). The cheese blocks obtained were allocated into three groups, which were salted in static brine with 20% (w/v) NaCl, 0.5% (w/v) CaCl2, and pH 5.5 (adjusted with 1 M HCl). The volume of brine was 3.5 times greater than the volume of cheese, and was kept at 4  C throughout the salting period. The three treatments were only differentiated by the salting times, which were 12 h for the control cheese, and 6 and 3 h for the cheeses with 25% and 50% salt reduction, respectively. The cheeses were then stored for 48 h at 12  C, vacuumpacked into heat-shrinkable plastic bags, and stored for 60 days at 12  C. One cheese block was used for sampling at each evaluation day (1, 15, 30, 45 and 60 days of storage). Eight cylindrical samples of 20 mm diameter and 24 mm height were sampled for texture analysis and the remaining cheese was grated, mixed and used for physicochemical composition analysis and proteolysis evaluation. 2.3. Physicochemical composition of milk and cheeses The heat-treated milk was characterised for pH, titratable acidity (AOAC, 2006j), total solids (AOAC, 2006a), ash (AOAC, 2006d), total nitrogen (AOAC, 2006f), and fat by the method of Gerber (AOAC, 2006b). The protein content was determined by multiplying the total nitrogen by the conversion factor 6.38. Milk heat treatment efficiency was evaluated by the activity of the enzymes alkaline phosphatase (AOAC, 2006h) and peroxidase (LANARA, 1981). One day after storage, the cheeses were characterised for pH with the use of a potentiometer, titratable acidity (AOAC, 2006i), fat by the method of Gerber (BSI, 1989), ash (AOAC, 2006c), total nitrogen (TN) (AOAC, 2006e), pH 4.6 (pH 4.6 SN) and 12% TCA (12% TCA-SN) soluble nitrogen by Kjeldahl method according to Bynum and Barbano (1985). Fifteen days after storage, the cheeses were analysed for moisture (AOAC, 2006g), NaCl content by the method of Volhard (Richardson, 1985) and sodium. For sodium determination, the samples were incinerated in a muffle (550  C for 16 h), and ash was diluted in 3% HNO3, and the sodium level was deter~o Paulo, SP, Brazil). mined in a flame photometer (Digimed DM62, Sa 2.4. Evaluation of cheeses during ripening The proteolysis was evaluated after 1, 15, 30, 45, and 60 days of storage by the non-specific method through the nitrogen fractionation. Total nitrogen (TN), and pH 4.6-soluble and 12% TCAsoluble nitrogen were determined as previously described and expressed as pH 4.6-soluble and 12% TCA-soluble nitrogen in relation to total nitrogen [NS pH 4.6 (%TN) and NS TCA 12% (%TN), respectively]. The hydrolysis profile of caseins was analysed by CE and the peptides formed were evaluated by MALDI-TOF-MS. The firmness was measured by texture profile analysis (TPA). 2.4.1. Fractionation of peptides by pH 4.6-soluble and 70% ethanolsoluble fractions Grated cheese samples (40 g) were homogenised with 80 mL of water for 10 min in a Stomacher 400 (Seward Laboratory, UK), as described by Kuchroo and Fox (1982).

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The fractionation of peptides was performed according to the methodology described by Piraino et al. (2007): the mixture was acidified to pH 4.6 with 1.0 M HCl, kept at room temperature for 30 min, and then the pH was adjusted to 4.6. The solution was maintained at 40  C for 1 h and centrifuged at 3000  g for 30 min at 4  C in a centrifuge R64 Allegra (Beckman Coulter,
polis, IN, USA). The supernatant was filtered through glass Indiana wool and Whatman filter paper no. 113 (pore size of 30 mm), and frozen at 80  C. The resulting pellet (pH 4.6-insoluble fraction), was analysed by CE, and the supernatant (pH 4.6-soluble fraction) was subjected to freeze-drying. Duplicate aliquots of the freezedried samples (10 mg) were dissolved in 1 mL of 70% ethanol, kept at room temperature for 30 min and centrifuged at 13,000  g for 10 min. The resulting supernatant, pH 4.6-soluble and 70% ethanol-soluble fraction, was separated for MALDI-TOFMS analysis. 2.4.2. Analysis of pH 4.6-insoluble fraction by capillary electrophoresis The pH 4.6-insoluble fraction was analysed by capillary electrophoresis (CE) according to Ortega, Albillos, and Busto (2003) and Otte, Zakora, Kristiansen, and Qvist (1997) with modifications as described by Alves, Merheb-Dini, Gomes, Da Silva, and Gigante (2013). The separation was performed on a fused silica capillary column of 57 cm (50 cm effective length)  75 mm, in P/ACE MDQ system (Beckman Coulter, Santana de Parnaiba, SP, Brazil) and Karat software (Beckman Coulter). For that, 20 mg of the pH 4.6-insoluble fraction were dissolved in 1 mL buffer solution containing 10 mM sodium phosphate, 8 M urea, and 10 mM dithiothreitol (DTT) at pH 8.0, and kept at rest for 1 h. The samples were filtered through a 0.45 mm membrane filter and injected for 5 s at 3.45  103 Pa. The separation occurred at 18.5 kV and 23  C, and the detection was performed at 214 nm for 60 min. Between injections, the capillary was washed with 0.5 M NaOH for 5 min, pure water for 5 min, and running buffer (10 mM sodium phosphate, 6 M urea, and 0.05% hydroxypropyl methylcellulose) with pH adjusted to 3.0 for 5 min. For peak identification, a-CN, b-CN and k-CN standards (SigmaeAldrich, St. Louis, MO, USA) were used. 2.4.3. Analysis of pH 4.6-soluble and 70% ethanol-soluble fraction by MALDI-TOF-MS Acetonitrile HPLC grade (99.9%), a-cyano-4-hydroxycinnamic acid (CHCA, 99%) and trifluoroacetic acid (99%) were purchased from SigmaeAldrich, and the peptide calibration standard II was purchased from Bruker Daltonics (Bremen, Germany), which is composed of 9 peptides: bradykinin 1e7, m/z 757.3992 [M þ H]þ; angiotensin II, m/z 1046.5418 [M þ H]þ; angiotensin I, m/z 1296.6848 [M þ H]þ; substance P, m/z 1347.7354 [M þ H]þ; bombesin, m/z 1619.8223 [M þ H]þ; renin substrate, M þ H m/z 1758.9326 [M þ H]þ; ACTH clip 1e17, m/z 2093.0862 [M þ H]þ; ACTH clip 18e39, m/z 2465.1983 [M þ H]þ; somatostatin 28, m/z 3147.4710 [M þ H]þ. The mass spectra were acquired in an MALDI-TOF instrument Autoflex III (Bruker Daltonics) equipped with a Smart Beam laser, according to the conditions described by Baptista, Araújo, Eberlin, and Gigante (2017) with modifications. Samples (1 mL) were applied on a steel target plate (MSP 96 polished-steel target, Bruker Daltonics), dried at room temperature, and covered with matrix solution (1 mL) previously prepared from a-cyano-4hydroxycinnamic acid (CHCA) dissolved in 50% acetonitrile and 2.5% trifluoroacetic acid. Subsequently, the droplet was fully evaporated, and the plate inserted into the mass spectrometer. The equipment was operated in positive ion reflector mode with mass range of m/z 600e3500, controlled by Flex Control 3.3 software (Bruker Daltonics). The laser power was adjusted to 50e80% and

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ion source 1, ion source 2, lens, reflector, and reflector 2 voltages were 20.00, 17.77, 7.90, 21.95, and 10.03 kV, respectively. The pulsed ion extraction time was 30 ns, and the mass deletion threshold was set at m/z 600. The instrument was calibrated using external peptide calibration standard II. Each sample was analysed in quintuplicate. 2.4.4. Data processing and chemometric analysis The mass spectra data were preprocessed using the software Flex Analysis 3.4 (Bruker Daltonics), which included baseline subtraction and normalisation along the m/z axis. For each spectrum, a list of ion peaks corresponding to relative intensities (%) (ratio of the ion peak intensity to that of the base peak) was exported to Excel, and the data matrix was assembled. To reduce noise and low abundance metabolites, only the variables (ion peaks) with relative intensity higher than 10% were considered. The online MetaboAnalyst 3.0 software was used for chemometric analysis (Xia, Mandal, Sinelnikov, Broadhurst, & Wishart, 2012), using the Pareto method for data pretreatment. Principal component analysis (PCA) was the chemometric tool used to analyse the mass spectra data of pH 4.6 and 70% ethanol soluble fractions, which is an unsupervised technique for pattern recognition. 2.5. Firmness The firmness of cheeses was measured by texture profile analysis (TPA) in a TA-XT2 texture analyser, with an aluminium probe of 35 mm in diameter. Cylindrical samples of 20 mm diameter and 24 mm height were sampled, as described by Mazal et al. (2007). The cylindrical samples were packed in plastic film, stored in plastic bags (low density polyethylene, LDPE; Spelvac CS, Spel, AtibaiaeSP, Brazil) and kept in an ice-water bath (10  C) for a minimum of 4 h to stabilise the temperature. The test speed was 100 mm min1, with compression of 40% of the initial height of the cheese cylinder, with repetition after 5 s. 2.6. Sensory evaluation Sensory evaluation was performed after approval by the Research Ethics Committee of UNICAMP (University of Campinas, Campinas, SP, Brazil, CAAE: 39693114.5.0000.5404). Cheeses were evaluated after 30 and 60 days of ripening by 120 untrained assessors. The samples were evaluated for overall acceptance, appearance, aroma, flavour, and texture, using a 9 cm unstructured linear hedonic scale between the anchors “I disliked it very much” and “I liked it very much” (Stone & Sidel, 2004). The tests were performed in individual cabins under white light. One cube of cheese from each treatment was served in a plastic container coded with three-digit numbers. The order of presentation was randomised, and cracker biscuits and mineral water at room temperature were served to assessors for palate cleansing between samples. 2.7. Experimental design and data analysis A 3  5 factorial experimental design in completely randomised blocks was used, with three replicates. The treatment had three levels of variation (control cheese, cheese with 25% salt reduction, and cheese with 50% salt reduction), and the storage time with 5 levels of variation (1, 15, 30, 45, and 60 days of storage). The effect of salt content and ripening period, as well as the interaction between these factors, were evaluated by Analysis of Variance (ANOVA). In case of difference, the averages were compared by the Tukey's test considering a level of significance of 5%. The results were analysed using the software Statistica 7.0.

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3. Results and discussion 3.1. Physicochemical characterisation of milk and cheeses The heat-treated milk presented on average 12.23 ± 0.08% total solids, 3.51 ± 0.02% fat, 3.43 ± 0.26% protein, 0.72 ± 0.09% ash, and pH 6.76 ± 0.03. The milk showed positive result for the peroxidase activity and inactivation of alkaline phosphatase, revealing the efficiency of the heat treatment. As shown in Table 1, the treatments significantly affected the salt content, salt-in-moisture ratio (S/M), and sodium levels of the cheeses. As the cheese manufacture procedure was identical for all cheeses until the brining step, other compositional factors were not impacted. Cheeses subjected to 6 and 3 h of salting presented 26.8% and 49.9% of salt reduction in relation to the control cheese, respectively. Regarding the sodium reduction, the same treatments presented 27.3% and 47.9% sodium reduction when compared with the control. 3.2. Effect of salt reduction and storage time on Prato cheese proteolysis Proteolysis monitoring through the formation of nitrogen compounds revealed that salt reduction did not significantly affect pH 4.6 SN (%TN) (p ¼ 0.8128) and TCA 12% SN (%TN) (p ¼ 0.9941), which represents no impact of salt reduction on chymosin, plasmin or starter activities. Storage time affected proteolysis of cheese which showed an increase in nitrogen fractions (p < 0.0001) during 60 days of storage. As observed in Fig. 1, the control cheese and cheeses with 25 and 50% salt reduction showed similar casein degradation profiles, showing the para-kcasein formed during coagulation, cleavage of aS1-casein at the Phe23ePhe24 bond by the residual chymosin, and slower degradation of b-casein. The hydrolysis of aS1-casein Phe23ePhe24 is responsible for the softening of cheeses and leads to the formation of the peptides aS1-CN (f1e23) and aS1-CN (f24e199) (aS1-I-CN) (Upadhyay, McSweeney, Magboul, & Fox, 2004). The formation of the fraction aS1-CN (f24e199), insoluble at pH 4,6, was evidenced in all cheeses after 1 day of storage due to the appearance of aS1-I-CN 8P and aS1-I-CN 9P peaks, which was more evident after 15 days of ripening. The b-casein degradation was evidenced in all cheeses by the reduction of the intensities of the peaks of b-CN A1 and b-CN A2 variants. The analyses of pH 4.6 and 70% ethanol-soluble peptides by MALDI-TOF-MS revealed a very complex peptide profile. For all cheeses, including those with 25% and 50% salt reduction, 183 peaks were detected throughout 60 days of ripening (Supplementary material: Figs. S1eS3). The peptide identifications were

performed through the comparison of the mass-to-charge ratio (m/ z) values obtained from the mass spectra for the protonated molecules and those from peptides previously identified in cheese (Addeo et al., 1994, 1992; Alli, Okonieska, Gibbs, & Kunishi, 1998; Broadbent et al., 1998; Exterkate, Lagerwerf, Haverkamp, &
, Herrouin, & Le
onil, 2001; Schalkwijk, 1997; Gagnaire, Molle Gouldsworthy, Leaver, & Banks, 1996; Soeryapranata, Powers, Weller, Hill, & Siems, 2004; Soeryapranata et al., 2008). Thirty-six peaks corresponded to m/z ratios already reported in the literature as peptides derived from casein hydrolysis in cheeses (Supplementary material: Tables S1eS3). A selection criterion was required to analyse the data set, due to the diversity and complexity of peptides formed during cheese ripening. When analysing mass spectral data by MALDI-TOF-MS, some authors (Piraino et al., 2007; Rossano et al., 2005) have discarded peaks with a relative intensity lower than 10% of the intensity of the highest peak. Thus, Table 2 exhibited the peptides with a relative intensity greater than 10% for the control, cheese with 25% salt reduction, and 50% salt reduction. Despite a large number of peaks detected in both the control cheese and those with salt reduction during ripening, only 11 peptides, originating mainly from the aS1-casein, b-casein and possibly k-casein fractions, exhibited relative intensities greater than 10%. The pH 4.6 and 70% ethanol soluble peptide b-CN (f193e209) (m/z 1881) was detected in all cheeses on the first day of ripening. This peptide was the most intense signal at all periods evaluated and it was used as a reference for calculating the relative intensity of the other ion peaks. This result indicates that there was little enzymatic degradation of the peptide b-CN (f193e209) (m/z 1881) over the 60 days of ripening. This peptide, composed of 13 hydrophobic amino acids (AAs) in a total of 17 AAs, is recognised as a bitter-tasting peptide, whose accumulation in cheeses may lead to a defect in taste (Soeryapranata et al., 2002). The peptides aS1-CN (f1e9) (m/z 1141) and aS1-CN (f1e13) (m/z 1536) are also recognised as bitter-tasting peptides in cheeses (Soeryapranata et al., 2008). In the present study, the reduction of 25% and 50% salt in Prato cheese did not lead to an increase in relative intensity of these bittertasting peptides. The salt concentration did not affect the signal intensity of the peptide b-CN (f193e209) (m/z 1881), which remained at high signal intensity during ripening for both the control and cheeses with salt reduction. In addition, the peptide aS1-CN (f1e9) (m/z 1141) appeared only in the control cheese after 45 and 60 days of ripening, while the peptide aS1-CN (f1e13) (m/z 1536) appeared in higher intensity in the control cheese throughout the ripening period (Table 2). Møller et al. (2012) observed an inverse relation between the concentration of the majority of the identified peptides and salt content in Cheddar cheese. The present study showed that the salt reduction of up to

Table 1 Physicochemical composition of cheeses.a

pH Acidity (%) Moisture (%) FDM (%) PDM (%) pH 4.6 SN (% TN) TCA 12% SN (% TN) Salt (%) S/M (%) Sodium (mg 100 g1)

Control cheese

Cheese with 25% salt reduction

Cheese with 50% salt reduction

p-Value

5.08 ± 0.02a 0.88 ± 0.05a 39.69 ± 0.43a 52.63 ± 2.82a 41.96 ± 0.96a 5.80 ± 0.56a 2.54 ± 0.22a 1.68 ± 0.04a 4.25 ± 0.12a 547.35 ± 33.10a

5.06 ± 0.04a 1.07 ± 0.11a 39.80 ± 0.81a 52.53 ± 2.61a 42.07 ± 1.42a 6.27 ± 0.23a 2.57 ± 0.14a 1.23 ± 0.04b 3.09 ± 0.06b 397.99 ± 28.89b

5.05 ± 0.04a 0.98 ± 0.14a 39.82 ± 0.98a 53.54 ± 2.93a 42.81 ± 1.24a 6.30 ± 0.84a 2.67 ± 0.22a 0.84 ± 0.02c 2.12 ± 0.04c 285.27 ± 17.47c

0.6692 0.2049 0.9273 0.8903 0.6677 0.5589 0.7535 <0.0001 <0.0001 <0.0001

a Abbreviations are: FDM, fat in dry matter; PDM, protein in dry matter; S/M, salt in moisture. Moisture, salt, S/M and sodium were determined after 15 days of storage, the other physicochemical parameters were determined after 1 day of storage. Values are means ± standard deviation (n ¼ 3); means with different superscript letters in the same line are significantly different by the Tukey's test (p < 0.05).

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Fig. 1. Capillary electropherograms of the pH 4.6-insoluble fractions of Prato cheeses (A, control; B and C, cheeses with 25% and 50% salt reduction, respectively) showing the hydrolysis profiles of the caseins during storage. Identification and labelling of peaks according to sigma standards and previous findings (Alves et al., 2013; Otte et al., 1997; Rehn,
n, & Ardo € , 2010). AU: arbitrary unit. Petersen, Saede

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Table 2 Peptides with relative intensity greater than 10% of the highest peak detected by MALDI-TOF-MS in the pH 4.6 and 70% ethanol soluble fractions of Prato cheeses during ripening.a m/z

906 1141 1247 1536 1665 1718 1877 1881 1899 1903 1991

Suggested peptide

aS1-CN f17e23/k-CN f161e169 aS1-CN f1e9 aS1-CN f14e23 aS1-CN f1e13 aS1-CN f1e14 b-CN f194e209 aS1-CN f1e16 b-CN f193e209 * *

aS1-CN f1e17

Control cheese

Cheese with 25% salt reduction

Cheese with 50% salt reduction

Day 1

Day 15 Day 30 Day 45 Day 60 Day 1 Day 15 Day 30 Day 45 Day 60 Day 1 Day 15 Day 30 Day 45 Day 60

20 (11) <10 11 (3) 18 (8) <10 25 (5) 86 (12) 89 (11) 15 (6) 18 (4) 18 (4)

26 (6) <10 14 (2) 24 (8) <10 <10 76 (21) 91 (15) 16 (4) 23 (6) 19 (7)

16 (7) <10 <10 29 (15) 10 (5) <10 47 (12) 100 (0) 11 (4) 28 (3) 20 (8)

14 (8) 13 (7) <10 42 (25) 15 (10) <10 35 (16) 100 (1) <10 27 (1) 23 (11)

<10 11 (5) <10 36 (15) 14 (7) <10 20 (6) 100 (0) <10 26 (3) 20 (7)

23 (5) <10 10 (2) 15 (5) <10 26 (5) 91 (7) 91 (8) 18 (5) 20 (4) 17 (3)

20 (6) <10 <10 13 (2) <10 <10 74 (6) 93 (12) 15 (2) 22 (5) 15 (1)

17 (5) <10 <10 20 (7) 11 (4) <10 58 (15) 100 (0) 13 (4) 27 (1) 18 (7)

<10 <10 <10 18 (8) 11 (5) <10 35 (8) 100 (0) <10 29 (3) 16 (6)

<10 <10 <10 14 (3) 10 (3) <10 19 (2) 100 (0) <10 30 (2) 12 (2)

19 (6) <10 <10 10 (4) <10 23 (5) 91 (9) 93 (9) 18 (4) 20 (4) 15(4)

20 (7) <10 <10 <10 <10 <10 78 (16) 93 (11) 18 (4) 25 (6) 14 (3)

12 (4) <10 <10 10 (5) <10 <10 52 (4) 100 (0) 11 (2) 27 (2) 13 (4)

<10 <10 <10 11 (1) 11 (1) <10 34 (7) 100 (0) <10 29 (3) 12 (1)

<10 <10 <10 <10 <10 <10 19 (3) 97 (6) <10 27 (2) 10 (4)

a

Values are mean relative intensity with standard deviation in parenthesis. Abbreviations are: m/z, mass-to-charge ratio; CN, casein. Peptides are suggested according to the comparison between the m/z values obtained in the mass spectra for the protonated molecules and those from peptides previously identified in cheese; an asterisk indicates unidentified.

50% in Prato cheese did not lead to an increase in relative intensities of known bitter tasting peptides. Fig. 2 shows the score and loading plots of PCA applied to the mass spectra data of pH 4.6 and 70% ethanol soluble fractions of control cheese and cheeses with 25% and 50% salt reduction. Fig. 2a, c, and e display the information related to the cheeses in the form of score plots. The loading plots (Fig. 2b, d, and f) show the variables that describe the samples in the score plots, and the most important variables for samples discrimination are those with higher positive and negative loadings. The PCA of both the control cheeses (Fig. 2a) and those with 25% salt reduction (Fig. 2c) revealed that they can be distinguished from each other based on the first two PCs, explaining 86.3% and 85.8% of the total variance, respectively. In both cases, the discriminations of the samples were not observed in PC2 alone. The PCA of the control cheese (Fig. 2a and b) showed that the aS1-CN (f1e16) peptide (m/z 1877) corresponded to the variable with the highest positive loading (Fig. 2b) for discriminating the samples to the right of the score plots (Fig. 2a), representing the cheeses with a ripening period of 1 and 15 days. In contrast, control cheeses with a longer ripening period, 45 and 60 days, presented lower relative intensity of this peptide and were characterised by the high relative intensity of the aS1-CN (f1e13) peptide (m/z 1536) (Fig. 2a and b; Table 2). The effective separation of the samples indicates an evolution of the proteolysis of this cheese variety, with the presence of a characteristic peptide profile for each ripening period evaluated. In the PCA of the Prato cheese with 25% salt reduction (Fig. 2c and d), an unidentified peptide (m/z 1903) displays the highest positive loading in PC1 (Fig. 2d), presenting high relative abundance in cheeses with 60 days of ripening. The aS1-CN (f1e16) peptide (m/z 1877) was the variable with the highest negative loading in PC1, showing higher relative abundance in the cheese samples with 1 and 15 days of ripening. The Prato cheese samples with 50% salt reduction were better grouped in PC1 versus 3 (Fig. 2e), which accounted for 68% of the multivariate information. The peptide associated with the ion of m/ z 1877 (aS1-CN (f1e16)) showed high positive loading in PC1 (Fig. 2f), with high relative abundance in the samples with 1 and 15 days of ripening. The separation was observed only between the cheeses at 1 and 60 days of ripening, the samples from other ripening periods did not present good separation behaviour. This result suggests a more disordered proteolysis in cheeses with 50% salt reduction when compared with the other cheeses (control and cheese with 25% salt reduction), possibly due to the higher microbial and enzymatic activities at lower salt content.

In addition, chemometric analyses of mass spectra data of pH 4.6 and 70% ethanol soluble fractions were performed for the three cheese treatments together in specific ripening periods (1, 30, and 60 days) to compare the evolution of cheese proteolysis. The PCA of the cheeses after 1, 30, and 60 days of ripening showed that the first two PCs (Fig. 3) explain 82.9%, 82.3%, and 83.9% of the total variance, respectively, although the samples were not well discriminated, suggesting a minimal effect of salt reduction on peptide profile formed during Prato cheese ripening. The higher relative abundance of the aS1-CN (f1e13) peptide (m/ z 1536) in the control cheese (Table 2) throughout storage suggests greater activity of the lactic acid culture and, possibly NSLAB, in cheeses with lower salt content, leading to the production of proteolytic enzymes that degrade this fraction. According to Upadhyay et al. (2004), different strains of Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris, which were the lactic acid cultures used in the manufacture of the cheeses in this study, produce peptidases that hydrolise the fraction aS1-CN f1e13 (m/z 1536). The lower hydrolysis of this fraction in the control cheese was evidenced by the accumulation of this peptide during ripening, with a relative intensity of 35.85% in the control cheese, 14.43% in the cheese with 25% salt reduction and lower than 10% in the cheese with 50% salt reduction after 60 days of ripening (Table 2).

3.3. Effect of treatments on the firmness of Prato cheese The cheese with 50% salt reduction was significantly less firm (p ¼ 0.0021) than both the control and cheese with 25% salt reduction. Control cheese and cheeses with 25% and 50% salt reduction showed average firmness values of 3332.95 ± 301.33 g, 3196.06 ± 386.09 g, and 2936.45 ± 498.77 g, respectively. Lower firmness was also reported by Rulikowska et al. (2013) in Cheddar cheese with salt reduction. The authors attributed the lower firmness to the greater proteolysis and changes in the hydration of the protein matrix due to salt reduction. In our study, the salt reduction did not increase the proteolysis, suggesting that changes in firmness were exclusively due to the physicochemical changes of the protein matrix. The effect of salt on protein hydration is conditioned to the salt content used in the manufacturing process. Low salt concentrations can favour casein hydration (salting-in), while high salt contents lead to protein dehydration (salting-out) (Guinee & Fox, 2004). The reduction of 50% salt in cheeses may have resulted in lower water holding capacity and, consequently, more free water in the system resulting in less firmness.

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Fig. 2. Principal component analysis (PCA) of mass spectra data of pH 4.6 and 70% ethanol soluble fractions. Score plots (A, C, E) and loading plots (B, D, F) of control cheese (A, B) and cheeses with 25% (C, D) and 50% (E, F) salt reduction.

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Fig. 3. Principal component analysis (PCA) of mass spectra data of pH 4.6 and 70% ethanol soluble fractions. Score plots (A, C, E) and loading plots (B, D, F) of cheeses after 1 day (A, B), 30 days (C, D), and 60 days (E, F) of ripening.

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References

Table 3 Scores for the sensory attributes.a

30 days of ripening Appearance Aroma Flavour Texture Overall acceptance 60 days of ripening Appearance Aroma Flavour Texture Overall acceptance

109

Control cheese

Cheese with 25% salt reduction

Cheese with 50% salt reduction

p-Value

7.35 7.10 6.81 7.25 7.13

7.25 7.01 6.81 6.93 6.91

7.24 6.98 6.54 6.99 6.78

0.8029 0.8368 0.3442 0.2741 0.1810

7.19 7.00 6.79a 7.11a 6.94a

7.16 6.83 6.16b 6.78ab 6.52ab

7.13 6.89 5.70b 6.40b 6.14b

0.9488 0.7237 0.0002 0.0080 0.0018

a Scores were obtained using a 9-cm unstructured linear hedonic scale with the anchors “I disliked it very much” and “I liked it very much”; values are means (n ¼ 120), means with different superscript letters in the same line showed significant difference by the Tukey's test (p < 0.05).

3.4. Sensory evaluation Table 3 shows the results of the sensory evaluation of cheeses after 30 and 60 days of ripening. The Brazilian legislation has established the ripening period for Prato cheese to be at least 25 days (Brasil, 1997). After 30 days, no significant differences were observed in appearance, aroma, flavour, texture, and overall acceptance of the cheeses, indicating that cheeses with salt reduction and control cheeses were equally accepted. After 60 days of ripening, the cheeses with salt reduction showed a different behaviour regarding the sensory parameters evaluated. The reduction of 25% salt content did not affect the overall acceptance of the cheese, even though cheeses with 25% salt reduction were less acceptable than the control cheese regarding the flavour attribute. Cheeses with 50% salt reduction were less acceptable than the control for the attributes flavour, texture, and overall acceptance. 4. Conclusion Salt reduction did not affect the proteolysis evaluated by both non-specific and the applied specific methods. The reduction of 25 and 50% salt content in Prato cheese did not lead to the formation of known bitter tasting peptides, as observed in the analysis of peptides by MALDI-TOF-MS. Further studies on the identification of peaks not identified in our study (e.g., m/z 1899 and 1903) and the evaluation of the fraction pH 4.6 soluble-70% ethanol insoluble are needed. The sensory evaluation indicated the possibility of reducing 25% salt in Prato cheese, without significantly affecting the global acceptance, which makes Prato cheese a more suitable matrix for salt reduction than other cheese varieties. A more robust sensory evaluation is required to better understand the lower acceptance of cheese with reduction of 25% salt content for the attribute flavour when compared with the control after 60 days of ripening. Acknowledgements The authors thank the Higher Education Personnel Improvement Coordination (CAPES) for granted scholarship. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.idairyj.2017.08.001.

Addeo, F., Chianese, L., Sacchi, R., Musso, S. S., Ferranti, P., & Malorni, A. (1994). Characterization of the oligopeptides of Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic acid/L. Journal of Dairy Research, 61, 365e374. Addeo, F., Chianese, L., Salzano, A., Sacchi, R., Cappucio, U., Ferranti, P., et al. (1992). Characterization of the 12% trichloroacetic acid-insoluble oligopeptides of Parmigiano-Reggiano cheese. Journal of Dairy Research, 59, 401e411. Alli, I., Okonieska, M., Gibbs, B. F., & Kunishi, Y. (1998). Identification of peptides in Cheddar cheese by electrospray ionisation mass spectrometry. International Dairy Journal, 8, 643e649. Alves, L. S., Merheb-Dini, C., Gomes, E., Da Silva, R., & Gigante, M. L. (2013). Yield, changes in proteolysis, and sensory quality of Prato cheese produced with different coagulants. Journal of Dairy Science, 96, 7490e7499. AOAC. (2006a). Direct forced air oven drying, method no. 990.19. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., p. 35). Gaithersburg, MD, USA: AOAC International. AOAC. (2006b). Gerber method, method no. 2000.18. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., pp. 21e23). Gaithersburg, MD, USA: AOAC International. AOAC. (2006c). Gravimetric method, method no. 935.42. In W. Horwitz (Ed.), Official methods of analysis of AOAC International (18th edn., pp. 72e73). Gaithersburg, MD, USA: AOAC International. AOAC. (2006d). Gravimetric method, method no. 945.46. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., p. 10). Gaithersburg, MD, USA: AOAC International. AOAC. (2006e). Kjeldahl method, method no. 2001.14. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., pp. 72e73). Gaithersburg, MD, USA: AOAC International. AOAC. (2006f). Kjeldahl method, method no. 991.20. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., pp. 10e12). Gaithersburg, MD, USA: AOAC International. AOAC. (2006g). Method I, method no. 926.08. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., pp. 68e69). Gaithersburg, MD, USA: AOAC International. AOAC. (2006h). Method V, method no. 979.13. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., pp. 37e38). Gaithersburg, MD, USA: AOAC International. AOAC. (2006i). Titrimetric method, method no. 920.124. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., p. 73). Gaithersburg, MD, USA: AOAC International. AOAC. (2006j). Titrimetric method, method no. 947.05. In W. Horwitz (Ed.), Official methods of analysis of AOAC international (18th edn., p. 7). Gaithersburg, MD, USA: AOAC International. Baptista, D. P., Araújo, F. D. S., Eberlin, M. N., & Gigante, M. L. (2017). A survey of the peptide profile in Prato cheese as measured by MALDI-MS and capillary electrophoresis. Journal of Food Science, 82, 386e393.
ria e Abastecimento e MAPA. Portaria n Brasil. (1997). Minist
erio da Agricultura Pecua ~o de 358, de 04 de setembro de 1997, que aprova o Regulamento T
ecnico para Fixaça
rio Oficial da Unia ~o: Brasília, Distrito Identidade e Qualidade do Queijo Prato. Dia ~o 1 [Ministry of Agriculture, Livestock and Federal, em 08 de setembro de 1997. Seça Food Supply e MAPA. Ordinance no. 358, September 4, 1997. Technical rules for identity and quality of Prato cheese. Official gazette: Brasilia, Federal District, September 08, 1997. Section 1]. Broadbent, J. R., Strickland, M., Weimer, B. C., Johnson, M. E., & Steele, J. L. (1998). Peptide accumulation and bitterness in Cheddar cheese made using singlestrain Lactococcus lactis starters with distinct proteinase specificities. Journal of Dairy Science, 81, 327e337. BSI. (1989). Determination of fat content of milk and milk products (Gerber methods). Methods (p. 12). London, UK: British Standard Institution. Bynum, D. G., & Barbano, D. M. (1985). Whole milk reverse osmosis retentate for Cheddar cheese manufacture: Chemical changes during aging. Journal of Dairy Science, 68, 1e10. Doyle, M. E., & Glass, K. A. (2010). Sodium reduction and its effect on food safety, food quality, and human health. Comprehensive Reviews in Food Science and Food Safety, 9, 44e56. Exterkate, F. A., Lagerwerf, F. M., Haverkamp, J., & Schalkwijk, S. V. (1997). The selectivity of chymosin action on aS1- and b-caseins in solution is modulated in cheese. International Dairy Journal, 7, 47e54. Farkye, N. Y. (2004). Cheese technology. International Journal of Dairy Technology, 57, 91e98. Fox, P. F., Guinee, T. P., Cogan, T. M., & McSweeney, P. L. H. (2000). Biochemistry of cheese ripening. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), Fundamentals of cheese science (pp. 236e281). Gaithersburg, MD, USA: Aspen Publishers. Fox, P. F., McSweeney, P. L. H., & Singh, T. K. (1995). Methods for assessing proteolysis in cheese during ripening. In E. L. Malin, & M. L. Tunick (Eds.), Chemistry of structure/function relationships in cheese (pp. 161e194). New York, NY, USA: Plenum Press.
, D., Herrouin, M., & Le
onil, J. (2001). Peptides identified during Gagnaire, V., Molle Emmental cheese ripening: Origin and proteolytic systems involved. Journal of Agriculture and Food Chemistry, 49, 4402e4413.

110

D.P. Baptista et al. / International Dairy Journal 75 (2017) 101e110

Ganesan, B., Brown, K., Irish, D. A., Brothersen, C., & McMahon, D. J. (2014). Manufacture and sensory analysis of reduced- and low-sodium Cheddar and mozzarella cheeses. Journal of Dairy Science, 97, 1970e1982. Gouldsworthy, A. M., Leaver, J., & Banks, J. M. (1996). Application of a mass spectrometry sequencing technique for identifying peptides present in Cheddar cheese. International Dairy Journal, 6, 781e790. Grummer, J., Bobowski, N., Karalus, M., & Vickers, Z. (2013). Use of potassium chloride and flavor enhancers in low sodium Cheddar cheese. Journal of Dairy Science, 96, 1401e1418. Grummer, J., Karalus, M., Zhang, K., Vickers, Z., & Shoenfuss, T. C. (2012). Manufacture of reduced-sodium Cheddar-style cheese with mineral salt replacers. Journal of Dairy Science, 95, 2830e2839. Guinee, T. P., & Fox, P. F. (2004). Salt in cheese: Physical, chemical and biological aspects. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), General aspects: Vol. 1. Cheese e Chemistry, physics, and microbiology (3rd edn., pp. 347e360). London, UK: Elsevier Academic Press. Guinee, T. P., & Sutherland, B. J. (2011). Salting of cheese. In J. W. Fuquay, P. F. Fox, & P. L. H. McSweeney (Eds.), Encyclopedia of dairy sciences (2nd edn., pp. 595e606). London, UK: Elsevier Academic Press. Kuchroo, C. N., & Fox, P. F. (1982). Soluble nitrogen in cheddar cheese: Comparison of extraction procedures. Michwissenschaft, 37, 331e335.
ria. M.A. M
LANARA. (1981). Secretaria de Defesa Agropecua etodos Analíticos Oficiais de Controle de Produtos de origem animal e seus Ingredientes. II-M
etodos Físicos e Químicos [Secretariat of Agriculture Defense. Official Methods for examination of animal products. II-physical and chemical methods]. Brasilia-DF: Ministry of Agriculture, Livestock and Supply. Mazal, G., Vianna, P. C. B., Santos, M. V., & Gigante, M. L. (2007). Effect of somatic cell count on Prato cheese composition. Journal of Dairy Science, 90, 630e636. Mistry, V. V., & Kasperson, K. M. (1998). Influence of salt on the quality of reduced fat cheese. Journal of Dairy Science, 81, 1214e1221. € , Y. (2012). Manufacture and biochemical Møller, K. K., Rattray, F. P., Høier, E., & Ardo characteristics during ripening of Cheddar cheese with variable NaCl and equal moisture content. Dairy Science and Technology, 92, 515e540. Ortega, N., Albillos, S. M., & Busto, W. D. (2003). Application of factorial design and response surface methodology to the analysis of bovine caseins by capillary zone electrophoresis. Food Control, 14, 307e315. Otte, J., Zakora, M., Kristiansen, K. R., & Qvist, K. B. (1997). Analysis of bovine caseins and primary hydrolysis products in cheese by capillary zone electrophoresis. Lait, 77, 241e257. Piraino, P., Upadhyay, V. K., Rossano, R., Riccio, P., Parente, E., Kelly, A. L., et al. (2007). Use of mass spectrometry to characterize proteolysis in cheese. Food Chemistry, 101, 964e972.
n, K. H., & Ardo € , Y. (2010). Ripening of extra-hard Rehn, Y., Petersen, M. A., Saede cheese made with mesophilic dl-starter. International Dairy Journal, 20, 844e851.

Richardson, G. H. (1985). Standard methods for examination of dairy products. Washington, DC, USA: American Public Health Association. Rossano, R., Piraino, P., D'Ambrosio, A., O'Connell, O. F., Ungaro, N., McSweeney, P. L. H., et al. (2005). Proteolysis in miniature cheddar-type cheeses manufactured using extracts from crustacean Munida as coagulant. Journal of Biotechnology, 120, 220e227. Rulikowska, A., Kilcawley, K. N., Doolan, I. A., Alonso-Gomez, M., Nongonierma, A. B., Hannon, J. A., et al. (2013). The impact of reduced sodium chloride content on Cheddar cheese quality. International Dairy Journal, 28, 45e55. Schornsteiner, E., Mann, E., Bereuter, O., Wagner, M., & Schmitz-Esser, S. (2014). Cultivation-independent analysis of microbial communities on Austrian raw milk hard cheese rinds. International Journal of Food Microbiology, 180, 88e97. Soeryapranata, E., Powers, J. R., Fajarrini, F., Weller, K. M., Hill, H. H., & Siems, W. F. (2002). Relationship between MALDI-TOF analysis of b-CN f193e209 concentration and sensory evaluation of bitterness intensity of aged Cheddar cheese. Journal of Agriculture and Food Chemistry, 50, 4900e4905. Soeryapranata, E., Powers, J. R., & Ünlü, G. (2008). Degradation of aS1-CN f1-23 by aminopeptidase N and endopeptidases E, O, O2, and O3 of Lactobacillus helveticus WSU19 under cheese ripening conditions. International Dairy Journal, 18, 178e186. Soeryapranata, E., Powers, J. R., Weller, K. M., Hill, H. H., & Siems, W. F. (2004). Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Lebensmittel-Wissenschaft þ Technologie, 37, 17e22. Sousa, M. J., Ardo, Y., & McSweeney, P. L. H. (2001). Advances in the study of proteolysis during cheese ripening. International Dairy Journal, 11, 327e345. Stone, H., & Sidel, J. L. (2004). Sensory evaluation practices (3rd edn.). San Diego, CA, USA: Elsevier Academic Press. Thibaudeau, E. T., Roy, D., & St-Gelais, D. (2015). Production of brine-salted Mozzarella cheese with different ratios of NaCl/KCl. International Dairy Journal, 40, 54e61. Upadhyay, V. K., McSweeney, P. L. H., Magboul, A. A. A., & Fox, P. F. (2004). Proteolysis in cheese during ripening. In P. F. Fox, P. L. H. McSweeney, T. M. Cogan, & T. P. Guinee (Eds.), General aspects: Vol. 1. Cheese e Chemistry, physics, and microbiology (3rd edn., pp. 391e433). London, UK: Elsevier Academic Press. Visser, S. (1993). Proteolytic enzymes and their relation cheese ripening and flavor. Journal of Dairy Science, 76, 329e350. WHO. (2012). Guideline: Sodium intake for adults and children. Geneva, Switzerland: World Health Organisation. Xia, J., Mandal, R., Sinelnikov, I. V., Broadhurst, D., & Wishart, D. S. (2012). MetaboAnalyst 2.0dA comprehensive server for metabolomics data analyst. Nucleic Acids Res, 40, W127eW133.