Repositorio Institucional

Czech J. Food Sci., 34, 2016 (5): 445–455 FFood Technology and Economy, Engineering and Physical Properties

doi: 10.17221/567/2015-CJFS

Use of an Exogenous Carboxypeptidase to Accelerate Proteolysis in Reggianito Cheese

Roberto J. Ceruti1, María B. Pirola2, Elisabet Ramos2, Laura Robert2, Amelia C. Rubiolo1 and Guillermo A. Sihufe1

1Instituto de Desarrollo Tecnológico para la Industria Química, Consejo Nacional de Investigaciones Científicas y Técnicas ‒ Universidad Nacional del Litoral, Santa Fe, Argentina;

2Instituto Nacional de Tecnología Industrial (INTI ‒ Lácteos) Rafaela, Argentina

Abstract

Ceruti R.J., Pirola M.B., Ramos E., Robert L., Rubiolo A.C., Sihufe G.A. (2016): Use of an exogenous carboxypeptidase to accelerate proteolysis in Reggianito cheese. Czech J. Food Sci., 34: 445–455.

The effect of an exogenous commercial carboxypeptidase on the proteolysis of Reggianito cheese was evaluated. Cheeses were manufactured using 4 concentrations of enzyme: 0, 5, 10, and 20 g/100 l milk. Cheese samples were analysed at 0, 60, 90, 120, 150, 180, and 210 days of ripening. Nitrogen content values increased during ripening, but no clear effect due to the enzyme addition was observed. A profound degradation of β-casein was observed during the first 2 months of ripening in cheeses with the highest rates of enzyme addition. An increase of amino acid concentrations was clearly observed in some cheeses manufactured with exogenous enzymes compared with control cheeses at the same ripening time. However, the principal component analysis showed that experimental cheeses had a slight increase in the rate of formation and/or degradation of proteolysis products. Our results represent an important contribution to select new alternatives for enzyme addition during the manufacture of hard cheese.

Keywords: Reggianito; cheese; proteolysis; ripening; exogenous enzymes



Proteolysis is the most important and complex biochemical event that occurs during cheese maturation (McSweeney & Fox 1997). The pattern of proteolysis is very variable and it is essentially unique to a particular cheese variety. The observed differences are principally due to different moisture content, levels of NaCl, pH, cheese microflora, among many important factors. Therefore, differences in enzyme activities may be responsible for the modification of traditional characteristics, appearance, texture, and flavour of the product.

Most rennet-coagulated cheeses are ripened after manufacture for periods ranging from a couple of weeks to more than two years. Consequently, as the process can be slow, it can be expensive due to the cost associated with holding a large amount of cheese in adequate ripening facilities (Upadhyay & McSweeney 2003). Various approaches have been used to accelerate cheese ripening, including



the use of elevated ripening temperatures, addition of exogenous enzymes or attenuated starters, use of adjunct cultures, genetic modification of starter bacteria, and high-pressure treatments (McSweeney 2004; Azarnia et al. 2006; El Soda & Awad 2011).

Particularly, addition of exogenous enzymes to cheese increases the enzyme pool accelerating the rate of certain reactions in cheese in contrast to elevated temperature which results in an increase of the rate of all reactions (Upadhyay & McSweeney 2003). In addition, the use of exogenous enzymes is at present an interesting way to accelerate ripening in different cheese varieties. For example, commercial proteinase preparations are used to accelerate ripening in Cheddar and Dutch cheeses (Wilkinson & Kilcawley 2005).

There are four main points for the addition of exogenous enzyme in cheese manufacture: with milk before cheese manufacture, with the starter



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Food Technology and Economy, Engineering and Physical Properties Czech J. Food Sci., 34, 2016 (5): 445–455



doi: 10.17221/567/2015-CJFS



culture or coagulant, at dry salting, and directly into a cheese block (Wilkinson & Kilcawley 2005). Addition to cheese milk appears to be the best stage for enzyme incorporation due to the homogeneous mixing of the enzyme with the milk and its subsequent transfer to cheese curd. However, most of the enzyme added to the milk is lost in the whey and proteolytic enzymes degrade caseins to peptides that are lost in the whey, which results in a reduction of cheese yield (Upadhyay & McSweeney 2003). Some commercial products are available for accelerating cheese ripening, however little data exist in relation to their partitioning or retention within cheese curd during ripening (Wilkinson & Kilcawley 2005; Doolan et al. 2014).

Various researchers have focused their work on accelerating lipolysis in Cheddar cheese by the use of exogenous lipases or enzyme preparations containing lipases (Upadhyay & McSweeney 2003). On the other hand, considering that proteolysis is the major biochemical process occurring during the ripening of some important cheese varieties such as Cheddar, Gouda, and Italian-type cheeses, most enzyme preparations used to accelerate ripening contain proteinases and peptidases (Upadhyay & McSweeney 2003). Most peptidases used to date are aminopeptidases, but few carboxypeptidases have been assessed in cheese ripening which cleave amino acids from the C-terminus of peptides (Kilcawley et al. 2002).

Reggianito is the most important hard cheese produced in Argentina and is one of the most frequently exported cheeses to different countries such as USA, Brazil, Chile, and Russia (M.A.G. y P. 2010; http:// www.minagri.gob.ar/site/index.php). Italian immigrants in the late 19th and early 20th centuries developed a distinctive product inspired by Italian hard cheeses. Reggianito cheese has higher moisture and fat contents, and a shorter ripening period than Italian hard cheeses (Zalazar et al. 1999), and it is manufactured with pasteurised cow’s milk and natural whey starter mainly composed by Lactobacillus helveticus (66%) and Lactobacillus delbrueckii subsp. lactis (33%) (Reinheimer et al. 1996). It is a cheese generally ripened at 11–13°C at 82–85% relative humidity for six months. Studies have been carried out to accelerate Reggianito cheese ripening, principally by elevating the storage temperature (Sihufe et al. 2007, 2010a, b, c). These studies identified that the optimal time for Reggianito Argentino cheese at 18°C ranged between 2 and 3 months. However,



information about the use of exogenous enzymes to accelerate the ripening of Argentinean hard cheeses is limited. The use of carboxypeptidase may offer an interesting alternative to favour the pathways for the amino acid formation, and therefore to accelerate flavour development.

The objective of this study was to evaluate the effect of the addition of an exogenous carboxypeptidase on Reggianito cheese proteolysis.

Material and methods

Cheesemaking. Four different cheese batches were prepared in a pilot plant of INTI (National Institute of Industrial Technology, Rafaela, Argentina) by experienced technicians and following the procedure proposed by Gallino (1994) using pasteurised cow’s milk (fat 21.7 ± 1.2 g/l; protein 31.9 ± 0.0 g/l; lactose 46.8 ± 0.0 g/l; pH 6.60 ± 0.01). Commercial chymosin was used as a rennet (13 g/1000 l of milk). The starter medium is whey-based and is mainly composed of Lactobacillus helveticus and L. delbrueckii subsp. lactis (Reinheimer et al. 1996). The general procedure to obtain this natural whey starter and the volume used to inoculate milk were detailed by Sihufe et al. (2012). Free enzyme preparations (Accelerzyme® CPG, purified enzyme preparation derived from Aspergillus niger, > CPGU/g; DSM Food Specialties, Heerlen, The Netherlands) were added to cheese milk with the starter culture. The following enzyme treatments were used: control cheeses without enzyme addition (C), cheeses with 5 g enzyme/100 l milk (E1), cheeses with 10 g enzyme/100 l milk (E2), and cheeses with 20 g enzyme/100 l milk (E3). Cheeses (cylindrical shape and approximately 8 kg weight, 24 cm diameter, and 12 cm height) were salted by immersion in a saturated NaCl solution at 12°C for 8 days and ripened at 13°C and 85% RH. Samples were obtained at 0, 60, 90, 120, 150, 180, and 210 days of ripening.

Initial composition. After manufacturing (day 0), cheeses were analysed to determine their initial composition: moisture (ISO 2004), NaCl (ISO 2006), protein (ISO 2001), fat (IRAM 1988), and pH (ISO 2004).

Sample analysis. Samples were analysed to determine pH and moisture content (ISO 2004). Maturation was evaluated by the determination of water-soluble nitrogen at pH 4.6 (WSN) and nitrogen soluble in 2.5 g/100 ml phosphotungstic acid (PTA-N) (Gripon et al. 1975). All results obtained from nitrogen frac-



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Czech J. Food Sci., 34, 2016 (5): 445–455 FFood Technology and Economy, Engineering and Physical Properties



doi: 10.17221/567/2015-CJFS



Table 1. Initial composition (Day 0) of Reggianito cheeses (average values ± SD)



Parameter Moisture (g/100 g cheese) NaCl (g/100 g cheese) Protein (g/100 g cheese) Fat (g/100 g cheese) pH



Cheese C 37.31 ± 0.28

2.35 ± 0.13 30.71 ± 0.05 25.00 ± 0.00

5.32 ± 0.03



Cheese E1 37.92 ± 0.21

2.33 ± 0.06 31.22 ± 0.06 24.75 ± 0.35

5.43 ± 0.02



Cheese E2 37.79 ± 0.09

2.33 ± 0.01 32.28 ± 0.12 23.25 ± 0.35

5.31 ± 0.01



Cheese E3 37.57 ± 0.21

2.42 ± 0.01 31.95 ± 0.17 24.25 ± 0.35

5.34 ± 0.01



tions were expressed as a percentage of total nitrogen (TN). All determinations were carried out at least in duplicate.

Additionally, proteolysis was studied by electrophoretic, peptide, and amino acid analysis as described by Sihufe et al. (2010a). Some chromatographic conditions for the amino acid analysis were modified as follows. Soluble fractions in 2.5 g/100 ml sulfosalicylic acid (SSA-SF) were obtained from the water-soluble fraction at pH 4.6. Free amino acids were determined in SSA-SF using the derivatising procedure with o-phthaldialdehyde. The resulting solution was filtered through a disposable 0.2 µm filter before 10 µl were injected. An ISCO chromatography system (Isco, Inc., Lincoln, USA) was used, which consisted of model 2350 HPLC pump, model 2360/2361 gradient programmer, FL-2 fluorescence detector, and Chem Research 150 chromatographic data management/ system controller. A VARIAN (250 × 4.6 mm) C18, 100 Å column (Varian, Inc., Palo Alto, USA) at 40°C was used for chromatographic separations. Separations were carried out at a flow rate of 1.3 ml min using solvent A/tetrahydrofuran/methanol/0.05 mol/l sodium acetate(1 : 19 : 80), pH 5.9 , and solvent B: methanol/0.05 mol/l sodium acetate (80 : 20), pH 5.9 (Jones et al. 1981). The gradient program was: initial composition 0% B, isocratic step at 0% B for 1 min, linear step to 14% B in 5 min, isocratic step at 14% B for 5 min, linear step to 50% B in 5 min, isocratic step at 50% B for 4 min, linear step to 75% B in 6 min, isocratic step at 75% B for 4 min, linear step to 100% B in 6 min, isocratic step at 100% B for 4 minutes. Amino acids were identified according to their retention times by comparison with a standard solution chromatogram.

Statistical analysis. ANOVA was based on the replicates of determinations. The enzyme treatment and the ripening time were selected as main factors for ANOVA. When differences between treatment effects were significant (P < 0.05), a multiple comparison of means was performed using the least significant difference (LSD) test. Principal component



analysis (PCA) was used to reduce the dimensionality of the data obtained. Statistical analysis was carried out using the Minitab software (Minitab Inc., State College, USA).



Results and discussion

Gross composition. Table 1 shows the initial composition of the 4 cheese enzyme treatments studied. The moisture content decreased with ripening time as expected for cheeses ripened without wrapping (Simal et al. 2001; Sihufe et al. 2007). The direct addition of proteinases may increase the syneresis of curd, therefore it may decrease cheese yield (Mohedano et al. 1998). However, in this case, although the level of the added enzyme significantly affected

30



WSN/TN (%)



20



10



0



0



60



90 120 150 180 210



30



cheeses C



Ripening time (days)



cheeses E1



cheeses E2



20



cheeses E3



10



PTA-N/TN (%)



0 0



60



90 120 150 180 210



Ripening time (days)



Figure 1. Profiles of (A) WSN/TN and (B) PTA-N/TN during the ripening of Reggianito cheese (results are shown as mean value with standard deviation)



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Food Technology and Economy, Engineering and Physical Properties Czech J. Food Sci., 34, 2016 (5): 445–455



doi: 10.17221/567/2015-CJFS



Table 2. Average values and standard deviations of moisture and pH corresponding to the studied Reggianito cheese samples



Cheese



Ripening time Moisture (days) (g/100 g cheese)



0



37.31 ± 0.28m



60



34.31 ± 0.38ijkl



90



34.15 ±0.06hijk



C



120



33.75 ± 0.04efgh



150



32.58 ± 0.01d



180



31.99 ± 0.16bc



210



31.52 ± 0.06ab



0



37.92 ± 0.21n



60



34.55 ± 0.38kl



90



33.75 ± 0.18efgh



E1



120



33.30 ± 0.02e



150



32.59 ± 0.20d



180



33.31 ± 0.29e



210



32.50 ± 0.13d



0



37.79 ± 0.09mn



60



34.58 ± 0.31kl



90



34.02 ± 0.08ghij



E2



120



34.11 ± 0.07ghijk



150



33.82 ± 0.11fgh



180



34.34 ± 0.04jkl



210



31.99 ± 0.06bc



0



37.57 ± 0.21mn



60



34.68 ± 0.03l



90



33.62 ± 0.28efg



E3



120



33.37 ± 0.01ef



150



32.27 ± 0.14cd



180



33.83 ± 0.79fghi



210



31.44 ± 0.25a



Enzyme level



*



Ripening time



*



Interaction



*



pH

5.32 ± 0.03 5.31 ± 0.04 5.32 ± 0.00 5.33 ± 0.01 5.43 ± 0.06 5.40 ± 0.11 5.44 ± 0.06 5.43 ± 0.02 5.47 ± 0.06 5.44 ± 0.08 5.50 ± 0.07 5.50 ± 0.03 5.29 ± 0.01 5.29 ± 0.01 5.31 ± 0.01 5.29 ± 0.01 5.31 ± 0.02 5.30 ± 0.00 5.33 ± 0.03 5.31 ± 0.03 5.36 ± 0.01 5.34 ± 0.01 5.35 ± 0.02 5.35 ± 0.00 5.34 ± 0.01 5.33 ± 0.00 5.35 ± 0.00 5.35 ± 0.01

ns * ns



a–naverage values in the same column with different letters are significantly different (P < 0.05); last rows show the ANOVA result for the different factors analysed; *significant effect (P < 0.05); ns – no significant effect (P > 0.05)



levels of proteolysis (Mohedano et al. 1998). In this case, the pH values were not significantly affected by the level of carboxypeptidase. The pH values for all assayed samples were in the range of 5.29–5.50 (Table 2), which were similar to those reported by other researchers for this type of cheese (Candioti et al. 2002; Hynes et al. 2003; Perotti et al. 2004).

Nitrogen fraction analysis. The extent of proteolysis was evaluated by determining WSN and PTA-N as a percentage of TN (Figure 1). The fraction associated with WSN contains proteins, peptides, and amino acids, while nitrogen soluble in PTA consists of free amino acids and small peptides. Values determined in control cheeses were similar to those reported by other authors for Reggianito cheese (Candioti et al. 2002; Hynes et al. 2003; Perotti et al. 2004).

Various authors used nitrogen indices to study the effect of enzyme addition on cheese proteolysis. Azarnia et al. (2010) investigated the proteolysis in enzymemodified Cheddar cheese with added natural enzyme or recombinant aminopeptidase in the presence of a commercial proteinase. These authors did not observe any significant differences in WSN fraction between the control and experimental cheeses. However, they observed higher levels of PTA-N fraction in cheeses manufactured with exogenous enzymes. Azarnia et al. (2011) also studied the effect of free and encapsulated recombinant aminopeptidase on the Cheddar cheese proteolysis. They did not observe any significant differences in WSN fraction between the studied cheeses but they observed higher levels of PTA-N fraction in experimental cheeses, particularly in those with encapsulated enzymes. Kilcawley et al. (2012) evaluated the effect of the addition of enzyme preparations on Cheddar cheese ripening. They found that AccelerzymFiegu®reC2PG



12



34



5



6



7



8 9 10



γ-caseins



β-casein



F1 and F2

α -casein

s1

αs1-I-casein F3



the moisture content, no clear trend was observed. It is worth mentioning that the moisture content values for control and experimental cheeses were similar at the end of the studied ripening period (Table 2).

The pH value may be increased in cheeses manufactured with enzyme addition due to the higher



Figure 2. Urea-PAGE electrophoretogram for Reggianito cheeses manufactured with different amounts of carboxypeptidase and ripened during 60 days 1 = as1-casein standard; 2–3 = cheese C; 4–5 = cheese E1; 6–7 = cheese E2; 8–9 = cheese E3; 10 = b-casein standard



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Czech J. Food Sci., 34, 2016 (5): 445–455 FFood Technology and Economy, Engineering and Physical Properties



doi: 10.17221/567/2015-CJFS

Table 3. Average values and standard deviations corresponding to the IOD/g cheese of the electrophoretic fractions determined in Reggianito cheese



Cheese



Ripening time (days)



γ-CN



β-CN



0



13.4 ± 1.9 128.7 ± 4.2k



60



15.2 ± 2.3  72.7 ± 5.4h



90



  8.9 ± 4.4  18.5 ± 3.1e



C



120



ND



    8.9 ± 10.5abcd



150



ND



14.0 ± 1.0de



180



22.5 ± 3.6 11.1 ± 1.8cd



210



15.3 ± 13.7 12.0 ± 0.7cd



0



10.3 ± 2.0 124.2 ± 4.9ij



60



  6.0 ± 1.1 65.5 ± 3.3g



90



12.9 ± 3.5 9.8 ± 5.6bcd



E1



120



ND



27.6 ± 2.3f



150



ND



9.7 ± 2.6bcd



180



  9.5 ± 4.7 1 0.5 ± 1.2bcd



210



14.7 ± 7.7 12.1 ± 0.7cde



0



19.5 ± 5.4 73.9 ± 3.9h



60



  4.6 ± 0.9 5.6 ± 0.6abc



90



  4.2 ± 2.8 3.1 ± 2.0a



E2



120



ND



8.2 ± 10.1abcd



150



ND



12.1 ± 1.9de



180



13.9 ± 3.2 12.3 ± 0.8de



210



18.9 ± 11.9 12.0 ± 1.1cd



0



11.0 ± 2.2 120.6 ± 9.2i



60



13.3 ± 12.2   8.8 ± 1.7abcd



90



  5.0 ± 6.6 11.0 ± 3.7cd



E3



120



ND



  4.2 ± 0.8ab



150



ND



  9.7 ± 0.9bcd



180



16.0 ± 9.1 11.7 ± 0.6cd



210



13.4 ± 8.8 12.1 ± 1.2cde



Enzyme level



ns



*



Ripening time



*



*



Interaction



ns



*



F1

19.7 ± 1.3 8.4 ± 0.8 12.4 ± 0.7 10.1 ± 6.3 13.0 ± 2.1 15.6 ± 5.9 16.5 ± 11.4 2.1 ± 1.2 9.0 ± 0.7 11.1 ± 1.8 12.1 ± 2.1 9.9 ± 4.6 10.1 ± 9.0 19.6 ± 11.4 14.0 ± 0.9 4.8 ± 3.1 11.2 ± 3.1 10.5 ± 2.8 12.3 ± 1.6 12.8 ± 15.4 8.5 ± 4.2 19.7 ± 2.0 7.5  ± 0.6 11.0 ± 1.9 11.6 ± 1.6 14.8 ± 2.4

ND 15.8 ± 3.3

ns * ns



F2



as1-CN



25.4 ± 1.9i 151.1 ± 4.6m



17.4 ± 16.1g 128.5 ± 2.7k



7.7 ± 1.0cde 81.4 ± 2.5d



5.8 ± 1.1abcde 66.0 ± 2.1bc



5.5 ± 3.8abcde 66.5 ± 3.6bc



ND



68.5 ± 3.9c



2.0 ± 2.4abc 92.5 ± 2.2fg



19.5 ± 13.1ghi 145.1 ± 2.2lm



10.8 ± 13.2ef 124.5 ± 1.3k



7.9 ± 3.9cde 79.6 ± 1.0d



1.2 ± 2.5ab 78.7 ± 3.9d



5.8 ± 1.4abcde 64.5 ± 4.0bc



6.0 ± 7.0abcde 88.3 ± 5.9ef



ND



104.1 ± 8.2i



9.0 ± 0.5de 105.5 ± 3.2ij



15.2 ± 1.6fg 107.3 ± 1.5ij



5.7 ± 1.9abcde 69.4 ± 1.8c



2.9 ± 5.8abcd 61.7 ± 2.4ab



6.7 ± 3.1bcde 69.2 ± 1.29c



0.8 ± 1.6ab 83.4 ± 9.8de



2.4 ± 5.0abc 95.3 ± 4.9gh



24.4 ± 1.8hi 143.8 ± 3.5l



18.4 ± 7.4gh 111.9 ± 1.3j



1.2 ± 2.5ab 55.2 ± 1.2a



4.5 ± 3.1abcd 57.3 ± 2.0a



6.8 ± 1.7bcde 65.0 ± 2.4bc



11.2 ± 6.2ef 85.1 ± 7.5de



3.5 ± 4.4abcd 101.9 ± 4.7hi



*



*



*



*



*



*



as1-I-CN



F3



16.1 ± 0.2ij 7.0 ± 0.7 22.4 ± 15.0n 14.7 ± 3.4 13.8 ± 1.2h 5.2 ± 1.2 11.1 ± 0.8ef 20.8 ± 11.7 10.3 ± 1.0cde 15.2 ± 13.7 17.3 ± 1.5j 9.0 ± 2.6 13.2 ± 0.6gh 9.0 ± 1.8 10.2 ± 1.2cde 5.91 ± 1.7 28.2 ± 0.9m 11.7 ± 1.2 8.3 ± 0.1ab 14.4 ± 12.0 9.7 ± 0.3cd 17.14 ± 15.8 7.7 ± 0.4a 21.7 ± 12.3 13.2 ± 0.4gh 6.0 ± 1.2 14.0 ± 1.2h 7.4 ± 0.5 15.3 ± 0.9i 11.9 ± 2.5 23.3 ± 1.0k 21.8 ± 2.0 9.5 ± 0.2cd 6.1 ± 0.4 9.3 ± 0.6bc 9.9 ± 13.0 10.7 ± 0.2de 9.6 ± 14.3 13.8 ± 0.3h 7.7 ± 2.1 12.4 ± 0.9g 8.2 ± 1.0 12.8 ± 1.0gh 10.1 ± 3.1 26.0 ± 0.7l 22.3 ± 1.4 7.6 ± 0.2a 6.2 ± 1.0 7.8 ± 0.4a 12.9 ± 11.4 7.8 ± 0.4a 18.2 ± 10.7 13.3 ± 0.3gh 7.2 ± 1.5 12.1 ± 1.2fg 6.6 ± 1.2



*



ns



*



*



*



ns



a–naverage values in the same column with different letters are significantly different (P < 0.05); ND – not detected; the last rows show the ANOVA result for different analysed factors; *significant effect (P < 0.05); ns – no significant effect (P > 0.05);



added immediately after rennet addition at a dose rate of 7.5 ml per 100 l of milk (CPG cheeses) did not impact on the levels of WSN and PTA-N in comparison with a control at any ripening time studied. In our study, it was in agreement with this data.

Electrophoretic analysis. A typical urea-PAGE electrophoretogram is shown in Figure 2. Casein fractions are labelled according to Sihufe et al. (2010a). Standards allowed the identification of the



as1- and b-casein fractions by mobility comparison, and g- and as1-casein f24-199 (or as1-I-casein) were identified according to Marcos et al. (1979) and

McSweeney et al. (1994). Table 3 shows the average

values and standard deviations of integrated optical

density (IOD) for all the fractions evaluated during

the ripening of Reggianito cheese. According to

previous works (Sihufe et al. 2003, 2010a), F1 and F2 may be products of b-casein degradation, while



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Food Technology and Economy, Engineering and Physical Properties Czech J. Food Sci., 34, 2016 (5): 445–455

doi: 10.17221/567/2015-CJFS Table 4A. Average concentrations and standard deviations (mg/100 g cheese) of free amino acids (aspargine, glutamic acid, asparagine, serine, histidine, glutamine, glycine, threonine, arginine) determined during ripening of Reggianito cheese



Cheese Ripening  time (days)



Asp



Glu



Asn



Ser



His



Gln



Gly



Thr



Arg



0 60 90 C 120 150 180 210 0 60 90 E1 120 150 180 210 0 60 90 E2 120 150 180 210 0 60 90 E3 120 150 180 210



15.8 ± 0.1a 59.2 ± 0.7a 24.0 ± 0.2a 17.6 ± 0.3a



ND



26.6 ± 0.3a 10.9 ± 0.1a 24.5 ± 0.1b 28.4 ± 0.6a



38.7 ± 0.7bc 227.8 ± 2.9bc 87.1 ± 1.9bc 55.4 ± 1.8bc 52.7 ± 7.6hijkl 83.4 ± 1.9c 33.8 ± 1.2bc 49.5 ± 1.0c 93.0 ± 1.6cd



52.0 ± 2.1ef 298.6 ± 4.5efg 113.6 ± 1.7e 70.9 ± 0.7de 44.9 ± 21.3fghij 93.75 ± 2.2def 43.7 ± 0.9fg 62.3 ± 0.7e 108.2 ± 12.0efg



65.9 ± 0.0hi 368.2 ± 3.1hi 140.0 ± 0.0gh 91.5 ± 0.7ghi 63.4 ± 1.2ijkl 96.3 ± 1.2defg 46.2 ± 3.1gh 66.0 ± 0.3e 119.8 ± 1.5ghi



76.5 ± 0.8kl 423.0 ± 1.8jk 156.4 ± 2.9ij 110.8 ± 9.6jk 34.3 ± 3.3efgh 105.1 ± 1.3ghij 66.1 ± 3.5lm 92.0 ± 2.2gh 122.5 ± 3.8hi



91.9 ± 0.4op 561.6 ± 2.3op 201.3 ± 0.1op 139.6 ± 4.1no 46.7 ± 1.1ghijk 124.0 ± 0.7lm 80.0 ± 0.0op 116.3 ± 0.1n 154.4 ± 1.0hl



75.5 ± 7.3kl 495.4 ± 57.1m 166.7 ± 16.5jklm 135.5 ± 16.3mno 63.4 ± 24.4ijkl 105.4 ± 11.1hij 83.7 ± 9.0p 101.4 ± 4.0ijk 157.5 ± 17.9l



15.3 ± 0.1a 57.3 ± 0.4a 21.9 ± 0.3a 15.8 ± 0.8a



ND



27.4 ± 0.2a 10.9 ± 0.1a 23.8 ± 0.3b 26.9 ± 0.1a



49.7 ± 3.3e 278.9 ± 12.3def 105.5 ± 5.0de 66.7 ± 2.0cde 43.4 ± 4.4fghi 100.8 ± 2.4fghi 40.6 ± 1.9def 61.4 ± 0.0e 108.4 ± 2.6efg



48.1 ± 0.28de251.9 ± 1.4cd 92.6 ± 0.3bcd 56.7 ± 3.0bc



ND



70.6 ± 0.2b 37.5 ± 0.9cde 59.7 ± 0.2de 78.1 ± 2.0b



69.7 ± 0.4ij 397.4 ± 4.6ij 145.3 ± 1.9hi 101.3 ± 1.9ij 32.0 ± 1.9efgh 98.0 ± 1.9efgh 51.5 ± 0.3hi 80.6 ± 0.0f 116.1 ± 1.7fgh



89.3 ± 4.2no 511.3 ± 28.9mn 177.1 ± 8.0klm 129.1 ± 9.7lmn 45.3 ± 22.1fghij 118.9 ± 6.1kl 70.4 ± 4.2mn105.3 ± 1.8jk 143.7 ± 7.4jk



90.8 ± 0.6o 545.0 ± 7.3nop 178.9 ± 2.2lm 132.5 ± 3.0lmno 31.0 ± 0.7efgh 112.1 ± 0.9jk 75.2 ± 0.1no 107.9 ± 0.6kl 148.7 ± 0.5kl



81.2 ± 5.8lm 542.2 ± 26.9nop 179.3 ± 12.4mn 141.6 ± 6.2no 71.9 ± 29.9lm 108.5 ± 4.5ij 80.8 ± 1.5p 94.4 ± 17.3ghi174.4 ± 6.8m



13.1 ± 0.1a 67.6 ± 0.1a 26.3 ± 0.2a 2 1.1 ± 1.0a 13.4 ± 1.9cde 34.2 ± 0.6a 10.8 ± 0.4a 16.0 ± 0.1a 35.6 ± 1.3a



43.1 ± 0.1cd 263.3 ± 4.6cde 97.2 ± 1.2cd 64.2 ± 0.4cd 70.4 ± 5.3klm 91.4 ± 1.1cde 36.7 ± 0.5cd 53.4 ± 0.3cd 102.1 ± 1.7de



60.3 ± 1.6gh 338.7 ± 6.9gh 129.5 ± 0.9fg 78.6 ± 1.9efg 34.3 ± 6.3efgh 97.9 ± 2.4efgh 43.0 ± 0.8efg 64.5 ± 1.1e 103.9 ± 3.13def



79.5 ± 3.6lm 444.9 ± 11.3kl 165.8 ± 2.0jkl 111.0 ± 9.6jk 22.1 ± 16.5def 111.3 ± 2.2jk 56.6 ± 2.4ij 97.8 ± 0.9hij 132.6 ± 2.9ij



84.1 ± 6.8mn 512.0 ± 56.5mn 178.7 ± 18.9lm 124.0 ± 15.9klm 37.6 ± 16.6efgh 124.7 ± 14.6lm 71.4 ± 5.8mn108.2 ± 5.7klm 152.8 ± 17.0kl



93.9 ± 0.8op 583.1 ± 9.3pq 192.5 ± 2.8no 144.6 ± 0.9op 43.7 ± 0.5fghi 125.2 ± 2.2lm 82.2 ± 1.1p 113.1 ± 0.1lmn 154.0 ± 2.0kl



77.4 ± 0.8kl 524.0 ± 25.2mno 168.6 ± 7.0jklm 134.9 ± 7.0lmno 90.1 ± 16.8m 108.7 ± 4.7ij 83.0 ± 0.0p 80.6 ± 2.3f 171.1 ± 7.8m



11.9 ± 0.0a 51.3 ± 0.7a 19.9 ± 0.0a 14.8 ± 0.3a 4.5 ± 1.7bcd 25.4 ± 0.3a 8.4 ± 0.1a 14.8 ± 0.1a 28.1 ± 0.9a



35.9 ± 0.3b 203.1 ± 0.4b 79.9 ± 1.6b 48.7 ± 1.3b 31.2 ± 3.8efgh 73.1 ± 0.1b 31.0 ± 0.2b 47.8 ± 0.1c 84.5 ± 0.6bc



56.3 ± 0.3fg 305.4 ± 4.0fg 116.1 ± 0.9ef 72.9 ± 0.4def 25.3 ± 5.0defg 89.2 ± 0.7cde 40.0 ± 0.1def 62.2 ± 0.3e 93.6 ± 1.0cd



71.7 ± 0.1jk 412.3 ± 16.5jk 146.5 ± 5.4hi 97.1 ± 2.1hi 25.2 ± 2.9defg 103.3 ± 3.8ghij 59.4 ± 0.9jk 88.7 ± 0.8g 123.4 ± 3.7hi



66.2 ± 2.9ij 361.3 ± 21.6hi 129.4 ± 4.9fg 84.5 ± 4.7fgh



ND



87.6 ± 3.4cd 55.8 ± 1.5ij 90.9 ± 0.1gh 104.0 ± 4.2def



97.4 ± 4.1p 607.9 ± 35.3q 207.0 ± 10.5p 156.2 ± 8.0p 68.0 ± 13.0jklm 132.5 ± 7.2m 85.4 ± 4.9p 115.8 ± 5.3mn 172.6 ± 10.5m



88.1 ± 2.0no 486.3 ± 18.0lm 164.1 ± 9.0jk 121.7 ± 11.6kl 63.0 ± 1.9ijkl 91.7 ± 4.1cde 63.8 ± 3.3kl 78.1 ± 0.6f 149.0 ± 6.4kl



Enzy-



me



*



*



*



*



*



*



*



*



*



 level



Ripening time



*



*



*



*



*



*



*



*



*



Interaction



*



*



*



*



*



*



*



*



*



ND – not detected; the last rows show the ANOVA result for different analysed factors; *significant effect (P < 0.05);  a–naverage  values in the same column with different letters are significantly different (P < 0.05)



the fraction F3 may be associated with products

of as1- or as1-I-casein degradation. Fractions corresponding to the most important caseins (as1-, as1-I, or b-casein) were significantly affected by the



levels of added enzyme and ripening time, the most extensive degradation being observed for b-casein during the first 2 months of ripening (Figure 2 and Table 3). These results are very interesting taking



450



Czech J. Food Sci., 34, 2016 (5): 445–455 FFood Technology and Economy, Engineering and Physical Properties

doi: 10.17221/567/2015-CJFS Table 4B. Average concentrations and standard deviations (mg/100 g cheese) of free amino acids (alanine, tyrosine, methionine, phenylalanine, isoleucine, leucine, lysine) and total amino acids determined during the ripening of Reggianito cheese



Cheese Ripening  time (days)



Ala



Tyr



Met



Val



Phe



Ile



Leu



Lys



Total



0 14.0 ± 0.1a 14.1 ± 0.3a 9.1 ± 0.1a 24.9 ± 0.2a 14.3 ± 0.2a 16.2 ± 0.2a 48.9 ± 0.7a 80.2 ± 0.4a 419.7 ± 5.4a 60 37.4 ± 1.3bcd41.1 ± 2.0bcd 33.1 ± 0.8b 92.0 ± 0.9b 57.5 ± 1.4bc 66.4 ± 0.6bc 181.5 ± 4.9bc 288.1 ± 5.6bc 1518.7 ± 36.9bc 90 43.1 ± 1.5cde47.0 ± 1.5def 42.1 ± 1.7cd 124.4 ± 3.9de 73.7 ± 4.1def 92.1 ± 3.8ef 222.5 ± 13.3d 347.3 ± 26.2def1880.8 ± 98.0de C 120 52.5 ± 0.7fg 54.1 ± 0.2gh 48.8 ± 0.8e 150.3 ± 3.1fg 87.9 ± 0.1ghi 113.8 ± 3.3hi 261.2 ± 5.9e 348.5 ± 15.3ef 2174.4 ± 37.8fg 150 63.1 ± 4.7hi 66.0 ± 3.5j 58.1 ± 2.3fgh 172.4 ± 6.4hi 108.3 ± 3.6jk 128.5 ± 4.5jk 304.7 ± 10.4fg465.5 ± 4.3gh 2553.4 ± 68.9ij 180 74.2 ± 1.6jk 79.6 ± 0.8klm 72.6 ± 0.9lm 225.7 ± 1.8l 133.3 ± 0.4n 168.1 ± 1.3o 375.1 ± 7.1j 578.8 ± 7.7jkl 3223.4 ± 0.20mn 210 73.3 ± 11.4jk80.6 ± 10.5lm 73.1 ± 6.1lmn181.8 ± 16.7ij 130.3 ± 14.2mn145.5 ± 14.5l 359.9 ± 39.1ij 579.8 ± 71.4kl 3008.8 ± 347.7lm 0 14.6 ± 0.0a 13.9 ± 0.5a 9.0  ±  0.4a 22.6 ± 0.2a 13.3 ± 0.2a 14.5 ± 0.3a 44.4 ± 0.5a 82.2 ± 1.4a 413.6 ± 6.3a 60 47.0 ± 1.3ef 48.7 ± 0.9efg 42.1 ± 1.1cd 112.7 ± 2.8cd 66.5 ± 0.0cd 82.5 ± 1.2de 212.9 ± 0.2d 336.9 ± 2.4cde 1804.8 ± 38.6de 90 36.2 ± 2.2bc 37.7 ± 0.0bc 34.4 ± 0.2b 99.6 ± 0.4bc 53.4 ± 0.2b 74.2 ± 0.78cd 165.11 ± 0.2b 269.6 ± 3.5b 1459.9 ± 9.50b  E1 120 58.4 ± 1.9gh 56.5 ± 1.1hi 54.3 ± 0.5f 161.7 ± 4.8gh 90.3 ± 0.9hi 118.2 ± 1.7ij 260.9 ± 4.9e 354.9 ± 2.5ef 2247.2 ± 32.1gh 150 72.0 ± 6.7kl 73.3 ± 7.4k 67.0 ± 3.2jk 197.4 ± 9.8k 114.8 ± 11.1kl 146.5 ± 5.7l 325.6 ± 26.2gh514.4 ± 60.4hi 2901.6 ± 223.0kl 180 71.3 ± 0.8jkl 77.1 ± 0.6kl 69.8 ± 0.8kl 205.8 ± 1.2k 121.0 ± 0.8lm 158.5 ± 1.3mno 337.3 ± 1.0hi 547.1 ± 6.5ijk 3010.0 ± 25.5lm 210 76.6 ± 1.6kl 84.8 ± 2.2mn 76.7 ± 2.2mn200.7 ± 8.5k 134.1 ± 1.8n 165.9 ± 4.8no 370.5 ± 3.0j 604.4 ± 5.2l 3188.1 ± 67.4mn 0 19.2 ± 1.3a 17.5 ± 1.3a 11.2 ± 0.4a 29.1 ± 0.8a 16.9 ± 1.1a 19.3 ± 0.5a 58.6 ± 3.6a 98.6 ± 8.0a 508.6 ± 22.8a 60 44.4 ± 1.5de 46.2 ± 1.9de 39.4 ± 1.3c 107.8 ± 2.3c 65.5 ± 2.2cd 76.1 ± 1.7cd 205.1 ± 5.3cd 334.9 ± 10.0cde1741.1 ± 41.0cd 90 48.6 ± 0.0ef 49.7 ± 0.2efgh 46.3 ± 0.3de 138.9 ± 2.2ef 78.4 ± 1.0efg 99.1 ± 0.5fg 231.3 ± 2.3d 322.9 ± 5.8b 1966.1 ± 37.0def E2 120 64.4 ± 4.2hij 63.0 ± 2.5ij 60.2 ± 2.1hi 179.7 ± 5.0ij 101.9 ± 5.0j 131.9 ± 2.6k 296.3 ± 8.4f 395.3 ± 14.6f 2514.3 ± 95.7ij 150 69.3 ± 8.6ijk 74.6 ± 7.2kl 67.2 ± 7.2jk 201.6 ± 20.6k 118.1 ± 13.2kl 148.1 ± 15.1lm 335.7 ± 35.9hi528.4 ± 58.9ij 2936.3 ± 324.8kl 180 77.4 ± 0.1kl 81.0 ± 0.1lm 76.8 ± 0.5mn224.5 ± 1.3l 135.1 ± 1.5n 166.4 ± 0.05o 375.8 ± 3.9j 602.5 ± 7.5l 3271.9 ± 11.8n 210 76.3 ± 1.6jk 81.3 ± 3.5lm 77.9 ± 2.8n 203.1 ± 9.0k 135.3 ± 4.5n 162.1 ± 6.1no 367.7 ± 17.1j 606.4 ± 24.3l 3148.6 ± 142.8lmn 0 13.8 ± 0.4a 13.1 ± 0.2a 8.8 ± 0.2a 22.2 ± 0.7a 12.7 ± 0.6a 14.4 ± 0.4a 44.8 ± 2.0a 79.3 ± 5.3a 388.0 ± 13.8a 60 32.5 ± 0.2b 35.4 ± 0.4b 30.7 ± 0.1b 87.4 ± 0.8b 49.2 ± 0.6b 62.4 ± 0.5b 157.8 ± 3.5b 254.7 ± 9.5b 1345.3 ± 19.6b 90 44.2 ± 0.2de 44.5 ± 0.4cde 42.3 ± 0.1cd 123.9 ± 2.0d 69.6 ± 0.8de 89.2 ± 1.6ef 207.2 ± 1.9cd 296.6 ± 3.2bcd 1778.7 ± 3.6d E3 120 60.9 ± 2.3h 62.0 ± 2.8ij 55.7 ± 3.3fg 164.6 ± 7.9gh 97.6 ± 7.7ij 123.3 ± 5.5ijk 283.5 ± 14.8ef452.2 ± 30.1g 2427.5 ± 111.2hi 150 46.3 ± 2.1ef 53.4 ± 2.8fgh 46.6 ± 2.1de 141.3 ± 7.5f 80.5 ± 4.9fgh 106.3 ± 4.1gh 227.9 ± 12.7d 374.0 ± 23.0ef 2033.3 ± 118.9efg 180 84.1 ± 4.6l 90.2 ± 4.0n 84.2 ± 3.0o 241.4 ± 11.3m 149.5 ± 6.7o 186.1 ± 5.4p 408.2 ± 18.0k 673.3 ± 30.9m 3559.8 ± 182.9o 210 63.5 ± 5.3hi 65.5 ± 5.0j 62.7 ± 3.5ij 191.1 ± 10.5jk 107.7 ± 6.2jk 155.6 ± 10.3lmn304.9 ± 21.0fg469.4 ± 26.7gh 2726.5 ± 145.5jk



Enzy-



me



*



*



*



*



*



*



*



*



*



 level



Ripening time



*



*



*



*



*



*



*



*



*



Interaction



*



*



*



*



*



*



*



*



*



ND – not detected; the last rows show the ANOVA result for different analysed factors; *significant effect (P < 0.05); a–qaverage values in the same column with different letters are significantly different (P < 0.05)



into account that carboxypeptidases are essential for promoting extensive protein hydrolysis and prevention of bitterness (Gobbetti et al. 1997; Forde & Fitzgerald 2000). It is worth mentioning that urea-



PAGE helps detecting changes in the intact caseins and their primary proteolytic degradation products during ripening. In this case, it can be hypothesised that carboxypeptidase catalyses the hydrolysis of



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Food Technology and Economy, Engineering and Physical Properties Czech J. Food Sci., 34, 2016 (5): 445–455



doi: 10.17221/567/2015-CJFS



b-casein from the C-terminal and changes its charge, hydrophobicity, and size. Unfortunately, there is a lack of information about urea-PAGE to study b-casein degradation by the action of carboxypeptidase.

Peptide analysis by RP-HPLC. The chromatograms of the water soluble fraction at pH 4.6 are often referred to as ‘finger prints’ of the cheese proteolysis (Pripp et al. 1999). In our case, the regular peptide profile of Reggianito cheese was not substantially modified by the carboxypeptidase action. Twentyeight chromatographic peaks were determined by RP-HPLC, which were present in all the analysed samples. Sixteen peaks were significantly affected by the amount of enzyme added during cheese manufacture, while all 28 peaks were significantly affected by ripening time. This result indicates the significance of the influence of the amount of added enzyme. The obtained large multivariate data set will be more easily interpreted by principal component analysis.

Free amino acid analysis. Seventeen amino acids were determined in 40 min of the chromatographic run. ANOVA highlighted that the ripening time and the amount of enzyme used during cheese manufacture significantly affected the concentrations of all the amino acids studied. Similar to Sihufe et al. (2010a), amino acids Lys, Glu, Leu, and Val were present at the highest concentrations and represented more than 50% of total amino acids at the end of the ripening period in all the cheeses analysed.

High amino acid concentrations were observed in some cheeses manufactured with exogenous enzymes compared with control cheeses at the same ripening time (Table 4A and B). Such behaviour can be clearly observed in cheeses E1 at 60 and 150 days of ripening, in cheeses E2 at 120 and 150 days of ripening, and in cheeses E3 at 120 and 180 days of ripening. These results are in agreement with those reported by Azarnia et al. (2011), who observed increased levels of free amino acids in Cheddar cheeses manufactured with free exogenous aminopeptidases. Kilcawley et al. (2012) concluded that the carboxypeptidase activities during ripening in cheese with added carboxypeptidase were significantly higher than in control cheese. However, the levels of total free amino acids of CPG cheeses were not significantly different from those corresponding to control cheeses at the same ripening time.

It is worth mentioning that the comparison of amino acid concentrations should be analysed taking into account that amino acids can also be degraded to catabolic products after their formation. There-



fore, it may be difficult to observe higher amino acid concentrations in cheeses manufactured with exogenous enzymes at all ripening times. Thus, in the case of the cheeses manufactured with the higher enzyme level, low amino acid concentrations were also clearly observed in some cases (cheeses E3 at 150 and 210 days of ripening) compared with control cheeses at the same ripening time (Table 4).

Principal component analysis. Principal component analysis (PCA) was used to summarise the data set and to detect possibly unanticipated patterns in the data. PCA was applied using the nitrogen content of the 2 studied fractions (WSN & PTA), 7 electrophoretic fractions analysed by urea-PAGE (γ-caseins, β-casein, F1, F2, αs1-casein, αs1-I-casein, and F3), 28 chromatographic peaks from the RP-HPLC analysis of the water-soluble fraction at pH 4.6, and of the 17 free amino acids studied. After using PCA with all samples, a subset of samples was selected excluding samples corresponding to 0 and 210 days of ripening to improve the analysis of the information. The first 4 principal components represented more than 70% of the total variance (73.2% VAR).

A biplot of the first 2 principal components (61.1% VAR) is shown in Figure 3. The first principal component PC1 (that explains 50.1% of the variance) can be interpreted by the behaviour of the chromatographic areas of most of the peptides and the concentration of the free amino acids which can clearly be related to the ripening process. Those variables increased with ripening time within the same treatment (in Figure 3, samples corresponding to the same treatment spread from left to right as the ripening time increases). Moreover, this effect can be observed in a cluster structure since all samples corresponding to the same ripening time but to different treatments are positioned close together.

The second principal component PC2 (that explains 11% of the variance) has a quadratic effect with respect to the PC1, having higher score values at the beginning and at the end of the process with a minimum in the middle region (Figure 3). This second component differentiates the treatments. Control cheeses at 60 days of ripening showed notably higher β-casein IOD values than those values corresponding to experimental cheeses at the same ripening time (Table 3). Similarly, but less pronounced, αs1-casein IOD values were higher in control cheeses, while αs1-I-casein IOD values were lower in control cheeses than in experimental ones (Table 3). However, the association of PC2 with the level of added enzyme



452



Czech J. Food Sci., 34, 2016 (5): 445–455 FFood Technology and Economy, Engineering and Physical Properties

doi: 10.17221/567/2015-CJFS

180



PC2 (11% VAR)



180 60 60



60



60 60



60 60

60

90



αs1-cαass1e-Iin-casein F2 β-casein

p15



90



90



F3



p1 90



90 120



90 90 p5 120 150



γ-caseiHnis



0



p23 GLAlynAMrWsgSTAlGayeSesrtlNpur/TN180



p182102012105102Fp011p32pp611252700p11540p20150pp1121p3dP115T5p00A4 -N/TN



180 150



181080



180



90



120



PC1 (50.1% VAR)



Figure 3. A biplot of scores and loadings of data obtained from different analyses used to study the proteolysis of Reggianito cheeses (■) cheeses C; (□) cheeses E1; (●) cheeses E2; (○) cheeses E3; numbers correspond to days of ripening; labels starting with p followed by a number indicate peaks from the peptide chromatographic analysis



in the case of cheeses E3 at 180 days of ripening was not definite.

Finally, the biplot also showed that for the same ripening time, some cheeses made with carboxypeptidase are slightly on the right from the samples corresponding to control cheeses, indicating an increase in the rate of formation and/or degradation of proteolysis products. Kilcawley et al. (2012) studied the effect of enzyme addition on rheological properties of cheese by the texture profile analysis and found no statistical difference between control and CPG cheeses for any sensory texture attribute. They found that CPG cheeses were associated with sensory characteristics of aged Cheddar. Moreover, the authors related the greater diversity of volatile compounds in the experimental cheeses partially to the enhanced levels of secondary proteolysis that provided an additional nitrogenous substrate. Therefore, in our case no adverse impact on cheese sensory characteristics was expected because the secondary proteolysis was not extremely accelerated.

At present, little is known about the manufacture of Reggianito cheese with the addition of exogenous enzymes. Therefore, this study can be considered as the first early stage and it will allow focusing on



complementary studies (sensory studies, rheological studies, etc.) and on confirmatory experiments more easily.

Conclusions

The impact of the addition of an exogenous carboxypeptidase enzyme during the manufacture of Reggianito cheese was evaluated by assessing its impact on composition and proteolysis during ripening. Fractions corresponding to the most important caseins were significantly affected by the level of added enzyme and ripening time, the most important effect being observed for b-casein in the first 2 months of ripening. The amino acids Lys, Glu, Leu, and Val were present at the highest concentrations in all analysed cheeses. An increase of amino acid concentrations was clearly observed in some cheeses manufactured with exogenous enzymes compared with control cheeses at the same ripening time. The principal component analysis summarised the information related to proteolysis determinations. The first principal component accounted for 50.1% of the total variance and it was clearly related to the



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Food Technology and Economy, Engineering and Physical Properties Czech J. Food Sci., 34, 2016 (5): 445–455



doi: 10.17221/567/2015-CJFS



ripening process. Useful information was obtained from the biplot of the first 2 principal components. A slight acceleration in proteolysis was observed when comparing the position of samples corresponding to control and experimental cheeses. The use of carboxypeptidase appears to be an interesting option to accelerate ripening of Reggianito cheese, but complementary studies such as sensory evaluation are necessary. This study will allow focusing on confirmatory experiments more easily regarding the enzyme addition to accelerate the ripening of hard cheeses.

Acknowledgements. The authors thank Dr Liliana Forzani for her advice on statistical analysis and Daniel De Piante Vicín, Jorge Hajduczyk, and Leandro Aguilar for their technical assistance. Experiences of cheese manufacture and analysis of different obtained samples were performed in 2010–2011.

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Azarnia S., Lee B.H., Yaylayan V., Kilcawley K.N. (2010): Proteolysis development in enzyme-modified Cheddar cheese using natural and recombinant enzymes of Lactobacillus rhamnosus S93. Food Chemistry, 120: 174–178.

Azarnia S., Lee B., St-Gelais D., Kilcawley K., Noroozi E. (2011): Effect of free and encapsulated recombinant aminopeptidase on proteolytic indices and sensory characteristics of Cheddar cheese. LWT-Food Science and Technology, 44: 570–575.

Candioti M.C., Hynes E., Quiberoni A., Palma S.B., Sabbag N., Zalazar C.A. (2002): Reggianito Argentino cheese: influence of Lactobacillus helveticus strains from natural whey cultures on cheese making and ripening processes. International Dairy Journal, 12: 923–931.

Doolan I.A., Nongonierma A.B., Kilcawley K.N., Wilkinson M.G. (2014): Partitioning of starter bacteria and added exogenous enzyme activities between curd and whey during Cheddar cheese manufacture. International Dairy Journal, 34: 159–166.

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Forde A., Fitzgerald G.F. (2000): Biotechnological approaches to the understanding and improvement of mature cheese flavour. Current Opinion in Biotechnology, 11: 484–489.



Gallino R.R. (1994): Queso Reggianito Argentino: tecnología de fabricación. In: Ciencia y Tecnología de los Productos Lácteos. Diagramma S.A., Medios Audiovisuales y Gráficos – CERIDE, Santa Fe: 244–287.

Gobbetti M., Burzigotti R., Smacchi E., Corsetti A., De Angelis M. (1997): Microbiology and biochemistry of Gorgonzola cheese during ripening. International Dairy Journal, 7: 519–529.

Gripon J.C., Desmazeaud M.J., Le Bars D., Bergère J.L. (1975): Étude du rôle des microorganismes et des enzymes au cours de la maturation du fromage. Lait, 55: 502–516.

Hynes E., Bergamini C.V., Suárez V.B., Zalazar C.A. (2003): Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus. Journal of Dairy Science, 86: 3831–3840.

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Received: 2015–12–14 Accepted after corrections: 2016–09–15



Corresponding author:

Dr Guillermo Adrian Sihufe, Consejo Nacional de Investigaciones Científicas y Técnicas – Universidad Nacional del Litoral, Instituto de Desarrollo Tecnológico para la Industria Química, Güemes 3450, S3000GLN Santa Fe, Argentina; E-mail: gsihufe@intec.unl.edu.ar



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