Effect of natamycin-containing coating on the evolution of biochemical and microbiological parameters during the ripening and storage of ovine hard-Gruyère-type cheese

International Dairy Journal 50 (2015) 1e8

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Effect of natamycin-containing coating on the evolution of biochemical and microbiological parameters during the ripening and
re-type cheese storage of ovine hard-Gruye Golfo Moatsou*, Ekaterini Moschopoulou, Antigoni Beka, Paraskevi Tsermoula, Dimosthenis Pratsis Laboratory of Dairy Research, Department of Food Science and Human Nutrition, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 January 2015 Received in revised form 24 May 2015 Accepted 25 May 2015 Available online 16 June 2015


re-type cheese The effect of a coating containing natamycin on the ripening course of the hard-Gruye Graviera Kritis was assessed. A single treatment at an early stage of ripening was carried out; samples from natamycin-treated (NT) and control cheeses (CTR) were then taken throughout a 12 month ripening and storage period. Coating gave a statistically significant reduction in the yeasts and moulds counts in the cheese rind. It did not influence the counts and the evolution of the thermophilic bacteria related to starter or of the propionic acid bacteria, nor did it affect the associated aminopeptidase activities. Gross composition of NT cheeses did not differ significantly from that of the control cheeses; the same was also true for proteolysis. Natamycin in the cheese rind after the removal of coating was lower than 0.1 mg dm
2 at all stages of ripening and no migration to the cheese interior was observed. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Moulds and yeasts on cheese rind can be a major threat for all cheese varieties, in particular for hard- and semi-hard-type cheeses ripened for a considerable amount of time. Moulds can induce spoilage destabilising cheese by their lipases and proteinases, and they can threaten safety due to their potential to produce mycotoxins (Lund, Filtenborg, & Frisvad, 1995). Moreover, elevated labour-costs are necessary for the treatment of cheeses throughout ripening to avoid or control fungal growth on cheese surface. Stark (2007) reviewed fungal problems related to cheese and reported that the main sources of fungal contamination of cheeses are the processing equipment, shelves, air and brining baths, while, on the other hand, the raw materials do not contribute. Brining baths may contain up to 106 mL
1 salt-tolerant yeasts, resulting in a contamination of 106 yeasts dm
2 of cheese surface. In addition, moulds developing on the side of the brining bath may contaminate the brine up to 103 cfu mL
1. Moulds are always present inside wooden shelves, despite the application of cleaning

* Corresponding author. Tel.: þ30 210 5294640. E-mail address: [email protected] (G. Moatsou). http://dx.doi.org/10.1016/j.idairyj.2015.05.010 0958-6946/© 2015 Elsevier Ltd. All rights reserved.

and disinfection. Mycelium fragments and mould spores may be circulated by the air-conditioning systems of the ripening rooms. Therefore, fungicides have been employed in cheese production. The most common are sorbate and natamycin. Natamycin (E235, pimaricin) is a fungicide of the polyene macrolide group. According to European Directive 95/2/EC (EC, 2006), it may be used for the surface treatment of hard, semi-hard and semi-soft cheese at a maximum level of 1 mg dm
2 and not be present at a depth of 5 mm. The advantages of natamycin were reviewed by Stark (2007). Natamycin is colourless, odourless and without taste, and has a broad specificity against moulds and yeasts. It binds to the ergosterol in the fungal cell membrane inducing thus cell death. It forms micelles even at low concentrations, which, however, are very effective against contacted fungal cells. It is released slowly due to its low water solubility, i.e., 30e50 ppm. The application of natamycin on the cheese surface can be carried out repeatedly by spraying, dipping or by means of its incorporation in various coatings in concentrations from 100 to 750 ppm. The interaction with fungal cells, hydrolysis and light eliminate natamycin. The growth of most moulds and yeasts is prevented at concentrations of active natamycin < 10 ppm, whereas 30 ppm are effective against fungal growth on cheeses (Stark, 2007). In the last decade, several publications dealt with the incorporation of natamycin in various polymer materials and protein- or

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polysaccharide-based edible films for cheese coating. They are mainly studies on diffusion and/or antimicrobial activity of natamycin in model systems. There is also another group of articles, which are studies on the effect of treatment with natamycin, alone or in combination with other antimicrobial agents on cheese featima Ferreira Soares, Pereira, & De tures (e.g., De Oliveira, De Fa Freitas Fraga, 2007; Dos Santos Pires et al., 2008; Fajardo et al., , Dobia s, & Voldrich, 2012; Hanusov 2010; Hanusova a et al., 2010; Kallinteri, Kostoula, & Savvaidis, 2013; Mann & Beuchat, 2008;  Resa, Gerschenson, & Jagus, 2014a; Olle  Resa, Jagus, & Olle Gerschenson, 2014b; Ramos et al., 2012; Su arez, Tremmel, Rivera, Reinheimer, & Meinardi, 2012; Ture, Eroglu, Ozen, & Soyer, 2011; Var, Erginkaya, Güven, & Kabak, 2006). The major part of publications report on the effect of coatings with natamycin on cheese microbiological characteristics and there is scarce information about their impact on the course of ripening. The aim of the present study was to assess the effect of a coating containing natamycin on the ripening course of the
re-type hard-type cheese Graviera Kritis. Graviera Kritis is a Gruye cheese, although normally propionibacteria are not added. It is made from curds scalded up to 50  C at pH 6.2e6.3, pressed, salted in brine and ripened for at least three mo. Mean moisture content is about 33e35%, fat-in-dry matter 53e58%, protein 25e27%, saltin-moisture 4% and pH 5.5e5.8 (Kandarakis, Moschopoulou, Moatsou, & Anifantakis, 1998; Moatsou, Moschopoulou, & Anifantakis, 2004; Nega & Moatsou, 2012). The long storage of cheeses in ripening rooms with high-relative humidity and subsequent storage at low temperatures favours fungal growth on cheese surface. Although Graviera is not a surface-ripened cheese, it has been considered that the smear on its rind contributes to ripening. It is well-known that the microbiological profile of cheese rind is different from that of cheese interior. Yeasts and moulds metabolise lactic acid and produce ammonia as a result of proteolysis. Thus, surface pH rises from 4.8e5.2 to up to 6e8.2 that favours the growth of salt-tolerant and acid-sensitive bacteria (Montel et al., 2014). Since treatment with natamycin was expected to change rind ecology, the present research work was planned to examine if cheese characteristics also change and which stage of ripening may be affected. Considering the long ripening and a possible long storage period, cheese samples were taken up to 12 months. The tools for this investigation were the study of the evolution of various microbial groups, of bacterial aminopeptidases, of gross composition and of proteolysis, which is the most important biochemical pathway during cheese ripening. For this purpose natamycin-treated and non-treated (control) cheeses were manufactured. In addition to biochemical and microbiological analyses, the natamycin content of cheese rind was estimated and possible diffusion in the inner layer of cheese was investigated. 2. Materials and methods 2.1. Cheesemaking and application of cheese coating Cheese was manufactured from ewes' milk containing approximately 1% goats' milk. A thermophilic starter culture consisting of Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus helveticus was added to cheese milk at 34  C and calf rennet powder was used according to the instructions of the manufacturer. Thirty minutes after the addition of rennet, the curd was cut into 2 cm cubes. After a rest period of 15 min, curd cubes were further cut into small pieces of the size of corn grains. Curd pieces were scalded up to 47  C and then they were transferred into cylindrical moulds. Pressure by means of weight equal to fresh cheese weight was applied for 15 min and for another 15 min after inversion of

moulds with cheese. The next day, seventeen cheese wheels of about 1.5 kg each were salted in brine bath with 18% NaCl. The first stage of ripening was carried out at 13  C. At 8 d, after cheesemaking, two groups with eight cheese wheels each were formed. The cheese coating CESKA-COAT with 0.05% natamycin (CSK Food Enrichment BV, De Hoef, The Netherlands) was manually applied using a brush onto the surface of cheeses of one group. Control cheeses without coating were designated as CTR and the cheeses ripened under natamycin coating as NT. At 14 d, all cheeses were transferred to 17  C, at 60 d to 9  C and at 90 d to 4  C. The experiment was carried out three times, i.e., in three consecutive weeks. Samples were taken from all three experiments at 8 d, just before the application of the coating. Then, samples of NT and CTR cheeses were taken at 60 and 90 d of ripening and at 210 and 360 d during storage. Two cheese wheels, weighing about 1.3 kg each, were taken from each group of cheeses, at each sampling point. One of them was used for the sensory assessment and the other was divided to sub-samples utilised for analyses. 2.2. Microbial groups Microbial counts were determined in both the cheese rind and interior by the pour plate method using selective growth media (Drinan & Cogan, 1992; Moschopoulou, Anisa, Katsaros, Taoukis, & Moatsou, 2010) after aseptic dilution of samples in trisodium citrate 4% (w/v). Cheese rind was the outer layer of cheese of thickness 5 mm. The following microbial groups were enumerated, using the pour plate method: a) yeasts and moulds (YMC) on yeast glucose chloramphenicol (YGC) agar (Merck KGaA, Darmstadt, Germany) incubated at 25  C for 5 d; b) total mesophilic microflora (TMC) on plate count agar (PCA) (Biokar Diagnostics, Beauvais, France) incubated at 30  C for 72 h; c) thermophilic lactococci (THCC) on M17 agar (LabM Limited, Heywood, UK) incubated at 42  C for 48 h; d) thermophilic lactobacilli (THBC) on acidified MRS agar (Biokar Diagnostics), pH 5.4, incubated anaerobically at 42  C for 72 h; e) non-starter lactic acid bacteria (NSLAB) on Rogosa agar (Biokar Diagnostics), pH 5.5, incubated at 30  C for 4 d; f) propionic acid bacteria on sodium lactate cloxacillin (SLAC) agar containing 1.35% (w/v) sodium lactate, 1% (w/v) yeast extract (Oxoid Ltd, Basingstoke, UK), 1% (w/v) tryptone (Oxoid Ltd) and 0.5% (w/v) KH2PO4, incubated anaerobically at 30  C for 7 d; g) enterobacteriaceae on violet red bile lactose (VRBL) agar (AppliChem GmbH, Darmstadt, Germany) incubated anaerobically at 37  C for 24 h; h) enterococci on Kanamycin Aesculin Azide (KAA) agar (Oxoid Ltd) incubated at 37  C for 48 h; i) anaerobic bacteria on reinforced clostridial medium (RCM) agar (LabM Limited) incubated at 37  C for 5 d. 2.3. Aminopeptidase activities The substrates utilised were according to Gatti et al. (2008), i.e., Lys-b-naphthylamide (Lys-bNA) for broad-specificity aminopeptidase N (PepN), Arg-bNA for broad specificity aminopeptidase C (PepC), Pro-bNA for proline iminopeptidase (PepI), Glu-bNA for glutamyl aminopeptidase A (PepA), Leu-bNA for peptidase with high specificity for leucine and alanine (PepL) and Gly-Pro-bNA for X-prolyl dipeptidyl aminopeptidase (PepX) activities. Substrates except for Gly-Pro-bNA were from Bachem AG (Bubendorf, Switzerland); Gly-Pro-bNA was from SigmaeAldrich Chemie GmbH (Steinheim, Germany). Aminopeptidase activities were determined in duplicate in cheese extracts, which were prepared similarly to Moschopoulou et al. (2010) with a modification of cheese to buffer ratio. In the present work 10 g cheese were diluted in 40 mL 100 mM sodium phosphate buffer, pH 7.0. Substrate dilutions were 1.05 mM in 50 mM sodium phosphate buffer, pH 7.0. The filtrates were assayed

G. Moatsou et al. / International Dairy Journal 50 (2015) 1e8

in microtitre plates. The assay mixture was 125 mL filtrate and 125 mL of each substrate dilution incubated at 37  C. The reaction product was determined at 580 nm. Preliminary analyses showed that maximum absorbance at 580 nm for Lys-bNA, Arg-bNA and Leu-bNA substrates was at 10 min and for Pro-bNA, Glu-bNA and Gly-Pro-bNA at 20 min of reaction. 2.4. Gross composition Cheese moisture, fat, ash, pH, protein and the N content of water soluble fractions of cheeses (SN) were determined as described by Zoidou, Plakas, Giannopoulou, Kotoula, and Moatsou (in press). 2.5. Proteolysis Twenty grams of cheese were dispersed in 100 g distilled water at 40  C using a Stomacher apparatus (Seward Medical, London, UK); the cheese dispersion was moderately stirred for 60 min and pH was adjusted at pH 4.4 using 1 mol L
1 HCl. The suspension was left for 60 min at 4  C and then it was filtered through a Whatman No. 1 filter paper. Twenty millilitres of the filtrate designated SN (soluble nitrogen) were analysed by the Kjeldahl method, in duplicate. Analyses of 100 mL SN extracts by reversed phased high performance liquid chromatography (RP-HPLC) were carried out (Nega & Moatsou, 2012). Free amino groups (FAG) were estimated by the TNBS (trinitrobenzenesulphonic acid) method as reported by Polychroniadou (1988). One g of cheese was dispersed in 20 mL of borax buffer (0.1 M Na2B4O7 in 0.1 M NaOH, pH 9.5) at 45  C for 15 min. After centrifugation at 3000  g for 20 min, 3 mL of the supernatant were diluted with distilled water to 50 mL. One mL of this dilution, one mL of borax buffer and 2 mL of TNBS (1 mg mL
1 in water) were mixed thoroughly and incubated at 37  C for 60 min. The reaction was terminated with 4 mL of 0.1 M NaH2PO4, 1.5 M NaSO3 and the absorbance at 420 nm was recorded. Quantification was carried out by means of a standard curve, which was prepared using 0e0.6 mM glycine dilutions instead of cheese extract. 2.6. Determination of natamycin The determination of natamycin concentration was carried out in the outer layer of the cheeses, of thickness 5 mm with and without excluding the coating layer. Moreover, a slice of about 1 mm taken after removing the rind from the outer section of the samples was analysed. Treatment of samples and analysis was according to the International Standard IDF 140-2/ISO 9233-2 HPLC method (ISO/IDF, 2007). In most determinations, the methanolic extracts of cheeses were concentrated by means of reversed phase solid phase extraction cartridges (Sep-Pak C18 WAT051910 cartridges, Waters, Dublin, Ireland). 2.7. Sensory evaluation Cheeses were graded at 60, 90, 210 and 360 d by a panel of eight non-trained laboratory staff members, who were familiar with cheese grading. Cheeses were presented in random order and graded for appearance, texture and flavour on a 0e10 point scale. The three scores were multiplied by 1, 4 and 5 respectively, considering their relative contribution to the sensory characteristics of the cheeses. 2.8. Statistical analysis Two-way analysis of variance (ANOVA) was applied to test the effect of treatment with natamycin and of the stage of ripening on

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cheese ripening parameters. The contribution of each factor was measured after having removed the effects of all other factors and the differences were tested using the least significance difference method (LSD) at P < 0.05. The software Statgraphics Centurion XVI (Manugistics, Inc., Rockville, MA, USA) was used. 3. Results and discussion 3.1. Gross composition The gross composition parameters of cheeses and the effect of coating and stage of ripening on them are shown in Table 1. According to statistical analysis the treated cheeses NT did not differ (P > 0.05) from their counterpart control cheeses CTR with regard to gross composition, which was significantly affected by ripening (P < 0.05). The significant decrease (P < 0.05) of moisture during ripening caused a statistically significant increase of proteins, fat and ash contents of cheeses. As shown in Table 1, the moisture of Graviera Kritis in the present experiment was approximately 33% at 90 d and 28% at 210 d. Both the interior and the rind moisture contents continually decreased during ripening, since no packaging material was utilised. As expected, the rind became extremely dehydrated. Average moisture contents of the rind of CTR and NT cheeses at 90 d, were 19.03 ± 0.55% and 21.01 ± 0.28% respectively. At 210 d, they were decreased to 16.34 ± 0.68% and 17.73 ± 1.32%. Finally, at 360 d, the respective values were 15.72 ± 0.47% and 15.47 ± 0.85%. Moisture of the cheeses in the present study up to 90 d was similar to values reported by other studies on Graviera Kritis (Kandarakis et al., 1998; Nega & Moatsou, 2012). Moisture at 210 d was approximately 28%, which was lower than the 33e34% reported by Moatsou et al. (2004). Apparently, the small size of cheese wheels in the present study facilitated dehydration. Cheese pH was not affected significantly by natamycin coating. Ripening induced a significant decrease in pH (P < 0.05) from 8 to 60 d, which did not change thereafter. The low moisture of the mature cheeses enhanced their buffering capacity that did not favour further pH changes (Cogan & Beresford, 2002). Moreover, in these cheese types, lactic acid is catabolised further during the propionic acid fermentation, which is favoured after two weeks of warm room ripening (Carcano, Todesco, Lodi, & Brasca, 1995; Choisy et al., 1987). Salt content of cheeses was <2%, in accordance with the legislation standards for this cheese type.

Table 1 Effect of the factors of the experiment on gross composition parameters of Graviera Kritis cheeses, analysed by ANOVA.a Factor

Moisture (%)

Protein (%)

Fat (%)

Ash (%)

NaCl (%)

Treatment CTR 5.61 NT 5.62 LSD 0.05

pH

31.70 32.22 0.65

27.10 26.91 0.30

38.18 38.41 0.89

4.64 4.63 0.13

1.60 1.61 0.13

Days 8 60 90 210 360 LSD

38.09D 33.70C 33.01C 28.71B 26.31A 1.03

23.73A 25.29B 25.69B 28.18C 32.14D 0.48

36.83A 37.84A,B 37.79A,B 38.65B 40.39C 1.41

4.08A 4.56B 4.58B 4.84C 5.09D 0.20

1.33A 1.74B,C 1.61B 1.84C 1.52A,B 0.21

5.76B 5.54A 5.59A 5.59A 5.59A 0.08

a The values of the factor “treatment” are means of 15 determinations (five ripening stages  three replications) and the values of the factor “ripening days” are means of six determinations (two treatments  three replications); means with different superscript letters in the same column of each factor were significantly different (P < 0.05), LSD denotes least significance difference (P < 0.05). CTR and NT denote cheeses ripened without (control) and with natamycin-containing coating, respectively.

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3.2. Microbial counts and aminopeptidase activities The evolution of microbial groups in both cheese rind and interior during cheese ripening is shown in Fig. 1. It was evident that viable counts were higher in cheese interior except for TMC and YMC. The above-mentioned intense reduction of cheese rind moisture resulted in reduction of most microbial groups during ripening. According to least significant difference test, the application of natamycin coating affected significantly (P < 0.05) only the YMC in the cheese rind and caused at 360 d a 3.7 log cfu g
1 reduction in the rind of cheese NT compared with control cheese CTR. In fact, from 30 to 210 d, the YMC of the rind of cheese NT was 1.5e1.9 log cfu g
1 less than that of CTR. Apart from lower YMC, the rind of cheese NT at 360 d had higher NSLAB and propionibacteria counts by 2.4 and 2.3 log cfu g
1, respectively. Thermophilic counts were not affected. Apparently, yeasts and moulds metabolism affected the microflora profile of CTR cheese surface. Reduction of YMC in the interior of cheese NT was also observed, which at 360 d was 1.5 log cfu g
1 lower than that of control cheese CTR. Total mesophilic counts remained steadily high during ripening. Interestingly, THCC and THBC that were assigned to starter culture were detected in high numbers in the cheese interior even after 11 months of ripening in both CTR and NT cheeses; a reduction of about 1.3e1.4 log cfu g
1 and 0e0.2 log cfu g
1 for THCC and THBC, respectively, was observed. Considering that starter counts in similar cheese varieties reach 9 log cfu g
1 at day one (Cogan & Beresford, 2002) and that starter autolysis occurs during the first four weeks of ripening (Wilkinson, Guinee, O'Callaghan, & Fox, 1994), it appears that these high counts can be due to NSLAB

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numbers increasing from the first month to 210 d of ripening (Table 1). NSLAB counts increased steadily up to 8 log cfu g
1 within 2e4 months of cheese ripening favoured by the high cheese moisture and high ripening temperatures (Cogan & Beresford, 2002). The evolution of propionibacteria counts was very interesting, since they were not included in starter mixture. Therefore, they came from the native microflora of the cheese milk. They were found in high numbers in the cheese interior and they were >8 log cfu g
1 at the end and after the warm room ripening, i.e., between 60 and 90 d. Propionic acid bacteria can survive HTST pasteurisation at 72  C for 15 s and grow at salt concentrations <2%. They can increase up to >8e9 log cfu g
1 during warm room  at 20e22 and 16e18  C respecripening of Emmental and Comte tively (Carcano et al., 1995; Choisy et al., 1987). Therefore, natamycin coating did not influence the counts and the evolution of microbial groups in the cheese interior that are important for the ripening of this cheese variety, i.e., the thermophilic cocci and bacilli related to starter and the propionic acid bacteria. Coliforms at 8 d, were 5.9 log cfu g
1 and decreased to 3.5 and 3.3 log cfu g
1 in CTR and NT cheeses respectively, at 360 d. Initial high numbers of this group are due to the sub-pasteurisation of cheese milk. Although coliforms are suppressed by the growth of starter bacteria, they can easily re-contaminate cheeses through the environment. Enterococci counts at 8 d were 7.8 log cfu g
1 and remained almost constant in the cheese interior. At 360 d, they were 7.3 and 7.5 log cfu g
1 in CTR and NT cheeses, respectively. Similar enterococci counts have been reported for Graviera Kritis by Kandarakis et al. (1998). Enterococci counts of the present cheeses

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Fig. 1. Evolution of the counts of microbial groups in the rind (>, ) and in the cheese interior ( , B) throughout ripening of Graviera Kritis cheese with ( , B) and without (>, ) natamycin coating: A, yeasts and moulds; B, thermophilic bacilli; C, total mesophiles; D, thermophilic cocci; E, non-starter lactic acid bacteria; F, propionic acid bacteria counts.

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were one log lower in the rind than in the cheese interior apparently due to lower moisture content of the former. At 8 d, anaerobic bacteria in the cheese interior were 7.0 log cfu g
1. At 90 d, they increased by >1 log cfu g
1 and remained constant thereafter. At 360 d, they were 8.0 and 8.4 log cfu g
1 in CTR and NT cheeses, respectively. There are various reports about the effect of natamycin on cheese microflora. Var et al. (2006) found that natamycin application alone or in combination with packaging materials at the start of ripening had no effect on total mesophilic and yeast counts of Kashar cheese. They also reported that its anti-mould effect was observed in the first two mo of ripening. On the other hand, Dos Santos Pires et al. (2008) reported a decrease of yeasts and moulds by 2 log cfu g
1 after 9 d of storage of sliced Mozzarella covered with natamycin containing film. Fajardo et al. (2010) found that after 27 d of storage of semi-hard cheeses coated with chitosan containing 0.5 mg mL
1 natamycin, the yeasts and moulds were by 1.1 log cfu g
1 lower compared with control. They also reported that at 37 d, the mesophilic bacteria counts were 0.7 log cfu g
1 higher in coated than in the uncoated samples. According to Ramos et al. (2012), natamycin incorporated at 0.05 g L
1 in edible films, displayed a strong inhibitory effect against yeasts, whereas it was not active against the bacteria Escherichia coli and Staphylococcus aureus. Natamycin added into the mass of soft cheese Galotyri at 0.01 and 0.02% unexpectedly reduced significantly lactobacilli counts by about one log cfu g
1 between day 21 and 28. Moreover, 0.02% natamycin significantly suppressed the yeasts throughout the entire storage period, resulting in counts ranging from 3.0 to 4.1 log cfu g
1 (Kallinteri et al., 2013).

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The evolution of various aminopeptidase activities is shown in Fig. 2. According to Gatti et al. (2008) these aminopeptidases presented their maximum activity at pH 5.5, 19  C and 3% NaCl. These conditions are similar to those in the cheeses in the present study. Aminopeptidase activities were not detected at 8 d, apparently because lysis of starter bacterial cells did not occur massively during the first week of ripening. In all cases, the highest activity was observed after 12 months of ripening. But, from 8 to 60 d, both CTR and NT cheeses were enriched with aminopeptidases. Thus, a great part of autolysis was carried out within this period, confirming the evolution of microbial groups showed in Fig. 1. The increase of activities during the first two months of ripening can be attributed to starter autolysis. The increase of activities observed between 210 and 360 d, which antiparalleled the decrease of NSLAB counts in the same period (Fig. 1), was consistent with NSLAB autolysis. The treatment of cheeses with natamycin did not affect significantly (P > 0.05) these aminopeptidase activities. However, up to 60 d they were released more slowly in NT than in CTR cheese. 3.3. Proteolysis The proteolysis indices and the effect of experimental factors on them are presented in Table 2. The proteolysis rate was assessed by the evolution of the SN fraction and the FAG. The SN fraction increases as casein hydrolysis in cheese proceeds. It includes whey proteins retained in cheese, peptides soluble at pH 4.4 and free amino acids. The expression of SN on total nitrogen of cheese (SN/ TN) is considered as an index for cheese ripening. FAG determined

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Fig. 2. Evolution of aminopeptidase activities (change of enzyme activity per minute; DE min
1) throughout ripening of Graviera Kritis cheese with ( ) and without (>) natamycin coating: A, general aminopeptidase A (PepN); B, aminopeptidase L (PepL); general aminopeptidase C (PepC); D, proline specific aminopeptidase I (PepI); E, narrow-specific aminopeptidase A (PepA); F, X-prolyl dipeptidyl aminopeptidase (PepX).

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Table 2 Effect of the factors of the experiment on proteolysis parameters of Graviera Kritis cheeses, analysed by ANOVA.a Factor

SN (%)

SN/TN (%)

FAG (mM Gly)

Area ratios (HB/HL) 55e100 min/ 0e55 min

40e100 min/ 10e40 min

Treatment CTR 0.58B NT 0.55A LSD 0.03

13.17 12.65 0.58

0.50 0.51 0.04

1.58 1.48 0.16

5.11 4.81 0.47

Days 8 60 90 210 360 LSD

6.90A 11.52B 12.35B 16.45C 17.34C 0.92

0.15A 0.40B 0.48C 0.57D 0.94E 0.06

1.99C 1.58C 1.61B 1.21B 1.24A 0.25

7.10C 5.28B 5.15B 3.60A 3.70A 0.74

0.26A 0.46B 0.50C 0.73D 0.87E 0.04

a The values of the factor “treatment” are means of 15 determinations (five ripening stages  three replications) and the values of the factor “ripening days” are means of six determinations (two treatments  three replications); means with different superscript letters in the same column of each factor were significantly different (P < 0.05), LSD denotes least significance difference (P < 0.05). Abbreviations are: SN, soluble nitrogen; TN, total nitrogen; FAG, free amino groups; HB/HL, ratio of areas of hydrophobic and hydrophilic peptides eluted from reverse phasehigh performance liquid chromatography within the defined time windows; CTR, cheeses ripened without natamycin-containing coating (control); NT, cheeses ripened with natamycin-containing coating.

through the reaction of TNBS with a-amino groups was expressed as mM Gly. According to statistical analysis, the treatment with natamycin affected significantly (P < 0.05) the SN expressed as percentage of cheese mass. Since SN/TN did not differ significantly, the higher SN content of control cheese CTR was due to its higher protein and lower moisture content. Ripening affected all ripening parameters significantly (P < 0.05). The SN and FAG courses paralleled each other. SN was almost doubled between 8 and 60 d of ripening, whereas FAG increased even more. Ripening index SN/TN was not affected by moisture changes. It was increased at a slower pace, but very intensively. Proteolysis proceeded slowly between 60 and 90 d. Moreover, all proteolysis parameters were increased slowly from 3 to 12 months, during the storage of cheeses, at 4  C. The strong increase observed in the first two months was apparently due to the warm ripening period. Also, as reported earlier, autolysis of starters occurring during the first four weeks of ripening enriched cheeses with peptidolytic enzymes. Indeed, within this time interval, the SN and mM Gly courses anti-paralleled the log counts of two major microbial groups, i.e., total mesophilic and thermophilic cocci

counts (Fig. 1). Moreover, between 8 and 90 d, all aminopeptidase activities portrayed in Fig. 2 were augmented intensively. The accumulation of proteolysis products is initially driven by caseinolysis. At later stages of ripening, proteolysis products are precursors of volatile compounds. The increase of SN and free amino groups after several months of ripening/storage indicated that cheeses were constantly enriched with bacterial enzymatic activities in accordance with the evolution of aminopeptidase activities shown in Fig. 2. At all sampling points, the coating with natamycin did not cause any significant difference. In general, at 90 d, the SN/TN ratios of the present study were lower than those reported for this cheese type by Moatsou, Kandarakis, Georgala, Alichanidis, and Anifantakis (1999). Average SN/TN values for marketed Graviera Kritis cheeses was 17.3 ± 3.8% (Nega & Moatsou, 2012) and values for this index approximately 20% or more have been reported for experimental cheeses (Moatsou et al., 1999, 2004). The qualitative characteristics of proteolysis of CTR and NT cheeses were studied by means of the chromatographic profiles of SN fractions (Fig. 3). Profiles were divided in two parts, i.e., hydrophilic (HL) and hydrophobic (HB) according to elution time, which was related to the percentage of acetonitrile in the mobile phase. In the latter part of the chromatograms, at >85 min the major whey proteins were eluted, whereas in the first 10 min free amino acids or non-nitrogen substances of the sample appeared. Also, the ratio of peptide areas eluted within 40e100 to those eluted within 10e40 min was calculated. It has been suggested that the 10e40 min part of the profiles is very important for cheese types similar to Graviera Kritis (Nega & Moatsou, 2012). Both HB/HL and 40e100/10e40 ratios decreased significantly (P < 0.05) during ripening and storage, as a result of further hydrolysis of paracasein and peptides. The more intense changes were observed between 8 and 90 d. Natamycin-containing coating had no significant effect on these ratios (P > 0.05). 3.4. Sensory evaluation According to the analysis of the results of sensory evaluation (Table 3), the natamycin-containing coating did not affect the total score of cheeses statistically significantly (P < 0.05). The only parameter affected was the appearance, which gained higher scores for NT cheese. Although, moulds were removed from the surface of CTR cheeses before testing, the difference in rind appearance between the two cheeses was noticed by the members of the panel. The factor ripening caused significant differences in the scores of cheeses; cheeses up to 90 d gained the highest scores.

Fig. 3. Reverse phase-high performance liquid chromatography profiles of the soluble nitrogen extracts of Graviera Kritis cheese with (lower panel) and without (upper panel; control) natamycin coating at 90 d of ripening.

G. Moatsou et al. / International Dairy Journal 50 (2015) 1e8 Table 3 Effect of the factors of the experiment on the scores of the sensory evaluation of Graviera Kritis cheeses, analysed by ANOVA.a Factor

Appearance (0e10 points)

Texture (0e40 points)

Flavour (0e50 points)

Total score (0e100 points)

Treatment CTR 8.39A NT 8.79B LSD 0.295

34.06 34.64 1.189

42.31 42.98 1.970

84.67 86.41 2.974

Days 60 90 210 360 LSD

35.52C 35.92B,C 34.00B 31.97A 1.610

42.80 42.72 43.50 41.58 2.667

87.15B 87.52B 85.98A,B 81.71A 4.027

8.83B 8.88B 8.48A,B 8.16A 0.399

Units mg mg mg mg mg mg mg mg

90 210 360 a

kg
1 dm
2 kg
1 dm
2 kg
1 dm
2 kg
1 dm
2

Without coating 1.09 0.08 1.41 0.08 1.36 0.09 1.42 0.08

± ± ± ± ± ± ± ±

0.09 0.01 0.30 0.02 0.41 0.02 0.60 0.05

With coating 8.73 0.68 34.8 2.19 8.01 0.59 26.31 1.43

± ± ± ± ± ± ± ±

Acknowledgements

References

Table 4 Determination of natamycin in the rind of cheeses with natamycin-containing coating (NT) by means of high performance liquid chromatography.a

60

various aminopeptidase activities as well as for the counts and the evolution of microbial groups relevant to this cheese type.

The authors thank the Greek Milk and Meat Organisation (www. elogak.gr) and the Union of Cheesemakers of Rethimnon for their financial contribution to the present study (Research grants 33.02.04 and 33.02.06) and the cheese plant Tzourbaki Bros at Karines Village of Rethimnon Crete, in which the experimental cheesemaking took place.

a The values of the factor “treatment” are means of 12 determinations (four ripening stages  three replications) and the values of the factor “ripening days” are means of six determinations (two treatments  three replications); means with different superscript letters in the same column of each factor were significantly different (P < 0.05), LSD, least significance difference (P < 0.05). CTR and NT denote cheeses ripened without (control) and with natamycin-containing coating, respectively.

Ripening (days)

7

0.41 0.04 7.12 0.53 0.49 0.05 10.30 0.26

Values are means of three experiments ± standard deviation.

3.5. Presence of natamycin The results of the HPLC method for natamycin determination in the rind of NT cheeses is presented in Table 4. No migration into the cheese interior was observed since no natamycin was detected in the slice of about 1 mm taken after removing the outer 5 mm rind of samples. Due to manual application of coating, rather high standard deviations were observed in some cases, especially when coating was not removed. Between 60 and 90 d, there was an increase in average concentrations following the intense decrease in rind moisture. Nevertheless, after coating removal, which is suggested in the International Standard (ISO/IDF, 2007), the concentration of natamycin was always <0.1 mg dm
2. Therefore, the mode and time of application of coating did not favour high concentrations in the cheese rind and migration into the cheese interior, although it remained effective in controlling the fungal growth on the cheese surface.

4. Conclusions A single application of a natamycin-containing coating on the
re-type cheese, at an early stage of ripening surface of a hard-Gruye was appropriate for the control of moulds. No migration into the cheese interior was observed and its concentration in the cheese rind was <0.1 mg dm
2 at all stages of ripening. The gross composition and the proteolytic pattern of cheeses throughout ripening were not significantly affected. The same was also true for

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