Casein/natamycin edible films efficiency for controlling mould growth and on microbiological, chemical and sensory properties during the ripening of Kashar cheese

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Received: 29 January 2015 Revised: 9 July 2015 Accepted article published: 22 July 2015 Published online in Wiley Online Library: 21 August 2015

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7348

Casein/natamycin edible ﬁlms eﬃciency

for controlling mould growth and on

microbiological, chemical and sensory

properties during the ripening of Kashar

cheese

Filiz Yangılar

a*

and Pınar O ˘guzhan Yıldız

b

Abstract

BACKGROUND: The objective of the present study was to investigate the eﬀects of the dipping application of coating materials such as casein (Cas), casein/natamycin (Cas/N) and natamycin (N) solutions on chemical (e.g. pH, dry matter, fat, acidity, salt, protein, water-soluble nitrogen, ripening index, 12% trichloroacetic acid-soluble nitrogen and pH 4.6 soluble nitrogen), microbial (e.g. total number of aerobic mesophilic bacteria, lactic acid bacteria in MRS, lactic acid bacteria in M17, coliforms and mould) and organoleptic properties of Kashar cheese samples during ripening (3, 30, 60 and 90 days).

RESULTS: The diﬀerence in microbiological and chemical changes between samples were found to be signiﬁcant (P< 0.05) during the ripening period. The highest and lowest mould counts were determined for the control (2.87 log cfu g−1) and the Cas/N (<1 log cfu g−1) samples, respectively, at 90 days of ripening. Compared to other cheeses, control and Cas/N-coated cheeses had higher levels of water-soluble nitrogen and ripening index at in the end of storage. The Cas/N-coated cheese samples were preferred more by the panellists, while N-coated cheese samples received the lowest scores.

Keywords: casein/natamycin; edible coating; shelf life; Kashar cheese

INTRODUCTION

Traditional dairy products, including cheese, have an important place in rural food culture, thus contributing to the formation of a whole country’s culture. Today, most of cheese types have reg-istered trademarks and have protected geographic indications.1,2

Kashar cheese is one of the most important cheese types in Turkey and it can also be seen as the most typically stretched curd or pasta-ﬁlata cheese.3_{Kashar cheese is traditionally made from raw}

sheep’s or cow’s milk or their mixture, and the natural flora of milk is effective in the maturation process. According to Turkish Stan-dards Code 3272, this cheese type is classified as ‘fresh or aged (i.e. ripened) Kashar’. Fresh Kashar should be ripened within a period of 90 days to obtain aged Kashar, according to the related standards.4

Microbial growth and oxidation reactions occurring on the food surface are two of the main causes of deterioration and loss of fresh and processed solid food products. In order to prolong shelf life, some types of preservatives are added widely to most types of food, including cheese, at the rate required (by legislation on food additives)5,6_{to control degradation processes such as growth}

of natural microﬂora or oxidation of native vitamins, colorants and ﬂavours etc.

Being among the ready-made food types, cheese may be quickly and easily contaminated with harmful microorganisms, some of

which can spoil, causing undesired appearance and economic losses. Among such spoiling microorganisms are Yarrowia

lipoly-tica, Pseudomonas aeruginosa and Penicillium spp., while those

such as Listeria monocytogenes are pathogenic and may cause foodborne listeriosis.7,8 _{Packaging ﬁlms based on antimicrobial}

polymers could prove to be more eﬃcient by maintaining high concentrations of the active substance on the food surface while preventing its migration, thereby maintaining a critical concen-tration for an extended period of time.9,10_{Natamycin (pimaricin)}

is an agent showing antifungal features and is categorized as a polyene macrolide antibiotic. Natamycin is produced as the result of fermenting aerobically Streptomyces natalensis and other similar species which can be used as a food additive (E235) for the

∗ _{Correspondence to: Filiz Yangılar, Department of Nutrition and}

Dietet-ics, School of Health, Erzincan University, 24000 Erzincan, Turkey, E-mail: [email protected]

a Department of Nutrition and Dietetics, School of Health, Erzincan University,

24000 Erzincan, Turkey

b Department of Food Engineering, Faculty of Engineering, Ardahan University,

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Casein/natamycin coatings in production of Kashar cheese www.soci.org

surface treatment of hard, semi-hard and semi-soft cheese types and dry-cured sausages because of its activity against yeasts and moulds.11–13

It has recently been stated that antimicrobial ﬁlms and coatings, including organic acids, enzymes, fungicides and natural antimi-crobial compounds such as spices, are used for various types of cheese.14–16_{Several studies have been conducted on the}

applica-tion of various antimicrobial films in Kashar cheese in the literature, however; only a few of them dealt with the use of coating mate-rial. From this point of view, the objectives of the present study were to determine the effectiveness of casein, casein/natamycin and natamycin solution-based film coatings in the prevention of mould growth and to investigate their effects on microbiological and chemical properties, proteolysis levels and sensory evaluation of traditional Kashar cheese during ripening.

MATERIALS AND METHODS

Materials

Cow’s milk was obtained from a livestock farm in Göle district, Ardahan province. Microbial rennet (1:15 000) was purchased from Mayasan Co., Istanbul, Turkey, while the analytical grade reagents (casein and natamycin) were obtained from Merck (Darmstadt, Germany) and Sigma Chemical Co. (St Louis, MO, USA).

Production of traditional Kashar cheese

Cheese was manufactured in Alibey Plant in Göle, Ardahan. Cheese was produced in the traditional way from raw cow’s milk without using starter culture. In the production of cheese, raw cow’s milk (400 kg) was filtrated and heated to 33 ∘C, and rennet diluted with water (1/10) was added to the milk for coagulation. When curds attained sufficient maturity they were cut automatically in the process tank. Whey was removed from the cheese in a press cloth and the fresh cheese was taken to the press and cut into blocks. The blocks were then grated and kept at room temperature for 6–8 h to remove whey-developed acid. Thereafter, the fresh cheese was again cut into blocks and grated. After this process, the cheese sample was examined to determine if it had reached a suitable level for boiling by removing whey and developing acidity. Cheese suitable for boiling was weighed and 2 kg sam-ples were placed into baskets and boiled at 69–80 ∘C in 8% salt solution for 2–2.5 min. Boiled fresh cheese sample was plasti-cized and placed into moulds, which were sometimes reversed and allowed to gain suitable shapes. Kashar dough forming air bubbles was pricked with a probe and air was removed. Moulded cheese samples were stored in the mould for 12 h, then taken from moulds to the pre-ripening room. Kashar cheese samples manufactured traditionally were stored for 20 days to ripen at ambient temperature and 60–70% relative humidity. Following pre-ripening, samples were stored for an additional period of 70 days in four blocks at 4 ± 1 ∘C and 70–80% relative humidity. Thus cheese samples were ripened within a total of 90 days. Follow-ing the pre-ripenFollow-ing process, cheese samples were divided into four groups: control (C; without edible films); Cas (covered with casein solution); Cas/N (covered with casein solution containing natamycin; and N (covered with natamycin solution). Cheese sam-ples were first analysed at the end of the pre-ripening process and before coating. Other analyses were carried out at 30-day inter-vals (i.e. on the 3rd, 30th, 60th and 90th days) by duplicating all the analyses.

Preparation of casein/natamycin ﬁlm-forming solution In the preparing the coating solution, a modiﬁed procedure of Chick and Hernandez17_{and Mezgheni et al.}18_{was used, in which}

acid casein (7.5%, w/w) was dispersed in deionized water, the pH was adjusted to 10.0 with 0.1 mol L−1 _{NaOH and heated at}

75 ∘C for 20 min. The solution was then cooled to 25 ∘C, pH was adjusted to 7.0 and 2.5% (w/w) glycerol, 0.0125% (w/w) CaCl2and

0.07% (w/w) natamycin were added.19_{This solution was applied}

to cheese sample groups by dipping the cheese wheels twice for 60 s only and then left to dry for 2 h. The residual natamycine was allowed to drip oﬀ and then the cheeses were dried for 1 h at 22 ∘C to allow the coating to cake.

Microbiological analysis

The dried coating solution was removed from each cheese wheel using sterile gloves and the sample groups were subjected to microbiological analysis. For each sample, 11 g cheese was taken and diluted in 99 mL of 0.85% (w/v) sterile saline solution. After that, a homogenizer (Lab. Stomacher Blender 400 BA 7021, Swardmedical) was used to homogenize the samples in a ster-ile polyethylene bag for 1.5 min. A sterster-ile NaCl solution (9 mL 0.85%, w/v) was used for dilution and the number of total aer-obic mesophilic bacteria (TAMB; Merck, at 30 ± 1 ∘C for 72 h),20

lactic acid bacteria (LAB) (in MRS; Merck, at 30 ∘ C for 48 h under anaerobic conditions),21 _{LAB (in M17; Merck, at 30 ∘C for 48 h),}22

coagulase-positive staphylococci on Baird–Parker agar with egg yolk tellurite enrichment (Merck, at 37 ∘C for 24 h),20_coliforms

(Vio-let Red Bile Agar, Oxoid, at 35 ± 2 ∘C for 48 h)21_{and moulds (Potato}

Dextrose Agar, Oxoid, at 25 ∘C for 5–7 days)23_{were counted.}

Chemical analysis

After the removal of coating materials, each of the cheese samples was fully shredded. Parameters of moisture, fat and salt were con-veniently measured by the method of Kurt et al.,24_{while titratable}

acidity was detected by AOAC Oﬃcial Method 920.124.25_The

Kjel-dahl method26_{was used to determine total nitrogen (TN), while}

pH was measured using a pH meter (WTW 340-1) as in the study of Savello et al.27

Nitrogen fractions

Water-soluble nitrogen (WSN), pH 4.6 soluble-nitrogen (pH 4.6-SN) and 12% trichloroacetic acid-soluble nitrogen (TCA-SN) nitrogen fractions as percent of total nitrogen of the cheese were deter-mined by Kuchroo and Fox.28_{A 20 g grated cheese sample was}

homogenized in 40 mL H2O for 2 min using an Ultra Turrax blender

(IKA, Wilmington, NC, USA); stored at 40 ∘C for 1 h; centrifuged at 3000 × g for 30 min at 4 ∘C; then its fatty layer was removed and the supernatant was filtered with filter paper (Whatman 113). A 25 mL extract prepared for WSN was taken at an equal volume of 24% (w/v) and TCA was added for further fractionation of the nitrogenous compounds. The mixtures were incubated for 2 h at room temperature. Precipitates were filtered through filter paper.29_{The contents of WSN, TCA-SN and pH 4.6-SN were}

determined using the Kjeldahl method. The ripening index (RI) was determined using the formula WSN/TN × 100.

Sensory evaluation

Eight panellists experienced in the sensorial evaluation of Kashar cheese assessed the cheese samples on the 3rd, 30th, 60th and 90th days of ripening, employing the method of Bodyfelt

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Figure 1. Changes in total aerobic mesophilic bacteria counts (log cfu g−1_{) after coating Kashar cheese samples during ripening. Diﬀerent letters above}

the bars indicate signiﬁcant diﬀerences by Duncan’s multiple comparison test (P< 0.05).

et al.30_{and modifying the sensory criteria for the characteristics of}

Kashar cheese. Samples were scored considering the ﬁve sensorial features – colour–appearance, texture, taste, odour and general acceptability – ranging from 1 (poor) to 9 (excellent).

Statistical analysis

The experimental design consisted of a completely randomized design in a factorial arrangement: four treatments of Kashar cheese (C, Cas, Cas/N and N), four ripening times (3, 30, 60 and 90 days) and two replicates. All statistical calculations were performed using SAS Statistical Software (SPSS 17.0; Chicago, IL, USA).31_Duncan’s

multiple range test and variance analysis were used to evaluate the signiﬁcance level (P< 0.05) for statistical diﬀerences.

RESULTS AND DISCUSSION

Dry matter, fat and protein contents and acidity, SH and pH rates of the cow’s milk used in the manufacturing process of Kashar cheese samples were 12.71 ± 0.28%, 3.5 ± 1.24%, 3.21 ± 0.11%, 5.8 ± 0.04 and 6.52 ± 0.07, respectively. Where ∘SH is the Soxhlet Henkel degrees, obtained by titrating 100 ml of milk with N/4 NaOH, using phenolphtalein as the indicator.

Microbiological evaluation

The results of the microbiological analysis carried out over cheese samples are given in Figs 1–5. Microbiological counts in cheese samples were found to be signiﬁcant (P< 0.05) during the ripening period. TAMB counts of C samples were determined to be higher than the others on the 90th day of ripening. As can be seen in Fig. 1, total numbers of mesophilic aerobic bacteria in cheese samples were determined to range from 7.04 to 8.05 log cfu g−1_{. Fajardo}

et al.10_{found mesophilic bacteria counts to be between 6.29 and}

7.51 log cfu g−1_{in natamycin-coated Saloio cheese. Such results}

found in previous studies are consistent with the present study. Generally, TAMB counts showed an increase for all samples during the 90th day in the present study. Similar results were obtained by several other researchers.32,33

Counts of LAB in MRS were determined in cheese samples to range between 6.99 and 8.14 log cfu g−1_{, which were found to be}

signiﬁcantly lower (P< 0.05) in control and N samples on the 3rd

and 30th days of ripening, respectively. These results were found to be consistent with the values found in previous studies.33 _Di

Pierro et al.34_{found LAB (MRS) to be in the range of 5–6 log cfu g}−1

for the control on the14th day and for the chitosan/whey protein ﬁlm-coated cheese samples on the 30th day, which are lower than the ﬁndings in the present study.

As can be seen in Fig. 3, the LAB (in M17) values found in cheese samples were between 6.90 and 7.89 log cfu g−1_{. In particular, LAB}

counts of N samples were found to be 7.47 on the 60th and 6.93 on the 90th day of ripening. Such results showed that coating with natamycin had a negative eﬀect on the growth of microorganisms compared with other coating materials. Yılmaz and Dagdemir33

obtained similar results, of between 6.73 and 8.13 for 120 days for beeswax-coated Kashar cheese samples. Lucera et al.35_stated

that the total bacterial counts showed an increasing trend in fresh mozzarella cheese. The initial microbial count between samples was about 4.5 log cfu g−1_{and slightly faster in control samples than}

in another cheese coated with potassium sorbate. Such results found in previous studies are not consistent with the present study. These results showed that coating with casein, casein/natamycin and natamycin solutions did not have any negative inﬂuences on the growth of microorganisms necessary for the maturation of cheese.

The treatment and ripening processes were found to aﬀect sig-niﬁcantly the moulds (P< 0.05) in cheese samples, being between

<1 and 4.32 log cfu g−1_{. Microbiological analyses showed that}

sam-ples coated with casein, casein/natamycin and natamycin solu-tions exhibited a decrease in moulds compared to control after 90 days of storage, while the Cas sample represented higher mould counts than the sample Cas/N and N at the end of the ripening period. Similar results have been reported by several studies.32,33_{Baseline mould counts were reported to be 10}2_{cfu g}−1

for all samples in Yılmaz and Dagdemir,33_{but increased in}

con-trol sample from 2.75 to 4.60 log cfu g−1_{in the storage period}

and from the 15th day of ripening. Sarıoglu and Oner32 _stated

that mould and yeast could not be counted in Na-caseinate ﬁlm-coated Kashar cheese samples from the 60th day while in uncoated samples counting could not be done from the 90th day. Fajardo et al.10_{reported the counts of mould and yeast to be}

4.53 and 6.06 (log cfu g−1_{), respectively, and that on the 27th day}

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Figure 2. Changes in LAB (in MRS agar) counts (log cfu−1_{) after coating Kashar cheese samples during ripening. Diﬀerent letters above the bars indicate}

signiﬁcant diﬀerences by Duncan’s multiple comparison test (P< 0.05).

Figure 3. Changes in LAB (in M17 agar) counts (log cfu−1_{) after coating Kashar cheese samples during ripening. Diﬀerent letters above the bars indicate}

significant differences by Duncan’s multiple comparison test (P< 0.05). mould/yeast rate (4.95 ± 0.27). Ramos et al.36_{studied the efficacy}

of edible films produced from whey protein isolate, glycerol and natamycin as antimicrobial agent. The authors showed through the viable cell count assay that natamycin incorporated in the film displayed a cidal effect against Yarrowia lipolytica. Balaguer et al.37

observed no fungi in the cheese samples packaged with the active ﬁlm on the 26th day of storage at 4 ∘C, while there was fungal growth in control samples on the16th day of storage. Ollé Resa

et al.38_{studied the eﬀectiveness of natamycin against yeast in Port}

Salut cheese and found that it exerted an initial fungicidal effect against Saccharomyces cerevisiae. The barrier test was performed on cheese with Cas/N films, which means that the edible film exerted a fungicidal effect against moulds. Additionally, coated materials prevented contamination of the cheese by microorgan-isms inoculated in the dipping.

Counts of coliform bacteria found in cheese samples were between 0.90 and 4.10 log cfu g−1_{in the present study, while it}

was<1 log cfu g−1_{in Yılmaz and Dagdemir’s study.}33_{Sarıoglu and}

Oner32_{stated that counts of coliform microorganisms could not}

be detected in Na-caseinate ﬁlm-coated Kashar cheese on the 90th day, while it was possible to count them in control samples on the 60th day.

The presence of human- or animal-sourced Staphylococcus in food at a high level indicates that the procedures of sanitation and heating are not enough. The use of antimicrobial ﬁlms was also highly eﬀective in inhibiting Staphylococcus aureus during ripen-ing time. Staphylococcus aureus count was below detectable levels (2 log cfu g−1_{) in all samples, which can be attributed to the}

scald-ing process applied in the production of traditional Kashar cheese. A similar result was also reported by Yılmaz and Dagdemir.33

Pra-noto et al.39_{determined the inhibitor eﬀect of antimicrobial}

algi-nate ﬁlm containing 0.4% garlic oil on Staph. aureus. Torlak and Nizamoglu40_{reported that in Kashar cheese samples coated with}

renewable ﬁlms the counts of Staph. aureus decreased on 14th day compared to the control group between 0.90–2.66 log cfu g−1_and

all the film types exhibited an antimicrobial effect at a significant level compared to the control group (P< 0.05). Furthermore, Mei

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Figure 4. Changes in mould counts (log cfu−1_{) after coating Kashar cheese samples during ripening. Diﬀerent letters above the bars indicate signiﬁcant}

diﬀerences by Duncan’s multiple comparison test (P< 0.05).

Figure 5. Changes in coliform counts (log cfu−1_{) after coating Kashar cheese samples during ripening. Diﬀerent letters above the bars indicate signiﬁcant}

diﬀerences by Duncan’s multiple comparison test (P< 0.05).

storage of Mongolian cheese for controlling microbial populations was eﬀective.

Chemical evaluation

The chemical composition of the cheese samples is given in Table 1. Chemical changes in samples were found to be statis-tically signiﬁcant (P< 0.05) during a ripening period of 90 days. The dry matter values of cheese samples signiﬁcantly increased. Dry matter of the samples was found to change between 56.73% and 64.61%. This increase was higher in the cheese samples without coating. The results also showed that the coating process with casein, casein/natamycin and natamycin solution might have delayed moisture losses compared to control. Fajardo

et al.10 _{reported the moisture content of coated Saloio cheese}

before storage to be signiﬁcantly higher than that of uncoated cheese samples, resulting mainly from the water content of the coating itself, while this diﬀerence was valid at 4 ∘C only for natamycin + chitosan-coated cheese samples. Yıldırım et al.42

found that the dry matter content in Kashar cheese samples A (control), C (coating with casein solution), D (coating with casein solution containing natamycin) and E (dipping in natamycin

solution) increased until the 60th day of storage (P< 0.05), after which it did not change signiﬁcantly (P> 0.05).

The pH values of control and coated Kashar cheese samples during ripening are shown in Table 1. pH values of the coated samples decreased linearly during ripening. Fluctuations in pH during ripening were reported by Yıldırım et al.42_{in Kashar cheese}

coated with casein solution. It was found by Di Pierro et al.34_that

pH values of CWP-coated Ricotta cheese decreased after 7 days of storage. These ﬁndings are consistent with those in the present study.

Both the lowest (24.06%, w/w) and the highest (31.33%, w/w) fat contents were observed in the control sample. In coated Kashar cheese samples, Sarıoglu and Oner32_{reported that the lowest and}

highest fat rates were 31.37% and 43.25%, while in the control group they were 30.5% and 42.25%. Similar results were obtained by Yılmaz and Dagdemir33_{and Yıldırım et al.}42

Our data demonstrated that salt values found in the samples were between 2.22% and 4.74%. Gulec et al.43 _{also found the}

lowest and highest salt rates of casein-coated Kashar cheese samples and control to be from 1.54% to 2.54% and from 1.54% to 2.43%, respectively.

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T able 1. T h e changes in chemical charac te ristics o f K ashar cheeses during ripening Cheese sample Ri p e n in g (da ys) Dr y m att e r (%) Fa t (%) Salt (%) p H A cidit y (%) Pr o te in (%) WSN (%) R ipening index (%) TC A -S N (%) pH 4.6-SN (%) C 3 59.17 ± 0.49d 24.06 ± 0.09d 2.22 ± 0.12a 5.05 ± 0.05a 1.22 ± 0.02b 24.51 ± 0.34a 3.44 ± 0.13b 14.04 ± 0.34c 4.52 ± 0.04b 5.94 ± 0.04c 30 59.53 ± 0.09b 29.87 ± 0.06c 3.08 ± 0.08a 5.17 ± 0.02a 1.30 ± 0.02a 27.11 ± 0.62a 3.94 ± 0.02b 14.55 ± 0.25c 5.51 ± 0.19c 6.74 ± 0.53c 60 60.09 ± 0.08a 30.19 ± 0.08d 3.30 ± 0.01c 5.29 ± 0.01a 1.50 ± 0.04a 28.15 ± 0.01a 4.27 ± 0.03c 15.18 ± 0.07b 6.39 ± 0.27c 9.94 ± 0.11d 90 64.48 ± 0.46b 31.33 ± 0.24d 4.74 ± 0.05d 5.31 ± 0.03c 2.82 ± 0.01c 28.49 ± 0.21a 5.34 ± 0.76b 18.71 ± 2.47b 7.25 ± 0.08d 10.59 ± 0.20d C a s 3 58.79 ± 0.16c 24.43 ± 0.01a 2.75 ± 0.62ab 5 .30 ± 0.01b 1.10 ± 0.01a 28.52 ± 0.10c 2.20 ± 0.01a 7.67 ± 0.02a 2.50 ± 0.12a 3.22 ± 0.02a 30 60.64 ± 0.17c 24.73 ± 0.59a 2.84 ± 0.14a 5.32 ± 0.08b 1.21 ± 0.04a 29.21 ± 0.13b 3.31 ± 0.14a 10.95 ± 0.09a 3.80 ± 0.13b 3.88 ± 0.05a 60 62.29 ± 0.01c 25.52 ± 0.03a 3.04 ± 0.01a 5.24 ± 0.06a 1.70 ± 0.10b 31.12 ± 0.17c 3.23 ± 0.02b 10.39 ± 0.05a 4.58 ± 0.08b 4.89 ± 0.01a 90 62.89 ± 0.05a 25.92 ± 0.06a 3.25 ± 0.02b 5.19 ± 0.02b 1.89 ± 0.01a 29.22 ± 0.22b 4.54 ± 0.21b 15.18 ± 0.89b 4.63 ± 0.06a 5.46 ± 0.19a C a s/N 3 56.73 ± 0.12a 26.40 ± 0.14c 3.17 ± 0.01b 5.47 ± 0.02c 1.56 ± 0.03d 28.13 ± 0.02c 3.93 ± 0.02c 13.98 ± 0.06c 2.63 ± 0.02a 3.49 ± 0.09b 30 58.94 ± 0.02a 26.75 ± 0.07b 3.42 ± 0.02b 5.22 ± 0.03ab 1 .74 ± 0.02c 29.59 ± 0.01b 4 ± 0.01b 13.51 ± 0.05b 3.89 ± 0.01b 6.36 ± 0.09bc 60 61.89 ± 0.02b 27.15 ± 0.06c 3.74 ± 0.04d 5.22 ± 0.01a 2.10 ± 0.03c 29.67 ± .0.26b 4.38 ± 0.16c 14.77 ± 0.41b 4.56 ± 0.21b 8.48 ± 0.26c 90 62.65 ± 0.19a 28.57 ± 0.03c 4.14 ± 0.02c 5.07 ± 0.06a 2.36 ± 0.14b 30.09 ± 0.02c 4.66 ± 0.72b 16.23 ± 1.63c 6.84 ± 0.09c 9.77 ± 0.03c N 3 57.85 ± 0.36b 25.71 ± 0.02b 2.76 ± 0.02ab 5 .62 ± 0.03d 1.34 ± 0.04c 26.28 ± 0.01b 2.66 ± 0.02b 10.14 ± 0.08b 2.47 ± 0.08a 3.19 ± 0.03a 30 62.41 ± 0.02d 26.40 ± 0.56b 3.01 ± 0.03a 5.31 ± 0.06b 1.47 ± 0.02b 27.81 ± 0.03a 3.11 ± 0.02a 11.31 ± 0.36a 3.19 ± 0.01a 5.82 ± 0.02b 60 62.83 ± 0.04d 26.65 ± 0.21b 3.12 ± 0.02b 5.21 ± 0.03a 1.66 ± 0.03ab 28.45 ± 0.02a 2.65 ± 0.28a 9.32 ± 1.01a 2.79 ± 0.04a 6.08 ± 0.02b 90 64.61 ± 0.05b 27.36 ± 0.14b 3.18 ± 0.04a 5.12 ± 0.02b 1.84 ± 0.03a 29.58 ± 0.05bc 2 .83 ± 0.04a 9.56 ± 0.06a 6.38 ± 0.15b 8.32 ± 0.03b M e an values fo llo w e d b y d iff er en t lett e rs in the same column a re sig n ifican tly diff e re n t (P < 0.05). C o n tr o l( C; without e dible fi lms); C as (0.8% w/v casein solution); C as/N (0.8% w/v casein solution con taining n atam yc in); N (0.8% w/v natam ycin solut ion).

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Table 2. Changes in sensory characteristics of Kashar cheeses during ripening

Cheese sample Ripening (days) Colour and appearance Texture Taste Odour General acceptability

C 3 8.30 ± 0.16c 7.64 ± 0.22c 7.53 ± 0.24ab 7.11 ± 0.08ab 7.93 ± 0.08b

30 8.76 ± 0.19d 7.05 ± 0.12b 7.15 ± 0.17a 7.26 ± 0.15a 7.51 ± 0.11b

60 7.48 ± 0.20b 7.65 ± 0.23b 7.58 ± 0.29a 7.86 ± 0.30d 7.39 ± 0.07a

90 7.37 ± 0.23b 7.06 ± 0.13a 7.25 ± 0.17b 7.47 ± 0.39a 7.34 ± 0.23a

Cas 3 6.97 ± 0.05a 6.71 ± 0.15a 7.36 ± 0.35a 7.37 ± 0.28b 7.51 ± 0.25a

30 6.50 ± 0.28a 6.27 ± 0.15a 7.60 ± 0.26b 7.31 ± 0.15a 6.97 ± 0.05a

60 6.34 ± 0.34a 6.20 ± 0.17a 7.56 ± 0.13a 7.17 ± 0.13a 7.46 ± 0.27a

90 7.33 ± 0.11b 7.32 ± 0.29ab 6.93 ± 0.08a 7.40 ± 0.26a 7.55 ± 0.29c

Cas/N 3 8.51 ± 0.19c 8.46 ± 0.36d 7.09 ± 0.10a 6.94 ± 0.07a 7.93 ± 0.41b

30 8.06 ± 0.07c 8.81 ± 0.20d 7.04 ± 0.19a 7.52 ± 0.34ab 7.54 ± 0.53b 60 7.50 ± 0.28b 8.31 ± 0.10c 7.41 ± 0.32a 7.48 ± 0.15b 7.96 ± 0.52b 90 7.31 ± 0.13b 8.06 ± 0.07c 7.38 ± 0.37c 7.57 ± 0.28ab 7.48 ± 0.46b N 3 7.51 ± 0.07b 7.28 ± 0.04b 7.96 ± 0.05b 7.75 ± 0.21c 8.11 ± 0.10c 30 7.43 ± 0.44b 8.63 ± 0.17c 7.34 ± 0.27a 7.81 ± 0.44b 7.45 ± 0.22b 60 7.98 ± 0.10c 7.71 ± 0.34b 7.35 ± 0.12a 7.57 ± 0.17c 7.97 ± 0.04b 90 7.09 ± 0.06a 8.20 ± 0.24d 7.20 ± 0.15b 7.65 ± 0.26b 7.58 ± 0.68d

Mean values followed by different letters in the same column are significantly different (P< 0.05).

Control (C; without edible ﬁlms); Cas (0.8% w/v casein solution); Cas/N (0.8% w/v casein solution containing natamycin); N (0.8% w/v natamycin solution).

As can be seen in Table 1, treatment and storage signiﬁcantly aﬀected pH values (P< 0.05). Gulec et al.43_{stated that pH ranged}

from 5.14 to 5.25 in casein-coated and uncoated Kashar cheese samples on the 90th of storage, which is consistent with the present study. Lucera et al.35_{determined that the pH of mozzarella}

cheese, monitored during the entire observation period, ranged between 6.50 and 6.30.

Acidity rates of the samples varied between 1.10% and 2.82%. Di Pierro et al.34_{reported that titratable acidity of Ricotta cheese,}

coated with a chitosan/whey protein ﬁlm, reached the same level as measured in the control sample at the end of storage.

It was observed that protein values of the samples were between 24.51% and 31.12%. Gulec et al.,43_{reported the lowest and highest}

rates of protein in cheese samples and control Kashar samples to be from 24.5% to 31.36% and from 24.5% to 31.28%, respectively, while Sarıoglu and Oner32_{found this rate in Kashar samples and}

the control group to be from 27.70% to 36.64% and from 27.25% to 34.40%, respectively.

The rate of WSN increased during the ripening period in all cheese samples except in N ﬁlm-coated Kashar cheese. Yılmaz and Dagdemir33_{found WSN to be signiﬁcantly higher in control cheese}

until day 90, followed by BW1 (single-layer coating). In the evalua-tion of the ripening index, the value ranged from 14.04% to 18.71% for control sample, from 7.67% to 15.18% for Cas-coated sample, from 13.51% to 16.23% for Cas/N-coated sample and from 9.32% to 11.31% for N-coated sample. While the lowest percentage of ripening index was determined in N and Cas cheeses, C and Cas/N cheeses showed the highest percentages. Also the ripening index values obtained at 90 days were diﬀerent statistically. A decreasing trend was observed in ripening index levels of Kashar cheeses from the eﬀect of coated materials. These results were similar to those of Yılmaz and Dagdemir.33_{In Yıldırım et al.,}42_{the ripening}

index in Kashar cheese samples increased steadily until the 60th day of ripening. Gulec et al.43_{reported that ripening index values}

of all samples were similar until the 60th day, while on the 90th day ripening index values of control increased more than coated sam-ples. A balanced degradation of proteins into peptides and amino

acids is necessary for the development of texture, taste and aroma characteristics of ripened cheese samples. Proteolysis in cheese is often measured by means of quantiﬁcation of WSN fraction of cheese, which consists of whey proteins, medium- and small-sized peptides from the degradation of caseins and free amino acids.33,44

The eﬀects of the coating with these materials on the rate and extent of proteolysis were estimated quantitatively by the level of soluble nitrogen components in cheese during ripening.

As can be seen in Table 1, values of 12% TCA-SN in the cheese samples increased with increasing ripening time (P< 0.05). The values of 12% TCA-SN, expressed as a percentage, showed a signif-icant increase (P< 0.05) during the ripening process, similar results were obtained by Guven et al.33,45 _Aydemir46 _{reported that the}

increase in WSN%TN and TCA%TN values were low when Kashar cheese samples were ripened at 4 ± 1 ∘C following pre-ripening.

pH 4.6-SN ranged from 5.94% to 10.59% of the control samples, from 3.22% to 5.46% for Cas, from 3.49% to 9.77% for Cas/N and from 3.19% to 8.32% for N. pH 4.6-SN was signiﬁcant (P< 0.05) during the ripening time. The pH 4.6-SN results were aﬀected by the coated materials and their interactions. Values of pH 4.6-SN were found to be 10.72–23.76% of the sample of Kashar cheese samples in Hayaloglu.47

Sensory evaluation

The results of the sensory evaluation of cheese samples on a scale from 1 (poor) to 9 (excellent) are shown in Table 2. A sig-nificant difference (P< 0.05) was found to be among samples for colour–appearance, taste, texture, odour and general acceptabil-ity. The natamycine-coated cheese (N) received the highest texture score (8.20), while control cheese (C) had the lowest score (7.06). Sample Cas had the lowest odour (7.40) and taste scores (6.93). N and Cas/N samples were mostly preferred by the panellists. In particular, high dry matter and protein content of N sample might have affected the panellists’ scores. Cas sample was given the lowest scores by the panellists. The use of packaging films could prove more sensory qualification during ripening. Yıldırım et al.42

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Kashar cheese compared to control and those coated with casein. Di Pierro et al.34_{stated that, compared to control, Ricotta cheese}

exhibited better texture conditions when it was coated with chitosan/whey protein film, while no difference was found in visual appearance, texture, flavour and odour between uncoated and chitosan/whey protein film-coated Ricotta cheese samples. Cetinkaya et al.48_{reported significant differences between Kashar}

samples coated with beeswax in terms of aroma, ﬂavour, colour, appearance and texture.

CONCLUSIONS

The coating process significantly reduced the mould growth dur-ing the ripendur-ing process and has extended the shelf-life when compared to control. It is also indicated from the results that the use of casein coating together with natamycin can suppress mould growth during the ripening time without any adverse effects on cheese quality. An decreasing trend was observed in ripening index values of Kashar cheese samples coated with these mate-rials during ripening. In terms of sensorial evaluation, panellists gave the highest scores especially to the N group. This study is rel-evant since it indicates successfully through samples coated with casein, casein/natamycin and natamycin solutions that the men-tioned materials could be used as effective coating materials in the manufacture of traditional Kashar cheese.

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