1. INTRODUCTION
⌅Kashar cheese is rich in biochemicals and is highly appreciated in Turkey. It is also manufactured in some European and Balkan nations under various names. Kashar cheese is classified as a Pasta-Filata (plastic curd) in the classification made by taking into account specific elements of its processing (coagulation with rennet, boiling the curd, hand molding, and not being pressed) and according to the humidity amount, it belongs to the semihard cheese group. A variety of cheeses known as Pasta Filata are made in Italy, Greece, the Balkans, Turkey, and Eastern Europe. (Çetinkaya, 2020Çetinkaya A, 2020. Effect of Different Preservation and salting methods on some volatile compounds and sensory properties of kashar cheese. Kafkas Universitesi Veteriner Fakültesi Dergisiv. 26 (3), 435–444, 2020, doi: 10.9775/kvfd.2019.23508; Topcu et al., 2020Topcu A, Bulat T, Ozer T. 2020. Process design for processed Kashar cheese (a pasta-filata cheese) by means of microbial transglutaminase: Effect on physical properties, yield and proteolysis. Food Sci. Technol. 125. 109226. https://doi.org/10.1016/j.lwt.2020.109226). The ripening period is crucial for producing the desired aroma of the cheese because aromatic compounds change during ripening (Eroğlu et al., 2016Eroglu, A., Toker, O. S., Dogan, M. 2016. Changes in the texture, physicochemical properties and volatile compound profiles of fresh Kashar cheese (< 90 days) during ripening. Internat. J. Dairy Technol. 69, 243–253.). However, if conditions in the ripening rooms, such as temperature and relative humidity, are not strictly controlled, the prolonged ripening period could lead to increased surface contamination.
Late blowing (LBD), which results from butyric acid fermentation and affects the aroma and structure of cheese, is the main issue with hard and semihard cheeses during storage. Anaerobic spore-forming bacteria like Clostridium produce certain organic acids (butyric and acetic acid) and gases (H2 and CO2) during the ripening process of hard and semihard cheeses and are undesired metabolites in cheese. These products lead to cheese that has undesirable quality flaws such as swelling, uneven eye development, cracks, and taste disturbance. This is a significant economic issue for cheese production (Brandle et al., 2016Brandle J, Domig KJ, Kneifel W. 2016. Relevance and analysis of butyric acid producing clostridia in milk and cheese. Food Control 67, 96–113. https://doi.org/10.1016/j.foodcont.2016.02.038). These bacteria have the ability to generate spores that withstand the thermal processing used to create hard cheeses, which can then germinate and produce the defect-causing gas (D’Incecco et al., 2016D’Incecco P, Gatti M, Hogenboom JA, Bottari B, Rosi V, Neviani E, Pellegrino L. 2016. Lysozyme affects the microbial catabolism of free arginine in raw-milk hard cheeses. Food Microbiol. 57, 16–22. Doı: 10.1016/j.fm.2015.11.020.).
Many methods, including lysozymes, a naturally occurring antibacterial and antiviral agent, were suggested to prevent LBD in cheese. Along with g-type (bacterial lysozyme) and i-type (chicken or conventional lysozyme), c-type (chicken or conventional) lysozymes are among the principal types found in the animal kingdom (invertebrate lysozyme). (Callawaert and Michiels, 2010Callawaert L, Michiels CW. 2010. Lysozymes in the animal kingdom. J. Biosci. 35, 127-160.). In-depth research has been done on the impact of lysozyme generated from eggs on the physical and chemical characteristics of semihard and hard cheeses (Conte et al., 2011Conte A, Brescia I, Del Nobile MA, 2011. Lysozyme/EDTA disodium salt and modified-atmosphere packaging to prolong the shelf life of burrata cheese. J. Dairy Sci. 94 (5), 289–5297 doi: 10.3168/jds.2010-3961; Urbienė and Sasnauskait, 2010Urbienė S, Sasnauskaitė L. 2010. Influence of lysozyme on the quality of cottage cheese during storage Zemės ūkio mokslai. T. 17 (1–2), 60–67.). However, there is currently no information on the impact of bacterial lysozyme on cheese storage and ripening.
In light of these facts, the purpose of this study was to use bacterial lysozyme and egg lysozyme in various forms (liquid and powder). Since Kashar cheese is a high-cooked pasta-filata variation, the manufacturing procedures for other high-cooked pasta-filata cheeses, like Kashkaval, Mozzarella, or Provalone, could be modified to use bacterial lysozyme for Kashar cheese in order to prevent LBD and enhance physical features.
2. MATERIALS AND METHODS
⌅2.1. Cheese making
⌅Kashar cheese production took place at a nearby dairy facility (Mega Süt Company, Aydın, Turkey). Figure 1 shows the processing procedures for producing Kashar cheese. LysochTM G4 (Code: 0402) is a powdered bacterial lysozyme concentration made by Stretomyces sp. (Handary S.A., 2016, Bruxelles, Belgium). Kashar cheese was produced in three replicas, weighing 500 g, and was vacuum packed. It was kept in storage for 90 days. The supplier of the lysozyme was FMI Gıda Kimya İthalat-İhracat Sanayi ve Ticaret Ltd. in İzmir, Turkey. The Megasüt Dairy Company provided the rennet (Maysa, İzmir), calcium chloride (Merck), polyethylene plastic vacuum packing, rock salt, and other ingredients needed for processing.
2.2. Methods
⌅2.2.1. Gross compositional and textural analyses
⌅Using a pH meter (made by Mettler Toledo), the pH of the milk samples was calculated, and the acidity was assessed using the titrimetric method. Dry matter and ash values were evaluated using the graphimetric method, and the fat contents in the milk samples were determined using the Gerber method (ISO/IDF, 2008ISO/IDF 2008. ISO 2446:2008. International Organization for Standardization. IDF 226:2008. Interantional Dairy Federation 2008. Milk - determination of fat content.). The Gerber test involved visually inspecting the fat content within a Gerber milk-tester butyrometer column. This examination occured after the sulfuric acid hydrolysis of milk components and centrifugation.The Micro Kjeldahl technique was used to determine the total nitrogen content. The total nitrogen amount was multiplied by a coefficient of 6.38 to determine the protein content. At 15 °C, specific gravity values were determined using a lactodensimeter. Standard culture enumeration techniques were used to determine the microbial counts in raw milk samples (Halkman and Ayhan, 2000Halkman AK, Ayhan K. 2000. Gıdaların mikrobiyolojik analizi 2. Mikroorganizma sayımı. Gıda Mikrobiyolojisi ve Uygulamaları. Sim Matbaacılık LTD. ŞTİ. 2.basım. 513s., Ankara.). Sporebacteria were also enumerated on Tryptose Sulfite Cyclocerine Agar (Merck, Germany), incubating at 37 °C for 24-48 h under anaerobic conditions. After 48 hours of incubation at 28-30 °C, the total number of aerobic mesophilic bacteria was counted on Plate Count Agar (PCA) (Merck, Germany). E. coli and total coliform bacteria were counted on Violet Red Bile Agar (VRB) (Merck, Germany) after being incubated for 24 and 48 hours at 35 to 37 °C in an aerobic environment. After 3-5 days of incubation at 25 °C, yeast and mold were counted on Dichloran Rose Bengal Chloramphenicol Agar (DRCB) (Merck, Germany) (VRB). Lactic acid bacteria were counted on MRS Agar and M17 Agar (Merck, Germany) for 48 hours under aerobic conditions at 35-37 °C. Spore bacteria were counted on Tryptose Sulfite Cyclocerine Agar (Merck, Germany), which was incubated at 37 °C for 24-48 hours under anaerobic conditions.
The dry matter and fat contents in the cheese samples were measured using the Gerber (ISO 3433, 2008ISO 3433. 2008. Cheese-Determination of fat content-Van Gluik Method. The International Organization for Standardization.) and conventional gravimetric (ISO 5534ISO 5534. 2004. Cheese and processed cheese- Determination of the Total Solid Content (Reference method). The International Organization for Standardization Jakob, 2011.) techniques. Lactic acidity (LA, %) was analyzed according to AOAC (1995AOAC. 1995. Official methods of analysis (16th edn., Vol. II). Arlington, VA, USA: Association of Official Analytical Chemists International.). A pH meter was used to measure the pH levels of the cheese samples (Mettler Toledo). The micro-Kjeldahl method was used to measure the nitrogen content in cheese in terms of total nitrogen (TN), water-soluble nitrogen (WSN), and non-protein nitrogen (NPN). (WSN/TN) x 100 was used to generate ripening index (RI) values. For calculating salt (%), the Mohr method was applied. Six different metrics were used to examine the microbial composition of the cheese samples under the same circumstances as those used for raw milk samples (Halkman and Ayhan, 2000Halkman AK, Ayhan K. 2000. Gıdaların mikrobiyolojik analizi 2. Mikroorganizma sayımı. Gıda Mikrobiyolojisi ve Uygulamaları. Sim Matbaacılık LTD. ŞTİ. 2.basım. 513s., Ankara.).
Texture profile analyses (TPA) were carried out using a Texture Analyzer (TA-XT plus, Stable Micro Systems, Godalming, UK), (Bourne, 1978). Samples were roughly cut into 2.5 cm cubes before analysis, wrapped in airtight plastic wrap to prevent moisture loss, and brought to assay temperature (25 ºC). The test speed was set at 1.5 mm/sec, and each cut sample was compressed and set to 60%. 2.00 mm/sec and 10.0 g were chosen as the pre-test speed and trigger force, respectively. Values from the texture analysis are the average of three replicates.
2.2.2. Free fatty acid composition and volatiles
⌅Using an Agilent GC (model GC 6890N) equipped with a capillary column (300X250mX0.25m, Agilent 19091F-433 HP-FFAP,CA, USA), the free fatty acid composition was evaluated as ppm in GC in accordance with the capillary gas chromatography method recommended by Deeth et al., 1983Deeth HC, Fitz-Gerald CH, Snow AJ. 1983. A gas chromatographic method for the quantitative determination of free fatty acids in milk and milk products. New Zealand J. Dairy Sci. Technol. 18, 13–20.. There are two steps to extraction. First, free fatty acids were extracted (using 0.3-0.5 g sample) along with all other lipids in an acidic hexane/diethyl ether solution. Next, free fatty acids were separated from their triglycerides using neutral alumina chromatography on neutral aluminum oxide. Finally, formic acid in ether was used to remove aluminum oxide from the process. The kind and quantity of free fatty acids in cheese samples were lastly identified by injecting them into the gas chromatography apparatus.
Stashenko and Martinez’s (2007)Stashenko EE, Martínez JR. 2007. Sampling volatile compounds from natural products with headspace/solid-phase micro-extraction. J. Biochem. Biophys. Methods. 70 (2), 235–42. https://doi.org/10.1016/j.jbbm.2006.08.011 description of the static headspace solid phase micro-extraction (SPME) method was used to analyze the volatiles with Agilent GC 7820A gas chromatography-mass spectrometry equipment. For this purpose, 10 ml of sample were kept at 40 °C for 30 minutes in the SPME extraction system. Then the sample was extracted using 50/30 µm Divinylbenzene/ Carboxen/ Polydimethylsiloxane (DVB/CAR/PDMS, Agilent, USA) fiber for another 30 minutes. Desorption was carried out at 250 °C for 10 minutes. The separation was carried out using a DB WAX column (122-7032, Agilent Technologies, USA; 30 m×0.25 mm i.d; 0.25 μm film thickness). Column temperature programme was as folllows: 40 °C (for 5 min) → to 100 °C at 10 °C/min → to 100 °C at 20 °C/min (10 min) NIST/Flavournet library was used to identify the volatile compounds.
Each of the analytical determinations was made in three replicates.
2.2.3. Statistical analyses
⌅One-way analysis of variance (ANOVA) was used to analyze variance and find significant differences. Biochemical and textural data were subjected to an Anova analysis of variance with the following factors: (1) control or lysozyme addition; and (2) storage term. Duncan’s test was used to compare significant means (significance P < 0.05). The number of replicates was three. The Tukey test was used to compare significant means primarily for textural characteristics (significance P < 0.05). Data were examined using SPSS 15.0. (SPSS Inc., Chicago, USA). The 95% confidence interval (p < 0.05) was used to signify statistical significance, which was also expressed in terms of P values.
3. RESULTS AND DISCUSSION
⌅3.1. Gross composition analysis of cheese
⌅Table 1 lists the gross composition of raw cow’s milk. According to the literature (Say and Güzeler, 2008Say D, Güzeler N. 2008. Taze Kaşar peynirlerinin randıman, bileşim ve duyusal özellikleri üzerine haşlama suyunun tuz konsantrasyonunun etkisi.Çukurova Üniversitesi Fen Bilimleri Enstitüsü 19 (3), 30–41.; Göncü et al., 2017Göncü B, Çelikel A, Akın MB, Akın MS. 2017. Şanlıurfa’da satışa sunulan sokak sütlerinin bazı kimyasal ve mikrobiyolojik özelliklerinin belirlenmesi üzerine bir araştırma. Harran Üniversitesi (HU) Muh. Der. 02, 15–23.), the general characteristics of raw milk were consistent.
| Parameters | Raw milk |
|---|---|
| pH | 6.56±0.04 |
| LA (%) | 0.13±0.00 |
| Dry matter (%) | 11.54±0.08 |
| Protein (%) | 3.05±0.02 |
| Fat (%) | 3.36±0.03 |
| Ash (%) | 0.69±0.01 |
| TAMB (log cfu/g) | 6.38±0.01 |
| Coliform (log cfu/g) | 4.44±0.01 |
| E. coli (log cfu/g) | 4.09±0.03 |
| Yeast and mold (log cfu/g) | 5.71±0.14 |
| Sporatic bacteria (log cfu/g) | 2.13±0.04 |
| Lactobacillus (log cfu/g) | 7.21±0.06 |
Table 2 displays the findings from the examination of the gross compositionand microbiological makeup of the cheese when it was stored. The inclusion of lysozyme had a considerable impact on a number of characteristics. The titratable acidity (measured as g of lactic acid per 100 g of cheese) of 1-day-old Kashar cheese ranged from 1.12 to 1.48%. At the same time, other chemical components were measured, including total solids at 60.30 to 66.43%, protein at 22.18 to 23.6%, fat at28.76 to 34.11%, and salt at 1.51 to 2.86%. The titratable acidity, pH, spore bacteria, coliforms, and E. coli contents in the chese were not significantly impacted by the application of various types and forms of lysozyme (P > 0.05). For Kashar cheese, suitable levels of moisture and salt were discovered. The C and MLL in the chese showed the greatest and lowest total solid and protein levels, respectively.
| Samples | Day 1 | Day 30 | Day 60 | Day 90 | |
|---|---|---|---|---|---|
| pH | C | 5.40±0.29Aa | 5.63±0.12Aa | 5.33±0.12Aa | 5.37±0.05Aa |
| ELP | 5.43±0.05Aa | 5.30±0.14Aa | 5.37±0.09Aa | 5.20±0.16Aa | |
| MLP | 5.50±0.24Aa | 5.40±0.14Aa | 5.33±0.09Aa | 5.17±0.05Aa | |
| MLL | 5.37±0.12Aa | 5.30±0.08Aa | 5.43±0.12Aa | 5.27±0.12Aa | |
| Titratable acidity LA % | C | 1.48±0.32Aa | 1.48±0.24Aa | 1.84±0.08Aa | 1.48±0.28Aa |
| ELP | 1.31±0.13Aa | 1.48±0.11Aa | 1.49±0.04Aa | 1.40±0.45Aa | |
| MLP | 1.12±0.02Aa | 1.26±0.08Aa | 1.26±0.15Aa | 1.76±0.11Aa | |
| MLL | 1.21±0.06Aa | 1.41±0.07Aa | 1.32±0.03Aa | 1.53±0.04Aa | |
| Drymatter % | C | 62.19±0.94ABb | 63.77±1.39ABb | 63.08±0.88ABb | 68.52±0.99ABa |
| ELP | 66.43±3.05Ab | 63.82±2.32Ab | 65.98±3.04Ab | 69.82±0.94Aa | |
| MLP | 60.36±2.06Bb | 60.50±1.23Bb | 62.02±1.29Bb | 66.50±1.13Ba | |
| MLL | 60.30±0.91Bb | 62.65±1.65Bb | 63.75±2.57Bb | 66.54±1.79Ba | |
| Ash % | C | 4.22±0.10Ca | 3.91±0.05Ca | 3.91±0.15Ca | 3.66±0.14Ca |
| ELP | 3.93±0.07Ca | 3.99±0.08Ca | 4.08±0.16Ca | 3.99±0.07Ca | |
| MLP | 5.54±0.18Aa | 5.21±0.04Aa | 5.19±0.09Aa | 5.41±0.26Aa | |
| MLL | 4.36±0.24Bb | 4.41±0.07Bb | 4.24±0.16Bb | 4.69±0.68Bb | |
| Fat % | C | 32.80±1.79Bb | 32.93±0.32Bb | 30.81±0.31Bb | 37.23±1.75Ba |
| ELP | 34.11±3.49Ab | 35.45±2.04Ab | 36.52±2.58Ab | 37.77±1.29Aa | |
| MLP | 28.76±2.42Bb | 30.09±1.47Bb | 31.31±1.18Bb | 35.62±1.30Ba | |
| MLL | 31.68±0.89Bb | 32.58±1.76Bb | 32.38±2.68Bb | 34.11±1.47Ba | |
| Protein % | C | 23.66±0.64Ab | 25.33±1.29Aab | 27.69±1.67Aa | 25.01±1.93Aa |
| ELP | 22.69±1.92Bb | 22.56±0.78Bab | 23.15±0.52Ba | 25.73±0.51Ba | |
| MLP | 23.20±1.56Bb | 22.50±0.24Bab | 23.07±0.76Ba | 23.07±1.28Ba | |
| MLL | 22.18±0.21ABb | 23.58±0.27ABab | 25.01±0.63ABa | 25.01±0.96ABa | |
| TN % | C | 3.60±0.22Ab | 3.97±0.20Aab | 4.34±0.26Aa | 3.92±0.30Aa |
| ELP | 3.56±0.30Bb | 3.54±0.12Bab | 3.52±0.07Ba | 4.03±0.08Ba | |
| MLP | 3.64±0.24Bb | 3.53±0.04Bab | 3.62±0.12Ba | 3.62±0.20Ba | |
| MLL | 3.48±0.03ABb | 3.70±0.04ABab | 3.92±0.10ABa | 3.92±0.15ABa | |
| WSN % | C | 0.43±0.02Ac | 0.56±0.04Ab | 0.69±0.07Aa | 0.70±0.08Aa |
| ELP | 0.39±0.02Bc | 0.43±0.04Bb | 0.47±0.06Ba | 0.53±0.06Ba | |
| MLP | 0.34±0.01Cc | 0.36±0.01Cb | 0.38±0.03Ca | 0.42±0.04Ca | |
| MLL | 0.35±0.01 Bc | 0.43±0.03 Bb | 0.52±0.05 Ba | 0.62±0.01 Ba | |
| Ripening index % | C | 12.00±1.10Ac | 14.19±1.67Abc | 16.06±2.07Aab | 17.85±1.24Aa |
| ELP | 10.97±1.32Bc | 12.06±0.91Bbc | 13.36±1.92Bab | 13.24±1.58Ba | |
| MLP | 9.38±0.46Cc | 10.21±0.26Cbc | 10.62±0.98Cab | 11.59±0.43Ca | |
| MLL | 10.07±0.32Bc | 11.73±0.84Bbc | 13.39±1.62Bab | 15.94±0.93Ba | |
| Salt % | C | 1.51±0.44Ca | 1.60±0.13Ca | 1.66±0.16Ca | 1.95±0.29Ca |
| ELP | 1.69±0.12BCa | 1.82±0.24BCa | 2.24±0.06BCa | 2.32±0.12BCa | |
| MLP | 2.86±0.14Aa | 2.70±0.05Aa | 2.44±0.11Aa | 2.40±0.43Aa | |
| MLL | 2.08±0.14ABa | 2.07±0.09ABa | 2.13±0.03ABa | 2.73±0.40ABa | |
| TMAB log cfu/gr | C | 5.01±0.41Aab | 5.71±0.32Ab | 7.00±0.12Aab | 6.52±0.16Aa |
| ELP | 5.01±0.41Aab | 5.28±0.56Ab | 5.87±0.11Aab | 6.29±0.16Aa | |
| MLP | 4.10±0.15Bab | 3.61±0.50Bb | 3.99±0.74Bab | 4.46±0.24Ba | |
| MLL | 5.58±0.04Bab | 3.95±0.46Bb | 4.66±0.33Bab | 4.89±1.13Ba | |
| Coliform cfu | C | 4.20±0.48Ab | 1.81±0.58Ac | 3.55±0.11Aab | 4.23±0.32Aa |
| ELP | 3.98±0.15Ab | 1.59±0.30Ac | 3.67±0.11Aab | 4.28±0.49Aa | |
| MLP | 3.28±0.20Ab | 2.13±0.40Ac | 4.11±0.31Aab | 4.70±0.07Aa | |
| MLL | 3.58±0.08Ab | 2.14±0.20Ac | 4.17±0.43Aab | 4.04±0.22Aa | |
| Total yeast and mold count (TYMC) cfu | C | 2.00±0.00Ab | 2.61±0.32Aa | 2.87±0.26Aa | 2.85±0.21Aa |
| ELP | 0.87±1.23ABb | 2.68±0.08ABa | 2.77±0.07ABa | 2.62±0.23ABa | |
| MLP | 0.00±0.00Cb | 1.63±0.05Ca | 2.37±0.30Ca | 1.90±0.47Ca | |
| MLL | 0.43±0.61BCb | 2.42±0.19BCa | 2.69±0.17BCa | 2.52±0.20BCa | |
| Lactobacillus cfu | C | 6.65±0.12Ac | 6.48±0.11Abc | 7.11±0.07Ab | 7.25±0.12Aa |
| ELP | 4.83±0.28Ac | 6.01±0.09Abc | 6.91±0.25Ab | 7.27±0.03Aa | |
| MLP | 3.67±0.14Cc | 3.83±0.12Cbc | 3.77±0.53Cb | 6.03±1.07Ca | |
| MLL | 4.33±0.23Bc | 4.68±0.21Bbc | 5.44±0.39Bb | 6.24±0.65Ba | |
| Sporadic microorganism cfu | C | ˂1 | ˂1 | ˂1 | ˂1 |
| ELP | ˂1 | ˂1 | ˂1 | ˂1 | |
| MLP | ˂1 | ˂1 | ˂1 | ˂1 | |
| MLL | ˂1 | ˂1 | ˂1 | ˂1 | |
| E. coli cfu | C | ˂1 | ˂1 | ˂1 | ˂1 |
| ELP | ˂1 | ˂1 | ˂1 | ˂1 | |
| MLP | ˂1 | ˂1 | ˂1 | ˂1 | |
| MLL | ˂1 | ˂1 | ˂1 | ˂1 |
*C:
Control, ELP: cheese treated with eggwhite lysozyme, MLP: cheese
treated with microbial lysozyme powder, MLL: cheese treated with
microbial lysozyme liquid
The Duncan test was used to compare significant
means. Different lowercase superscript letters in the same row indicate
significant differences during ripening (P < 0.05); different
uppercase superscript letters in the same column indicate significant
differences among sample groups (P < 0.05).
The microbiological quality of the cheese affects how pH and titratable acidity grow. Cheese-treated bacterial lysozyme (MLL, MLP) revealed lower initial microbial counts in TMAB, yeast, mold, and coliform levels. The addition of bacteria which produce lysozyme caused the pH to drop and the acidity to rise, which in turn prevented the growth of coliform bacteria, yeast, and TMAB. The pH value did not change as much since there were fewer living bacteria, which led to less CO2 being produced (Al Baarri et al., 2018Al-Baarri ANM, Legowo AM, Arum SK, Hayakawa S. 2018. Extending shelf life of Indonesian soft milk cheese (dangke) by lactoperoxidase system and lysozyme. Int. J. Food Sci. ID 4305395, 1–7. https://doi.org/10.1155/2018/4305395). In essence, the cheese treated with bacterial lysozyme had a lower bacteria count than the control cheese, which was proven to be a significant difference (MLP and MLL). This circumstance demonstrates the antimicrobial effectiveness of lysozyme in Kashar cheese. By dissolving the structural elements on the cell walls of bacteria and fungi, lysozyme is known to exert its antimicrobial effect against these microorganisms as well as viruses and protozoa (Al Baarri et al., 2018Al-Baarri ANM, Legowo AM, Arum SK, Hayakawa S. 2018. Extending shelf life of Indonesian soft milk cheese (dangke) by lactoperoxidase system and lysozyme. Int. J. Food Sci. ID 4305395, 1–7. https://doi.org/10.1155/2018/4305395). However, the bacterial lysozyme treatment substantially reduced the lactic acid bacteria count in the cheese. The lowest lactic acid bacteria counts were seen in MLP and MLL in the current study at both the beginning and end of ripening. This might be because high salt concentration inhibits LAB (MLP: 2.86-2.40% and MLL: 2.08-2.73%), and lysozyme has a substantial inhibitory effect on all lactic acid bacteria (Silvetti et al., 2017Silvetti T, Morandi S, Hintersteiner M, Brasca M. 2017. Egg innovations and strategies for improvements. 233–241. Ed: P. Hester. http://dx.doi.org/10.1016/B978-0-12-800879-9.). Badem and Uçar (2016)Badem A, Ucar G. 2016. Changes in chemical and microbiological properties of Kashar Cheese produced without starter culture during ripening. Euras. J. Veter. Sci. 32, 3, 188–192 DOI: 10.15312/EurasianJVetSci.2016318399 discovered, in contrast to our findings, that LAB activity was higher at the start of ripening, and the LAB number dropped on the 90th day of Kashar ripening since lysozyme was not used in the study.
Very comparable trends were seen in the fat content of the cheese, which is consistent with the structural impact of egg white lysozyme. When ELP was ripening, the maximum fat percentage was detected (34.11% and 37.77%, respectively). Our results were better than those of previous researchers who studied Kashar cheese (Eroğlu et al., 2016Eroglu, A., Toker, O. S., Dogan, M. 2016. Changes in the texture, physicochemical properties and volatile compound profiles of fresh Kashar cheese (< 90 days) during ripening. Internat. J. Dairy Technol. 69, 243–253.; Ozturkoglu-Budak et al., 2021Ozturkoğlu-Budak S, Akal HC, Bereli N, Cimen D, Akgonullu S. 2021. Use of antimicrobial proteins of donkey milk as preservative agents in Kashar cheese production. Internat. Dairy J. 120, 105090. https://doi.org/10.1016/j.idairyj.2021.105090; Yalman et al., 2017Yalman M, Güneşer O, Karagül Yüceer Y. 2017. Evaluation of some physical, chemical and sensory properties of kasar cheese and its processed and analogue types. J. Agric. Sci. 23, 63–75.). The composition of the cheese milk and the cheese-making process may be the cause of the variations in the dry matter and fat contents of the Kashar cheese samples.
The cheese (MLP and MLL) treated with bacterial lysozyme showed the highest salt level at 1 and 90 days, while the control had the lowest salt content. The measured values are in line with previous studies, which reported that Kashar cheese has a salt level of 1.16% (Yalman et al., 2017Yalman M, Güneşer O, Karagül Yüceer Y. 2017. Evaluation of some physical, chemical and sensory properties of kasar cheese and its processed and analogue types. J. Agric. Sci. 23, 63–75.) and 1.22-1.37% (Badem and Uçar, 2016Badem A, Ucar G. 2016. Changes in chemical and microbiological properties of Kashar Cheese produced without starter culture during ripening. Euras. J. Veter. Sci. 32, 3, 188–192 DOI: 10.15312/EurasianJVetSci.2016318399).
The highest protein contents were found in the control cheese (23.66% on day 1, 27.69% on day 60), which showed a drop in protein contents with lysozyme treatments and an increase in protein contents throughout ripening (p < 0.05). The protein contents in the cheese showed slight changes as they ripened. As a result of our investigation, it is believed that lysozyme’s inhibitory action on microorganisms such as TMAB, coliforms, yeast, and mold, as well as the inhibitory effect of salt on LAB ,is to blame for this condition. These findings were in line with a previous study using four distinct types of Kashar samples that had matured for 90 days (Eroğlu et al., 2015Eroglu A, Dogan M, Toker OS, Yilmaz MT. 2015. Classification of Kashar cheeses based on their chemical, color and instrumental textural characteristics using principal component and hierarchical cluster analysis. Int. J. Food Propert. 18, 909–921.).
In terms of the lysozyme treatment, these values were considerably higher in C and lower in the cheese treated with bacterial lysozyme (MLL and MPP) at the start and end of ripening, respectively (p < 0.005). This outcome appears to be connected to lysozyme’s inhibitory effect and reduced levels of LAB and salt in MLL and MPP. In comparison to treated cheese, the control cheese may have had a higher proteolysis level and RI value due to a higher microbial count (Ozer and Kesenkas, 2019Ozer E, Kesenkas H. 2019. The effect of using different starter culture combinations on ripening parameters, microbiological and sensory properties of Mihalic cheese. J. Food Sci. Technol. 56, 1202-1211. doi: 10.1007/s13197-019-03583-2). All samples underwent the anticipated formation of WSN and TCA-SN,a rise in RI during ripening, and other authors (Sulejmani and Hayaloglu, 2016Sulejmani E, Hayaloglu AA. 2016. Influence of curd heating on proteolysis and volatiles of Kashkaval cheese. Food Chem. 211, 160–170. DOI: 10.1016/j.foodchem.2016.05.054; Ozturkoglu-Budak et al., 2021Ozturkoğlu-Budak S, Akal HC, Bereli N, Cimen D, Akgonullu S. 2021. Use of antimicrobial proteins of donkey milk as preservative agents in Kashar cheese production. Internat. Dairy J. 120, 105090. https://doi.org/10.1016/j.idairyj.2021.105090) noted the same trend for Kashar cheese.
In relation to the textural analysis, Table 3 provides changes in the textural characteristics of the cheese as it ripens. Overall, the results show that storage and lysozyme treatment considerably changed the textural characteristics of kashar cheese (P 0.05). The highest hardness and cohesiveness values were found in cheese that had been treated with microbial powder lysozyme (MLP). However, the lowest springiness, gumminess, and resilience values were also found. At the end of storage, a similar tendency was seen in all the cheese.
| Samples | Day 1 | Day 90 | |
|---|---|---|---|
| Hardness (g) | C | 5575.55± 308.99ABa | 5647.45± 840.17ABb |
| ELP | 4232.1± 381.6 Ca | 1468.52±199.47Cb | |
| MLP | 6405.08±843.52 Aa | 6977.55±404.97Ab | |
| MLL | 6100.56±385.76 Ba | 3153.11±509.48Bb | |
| Adhesiveness (g s) | C | -64.11± 18.11Ba | -124.79±15.01Bb |
| ELP | -52.3 ± 6.63ABa | -63.28±34.52ABb | |
| MLP | -23.23 ±17.75Aa | -13.83±9.67Ab | |
| MLL | -9.97±7.12ABa | -121.57±46.96ABb | |
| Springiness (cm) | C | 0.66±0.03Ba | 0.55±0.02Bb |
| ELP | 0.77±0.02Aa | 0.62±0.02Ab | |
| MLP | 0.33±0.07Ca | 0.34±0.03Ca | |
| MLL | 0.76±0.04ABa | 0.55±0.03ABb | |
| Cohesiveness | C | 0.49±0.04Ba | 0.42±0.01Ba |
| ELP | 0.53 ± 0.05Aa | 0.55 ±0.02Aa | |
| MLP | 0.86±0.01Aa | 0.57±0.03Ab | |
| MLL | 0.37 ±0.03Ba | 0.46±0.02Ba | |
| Gumminess (g) | C | 2744.98±192.18Aa | 2338.79 ±394.73Ab |
| ELP | 2219.61±280.15Ba | 817.31±141.13Bb | |
| MLP | 2097.34±400.66Aa | 1355.61±93.66Ab | |
| MLL | 2303.34±323.83ABa | 1464.54±235.72ABb | |
| Chewiness (g cm) | C | 1820.11±106.84Aa | 1275.93±214.31Ab |
| ELP | 1705.92±233.53Ba | 504.58±75.07Bb | |
| MLP | 2097.34±400.66Aa | 1355.61±93.66Ab | |
| MLL | 1747.57± 321.14ABa | 806.68±97.59ABb | |
| Resilience | C | 0.19±0.02Ba | 0.15±0.01Bb |
| ELP | 0.26±0.03Aa | 0.23±0.03Ab | |
| MLP | 0.15±0.01Ca | 0.13±0.02Cb | |
| MLL | 0.22±0.02Ba | 0.17±0.01Bb |
C:
Control. ELP: cheese treated with eggwhite lysozyme, MLP: cheese
treated with microbial lysozyme powder, MLL: cheese treated with
microbial lysozyme liquid
The Tukey test was used to compare significant
means. Different lowercase superscript letters in the same row indicate
significant differences during ripening (P < 0.05); different
uppercase superscript letters in the same column indicate significant
differences among sample groups (P < 0.05).
3.2. Free fatty acids and volatiles in cheese
⌅Table 4 contains the FFA profiles of the cheese after 90 days of ripening. The results show that short-chain fatty acids (C4 to C10, SCFA), medium-chain fatty acids (C12 and C14, MCFA), and long-chain fatty acids (C16, C18, and C18:1, LCFA) were all significantly affected by the lysozym treatment of Kashar cheese, with the exception of butyric acid (C4:0), myristic acid (C14:0), and palmitic acid (C16:0). All samples had significantly greater levels of LCFA overall than short- and medium-chain free fatty acids (MCFA an SCFA). Linoleic acid (C18:1), palmitic acid (C16), and butyric acid (C4) were the three free fatty acids found in the highest concentrations, respectively. These outcomes might be explained by lysozym’s inhibiting impact on the cheese’s microbial population. Cheese treated with egg white lysozym (ELP) had much higher levels of the enzyme than cheeses treated with bacterial lysozym (MLP, MLL). This observed value is consistent with the fat % result presented in Table 2, where the highest ELP was discovered (34.11% on day 1 and 37.77% on day 90). This conclusion is consistent with that of Urbienė and Sasnauskait (2010)Urbienė S, Sasnauskaitė L. 2010. Influence of lysozyme on the quality of cottage cheese during storage Zemės ūkio mokslai. T. 17 (1–2), 60–67., who studied the effects of 0.01% lysozyme in 18-day-old cottage cheese. As can be seen in Table 4, there were generally no appreciable variations in the FFA profiles of the samples during ripening; the same pattern was noted in all the cheese. Güler (2005)Güler Z, 2005. Quantification of free fatty acids and flavor characteristics of Kasar cheeses. J. Food Lipids 12, 209–221. https://doi.org/10.1111/j.1745-4522.2005.00018.x claims that when Kashar cheese’s short-chain FFA concentrations were high, flavor intensity increased, but flavor quality was inconsistent. Despite the fact that palmitic, stearic, and oleic acids were the most prevalent FFAs in Kashar cheese, they did not add as much to the cheese’s flavor and aroma as short- and medium-chain fatty acids, which both had very high perception threshold values and a less distinctive flavor.
| Samples | Day 1 | Day 30 | Day 60 | Day 90 | |
|---|---|---|---|---|---|
| C4 Butyric acid |
C | 370.98±66.65Aa | 336.27±31.78Aa | 296.61±38.62Aa | 387.29±24.25Aa |
| ELP | 182.09±17.60Aa | 365.64±82.46Aa | 456.12±99.39Aa | 465.68±153.61Aa | |
| MLP | 283.22±106.12Aa | 347.77±33.34Aa | 247.15±48.68Aa | 351.63±26.46Aa | |
| MLL | 347.35±68.12Aa | 312.99±45.37Aa | 410.40±132.89Aa | 361.02±50.86Aa | |
| C6 Caproic acid |
C | 28.28±11.93ABa | 33.71±3.42ABa | 41.95±1.34ABa | 50.88±11.81ABa |
| ELP | 55.72±17.81Aa | 29.85±5.81Aa | 43.78±10.59Aa | 39.60±2.04Aa | |
| MLP | 14.75±1.99Ca | 27.41±9.51Ca | 19.32±1.61Ca | 24.29±2.94Ca | |
| MLL | 16.01±4.06BCa | 27.50±4.06BCa | 31.07±1.18BCa | 39.27±2.44BCa | |
| C8 Caprylic acid |
C | 11.64±2.19Ab | 13.60±0.77Ab | 26.63±0.62Aa | 31.60±1.21Aa |
| ELP | 10.44±1.27ABb | 11.34±3.01ABb | 23.93±11.62ABa | 19.89±0.87ABa | |
| MLP | 10.51±2.26Bb | 11.01±0.25Bb | 17.22±1.68Ba | 20.79±0.82Ba | |
| MLL | 9.05±2.74ABb | 11.15±0.32ABb | 23.50±0.55ABa | 24.83±0.29ABa | |
| C10 Capric acid |
C | 26.09±7.65Ab | 31.80±1.83Ab | 48.75±1.39Aa | 61.86±1.44Aa |
| ELP | 13.00±0.80Bb | 22.73±3.94Bb | 44.78±22.98Ba | 33.65±2.18Ba | |
| MLP | 22.79±2.81Bb | 22.99±2.10Bb | 35.21±4.28Ba | 41.32±3.18Ba | |
| MLL | 15.83±9.37ABb | 26.74±1.55ABb | 44.37±0.51ABa | 48.69±0.83ABa | |
| C12 Lauric acid |
C | 43.44±13.23Ab | 47.53±4.26Ab | 59.88±1.37Aa | 77.76±0.79Aa |
| ELP | 31.54±6.67Bb | 38.91±5.18Bb | 59.53±26.15Ba | 43.59±2.99Ba | |
| MLP | 30.09±7.14Bb | 40.49±0.60Bb | 47.56±5.76Ba | 56.35±5.38Ba | |
| MLL | 26.77±11.06ABb | 44.06±0.23ABb | 55.80±1.98ABa | 63.66±1.26ABa | |
| C14 Myristic acid |
C | 143.54±50.54Aa | 134.53±19.03Aa | 149.41±6.44Aa | 198.97±3.41Aa |
| ELP | 199.28±88.85Aa | 124.77±19.41Aa | 155.81±59.72Aa | 111.11±9.54Aa | |
| MLP | 88.27±11.32Aa | 130.89±12.56Aa | 119.35±14.36Aa | 147.02±15.70Aa | |
| MLL | 85.58±17.84Aa | 125.97±6.87Aa | 139.96±7.90Aa | 165.16±3.24Aa | |
| C16 Palmitic acid |
C | 609.42±242.27Aa | 405.50±52.42Aa | 411.54±10.26Aa | 525.01±2.99Aa |
| ELP | 495.66±149.39Aa | 481.06±145.21Aa | 454.23±140.49Aa | 323.45±32.85Aa | |
| MLP | 287.48±17.83Aa | 391.92±24.98Aa | 373.83±44.37Aa | 434.91±45.82Aa | |
| MLL | 304.02±48.61Aa | 387.46±10.78Aa | 394.80±26.47Aa | 460.31±4.37Aa | |
| C18 Stearic acid |
C | 283.28±103.26Aa | 117.23±9.47Aa | 132.50±3.17Aa | 144.31±8.51Aa |
| ELP | 108.10±11.35Aa | 175.90±64.78Aa | 139.80±27.32Aa | 106.02±11.98Aa | |
| MLP | 101.45±6.33Aa | 112.44±2.03Aa | 121.42±12.50Aa | 133.81±13.39Aa | |
| MLL | 122.77±19.21Aa | 120.97±3.07Aa | 120.50±6.10Aa | 135.08±3.39Aa | |
| C18:1 Oleic acid |
C | 861.59±228.62Aa | 477.06±76.89Aa | 457.11±1.99Aa | 549.39±27.60Aa |
| ELP | 499.64±168.03ABa | 587.75±189.12ABa | 495.92±114.50ABa | 367.88±44.06ABa | |
| MLP | 310.77±9.49Ba | 412.03±25.38Ba | 406.98±42.07Ba | 478.54±49.42Ba | |
| MLL | 376.31±63.09ABa | 425.99±13.78ABa | 426.20±23.62ABa | 501.86±10.50ABa | |
| C18:2 Linoleic acid |
C | 221.64±72.97Aa | 78.37±7.17Aa | 80.72±3.62Aa | 81.92±3.92Aa |
| ELP | 74.98±13.85ABa | 119.59±48.45ABa | 79.19±9.10ABa | 59.21±4.56ABa | |
| MLP | 66.77±5.66Ba | 69.41±1.39Ba | 64.81±5.32Ba | 70.85±5.60Ba | |
| MLL | 85.81±15.47ABa | 74.35±1.53ABa | 68.57±2.52ABa | 75.44±1.59ABa |
C:
Control, ELP: cheese treated with eggwhite lysozyme, MLP: cheese
treated with microbial lysozyme powder, MLL: cheese treated with
microbial lysozyme liquid
Duncan test was used to compare significant
means. Different lowercase superscript letters in the same row indicate
significant differences during ripening (P < 0.05); different
uppercase superscript letters in the same column indicate significant
differences among sample groups (P < 0.05).
In that order, butyric acid, caproic, capric, and caprylic acids were the most prevalent short-chain fatty acids (FFA). Although the pattern was not continuous in every instance, the levels of each unique SCFA significantly increased during the course of the storage period and decreased with lysozym treatment (p < 0.05). At the end of ripening, the control (C) cheese had the greatest FFA concentrations. These ratios were C4 (387.29), C6 (50.88), C8 (31.60), and C10 (61.86), respectively. This outcome can be explained by the increased proliferation of lactobacilli and yeast during storage (as noted in Table 2). The breakdown of amino acids and the lipolysis of milk fat may produce fatty acids with four or more carbons (Urbach, 1993Urbach G. 1993. Relations between cheese flavour and chemical composition. Internat. Dairy J. 3, 389–422.). For Kashar cheese and Muenster-type cheese, De Leon-Gonzalez et al. (2000)De Leon-Gonzalez LP, Wendorff WL, Ingham BH, Jaeggi JJ, Houck KB. 2000. Influence of salting procedure on the composition of muenster-type cheese. J. Dairy Sci. 83, 1396–1401. https://doi.org/10.3168/jds.S0022-0302(00)75008-9. and Temiz et al. (2010)Temiz A, 2010. Effect of modified atmosphere packaging on characteristics of sliced Kashar cheese. J. Food Process. Preserv. 34, 926–943. https://doi.org/10.1111/j.1745-4549.2009.00431.x reported that the concentration of FFAs, namely, C4, C8, and C10 acids, considerably rose with the storage period.
Initial lauric acid (C12 ratio) and myristic acid (C14 ratio) levels for medium-chain fatty acids (C12 and C14, MCFA) were 43.44-26.77% and 199.28 -85.58%, respectively. The control cheese finished ripening with higher levels of C12 and C14 than other cheese (MLP, MLL, ELP). According to these data, lauric acid (C12) was strongly affected by the treatments (lysozym and storage duration) in MCFA (p 0.05). According to Temiz (2010)Temiz A, 2010. Effect of modified atmosphere packaging on characteristics of sliced Kashar cheese. J. Food Process. Preserv. 34, 926–943. https://doi.org/10.1111/j.1745-4549.2009.00431.x the FFAs in this situation might have hydrolyzed into specific molecules such as methyl ketones, alcohols, lactones, aldehydes, etc..
Long-chain fatty acids (C16, C18, and C18:1) declined significantly during ripening in the current study, and the cheese treated with lysozyme likewise displayed lower values than the control cheese at both the beginning and the end of the ripening. All the cheese included long-chain fatty acids, but oleic (C18:1), palmitic (C16:0), and stearic (C18:0) acids predominated. Furthermore, different lysozyme treatments had no appreciable impact on the samples of Kashar cheese’s palmitic acid (C16:0) or stearic acid (C18:0) (p > 0.05). However, lysozyme caused a decrease in oleic acid (C18:1) and linoleic acid (C18:2) (p < 0.05). Because of this, the cheese treated with bacterial lysozym (MLP) had the lowest amount of oleic acid (C18:1) and linoleic acid (C18:2) at the start of storage (861.59 and 221.64), while cheese under the control (C) conditions had the largest amount of these acids (310.77 and 66.77). After 90 days of storage, the amounts of palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) acids in all Kashar cheese dropped. When compared to Kashar cheese which had been treated with lysozyme at the beginning and final stages of storage, the control sample (C) had the highest LCFA values. During ripening, longer free fatty-acid chains are broken down into shorter ones, and methyl ketones are produced, which may be the cause of the shift in fatty acid composition (Caglar and Cakmakci 1998Caglar A, Cakmakci S. 1998. Use of protease and lipase enzymes by different methods to accelerate Kashar cheese ripening. Gıda, 23, 291– 301.).Temiz et al. (2010)Temiz A, 2010. Effect of modified atmosphere packaging on characteristics of sliced Kashar cheese. J. Food Process. Preserv. 34, 926–943. https://doi.org/10.1111/j.1745-4549.2009.00431.x found similar findings for Kashar cheese that had matured for 120 days; while Güler (2005)Güler Z, 2005. Quantification of free fatty acids and flavor characteristics of Kasar cheeses. J. Food Lipids 12, 209–221. https://doi.org/10.1111/j.1745-4522.2005.00018.x reported similar findings for Kashar cheese that had been in the market for at least three months.
Tables 5a and 5b display the volatile composition range of the Kashar cheese samples at the start and the completion of the ripening process. 37 distinct volatile chemicals were identified in all, including acids, alcohols, esters, terpenes, and hydrocarbons. Kashar cheese included 10 hydrocarbons, 7 terpenes, 6 alcohols, 7 esters, 7 terpenes, and 7 acids, among their volatile constituents. Other researchers have previously examined the volatile compounds in Kashar cheese (Eroğlu et al., 2016Eroglu, A., Toker, O. S., Dogan, M. 2016. Changes in the texture, physicochemical properties and volatile compound profiles of fresh Kashar cheese (< 90 days) during ripening. Internat. J. Dairy Technol. 69, 243–253.; Sulejmani and Hayaloğlu, 2016Sulejmani E, Hayaloglu AA. 2016. Influence of curd heating on proteolysis and volatiles of Kashkaval cheese. Food Chem. 211, 160–170. DOI: 10.1016/j.foodchem.2016.05.054). However, the lysozyme treatment of Kashar cheese has not been examined. Therefore, the present study’s results may be considered valuable information for future studies of this sort of processed dairy product. The type of lysozyme treatment and cheese age impacted some chemicals, but not all of them in a statistically significant way.
| Compound | Day 1 | Day 30 | ||||||
|---|---|---|---|---|---|---|---|---|
| C | ELP | MLP | MLL | C | ELP | MLP | MLL | |
| Acids | ||||||||
| Hexanoic acid | 1.27±1.53Aa | 4.22±4.60Aa | 2.24±0.29Aa | 2.58±0.64Aa | 2.25±1.10Aa | 1.20±0.35Aa | 0.93±0.26Aa | 3.55±0.98Aa |
| Octanoic acid | 2.70±1.77Aa | 4.95±6.22Aa | 1.09±0.14Aa | 1.51±0.17Aa | 0.79±0.35Aa | 0.66±0.15Aa | 0.50±0.15Aa | 1.64±0.37Aa |
| n-Decanoic acid | 2.74±1.72Aa | 4.55±5.88Aa | 0.71±0.08Aa | 1.08±0.37Aa | 0.43±0.17Ab | 0.41±0.09Ab | 0.33±0.11Ab | 0.77±0.40Ab |
| Tetradecanoic acid | NA | 0.84±7.66Aa | NA | NA | NA | NA | NA | NA |
| Propanoic acid, 2-methyl- | NA | NA | NA | NA | 0.07±0.10 | NA | NA | NA |
| Butanoic acid | NA | 0.42±0.40Ab | 1.38±0.04Ab | 2.44±0.34Ab | 2.67±0.75Aab | 0.98±0.29Aab | 1.19±0.46Aab | 3.45±0.56Aab |
| Butanoic acid, 3-methyl- | NA | NA | NA | NA | 1.53±1.23 | NA | NA | NA |
| Alcohols | ||||||||
| 1-Hexadecanol | NA | 1.90±1.90 | NA | NA | NA | NA | NA | NA |
| 1-Butanol, 3-methyl- | NA | NA | NA | NA | 3.73±1.38Ab | 0.46±0.46Aa | NA | 1.80±0.73Aa |
| Linalool | NA | NA | NA | NA | NA | NA | 0.08±0.11Abc | NA |
| Benzylalcohol | NA | NA | NA | NA | NA | NA | NA | 0.16±0.00Aa |
| PhenylethylAlcohol | NA | NA | NA | 0.50±0.10ABb | 10.09±2.02Aa | 0.66±0.15Ba | 2.52±0.74Ba | 3.37±0.86ABa |
| Silanediol, dimethyl- | 6.54±4.53Aa | 10.65±14.46Aa | 1.05±0.05Aa | 1.02±0.74Aa | 0.27±0.04Aa | 0.34±0.05Aa | 0.34±0.06Aa | 0.30±0.04Aa |
| Esters | ||||||||
| Arsenousacid. tris (trimethylsilyl) ester | NA | NA | 0.43±0.61Aa | 0.92±0.00Aa | NA | NA | 0.33±0.00Aa | 0.53±0.00Aa |
| Decanoicacid, methyl ester | NA | NA | NA | NA | 1.54±0.88A | 0.57±0.13A | 0.35±0.20A | 2.01±1.17A |
| Decanoicacid, ethyl ester | NA | NA | NA | NA | NA | NA | NA | NA |
| Hexanoicacid, methyl ester | NA | NA | NA | NA | 0.44±0.35a | NA | NA | NA |
| Hexanoicacid, ethyl ester | NA | NA | NA | NA | 0.10±0.14Aa | NA | NA | NA |
| Octanoicacid, ethyl ester | NA | NA | NA | NA | 0.09±0.12Aa | NA | NA | NA |
| Octanoicacid, methyl ester | NA | 0.13±0.19Bb | 0.19±0.26Bb | 1.09±0.35Ab | 3.05±1.29Ba | 1.75±0.38Ba | 2.09±0.90Ba | 5.05±0.57Aa |
| Methyltetradecanoate | NA | NA | NA | NA | NA | NA | NA | NA |
| Terpenes | ||||||||
| p-Xylene | 13.51±19.10Aa | 8.89±8.89Aa | 1.56±1.32Aa | NA | 2.05±2.23Aa | 2.85±2.41Aa | 3.81±3.05Aa | NA |
| o-Xylene | 0.57±0.80Aa | NA | NA | NA | 0.49±0.42Aa | NA | NA | NA |
| D-Limonene | 12.86±8.19Ac | 10.44±14.77Ac | 22.60±18.69Ac | 28.66±13.19Ac | 27.06±7.86Ab | 24.47±3.97Ab | 56.21±14.86Ab | 42.35±17.91Ab |
| p-Cymene | NA | NA | 0.38±0.54Aa | 1.22±0.00Aa | NA | 0.39±0.55Aa | 1.07±0.77Aa | 1.23±0.06Aa |
| β-Pinene | NA | NA | NA | NA | 0.51±0.36Ba | 0.55±0.39Ba | 1.35±0.36Aa | 0.74±0.11Aa |
| γ-Terpinene | NA | 0.57±0.40ABd | 0.85±0.06Ad | 0.75±0.00Bd | 1.14±0.01Cb | 1.40±0.10Ab | 1.69±0.23Ab | 1.14±0.09Bb |
| o-Cymene | NA | 0.69±0.49Aa | NA | NA | 0.90±0.63Aa | 0.65±0.65Aa | NA | NA |
| Hydrocarbons | ||||||||
| Ethylbenzene | 4.60±6.50Aa | 1.69±1.69Aa | 0.41±0.58Aa | NA | 0.69±0.49Aa | NA | 0.72±1.01Aa | 0.97±0.69Aa |
| 1.3 dimethyl-benzene | 5.13±3.67A | 7.23±9.36A | 4.29±2.85A | 3.42±2.43A | 2.01±1.94A | 3.08±2.52A | 5.81±2.19A | 2.40±2.22A |
| Bicyclo[3.1.0]hexane. 4-methylene-1-(1-methylethyl)- | NA | NA | 0.16±0.22Ab | NA | NA | NA | 0.43±0.43Ab | NA |
| Oxime-. methoxy-phenyl-_ | 3.91±5.27Aa | 6.64±8.59Aa | 1.79±0.52Aa | 2.05±0.50Aa | 0.53±0.07Aa | 1.13±0.89Aa | 0.86±0.47Aa | 0.59±0.08Aa |
| Styrene | NA | NA | NA | NA | 0.39±0.08Aa | NA | 0.24±0.18Aa | 0.43±0.00Aa |
| 3.3-Dimethyl-1,2-epoxybutane | NA | NA | NA | 3.25±0.00Aa | NA | NA | NA | NA |
| Cyclododecane | NA | 1.37±8.47Aa | NA | NA | NA | NA | NA | NA |
| Cyclotetradecane | NA | 1.37±8.47Aa | NA | NA | NA | NA | NA | NA |
| Benzene. 1,2-dichloro | NA | 0.91±0.67a | NA | NA | 0.99±0.70a | 2.33±0.08a | NA | NA |
| Benzene. 1,3-dichloro | NA | NA | NA | NA | NA | NA | 1.41±1.07A | 2.05±0.51A |
*C: Control. ELP: cheese treated with eggwhite lysozyme. MLP: cheese treated with microbial lysozyme powder. MLL: cheese treated with microbial lysozyme liquid. Duncan’s test was used to compare significant means. Different lowercase superscript letters in the same row indicate significant differences during ripening (P < 0.05); different uppercase superscript letters in the same column indicate significant differences among sample groups (P < 0.05).
| Compound | Day 60 | Day 90 | ||||||
|---|---|---|---|---|---|---|---|---|
| C | ELP | MLP | MLL | C | ELP | MLP | MLL | |
| Acids | ||||||||
| Hexanoic acid | 2.76±0.46Aa | 0.84±0.13Aa | 1.18±0.07Aa | 1.40±0.07Aa | 5.03±2.10Aa | 6.76±7.32Aa | 2.52±0.87Aa | 2.40±0.57Aa |
| Octanoic acid | 1.07±0.66Aa | 0.51±0.07Aa | 0.38±0.27Aa | 0.77±0.01Aa | 1.91±0.75Aa | 2.41±2.36Aa | 1.03±0.29Aa | 0.96±0.19Aa |
| n-Decanoic acid | 0.52±0.01Ab | 0.17±0.01Ab | 0.13±0.10Ab | 0.22±0.02Ab | 0.62±0.31Ab | 1.33±1.33Ab | 0.34±0.15Ab | 0.37±0.10Ab |
| Tetradecanoic acid | NA | NA | NA | NA | NA | NA | NA | NA |
| Propanoic acid. 2-methyl- | NA | NA | NA | NA | NA | NA | NA | NA |
| Butanoic acid | 2.93±0.80Ab | 0.45±0.07Ab | 0.76±0.13Ab | 0.96±0.03Ab | 5.07±2.67Aa | 4.58±5.08Aa | 1.72±0.65Aa | 2.42±0.75Aa |
| Butanoic acid. 3-methyl- | NA | NA | NA | NA | NA | NA | NA | NA |
| Alcohols | ||||||||
| 1-Hexadecanol | NA | NA | NA | NA | NA | NA | NA | NA |
| 1-Butanol. 3-methyl- | NA | NA | 0.15±0.21Ab | NA | 0.63±0.63Aab | NA | 0.55±0.78Aab | 0.91±0.00Aab |
| Linalool | NA | NA | 0.36±0.13Aa | 0.28±0.05Aa | NA | NA | 0.21±0.16Aab | 0.29±0.00Aab |
| Benzylalcohol | NA | 0.05±0.04Ba | 0.02±0.03Ba | 0.12±0.00Aa | NA | NA | NA | 0.23±0.00Aa |
| Phenylethyl Alcohol | 0.80±0.13Ab | 0.11±0.03Bb | 0.24±0.11Bb | 1.24±0.15ABb | 0.57±0.17Ab | 0.82±0.82Bb | 0.48±0.20Bb | 0.54±0.22ABb |
| Silanediol. dimethyl- | 0.68±0.10Aa | 0.35±0.10Aa | 0.36±0.07Aa | 0.40±0.08Aa | 1.09±0.34Aa | 3.15±3.20Aa | 0.79±0.44Aa | 1.22±0.67Aa |
| Esters | ||||||||
| Arsenousacid. tris (trimethylsilyl) ester | NA | NA | 0.17±0.00Aa | 0.30±0.00Aa | NA | NA | 0.42±0.13Aa | 0.25±0.00Aa |
| Decanoic acid. methyl ester | NA | NA | NA | NA | NA | NA | NA | NA |
| Decanoic acid. ethyl ester | 0.06±0.08 | NA | NA | NA | NA | NA | NA | NA |
| Hexanoic acid. methyl ester | NA | 0.16±0.00Aa | NA | NA | NA | NA | NA | NA |
| Hexanoic acid. ethyl ester | 0.48±0.11a | NA | NA | NA | 0.96±0.62a | NA | NA | NA |
| Octanoic acid. ethyl ester | 0.29±0.26Aa | NA | NA | NA | 0.08±0.08Aa | NA | NA | NA |
| Octanoicacid. methyl ester | 0.49±0.11Bb | NA | 0.14±0.19Bb | 0.67±0.38Ab | NA | 0.10±0.10Bb | 0.22±0.19Bb | 0.22±0.00Ab |
| Methyltetradecanoate | NA | NA | NA | NA | NA | NA | NA | NA |
| Terpenes | ||||||||
| p-Xylene | 9.29±3.47Aa | 6.51±5.54Aa | 9.69±6.05Aa | NA | 0.92±0.92Aa | NA | 7.11±3.82Aa | NA |
| o-Xylene | 0.25±0.35Aa | 1.36±0.07Aa | NA | NA | 0.87±0.61Aa | 0.43±0.61Aa | NA | NA |
| D-Limonene | 62.34±3.87Aa | 65.35±3.01Aa | 45.77±32.37Aa | 63.01±1.33Aa | 39.94±9.31Ab | 41.44±9.33Ab | 31.32±19.90Ab | 34.52±24.01Ab |
| p-Cymene | NA | 1.02±0.74Aa | 0.14±0.20Aa | 1.47±0.21Aa | NA | 0.74±0.00Aa | 0.78±0.56Aa | NA |
| β-Pinene | NA | NA | 1.09±0.77Aa | 1.60±0.02Aa | 0.36±0.51Bab | NA | 0.29±0.42Aab | 1.06±0.19Aab |
| γ-Terpinene | 1.50±0.14Ca | 2.09±0.10ABa | 1.93±0.08Aa | 1.62±0.20Ba | 0.91±0.17Cc | 0.99±0.50ABc | 1.21±0.10Ac | 1.20±0.15Bc |
| o-Cymene | 0.16±0.23Aa | 0.86±0.86Aa | NA | NA | 0.65±0.47Aa | 0.72±0.72Aa | NA | NA |
| Hydrocarbons | ||||||||
| Ethylbenzene | 1.42±1.23Aa | 3.20±0.42Aa | NA | 3.66±0.00Aa | 1.58±1.12Aa | 2.40±1.74Aa | 2.33±0.03Aa | 2.50±0.02Aa |
| 1.3 dimethyl-benzene | 1.86±0.86A | 10.96±1.33A | 8.38±4.00A | 16.79±2.58A | 7.27±5.15A | 1.12±1.58A | 4.80±3.96A | 3.18±1.51A |
| Bicyclo[3.1.0]hexane. 4-methylene-1-(1-methylethyl)- | NA | NA | 1.73±0.06Aa | NA | NA | NA | 0.56±0.48Ab | NA |
| Oxime-. methoxy-phenyl- | 0.78±0.23Aa | 0.29±0.02Aa | 0.31±0.04Aa | 0.53±0.12Aa | 1.07±0.43Aa | 3.71±4.22Aa | 0.66±0.35Aa | 0.95±0.47Aa |
| Styrene | NA | NA | NA | NA | NA | NA | NA | 1.20±0.00Aab |
| 3.3-Dimethyl-1.2-epoxybutane | NA | NA | NA | 0.51±0.51Aa | NA | NA | 0.13±0.18Aa | NA |
| Cyclododecane | NA | NA | NA | NA | NA | NA | NA | NA |
| Cyclotetradecane | NA | NA | NA | NA | NA | NA | NA | NA |
| Benzene. 1.2-dichloro- | NA | 0.26±0.22a | NA | NA | 0.10±0.13a | 2.22±2.22a | NA | NA |
| Benzene. 1.3-dichloro- | NA | NA | 0.20±0.15A | 0.46±0.03A | NA | NA | 0.07±0.10A | NA |
*C: Control. ELP: cheese treated with eggwhite lysozyme. MLP: cheese treated with microbial lysozyme powder. MLL: cheese treated with microbial lysozyme liquid. Duncan test was used to compare significant means. Different lowercase superscript letters in the same row indicate significant differences during ripening (P < 0.05); different uppercase superscript letters in the same column indicate significant differences among sample groups (P < 0.05).
Acids. The highest concentration of the seven acids observed in the Kashar cheese was butanoic (butyric), and after 30 days of ripening, there were significant differences between its concentrations and those of the other cheeses (p < 0.05). In addition, the most prevalent acids in all samples at day 90 were butyric and hexanoic acids, which contrasts with the findings of Eroğlu et al. (2016)Eroglu, A., Toker, O. S., Dogan, M. 2016. Changes in the texture, physicochemical properties and volatile compound profiles of fresh Kashar cheese (< 90 days) during ripening. Internat. J. Dairy Technol. 69, 243–253. but is consistent with research of Suleymani and Hayaloğlu (2016)Sulejmani E, Hayaloglu AA. 2016. Influence of curd heating on proteolysis and volatiles of Kashkaval cheese. Food Chem. 211, 160–170. DOI: 10.1016/j.foodchem.2016.05.054.
Alcohols. The alcohols with the highest concentrations in Kashar cheese after ripening were 3-methyl-1-butanol, linalool, phenylethyl alcohol, and silanediol dimethyl. The kind of lysozyme treatment or ripening period had no effect on the concentrations of these alcohols. In general, the relative abundance of alcohols declined at the end of ripening and was higher in the control cheese than in other types. This might be connected to how lysozyme treatment affects the formation of alcohol in cheese.
Esters. The ester makeup of the cheese samples remained unchanged after the lysozyme and ripening procedures. This could be because there were not any appreciable variations in any of the pH samples throughout ripening (Table 2), which shows a similar pH trend for all samples. Eroğlu et al. (2016)Eroglu, A., Toker, O. S., Dogan, M. 2016. Changes in the texture, physicochemical properties and volatile compound profiles of fresh Kashar cheese (< 90 days) during ripening. Internat. J. Dairy Technol. 69, 243–253. hypothesized that the composition of milk and reaction parameters like pH might significantly impact the synthesis of ester compounds.
Terpenes: The volatile percentage of Kashar cheese contained seven terpenes, of which pinene, cymene, terpinene, and xylene derivatives were the main ones (Table 4). These substances were detected in significant amounts in the cheese and the limonene. Terpenes in milk are derived from plants found in the feed combination or pasture (Curioni and Bosset, 2002Curioni PMG, Bosset JO. 2002. Key odorants in various cheese types as determined by gas chromatography-olfactometry. Int. Dairy J. 12, 959–984. https://doi.org/10.1016/S0958-6946(02)00124-3), and the kind and quantity of cheese are mostly dependent on the quality of the milk used to make it. On the other hand, during the ripening and lysozyme treatments, the concentrations of hydrocarbons fluctuated, but their abundance did not statistically alter.
4. CONCLUSIONS
⌅The findings of this study demonstrate how the use of bacterial and egg lysozymes in Kashar cheese altered the cheese’s quality, whether in liquid or in powder form. The application of milk lysozyme did not significantly change the cheese’s titratable acidity, pH, spore bacteria, coliforms, or E. coli contents, according to physicochemical data (P > 0.05). However, the microbial analysis showed that cheeses treated with bacterial lysozyme (MLL, MLP) were effective against TMAB, yeast and mold, coliform levels, and lactic acid bacteria counts. When compared to control cheese, significant variations in microbe counts during ripening were found. The texture profile showed that lysozyme-treated cheese had improved textural characteristics, with a notable increase in hardness and cohesiveness but a decrease in springiness, gumminess, and resilience during storage, especially in lysozyme-treated cheese made with microbial powder (MLP). It is believed that the decrease in fatty acids observed in the lysozym-treated cheese throughout the ripening phase is connected to the microbial population of the cheese due to the inhibitory impact of lysozym. Of all the samples, the control cheese scored the highest in terms of free fatty acids. With the exception of C4, C14, C16, and C18, the free fatty acid fractions generated in kashar cheese’s short chains (SCFA), long chains (LCFA), and fatty acids were all decreased by the application of lysozyme forms. Ripening and lysozme variables did not significantly affect the majority of the volatile profiles.
As a result, we firmly advocate the use of lysozyme, particularly microbial powder lysozyme, as a feasible substitute for enhancing the physical, textural, and compositional aspects of Kashar cheese production as well as other highly-cooked pasta filata type cheese like Mozzarella or Provalone. In addition, the present work provides precise knowledge on the types of lysozyme to be employed while producing cheese that could inhibit microbial proliferation while also improving cheese quality and ensuring consumer safety and health when cheese is ripening. To make Kashar cheese with a longer shelf life, the viability of the solutions suggested in this work could be encouraged at an industrial level.