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.

Table 1 lists the gross composition of raw cow’s milk. According to the literature (Say and Güzeler, 2008; Göncü et al., 2017), the general characteristics of raw milk were consistent.

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.

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., 2018). 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., 2018). 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., 2017). Badem and Uçar (2016) 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., 2016; Ozturkoglu-Budak et al., 2021; Yalman et al., 2017). 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., 2017) and 1.22-1.37% (Badem and Uçar, 2016).

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., 2015).

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, 2019). All samples underwent the anticipated formation of WSN and TCA-SN,a rise in RI during ripening, and other authors (Sulejmani and Hayaloglu, 2016; Ozturkoglu-Budak et al., 2021) 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.

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), 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) 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.

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, 1993). For Kashar cheese and Muenster-type cheese, De Leon-Gonzalez et al. (2000) and Temiz et al. (2010) 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) 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 1998).Temiz et al. (2010) found similar findings for Kashar cheese that had matured for 120 days; while Güler (2005) 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., 2016; Sulejmani and Hayaloğlu, 2016). 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.

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) but is consistent with research of Suleymani and Hayaloğlu (2016).

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) 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, 2002), 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.