Physicochemical, Microbiological, and Sensory Characterization of Halloumi Cheese Fortiﬁed with Garlic ( Allium sativum ) and Pepper ( Piper nigrum ) †

: This study aimed to develop a Halloumi cheese using cow’s milk and fortify it with garlic and pepper to evaluate its physicochemical, microbiological, and sensory properties. The developed Halloumi cheese showed a signiﬁcant increase ( p < 0.05) in its total solids, protein, fat, and ash contents, and a signiﬁcant decrease ( p < 0.05) in moisture and pH during a 35-day storage period. The textural properties of the cheese were signiﬁcantly affected ( p < 0.05) by the storage period. The microbiological shelf life of the Halloumi cheese was 21 days in refrigerated conditions. In sensory evaluation, the cheese fortiﬁed with the spice powder mixture received higher consumer acceptance than nonfortiﬁed Halloumi cheese.


Introduction
Halloumi is the traditional cheese of Cyprus.It is widely popular in Cyprus and other countries of the Eastern Mediterranean.Recently, it has gained international acceptance and recognition [1].Halloumi cheese is a semi-hard to hard unripened cheese, with a unique sensory and texture profile obtained by cooking (90-95 • C) the pressed curd in the whey for more than 30 min.The color varies from white (if a mixture of ovine and caprine milk is used) to yellowish (if bovine milk is used) [2].This fresh Halloumi cheese has a characteristic aroma; its texture is elastic and compact with no holes and it is easy to cut.Halloumi has a greater melting point than usual cheese; therefore, it is one of the few cheeses that can be grilled or fried [3].Traditional Halloumi cheese has been produced from raw ovine or caprine milk.However, the rising demand has meant that bovine milk has become readily available in order to prepare Halloumi.The compositional changes due to the use of bovine milk have inevitably led to some modifications being made to the traditional Halloumi cheese-making procedure [1].The objective of this study was to produce Halloumi cheese from cow's milk with added value, fortified with garlic and pepper, and to assess its sensory, microbiological, and physicochemical characteristics.

Manufacture of Halloumi Cheese Fortified with Garlic and Pepper
The Halloumi cheese fortified with garlic and pepper was manufactured according to the procedure described by Mehyar et al. [4].

Physicochemical Analysis
The Halloumi cheese samples were prepared according to the method described by Milci et al. [2].The total solid and moisture contents of the Halloumi cheeses were determined using the oven drying method (AOAC, 2000; method 925.23 and method 948.12, respectively).The fat content was determined using the Soxhlet method (AOAC, 2000; method 963.15).The ash content was determined by ashing the sample to a constant weight at 550 • C for 2 h (AOAC, 2000; method 942.05).The Kjeldahl method was used to determine the protein content (AOAC, 2000; method 978.02).The pH of the cheese samples was measured using a digital pH meter (ST 3000-Ohaus Corporation-Parsippany, NJ, USA).
The texture profile of fortified Halloumi cheese samples (hardness, gumminess, cohesiveness, and chewiness) was evaluated using a TX 700 texture analyzer (Shimadzu, Kyoto, Japan) according to the methods of Zheng et al. [5].
A Hunter Lab color meter (KONICA MINOLTA INC.Osaka, Japan) was used to determine L* (lightness), a* (green-red value), and b* (blue-yellow value).The relevant values were auto-generated by a software [6].The differences between the samples were evaluated using individual parameters (L* a* b*), and the total color difference (∆E*) according to the method described by Nedomová et al. [6].

Microbiological Analysis
In total, 10 g of cheese was blended with 90 mL of sterile peptone water in a stomacher to obtain a homogeneous mixture.Serial dilutions down to 10 −6 were made, by means of 1 mL transfers into 9 mL of sterile peptone water [7].Counts of total bacteria, Escherichia coli, yeasts and molds, Staphylococcus aureus, and lactic acid bacteria were enumerated via the spread plate technique using plate count agar, MacConkey agar, potato dextrose agar, mannitol agar, and de Man, Rogosa and Sharpe (MRS) agar, respectively.The total bacteria were determined at 35 ± 1 • C using an incubation period of 48 ± 2 h.Escherichia coli were determined at 35 ± 1 • C for 24 ± 2 h.Yeasts and molds were incubated at 35 ± 1 • C for 48 ± 2 h.Lactic acid bacteria were incubated at 35 ± 1 • C for 72 ±2 h and Staphylococcus aureus were determined at 35 ± 1 • C for 24 ± 2 h.All microbiological analyses were performed over a 40-day period with 10 days of intervals [4].

Sensory Evaluation
A sensory assessment was conducted on two types of cheese samples: nonfortified Halloumi cheese (control sample) and Halloumi cheese fortified with garlic and pepper (fortified sample).Based on a 9-point hedonic scale, the evaluation was performed by 30 untrained panelists who rated the samples' appearance, aroma, color, before taste, after taste, texture, and overall acceptability.

Statistical Analysis
In this experiment, triplicate samples were taken for all the analyses.The significance was evaluated using analysis of variance (ANOVA) in regard to the physicochemical and microbiological properties of the Halloumi cheese samples.All statistical calculations were performed using SPSS software version 16.0.A level of confidence of 95% (p < 0.05) was used.The results from the sensory panel were analyzed using XLSTAT software (version 2022.4).

Physicochemical Characteristics
Chemical composition is one of the most significant factors influencing cheese production [8].According to Table 1, the moisture content and pH values of fortified Halloumi cheese significantly decreased (p < 0.05) during the storage period.The decrease in the moisture content of produced cheeses during the storage period might be due to a loss of moisture and a decrease in the pH content might be due to the increase in lactic acid formation with storage time.The pH fluctuations in the current study seem to be consistent with Milci et al. [2].The total solid, protein, fat, and ash contents of the cheese samples were found to be significantly increased (p < 0.05) during the storage period.The cheese's texture is assumed to be one of the significant determinants of the consumer acceptability of the cheese [5].It was observed that fortified Halloumi cheese hardness (from 4.20 N to 6.11 N), gumminess (from 1.99 N to 3.57 N), and chewiness (from 2.31 N to 3.34 N) significantly increased (p < 0.05) and cohesiveness (from 0.85 to 0.74) significantly decreased (p < 0.05) during the storage period (Table S1).These findings contradict Ayyash et al. [9], who reported no significant differences (p > 0.05) in hardness, cohesiveness, and gumminess among the experimental cheeses during the entire 56-day storage period evaluated.The increases in the hardness and chewiness values of developed Halloumi cheese in the current study may be due to a decrease in cheese moisture content.The gumminess in Halloumi cheese was related to moisture content and hardness.Also, water, protein, and fat are the main factors affecting cheese hardness [6].Cheese cohesiveness usually decreases as cheese moisture content decreases [10].
The results in Table 2 indicate that the L* values of fortified Halloumi cheese decreased significantly (p < 0.05) during storage.In the fortified Halloumi cheese, due to moisture loss during storage, the bright white color intensity reduced and the color became a dull yellow, which can be attributed to a decrease in L* values [11].During the storage period, the fortified Halloumi sample's a* and b* mean values significantly increased (p < 0.05).The results were in agreement with Nedomová et al. [6].An increase in b* values could be explained by a loss of moisture during storage [10].Over the 35-day storage period, the fortified Halloumi cheese's total color difference (∆E*) increased from 2.07 to 2.11.Notably, a significant color difference (5.99) was observed on the 21 st day of storage.Values are mean ± SD (n = 3).Means with different superscript lowercase letters in the same row are significantly different (p < 0.05).

Growth Pattern of Microorganisms
According to Figure 1 and Table S2, the total bacteria count increased slowly in the early stages of storage and exhibited a sharp increase after that, reaching 4.78 ± 0.02 log CFU/g at the end of the 40-day storage period.These results agree with the findings of Kamleh et al. [7].The Escherichia coli count in fortified Halloumi cheese samples increased significantly (p < 0.05) as the storage progressed.However, the total coliforms count in the Halloumi cheese sample was lower than those mentioned by Kamleh et al. [7].The processing environment, biofilm development in the post-heating equipment or sources, and heat treatment failure could be the reasons for this significant growth of E. coli.The growth of yeasts and molds was significantly different (p < 0.05) during the 40-day storage time.The results differ from those reported by Papademas and Robinson [1] in fresh Halloumi cheese made from bovine milk.The reason for this significant growth of yeasts and molds may be the increased acidity throughout the storage time of Halloumi cheese.The Staphylococcus aureus count decreased significantly (p < 0.05) during the 40-day storage time.Similarly, a significant decrease (p < 0.05) in Staphylococcus aureus count during the storage period of 28 days was reported [12].The lactic acid bacteria count was not significantly different (p > 0.05) during the storage.

Microbiological Shelf Life
In the current study, the total bacteria required 32 days to reach their 4.0 log CFU/g maximum permissible limit (Figure 1a).Escherichia coli required 33 days to reach their 2.0 log CFU/g maximum permissible limit (Figure 1b), yeasts and molds exceeded their 1.5 log CFU/g maximum permissible limit at 21 days of storage (Figure 1c), Staphylococcus aureus required more than 40 days to reach their 2.0 log CFU/g maximum permissible limit (Figure 1d), and lactic acid bacteria exceeded their 2.6 log CFU/g maximum permissible limit at 25 days of storage (Figure 1e) [13].

Conclusions
This study indicated that the fortified Halloumi cheese sample's pH value and moisture content significantly declined and the total solid, fat, protein, and ash contents were significantly increased.The microbiological shelf life of functional Halloumi cheese fortified with garlic and pepper was detected to be 21 days at 10 ± 1 • C, which is an indication of the shortest time required for yeasts and molds to exceed their maximum permissible limits in the Halloumi cheese.In the sensory evaluation, the spice powder mixture fortified Halloumi cheese had higher consumer acceptance than the control unfortified Halloumi cheese.

Figure 1 .
Figure 1.Growth curves of total bacteria (a) Escherichia coli (b) yeasts and molds (c) Staphylococcus aureus (d) and lactic acid bacteria (e) during storage time.(Black Solid Line: Maximum permissible limit; Black dotted line: minimum permissible limit; Orange line: count of microorganism).Values are mean ± SD (n = 3).Means with different superscript lowercase letters are significantly different (p < 0.05).

Table 1 .
Chemical composition of Halloumi cheese fortified with garlic and pepper powder during storage at 10 ± 1 • C for 35 days.
Values are mean ± SD (n = 3).Means with different superscript lowercase letters in the same row are significantly different (p < 0.05).

Table 2 .
Color properties of Halloumi cheese fortified with garlic and pepper powder during storage at 10 ± 1 • C for 35 days.