EVALUATION OF THE EFFECT OF THYME OIL NANOPARTICLES ON THE SHELF LIFE OF KARISH CHEESE

The most widely consumed soft cheese in Egypt, particularly in the rural, is karish cheese. When cheese is produced, handled, distributed, or stored in unsanitary conditions, it could become contaminated by many types of microorganisms. The spoilage of cheese and/or foodborne diseases are caused by contamination with various microorganisms. A total of 120 Karish cheese samples were collected from El Sharkia City supermarket, Egypt. The microbiological tests performed were: aerobic plate count (APC), Escherichia coli ( E. coli ) and Staphylococcus aureus ( Staph. aureus ) counts. The recorded results showed that the mean of APC, E. coli and Staph. aureus counts were 6.9, 3.45 and 4.3 log 10 cfu/g, respectively. Researchers have investigated edible packaging materials for essential oils encapsulated in biopolymers to offer an effective and secure method of food preservation. In this study, to increase the shelf-life of Karish cheese, chitosan-based emulsions consisting of liposomes infused with thyme essential oil (TEO) were explored. Antimicrobial activities were assessed via Staph. aureus and E . coli counts over 4 weeks. High counts of Staph . aureus and E . coli recorded by the fourth week, for all cheese samples either coated or not coated with chitosan solutions. However, samples coated with TEO 1% and liposomal chitosan emulsions encapsulated TEO 0.5 and 1% v/v decreased in microbial counts up to 4 weeks, while samples coated with liposomal chitosan emulsions encapsulated TEO 2% occurred complete absence of Staph. aureus and E. coli at the end of the fourth week of storage. According to the findings, TEO 1% and liposomal chitosan emulsion encapsulated TEO 2% v/v may be promising natural solutions, with a satisfactory appearance to extend the shelf life, in addition to preserving the flavour of Karish cheese.


INTRODUCTION
Due to its great nutritional value and low price, Karish cheese is a very popular soft cheese in Egypt, besides its low-fat content because it is made of raw skim milk.The methods of manufacturing such type of cheese by native ways are still unhygienic, the finished products are exposed to contamination from a variety of microorganisms (Deeb et al., 2004).The primary causes of Karish cheese's short shelf life are its high water level and minimal salt content.(Delacroix-Buchet et al., 2005).Karish cheese is mostly contaminated with food-borne pathogens incriminated in poisoning from food and the finished product with poor quality (Todaro et al., 2013).E coli is one of the most serious bacteria, especially pathogenic types found in milk and its products.E. coli often lives in the intestinal tract of humans and animals.Some species possess virulent genes that pose a risk to both humans and animals.(Malik and Memona 2010).Staph.aureus is a different sort of bacteria that typically lives in both humans' and animals' skin, nose and throat.Enterotoxins produced by Staph.aureus results in food poisoning illnesses (Orwin et al., 2003).
Currently, chemical food preservatives are not preferred by consumers worldwide.As a result, consumers are demanding the use of natural antibacterial agents for preservation (Weiss et al., 2015).Numerous essential oils have been investigated for their potent antibacterial properties against a wide range of pathogens linked to food illness or spoilage.At the same time, they should not have any adverse effect on the sensory characters of the product.Numerous studies have found that using essential oils as organic preservatives in the food industry is beneficial (Hyldgaard et al., 2012).However, due to their limited water solubility, potential interactions with other food matrix ingredients, and even evaporation into the packaging headspace, the direct mixing of essential oils with the product may make them less effective (Gill et al., 2002;Devlieghere et al., 2004;Gutierrez et al., 2008;Fernandez-Pan et al., 2014).Additionally, when essential oils are used as food additives, the primary taste of the product is changed.(Hayouni et al., 2008).
Nanotechnologies have been developed to achieve this goal by enhancing bioactivities while simultaneously reducing unfavorable organoleptic effects (Guo et al., 2018).According to previous studies, nanoemulsions have stronger bioactivity and antibacterial effectiveness than conventional carrier agents due to their nanometer-sized particles and improved stability (Donsì and Ferrari 2016;Chuacharoen and Sabliov 2019).Encapsulating essential oils has many benefits, including good antimicrobial packaging that eliminates their problem with strong odors (Al-Moghazy et al., 2021).Some kinds of hard cheese and minced beef have been prepared effectively using Liposome encapsulated essential oils (Khosravi-Darani et al., 2016;Lin et al., 2016;Cui et al., 2017).Thyme essential oil (TEO) and its primary component, thymol, are utilized as antioxidants, antiinflammatory agents, topical anesthetics and scar-curing agents.They are also antibacterial agents against infections and food spoilage, in addition to their advantages for the circulatory system (Marchese et al., 2016).
In this work, to provide a secure and efficient coating for Karish cheese, TEO was encapsulated into a coating emulsion consisting of chitosan and liposomes, and to distinguish the effectiveness of TEO and its antimicrobial activities against different microorganisms.

Samples collection:
Researchers collected 120 samples of Karish cheese from EL Sharkia city supermarkets, Egypt.The obtained samples were immediately brought into the lab under strictly aseptic circumstances, where they underwent the subsequent analysis.

Aerobic plate count (APC) (FDA, 2013):
Pipetting two identical sterile Petri dishes with one milliliter of each dilution was done.The Petri plates were then filled with 20 ml of melted sterile plate count agar, and the contents were properly mixed by spinning the plate several times.The plates were inverted and incubated at 35°C for 48 hours after the media had solidified.Using a Quebec counter, plates with 25-250 colonies were counted.The average number per plate was then multiplied by the dilution factor to determine the total colony forming units cfu/g.

E. coli count (ISO, 16649/2-2001):
Aseptically, 1 ml of each dilution from the previously made homogenate was added to a sterile Petri dish, and 10 ml of Tryptone Bile X-Glucuronide (TBX) agar was then added.The dish was then incubated at 44 o C for 24 hours.Blue to blue-green colonies with a bluish halo zone were E. coli's distinctive colonies.To identify the isolated strains, biochemical tests such as Triple Sugar Iron (TSI), Indol, Methyl Red, Voges-Proskaur, and Citrate Utilization were utilized.(A.P.H.A., 2004) Staph.aureus count (US-FDA, 2016): One ml sample suspension was aseptically transferred to 3 plates of Baird-Parker agar from the previously made homogenate, dividing the 1 ml inoculum evenly among the 3 plates (i.e., 0.4, 0.3, and 0.3 ml).A sterilized bent glass streaking rod was used to evenly distribute the inoculum over the surface of the agar plates.48 hours were spent incubating plates at 35-37°C.Staph.aureus colonies are circular, smooth, convex, moist, ranging in color from gray to jet black, usually with a light-colored (offwhite) edge, surrounded by an opaque zone, and frequently with an outer transparent zone.They measure 2-3 mm in diameter on plates.For counting, choose plates with 20-200 colonies.Staph.aureus was confirmed by tube coagulase (coagulase positive).In brief, suspicious colonies were introduced into rabbit plasma, and the tube was then incubated for three hours at 37 °C.Re-incubate for 18 hours at room temperature if the results are negative.Staph.aureus was thought to be present in coagulase-positive cultures.

Thyme essential oil preparation (Ozcan et al., 2003)
Thyme essential oil was obtained by steam distillation from the oil extraction unit at the National Research Center, Cairo, Egypt.The essential oils diluted to 1% by mixing with ethylene glycol in the sterilized distilled water as an inert substance act as a dissolving agent for oil without any harmful effects on the food.

Chitosan-TEO coating preparation (Al-Moghazy et al., 2020)
The Chitosan layer was created by mixing 2% acetic acid solution with medium molecular weight Chitosan (2 g; Sigma-Aldrich, Germany).The constant stirring persisted until a clear solution was obtained.The previously produced solution was then supplemented with TEO in three different concentrations (0.5, 1 and 2% v/v).Final solutions were homogenized by a CAT Unidrive homogenizer (12000 rpm for 2 min).

Chitosan-liposomal coating solutions preparation
By mixing 2 g of lecithin (Lio, Turkey) with 0.25, 0.5, and 1 g of steam-distilled TEO (provided by the oil extraction unit, National Research Centre, Egypt) and 50 mL of chloroform (Acros Organics, Belgium), three concentrations were created: 0.5%, 1%, and 2%.After 30 minutes of stirring at 22°C, 50 mL of chitosan/acetic acid was added.At 60°C, stirring was then carried out for an additional 30 minutes.Using compact pressure at 50°C for 2 hours, the suspension became milky and concentrated (Al-Moghazy et al., 2020).The inoculated milk was incubated at 42 • C until curding was completed.The formed cheese block was cut into similar cubes with approx.10 g each.Karish cheese cubes were infected by immersing them in a culture of Staph.aureus and other E.coli containing 10 3 cfu/ml for 10 seconds.The extra liquid was then squeezed with a sterile cotton pad.Each cheese cube was placed in thyme essential oil at 1% (T1), CL-TEO .5% v/v (T2), CL-TEO 1% v/v (T3) and CL-TEO 2% v/v (T4) separately. 2 mL of a special coating solution was used to coat cheese cubes.Without whey, treated Karish cheeses were kept at 4°C for four weeks.At 0, 1, 2, 3, and 4 weeks of storage, microbiological studies were conducted (Al-Moghazy et al., 2021).

Evaluation of the bacteriological quality of karish cheese
During their four-week cold storage phase, qualitatively in a stomacher, 10 g of each sample were aseptically homogenized for one minute in 90 ml of a sterile 2% sodium citrate solution.The samples were exposed for counting of Staph.aureus and E. coli weekly, as mentioned before.
Sensory evaluation of treated Karish cheese (Effat et al., 2012) Karish cheese samples were divided into pieces measuring approximately 5x5 cm and arranged on white plates.At room temperature (20.0°C), samples were tempered before being presented to the panelists in random order.Nine laboratory staff members, who were familiar with Karish cheese, evaluated the cheeses organoleptically at 0, 1, 2, 3, and 4 weeks of ripening at the dairy control department of the Animal Health Research Institute.Cheese was graded by the panelists on three criteria: flavor (50 points), body and texture (40 points), and appearance (10 points).The three sensory features were given scores.According to the scores recorded for flavor, body and texture, color and appearance, and overall acceptability, adding T1 and T4 to Karish cheese had a minor impact on the sensory qualities (Table 3).Karish cheese treatments' flavor ratings slightly declined in T1 and T4 treatments.On average, T1 and T4 had respectable to good flavor.Actually, EOs should only be used sparingly because of their negative effects on cheese's flavor, where the flavor of thyme-fortified soft cheese scored 35 until the end of the second week, 32 in the third week, and 29 in the fourth week of storage with a faint bitter almond flavor (Samah and Ahmed, 2019).Furthermore, Han et al. (2015) found that thyme oil treatment significantly (p>0.05)altered the flavor of shredded cheese, resulting in a less favored flavor compared to the control.As a result, this study demonstrated the benefit of essential oil encapsulation in maintaining palatable cheese flavor.

RESULTS
Body and texture, in addition to storage time, were among the sensory qualities that changed somewhat between all treatments.Results from El-Sayed and El-Sayed (2021) were consistent with those of the current investigation, although UF Labneh's body and texture were only marginally impacted by EO treatments.Throughout the second, third, and fourth weeks of storage, there was no statistically significant variance between the mean color and appearance scores (p>0.05).Additionally, the treated samples had the same hue as the control samples, which had a clear, natural white appearance, without changing the significance of the difference as indicated in Table (3).On the other hand, labneh treated with TEO nanomulsions had uniformity and a velvety sensation in color and appearance scores.
Even though the Karish cheese was treated with T1, or T4, its general acceptance in this study was only slightly impacted during the storage period.Accordingly, fresh soft cheese produced in a lab that had been supplemented with 0.01% ginger and thyme oils received a grade A for general sensory acceptance after being stored at 4°C for a month, with the highest scores coming in the first two weeks (Ahmed et al., 2021)

CONCLUSION
In this study, the microbiological quality of Karish cheese showed high APC, Staph.aureus and E. coli counts that suggest that this kind of cheese was not handled or manufactured with enough hygienic practices.Also, the results of every microbiological examination conducted on Karish cheese demonstrate the effectiveness of chitosan, liposome, and TEO coatings in extending the cheese's shelf life and replicating it when compared to uncoated control samples.
The overall acceptability was marginally impacted during the storage period, regardless of whether the Karish cheese was treated with 1% TEO or 2% v/v TEO-containing chitosan.
Antimicrobial activity was found when coating Karish cheese with chitosan containing TEO at 0.5 and 1% v/v reduced the amounts of E. coli and Staph.aureus, and this impact outperformed that of TEO by 1%.Whereas a 2% v/v concentration of TEO in chitosan results in a complete reduction of E. coli and Staph.aureus, Karish cheese's shelf life has increased from two to four weeks as a result.

REFERENCES
Karish cheese was prepared, according to Effat et al. (2012), as milk heated at 85 o C/5 min, cooled to 42 o C and then inoculated by a 2% active mixture starter of Streptococcus thermophilus & Lactobacillus bulgaricus.

Table 1 :
Statistical examination of APC, Staph.aureus and E. coli count (log10 cfu/g) of the examined samples.

Table 2 :
Classification of the examined 120 Karish cheese samples.

Table 3 :
Sensory evaluation of Karish cheese samples during four weeks of cold storage.

Table 4 :
Total Staph.aureus counts (log10 cfu/g) of different groups of Karish cheese samples over 4 weeks.

Table 5 :
Total E. coli counts (log10 cfu/g) of different groups of Karish cheese samples over 4 weeks The Egyptian Standards for Kariesh Cheese of the Egyptian government (E.S,1008-4,2005) categorize a sample as unfit for human consumption if it yielded any of the following (Any presence of E. coli and Staph.aureus in 1 g of food sample).
Additionally, Hachana et al. (2019)examined the impact of 1% v/w TEO on soft cheese, which causes a significant decrease in the total APC counts.A few other research(V'azquez  et al., 2001)looked into the use of pure thymol to improve cheese's microbiological quality.