Elaboration of an Antimicrobial , Biodegradable and Aromatic Film Against the Natural Contamination of Sliced Mozzarella Cheese

The search for foods that do not use chemical preservatives in their composition has been growing over the years, and as a result, studies on active packaging have been intensified, which has as principle, in addition to protecting, transporting and containing the food, interact with it. In this context, the present study has developed an active antimicrobial, aromatic and biodegradable packaging in order to reduce the natural contamination of sliced Mozzarella cheese, which has shelf life of five days after manipulation. For this, a film was developed containing cassava starch, a plasticizing agent, gum arabic and oregano essential oil, as an antimicrobial agent. The research used the serial dilution methodology for counting and to aid in the isolation of the product natural contamination. The diffusion methodology in Agar in PDA medium with chloramphenicol was used to identify the antimicrobial power of the polymer matrix and the Casting method for film manufacture. With the results, it was possible to identify that only the film with the addition of oregano essential oil showed fungicidal action when compared to the same film without it, forming an inhibition halo of about 25 mm against Hyphopichia burtonii yeast isolated from the product itself, when stored under conditions stipulated by the supplier (Styrofoam trays stored in conventional refrigerator).


I. INTRODUCTION
Mozzarella cheese is food rich in nutrients, which favors the proliferation of microorganisms that can lead to changes in the organoleptic characteristics of the product and / or food poisoning [1].In addition, cheese flavor is altered due to the production of compounds resulting from bacterial metabolism, such as acetic acid [2].
The main approaches to maintaining the quality of this food for longer are based on improved raw material quality, process innovations and the use of adequate storage conditions [3]- [6].In the case of storage, generally the technique used is freezing, which despite increasing the shelf life of the product, modifies its physical properties [7], [8].Thus, studies using active packaging are growing.In this innovative concept of food protection, antimicrobial agents are incorporated into polymer chains and can be slowly released onto the food surface, inhibiting microbial deterioration on the product surface [9].The controlled release of the active agent allows maintaining the critical concentration necessary to inhibit microbial multiplication and, consequently, the amount of additives added to the product is reduced [10].In addition, active antimicrobial packaging is an alternative method to overcome limitations of the direct addition of preservatives in food formulations, such as the reduction of its antimicrobial potential due to reactions and/or interaction with food matrix components [11].
Essential oils are complex, volatile, lipophilic, odoriferous and liquid substances, derived from the secondary metabolism of vegetables.They comprise terpene hydrocarbons, simple alcohols, aldehydes, ketones, phenols (high content of Carvacrol), esters, fixed organic acids, among others [11], [12].According to reference number, as in [15], it is a low-toxicity liquid, colorless, poorly soluble in water used in the food industry as a preservative due to its bactericidal, fungicidal action and anti-inflammatory and antioxidant characteristics.
The aim of this study was to develop an antimicrobial, aromatic and biodegradable active packaging for sliced Mozzarella cheese, which has shelf life of one week after being cut and placed in styrofoam trays through the incorporation of oregano essential oil.

A. Non-microbiological material
Sliced Mozzarella cheese was kindly provided by the Faculty of Thermomechanical Technology (FTT).In the Laboratory of analysis, it was separated into portions of 25 g, wrapped in styrofoam trays and sealed with plastic film.This packaging is similar to that acquired in the local retail trade.After packaging, the product was conditioned under refrigeration in conventional refrigerator in the same way final consumers store it in their residence.
For the formation of the biodegradable film, the antimicrobial agent was incorporated in cassava starch, sorbitol, gum arabic and water.Oregano essential oil, the antimicrobial agent, was kindly provided by Synthite ® Company.Cassava starch was purchased from local retailers, with no specific preference brand.[18], which consists of the preparation of a film-forming solution by the dissolution of cassava starch and other compounds in distilled water, used to give elasticity and resistance to the final film.
For the final formulation of the active film in distilled water, 1% cassava starch, 0.05% sorbitol, 0.25% gum arabic and 1.25% oregano essential oil were added (percentage that showed inhibition power against the microorganism in work done by reference number, as in [19]).The solution was homogenized by means of a magnetic stirrer for 5 minutes without heating.Stirring was maintained, and after that time, the heating process was started until temperature reached 68 °C.At this temperature, the mixture was left for 4 minutes until complete starch gelatinization.After this heating period, the mixture was allowed to cool to room temperature and then placed on the bottom of an acrylic Petri dish, which was oven conditioned with forced air circulation at 30 °C for about 7 hours.
For the production of the control film, oregano essential oil was not added, but the same production methodology was followed, with the addition of the same amount of distilled water replacing oregano essential oil.

2) Microbiological analysis in Mozzarella cheese
Samples of sliced cheese stored in styrofoam trays sealed with plastic film were conditioned in conventional refrigerator for 6 days.Every two days, a tray was removed for microbiological analysis.It is noteworthy that the sliced cheese trays were from the same piece of cheese.
For the microbiological analysis of the product, 25 g of Mozzarella cheese were weighed in a sterile Petri dish.Near the sterility zone, the 25 g of Mozzarella cheese were homogenized with 225 mL of sterile 0.85% saline solution.With the aid of a sterile pipette, 1 mL of solution was collected from the homogenizer and transferred to the first test tube containing 9 mL of 0.85% saline solution, also previously sterilized.Then, 1 mL from the first test tube was transferred to a second test tube, which also contained 9 mL of previously sterilized 0.85% saline solution.The same procedure was followed up to the fifth dilution successively.
With PDA (Potato Dextrose Agar -Difco™, EUA) medium with chloramphenicol ( Difco™, EUA), already solidified on Petri dishes, 0.1 mL of each of the dilutions was inoculated on each Petri dish and, with the help of a glass loop, the sample was spread in the medium until it was completely absorbed.After homogenization of the sample in the medium, inoculated Petri dishes were stored in an oven at 25 ºC for 5 days (Compendium of Methods for the Microbiological Examination of Foods - [20])), for later counting and isolation of the natural contamination.

3) Isolation of the Mozzarella cheese natural contamination
In order to obtain a pure fungal colony for subsequent inhibition analysis, recommendations of reference number, as in [21] with modifications.For this, with the aid of an inoculation loop, the point of the plate resulting from item B.2) was chosen, where there was a single colony, well separated from the others and uniform in order to avoid the dragging of more than one yeast species.This colony was then inoculated in Petri dishes containing solid PDA medium in the presence of antibiotic chloramphenicol.After inoculation, this Petri dish was stored in an oven for 5 days at 25 °C.After growth, an isolated colony was chosen again and replating and storage were performed under the same conditions previously used, which step was sufficient for the isolation of a single fungus species.The replating procedure was repeated every week of the experiment in order to have always fresh microorganism.

4) Antimicrobial Film Agar Diffusion Analysis
In order to test the antimicrobial activity of the best film prepared in item B.1), 15 Petri dishes containing PDAchloramphenicol culture medium were prepared, which after solidification, was inoculated with a 0.1 mL aliquot of pure culture previously isolated in item B.3), activated and standardized for number of cells of approximately 1 x 10 4 CFU.mL -1 [22].After the entire absorption of this fungic culture by the solid culture medium, films previously cut into 1 cm edge squares and sterilized in a UV lamp chamber (Prodicil, 110 V, 254 nm) for 10 minutes were placed in the center of the 15 aseptic-shaped Petri dishes.A set of 5 Petri dishes contained the same type of sample, totaling the 15 Petri dishes (5 negative control Petri dishes without film), five positive control Petri dishes (containing film, but without antimicrobial agent) and five Petri dishes containing the film of interest (plastic film containing oregano essential oil).Petri dishes were conditioned at 25-28 °C.After 24 hours, the diameter of halos formed around the film was measured with the aid of Mitutoyo S.A digital caliper in triplicate in three different positions.

5) Data analysis
All experiments were conducted in triplicate.Data obtained were analyzed using SPSS software v.13.0;Chicago, Inc., with application of ANOVA and Tukey test (5% significance level).

A. Elaboration of the active packaging using cassava starch.
To prepare the film, a total of 34 tests were performed.Among them, xanthan gum and glycerol were used, in addition to cassava starch.These two ingredients were then replaced by gum arabic and sorbitol, which gave better results in relation to film elasticity and incorporation of the oregano essential oil, which is a fully hydrophobic compound.
The first tests were carried out to verify the ideal amount of cassava starch that could be used in order to form a flexible, elastic and resistant plastic film.For this, tests were performed with concentrations ranging from 0.25 to 5%, where the ideal occurred at concentration of 1%.At this concentration, the film presented greater rigidity when compared to the other concentrations, being transparent, flexible and difficult to break.
Sorbitol was then added to this "ideal" film.This plasticizing agent has the function of providing greater elasticity to the film, as well as to aid in the incorporation of the essential oil, also providing gloss.It was used at concentrations of 0.01 to 1%.The ideal concentration was 0.01%, and the resulting film presented better gloss than the previous one, only with cassava starch, but the incorporation of oil in the film was shown to be very low.
For the incorporation of the essential oil, gums were used, where the first one to be used was xanthan, which did not provide satisfactory results.Therefore, the oil incorporation was accomplished with the addition of gum arabic.For these tests, concentrations ranging from 0.05 to 0.1% were used, and the effective concentration was 0.05%.Table I shows the ideal ready-made film as well as its final characteristics.

Film Characteristics
Adequate incorporation of the antimicrobial agent, not releasing antimicrobial agent when handling the sample; Resistance, gloss and malleability; Less adhesion of the film to the drying plate and, consequently, easier film removal;  Characteristic odor and adequate transparency.
The obtained film presented the desirable characteristics for biofilm with the addition of a technological adjuvant, in this case, oregano essential oil.The adequate incorporation of the antimicrobial agent was perceived in comparison to the formulations tested, as it did not release antimicrobial agent when handling the sample, with higher strength, gloss and malleability than the tested formulations, which were brittle, opaque and inflexible, with characteristic odor and adequate transparency.

B. Isolation and counting of Mozzarella cheese natural contamination
Microbiological analyses of molds and yeasts were carried out in order to verify the natural fungal contamination of sliced mozzarella cheese (as described in item B.2)).Analyses were performed in freshly sliced sample (time 0 Day) and then for another six days (with intervals of 2 days for each new analysis).This occurred to verify if during storage, the product acquired any other contaminant different from that demonstrated in the initial time of microbiological analysis (time 0 Day), as well as if during storage in conventional refrigerator, the natural contamination of the product could multiply.The contamination results are presented in Table II, and counting averages are expressed in logarithmic function at base 10.As can be verified from results shown in Table II, the concentration of microorganisms over time increased in the proportion of a logarithmic cycle, proving to be a microorganism capable of growing at cooling temperature (about 4 °C).These results were close to those presented by reference number, as in [23], where the contamination of Mozzarella cheese fluctuated between values of 6.07 and 7.64.This demonstrates why even when sliced Mozzarella is stored in refrigerator, it is capable of showing signs of deterioration over a period of five days.
Although there is no current legislation for mold and yeast count in this type of sample, reference number, as in [24] reported that high yeast and mold counts, such as those mentioned above, mainly reflect inadequate storage and/or product handling conditions.
Table III shows that over time, contamination remained being produced by the same microorganism of the initial moment of the microbiological analyses until the sixth day of analysis.In all cases, Petri dishes containing PDA medium with chloramphenicol had growth of regular and small gelatinous colonies, maintaining the color pattern.The identification of the microorganism, certified in report provided by the Faculty of Medicine of ABC, denominates the yeast, grown in Petri dishes, as being Hyphopichia burtonii.This microorganism can also be called as Pichia burtonii Boidin, Candida fibrae, Dematium chodati, Endomycopsis chodati, Trichosporon behrendii.
The genus Hyphopichia, according to reference number, as in [25], is inserted within the kingdom Fungi, phylum Ascomycota and family Ascoideaceae.This genus is formed by heterotalic fungi producing ascos and ascospores, being the species of the genus Hyphopichia burtonii.Hyphae are septate and these microorganisms have conidiogenic cells producing blastoconidia [26].
This yeast is a common soil inhabitant, and has already been isolated from food, animals and the intestinal tract and human skin [25].However, there are few reports of this species as a mycosis agent in humans [27].Its pathogenicity is still discussed [28] and according to reference number, as in [29], it is a eukaryotic microorganism with probiotic properties.

C. Antimicrobial power of the polymer matrix
To carry out the inhibition test, Petri dishes containing the film without the incorporation of oregano essential oil, another with the film containing oregano essential oil and the negative control without the incorporation of any film were used.As mentioned in item III-B, the microorganism used was the isolate of the product itself.A total of five Petri dishes from each of the samples were performed and their inhibition halos were measured with the aid of Mitutoyo S.A. d digital caliper in triplicate at different positions, and the results expressed in Table IV.By the ANOVA application, the resulting inhibition halo was about 25 mm (Table VI).It is worth mentioning that in Table IV, only the results of halos referring to film containing the inhibition oil are expressed, because in the other two cases (negative control and Petri dish containing film without the inhibition oil) the yeast growth was not inhibited (Table V).This result is important because it shows that the oregano essential oil makes the difference when incorporated into the film, making it clear that when applying the Tukey test at 95% confidence interval, there is a significant difference between sample containing film with the antimicrobial action and the other samples.
According to the methodology and results of reference number, as in [30], the antimicrobial efficiency of oregano essential oil was evaluated by the halo test in Aspergillus niger, with oil concentration of 10 μL and 20 μL, resulting in a halo of 18 mm and 23 mm, respectively, even incorporating the oregano essential oil into the film; but in our study, it managed to reach higher inhibition level than when used alone, in addition to the fact that lower concentration was used.Of course we cannot forget that this is another microorganism that may have different inhibition characteristics unlike the filamentous fungus studied in this research.In this study, it was possible to develop an active AAB (Antimicrobial, Aromatic and Biodegradable) package containing cassava starch, sorbitol and gum arabic for sliced mozzarella cheese using oregano essential oil, which proved to be effective in inhibiting Hyphopichia butonni at concentration of 1.25%.
• Antimicrobial (A): Active packaging containing oregano essential oil, a substance incorporated into its constitution that has the function of inhibiting the microorganism isolated from sliced Mozzarella cheese, demonstrated by the formation of inhibition halos of 25 mm in Petri dishes.• Aromatic (A): due to the presence of oregano essential oil, essence with strong and concentrated aroma.• Biodegradable (B): flexible films, made from natural materials.
TABLE V: ANTIMICROBIAL ACTION TEST OF HYPHOPICHIA BURTONII YEAST AGAINST FILMS ELABORATED IN THE PROJECT

TABLE I :
CHARACTERISTICS OF FILMS PREPARED FOR THE INCORPORATION OF OREGANO ESSENTIAL OIL, USING GUM ARABIC, SORBITOL AND CASSAVA STARCH.

TABLE II :
LOGARITHMIC QUANTIFICATION AT BASE 10 OF THE MICROBIAL CONCENTRATION OF SLICED MOZZARELLA SAMPLES OVER TIME.

TABLE III :
PETRI DISHES RESULTING FROM MICROBIOLOGICAL ANALYSES PERFORMED WITH SLICED MOZZARELLA STORED OVER 6 DAYS.