טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTamar Shragai-Vollach
SubjectDevelopment and Characterization of Antimicrobial Films for
Packaging of Pastrami
DepartmentDepartment of Biotechnology and Food Engineering
Supervisors Professor Emeritus Miltz Joseph
Professor Livney Yoav
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Antimicrobial (AM) packaging is a novel active packaging technique for food  preservation.

Most of the published articles in the field of active packaging focus on the strategy of placing a sachet containing preservatives like sodium metabisulphite, benzoates, sorbates and propionates, or scavengers, between the food and its primary package.

While preservatives, traditionally incorporated in the food formulation are effective, significant amounts of these antimicrobial substances are required, as they are dispersed throughout the food volume, a fact which increases costs and might also cause undesirable sensorial and health effects.

Since microbial contamination of food occurs primarily at the surface, there is often no need to incorporate preservative substances in food volume. Therefore, incorporation of antimicrobial compounds in the laminate is an attractive solution, which may also allow a more "friendly label", as it is usually only required to list ingredients of the product itself on the label. Food safety concerns, growing problems with microbial resistance and an increase of the production of minimally processed food together with green image perception policies, brought upon a renewed interest in natural preservatives in the food industry. This work focused on the incorporation of a natural antimicrobial agent, linalool, in the packaging film to improve effectiveness of current AM packaging technologies, as well as the appeal of the product ("all natural ingredients"). Linalool migrates slowly from the package onto the surface of the food' and maintains a high enough concentration of the AM agent only where it is required and thus preserves the product from spoilage. Linalool is known to work as a growth inhibitor for various types of microorganisms, among them many gram negative and gram positive bacteria, molds and yeasts. The FDA classified linalool as GRAS (Generally Recognized As Safe) and allowed its use for human consumption.

The purpose of this work was to investigate the incorporation of linalool in the

packaging film and to evaluate the ability of the package to inhibit bacterial growth.

The first chapter of this work presents the production process of the antimicrobial film and its characterization in terms of the remaining active substances in the created film. The AM package is made of a laminate composed of three polymer layers: a control layer, a matrix layer and a barrier layer. The polymer films were selected to allow a



slow and controlled release of the AM substance onto the food while preventing its escape out from the package into the environment. The matrix layer, which is sandwiched between the external barrier layer and the internal control layer, contains the active AM agent that inhibits the microorganism growth. The external barrier layer, which is designated to prevent the escape of the linalool to the atmosphere, is required to have low permeability coefficient for linalool, oxygen and moisture. The internal control layer sets the release rate of the AM agent from the matrix layer onto the food surface.

The matrix layer contains the active AM substance and allows it to pass through it. The basis of the matrix layer is Eudragit ? NE 40. Eudragit enables a slow and continuous release of the linalool, thus it prolongs the food preservation, after it was packed. The main advantages of Eudragit are: It can be easily mixed with other materials including ethanol and linalool, it can be easily processed and the final film is highly transparent.

The external barrier layer is composed of a Polyethylene-terephthalate (PET) and a Polyethylene (PE) laminate. The PET's low permeability serves as a barrier. The PE is used as an adhesive layer between the PET and the other layers. The control layer is composed of Ethylene Vinyl Acetate (EVA). The EVA is a thermoplastic copolymer, which readily processed into desirable shapes and thicknesses and it is highly transparent. Both Eudragit and EVA are used in the pharmaceutical industry as a controlled drug release materials.

The laminate produced, forms a suitable food packaging material. The package is relatively thin, has a high transparency and is also strong and flexible.

The formation of the multilayer film used in the current research (dissolution and evaporation instead of high temperature extrusion) demonstrates a progress relative to previous attempts. The linalool lost during the package production in the present work was much lower than in previously reported studies.

In the second part of this work, the effectiveness of the linalool and the film as an AM packaging for pastrami was evaluated. The packaging efficiency was compared with today's common market packaging technologies for pastrami.

The tests included: qualitative and quantitative examinations of linalool against microbial growth, examination of the microbial inhibition properties of the AM film

and accelerated shelf-life tests of the AM film effect on bacteria inoculated pastrami. Pseudomonas Pragi and Klebsiella pneumonia are common in pastrami flora.


Therefore, this work focused on their inhibition; however, several other microorganisms were tested as well.

The effect of linalool against microorganism was tested using the "agar disk diffusion" method. All the tested bacteria, including several types of pseudomonas, pathogenic bacteria and particularly resistant bacteria were inhibited by this AM agent. The efficiency of the linalool against bacteria was tested by performing growth experiments in liquid and solid media. In a solid medium, the bacteria, Klebsiella pneumonia, was completely inhibited and even exterminated by 0.7 mg/ml linalool. In a liquid medium, the same bacterium was completely destroyed at a linalool concentration of 0.8 mg/ml at 37°C. Inhibition signs were shown already at a concentration as low as 0.3 mg/ml linalool.

The linalool AM film was tested to evaluate the inhibition and extinction effects against Klebsiella   pneumonia. A  high initial bacteria concentration of 107 cell/ml was used for all the experiments. The results showed that higher linalool concentrations and lower temperatures yield better bacterial inhibition.

The accelerated shelf life experiments preformed with specially made pastrami with no added preservatives, which was separately inoculated with Pseudomonas fragi or Klebsiella pneumonia. The experiments included several types of packaging: AM films with 0.31% and 1% linalool with respect to the film weight, standard commercial PE packaging of pastrami (with preservatives in the product) and a control packaging (An identical laminate, only without linalool). Control experiments, with no inoculation, were placed for each type of package mentioned above. The experiments included microbial bacterial counting, pH measurements, organoleptic evaluations (by a sensory panel) and specimen color and lightness testing were carried out to indicate consequences of oxygen levels at the packaging head space. The 3% linalool packaging material was found to inhibit and even eliminate K. pneumonia, whereas the commercial packaging showed an increase in the bacteria concentration after day 18. In comparison between the controls without inoculation (3% linalool packaging and commercial packaging of pastrami) there was no evidence of bacteria in the 3% linalool pastrami packaging. However, pseudomonas bacteria were found in the commercial pastrami packaging already at the beginning of the experiment.

In conclusion, the pastrami packages including 3% linalool have a better preservation effect with comparison to common preservation methods with regards to the two selected bacteria (Pseudomonas fragi or Klebsiella pneumonia).



In the future, it is recommended to expand the research over a wider variety of microorganisms. These tests should be performed with different initial concentrations of bacteria. The shelf life should be evaluated in both real time and accelerated experiments. In addition, the preservation effect in this method (active packaging with an AM agent) has another advantage in comparison to the common modified atmosphere packaging method: the preservation effect continues even after the first opening of the package; it allows a safe reopening. This advantage has to be further investigated.

A further research direction of shelf life extension can be the use of multiple function active systems, for example, the combination of oxygen scavengers with antimicrobial compound releasing systems.