Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Olive leaf extract and usage for development of antimicrobial food packaging Z.Ö. Erdohan and K.N. Turhan, Department of Food Engineering, University of Mersin, 33343, Çiftlikköy, Mersin, Turkey. In recent years, studies on usage of antimicrobial plant extracts, like thyme, sage and olive leaf extracts, have gained acceleration since these are generally classified as GRAS (generally recognized as safe). As an important part of these studies is focused on medical beneficences, the rest of them are focused on alternative usage of the extracts. Antimicrobial packaging applications are really interesting field among these studies. This part of active packaging prolongs shelf life and maintains quality and safety of food products by extending the lag phase and reducing the growth rate of food spoilage microorganisms or food pathogens. This chapter reviews antimicrobial efficiency of olive leaf extract against some food pathogens and usage of this extract in antimicrobial packaging materials and inhibitory efficiencies against Staphylococcus aureus. Keywords: Olive leaf extract, methylcellulose, polylactic acid, antimicrobial packaging, Staphylococcus aureus

1. Introduction The knowledge of the medicinal properties of the olive tree (Olea europaea) date back to the early 1800's where it was used in liquid form as a very effective treatment for malarial infections. Pulverized leaves were used in a drink to lower fevers and a few decades later, green olive leaves were used in tea as a treatment for malaria [1]. According to Pharmaceutical Journal of Olive (1854), olive leaves have been recognized for providing many benefits in food as well as cosmetic and mostly nutraceutical applications. Now, olive leaf extract (OLE) is known with its high antioxidant, antimicrobial and antibacterial activity. OLE is very effective activity against various diseases, such as coronary artery disease, hypertension, high cholesterol level, arrhythmia, cancer, diabetes, overweight, osteoporosis, herpes, flu and colds, and some bacterial, fungus and yeast infections. It is also a natural, genetically modified organism free and allergen free product. Thus, OLE has been served in capsule form to make it easily taken recently [2]. The antioxidant and antimicrobial efficiency of the OLE are directly related with its polyphenols. There are some studies in literature revealing that polyphenols can inhibit the sporulation of Bacillus cereus and growth of Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Vibrio parahaemolyticus and Staphylococcus aureus, that all known as food pathogenes. To prevent of these pathogens, heat treatments, cold pasteurization techniques, addition of preservatives and some antimicrobials in food formulation or spread these additives on food surfaces may be applied. Antimicrobial packaging is an innovative way of inhibiting microbial growth on the foods while maintaining quality, freshness, and safety. Although there has been a rising interest in the researches in this field, availability of antimicrobials and new polymeric materials, regulatory concerns, and appropriate testing methods limit the developments. Antimicrobial packaging is highly regulated around the world and researchers must take these regulations into consideration [3].

2. Effects of Olive Leaf Extract on Food Pathogens Olive and its products are an important part of the Mediterranean diet and olive leaf is the by-product of the olive oil industry. The leaves are rich in polyphenols, namely oleuropein, tyrosol, hydroxytyrosol, rutin, verbacoside, apigenin7-glucoside and luteolin-7-glucoside [4, 5]. There are some studies in the literature showing that polyphenols are responsible for the functional properties especially antimicrobial activity [5-8]. In these studies, the phenolics are extracted with different solvents, and isolation or purification can also be applied for specific one. The choice of solvents used in extracts, cultivars of olives, crop origin, harvesting time and climate may all change the leaf composition, which could influence antibacterial activities of extracts [5, 9]. The solvent type is the most important factor affecting the efficiency of liquid-solid extraction [10]. There are many studies in the literature showing the effect of solvents used in extraction on the phenolics distribution and total phenol content in the OLEs [6, 10, 11]. An HPLC chromatogram of phenolics in the chloroform/methanol extracted OLE is shown in Figure 1 and the phenolic compounds distributions in different OLEs are given in Table 1.

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Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Méndez-Vilas (Ed.)

DAD1 A, Sig=280,4 Ref=400,100 (SIG00610.D) mAU

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Figure 1 HPLC chromatogram of phenolics in the chloroform/methanol (50/50, v/v) extracted OLE [9].

Oleuropein is the most abundant compound in OLEs given in Table 1 and followed by apigenin-7-glucoside for Extract A; hydroxytyrosol for Extract B and Extract D, and verbascoside for Extract C. Difference in the phenolics distribution may caused by the solvent type with the other factors mentioned above. Altıok and others (2008) examined the effect of different solvents on the total phenolic content of OLEs and oleuropein abundance in extracts. They obtained an extract with highest total phenolics (10.3 mg total phenolics/g leaf) and oleuropein (92 mg oleuropein/g leaf) content using 70% ethanol [10]. Pereira and others (2007) used water as solvent to obtain OLE and they detected 26.5 mg oleuropein/g lyophilized olive leaves extract [6]. Le Floch and others (1998) compared phenol yields of OLE obtained by supercritical fluid extraction and sonication. They used methanol, n-hexane, diethyl ether, ethyl acetate and carbon dioxide modified with 10% methanol as solvent for extraction. They obtained the highest and lowest phenol yields with methanol and n-hexane extraction, respectively. They also revealed supercritical fluid extraction was much successive than sonication extractions with n-hexane, diethyl ether and ethyl acetate [11]. Table 1 Peak numbers and abundances (peak area, %) of the main phenolic compounds present in OLE solutions [1, 4, 10].

Peak no

Phenolic Compounds

1 2 3 4 5 6 7 8 9 10 11 12 13

Hydroxytyrosol Tyrosol Catechin Caffeic acid Vanilic acid Vanilin Rutin Luteolin-7-glucoside Verbascoside Apigenin-7- glucoside Diosmetin-7- glucoside Oleuropein Luteolin

Extract A [4] 4.33 TA 1.35 0.94 2.55 2.43 5.00 2.56 7.11 39.26 -

Peak Area (%) Extract B [4] Extract C [10] 5.17 2.27 2.20 1.85 2.11 2.23 1.43 1.09 2.33 3.08 2.17 2.52 3.64 4.66 2.41 1.92 6.1 3.89 2.3 25.55 29 0.8

Extract D [1] 1.46 0.71 0.04 0.34 0.63 0.05 0.05 1.38 1.11 1.37 0.54 24.54 0.21

Extract A: Chloroform/Methanol (50/50, v/v), (2% OLE, w/v), Extract B: Water (2% OLE, w/v), Extract C: Ethanol/Water (70/30, v/v), (2% OLE, w/v), Extract D: Commercial (0.5% OLE, w/v)

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Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Table 2 Antimicrobial efficiency of OLEs extracted with different solvents against S. aureus [9].

Solvent Water Ethanol Methanol Chloroform Chloroform/Ethanol (50/50, v/v) Chloroform/Methanol (50/50, v/v)

Inhibition Zone Diameter (mm) 29.4±1.1 a 24.8±0.7 c 25.3±1.0 c 16.7±0.7 d 27.3±1.2 b 27.1±0.9 b

Different superscript letters in each column are significantly different (p