Effect of Oregano Essential Oil and Tannic Acid on Storage Stability and Quality of Ground Chicken Meat Marwan Alhijazeen Iowa State University

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2014 Effect of oregano essential oil and tannic acid on storage stability and quality of ground chicken meat Marwan Alhijazeen Iowa State University

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Effect of oregano essential oil and tannic acid on storage stability and quality of ground chicken meat

Marwan Alhijazeen

A dissertation submitted to the graduate faculty In partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Meat Science

Program of Study Committee:

Ahn Dong UK, Major Professor

Sebranek Joseph G

Cordray Joseph C

Dickson James S

Mendonca Aubrey F

Iowa State University Ames, Iowa 2014

TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS…………………………………………………………. iv

ABSTARCT…………………………………………………………………………...v

CHAPTER 1: INTRODUCTION……………………………………………………..1 CHAPTER 2: LITERATURE REVIEW……………………………………………...6 References…………………………………………………………………………....31

CHAPTER 3: THE EFFECT OF OREGANO ESSENTIAL OIL (origanuim vulgare subsp.hirtum) ON THE STORAGE STABILITY AND QUAILTY CHARACTERISTICS OF GROUND CHICKEN MEAT…………………………...51

Abstract………………………………………………………………………………52 Introduction…………………………………………………………………………..53 Materials and Methods……………………………………………………………….55 Result and Discussion………………………………………………………………..59 Conclusion…………………………………………………………………………...64 References……………………………………………………………………………65

CHAPTER 4: THE EFFECT OF ADDING DIFFRENT LEVEL OF TANNIC ACID ON THE STORAGE STABILITY AND QUALITY CHARACTERISTICS OF GROUND CHICKEN MEAT…………………………...81

Abstract………………………………………………………………………………82 Introduction…………………………………………………………………………..83 Materials and Methods……………………………………………………………….85 Result and Discussion………………………………………………………………..89 Conclusion……………………………………………………………………….…..94 References……………………………………………………………………………95

CHAPTER 5: THE EFFECT OF OREGANO OIL AND TANNIC ACID COMBINATIONS ON THE QUALITY AND SENSORY CHARACTRISTICS OF GROUND CHICKEN MEAT……………………………………..……………109

Abstract……………………………………………………………………………..110 Introduction…………………………………………………………………………111 Materials and Methods……………………………………………………………...113 Result and Discussion………………………………………………………………119 Conclusion………………………………………………………………………….124 References…………………………………………………………………………..125

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CHAPTER 6: SURVIVAL AND GROWTH OF NATURAL BACTERIAL FLORA AND HUMAN ENTERIC PATHOGENS IN GROUND CHICKEN MEAT WITH ADDED OREGANO ESSENTIAL OIL OR TANNIC ACID, ALONE OR IN COMBINATION…………………………………………………..147

Abstract……………………………………………………………………………..148 Introduction…………………………………………………………………………149 Materials and Methods……………………………………………………………...152 Result and Discussion………………………………………………………………156 Conclusion………………………………………………………………………….160 References…………………………………………………………………………..161

CHAPTER 7: SUMMARY AND CONCLUSIONS……………………………….174

AKNOWLEDGMENTS

My deepest gratitude goes to my major professor Dr. Dong Ahn for his continuous encouragement, guidance, warmth and support through out my PhD program at Iowa State University. Completing this dissertation would have not been possible without his patience, kindness and help. I am truly grateful to my POS committee members, Dr. Joseph G. Sebranek, Dr.

Aubrey F. Mendonca, Dr. Joseph C. Cordray, and Dr. James S. Dickson, for their commitment, advice, and help through out my PhD program. In addition, I would also like to thank my friends, colleagues, the department faculty and staff for making my time at Iowa State University a wonderful experience. I want to also offer my appreciation to those who were willing to participate in my research observations, without whom, this thesis would not have been possible. My deepest appreciation goes to Dr. Aubrey Mendonca and his research group at the Department of Food Science and Human Nutrition (Food safety laboratory), without his great commitment and help, completing the work related to the microbial part of this desertion would not have been possible. My deepest gratitude goes to my wife Rania Ibrahim for her encouragement, hours of patience, respect and love. Thanks to my family and my relatives for being with me. I’m very much grateful to all my friends in my life for all the love, care, and help given to me and for always being with me. Finally I would like to thank my sponsor Mu’tah University (Jordan) for their help and support during my study.

Thank you so much!!

ABSTRACT

Lipid and protein oxidations are most important factors that can cause quality deterioration in fresh and cooked poultry meat during storage because many secondary compounds formed by lipid and protein oxidation can cause off-odor and off-flavor production in meat. Several synthetic antioxidants such as butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) have been successfully used to prevent the oxidative changes in meat. In recent years, however, growing number of consumers are more interested in natural antioxidants than synthetic ones because of the potential carcinogenic effects of the latter. Plant polyphenol extracts are known to have strong antioxidant and antimicrobial characteristics and have been used as preservatives in meat. Oregano essential oil and tannic acid are two examples of these polyphenol plant extracts that have strong antioxidant and antimicrobial effects. The aims of this study were: 1) to evaluate the antioxidant effect of adding oregano essential oil, tannic acid, and/or their diffrent combinations to ground chicken meat, 2) to evaluate the antimicrobial effect of oregano essential oil, tannic acid, and/or their combinations in ground chicken meat, and 3) to evaluate the sensory characteristics of combined oregano oil and tannic acid in ground chicken meat. Ground chicken meat was prepared to study the effect of adding oregano oil and tannic acid in both raw and cooked meat patties. Several oregano oil and tannic acid concentrations were prepared to study their single and combination effects on meat quality. For the raw meat study, samples were individually packaged in oxygen-permeable bags and stored in a 4 °C cooler for up to 7 days. On the other hand cooked meat samples were packaged in oxygen-impermeable vacuum bags and then cooked in-bag reaching the internal temperature of 75 °C. After cooling to room temperature, the cooked meat was transferred to new oxygen-permeable bags and stored at 4 °C for up to 7 days. Chicken meat patties were analyzed for lipid and protein

oxidation, color, and volatiles at 0, 3, and 7 days of storage. Parts of the samples prepared were also used for microbial studies. Oregano essential oil at level 400 ppm significantly reduced (p < 0.05) lipid oxidation, protein oxidation and off-odor volatiles, but improved color stability of raw ground meat. The effects of oregano oil on cooked meat were similar to that of the raw meat but the effects were greater than the raw meat. Hexanal was the major aldehyde and was decreased significantly (P < 0.05) by oregano oil treatment in cooked meat and the differences among the treatments were clearer in cooked meat than in raw meat. Overall, the addition of oregano essential oil at 100 - 300 ppm was a good way of preserving meat and could replace the synthetic antioxidant, BHA. Both raw and cooked chicken breast meat added with 10 ppm of tannic acid had significantly (P < 0.05) lower lipid and protein oxidation than other treatments during storage. In addition, tannic acid at 10 ppm level maintained the highest color a*- and L*-values during storage. Cooked chicken breast meat added with > 5 ppm tannic acid had significantly (p < 0.05) lower amounts of off-odor volatiles than control. Among the volatile compounds, the amount of hexanal in cooked meat increased rapidly during storage. Therefore, tannic acid at > 5 ppm could be used as a natural preservative in ground chicken meat to improve its quality during storage. The combination of 200 ppm oregano oil and 10 ppm tannic acid showed the highest effect (P < 0.05) on TBARS, total carbonyl, and off odor volatile formation in both breast and thigh meat. The combination of 200 ppm of oregano oil and 10 ppm of tannic acid also showed the highest effect in stabilizing color values of both raw breast and thigh meats. Sensory evaluation of thigh chicken meat also indicated that 200 ppm of oregano oil and 10 ppm of tannic acid combination treatment had positive effects on most of the attributes evaluated. In conclusion, the combination of 200 ppm oregano oil and 10 ppm tannic acid could be a good replacement for the synthetic antioxidant in ground chicken meat. Oregano oil, tannic acid, BHA treatments reduced the APC significantly (P < 0.05) when used separately. However, combination of oregano + tannic acid showed

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the strongest (P < 0.05) effect in reducing the APC in raw chicken breast meat during storage at 4 and 10 oC. The combination treatment also showed the strongest antimicrobial effects of all the treatments against Salmonella enterica, Listeria monocytogenes, Escherichia coli, and Enterobacteriacea (ENT) in raw meat during storage. In cooked chicken breast meat, oregano oil and oregano + tannic acid combination were efficient (P < 0.05) in suppressing the growth of aerobic bacteria in meat at all temperatures (10, 25, and 43 oC) tested. Oregano essential oil treatment was more effective than the BHA and tannic acid treatments in slowing down the growth and survival of Stapylococcus aureus in cooked meat during storage at 10, 25, and 43 oC, but oregano oil + tannic acid treatment was more effective than the oregano oil alone. Based on these results it is suggested that oregano oil has a strong antimicrobial activity but its effect can be further improved when it is combined with tannic acid. This study demonstrated that oregano oil + tannic acid combination could be a good natural antimicrobial treatment to suppress most of the food borne pathogens in chicken meat. In conclusion oregano essential oil at > 200 ppm significantly improved meat quality and prevented meat deterioration during storage. Tannic acid at 10 ppm also improved meat quality and safety significantly during storage. However, the combination of 200 ppm oregano essential oil and 10 ppm tannic acid showed higher effect on both meat quality and safety of ground chicken meat than using them singly. This combination could be an excellent replacement for synthetic antioxidants such as BHA, BHT and TBHQ, which are commonly used for meat preservation.

CHAPTER 1

INTRODUCTION

Oxidation process in fresh and cooked meats can be controlled by using artificial antioxidants such as butylatedhydroxyanisole (BHA), butylatedhydroxytoluene (BHT) and tertiary butylhydroquinone (TBHQ) (Monahan and Troy, 1997), but these could cause negative effects on human health. Therefore, many studies tested naturally-occurring antioxidants such as ascorbic acid, vitamin E and certain crude plant extracts as a replacements for synthetic antioxidants. Recently, researchers have been more interested in natural than synthetic antioxidants because consumers and industry prefer foods with natural antioxidants to synthetic ones (Kosar et al., 2005; Monahan and Troy, 1997; Solomakos et al., 2008). During past two decades, many researchers tested herbs and plant extracts like oregano, sage, rosemary, and grape seed (Economou et al., 1991; Man and Jaswir, 2000; Yanishlieva and Marinova, 1995) because they contain chemical compounds that can increase or enhance shelf-life of foods, make the them more resistant to oxidation, slow down the microbial or fungal growth, and show positive effect on their physiochemical properties (Shahdidi et al., 1992 ; Brannan, 2008; Abdel-Hamied et al., 2009). Lipid oxidation is one the most important problems that affect the quality and shelf life of meat, especially in poultry meat. It produces many volatile compounds such as aldehydes, which are responsible for off-odor (Ahn et al., 1998, Ahn and Lee, 2002), off-flavor (Dietze et al., 2007) and rancidity (Campo et al., 2006; Ahn et al., 2002; Byrne et al., 2002), and change meat color (Guillen and Guzman, 1998). Oxidation of lipids can occur in both fresh and cooked meats (Min and Ahn, 2005; Jo et al., 2006), and can have significant impact to meat industry. Many factors are influencing the development of lipid oxidation in meat, but the amount of fat, packaging, metal catalysts and degree of polyunsaturation in the lipids of meat play the most important roles (Gray and Pearson, 1987; Calkins and Hodgen, 2007)

Protein oxidation is also considered as an important factor that can cause changes in meat quality. As in lipid oxidation, protein oxidation is induced by several free radical species such as hydroxyl radicals, peroxyl and alkoxyl radicals as well as superoxide anions, hydrogen peroxide, and nitric oxide (Lawrie, 1998; Ouali et al., 2006; Hopkins and Geesink, 2009). The oxidation process in proteins leads to the destruction of amino acids and forms carbonyl compounds, which will affect protein functionality such as gel-forming ability, water-binding capacity, emulsifying capacity, solubility, and viscosity (Howell et al., 2001). The mechanisms of protein oxidation are very similar to lipid oxidation, but protein oxidation produces more complex oxidation products than lipid oxidation: In protein oxidation, hydrogen atom abstraction by ROS produces a protein carbon-centered radical (P·). In the presence of oxygen this carbonyl radical will be converted to peroxyl radical (POO·), and then converted to peroxide (POOH), alkoxyl radical (PO·) and its derivatives (POH) which are the final primary protein oxidation products (Lund et al., 2011; Estevez, 2011). Finding natural antioxidants to resolve the lipid and protein oxidation is of great interest recently (Brewer, 2011). Oregano essential oil is one of the most popular herb extracts that can be used as a meat preservative. Oregano is commonly used as a spice in the Mediterranean cuisine, and is produced by drying leaves and flowers of Origanum vulgare subsp. Hirtum plants. Oregano is well known for its antioxidant activity (Economou et al., 1991) because of its high phenolic content. There are two major phenols in oregano: carvacrol and thymol, which constitute about 78-82% of the essential oils in oregano, and are responsible for most of the antioxidant activity of oregano oil (Adam et al., 1998; Yanishlieva et al., 1999). Other phenolic compounds such as γ-terpinene and p-cymene take about 5 to 7 % of the total oil, and they also have strong antioxidant activities. The essential oil of oregano is also known for its antimicrobial effects. Sivropoulou et al. (1996) reported that the three species of oregano essential oils they tested showed strong antimicrobial activities against eight different strains of gram-positive and negative bacteria. Chouliara et al. (2007) studied the combined

effect of oregano essential oil on the shelf-life of fresh chicken breast meat in modified atmosphere packaging and found that the microbial populations (lactic acid bacteria, TVC, pseudomonas spp., yeast) and lipid oxidation significantly decreased by adding oregano oil. They indicated that thymol and carvacrol damaged bacterial cell membranes, destabilized the operation of phospholipids bilayer of bacteria, entered into the bacterial cell, and changed cell processes (Kalemba and Kunicha, 2003; Cristani et al., 2007; Xu et al., 2008; Di Pasqua et al., 2010). Another natural antioxidant found in most plant foods is tannic acid or tannins. Tannic acid is widely used in the process of converting animal skins to leather. Tannic acid is a water–soluble polyphenol found in a wide variety of plants in different concentrations (Chung et al., 1998a). For example, food grains such as millets, barley, peas, fababean, legumes, sorghum, and millets (Chaven et al., 1977; Hulse, 1980; Price and Butler, 1980; Salunkhe et al., 1982; Deshpande et al., 1984), fruits such as bananas, apples, grapes, and strawberries have a good quantity of tannins (Lease and mitchell, 1940; Reeve, 1959; Sanderson et al., 1975; Jones and Mangan, 1977). Wines and tea also have tannins (Lyoyd, 1911; Reeve., 1959; Hoff and Singleton, 1977). Tannins are classified into two categories: hydrolysable and non-hydrolysable (condensed) tannins (Freudenberg, 1920). The hydrolysable types contain polyhydric alcohol and hydroxyl groups, which are connected to gallic acid such as gallotannins or hexahydroxydiphenic acid (ellagitannins) by ester bonds. After hydrolysis by enzymes or acid/base conditions, gallotannins produce glucose and gallic acid (Hagerman, 2010; Salminen et al., 2011). The FDA categorized tannic acid as a generally recognized as safe (GRAS) food additive, which is allowed to use at 400 ppm in frozen dairy desserts and mixes, 50 ppm in non-alcoholic beverages and beverage bases, 150 ppm in alcoholic beverages, 100 ppm in backed goods and backing mixes, and 10 ppm in meat products (FDA, 2011). The radical scavenging and antioxidant mechanisms of tannic acid are still not fully understood and need more investigations (Gulcin et al., 2010). It is generally

known that the antioxidant activity of tannic acid is coming through the inhibition of hydroxyl radical formation from the Fenton reaction by complexing with ferrous ions (Lopes et al., 1999). However tannic acid may act as a pro-oxidant in the presence of copper ions, causing DNA damages (Freguson, 2001). Tannic acid can cause DNA degradation or DNA base modification in the presence of Cu(II) due to the generation of reactive oxygen species (Baht and Hadi, 1994a, 1994b). Glucin et al. (2010) reported that the structural features of tannic acid are responsible for the antioxidant activity, but it also can contribute to the pro-oxidant activity of tannic acid. It can produce hydroxyl radical in the presence of metal catalyst such as Cu(II), but it also can chelate large number of free radicals (Khan et al., 2000). Tannic acid is used in many foods as flavoring agent and has pharmacological applications in medicinal products to treat burns, diarrhea and chemical antidotes in poisoning and astringents (Hirono and Yamada, 1987). Furthermore, tannic acid is used in brewing and wine industries as a clarifying agent, and flavoring agent in frozen dairy desserts, baked foods and meat products (Chung et al., 1998a). Tannins and their derivatives showed significant antimicrobial activity against several foodborne pathogens (Chung et al., 1998b; Kim et al., 2010). The antimicrobial mechanisms of tannins are as follow: 1) through the astringent effect of tannins to many microbial enzymes such as cellulase, pectinase and xylanase; 2) their toxicity on the cell membrane of microorganisms; and 3) their binding ability to metals (metal chelator), which reduce the availability of essential metal ions such as iron, which is important for the metabolic activities of microorganisms (Scalbert, 1991). However, the antimicrobial mechanisms of tannins still need more studies, and suitable levels that could be used in food processing also should be determined. Strong evidence indicated that tannic acid has anti-carcinogenic effects (Korpassy, 1961; Morton, 1972; Pamukcu et al., 1984; Salunkhe et al., 1990). Green tea, fruit, and vegetables, which contain high levels of tannins and some other phenolic compounds, inhibited the growth of cancer cells in the experimental animals such as rats (Stocks, 1970; Stich and Rosin, 1984; Das et al., 1989; Shi et al., 1994).

Over all it is very rare to find research studies on using tannic acid as meat preservative in the recent publications especially in poultry meat. Poultry meat is considered as one of the most popular food commodities around the world, and their consumption increased dramatically over the last two decades in many countries (Chouliara et al., 2007). Recently, shelf-life extension of poultry meat has become one of the main concerns in the industry (Chouliara and Kontominas, 2006; Chouliara et al., 2007). Use of multiple factors or technology combinations called “hurdle technology’’ to suppress microorganism in food is of great interest for many researchers (Jay, 2000). However, no study has been conducted using the combination of both oregano oil and tannic acid to improve the safety and quality of poultry meat. The over all objective of this study is to determine the suitable levels of these two natural plant extracts (oregano oil, tannic acid, and their combinations) as the replacement of synthetic antioxidants to improve the quality as well as the safety of poultry meat products. The specific objectives are: 1) to evaluate the antioxidant effect of adding oregano essential oil, tannic acid, and/or their combinations to the ground chicken meat, 2) to evaluate the antimicrobial effect of oregano essential oil, tannic acid, and/or their combinations in ground chicken meat, and 3) to evaluate the sensory characteristics of combined oregano oil and tannic acid in ground chicken meat.

CHAPTER 2 LITERATURE REVIEW

Oxidants and free radicals in body systems Oxygen is essential to human life. Yet, paradoxically, oxygen is also involved in toxic reactions, and thus is a constant threat to the well-being of all living things (Halliwell and Gutteridge, 1989). Unfortunately, human natural defense systems are imperfect; they limit the harm caused by oxygen but do not eliminate it completely. Many evidences suggested that as years go by oxygen-induced damage to body tissues may accumulate (Machlin and Bendich, 1987). This damage has been hypothesized to be a major contributor to aging and many of the degenerative diseases including cardiovascular diseases, cancers, cataracts, age-related decline in immune system, and degenerative diseases of nervous system (Langseth, 1995). Most of the potentially harmful effects of oxygen are due to the formation and activity of reactive oxygen species. Reactive oxygen species are produced continuously in the human body as a consequence of normal metabolic processes and act as oxidants. Many reactive oxygen species are free radicals (Halliwell and Gutteridge, 1989). A free radical is any chemical species that has one or more unpaired electrons. Many free radicals are unstable and highly reactive. The chemical reactivity of hydroxyl radical

. .- . (OH ), superoxide radical (O2 ), nitric oxide radical (NO ) and lipid peroxyl radical (LOO.) can damage all types of cellular macromolecules, including proteins, carbohydrates, lipids, and nucleic acids (Dalle-Donne et al., 2003), But they are not always harmful. For example, some free radicals by specialized blood cells like phagocytes play a role in the destruction of disease-causing microbes (Langseth, 1995; Hampton et al., 1998).

Lipid oxidation mechanism and its effect on meat quality Lipid oxidation is considered as the most important factor that causes meat deterioration in both fresh and cooked meat products (Ladikos and Lougovois, 1990,

Nam et al., 2002; Ahn et al., 2009). This process is responsible for the formation of free radicals that leads to form various volatile compounds including aldehydes, which participate in the development of rancid flavor, and change meat color (Guillen and Guman, 1998; Ahn et al., 2009). Poultry meat tissues have low lipid content but contain relatively high polyunsaturated fatty acids (Igene and pearson, 1979, Pikul et al., 1984). Polyunsaturated fatty acids are highly susceptible to free radical attack that leads to the production of hydroperoxides, which are responsible for the formation of secondary by-products such as aldehydes, ketones and other compounds that affect negatively on the over all food quality (Lund et al., 2011;Vercellotti et al., 1992). This quality change will cause loss of protein solubility, loss of color, reduce nutritive value of food, and loss of vitamins such as vitamin E (-tocopherol), D, A and C (Madhavi et al., 1996; Min and Ahn, 2005). In addition, lipid oxidation is also reported to affect meat texture and over all their safety (Gray and Monahan, 1992). The rate of lipid oxidation in fresh and cooked meat products depends on various internal factors such as fat content, fatty acid composition, and antioxidants, heme pigments and iron content (Addis, 1986; Du et al., 2000; Choe and Min, 2006; Min et al., 2008). The external conditions such as processing, packaging and storage conditions also play important roles in lipid oxidation process (Ahn et al., 2009). Lipid oxidation can occur by autoxidation, photoxidation, and enzymatic oxidation processes. The enzymatic oxidation process is facilitated by cyclooxygenase and lipoxygenase. Photoxidation is primarily found in milk powder and oils, and their effect on meat quality is minimal (Choe and Min, 2006; Ahn et al., 2009). However Autoxidation of oils requires radical forms of acylglycerols, on the other hand

1 photosensitized oxidation dose not require lipid radicals since O2 reacts directly with double bonds. Autoxidation is considered the major mechanism that causes lipid oxidation in food and meat products and is initiated by reactive oxygen species (ROS) (Gray and Monhan, 1992; Kanner, 1994; Min and Ahn, 2005). Autoxidation mechanism is well studied and summarized into three main steps - initiation, propagation, termination (Gray and Monahan, 1992; Min and Ahn, 2005;

Laguerre et al., 2007). The initiation step is the rate-limiting, but very important in lipid peroxidation process, this usually starts by reactive oxygen species that abstracts hydrogen atoms from the site of a fatty acid chain and form a lipid free radical (L.). This lipid free radical will react rapidly with oxygen to form a peroxyl radical (LOO.). A more detailed explanation of the initiation step reported that the formation of lipid free radicals increased with the increase in the total number of bis-allylic carbons, where the energy between C-H bond between two double bonds position is the weakest (75-90 Kcal/mol), and the other positions at allylic and alkyl C-H bond are 88 kcal/mol and 101 kcal/mol, respectively (Sevanian and Hochstein, 1985; Buettner, 1993; Wagner et al., 1994). Therefore, C-H bond between two double bonds is the most reactive site for hydrogen abstraction (Min and Ahn, 2005; Choe and Min, 2006). The peroxyl radical (LOO.) formed in the presence of oxygen abstracts hydrogen atom from another hydrocarbon chain, and yields hydroperoxide (LOOH) and a new free radical (L.), which participate in propagation chain reaction (Pearson et al., 1977; Enser, 1987). Lipid peroxyl radical and alkoxyl radical can also abstract hydrogen atom from another lipid molecule and initiate and propagate the chain reaction (Addis, 1986; Coyle and Puttfarcken, 1993; Min and Ahn, 2006). The final step is called termination step in which free radicals such as lipid peroxyl radicals (LOO.) react with each other to form non-radical products. Usually lipid hydroperoxide (LOOH) is stable at the physiological temperature, but will be decomposed at high temperature or when it is exposed to metal ions (Gruger and Tappel, 1970; Halliwell and Gutteridge, 1990; Choe and Min, 2006). The decomposition of hydroperoxides (ROOH) through free radicals by the homolytic and hetrolytic -scission, catalyzed by transitional metal ions, produces a variety of volatile and nonvolatile compounds (Choe and Min., 2006). The homolytic cleavage pathway of hydroperoxide is more likely to happen first due to the low activation energy (46 kcal/mol) between oxygen-oxygen bonds compared to the oxygen-hydrogen bonds (Hiatt et al., 1968). The homolytic -scission of carbon-carbon bond is the next step for the alkoxy radical converting to

oxo-copmpounds and other alkyl radicals (Choe and Min, 2006). Formation of peroxyl or an alkoxyl radical is considered the first step during the decomposition of hydroperoxides (Gardner, 1989). However, peroxyl and alkoxyl radicals are relatively difficult to generate from the structure of hydroperoxides: a ROO-H bond has bond dissociation energy about 90 kcal/mol and a RO-OH bond of about 44 kcal/mol (Gardner, 1989). Thus, the formation of peroxyl and alkoxyl radical is highly depending on the presence of catalyst and other favorable conditions such as light, oxygen availability, and heat (Schaich, 1992; Velasco et al., 2003). The decomposition progress of hydroperoxides is very slow in the presence of antioxidants, low temperature and the absence of catalyst (Gruger and Tappel, 1970). Hydroperoixde compounds may be converted to different monomeric decomposition products which are composed of different functional groups such as keto, hydroxyl and epoxy group (Schieberle et al., 1979; Frankel, 1987). The low molecular weight of volatile oxidation products are usually produced by the decomposition process of hydroperoxides. These compounds play important roles on the flavor and odor of foods (Grosch et al., 1981). The formation of volatile and non-volatile oxidation secondary products is highly dependant on the composition and isomeric distribution of monohydroperoxides and cleavage pathway processes (Frankel, 1987). In addition, polymeric oxidation products composed of two or more fatty acid units where they are connected together by peroxide or carbon-carbon linkage are the products of the hydroperoxide decomposition (Miyashita et al., 1984; Neff et al., 1988). In the meat system, the secondary products of lipid oxidation such as carbonyls (ketones and aldehydes), alcohols, hydrocarbons (alkane, alkene), and furans play an important role in meat flavor and rancidity development in food systems (Frankel, 1984; 1987). Lipid oxidation products also affect protein solubility, emulsification, water-binding capacity, texture and the other rheological properties through the interactions between lipid and protein oxidation products (Hall, 1987).

Protein oxidation and its effect on meat quality The physical, chemical and functional properties of proteins mainly depend on the amino acid structure and polypeptide backbone. These functional properties are very important for protein quality in the final products of processed meat. Gelation, solubility, viscosity, and emulsification properties can be altered when any changes in amino acid side chain or conformational shape of the final protein structure occur (Xiong, 2000). Furthermore, protein quality can be modified and changed by many reactions with reactive oxygen species (Nystrom, 2005). In meat tissues, myofibrillar proteins are the main components that affect protein quality and functionality. These proteins can be oxidized by ROS (reactive oxygen species) during the aging or storage period. The most common consequences of protein oxidation are as follow (Lund et al., 2011, Estevez, 2011): first the reactive oxygen species react with protein and peptides in the presence of oxygen from the back bone of amino acid side chains. Protein exposure to ROS, irradiation, light, metal catalyst, and peroxidation form protein radicals (P.). The results of these reactions include protein cross linking, modification of amino acid side chains, and protein fragmentation. These changes in proteins affect the hydrophobicity, conformation, and solubility, and alter their susceptibility to proteolytic enzymes. In addition, protein hydroperoxide (POOH), protein carbonyl (P=O), and protein sulfoxide or sulfone are produced. Lund et al. (2011) explained the mechanisms of protein oxidation in muscle as a three step process: In the initial step, ROS leads to abstraction of a hydrogen atom from a protein molecule and form a protein carbon-centered radical (p.). In the next step, this protein radical is converted to a peroxyl radical (POO.) in the presence of oxygen, and then to a hydroperoxide or alkyl peroxide (POOH) by abstracting a hydrogen atom from a different molecule. Finally protein oxidation products such as hydroxyl derivative (POH) and alkoxyl radical (PO.) are produced by the reactions of

. 2+ + POOH with ROS (HO2 radical) or catalyzed by reduced metals (Fe or Cu ). Oxidation in meat tissues has been measured using different methods depending on

the chemical reactions and products such as loss of sulfhydyle groups, loss of tryptophan fluorescence, formation of carbonyl derivatives, inter- and intra-molecular cross links between amino acid residues. DNPH (2,4-dinitrophenylhydrazine) is an earlier method that determines the carbonyl content (nmol/mg protein) in muscle samples (Lund et al., 2008). This method has been used in protein oxidation studies in foods, but is considered nonspecific for measuring the protein oxidation or the actual level of carbonyls in meat tissues (Cao and Culter, 1995). For example, protein oxidation reaction can form other compounds than carbonyls. In addition, the carbonyl moieties could be formed by other mechanisms than amino acid oxidation in their residues. Advanced methodologies to measure protein oxidation such as electron spin resonance spectroscopy, fluorescence spectroscopy, and HPLC coupled to fluorescence or MS were developed later. These methods are very useful to understand the mechanisms of protein oxidation, role of iron, myoglobin, lipids, and phenolic compounds through this process (Lund et al., 2011). Carbonylation is considered as one of the most remarkable chemical changes during protein oxidation. Using HPLC-MS coupled with flourcence, Estevies et al. (2011) identified a specific carbonyl compounds in oxidized myofibril proteins. They found that two semialdehydes, -aminoadpic (AAS) and -glutamic semialdehyde (GGS), were produced mainly by protein carbonylation. These semialdehydes could form around 70% of the total amount of protein carbonylation in oxidized animal proteins. The AAS and GSS are usually formed in the presence of ferric iron (Fe+3) and H2O2 by oxidizing amino acids such as lysine, praline and arginine in animal tissues. Carbonyls such as ketones and aldehydes can be formed through four pathways as reviewed by Eztevies (2011): 1) Directly by oxidation of side chain amino acid (lysine, threonine and proline), 2) Glycation by a non-enzymatic reaction in the presence of reducing sugars, 3) The oxidative cleavage of the peptide backbone via the -amidation pathway or via oxidation of glutamyl side chain, and 4) Covalent binding to non-protein carbonyl compounds such as 4-hydroxyl-2-nonenal or malondialdehyde (MDA).

Protein is the major component in muscle tissue that affects their nutritional value, functional properties, and sensory traits (Lawrie, 1998). Protein oxidation process affects the amino acid binding ability, decrease protein solubility (polymerization), decrease protolytic activity, and loss of their nutritional value (Lund et al., 2011). Possible mechanism on how protein carbonylation could affect technological and sensory traits of meat and their products were reviewed by Eztevez (2011), Lund et al. (2011), and Xiong (2000). They showed that carbonylation affected negatively on meat flavor, tenderness, and water holding capacity, juiciness, and protein functionality. These changes in meat quality caused by carbonylation decreased proteolytic enzyme activity (e.g., calpain system); cross linking which stabilize the aggregation, loss of ε-NH3 group, and alteration of the electrical arrangement of meat proteins. In addition, protein oxidation has a negative effect on human health: carbonylation is associated with human diseases such as Alzheimer’s, Parkinson’s, cancer, catacataractogenesis, diabetes, and sepsis (Levine, 2002; Dalle-Donne et al., 2003).

Antioxidants Halliwell and Gutteridge (1985, 1989) defined antioxidant as “any substance, when present at much low concentrations compared with that of an oxidizable substrate, that significantly delays or inhibits oxidation of that substrate”. One important line of defense in biological systems is enzymes, including glutathione peroxidases, superoxide dismutases and catalase, which decrease the concentrations of the most harmful oxidants. Nutrition (several essential minerals including selenium, copper, manganese and zinc) is involved in the structure or catalytic activity of these enzymes and plays a role in maintaining the body's enzymatic defense against free radicals. A second line of defense in human is small-molecular-weight compounds which act as antioxidants. They react with oxidizing chemicals, reducing their capacity for damaging effects. Compounds such as glutathione, ubiquinol and uric acid are produced by normal metabolism. Other

small-molecular weight antioxidants are found in diet, which include vitamin E, vitamin C, carotenoids, phenolic, and polyphenolic compounds (Langseth, 1995). Synthetic antioxidants such as butylatedhydroxytoluene (BHT), butylatedhydroxyanisole (BHA) and tert-butylhydroquinone (TBHQ) have been dominant (Dapkevicius et al., 1998), but they may be harmful to human health. Therefore, many studies focused on testing the effects of naturally occurring antioxidants such as ascorbic acid, vitamin E and certain crude plant extracts (Kosar et al., 2003, 2005).

Using natural antioxidants as meat preservatives to prevent lipid and protein oxidation Recently there have been many researches done using natural antioxidant from a plant origin. This growing interest was due to the possible carcinogenic effects from using the synthetic antioxidants; the assumption that food contains certain amount of phytochemicals can affect the etiology and pathology of chronic disease and aging process in human body (Dorman and Hiltunen, 2004; Shahidi, 2008). In addition, consumer demands of finding more natural and healthy food product and avoid using the synthetic ones have been increased. In the meat and chicken products, rosemary and sage extracts found to be effective in reducing the rancidity and improve shelf-life (Murphy et al., 1998). Many herbs such as oregano, sage, thyme, basil, black and white pepper have antioxidant activity in porcine meat when used at 0.5% to 2.5% level (Fasseas et al., 2007). Tea catechins and carotenoids also showed antioxidant effects (Tang et al., 2000, 2001; Staszewski et al., 2011). Bostoglou et al. (2002) studied the effect of dietary oregano oil on the performance of broiler chicken (1-day old) and on the iron-induced lipid oxidation of breast, thigh and abdominal fat tissues, and found that the birds’ performance was unaffected by the experimental diet. However, the essential oil showed a significant antioxidant effect in the tissues. Botsoglou et al. (2003c) reported that dietary oregano oil had a significant effect in reducing lipid oxidation in

turkey meat during frozen storage. Brannan (2008) reported that adding 1% of grape seed extract to the ground chicken meat reduced lipid oxidation in processed meat. Abdel-Hamied et al. (2009) reported that adding natural antioxidant (rosemary, sage and their combination) extracts reduced oxidative rancidity and microbial growth in minced meat during refrigerated storage. Du and Ahn (2002) studied the effect of adding natural antioxidants (vitamin E, sesamol, rosemary, gallic acid) on the quality of irradiated sausages prepared with turkey thigh meat, and found that the antioxidants reduced the redness of meat and total volatiles. However, they did not show significant effect on the off-flavor of turkey sausages induced by irradiation. Adding oregano essential oil to beef Longissimus dorsi and Semimembranosus muscles decreased lipid oxidation and extended their shelf life, but adding higher levels of oregano essential oil showed negative effect on the palatability of the beef steaks because of the strong flavor from the essential oil (Scramlin et al., 2010). Natural plant extracts such as rosemary contain many phenolic compounds including rosmarinic acid, carnosic acid, rosmaridiphenol, rosmariquinone, rosmanol and carnosot, which react with lipid hydroxyl radicals, stabilize them and, chelate ionic ions such as Fe+2. Protein oxidation also can cause changes in meat quality such as meat color because of the conversion of myolglobin to oxymyoglobin or metmyoglobin. This oxidation process is accelerated by free radical species such as hydroxyl radicals, peroxyl radicals, superoxide anions, hydrogen peroxide, and nitric oxide (Lawrie, 1998; Ouali et al., 2006; Ahn et al., 2009). Protein oxidation can lead to the destruction of amino acids and form a carbonyl compound which will affect the protein functionality such as gel-forming ability, water-binding capacity, emulsifying capacity, solubility, and viscosity (Howell et al., 2001). Several changes in muscle fiber proteins during the post mortem phase can affect meat tenderness, pH, and color. In addition proteolytic enzymes can be affected by the oxidation process, which may inhibit the activity of these enzymes and affect the degradation process during the postmortem (Guttmann and Johnson, 1998). Rowe et al. (2004) examined the effect of

early postmortem protein oxidation on the color and tenderness of beef steaks, and found that increased oxidation of muscle proteins early postmortem could have negative effects on fresh meat color and tenderness. Finding natural antioxidants used to prevent the protein and lipid oxidation in the meat system is well documented (Abdel-Hamied et al., 2009; Brewer, 2011). Dietary supplementation of natural antioxidants such as tocopherols, rosemary, green tea, grape seed, and tomato extracts increased the oxidative stability of broiler breast meat and decreased the protein and lipid oxidation (Smet et al., 2008). Furthermore, the natural antioxidants played important roles in inhibiting the oxidation process not only during storage but also during cooking of pork (Salminen et al., 2006). The mechanisms of these natural antioxidants are very similar to those of the synthetic ones, which scavenge the free radicals, absorb light in ultraviolet (UV) region (100 to 400 nm), and form chelating complex with transition metals. All of these properties can decrease the autoxidation process, off-odors, and off-flavor production (Brewer, 2011). In the meat industry, recently finding new combinations of natural plant extracts have been of great interest. These natural antioxidants were used directly adding to the meat product (Jo et al., 2006; Abdel-Hamied et al., 2009; Glucin et al., 2010) or dietary supplement to the animal diet (Botsoglou et al., 2003a,b,c; Jamilah et al., 2009; Ariza-Nieto et al., 2011). Due to their positive effects on the food quality, safety, and over all animal health researchers found them as good replacements for the synthetic antioxidants.

Oregano Oregano has been used for flavoring fish, meat and sauces since ancient times (Sahin et al., 2004). In the Middle East where it grows abundantly, it has the common name of Za'atar and is usually collected for human consumption (Daouk et al., 1995). There are two major phenolics in oregano: carvacrol and thymol, which constitute about 78-82% of the essential oil, and are responsible for the antioxidant activity (Adam et al., 1998; Yanishlieva et al., 1999). γ-Terpenene and p-cymene constitute

about 5 to 7% of the total oregano essential oil, and also have antioxidant activity. It is important to know that the composition, production and quality of oregano essential oil can vary depending upon many factors including genetic, seasonal, temperature, moisture, soil, and daylight length (Farooqi et al., 1999; Russo et al., 1998; Gonuz and Ozorgucu, 1999; Ceylan et al., 2003; oncer et al., 2009; Viuda-Martos et al., 2010). Their antioxidant (Burt, 2004; Skerget et al., 2005) or antimicrobial activity (Sivropoulou et al., 1996; Adam et al., 1998) are also highly dependent on their chemical phenolic composition mainly carvacrol and thymol.

Essential oil composition of Origanum The essential oil in the Lebanese Za’atar (Origanum syriacum L.) is characterized for its high thymol and carvacrol content (Daouk et al., 1995; Tepe et al., 2004). On the other hand, the essential oil (EO) of Origanum majorana L. is characterized by the dominant occurrence of linalool, terpinen-4-ol, and sabiene hydrate (Berna’th, 1997). It is used as a folk remedy against asthma, indigestion, headache and rheumatism (Jun et al., 2001). Origanum syriacum var. bevanii collected from Turkey contains 43% – 79% of carvacrol (Baser, 2002). Alma et al. (2003) found that the major components in the essential oil of Origanum syriacum grown in Turkey are high in -terpenene, carvacrol, paracymene and -caryophyllene. Thymol or 5-methyl-2-(1-methylethyl) phenol (Lee et al., 2003) is a phenolic compound and is one of the predominant components of oil derived from the thyme plant, Thymus vulgari, and from Oregano, Origanum vulgare (Hazzit et al., 2006). It has antimicrobial (Lamber et al., 2001), antioxidant, astringent and antibiotic properties (Braga et al., 2006), and has been used as an oral care product. Because of its distinctive sharp odor and pungent flavor, thymol has adverse effect on the sensory characteristics of products when used in high levels (Lee et al., 2008) (Figure 1). Carvacrol or 2-methyl-5-(1-methylethyl) phenol is a phenolic compound found in certain essential oils (EOs) and shows antimicrobial, antioxidant and antiviral activity. It is abundant in some oils and its level can reach to 75% of essential oil (Sokmen et

al., 2004) and shows highly specific activities as compared to other EO components (Dorman et al., 2000). p-Cymene is an aromatic hydrocarbon that occurs widely in tree leaf oils (Yatagai and Sato, 1985). Its chemical structure is similar to that of the styrene. Generally, a good solvent for a certain polymer has a solubility parameter value close to that of the polymer. p-Cymene can be used as an alternative shrinking agent for expanded polystyrene (Hattori et al., 2010). Terpinen-4-ol is widely distributed in various plants such as oranges and mandarins, origanum, the New Zealand lemonwood tree and the Japanese cedar to black pepper. With other terpenes, it is thought to contribute to the unique taste of the Cuban bullock’s heart fruit (Pino et al., 2003). Terpinen-4-ol has shown a strong antimicrobial and anti-inflammatory property (Hart et al., 2000). Carvone is known as a component of spearmint (Egawa et al., 2003; Roohi and Imanpoor, 2014). Carvone is a member of terpenoids (Simonsen et al., 1953) found in a small amount in oregano vulgare spp. hirtum (Russo et al., 1998). Carvone is found naturally in many essential oils but is the most abundant in the oils from seeds of caraway (Carum carvi) and dill. Carvone has two mirror image forms or enantiomers: S-(+)-carvone smells like caraway. Its mirror image, R-(–)-carvone, smells like spearmint (Figure 1). Both carvones are used in the food and flavor industry (De Carvalho and Da Fonesca, 2006). R-(-)-carvone is also used for air freshening products, and like many essential oils, oils containing carvones are used in aromatherapy and alternative medicines (Leitereg et al., 1971). The amounts and kinds of other constituents in Oreganuim depend on the species and method of analysis (GC, HPLC): many other terpenoids including gamma-terpenene, borneol, linalool, and alpha-terpenene are found in O. onites (Ceylan et al., 1999; Baydar et al., 2004; Kacar et al., 2006; Toncer et al., 2009). Sabinene, limonene, chrysoeriol, diosmetin, quercetin, eriodictyol, cosmoside, vicenin-2, thymoquinol glycosides, neoisodihydrocarvyl acetate, caryophyllene oxide, oleanolic acid, trans-carveol, bornyl acetate, lithospermic acid, aristolochic acids, and

alph-pinene are also found in Origanum vulgare Species (Adam et al., 1998; Antuono et al., 2000). Some minor components such as aristolochic acid are still under investigation (Brewer, 2011).

Antioxidant activities of oregano The antioxidant activity of natural sources, including Origanum species, has been investigated by many researchers. Al-Bandak (2007) investigated the antioxidant activity of O. syriacum in corn oil at concentrations of 200, 500 and 1000 ppm, and found that diethyl ether (D) and ethanol (E) extracts of O. syriacum have the highest protection against oxidation followed by ethyl acetate (EAc) and petroleum ether (P). Tepe et al. (2004) suggested that the essential oil and extracts from the herbal parts of O. syriacum could be used as natural preservatives in food industry. They found that the inhibitive effect of Origanum on linoleic acid oxidation might be promoted by the presence of non-polar phenolics because both hexane and dichloromethane extracts showed high antioxidant activities. Sahin et al. (2004) suggested that the essential oil of Origanum vulgare ssp. vulgare possesses compounds with antioxidant activity, and therefore can be used as a natural preservative in food and/or pharmaceutical industry. Capecka et al. (2005) found that the strongest inhibition of linolenic acid (LA) peroxidation was found in both fresh and dried O. vulgare. Kosar et al. (2003) investigated antioxidant compounds in Origanum vulgare L., O. onites L., O. minutiflorum and O. syriacum L., and found that rosmaric acid and carnosic acid were the dominant radical scavengers in the extracts. Purified compounds from O. majorana L. possessed both O2-scavenging activity and an inhibitory effect against TPA-induced O2 generation (Jun et al., 2001). The antioxidant capacity of essential oils and aqueous tea obtained from Origanum vulgare, Thymus vulgaris and Thymus serpyllum showed a dose-dependent protective effect against copper-induced LDL oxidation (Kulisic et al., 2007). The protective effect of essential oils was due to the presence of phenolic monoterpenes, thymol and carvacrol, which are identified as the dominant compounds in these

essential oils. The strong protective effect of aqueous tea was proposed to be the consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids. Tsimogiannis et al. (2006) examined Origanum heracleoticum as a potential source of phenolic antioxidants. They extracted the component from plant using a Soxhlet apparatus sequentially using solvents with different polarity and found that the activity of the extracts widely varied depending upon the solvents used. The prime justification for using an antioxidant in frying oils is to delay lipid oxidation during frying. Antioxidants retard oxidation and prolong the shelf-life of the product. Recently, research on natural antioxidants for frying purposes has increased. Plant extracts, especially those obtained from herbs and spices, have been proposed for stabilizing frying oils and fried products (Houhoula et al., 2003). The effect of Origanum vulgare on the oxidative stability of cottonseed oil during frying of potato chips and on the storage stability of the produced chips indicated that the oxidation rates of both frying oil and the fried product were decreased when Origanum vulgare was added to the frying oil (Houhoula et al., 2003). Tsimidou et al. (1995) tested dry Origanum vulgare for its antioxidant activity in mackerel oil stored at 40 °C in the dark and found that its effectiveness at 0.5% level was comparable to that of 200 ppm BHA and 0.5% (w/w) dry rosemary, and stronger than that of red chillies and bay leaf which did not improve the stability of the fish oil. In the meat products area, researchers extensively worked on two strategies to improve antioxidant stability of meat and their products. The first strategy was adding this natural antioxidant through the animal diet (Botsoglou et al., 2003a; Simitzis et al., 2008) and this has been confirmed in a broiler research experiments (Wong and Shibamoto, 1995; Sarraga and Reguerio, 1999; Botsoglou et al., 2003b). In this case they were trying to use this natural antioxidant through the incorporation into phospholipids membrane layer where it can work effectively at their reaction site location (Gary and Pearson, 1987; De Winne and Dirinck, 1996). The second strategy was adding the oregano essential oil directly in the meat surface (Goulas and Kontominas, 2007; Martin-Sanchez et al., 2011), ground meat (Fasseas et al., 2007;

Oral et al., 2009; Mastromatteo et al., 2009) or their brine solution (Scramlin et al., 2010) prior to processing in order to improve meat quality and extend their shelf-life. In addition, oregano essential oils have medical properties which help to improve human health. Origanum spp. has been reported to have several pharmaceutical uses such as an antibacterial, fungicidal, antiviral, biocidal, antioxidant (Berna’th, 1997), antimicrobial (Sahin et al., 2004), anti-hyperglycemic (Lemhadri et al., 2004) and antithrombic activities, and possess activity against cancer (Goun et al., 2002). It also has a nematicidal (Berna’th, 1997) and insecticidal activities (Traboulsi et al., 2002). In some countries such as Jordan, Origanum syriacum leaves are used to cure eye ailments, burns and stomach troubles, and the seeds are used as sedative (Oran and Al-Eisawi, 1998).

Antimicrobial Activity of Oregano Origanum essential oils have high levels of carvacrol, thymol, γ-terpenene and p-cymene, and showed a high level of antimicrobial activity against eight different strains of gram-positive and gram negative bacteria (Sivropoulou et al., 1996). Among the essential oil components, carvacrol and thymol have the highest activity against bacterial growth, but the other two pheonolics (γ-terpenene and p-cymene) have no activity against bacterial strains. In addition, essential oils from different strains of oregano showed different antimicrobial activities. Horosova et al. (2006) found that oregano oils showed strong antibiotic effects on lactobacilli and E. coli when combined with antibiotics such as apramycin, streptomycin, and neomycin. Chouliara et al. (2007) reported that oregano essential oil showed a shelf-life extension effect on fresh chicken breast meat when combined with modified atmosphere packaging. Recently researchers are studying the antimicrobial effects of essential oils and their active components, including thymol and carvacrol. Thymol and carvacrol are both effective against E. coli (Xu et al., 2008), but carvacrol is more effective than thymol (Cristani et al., 2007). They both appeared to harm bacterial cell wall, and enter into bacteria and change cell processes. These effects may account for their

antibacterial properties (Cristani et al., 2007; Xu et al., 2008; Di Pasqua et al., 2010). Different hypothesis were suggested about the antimicrobial mechanism of the phenolic compounds or the essential oil components on microorganisms. Ultee et al. (2002) found that both carvacrol and cymene caused destabilization of the membrane of bacteria cells (Bacillus cereus), and decreased membrane potential. So, the most probable mechanism for the antimicrobial activity of carvacrol could be through the decrease in pH gradient across the cytoplasmic membrane due to hydroxyl groups and delocalized electrons. So if the metabolism is aerobic, this will reduce a proton motive force and effect on ATP synthesis. The absence of ATP inside the microorganism will lead to change in the cell process and may cause cell death. In addition, researchers found that increasing in the membrane fluidity and leakage of protons and ions were observed when bacteria were exposed to antimicrobial compounds (Sikkema et al., 1995; Heipieper et al., 1996). However, certain concentration of these compounds is needed to cause membrane leakage (Mendoza-Yepes et al., 1997). The mode of action of these phenolic compounds still needs more investigation to understand their antimicrobial effects. Recently, food industry has been showing a great interest in using plant derived extract as antimicrobial compounds in the food to suppress the growth of foodborne pathogens. Carvacrol and thymol are the main components of the oregano essential oil responsible for their antimicrobial activity (Ultee et al., 2002; Nostro et al., 2007). Combination studies using more than one phenolic compound are conducted to maximize their effect through the synergistic philosophy. For example, the combination action of nisin and carvacrol were found to have a synergistic effect against Bacillus cereus and listeria monocytogenes (Pol and Smid, 1999). Oregano essential oil has been well documented as an antimicrobial agent for many foodborne pathogens such as Staphylococcus aureus, Vibrio parahaemolyticus, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli (Sivropouluo et al., 1996; Ozcan, 2001; Lin et al., 2004). However, Lambert et al. (2001) reported that the mixture of carvacrol and thymol at certain concentration increased the total inhibition of Stapylococcus aureus and Pseudomonas aeruginosa. Similar results were found on

the growth of Enterobacteriaceae, lactic acid bacteria, and pseudomonas spp when thymol and carvacrol were added to the poultry meat patties (Mastromatteo et al., 2009). The inhibition may be related to the cell membrane damage, pH homeostasis, and inorganic ions equilibrium. Carvacrol is a major component of essential oils that were extracted from plant sources such as (Thymus vulgaris) and oregano (Oreganum vulgare) (Adam et al., 1998; Burt, 2004). Its percentage in the oregano essential oil is highly dependent upon the Oreganuim species, the extraction method, storage conditions, and laboratory method analysis. Carvacrol [2-methyl-5-(1-methlethyl) phenol is a hydrophobic phenolic compound synthesized from p-cymene and -terpinene (Poulose and Rodney, 1978). Their antimicrobial activity against several types of pathogens such as Bacillus cereus (Beuchat et al., 1994; Ultee et al., 1998, 1999; 2000), Escherichia coli O157:H7 (Friedman et al., 2004; Perez-Conessa et al., 2011), Staphylococcus aureus (Lambert et al., 2001; Knowles et al., 2005; Nostro et al., 2004, 2007), Salmonella enterica (Friedman et al., 2002; Nazer et al., 2005; Gutierrez et al., 2008) are well documented.

Tannic Acid Plants produce a wide variety of “secondary compounds” including alkaloids, terpenes, and phenolics in their cells. These compounds play important roles in protecting plants from herbivores and diseases (Hagerman, 1987, 2002, 2010; Getachew et al., 2000). Tannin is one of these compounds that have metal ion chelating and antioxidant activities, influence protein digestibility, and precipitate proteins through the tannin protein interactions (Hagerman and Butler, 1980; Hagerman, 1992; Hagerman et al., 1998; Okuda and Ito, 2011). Tannins were defined by Bate-Smith and Swain (1962) as “water soluble phenolic compounds that have molecular weights between 500 and 3000 Da, and have special properties such as the ability to precipitate alkaloids, gelatin, and other proteins’’. These properties of tannin are based on their chemical structures which have two or three phenolic hydroxyl groups on phenyl ring (Fig.2). Tannins can be classified into two groups: pyrogallol

type and catechol types (or certain catechin type) according to the polyphenol group in their structure. However, tannins can also be classified into hydrolysable tannin and condensed tannins (Okuda et al., 1985; Haslam, 1989; Okuda, 1999; 2005), or polyphenols of constant chemical structure (Type A) and polyphenols of variable composition (Type B). Tannins or plant polyphenols are distinguished from other polyphenols found in plants by their characteristics such as protein interaction ability, antioxidant ability, metal chelating ability, basic compounds, large molecular weight compounds, and pigments attached (Haslam, 1989; 1996). The United State Department of Health and Human Services (Food and Drug Administration) defined tannic acid or hydrolysable gallotannins as a complex polyphenolic compound that yields gallic acid and either glucose or quinic acid as hydrolysis products. It has yellowish-white to light brown color, odorless or has a faint characteristic odor, and has astringent taste. Tannic acid can be obtained in two different ways: solvent extraction of nutgalls or excrescences from young twigs of Quercus infectoria Oliver, from solvent extraction of the seed pod of Tara (Caesalpinia spinosa), or the nutgalls of various sumac species, including Rhus semialata, R. coriaria, R. galabra, and R. typhia (FDA, 2011). The hydrolysable types of tannins contain polyhydric alcohol and hydroxyl groups, which are connected to gallic acid such as gallotannins or to hexahydroxy diphenic acid (ellagitannins) by ester bond. After hydrolysis by enzyme or acid/base conditions, gallotannins produce glucose and gallic acid. Example on the gallotannins is Chinese tannin, Turkish tannin, Tara tannin, Acer tannin, and hamaelis tannin. Ellagitannin (corilagin), Chebulagic acid, Chebulinic acid, and Chebulic acid are also examples of ellagatanins (Chung et al., 1998a). Condensed tannin types are more complex in their structure compared to hydrolysable tannins and they are mainly produced by two main polymerized products of flavan-3-ols and flavan-3,4-diols, or mixture of both (Hagerman and Butler, 1994; Hagerman et al., 1997; Chung et al., 1998a). Flavan-3-ols are

considered as catechins which have four possible isomers due to two asymmetrical carbon atoms at carbon number 2 and 3. These isomers are formed into (+) and (-) catechins, (-) epicatechin, (-) epigallocatechin, (+)-gallocatechin, and (+)-catechin (Chung et al., 1993, 1998a). Falvan-3,4-diols are classified as a type of compounds called leucoanthocyanins due to their properties after exposure to heat and acid conditions since they polymerize to phlobaphene-like products which will produce the anthocyanidines (Haslam, 1981; Zucker, 1983). Falvan-3,4 diols can form eight isomers from their molecule structure which depend on three asymmetric carbon atoms at C-2, C-3, and C-4; leucocyanidin, leucopelargonidin, leucodelphinidin, guibourtacacidin, (+)-leucorobinetinidin, (-)–melacacidin, and (-)–teracacidin (Chung et al., 1998a). Tannic acid as used in many studies is a specific commercial type of tannin with pKa around 6 due to their polyphenol structure (Fig. 2). It is also classified as an important gallotannins which belong to the hydrolysable class (Scalbert, 1991;

Miranda et al., 1996). The commercial formula for tannic acid is given as C76H52O46 and is composed of a mixture of polygalloyl glucose and polygalloyl quinnic acid ester depending on the plant extract source. The commercial form of tannic acid can be fractionated chromatographically to yield a specific galloyl ester or can be methanolyzed which produce homogenous pentagalloyl glucose (Hagerman, 2002; Salminen et al., 2011). Tannin purification method is also important because it affects product purity and chemical content. The percentage of condensed tannins and hydrolysable tannins highly depend on the method of purification from plant tissues such as tissue preservation, grinding and extraction. The purity of tannic acid products can be assessed by different methods such as TLC and HPLC. In addition, their structures can be studied by degradative functional methods or by spectroscopic methods (Hagerman, 2002).

Antioxidant Activity of Tannic acid Polyphenol tannic acid inhibits hydroxyl radical formation from the Fenton reaction by complexing with ferrous ions. When ferric ion (Fe3+) is added to sample containing tannic acid, they form Fe(II)n-TA complexes, which mean tannic acid is capable of reducing ferric ion (Fe3+) to ferrous ion (Fe2+). So, researchers proposed that Fe3+ complexes to tannic acid converts Fe3+ to Fe2+ and prevents ferrous ion (Fe2+) from participating in the Fenton reaction and so the hydroxyl radical formation (Lopes et al., 1999). Tannic acid chelates iron due to polyphenol groups (ten galloyl group) and decreases the intestinal non-heme iron absorption (Brune et al., 1989; Geisser, 1990). Tannic acid also protects DNA from oxidative damage by chelating Fe(II) and

. so prevents OH formation in the presence of H2O2 (Fenton Reaction) (Toyokuni, 1996; Meneghini, 1997). Therefore, the antimutagenic and anticarcinogenic ability of tannic acid could come from its ability to prevent or delay the Fenton reaction and hydroxyl radical formation (Lopes et al., 1999). In addition, lipid peroxidation could be decreased by tannic acid through its ability to chelate iron ions (Afanas’ev et al., 1989; Guo et al., 1996; Moran et al., 1997). Tannic acid showed antioxidant activity when added to samples containing copper due to the ability of tannic acid to complexes with copper, and this complex is less capable of inducing ascorbate oxidation (Khan et al., 2000; Andrade et al., 2005). Okuda et al. (1992) found that adding green tea (rich in tannins) increased ascorbic acid stability in the presence of oxygen and metal catalyst. In a recent study about the enhancing antioxidant capacity of tannic acid by using thermal processing, researchers found that thermal processing to hydrolysable tannins increased their antioxidant and antimicrobial activities (Kim et al., 2010). This improvement on the antioxidant capacity could be due to the hydroxyl groups of flavonoids and tocopherol formed by heat treatment (Kwon et al., 2003). The increase in the antioxidant capacity may also be explained by the hydrolysis of tannic acid, which produced one free gallic acid and a galloyl group on the remaining gallotanins (Kim et al., 2010). Gulcin et al. (2010) studied the radical scavenging and antioxidant activity of

tannic acid and found that tannic acid inhibited oxidation of linoleic acid emulsion by 97.7 % at 15 g/mL. In addition, they found that tannic acid was effective in DPPH (1,1-diphenyl-2-picry-hydrazyl free radical) scavenging, ABTS.+ 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging, superoxide anion radical scavenging and hydrogen peroxide scavenging, and had Fe+3-reducing power and metal chelating activities. Ho et al. (1999) concluded that the antioxidant activity of tannin components from Vaccinium vitis-idaea L. exhibited multiple antioxidant activity, and could be used to treat periodontal disease due to the complex interactions between pathogenic bacteria and the host’s immune response.

Antimicrobial activity of tannic acid The antimicrobial effect of tannins is well studied and documented. Many studies showed that tannins have the ability to inhibit the growth of many microorganisms such as fungi, yeast, bacteria, and viruses. The antimicrobial characteristics of tannins are related to the hydrolysis of ester linkage between gallic acid and polyols especially in the edible fruits. This natural defense by the tannins can be used in food processing to improve their shelf life and prevent microbial growth (Chung et al., 1993, 1998b). Tannin with unknown origin showed inhibitory effects on the growth of filamentous fungi Fomes annosus with a minimum inhibitory concentration (MCI) (Haars et al., 1981). The same effect was found by Wehmer (1912) on the growth of merulius lacrymans and penicillium species at 10 to 20 mg/L tannin concentration. Condensed tannins were found to have inhibitory effects on the growth of Colletorichum graminicola (Nicholson et al., 1986). In addition, many studies showed the inhibitory effects of different tannin sources on the filamentous fungi such as Coniophora olivaccea (Nicholson et al., 1986), Coriolus versicolor (Hart and Hilis, 1972), Crinipellis perniciosa (Brownlee et al., 1990), Trametes hirsute (Arora and Sandhu., 1984) and Trichoderma Viride (Arriet-escobar and Belin., 1982). Tannins also showed inhibitory effects on the growth of various yeast species

(Jacob and Pignal, 1972) and bacteria such as Staphylococus aureus, Streptocoucus punumonia, Bacilus anthracis, Shigella dysenteriae, and Salmonella senftenberg (Beuchat and Heaton, 1975; Kau and Wu, 1980; Akiyama et al., 2001). Tannic Acid and propyl gallate also showed inhibitory effects on the growth of food borne bacteria such as E. coli, Alcaligenes faecalis, Listeria monocytogeneses, Salmonella entritidis, S. paratyhi, Staphyl. Aureus, Strep. Faecalis, Strep. Pyogenes, and Yersinia enterocolitica (Scalbert et al., 1991; Chung et al., 1993). Tannins give more benefits to human health from their effect on the survival of viruses and their activities. For example, tobacco mosaic virus (Bawden and Kleczkowski, 1945; Thresh, 1956) and influenza virus (Carson and Frisch, 1953) were affected negatively by adding tannic acid to their environment. Tannic acid also has an inhibitory effect on many virus species such as echovirus, coxsackievrus, reoviruses, polioviruses, and herpes viruses (Kucera et al., 1967; Konowalchuk and Spiers, 1976). Also, tannic acid sulfate has inhibitory activity to the cytopathic effect of HIV (human immunodeficiency virus) at 6 g/ml level (Mizumo et al., 1992). The antimicrobial mechanisms of tannins are through the astringent effect of tannins by complex formation with enzymes and other substrates (Mason and Wasserman., 1987; Chung et al., 1998a), their effect on the membranes of microorganisms, metal complex formation (Chang et al., 1998b; Engels et al. 2011), and the hydrolysis of ester linkage between gallic acid and glucose. Chung et al. (1998b) reported that tannic acid works as a siderphore produced by bacteria to bind irons (Neilands, 1995) in the media which make it unavailable for microorganism to use in their metabolism and enzyme activity. The siderphores are low-molecular-weight carriers that have high iron (Fe+3) affinity, which help bacteria to make iron biologically available (Neiland, 1982). This iron complex structure is highly contributed in the antimicrobial activity of several gallatonnins (Engels et al., 2009; Cho et al., 2010). Other tannins compounds such as proyl gallate and methyl gallate (ester of gallic acid) are unable to bind iron efficiently. Therefore, their effects on bacterial growth should be explained by other mechanisms. In addition, tannic acid

does not affect lactic acid bacteria effectively compared to other intestinal bacteria because they do not have heme enzymes (Kabuki et al., 2000). However, the bacterial response to the gallotannins also depends on their membrane structure. The outside membrane of Gram-negative bacteria makes it more resistant to gallotannins compared to the Gram-postive bacteria (Tian et al., 2009; Engels et al., 2009, 2011).

Figure 1. Structure of A. thymol, B. carvacrol, C. p-cymene, D. terpenene-4-ol, E. the two mirror image form S and R-carvone, F. aristolochic acid.

Figure 2. Structure of tannic acid. T.J. Kim et al. Food Chem. (2010). 118: 740–746

Figure 3. The hydrolysis of tannic acid at an ester linkage between two gallic groups. T.J. Kim et al. Food Chem. (2010). 118: 740–746.

Figure 4. Chemical structure of gallic acid (3,4,5-trihydrox-ybenzoic acid) containing a galloyl group. K. South, and D. D. Miller, 1998. Food Chem.. 63 (2): 169.

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CHAPTER 3

THE EFFECT OF OREGANO ESSENTIAL OIL (ORIGANUIM VULGARE SUBSP. HIRTUM) ON THE STORAGE STABILITY AND QUALITY CHARACTERISTICS OF GROUND CHICKEN MEAT

Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,* 1Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and 2Department of Food Science and Human Nutrition, and 2Department of Animal Science, Iowa State University, Ames, IA 50011, USA.

*Corresponding author: D. U. Ahn Phone: (515) 294-6595, Fax: (515) 294-6595 Email: [email protected]

Abstract

The objective of this study was to investigate the effect of different levels of oregano essential oil on important meat quality parameters, including oxidative storage stability, of ground chicken meat. Five different treatments including 1) control (none added), 2) 100 ppm oregano essential oil, 3) 300 oregano essential oil, 4) 400 ppm oregano essential oil, and 5) 5 ppm butylatedhydroxyanisole (BHA) were added to ground boneless, skinless breast meat and used for both raw and cooked meat studies. For raw meat study, samples were individually packaged in oxygen-permeable bags and stored at 4 ℃ cooler for up to 7 days. For cooked meat study, the raw meat samples were packaged in oxygen impermeable vacuum bags and then cooked in-bag to the internal temperature of 75 °C. After cooling to room temperature, the cooked meat was transferred to a new oxygen-permeable bag and stored at 4 oC for up to 7 days. Both raw and cooked meats were analyzed for lipid and protein oxidation, color at 0, 3, and 7 days of storage. Volatiles profile of cooked meat was reported during storage time. Oregano essential oil treatments significantly reduced (p < 0.05) lipid oxidation, protein oxidation, and improved color stability of raw meat. However, oregano oil at 400 ppm showed the highest effect for all these parameters. No significant difference (P > 0.05) was seen on the a*-value of meat during storage. Cooked meat showed similar results to raw meat when oregano oil was added. Hexanal was the major aldehyde which decreased significantly (P < 0.05) by oregano oil treatment in cooked meat. The significant differences in the aldehydes formation among the treatments were clearer in cooked meat than in raw meat. Overall, oregano essential oil at level between 100-300 ppm could be a good meat preservative that can replace the synthetic antioxidant, BHA.

Key Words: Oregano essential oil, Butylatedhydroxyanisole, Ground meat, Lipid oxidation

Introduction

Poultry meat is among the most popular meats in the world because of its low price, short production time, and ease of preparation (Chouliara et al., 2007). Chicken meat contains high percentage of polyunsaturated fatty acids, which make it more susceptible to oxidative deterioration during storage (Ahn et al., 2002). Both raw and cooked poultry meats can be oxidized, but cooked meat is more sensitive to lipid oxidation than raw meat (Gray et al., 1996; Lee et al., 2003). Lipid oxidation produces many volatile compounds responsible for the off-flavor and rancidity in the meat (Ahn et al., 2009). Other quality characteristics, such as coloration and texture of meat, can also be influenced by the oxidation of meat (Jensen et al., 1997; Du et al., 2000). Several synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) have been used widely in the food industry to prevent the deteriorative changes. However, the use of synthetic antioxidants are discouraged due to their negative effects on human health, and the food industry is interested in finding new replacements for the synthetic ones (Solomakos et al., 2008). Natural antioxidants from plant origin are safe and can replace the synthetic ones. Herbs, spices and many plant extracts have been widely used in foods to improve their flavor, quality, and to extend their shelf-life (Abdel-hamied et al., 2009). Oregano essential oil is one of many plant extracts that have antioxidant effects when added to meat (Fasseas et al., 2007; Goulas and Kontominas, 2007; Scramlin et al., 2010). The antioxidant effect of oregano essential oil is due to its high poly-phenol content; Carvacrol, thymol, p-cymene and γ-terpinene are the major components responsible for the antioxidant activity of oregano essential oil (Kosar et al., 2003; Capecka et al., 2005; Al-Bandak, 2007). This type of plant extract could be used as a good replacement for the synthetic antioxidant in meat products. Most of the research that had been done on oregano essential oil were adding the oil directly to the diets of poultry (Botsoglou et al., 2003a,b;c), lamb (Simitizis et al., 2008), and swine

(Ariza-Nieto et al., 2011) to stabilize the meats from those animals. However, very little work has been done to add the oregano essential oil directly to meat to prevent oxidative changes in it (Fasseas et al., 2007; Chouliara et al., 2007; Scramlin et al., 2010). Many previous works showed that adding oregano essential oil directly into the meat products had positive effects on meat quality. Some research investigated the combination effects of carvacrol and thymol on the poultry meat patties and showed positive effect on their color and reduced off-odor formation (Mastromatteo et al., 2009). Sullivan et al. (2004) studied the effect of natural antioxidants such as rosemary, sage, and tea catechins, on the chicken nugget and found that the addition of these antioxidants decreased total lipid oxidation, and increased color stability and storage stability of the product. Keokamnerd et al. (2008) studied the effect of adding commercial rosemary oleoresin preparations on ground chicken meat which were packaged in a high-oxygen atmosphere and found a positive overall effect on raw meat appearance during storage and cooked meat flavor. Oxidation process was decreased in ground meat with added rosemary, and the color was more stable. Oregano essential oil containing high concentration of carvacrol (major antioxidant) showed higher antioxidant activity than other herbs extracts (Fasseas et al., 2007; Brewer et al., 2011). Oregano essential oil is one of the natural antioxidants that needs more research to determine its effect on food quality and preservation, especially for its optimal level to prevent oxidative changes while maintaining other quality parameters such as flavor. The objective of this study was to investigate the effect of different levels of oregano essential oil on important meat quality parameters, including storage stability, of ground chicken meat. It is also beneficial to compare the effect of oregano essential oil with a synthetic antioxidant BHA, in both raw and cooked meat.

Materials and Methods

1. Sample preparation: One hundred and twenty, 6-wk-old broilers raised on a corn-soybean meal diet were slaughtered using the USDA guidelines (USDA, 1982). The chicken carcass were chilled in ice water for 2 h and drained in a cold room, and the breast muscles were separated from the carcasses at 24h after slaughter. Boneless breast muscles were cleaned, skins removed, external fats trimmed off, and stored at -20 oC freezer until used. The frozen meats were thawed in a walk-in cooler (4 oC), ground twice a through a 10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Five different treatments including 1) (none added), 2) 100 ppm oregano essential oil, 3) 300 oregano essential oil, 4) 400 ppm oregano essential oil, and 5) 5 ppm butylatedhydroxy anisole (BHA) were prepared. The oregano essential oil was obtained from a certified company in Turkey (Healthy-Health, Oil of Oregano, Turkey). The GC/MS analysis of the oregano essential oil indicated that 80.12% of the essential oil was carvacrol. BHA powder (0.1 g) and oregano essential oil (1.25 g) were dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to make their stock solutions. The ethanol added was removed using a rotary evaporator (BUCH Rotavapor, Model R-200) at (70 °C, 175 mbar vacuum pressure) before adding the stock solution to meat samples. Each additive treatment was added to the ground breast meat and then mixed for 2 min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same amounts of mineral oil to provide the same conditions. For raw-meat study, the prepared meat samples (approximately 100 g each) were individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI), stored at 4 °C cooler for up to 7 days, and analyzed for lipid and protein oxidation, and color at 0, 3, and 7 days of storage. The same preparation method was used for cooked meat study, but the raw meat samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3

2 mL O2/m /24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were

cooked in-bag in a 90 °C water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA, USA) until the internal temperature of the meat reached to 75 °C. After cooling to room temperature, the cooked meat was transferred to a new oxygen-permeable bag (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at 4 °C for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3, and 7 days of storage.

2. 2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid oxidation was determined using a TBARS method (Ahn et al., 1998). Five grams of ground chicken meat were weighed into a 50-mL test tube and homogenized with 50μL BHT (7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron homogenizer (Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for 15s at high speed. One milliliter of the meat homogenate was transferred to a disposable test tube (13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA, 2 mL) was added. The mixture was vortex mixed and incubated in a boiling water bath for 15 min to develop color. Then samples were cooled in the ice-water for 10 min, mixed again, and centrifuged for 15 min at 2,500 x g at 4 oC. The absorbance of the resulting supernatant solution was determined at 532 nm against a blank containing 1 mL of DDW and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as mg of malondialdehyde (MDA) per kg of meat.

3. Color measurement (Konioka Minolta): The color of meat was measured on the surface of meat samples using a Konica Minolta Color Meter (CR-410, Konioka Minolta, Osaka, Japan). The colorimeter was calibrated using an illuminant source C (Average day light) on a standard white ceramic tile covered with the same film as the ones used for meat samples to negate the color and light reflectance properties of the packaging material. The color were expressed as CIE L*- (lightness), a*- (redness), and b*- (yellowness) values (AMSA, 1991). The areas selected for color measurement were free from obvious defects that may affect the uniform color readings. An average from two random readings on each side (upper and lower) of

sample surface was used for statistical analysis.

4. Volatile compounds: Volatiles of samples were analyzed using a Solatek 72 Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann, Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard Co., Wilmington, DE, USA) according to the method of Ahn et al. (2001). Sample (3 g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample vial, flushed with helium gas (40 psi) for 3 s, and then capped airtight with a Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA). Samples from different treatment were randomly organized on the refrigerated (4°C) holding try to minimize the variation of the oxidative changes in sample during analyses. The meat sample was purged with helium (40 mL/min) for 14 min at 20 °C. Volatiles were trapped using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and desorbed for 2 min at 225 °C, focused in a cryofocusing module (-70 °C), and then thermally desorbed into a capillary column for 2 min at 225 °C. An HP-624 column (7.5 m, 0.25 mm i.d., 1.4 m nominal), an HP-1 column (52.5 m, 0.25 mm i.d., 0.25m nominal), and an HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal) were connected using zero dead-volume column connectors (J &W Scientific, Folsom, CA, USA). Ramped oven temperature was used to improve volatile separation. The initial oven temperature of 25 °C was held for 5 min. After that, the oven temperature was increased to 85 °C at 40 °C per min, increased to 165 °C at 20 °C per min, and then increased to 230 °C at 5 °C per min and held for 2.5 min at that temperature. Constant column pressure at 22.5 psi was maintained. The ionization potential of MS was 70 eV, and the scan range was 20.1 to 350 m/z. The identification of volatiles was achieved by the Wiley Library (Hewlett-Packard Co.). The area of each peak was integrated using ChemStationTM software (Hewlett-Packard Co.) and the total peak area (total ion counts x 104) was reported as an indicator of volatiles generated from the samples.

5. Protein oxidation (total carbonyl): Protein oxidation was determined by the

method of Lund et al. (2008) with minor modifications. One gram of muscle sample was homogenized with a Brinkman Polytron (Type PT 10/35), Brinkman Instrument

Inc., Westbury, NY, USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10 mM Trizma-maleate), 100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol tetraacetic acid, pH 7.4). Two equal amounts of meat homogenate (2 mL) were taken from a sample, precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at 12,000 × g for 5 min at room temperature. After centrifugation, the pellet from 1 sample was treated with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M HCL, and the pellet from the other was incubated with 2 M HCL as a blank. During 30 min of incubation in the dark, samples were vortex-mixed for 10 s every 3 min. The protein was further precipitated with 2 mL of 20% trichloroacetic acid and centrifuged at 12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by washing 3 times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate, followed by centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium dihydrogen phosphate (pH = 2.3). The samples were kept at 5 °C overnight. In next day, the samples were centrifuged to remove insoluble materials. The absorbance of supernatant was read at 370 nm. The absorbance values for blank samples were subtracted from their corresponding sample values. Briefly, Protein concentration was measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA., Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).

Statistical analysis: Data were analyzed using the procedures of generalized linear model (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of the means (SEM) were reported. The significance was defined at P < 0.05 and Tukey test or Tukey’s Multiple Range test were used to determine whether there is a significant difference between the mean values.

Result and Discussion

Lipid Oxidation

Lipid oxidation is among the most critical quality parameters because it dose not only interacts with protein oxidation (Hall, 1987) that causes meat discoloration (Guillen-Sans and Guman-Chozas, 1998), but also is primarily responsible for producing many off-odor and rancid flavor in the meat during storage (Vercellotti et al., 1992; Min et al., 2005). The antioxidant activity of oregano essential oil in the preliminary studies indicated that there were no significant differences (p > 0.05) between treatments at day 0 in raw meat. TBARS values increased more in the cooked meat compared to the raw meat during storage. In general adding oregano essential oil for both raw and cooked ground chicken meat reduced the TBARS values (Tables 1 and 4). This result agreed with other studies that added oregano oil directly to the meat (Fasseas et al., 2007; Chouliara et al., 2007) or indirectly in the animal diet (Botsoglou et al., 2002; Botsoglou et al., 2003a, b). Among the treatments, oregano essential oil at 400 ppm showed the highest antioxidant effect. The TBARS values of raw meat were very low compared to the cooked meat. Cooked meat is more sensitive to oxidative changes than the raw meat because the antioxidant enzymes in meat are denatured during cooking, iron ions were released from the intracellular to extracellular compartment, membrane bi-layers became damaged and phospholipids were open to catalysts and oxygen during cooking and storage (Gray et al., 1996). However, the TBA values for the ground raw chicken meat showed very little changes during storage regardless of the oregano essential oil treatments. The raw meat values

are in agreement with those of Chouliara et al. (2007) who reported TBA values of 0.1-0.28 in fresh breast chicken meat when 0.1 % oregano oil was added and stored for 7 days. Kim et al. (2002) reported TBARS values of between 0.13-0.68 for turkey meat were found after 7 day of storage. All the oregano essential oil levels (100, 300, and 400 ppm) showed significant differences compared with the control for both raw and cooked meat at day 7 of storage. The oregano essential oil at 400 ppm, however, showed higher effect than BHA (5 ppm) especially on the cooked meat at day 7 (Table 4). The oregano essential oil at 100 and 200 ppm could be the minimum levels that can be used to slow down the oxidative changes in ground chicken meat.

Protein oxidation

The functional properties of proteins such as protein solubility, gelation, and emulsification capacity in the foods are highly dependent on their amino acid composition and structure (Uchida and Kawakishi, 1992; Xiong, 2000). The relationship between protein oxidation and overall food quality is also well documented. Ground meat is usually more susceptible to oxidation than the whole meat cuts due to their size and surface area that contact with the oxygen and the presence of primary free radicals. Several other factors that may affect or increase protein carbonyl formation in the meat system such as metal catalysts (iron, copper, hem and non-heme iron, and myoglobin), pH, temperature, and presence of other inhibitors (antioxidant phenolic compounds) should be considered (Xiong, 2000; Park et al., 2007; Estevez., 2011; Promeyrat et al., 2011;). In this study oregano essential oil significantly (P < 0.05) reduced the total carbonyl formation (nmol/mg of protein) especially in the cooked meat (Table 8). There was no significant difference (P > 0.05) in the total carbonyl values between the treatments in raw meat during the first 3 days of storage (Table 2). The changes in the total carbonyl content were very low in raw meat compared to the cooked meat during storage. This could have happened because the production of free radicals was higher in the cooked meat than in the raw meat. In addition, the total antioxidant capacity of the fresh chicken meat is usually higher than

that of the cooked meat. The reduction of antioxidant capacity in cooked meat is caused by the denaturation of antioxidant enzymes, exposure of amino acid side chains to the ROS, and loss of endogenous antioxidants (Fasseas et al., 2007; Serpen et al., 2012) by heat treatment. Generally, the formation of total carbonyl agrees with the TBA values in raw meat during storage. Oregano essential oil at 400 ppm showed the strongest effect in reducing the total carbonyl values (Tables 2 and 5). Requena et al. (2003) estimated that the total carbonyl content in various animal tissues is 1-2 nmol/mg protein. However, the result from the DNPH method from several studies varied widely (0.5-4.8 nmol/mg protein) depending on the meat type, animal species, experimental conditions, and analysis methods, which make it difficult to give a general conclusion. However, total estimated carbonyl contents falls in the range of 1-3 nmol/mg protein for raw meat and up to 5 nmol/mg protein for cooked meat products (Estevez et al., 2005; Sun et al., 2010; Estevea, 2011). The total carbonyl values agreed well with the TBA results, and meats with the highest level of oregano oil produced the lowest values. The result in this study agreed with Fasseas et al., (2007) who used oregano essential oil (3% w/w) in ground porcine and bovine raw or cooked meat. They found that the oregano oil showed higher antioxidant activity than sage and control treatments during the storage at 4 oC. There were no significant differences between oregano oil treatments at 100 and 300 ppm, and BHA for both raw and cooked meat at day 7 (Tables 2 and 5). The significant antioxidant effect of oregano oil was more prominent when cooked meat was compared with the raw meat.

Color values

Color is one of the most important quality parameters that determine retail sales and marketing strategies for raw ground chicken meat (Sandusky and Heath, 1998). In addition, meat color for various products is very important for the consumer’s decision for meat consumption (Hunt et al., 1993; Bekhit and Fasutman, 2005). The lightness (L*-values) of ground chicken meat significantly decreased during storage. Oregano essential oil added at level 400 ppm showed the highest stabilizing effects

for L*- and a*-values (Table 3). However, no significant difference (P > 0.05) among the treatments on the redness or a*-values during storage was detected. All treatments showed significant positive effects (P < 0.05) on L*-values compared to the control. These results are very similar to those of Chouliara et al. (2007) and Mastromatteo et al. (2009) who reported that oregano oil or their components had no significant effect on a*- but significantly preserved the L*-values at the end of storage. In general, b*-values decreased during storage time, but oregano oil maintained the b*-values of the meat after 7 days of storage, which agreed with the results of Choulira et al. (2007). There were no significant difference on a*-values between the treatments at day 7 of storage. This may be due to the low myoglobin content in the chicken breast meat (0.05 mg/g) that makes this change small. The same result was found by Ahn and Lee (2004) who reported that a*-values did not change much during 15 days of storage in both aerobic- and vacuum-packaged turkey breast meat. However, the a*-values of meat with oregano oil were the highest at day 7, which showed the role of oregano oil to stabilize the red color.

Volatiles production

Warmed-over flavor is the most critical problem affecting cooked meat products (Jo et al., 2006). The primary volatiles produced in raw and cooked meat can be further degraded into secondary products and cause rancid flavor. Adding oregano essential oil reduced the amount of off-odor volatiles in cooked chicken meat (Tables 6-8). The research on adding oregano oil to ground meat is rare and nobody has analyzed the effect of oregano oil on the volatiles (ketone, aldehydes, hydrocarbon and sulfur compounds) formation in chicken meat. The volatiles produced by the raw meat were very low and GC-MS did not detect any sulfur or aldehyde compounds during storage time. No significant differences for most of volatiles between treatments for both raw (data not shown) and cooked meat at day 0 of storage were found. Heptanal, dimethyl disulfide, and 1-butanol were found only in the control treatment at day 0 of storage time. This reflects lipid oxidation development in the

raw meat before cooking in the control treatment. However, oregano oil was able to decrease aldehydes formation especially in the cooked meat during storage. Oregano oil at 400 ppm showed the strongest effect in suppressing the volatile formation, especially aldehydes such as hexanal and pentanal. Oregano oil levels at 100 ppm and 300 ppm also significantly (P < 0.05) reduced the amounts of most of the aldehydes compared to the control in cooked meat. Lipid oxidation-dependent volatiles such as aldehydes (e.g., propanal, hexanal, pentanal and heptanal) and hydrocarbons (cyclohexane, hexane, heptane and octane) decreased by the addition of oregano oil during storage. Sulfur volatiles decreased after 3 days of storage in the control treatment. This agrees with several studies done using aerobically stored meat, from which most of the sulfur volatile compounds escaped or disappeared after 5 days of storage (Lee et al., 2003) due to their high volatility (Nam et al., 2002, 2003). Oregano oil at 300 and 400 ppm reduced the amount of dimethyl disulfide at days 0 and 3 of cooked meat compared to other treatments (Tables 6 and 7). Hexanal and pentanal were reported as the major indicators of lipid oxidation (Ahn et al., 1998; Shadidi and Pegg, 1994). In addition, positive relationships between aldehydes and TBARS values were reported (Du et al., 2003). Hexanal formation in the cooked meat increased rapidly during storage time (Tables 6-8), which reflected the relationship between total aldehydes and lipid oxidation status of ground meat. Volatile content of aerobically-packaged meat increased more rapidly than the vacuum-packaged meat during storage, indicating the role of oxygen in the formation of volatiles (Du et al., 2000). Minor compounds such as carbon disulfide, dimethyl sulfide, 2-butanone, ethyl acetate, ethyl propionate that did not appear consistently in the samples within the same treatment were not included in the tables. Samples from oregano oil treatment showed some terpenoids such as sabinene, paracymen, limonene, camphene, gamma terpinene, beta myrcene, and alpha terpinene in varying amounts. These compounds have significant impact on the odor and flavor of meat products. Finally GC-MS results did not show carvacrol and thymol probably due to their low volatility.

Conclusion

Oregano essential oil, at levels 300 and 400 ppm, showed the highest antioxidant effect to preserve the ground chicken meat. Adding >100 ppm of oregano essential oil improved color stability of raw meat and decreased off-odor volatile of cooked meats, but their effects were much clearer in cooked than in raw meat. Over all, oregano essential oil can be used in place of synthetic antioxidant (e.g., BHA) to prevent quality deterioration in raw and cooked meat during storage.

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Table 1. TBARS value of raw ground chicken breast meat at different storage time at 4 ℃. Time Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA ------TBARS (mg/Kg)meat ------az ay ay ax ax Day 0 0.13 0.12 0.11 0.11 0.13 0.005

ay by bcxy cx bcx Day 3 0.16 0.14 0.12 0.12 0.13 0.005

ax bx bx bx bx Day 7 0.23 0.15 0.13 0.12 0.14 0.006 SEM 0.005 0.008 0.006 0.006 0.009 a-c-Value with different letters within a row are significantly different (P < 0.05). n=4. x-z-Value with different letters within a column are significantly different (P < 0.05). TBARS value in mg malonaldehyde/kg meat. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 2. Effect of adding different level of oregano oil on protein oxidation of raw chicken breast patties during storage time. Time Control 100 ppm 300 ppm 400 ppm 5 ppm SEM

Without Oregano Oregano Oregano BHA

------Carbonyl (nmol/ mg of protein) ------

ay ax ax ax ax Day 0 0.7 0.69 0.69 0.65 0.69 0.04

axy ax ax ax ax Day 3 0.77 0.75 0.74 0.65 0.75 0.05

ax abx bx bx abx Day 7 0.94 0.79 0.77 0.69 0.80 0.03

SEM 0.05 0.04 0.04 0.04 0.06 a-c-Value with different letters within a row are significantly different (P < 0.05). n=4. x-z-Value with different letters within a column are significantly different (P < 0.05). Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 3. CIE Color value of ground chicken patties with different level of oregano oil during storage at 4℃. Time Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA L* Day 0 64.47abx 64.40abx 64.35bx 64.82abx 64.91ax 0.12 Day 3 63.49ay 63.74axy 63.84ay 64.04ax 63.89ay 0.15 Day 7 62.71bz 63.64ay 63.74ay 63.95ax 63.67ay 0.18 SEM 0.14 0.14 0.09 0.23 0.13 a* Day 0 8.65ax 8.62ax 8.55ax 8.62ax 8.59ax 0.10 Day 3 6.36ay 6.35ay 6.39ay 6.50ay 6.38ay 0.09 Day 7 6.16ay 6.26ay 6.39ay 6.48ay 6.37ay 0.11 SEM 0.07 0.16 0.1 0.08 0.06 b* Day 0 20.87cx 21.07cbx 21.27bx 21.85ax 21.70ax 0.09 Day 3 18.90cby 19.01cy 19.75cby 21.41ay 20.80bay 0.28 Day 7 18.62by 18.58by 18.68ay 19.29az 18.97abz 0.10 SEM 0.15 0.14 0.28 0.08 0.16 a-c- Value with different letters within a row are significantly different (P < 0.05). n=4. x-z- Value with different letters within a column are significantly different (P < 0.05). CIE Color L*, a*, b*-value of ground chicken patties with different level of oregano oil during storage at 4 ℃. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 4. TBA value of cooked ground chicken breast meat at different storage time at 4℃. Time Control 100 ppm 300 ppm 400 ppm BHA SEM without Oregano Oregano Oregano 5 ppm ------TBARS (mg/Kg)meat ------Day 0 0.26ay 0.14by 0.09by 0.08bz 0.11bz 0.01

Day 3 2.44ax 2.35ax 0.72cx 0.27dy 1.87by 0.06

Day 7 2.55ax 2.43bx 0.74dx 0.35ex 2.02cx 0.03

SEM 0.07 0.04 0.02 0.006 0.034

a-cValue with different letters within a row are significantly different (P < 0.05). n=4. x-zvalue with different letters within a column are significantly different (P < 0.05). TBA value in mg malonaldehyde/kg meat. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 5. Effect of adding different level of Oregano oil on protein oxidation of chicken breast patties during storage time/cooked meat. Time Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA

------Carbonyl (nmol/mg of protein)------

Day 0 0.69ay 0.44by 0.43bx 0.40bx 0.47by 0.041

Day 3 1.25ay 0.50by 0.45bx 0.42bx 0.48by 0.038

Day 7 1.90ax 0.95bcx 0.56bcx 0.44cx 1.00bx 0.12

SEM 0.15 0.074 0.056 0.046 0.032

a-cValue with different letters within a row are significantly different (P < 0.05). n=4. x-zValue with different letters within a column are significantly different (P < 0.05). TBA value in mg malonaldehyde/kg meat. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA

Table 6. Profile of volatiles cooked meat chicken patties with different level of oregano oil at day 0. Total Ion counts * 104

Compounds Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA 2-propanone 2758b 2552b 2466b 2733b 6001a 517 1-butanol 548a 0b 18b 0b 0b 36 2-propanol 381bc 319c 1371a 959ab 738bc 133 Hexane 1775a 336b 244b 243b 169b 203 Heptane 102a 59ab 12b 49ab 0b 19 1-propanol 2832a 2622a 1542b 1027b 1720b 174 Pentanal 215a 270a 125a 292a 110a 82 Heptanal 35a 0b 0b 0b 0b 2 Octane 86a 235a 406a 319a 165a 90 Cyclohexane 107a 63abc 86ab 30bc 0c 17 Dimethy-DS 507a 0b 0b 0b 0b 82 Hexanal 467a 167b 93b 91b 551a 66 α-Pinene 0b 265a 226a 362a 0b 36 Camphene 0b 256ab 457ab 715a 0b 142 Limonene 0b 72ab 173a 165a 0b 25 β-Myrcene 0c 162bc 1056ab 1891a 0c 226 γ-Terpinene 0b 285b 2159ab 4637a 0b 682 Sabinene 0c 0c 149b 354a 0c 20 a-dDifferent letters within a row are significantly different (P<0.05). n=4. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 7. Profile of volatiles cooked meat chicken patties with different level of oregano oil at day 3. Total Ion counts * 104

Compounds Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA 2-propanone 4106a 4854a 4530a 4517a 3458a 797 Pentane 580a 0b 0b 0b 0b 61 2-propanol 620b 3324ab 3643a 1337ab 905ab 668 Hexane 216a 20b 0b 0b 0b 15 Heptane 86a 0b 0b 0b 0b 7 1-propanol 2279a 1299b 2136a 1956a 1703ab 134 Propanal 58b 15b 0b 0b 144a 14 Pentanal 1065a 174bc 126bc 0c 466b 89 Heptanal 0 0 0 0 0 0 Octane 147a 204a 160a 175a 179a 43 Cyclohexane 31a 0b 0b 0b 0b 5 Dimethy-DS 23a 0b 0b 0b 0b 1 Hexanal 11371a 3194b 1341b 1448b 4167b 752 α-Pinene 0b 0b 475a 444a 0b 51 Camphene 0b 0b 299a 328a 0b 55 Limonene 0b 0b 381a 430a 0b 92 β-Myrcene 0b 0b 577ab 740a 0b 163 γ-Terpinene 0b 0b 1609ab 1984a 0b 396 Sabinene 0b 0b 180a 315a 0b 38 a-dDifferent letters within a row are significantly different (P<0.05). n=4. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA.

Table 8. Profile of volatiles cooked meat chicken patties with different level of oregano oil at day 7. Total Ion counts * 104

Compounds Control 100 ppm 300 ppm 400 ppm 5 ppm SEM without Oregano Oregano Oregano BHA 2-propanone 2307a 2031a 1966a 2065a 1224a 251 2-propanol 991a 909a 1008a 452a 951a 188 Hexane 47ab 172ab 0b 0b 209a 40 Heptane 160a 0b 0b 0b 132a 17 Acetaldehyde 90a 0b 0b 0b 0b 1 Propanal 297a 0b 0b 0b 0b 2 Pentanal 1153a 563b 305bc 297bc 254c 69 Heptanal 72a 0b 0b 0b 0b 2 Octane 322ab 137b 397ab 309ab 822a 128 Cyclohexane 127a 45b 45b 26b 184a 18 Hexanal 41262a 7887b 1252c 1215c 2095bc 1422 α-Pinene 0c 325bc 762a 378b 0c 85 Camphene 0b 258b 866a 503ab 0b 133 Limonene 0c 114bc 349ab 465a 0c 62 β-Myrcene 0b 335ab 640a 412a 0b 92 γ-Terpinene 0b 491ab 1326a 1020a 0b 200 Sabinene 0c 73bc 133b 355a 0c 23 a-dDifferent letters within a row are significantly different (P<0.05), n=4. Treatments: Control, 100 ppm oregano oil, 300 ppm oregano oil, 400 ppm oregano oil, and 5 ppm BHA

Preliminary study data.

Table 9. TBA value of cooked ground chicken breast meat at different storage time at 4℃.

------TBARS (mg/kg) ------

Time T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 SEM

Day 0 0.34a 0.20b 0.19bc 0.19bc 0.15c 0.15c 0.23b 0.38a 0.35a 0.35a 0.01

0.76ab 0.33cd 0.28dce 0.20de 0.16e 0.15e 0.37c 0.79a 0.71ab 0.70b 0.03 Day 3

Day 7 0.83a 0.37c 0.35cd 0.26ed 0.21e 0.18e 0.41c 0.81a 0.74a 0.64b 0.02

a-e Value with different letter within a raw are significantly different (P<0.05). Treatments : T1: Control; T2: 100 ppm Oregano;T3: 200 ppm oregano; T4:300 ppm oregano; T5:400 ppm oregano oil; T6: 500 ppm oregano; T7: 5 ppm BHA; T8: 2.5 ppm Tannic acid; T9:5 ppm Tannic acid; T10:10 ppm Tannic acid

Preliminary combination study

TBA Value

2.5 2 1.5 Day 0 Day 3 1 day 7

0.5 TBA Mg/kg meat Mg/kg TBA 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Treatment

Figure 1: TBA value of cooked meat at different levels of oregano and tannic acid in different combinations. The Antioxidant effect of tannic acid and oregano oil speratly or combined at different level (12 combinations) using 0, 100, 200, and 400 of oregano oil with 5 and 10 ppm of tannic acid.

Table 10. TBA value of cooked meat using different level and combinations of oregano oil and tannic acid at 4 °C. Treatment Day 0 Day 3 Day 7 SEM

1/control 0.27abz 1.78ay 2.19ax 0.022

2/ 5TA + 0 O 0.22bz 1.17by 1.87bx 0.005

3/ 10 TA + 0 O 0.23aby 0.82ex 0.93ex 0.047

4/0TA +100 O 0.23bz 1.04cdy 1.47cx 0.041

5/5TA + 100 O 0.25abz 0.98dy 1.38cx 0.028

6/10TA+ 100 O 0.26aby 0.35ghx 0.39fgx 0.018

7/0TA+ 200 O 0.26abz 0.54fy 1.17dx 0.019

8/5TA+ 200 O 0.35ay 0.41gxy 0.48fx 0.028

9/10TA+ 200 O 0.24aby 0.25hiy 0.29ghx 0.008

10/0TA+ 400O 0.29abx 0.32ghix 0.34fghx 0.015

11/5TA+ 400O 0.24aby 0.26hixy 0.29ghx 0.008

12/10TA+400O 0.21bx 0.22ix 0.24hx 0.005

13/BHA 5 ppm 0.29abz 1.14bcy 1.79bx 0.04 SEM 0.026 0.023 0.029 x-z-Value with different letters within a row are significantly different (P < 0.05). n=4. a-i-Value with different letters within a column are significantly different (P < 0.05). The Antioxidant effect of tannic acid and oregano oil speratly or combined at different level (12 combinations) using 0, 100, 200, and 400 ppm of oregano oil with 5 and 10 ppm of tannic acid.

CHAPTER 4 THE EFFECT OF ADDING DIFFRENT LEVEL OF TANNIC ACID ON THE STORAGE STABILITY AND QUALITY CHARACTERISTICS OF GROUND CHICKEN MEAT

*Corresponding author: D. U. Ahn Phone: (515) 294-6595, Fax: (515) 294-6595 Email: [email protected]

Abstract

The objective of this study was to determine the effect of tannic acid (TA) on the oxidative storage stability of ground chicken breast meat. Five different treatments including 1) control (none added), 2) 2.5 ppm TA, 3) 5 ppm TA, 4) 10 ppm TA, and 5) 5 ppm butylated hydroxyanisole (BHA) were added to boneless, skinless ground chicken breast meat, used for both raw and cooked meat studies. For the raw meat study, ground chicken breast meat was packaged in oxygen-permeable bags and stored at 4 ℃ cooler for up to 7 days. For the cooked study raw meat samples were packaged in oxygen-impermeable vacuum bags and then cooked in-bag to the internal temperature of 75 °C before transfer to the oxygen permeable bags for storage. Both raw and cooked meats were analyzed for lipid and protein oxidation, color, and volatiles at 0, 3, and 7 days of storage. Both raw and cooked chicken breast meat added with 10 ppm of TA had significantly (P < 0.05) lower lipid and protein oxidation than other treatments during storage. In addition, TA at 10 ppm level maintained the highest color a*- and L*-values during storage. In cooked chicken breast meat added with > 5 ppm TA had significantly (p < 0.05) lower amounts of off-odor volatiles than other treatments. Among the volatile compounds, the amount of hexanal increased rapidly during storage for cooked meat. However, meats added with > 5 ppm TA showed the lowest amount of hexanal and other aldehydes related to lipid oxidation, indicating a strong antioxidant effect of TA in ground chicken breast meat. Furthermore, the differences among the treatments were bigger in cooked meat than in raw meat, indicating that the antioxidant efficiency of TA in cooked meat was high. Therefore, TA at > 5 ppm can be used as a good natural preservative in ground chicken meat to improve its quality during storage. Key Words: Tannic acid, BHA, Lipid and protein oxidation, Volatiles, Chicken patties.

Introduction

Recently, the poultry meat industry has been increasingly seeking natural antioxidants to replace the synthetic antioxidants they currently use (Karre et al., 2013). Something that is safe to add, does not cause human health problems, and positively affect the consumers’ final purchase decisions (Solomakos et al., 2008; Naveena et al., 2008; Gulcin et al., 2010). Several studies using natural antioxidants from plant origins showed their positive effects on improving meat quality and extending shelf life (Abdel-hamied et al., 2009). Meat color and odor are considered the most important attributes affecting primary consumer evaluation for the meat quality (Liu et al., 1995; Mancini and Hunt, 2005). Usually fresh meat is characterized by cherry red color due to the formation of oxymyoglobin (OxyMb) pigment. However, farther oxidation will convert the meat into brown color due to metmyoglobin (MetMb) formation (Bekhit and Faustman, 2005; Bou et al., 2008). Lipid oxidation is considered as the major problem affecting meat quality (Hui et al., 2006; Ahn et al., 2007; Nam et al., 2007) because it changes color, generate off-odor (Gray and Monahan, 1992; Gray et al., 1996; Min and Ahn, 2005), and impair protein functionality (Hall, 1987). Protein functionality such as solubility, emulsification, water binding capacity, and texture are also affected by lipid oxidation secondary products and their interaction with protein oxidation products (Hall, 1987; Gray et al., 1996). The progress of oxidation in fresh meat depends on many internal factors such as catalyst (Iron Fe, Cu, etc), antioxidant capacity, pH, and free radical formation. However, many external factors such as high storage temperature, oxygen availability, meat processing method, and additives also can influence lipid and protein oxidation in meat (Ahn et al., 2009). Therefore, finding new natural antioxidants to resolve these problems in meat and other foods is important (Wong et al., 2006; Descalzo and Sancho, 2008). Herbs and plant extracts such as rosemary (Sebranek et al., 2005), oregano (Rojas and Brewer, 2008), grape seed extract (Brannan, 2008), plum (Lee and Ahn, 2005),

bearberry (Carpenter et al., 2007) and several oleoresin plant extracts such as garlic and onion (Nam et al., 2007) had been tested to prevent oxidation in meat because they contained high levels of antioxidants (Karre et al., 2013). Tannins are one of the secondary compounds produced by plants to protect themselves from external herbivores and diseases (Salminen et al., 2011). These compounds have biological, metal ion chelating and antioxidant (Hagerman, 2010), and protein precipitating activities (Hagerman et al., 1998). In addition tannins showed positive effect on meat color stability and extend their self life during refrigeration time (Luciano et al., 2009; Maqsood and Benjakul, 2010c). The tannins are defined as water soluble phenolic compounds with molecular weights between 500 and 3000 Da, and have special properties such as precipitating alkaloids, gelatin, and other proteins (Bate-Smith and Swain, 1962). These properties of tannins are based on their chemical structures which have two or three phenolic hydroxyl groups on phenyl ring. The antioxidant activity of tannic acid was explained by several researchers through their ability to prevent hydroxyl radical formation (Lopes et al., 1999), metal chelating activity (Okuda and Ito, 2011), radical scavenging activity (Glucin et al., 2010) and through tannic acid hydrolysis into gallic acid and a galloyl group (Kim et al., 2010). Tannins are classified into two different main groups - hydrolysable and condensed tannins. The hydrolysable type of tannins contains polyhydric alcohol and hydroxyl groups, which are esterifies by gallic acid (gallotannins) or to hexahydroxy diphenic acid (ellagitannins) (Okuda et al., 1985; Okuda, 2005; Haslam, 1989; Okuda and Ito, 2011). Condensed tannins are more complex in their structure compared to hydrolysable tannins. They are mainly produced by two main polymerized products of flavan-3-ols and flavan-3,4-diols, or mixture of both (Chung et al., 1998). Tannic acid is a specific commercial type of tannins that has a molecular formula C76H52O46, and 10 ppm is the maximum level allowed in meat products (FDA, 2011). Tannic acid is a yellowish-white to light-brown powder, and is soluble in water and alcohol. In addition, tannic acid is classified as an important gallotannins which

belong to the hydrolysable class (Miranda et al., 1996). These activities make the tannic acid, especially the hydrolysable type, a possible replacement for the synthetic antioxidants. Very little studies were done to determine the effect of tannic acid on the quality of meat products (Luciano et al., 2009; Maqsood and Benjakul, 2010a; b; c). Tannic acid is classified as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) and can be used directly in foods. However, little work has been done to determine their effect on the storage stability and quality characteristics in ground chicken meat. The objective of this study was to investigate the effect of adding tannic acid on stability and meat quality of ground chicken meat (raw and cooked meat) during storage.

Materials and Methods

Sample preparation: One hundred and twenty, 6-wk-old broilers raised on a corn-soybean meal diet were slaughtered using the USDA guidelines (USDA, 1982). The chicken carcass where chilled in ice water for 2 h and drained in a cold room, and the breast muscles were separated from the carcasses at 24h after slaughter. Boneless breast muscles were cleaned, skins removed, external fats trimmed off, and stored at

-20 ℃ freezer until used.

The frozen meats were thawed in a walk-in cooler (4 °C ), ground twice through a 10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Five different treatments including 1) (none added), 2) 2.5 ppm tannic acid, 3) 5 ppm tannic acid, 4) 10 ppm tannic acid, and 5) 5 ppm butylatedhydroxy anisole (BHA) were prepared. Tannic acid powder containing 90 % tannin was obtained from Sigma-Aldrich (St. Louis, MO, USA). The tannic acid contained gallic acid, monogalloyl glucose, digalloyl glucose, trigalloyl glucose, tetragalloyl glucose, pentagalloyl glucose, ESA galloyl glucose, EPTA galloyl glucose, and octagalloyl glucose. The product is hydrolyzable tannin obtained from oak gall nuts from Quercus

infectoria. Tannic acid (0.1 g) was dissolved in 50 ml of de-ionized distilled water (DDW) and stored in a dark area to prevent exposure to air and light. BHA powder (0.1 g) was dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to make their stock solution. The ethanol added was removed using a rotary evaporator (BUCH Rotavapor, Model R-200) at (70 °C, 175 mbar vacuum pressure) before adding the stock solution to meat samples. Each additive treatment was added to the ground breast meat and then mixed for 2 min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same amounts of mineral oil to provide the same conditions. For raw-meat study, the prepared meat samples (approximately 100 g each) were individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI), stored at 4 ℃ cooler for up to 7 days, and analyzed for lipid and protein oxidation, and color at 0, 3, and 7 days of storage. The same preparation method was used for cooked meat study, but the raw meat samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3

2 mL O2/m /24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were cooked in-bag in a 90 °C water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA, USA) until the internal temperature of the meat reached to 75 °C. After cooling to room temperature, the cooked meat was transferred to a new oxygen-permeable bag (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at 4 °C for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3, and 7 days of storage.

2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid oxidation was determined using a TBARS method (Ahn et al., 1998). Five grams of ground chicken meat were weighed into a 50-mL test tube and homogenized with 50μL BHT (7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron homogenizer (Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for 15s at high

speed. One milliliter of the meat homogenate was transferred to a disposable test tube (13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA, 2 mL) was added. The mixture was vortex mixed and incubated in a boiling water bath for 15 min to develop color. Then samples were cooled in the ice-water for 10 min, mixed again, and centrifuged for 15 min at 2,500 x g at 4 oC. The absorbance of the resulting supernatant solution was determined at 532 nm against a blank containing 1 mL of DDW and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as mg of malondialdehyde (MDA) per kg of meat.

Color measurement: The color of meat was measured on the surface of meat samples using a Konica Minolta Color Meter (CR-410, Konioca Minolta, Osaka, Japan). The colorimeter was calibrated using an illuminant source C (Average day light) on a standard white ceramic tile covered with the same film as the ones used for meat samples to negate the color and light reflectance properties of the packaging material. The color were expressed as CIE L*- (lightness), a*- (redness), and b*- (yellowness) values (AMSA, 1991). The areas selected for color measurement were free from obvious defects that may affect the uniform color readings. An average from two random readings on each side (upper and lower) of sample surface was used for statistical analysis.

Volatile analysis: Volatiles of samples were analyzed using a Solatek 72 Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann, Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard Co., Wilmington, DE, USA) according to the method of Ahn et al. (2001). Each sample (3 g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample vial, flushed with helium gas (40 psi) for 3 s, and then capped airtight with a Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA). Samples from different treatments were randomly organized on the refrigerated (4 ℃) holding try to minimize the variation of the oxidative changes in sample during

analyses. The meat sample was purged with helium (40 mL/min) for 14 min at 20 °C. Volatiles were trapped using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and desorbed for 2 min at 225 °C, focused in a cryofocusing module (-70 °C), and then thermally desorbed into a capillary column for 2 min at 225 °C. An HP-624 column (7.5 m, 0.25 mm i.d., 1.4 m nominal), an HP-1 column (52.5 m, 0.25 mm i.d., 0.25m nominal), and an HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal) were connected using zero dead-volume column connectors (J &W Scientific, Folsom, CA, USA). Ramped oven temperature was used to improve volatile separation. The initial oven temperature of 25 °C was held for 5 min. After that, the oven temperature was increased to 85 °C at 40 °C per min, increased to 165 °C at 20 °C per min, and then increased to 230 °C at 5 °C per min and held for 2.5 min at that temperature. Constant column pressure at 22.5 psi was maintained. The ionization potential of MS was 70 eV, and the scan range was 20.1 to 350 m/z. The identification of volatiles was achieved by the Wiley Library (Hewlett-Packard Co.). The area of each peak was integrated using ChemStationTM software (Hewlett-Packard Co.) and the total peak area (total ion counts x 104) was reported as an indicator of volatiles generated from the samples.

Protein oxidation: Protein oxidation was determined by the method of Lund et al. (2008) with minor modifications. One gram of muscle sample was homogenized with a Brinkman Polytron (Type PT 10/35), Brinkman Instrument Inc., Westbury, NY,

USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10 mM Trizma-maleate),

100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol tetraacetic acid, pH 7.4). Two equal amounts of meat homogenate (2 mL) were taken from a sample, precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at 12,000 × g for 5 min at room temperature. After centrifugation, the pellet from 1 sample was treated with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M HCL, and the pellet from the other was incubated with 2 M HCL as a blank. During 30 min of incubation in the dark, samples were vortex-mixed for 10 s every 3 min. The protein

was further precipitated with 2 mL of 20% trichloroacetic acid and centrifuged at 12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by washing 3 times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate, followed by centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium dihydrogen phosphate (pH = 2.3). The samples were kept at 5 oC overnight. In next day, the samples were centrifuged to remove insoluble materials. The absorbance of supernatant was read at 370 nm. The absorbance values for blank samples were subtracted from their corresponding sample values. Briefly, Protein concentration was measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA., Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).

Results and Discussion

Lipid Oxidation In the raw chicken meat there was no significant difference (P > 0.05) in TBARS between treatments at day 0. In addition tannic acid at 2.5 ppm did not show any significant antioxidant effect on the TBARS values of chicken breast meat during storage. However, 5 ppm BHA or tannic acid at 5 and 10 ppm showed significant antioxidant effects after three days of storage (Table 1). Tannic acid at 10 ppm showed

the strongest antioxidant effects of all during storage. This result is in agreement with that of Maqsood and Benjakul (2010a) who found that tannic acid at 100-200 ppm effectively decreased both peroxide value and TBARS values in catfish slices. They found that tannic acid exhibited the highest antioxidant activities (peroxide value, conjugated diene, TBARS values) in both fish oil emulsion and fish mince among the phenolic compounds (catechin, caffiec acid, ferulic acid, and tannic acid) they had tested (Maqsood and Benjakul, 2010b). They explained that the high antioxidant effect of tannic acid was due to its ability to chelate non-heme iron in the fish mince. Iron is considered a strong pro-oxidant transitional metal that can increase lipid oxidation in meat. Because of their chemical structure, polyphenol tannic acid inhibits hydroxyl radical formation from the Fenton reaction by complexing ferrous ions (Lopes et al., 1999). Lopes et al. (1999) found that the antioxidant activity of tannic acid is mainly due to iron chelating rather than ·OH scavenging activity. Maqsood and Benjakul (2010c) also reported that when tannic acid (200 mg/kg) were added to the refrigerated ground beef, its peroxide and TBARS values were significantly lower than that of the control. Research using tannic acid in cooked meat products has been very rare until recently. The initial TBARS values in cooked meat showed that all treatments added with tannic or BHA showed significantly antioxidant effects (Table 4). This could be explained by the development of lipid oxidation from raw meat as reviewed by Ahn et al. (2009). This was also in agreement with Rhee (1989) who reported that lipid oxidation of cooked meat products and their effect on meat flavors depends on the initial lipid oxidation in the raw meat. In addition, cooked meat is more susceptible to lipid oxidation than raw meat because the denaturation of antioxidant enzymes and the structural damages in membrane during cooking can expose phospholipids to pro-oxidant environment (Ahn et al., 1992; Gray et al., 1996). Therefore, lipid oxidation in cooked meat increased faster, and the variations between treatments were clearer than the raw meat. The highest antioxidant effect was observed when 10 ppm tannic acid was added to the meat. Maqsood and Benjaku (2010c) also found that

tannic acid can be a good additive that retards the initiation and propagation steps of lipid oxidation reaction when added to the ground beef meat. Antioxidant free radicals of tannic acid characterized as stable and have low energy or low activity to initiate oxidation of unsaturated fatty acid usually formed (Maqsood and Benjakul, 2010b). Tannic acid at 10 ppm had stronger antioxidant effect than 5 ppm tannic acid and 5 ppm BHA at day 7 of storage (Table 4). This strongly suggested that tannic acid could be a good replacement for the synthetic antioxidant in food containing fat. However, tannic acid at 10 ppm showed the strongest antioxidant (p < 0.05) effect of all during storage

Protein oxidation Protein and lipid oxidation are major concerns that affect meat quality during storage (Nieto et al., 2013). It has been reported that protein oxidation can lead to changes in overall properties of meat proteins such as gelation, viscosity, solubility, and emulsification etc. (Xiong, 2000). Little information about the relations between protein oxidation and their effect on some meat quality attributes are available. In this study different levels of tannic acid were investigated to study their effect on the total carbonyl formation (nmol/mg of protein). There were no significant differences (P > 0.05) among the tannic acid treatments in raw chicken breast meat during the first three days of storage (Table 2). The changes of protein oxidation values in raw chicken breast meat during the 7-day storage period were very low (0.54 at day zero to 0.82 at day 7 for control samples). This was in agreement with Xiao et al. (2011) who found that the total carbonyl content in raw chicken meat patties stored aerobically at 4 ℃ increased from 0.46 to 0.81 nmol/mg of protein. Tannic acid at level 10 ppm was the only treatment showed significant antioxidant effect (P < 0.05) at day 7 of storage. Maqsood and Benjakul (2010c) found that tannic acid reduced the degradation of myosin heavy chain and actin in meat by suppressing microbial growth during storage. This indicated that the antimicrobial effect of tannic acid could have contributed to the total carbonyl formation in meat with different levels of tannic acid.

On the other hand, the formation of secondary products (carbonyl and others) of protein oxidation is also related to the degree lipid oxidation because lipid oxidation is directly related to protein oxidation. In contrast to raw meat, total carbonyl values in cooked meat were much higher than those of raw meat and reached up to 2 nmol/mg protein after 7 days of storage (Table 5). Other researchers reported that total estimated carbonyl contents were in the range of 1-3 nmol/mg protein for raw meat and up to 5 nmol/mg protein for cooked meat products (Estevez et al., 2005; Sun et al., 2010; Estevea, 2011). Adding tannic acid at 10 ppm successfully delayed the total carbonyl formation in cooked meat during storage. However, tannic acid at 5 and 10 ppm were more effective than other treatments in inhibiting carbonyl formation during storage. These results are well correlated with the TBARS values of cooked ground chicken meat during storage. The mechanism of tannic acid in preventing protein oxidation still needs more investigation, but the iron-chelating activity of tannic acid could be major reason for delaying total carbonyl formation in meat (South and Miller, 1998; Lopes et al., 1999).

Color values Chicken breast meat contains much lower myoglobin content than meats from other animal species such as beef, goat, pork and sheep. Meat color is an important quality parameter that affects the consumer purchase decision (Bekhit and Faustman, 2005). However, very little research was done on the color of ground chicken meat (Sanduskey and Heath, 1998). Table 3 showed that L* values of meat regardless of treatments decreased significantly (P < 0.05) during storage. There were no significant differences (P > 0.05) in L*-values- among the treatments at days 0 and 3 of storage. Tannic acid showed the highest significant effect compared to the other treatments using level of 10 ppm. The breast meat added with 10 ppm tannic acid had the highest L*-value (lighter meat) and the control had the lowest value (darker meat). The meat with high lightness value is considered more acceptable by the consumer than the

darker one because it can be considered old or spoilage in process (Hood and Riordan, 1973). Regardless of treatments, a*-values decreased during storage. However, no significant difference was found (P > 0.05) between treatments at day 0. This agreed with the results of Xiao et al. (2011) who reported that a*- and L*-values of ground chicken meat decreased significantly after 7 day of refrigerated storage. Mancini and Hunt (2005) reported that the decrease of a*-value during storage is due to accumulation of metmyoglobin pigment. Tannic acid at 10 ppm showed the highest effect in preventing changes in a*-value of all treatments. This is in agreement with the results of Maqsood and Benjakul (2010c) who found that all ground beef samples treated with tannic acid had higher oxymyoglobin and a*-value, and received higher likeness score for color in sensory evaluation. However, no significant difference in a*-value between tannic acid level at 5, 10 ppm tannic acid, and 5 ppm BHA during storage. In comparison Luciano et al. (2009) found that dietary tannins improved lamb minced-meat color stability and their shelf life. Changes in b*-values in chicken breast meat during storage were not significant even though tannic acid -treated meat showed higher values than control.

Volatiles production No significant treatments effects on the volatile contents and profiles in raw meat were found (data not shown). In cooked meat, tannic acid at 5 and 10 ppm showed the highest significant effect on the formation of most of these volatiles after 3 days of storage (Tables 7 & 8). For example tannic acid at 10 ppm effectively decreased pentane, octane, hexanl, and pentanal formation during storage. Hexanal, which is considered as a good indicator for lipid oxidation (Shahidi et al., 1987; Ahn et al., 2000), was significantly affected by 10 ppm of tannic acid at Day 7 of storage (Table 8). Heptanal and nonanal were formed and increased only in control and 2.5 ppm tannic acid-added samples (Tables 7 & 8). The sulfur compounds formations were found to be inconsistent and disappear from the samples. These sulfur compounds such as dimethyl disulfide, dimethyl trisulfide were disappeared after the

first day of storage. Nam et al. (2002) and Lee et al. (2003) found that most of the sulfur volatile compounds in turkey breast escaped or disappeared during storage under aerobically packaging due to their high volatility. Cooked meat showed higher volatiles formation and more diversity compared with raw meat. At day 0, however, no significant differences in the amounts and profiles of volatiles in cooked meat among the treatments were found. Hexanal increased rapidly in cooked meat during storage due to high degree of lipid oxidation. Many lipid oxidation-related aldehydes such as propanal, hexanal, and pentanal were also detected during storage of cooked meat. Tannic acid at 10 ppm showed the strongest effect in preventing aldehydes formation in cooked meat. This agreed and reflected the positive relationships between the aldehydes and the degree of lipid oxidation as reported by Du et al. (2000) and Ahn et al. (1998) (Tables 10 & 11). Similar effects on the heptanal and nonanl formation were seen when 10 ppm of tannic acid was added to the cooked meat. Overall, the profile of volatiles indicated that tannic acid at 10 ppm effectively delayed the formation of lipid-oxidation-related volatiles in cooked chicken breast meat during storage.

Conclusion Tannic acid at 5 or 10 ppm could be effective in maintaining meat color and retarding lipid and protein oxidation and off-odor-related volatiles in fresh ground chicken breast during storage. Therefore, tannic acid could be a good candidate as a natural antioxidant to prevent oxidative changes and color changes in raw and cooked chicken breast meat during storage.

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Table 1. TBA value of raw ground chicken breast meat at different storage time at 4 ℃. Time Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM without Tannic Tannic Tannic BHA

------TBARS (mg/Kg) meat ------az az ay ax ay Day 0 0.14 0.13 0.13 0.12 0.13 0.007

ay ay abx bx ax Day 3 0.18 0.17 0.15 0.13 0.18 0.010

ax ax bcx cx bx Day 7 0.34 0.33 0.17 0.14 0.19 0.008 SEM 0.001 0.009 0.007 0.01 0.016 a-cValue with different letter within a row are significantly different (P<0.05). X-zValue with different letter within a column are significantly different (p<0.05). N=4.

TBA value in a mg Malonaldehyde per Kg meat. Treatment: Control, 2.5 ppm tanic acid, 5 ppm tannic acid, 7 ppm tannic acid, and 5ppm BHA.

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Table 2. Effect of adding different level of tannic acid on protein oxidation of chicken breast patties during storage time. Time Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM

without Tannic Tannic Tannic BHA

------Carbonyl (nmol/ mg of protein) ------

ay ay ay ax ay Day 0 0.54 0.54 0.52 0.52 0.53 0.025

ax ax ax ax ax Day 3 0.72 0.72 0.70 0.64 0.71 0.055

ax ax ax bx ax Day 7 0.82 0.81 0.77 0.66 0.77 0.020

SEM 0.040 0.040 0.034 0.040 0.033 a-cValue with different letter within a row are significantly different (P<0.05). X-zValue with different letter within a column are significantly different (p<0.05). N=4. Treatment: Control, 2.5 ppm tannic acid, 5 ppm tannic acid, 7 ppm tannic acid, and 5ppm BHA.

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Table 3. CIE color value of ground chicken patties with different level of tannic acid during storage at 4 ℃. Time Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA L* ax ax ax ax ax Day 0 64.52 64.28 64.32 64.40 64.32 0.17

ax ax ay ax ay Day 3 63.87 63.74 63.63 64.04 63.64 0.14

by aby az ay abz Day 7 61.44 62.45 62.58 63.27 62.44 0.24 SEM 0.29 0.16 0.14 0.18 0.08 a* ax ax ax ax ax Day 0 8.53 8.45 8.43 8.41 8.43 0.14

by by aby ay by Day 3 6.50 6.42 6.71 7.00 6.40 0.10

bz by aby ay aby Day 7 6.09 6.07 6.43 6.91 6.35 0.15 SEM 0.09 0.2 0.09 0.12 0.13 b* bx bx bx ax bx Day 0 20.09 20.14 19.63 21.33 20.21 0.15

cy cy cbx ax bax Day 3 19.03 19.02 19.75 21.41 20.43 0.27

abx abx bx ax abx Day 7 20.34 20.26 19.88 20.81 20.53 0.20 SEM 0.19 0.18 0.29 0.21 0.16 a-c Value with different letter within a row are significantly different (P<0.05). X-z Value with different letter within a column are significantly different (p<0.05). N=4. CIE Color L*,a*,b*-value of ground chicken patties with different level of oregano oil during storage at 4 C.

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Table 4. TBA value of cooked ground chicken breast meat at different storage time at 4℃. Time Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA ------TBARS (mg/km) meat ------az by bz bcz by Day 0 0.19 0.15 0.13 0.11 0.14 0.01

ay bx cy dy bx Day 3 1.42 1.08 0.57 0.28 0.97 0.06

ax bx cx dx cx Day 7 2.23 1.26 0.79 0.34 0.99 0.05 SEM 0.06 0.1 0.03 0.01 0.03 a-cValue with different letter within a row are significantly different (P<0.05). X-zValue with different letter within a column are significantly different (p<0.05). N=4.

TBA value in a mg Malonaldehyde per Kg meat.

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Table 5. Effect of adding different level of tannic acid on protein oxidation of chicken breast patties during storage time. Time Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA ------Carbonyl(nmol/mg of protein)------az ay ax ay ay Day 0 0.58 0.57 0.46 0.45 0.47 0.07

ay axy bx bxy bx Day 3 1.21 1.13 0.62 0.59 0.80 0.05

ax bx cx cx cx Day 7 2.01 1.38 0.64 0.60 0.82 0.10 SEM 0.03 0.14 0.08 0.04 0.03 a-cValue with different letter within a row are significantly different (P<0.05). X-zValue with different letter within a column are significantly different (p<0.05). N= 4.

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Table 6. Profile of volatiles cooked meat chicken patties with different level of Tannic acid at day 0. 4 ------Total Ion counts * 10 ------Compounds Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA Pentane 238a 240a 0b 0b 0b 38 2-propanone 5267a 3407a 3325a 4171a 1923a 1057 Ethanol 8757a 8805a 8302a 8483a 3557b 830 2-propanol 810a 551a 564a 494a 779a 142 Hexane 331b 277b 144bc 24c 830a 50 Heptane 56ab 50b 0c 0c 85a 7 Pentanal 566a 69b 0b 0b 0b 51 Octane 274a 148ab 143ab 54b 162ab 31 Hexanal 1761a 1447a 78b 0b 162b 174 a-cDifferent letter (a-c) within a row are significantly different (P<0.05), n=4.

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Table 7. Profile of volatiles cooked meat chicken patties with different level of tannic acid at day 3. 4 ------Total Ion counts * 10 ------Compounds Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA Pentane 912a 766a 0b 0b 0b 50 Propanal 1459a 981a 0b 0b 0b 152 2-propanone 3792b 2283b 5796a 5638a 2674b 403 Ethanol 5466a 6959a 4546ab 6540a 2845b 576 2-propanol 4826b 8903a 8552a 4842b 5671b 461 Heptane 410a 303a 0b 0b 0b 31 Pentanal 3916a 1732b 0c 0c 110c 115 Octane 171a 60b 61b 0b 149a 17 Hexanal 11755a 9757a 257b 0b 924b 559 Heptanal 64a 56a 0b 0b 0b 3 Nonanal 77a 47b 0c 0c 0c 4 a-cDifferent letter (a-c) within a row are significantly different (P<0.05), n=4.

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Table 8. Profile of volatiles cooked meat chicken patties with different level of tannic acid at day 7. 4 ------Total Ion counts * 10 ------Compounds Control 2.5 ppm 5 ppm 10 ppm 5 ppm SEM Without Tannic Tannic Tannic BHA Pentane 1195b 2349a 0c 0c 0c 101 Propanal 350a 310a 0b 0b 0b 19 2-propanone 6506a 4420ab 3682ab 3348b 3873ab 662 2-propanol 9122a 1354b 1435b 1438b 1364b 249 1-Propanol 2625a 1228b 1377b 1440b 1322b 172 Heptane 302a 144ab 0b 0b 0b 42 Pentanal 3849a 4148a 0b 0b 126b 225 Octane 776a 407b 56bc 0c 200bc 83 Hexanal 67351a 51368a 621b 0b 1328b 5374 Heptanal 252a 223a 0b 0b 0b 8 Nonanal 101a 76ab 0b 0b 0b 18 a-cDifferent letter (a-c) within a row are significantly different (P<0.05), n=4.

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CHAPTER 5 THE EFFECT OF OREGANO OIL AND TANNIC ACID COMBINATIONS ON THE QUALITY AND SENSORY CHARACTERISTICS OF GROUND CHICKEN MEAT

*Corresponding author: D. U. Ahn Phone: (515) 294-6595, Fax: (515) 294-6595 Email: [email protected]

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Abstract

The antioxidant effect of different combination levels of oregano essential oil and tannic acid on both breast and thigh ground chicken meat were studied. Six different treatments including 1) (none added), 2) 100 ppm Oregano essential oil + 5 Tannic acid, 3) 100 Oregano essential oil + 10 Tannic acid, 4) 200 ppm Oregano essential oil + 5 Tannic acid, 5) 200 Oregano essential oil + 10 tannic acid and 6) 5 ppm butylated hydroxy anisole (BHA) for breast or 14 ppm for thigh meat were prepared. For raw meat study, samples were individually packaged in oxygen-permeable bags and stored at 4 ℃ cooler for up to 7 days. On the other hand cooked meat samples were packaged in oxygen impermeable vacuum bags and then cooked in-bag to the internal temperature of 75 ℃. After cooling to room temperature, the cooked meat was transferred to new oxygen-permeable bags and stored at 4 ℃ for up to 7 days. Chicken meat patties were analyzed for lipid and protein oxidation, color, and volatiles at 0, 3, and 7 days of storage. The significant differences among the treatments were clearer in cooked meat than in raw meat. Thigh meat patties showed higher TBARS, total carbonyl, and volatiles values compared to the breast meat during storage. The combination of 200 ppm oregano oil and 10 ppm tannic acid showed the most significant effects (P < 0.05) on TBARS, total carbonyl, and off odor volatile formation for both breast and thigh meat. In addition, the combination of 200 ppm oregano oil and 10 ppm tannic acid showed the highest effect in stabilizing color for both raw breast and thigh meat. Oregano oil (200 ppm) and 10 ppm tannic acid combination also showed positive effects on the sensory scores of chicken thigh meat. In conclusion, the combination of 200 ppm oregano oil and 10 ppm tannic acid could be a good natural replacement for the synthetic antioxidants in the ground chicken meat.

Key Words: Oregano essential oil, butylated hydroxyanisole, ground meat, lipid oxidation, thigh meat.

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Introduction

Recently it has become very interesting and highly recommended to use natural antioxidants as food additives by the food industry (Solomakos et al., 2008; Karre et al., 2013). This new approach considers a good replacement for the synthetic antioxidant (butylated hydroxytoulene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroquinone (TBHQ)) which have a very limited usage (USDA, Food additive regulation; 9 CFR 318.7, and 381.147) in a maximum 0.02 % based on the fat content or meat preservative content (salt) in meat products such as dry sausage, fresh pork, beef sausage, dried meat, and various poultry products. On the other hand the natural antioxidant and particularly the plant sources have smaller limitation on their use in food or meat products. Several herbs and plant extracts were used in poultry meat to maintain their stability and extend shelf life (Fasseas et al., 2007; Brannan, 2008; Abdel-hamied et al., 2009). Lipid and protein oxidations are the major problems due to their effect on poultry meat quality and freshness. They are responsible for quality deterioration, including color, flavor, and nutritive values of meat (Lee et al., 2005a; Ahn et al., 2007; Nam et al., 2007). However, color and odor are the two main quality parameters that influence consumer’s final decision on purchasing meat (Liu et al., 1995; Mancini and Hunt, 2005). Natural antioxidants from plant origins are a safe and healthy replacement for the synthetic ones that can prevent oxidation in meat (Solomakos et al., 2008; Gulcin et al., 2010). Oregano essential oil and tannins are extracted from plants and have strong antioxidant properties (Goulas and Kontominas, 2007; Scramlin et al., 2010). The antioxidant effect of oregano essential oil is due to its high polyphenol content such as carvacrol, and thymol (Kosar et al., 2003; Capecka et al., 2005; Al-Bandak, 2007;). However, very little work has been done to add oregano essential oil directly to the meat to prevent oxidative changes in the meat (Fasseas et al., 2007; Chouliara et al., 2007; Scramlin et al., 2010). Tannins are secondary poly-phenol compounds produced by plants to protect

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themselves from external herbivores and diseases (Salminen et al., 2011). These compounds have biological, metal ion chelating, antioxidant (Hagerman, 2010; Okuda and Ito, 2011), and protein precipitating activities (Hagerman et al., 1998). In addition, tannins showed positive effect on meat color stability (Luciano et al., 2009; Maqsood and Benjakul, 2010c). These properties of tannins are due to their chemical structures which have two or three phenolic hydroxyl groups on phenyl ring. Hydrolysable tannins are more popular to use in the food preservation than the condensed type (Chung, 1998; Okuda and Ito, 2011). The hydrolysable type of tannins contains polyhydric alcohol and hydroxyl groups, which are esterified with gallic acid (Gallotannins) or to hexahydroxydiphenic acid (Ellagitannins) (Okuda et al., 1985; Okuda, 2005; Haslam, 1989; Okuda and Ito, 2011). Using one type of natural antioxidant to replace synthetic antioxidant is not enough as many researchers have reported. Therefore, a combination of antioxidants with different antioxidant mechanisms should be considered to increase their effectiveness (Brewer, 2011). The term Synergism is widely used to explain this effect between different combinations of antioxidants added to foods. For example, the effect of adding dietary synthetic antioxidant combined with vitamin E were found to be more effective in preventing lipid and protein oxidation in fresh frozen chicken patties than using one natural antioxidant (Smet et al., 2008). The synergistic effect can be explained by mixing the free radical acceptors for both antioxidants which will work synergistically to prevent the oxidation process. Aurand and Woods (1979) reported that the chemical associations between some acid compounds (e.g. citric acid) and phenolic antioxidants decreased the development of lipid oxidation. The antioxidant activity of phenolic compounds, the ability of acid compounds to bind metal ions, and the complex structure between them explained their synergistic effect. Furthermore, the combinations between the acid and antioxidant suppressed the role of antioxidant radicals to break down lipid peroxides. Lee et al. (2005b) found that the combination of chelators (soduim triphospahte or

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sodium citrate) with reductants (e.g erythrobate, and rosemary extract) were more effective in decreasing lipid oxidation and maintaining color of ground beef. Several studies found positive effect of using oregano oil and tannic acid in meat preservation (Chouliara et al., 2007; Maqsood and Benjakul, 2010c). Oregano oil were added in animal diet (Botsoglou et al., 2002) or added directly to meat during processing (Fasseas et al., 2007) to determine their effect on meat quality. Similar applications for tannic acid or tannins extracts also had been tested (Luciano et al., 2009; Lui et al., 2009; Maqsood and Benjakul, 2010a, b, c; Devatkal et al., 2010; Maqsood and Benjakul, 2011; Brogna et al., 2014). The antioxidant effects of various plants extracts such as green tea, fruit juices, and chestnut wood that contain high levels of tannins (Liu et al., 2009; Staszewski et al., 2011) were also tested in various foods and meat systems. Naveena et al. (2008) investigated the effect of adding natural plant resources containing high levels of tannins in chicken patties and found that 10 mg equivalent phenolics/100g meat would be sufficient to protect chicken patties from oxidative rancidity for period longer than the most commonly used synthetic antioxidant like BHT. There is no study conducted to evaluate the combination effect of tannic acid and oregano oil in meat quality and their storage stability. The objectives of this study were: 1) to evaluate combination effect of tannic acid and oregano oil on the quality and storage stability of raw and cooked ground chicken thigh or breast meat, and 2) to select the best tannic acid-oregano oil combinations to control lipid oxidation, protein oxidation, off-odor, and color in raw and cooked ground chicken meat.

Material and Methods

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Boneless muscles (breast and thigh) were cleaned, skins removed, external fats trimmed off, and stored at -20℃ freezer until being used.

The frozen meats were thawed in a walk-in cooler (4 ℃), ground twice a through a 10-mm and a 3-mm plates (Kitchen Aid, Inc., St. Joseph, MI, USA) before use. Six different treatments including 1) (none added), 2) 100 ppm Oregano essential oil + 5 Tannic acid, 3) 100 Oregano essential oil + 10 Tannic acid, 4) 200 ppm Oregano essential oil + 5 Tannic acid, 5) 200 Oregano essential oil + 10 tannic acid and 6) 5 ppm butylatedhydroxy anisole (BHA) for breast or 14 ppm for thigh meat (based on fat content) were prepared. The oregano essential oil was obtained from a certified company in Turkey (Healthy-Health, Oil of Oregano, Turkey). The GC/MS analysis of the oregano essential oil indicated that 80.12% of the essential oil was carvacrol. BHA powder (0.1 g) and oregano essential oil (1.25 g) were dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to make their stock solutions. The ethanol added was removed using a rotary evaporator (BUCH Rotavapor, Model R-200) at (70℃, 175 mbar vacuum pressure) before adding the stock solution to meat samples. Tannic acid powder containing 90 % tannin was obtained from Sigma-Aldrich (St. Louis, MO, USA). The tannic acid contained gallic acid, monogalloyl glucose, digalloyl glucose, trigalloyl glucose, tetragalloyl glucose, pentagalloyl glucose, ESA galloyl glucose, EPTA galloyl glucose, and octagalloyl glucose. The product’s name is hydrolyzable tannin obtained from oak gall nuts from Quercus infectoria. Tannic acid (0.1 g) was dissolved in 50 ml of de-ionized distilled water (DDW) and stored in a dark area to prevent exposure to air and light. Each additive treatment was added to the ground meat (breast or thigh) and then mixed for 2 min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same amount of mineral oil to provide the same conditions. For raw-meat study, the prepared meat samples (approximately 100 g each) were individually packaged in oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated

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Bag Co., Milwaukee, WI), stored at 4 ℃ cooler for up to 7 days, and analyzed for lipid and protein oxidation, and color at 0, 3, and 7 days of storage. The same preparation method was used for cooked meat study, but the raw meat samples were packaged in oxygen impermeable vacuum bags (O2 permeability, 9.3

2 mL O2/m /24 h at 0 °C, Koch, Koch, Kansas City, MO, USA), and the meats were cooked in-bag in a 90 ℃ water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA, USA) until the internal temperature of the meat reached to 75 ℃. After cooling to room temperature, the cooked meat was transferred to a new oxygen-permeable bag (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI, USA), and stored at 4 ℃ for up to 7 days, and analyzed for lipid and protein oxidation and volatiles at 0, 3, and 7 days of storage.

2. 2-Thiobarbituric acid-reactive substances (TBARS) measurement: Lipid oxidation was determined using a TBARS method (Ahn et al., 1998). Five grams of ground chicken meat were weighed into a 50-mL test tube and homogenized with 50μL BHT (7.2%) and 15 mL of deionized distilled water (DDW) using a Polytron homogenizer (Type PT 10/35, Brinkman Instruments Inc., Westbury, NY, USA) for 15s at high speed. One milliliter of the meat homogenate was transferred to a disposable test tube (13 × 100 mm), and thiobarbituric acid (15 mM TBA/15% TCA, 2 mL) was added. The mixture was vortex mixed and incubated in a boiling water bath for 15 min to develop color. Then samples were cooled in the ice-water for 10 min, mixed again, and centrifuged for 15 min at 2,500 x g at 4 ℃. The absorbance of the resulting supernatant solution was determined at 532 nm against a blank containing 1 mL of DDW and 2 mL of TBA/TCA solution. The amounts of TBARS were expressed as mg of malondialdehyde (MDA) per kg of meat.

3. Color measurement (Konioca Minolta): The color of meat was measured on the surface of meat samples using a Konica Minolta Color Meter (CR-410, Konioca Minolta, Osaka, Japan). The colorimeter was calibrated using an illuminant source C (Average day light) on a standard white ceramic tile covered with the same film as the

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ones used for meat samples to negate the color and light reflectance properties of the packaging material. The color were expressed as CIE L*- (lightness), a*- (redness), and b*- (yellowness) values (AMSA, 1991). The areas selected for color measurement were free from obvious defects that may affect the uniform color readings. An average from two random readings on each side (upper and lower) of sample surface was used for statistical analysis.

4. Volatile compounds: Volatiles of samples were analyzed using a Solatek 72 Multimatrix-Vial Auto-sampler/Sample Concentrator 3100 (Tekmar-Dohrmann, Cincinnati, OH, USA) connected to a GC/MS (Model 6890/5973; Hewlett-Packard Co., Wilmington, DE, USA) according to the method of Ahn and others (2001). Sample (3 g for raw meat and 2 g for cooked meat) was placed in a 40-mL sample vial, flushed with helium gas (40 psi) for 3 s, and then capped airtight with a Teflon*fluorocarbon resin/silicone septum (I-Chem Co., New Castle, DE, USA). Samples from different treatment were randomly organized on the refrigerated (4 ℃) holding try to minimize the variation of the oxidative changes in sample during analyses. The meat sample was purged with helium (40 mL/min) for 14 min at 20 ℃. Volatiles were trapped using a Tenax/charcoal/silica column (Tekmar-Dohrmann) and desorbed for 2 min at 225 °C, focused in a cryofocusing module (-70 ℃), and then thermally desorbed into a capillary column for 2 min at 225 °C. An HP-624 column (7.5 m, 0.25 mm i.d., 1.4 m nominal), an HP-1 column (52.5 m, 0.25 mm i.d., 0.25m nominal), and an HP-Wax column (7.5 m, 0.250 mm i.d., 0.25 m nominal) were connected using zero dead-volume column connectors (J &W Scientific, Folsom, CA, USA). Ramped oven temperature was used to improve volatile separation. The initial oven temperature of 25℃ was held for 5 min. After that, the oven temperature was increased to 85 ℃ at 40 ℃ per min, increased to 165 ℃ at 20 ℃ per min, and then increased to 230 ℃ at 5 ℃ per min and held for 2.5 min at that temperature. Constant column pressure at 22.5 psi was maintained. The ionization potential of MS was 70 eV, and the scan range was 20.1 to 350 m/z. The identification of volatiles was

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achieved by the Wiley Library (Hewlett-Packard Co.). The area of each peak was integrated using ChemStationTM software (Hewlett-Packard Co.) and the total peak area (total ion counts x 104) was reported as an indicator of volatiles generated from the samples.

5. Protein oxidation (total carbonyl): Protein oxidation was determined by the method of Lund et al. (2008) with minor modifications. One gram of muscle sample was homogenized with a Brinkman Polytron (Type PT 10/35), Brinkman Instrument

Inc., Westbury, NY, USA) in 10 mL of pyrophosphate buffer (2.0 mM Na4P2O7, 10 mM Trizma-maleate), 100 mM KCL, 2.0 mM MgCl2, and 2.0 mM ethylene glycol tetraacetic acid, pH 7.4). Two equal amounts of meat homogenate (2 mL) were taken from a sample, precipitated with 2 mL of 20 % trichloroacetic acid, and centrifuged at 12,000 × g for 5 min at room temperature. After centrifugation, the pellet from 1 sample was treated with 2 mL of 10 mM 2,4-dinitrophenylhydrazine dissolved in 2 M HCL, and the pellet from the other was incubated with 2 M HCL as a blank. During 30 min of incubation in the dark, samples were vortex-mixed for 10 s every 3 min. The protein were further precipitated with 2 mL of 20% trichloroacetic acid and centrifuged at 12,000 x g for 5 min. The 2,4-dinitrophenylhydrazine was removed by washing 3 times with 4 mL of 10 mM HCL in 1:1 (vol/vol) ethanol:ethyl acetate, followed by centrifuging at 12,000 × g for 5 min. The pellets were finally solubilized in 2 mL of 6.0 mM guanidine hydrochloride dissolved in 20 mM potassium dihydrogen phosphate (pH = 2.3). The samples were kept at 5℃ overnight. In next day, the samples were centrifuged to remove insoluble materials. The absorbance of supernatant was read at 370 nm. The absorbance values for blank samples were subtracted from their corresponding sample values. Briefly, Protein concentration was measured using Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) following Microplate Assay protocol at 280 nm absorbance (BioTek-Gen5 Microplate data collection & analysis software/BioTek Instruments, Inc., Model S4MLFPTA., Winooski, VT, USA). The carbonyl content was calculated as nmol/mg protein using

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absorption coefficient of 22,000/M/cm as described by Levine et al. (1994).

6. Sensory evaluation: Trained sensory panels were used to evaluate the sensory characteristics of the ground chicken thigh meat. Sensory panels were evaluated the color, aroma, and over all acceptability of both raw and cooked meat. Six treatments were prepared with the same method described in the quality analysis to evaluate the effect of adding different level of oregano oil and tannic acid combinations: control, combinations of 5 ppm tannic acid (TA) and 100 ppm oregano oil (Org), 10 ppm TA and 100 ppm Org, 5 ppm TA and 200 ppm Org, 10 ppm TA and 200 ppm Org, and 14 ppm BHA. The meat was refrigerated at 4 ℃ for three day before each evaluation session. Ten trained panelists (Iowa State University. Graduate Student and staff), were participated for each session. The evaluations were done twice after three day of storage time at 4 ℃. For training, 3 one-hour sessions were held using commercial and experimental products to develop descriptive terms for the desired attributes. Raw ground chicken meat was evaluated for color (redness), oregano aroma or spice odor, oxidative odor, and over all acceptability. Cooked meats were evaluated for cooked chicken, oxidative odor, spice odor or oregano odor, and over all acceptability.

All attributes were measured using a line scale without numbers (numerical value 9 units) with graduation from 0 to 9. For example overall acceptability; where 9 represented extremely desirable, and 0 represent extremely undesirable (9-extremely desirable, 8 very, 7 moderate, 6-slightly, 5-neithtly nor, 4 slightly undesirable, moderately, very, and extremely). Similar terminology (e.g detectable or undetectable) underline was used for the other attributes. Evaluation sessions for cooked and raw meat were done in a separate days to decrease any variability.

Refrigerated (4 °C) samples were evaluated by the panelists for each treatment. The panelist was served 1 glass vial, 20 ml volume, of each treatment to evaluate color and odor of raw thigh meat. All ground meat samples were packaged in oxygen

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permeable bags before evaluation session. The panelists were asked to evaluate the ground meat patties (color) and open the vials to evaluate odor. Raw meat samples of 10g each were placed in small vials (20-mL) capped with a septum (I-Chem CO.) in order to evaluate odor attributes. All samples vials were labeled by a three digit number selected randomly. For cooked meat, samples (4g) at 0 day of cooking were placed in a three digit coded 20-mL sample vial and capped with a septum (I-Chem Co.). Panelists were asked to smell samples in random order and record the intensity of odor or over all acceptability on the scale line.

Statistical analysis: Data were analyzed using the procedures of Generalized Linear Model (Proc. GLM, SAS program, version 9.3, 2012). Mean values and standard error of the means (SEM) were reported. The significance was defined at P < 0.05 and Tukey test or Tukey’s Multiple Range test were used to determine whether there is a significant difference between the mean values. Pearson correlation coefficient (CORR Procedure) was used to determine the relation between TBARS, total carbonyl, and hexanal.

Results and Discussion Lipid Oxidation The TBARS values of raw breast and thigh meat among the treatments at day 0 did not differ significantly (P > 0.05) (Table 1). However, the TBARS values of all treatments were significantly lower than that of the control (p < 0.05) at day 3 and 7. The highest combination effect was found when 200 ppm oregano oil and 10 ppm of tannic acid was used. Similar trends were found in thigh meat, but the TBARS values in thigh meat were higher than the breast meat (Tables 1). Several studies showed that oregano oil (Botsoglou et al., 2003a, b; Chouliara et al., 2007) and tannic acid (Luciano et al., 2009; Maqsood and Benjakul, 2010b; Maqsood and Benjakul, 2010c) have antioxidant effect when added directly or indirectly (animal diet) to the foods. It was also reported that a combination of antioxidants shows stronger antioxidant effects if the antioxidants work synergistically (Lee et al. 2005b; Brewer, 2011).

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Cooked chicken breast and thigh meats showed a clearer difference (p < 0.05) between treatments than the raw meat (Tables 5). Cooked meat is more susceptible to lipid oxidation than raw meat because of the denaturation of antioxidant enzymes and the structural damages in membrane, which can expose phospholipids to pro-oxidant environment in cooked meat (Ahn et al., 1992; Gray et al., 1996). The TBARS values of cooked ground thigh meat were higher than the cooked breast meat but the antioxidant effects were very similar in both breast and thigh muscle tissues. The higher TBARS values of thigh meat than breast are due to the higher fat and polyunsaturated fatty acid content, which increases their oxidation susceptibility to free radicals.

Protein Oxidation Total carbonyl (nmol/mg of protein) content of raw meat breast was lower than the thigh meat during storage (Table 2). Both ground chicken meat (raw breast and thigh meat) had no significant difference (P > 0.05) in total carbonyl values at day 0 of storage, and the formation of carbonyl was very slow in the raw meat during storage. This was in agreement with Xiao et al. (2011) who found that the total carbonyl content in raw chicken meat patties stored aerobically at 4 ℃ increased from 0.46 to 0.81 nmol/mg of protein. In thigh chicken meat, however, all treatment had significantly (P < 0.05) lower carbonyl content than the control at Day 3. Both breast and thigh meat treated with antioxidants (oregano, tannic acid, and BHA) had significantly (P > 0.05) lower carbonyl content than the control at day 7 of storage. However, combination of 200 ppm oregano oil and 10 ppm of tannic acid showed the highest effect in preventing carbonyl formation among the treatments. Both cooked breast and thigh meat showed higher total carbonyl values than the raw meat (Table 6) probably due to higher free radical formation in cooked meat than the raw meat. The highest total carbonyl value was found in cooked thigh meat (control), which reached to 4.58 nmol/mg protein. The results of total carbonyl formation were similar to those of other researchers who found that total estimated

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carbonyl contents were in the range of 1-3 nmol/mg protein for raw meat and up to 5 nmol/mg protein for cooked meat products (Estevez et al., 2005; Sun et al., 2010; Estevea, 2011). Again, the combination of 200 ppm oregano oil and 10 ppm tannic acid had the highest effect in preventing carbonyl formation in cooked meat. Generally a positive relation between TBARS values and total carbonyl formation were found during storage in both breast and thigh chicken meats. It is known that the antioxidant activity of oregano oil and tannic acid was due to their polyphenols, which works to prevent oxidation in the foods (Chung et al., 1998; Goulas and Kontominas, 2007; Okuda and Ito, 2011).

Color values There were no significant differences (P > 0.05) in color L*- and a*-values of breast meat among treatments at day zero (Table 3). However, significant differences in color L*- and a*-values were found after day 3 of storage. Both L*- and a*-values decreased, but the changes in b*-values were inconsistent during storage. Mancini and Hunt (2005) reported the decrease of a*-value during storage was due to the accumulation of metmyoglobin in meat. A strong relationship between Metmyoglobin (MetMb) accumulation and fresh meat discoloration were reported. However, several factors that can affect the MetMb reducing activity in fresh meat such as pH, storage temperature, storage time, lipid peroxidation, oxygen, ions and chemicals may also have been involved (Bekhit and Faustman, 2005). Since oregano oil and tannic acid slowed the oxidation in meat, they might have increased the MethMb reducing activity and stabilized meat color. The combination of 200 ppm oregano oil and 10 ppm TA acid showed the highest effect in stabilizing color values. This treatment had the highest L*- and a*-values at the end of storage time. This is in agreement with the results of others (Chouliara et al., 2007; Mastromatteo et al., 2009; Maqsood and Benjakul, 2010c) who found that oregano oil and tannic acid improved meat color stability. Thigh meat had lower L*-values, but higher a*-value than the breast meat (Tables 3 & 4). This agrees with the results of Sandusky and Heath (1998) who studied the differences between sensory and instrument-measured ground chicken

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meat color. The differences in a*- and L*-values between breast and thigh meat was due to their myoglobin content, water holding capacity, fatty acid content, and their freshness (Froning, 1995; Fletcher, 1999; Berri et al., 2005). The meat with high lightness value is considered more acceptable by the consumer than the darker ones because it is considered fresher (Hood and Riordan, 1973). The combination of 200 ppm of oregano oil and 10 ppm of tannic acid showed the highest effect in stabilizing color values. However, the differences between treatments and control were clearer in thigh meat than breast meat due to their higher myoglobin content than breast meat.

Volatiles production Generally, the amounts and number of volatiles compounds increased during storage time in both breast and thigh cooked meat patties. Raw meat patties showed very low volatiles production even after 7 days of storage (Data not shown). Among the carbonyl compounds, hexanal increased by five- to six-fold in untreated patties during the 7 day storage time. This was inagreement with Du et al. (2000) who found that the amount of total volatiles in aerobically-packaged chicken meat increased by five to six folds during 7 day of refrigerated storage conditions. However, in this study cooked thigh meat had higher volatiles values than the breast cooked meat (Tables 7-9 & Tables 10-12). The higher volatile values in thigh meat were due to their different chemical composition such as fat, water holding capacity, and polyunsaturated fatty acid. Control samples showed the highest aldehyde values among the treatments. Combination of 200 ppm oregano oil and 10 ppm of tannic acid showed the highest effects in inhibiting the volatile formation in both breast and thigh cooked meat during storage. Other treatments (oregano + tannic acid, and BHA) also decreased the volatile formation during storage. In addition, it showed significant effect (P < 0.05) on aldehydes, and other volatiles which are responsible for the off-odor and rancidity of ground chicken meat during storage time. Over all, this agrees with the TBARS values of ground cooked meat compared to the raw meat due to the higher oxidation susceptibility of cooked meat compared to the raw meat (Ahn

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et al., 1992; Gray et al., 1996). Lee et al. (2003) also found that the volatile aldehyde-reducing activity of antioxidants were consistent with the result of TBARS values in raw and cooked turkey breast. Similar relation was found between TBARS values and aldehydes formation in both breast and thigh meat. In addition, very high correlation coefficient between TBARS, total carbonyl, and hexanl values were found during storage time (Table 13). This explains the positive relationships between these three variables during storage time. Among aldehydes, hexanal and pentanal were the most predominant compounds affected by the combination of 200 ppm oregano oil and 10 ppm tannic acid. Jo et al. (2006) found that hexanal contents of control cooked chicken breast meat increased by about 2.7 times at day 1 compared with 0 day. Hexanal and pentanal are considered good indicators for lipid oxidation (Shahidi et al., 1987; Ahn et al., 2000) during storage time. The volatile profile also showed some terpenoids such as camphene and limonene when ground chicken meats mixed with oregano oil, and their amounts increased as the amount of oregano oil increased. These compounds are responsible for the spice odor of meat added with oregano oil. Combination of 200 ppm oregano oil and 10 ppm tannic acid also showed significant effect (P < 0.05) deteriorate most of aldhydes related-lipid oxidation such as propanal, hexanal, and pentanal in cooked thigh meat. Similar results were observed on the content of sulfur compounds (dimethyl trisulfide and dimethyl disulfide) and other volatiles which found in-consistant and in a very low concentration in the treated samples. Overall, very low amounts of sulfur volatiles were detected because of their high volatility, and susceptibility to escape from the oxygen permeable bags (Nam et al., 2002; and Lee et al., 2003). The combination (200 ppm oregano oil and 10 ppm tannic acid) treatment showed lower volatile formation than the BHA treatment in both breast and thigh cooked meat patties during storage.

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Sensory evaluation Panelists could not detect any significant treatment difference in color attribute of raw thigh chicken meat (Table 14). On the other hand, panelists easily distinguished oregano oil odor in raw thigh meat. However, sensory panels could not distinguish the treatment differences in cooked meat due to cooking effect on volatiles related to oregano odor (Table 15). Combination of 10 ppm of tannic acid and 200 ppm oregano oil treatment reduced oxidation odor score significantly ( P < 0.05). In addition, combination of 10 ppm of tannic acid and 200 ppm oregano oil treatment showed the highest score on the overall acceptability for both raw and cooked meat patties.

Conclusion

Tannic acid and oregano essential oil at level 200 ppm and 10 ppm showed positive effect on the decreasing TBARS values, total carbonyl, improve color stability, and deteriorate off-odor volatile formation. Data of lipid oxidation, protein oxidation, and color, volatile agreed with the sensory evaluation results. In conclusion the combination of 200 ppm oregano oil and 10 ppm tannic acid could be a good replacement for the synthetic antioxidant (BHA) in the ground chicken meat.

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Table 1. TBARS value of raw ground chicken breast and thigh meat added with different levels of oregano oil and tannic acid combinations during refrigerated storage at 4℃. Storage Control CM1 CM2 CM3 CM4 BHA SEM Time without 5 ppm

------TBARS (mg/Kg) ------Breast meat Day 0 0.12az 0.10az 0.11ay 0.09ay 0.10ax 0.10az 0.01 Day 3 0.19ay 0.16aby 0.14abcy 0.13cy 0.12cx 0.17aby 0.01 Day 7 0.34ax 0.26bcx 0.22cdx 0.17dex 0.14ex 0.28bx 0.01

SEM 0.01 0.01 0.01 0.02 0.01 0.01

Thigh meat 14 ppm

Day 0 0.25az 0.22ay 0.24ay 0.24ay 0.22ay 0.24ay 0.017 Day 3 0.90ay 0.30bcy 0.27bcy 0.25bcy 0.23cy 0.33by 0.022 Day 7 2.21ax 1.54bcx 1.16cdx 0.98dex 0.60ex 1.73bx 0.102 SEM 0.077 0.091 0.036 0.029 0.034 0.069 a-eValues with different letter within a row are significantly different (P < 0.05). x-zValues with different letter within a column are significantly different (P < 0.05). N= 4. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 2. Effect of adding different level of oregano oil and tannic acid on protein oxidation of raw chicken breast and thigh patties during storage time. Storage Control CM1 CM2 CM3 CM4 BHA SEM Time without 5 ppm

------Carbonyl (nmol/ mg of protein)------Breast meat Day 0 0.76ay 0.75ax 0.74ax 0.74ax 0.74ax 0.75ax 0.03 Day 3 0.84ay 0.80ax 0.77ax 0.75ax 0.74ax 0.82ax 0.04 Day 7 1.04ax 0.87abx 0.81bx 0.77bx 0.75bx 0.88abx 0.05 SEM 0.04 0.04 0.02 0.04 0.03 0.05 Thigh meat 14 ppm Day 0 0.86az 0.84ax 0.83ax 0.83ax 0.81ax 0.83ay 0.035 Day 3 1.27ay 0.94bx 0.87bx 0.83bx 0.83bx 0.95by 0.061 Day 7 1.68ax 1.11bcx 0.92bcx 0.86bcx 0.83cx 1.13bx 0.065 SEM 0.28 0.071 0.056 0.079 0.041 0.038 a-cValue with different letter within a row are significantly different (P<0.05). x-zValue with different letter within a column are significantly different (p<0.05). N = 4. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 3. *CIE color values of ground chicken breast patties with different level of oregano oil and tannic acid stored at 4℃. Time Control CM1 CM2 CM3 CM4 5 ppm SEM without BHA L* Day 0 63.0ax 62.7ax 63.2ax 62.9ax 62.9ax 62.8ax 0.18 Day 3 61.1by 61.5aby 61.7aby 61.7aby 62.1ay 61.1by 0.18 Day 7 60.6by 61.2aby 61.3aby 61.5ay 60.9abz 61.05aby 0.20 SEM 0.2 0.19 0.17 0.15 0.14 0.21 a* Day 0 8.28ax 8.24ax 8.31ax 7.84ax 8.24ax 8.40ax 0.21 Day 3 6.52by 6.39by 6.86aby 6.69by 7.32ay 6.64by 0.13 Day 7 5.89cy 6.12cby 6.24cbz 6.49aby 6.76az 6.43aby 0.12 SEM 0.20 0.20 0.10 0.09 0.13 0.18 b* Day 0 19.4by 18.9by 20.4ax 20.2ax 20.1ax 20.6ax 0.12 Day 3 19.9ay 19.4cx 19.6abcy 19.8aby 19.5bcy 19.9ay 0.09 Day 7 20.3ax 19.3bx 19.06bz 19.97ay 19.49by 20.32ax 0.10 SEM 0.14 0.08 0.06 0.14 0.11 0.11 a-cValue with different letter within a row are significantly different (P<0.05). x-zValue with different letter within a column are significantly different (p<0.05). N = 4. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 4. *CIE color value of ground chicken thigh patties with different level of oregano oil and tannic acid during storage at 4 ℃. Time Control CM1 CM2 CM3 CM4 14 ppm SEM without BHA L* Day 0 57.12ax 56.15bx 56.09bx 56.25abx 56.24abx 56.70abx 0.22 Day 3 55.02ay 54.78ay 54.85ax 55.09axy 54.56ay 55.02ay 0.25 Day 7 52.26bz 53.47abz 53.50aby 53.74aby 54.10ay 52.39bz 0.34 SEM 0.085 0.18 0.314 0.431 0.216 0.271 a* Day 0 10.03ax 9.85ax 9.78ax 9.62ax 9.63ax 10.0ax 0.10 Day 3 8.88by 9.43ax 9.48ay 9.46ax 9.85ax 9.52abx 0.08 Day 7 7.29cz 8.53aby 8.64abz 8.78aby 9.02ay 7.99bcy 0.22 SEM 0.138 0.199 0.076 0.124 0.064 0.221 b* Day 0 16.51ay 16.74axy 16.96ay 16.95ay 16.82ay 17.00ax 0.15 Day 3 17.16ax 16.26ay 16.32az 16.70ay 16.69ay 17.31ax 0.23 Day 7 17.33ax 17.54ax 17.36ax 17.42ax 17.65ax 17.57ax 0.08 SEM 0.151 0.284 0.097 0.147 0.108 0.144 a-c Value with different letter within a row are significantly different (P<0.05). X-z value with different letter within a column are significantly different (p<0.05). N=4. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 5. *TBARS values of cooked ground chicken meat with different combination level of oregano oil and tannic acid at 4℃. Time Control CM1 CM2 CM3 CM4 BHA Without 5 ppm SEM ------TBARS (mg/kg) meat ------Breast meat

Day 0 0.21az 0.18az 0.19ay 0.21az 0.19ay 0.21az 0.015

Day 3 1.36ay 0.87by 0.77bcx 0.74bcy 0.57cx 0.96by 0.066

Day 7 2.03ax 1.32bcx 0.97cdx 0.88dx 0.75dx 1.50bx 0.079

SEM 0.084 0.038 0.076 0.014 0.064 0.056

Thigh meat 14 ppm

Day 0 1.23az 1.10az 1.09az 0.97az 0.94ay 1.27az 0.074

Day 3 3.40ay 1.64cy 1.36dy 1.11ey 1.01ey 1.88by 0.036

Day 7 3.48ax 2.58bcx 2.32cdx 2.07dx 1.75ex 2.82bx 0.062

SEM 0.056 0.04 0.052 0.026 0.071 0.082 a-e Values with different letter within a row are significantly different (P < 0.05). n = 4. x-z Values with different letter within a column are significantly different (P < 0.05). *TBARS value in mg Malonaldehyde per Kg meat. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 6. Effect of adding different level of oregano oil and tannic acid on protein oxidation of cooked chicken patties during storage time. Time Control CM1 CM2 CM3 CM4 BHA SEM Without 5 ppm Breast ------Carbonyl (nmol/ mg of protein)------meat Day 0 1.00az 1.05ay 0.99ay 0.98ay 0.98az 1.01az 0.06 Day 3 1.69ay 1.45abxy 1.31bx 1.26bxy 1.15by 1.42aby 0.07 Day 7 2.15ax 1.75abx 1.41bx 1.34bx 1.30bx 1.76abx 0.11 SEM 0.04 0.16 0.04 0.08 0.03 0.07 Thigh 14 ppm Meat

Day 0 1.55az 1.45ay 1.42az 1.42az 1.37ay 1.48az 0.049

Day 3 3.10ay 2.63bx 2.16cy 1.75dy 1.51dxy 2.67by 0.088

Day 7 4.58ax 3.03bx 2.52cx 2.06dx 1.70dx 3.21bx 0.086

SEM 0.083 0.118 0.071 0.076 0.053 0.029 a-d Value with different letter within a row are significantly different (P<0.05). X-z Value with different letter within a column are significantly different (p<0.05). N = 4. Treatment: Control; Combination 1 (CM1) of 5 ppm tannic acid + 100 ppm oregano oil; CM2: 10 ppm tannic acid + 100 ppm oregano oil; CM3: 5 ppm tannic acid + 200 ppm oregano oil; CM4: 10 ppm tannic acid + 200 ppm oregano oil; BHA.

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Table 7. Profile of volatiles cooked breast meat chicken patties with different level of oregano oil and tannic acid at day 0. Total Ion counts*104 Compound Control CM1 CM2 CM3 CM4 5 ppm SEM without BHA Propanal 165a 0b 0b 0b 0b 0b 10 2-propanone 4388a 2677a 3127a 4125a 2627a 2100a 514 2-propanol 592a 611a 561a 615a 713a 848a 75 Hexane 177a 0b 0b 0b 0b 0b 5 Heptane 76a 0b 0b 0b 0b 0b 9 Pentanal 1043a 148b 0b 0b 0b 0b 66 Heptanal 41a 0b 0b 0b 0b 0b 1 Octane 333a 118b 82b 130b 94b 90b 17 Hexanal 3401a 561b 329b 335b 314b 316b 277 α-Pinene 0c 93bc 160ab 272a 229ab 0c 31 Camphene 0c 98bc 168b 296a 284a 0c 25 Limonene 0c 78b 51bc 167a 157a 0c 12 β-Myrcene 0b 0b 0b 199a 176a 0b 18 γ-Terpinene 0c 152b 128b 311a 286a 0c 21 Sabinene 0b 0b 0b 46a 52a 0b 4 Different letter (a-d) within a row are significantly different (P<

0.05), n=4. Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid, 100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.

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Table 8. Profile of volatiles cooked breast meat chicken patties with different level of oregano oil and tannic acid at day 3. Total Ion counts*104 Compound Control CM1 CM2 CM3 CM4 5 ppm SEM without BHA Pentane 536a 112b 0b 0b 0b 0b 43 Propanal 1736a 0b 0b 0b 0b 0b 153 2-propanone 4119ab 2921bc 2657c 2427c 2046c 4370a 304 2-propanol 278a 336a 466a 392a 385a 396a 48 Heptane 133a 58b 0c 0c 0c 0c 3 Pentanal 4385a 168b 62b 71b 91b 76b 158 Octane 259a 249a 93a 160a 109a 168a 39 Hexanal 39357a 2086b 701b 925b 870b 1136b 1778 Heptanal 132a 0b 0b 0b 0b 0b 15 α-Pinene 0b 126b 124b 372a 337a 0b 30 Camphene 0c 136b 123b 334a 330a 0c 20 Limonene 0c 94ab 34bc 107ab 136a 0c 16 β-Myrcene 0c 32bc 0c 188a 151ab 0c 27 γ-Terpinene 0c 77b 122b 337a 401a 0c 14 Sabinene 0c 20b 0c 0c 41a 0c 2 Different letter (a-d) within a row are significantly different (P<

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Table 9. Profile of volatiles cooked breast meat chicken patties with different level of oregano oil and tannic acid at day 7. Total Ion counts*104 Compound Control CM1 CM2 CM3 CM4 5 ppm SEM without BHA Pentane 697a 0c 0c 0c 0c 195b 25 Propanal 2156a 127b 0b 0b 0b 0b 38 2-propanone 1899a 2412a 2766a 2672a 2676a 2441a 198 2-propanol 274ab 172b 149b 394a 426a 152b 38 Heptane 137a 0c 0c 0c 0c 52b 10 Butanal 198a 0b 0b 0b 0b 0b 9 Pentanal 10241a 244bc 81c 91c 81c 994b 171 Octane 405a 348a 101c 98c 92c 227b 24 Hexanal 84538a 3741bc 1269c 1165c 1014c 9363b 1451 Heptanal 449a 0b 0b 0b 0b 0b 17 Nonanal 63a 0b 0b 0b 0b 0b 2 α-Pinene 0b 241a 216a 278a 282a 0b 29 Camphene 0d 243bc 165c 280ab 369a 0d 20 Limonene 0b 52b 55b 146a 196a 0b 14 β-Myrcene 0b 70b 57b 517a 433a 0b 35 γ-Terpinene 0b 203b 208b 1362a 1156a 0b 101 Sabinene 0b 0b 0b 140a 154a 0b 7

Different letter (a-d) within a row are significantly different (P< 0.05), n=4. Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid, 100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.

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Table 10. Profile of volatiles cooked thigh meat chicken patties with different level of oregano oil and tannic acid at day 0. Total Ion counts*104 Compound Control CM1 CM2 CM3 CM4 14 ppm SEM without BHA 2-propanone 4994a 4762ab 4183ab 3098b 3752ab 4336ab 416 2-propanol 819a 754a 693a 643a 816a 897a 89 Cyclohexane 106a 0b 0b 0b 0b 91a 8 Pentanal 691a 0c 0c 0c 0c 161b 9 Octane 306a 179ab 146b 163b 153b 212b 24 Hexanal 3395a 311c 427c 463c 213c 1360b 184 α-Pinene 0c 274b 383ab 282ab 416a 0c 30 Camphene 0c 284ab 278b 406a 358ab 0c 27 Limonene 0b 72a 69a 105a 107a 0b 15 β-Myrcene 0c 137b 98b 305a 329a 0c 21 γ-Terpinene 0c 486b 408bc 1081a 1416a 0c 100 Sabinene 0c 0c 0c 105a 83b 0c 5

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Table 11. Profile of volatiles cooked thigh meat chicken patties with different level of oregano oil and tannic acid at day 3. Total Ion counts*104 Compound Control CM1 CM2 CM3 CM4 14 ppm SEM without BHA Pentane 989a 0c 0c 0c 0c 307b 18 Propanal 1258a 0b 0c 0c 0c 598b 35 2-propanone 2069b 2114b 4777a 4392a 3827a 2140b 318 2-propanol 1649a 939a 1358a 1240a 1125a 1081a 188 Hexane 649a 403a 438a 0b 0b 572a 62 Cyclohexane 133a 98b 0c 0c 0c 149a 7 Pentanal 4358a 229c 285c 180c 0c 875b 88 Heptane 125a 0b 0b 0b 0b 0b 5 Octane 691a 438c 513abc 456bc 423c 672ab 51 Hexanal 54364a 2708c 1845c 1623c 565c 11797b 995 Heptanal 216a 0b 0b 0b 0b 0b 14 α-Pinene 0c 319b 382b 458b 687a 0c 50 Camphene 0d 169c 210c 411b 514a 0d 21 Limonene 0c 0c 43b 98bc 192a 0c 13 β-Myrcene 0b 0b 74b 199a 244a 0b 26 γ-Terpinene 0c 364b 250bc 1058a 874a 0c 72 Sabinene 0b 0b 0b 82a 86a 0b 7 Different letter (a-d) within a row are significantly different (P< 0.05), n=4. Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid, 100 ppm Oregano oil + 5 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.

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Table 12. Profile of volatiles cooked thigh meat chicken patties with different level of oregano oil and tannic acid at day 7. Total Ion counts*104 14 Compound Control CM1 CM2 CM3 CM4 SEM ppm without BHA Pentane 474a 0c 0c 0c 0c 176b 26 2-propanone 4064ab 4622a 2824bc 2807bc 2465c 3714abc 301 2-propanol 2983a 1620b 938b 809b 742b 629b 222 Hexane 243a 173ab 139bc 0d 0d 83c 17 Cyclohexane 152a 106b 0c 0c 0c 143a 7 Pentanal 2477a 375c 150c 44c 76c 1154b 89 Heptane 347a 0b 0b 0b 0b 57b 18 Octane 1337a 627bc 494c 478c 458c 850b 56 Hexanal 88685a 5573c 1871c 1652c 832c 25172b 1175 Heptanal 395a 0c 0c 0c 0c 135b 14 Octanal 70a 0b 0b 0b 0b 0b 4 α-Pinene 0d 434bc 333c 517ab 669a 0d 37 Camphene 0d 246c 283bc 449b 638a 0d 38 Limonene 0c 43b 53b 140a 144a 0c 5 β-Myrcene 0c 129b 100b 245a 280a 0c 9 γ-Terpinene 0c 319b 309b 638a 792a 0c 58 Sabinene 0c 0c 0c 41b 59a 0c 4 Different letter (a-d) within a row are significantly different (P< 0.05), n=4. Treatments: Control, Combination of 100 ppm oregano oil and 5 ppm Tannic acid, 100 ppm Oregano oil + 10 ppm Tannic acid, 200 oregano oil + 5 Tannic acid, 200 ppm oregano oil + 10 Tannic acid, and 5 ppm BHA.

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Table 13. Correlation Coefficients of TBARS, total carbonyl, hexanal values during storage time of control treatment. ______Pearson Correlation Coefficients, N = 12, Prob > | r | under HO: Rho = 0

Cooked Breast Cooked Thigh

Var TBA CAR HEX Var TBA CAR HEX TBA 1 0.98262* 0.94888* TBA 1 0.98085* 0.96005* CAR 0.98262* 1 0.97103* CAR 0.98085* 1 0.99116* HEX 0.94888* 0.97103* 1 HEX 0.96005* 0.99116* 1

TYPE TBA CAR HEX TYPE TBA CAR HEX

MEAN 1.1999 1.6147 42431.75 MEAN 3.8995 3.0758 48814.5 STD 0.8042 0.4976 35156.5 STD 2.517 1.3 36767.26

* P < 0.01 : Highly significant ** 0.01 < P < 0.05 : Strong significant difference *** 0.05 < P < 0.1 : Low significant difference **** P > 0.1 : No significant difference

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Table 14. Sensory attributes means of raw ground thigh chicken meat patties. b Sensory attributes Color Oregano Oxidation Over All TRTa redness Odor Odor Acceptability Control 6.9a 0.9c 3.8a 4.9b CM1 7.0a 2.3bc 3.6ab 5.3b CM2 7.4a 3.2b 2.8ab 5.8ab CM3 6.9a 5.6a 1.7ab 6.3ab CM4 8.0a 6.1a 1.1b 7.6a BHA 7.1a 1.2c 3.2ab 6.4ab SEMc 0.34 0.37 0.6 0.47 aTreatments: Control, Combination (CM) of 100 ppm oregano oil and 5 ppm Tannic acid; 100 ppm Oregano oil + 10 ppm Tannic acid; 200 oregano oil + 5 Tannic acid; 200 ppm oregano oil + 10 Tannic acid; and 5 ppm BHA. bSensory attributes: samples were evaluated on day 3. c SEM : Standard error of the means. d-fMean within same column with different superscripts are different (P < 0.05) n = 10.

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Table 15. Sensory attributes means of ground cooked thigh chicken meat patties. Sensory attributesb TRTa Cooked Oregano Oxidation Over All chicken Odor Odor Acceptability Control 4.6b 0.7d 5.3a 4.1c CM1 5.9ab 2.1cd 4.7ab 4.6c CM2 6.4ab 2.9bc 3.5ab 5.7bc CM3 6.7ab 4.7a 2.3b 7.5ab CM4 7.9a 4.3ab 2.6b 7.9a BHA 6.6ab 1.1d 3.1ab 7.3ab SEMc 0.51 0.37 0.6 0.44 aTreatments: Control, Combination (CM) of 100 ppm oregano oil and 5 ppm Tannic acid; 100 ppm Oregano oil + 10 ppm Tannic acid; 200 oregano oil + 5 Tannic acid; 200 ppm oregano oil + 10 Tannic acid; and 5 ppm BHA. bSensory attributes: samples were evaluated on day 0. c SEM: Standard error of the means. d-f Mean within same column with different superscripts are different (P < 0.05) n = 10.

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CHAPTER 6

SURVIVAL AND GROWTH OF NATURAL BACTERIAL FLORA AND HUMAN ENTERIC PATHOGENS IN GROUND CHICKEN MEAT WITH ADDED OREGANO ESSENTIAL OIL OR TANNIC ACID, ALONE OR IN COMBINATION

Marwan Al-Hijazeen1, Aubrey Mendonca2,* and Dong Uk Ahn1,*

1Department of Animal Science, Iowa State University, Ames, Iowa 50011, USA, and

2Department of Food Science and Human Nutrition, and 2Department of Animal

Science, Iowa State University, Ames, IA 50011, USA.

*Corresponding authors: Aubrey F. Mendonça and D. U. Ahn

Phone: (515) 294-9078. Fax: (515) 294-8181.

Email: [email protected] or [email protected]

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Abstract

The aim of this study was: 1) to evaluate the effect of oregano essential oil (EO) and tannic acid (TA) on the survival and growth of Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, Enterobacteriaceae, and total aerobic count in raw ground chicken meat, 2) to evaluate the effect of adding EO and TA on the survival and growth of Stapylococcus aureus, and aerobic plate count (APC) in cooked meat during storage at different temperature conditions. Five different treatments including 1) control (no antimicrobial agent), 2) 200 ppm EO, 3) 10 ppm TA, 4) 10 ppm TA + 200 ppm EO, and 5) 5 ppm butylated hydroxyanisole (BHA) were prepared. Oregano oil, tannic acid, BHA treatments showed significant (P < 0.05) growth inhibitory effects on the APC when used separately. However, the combination of EO + TA showed the strongest (P < 0.05) effect in reducing the APC of raw chicken breast meat during storage at 4 and 10 oC. Of all the treatments tested, the combination treatment also showed the strongest antimicrobial effects against Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, and Enterobacteriaceae (ENT) in raw meat during storage. In cooked chicken breast meat, EO and EO + TA combination were efficient (P < 0.05) in suppressing the growth of aerobic bacteria in meat at all temperatures (10, 25, and 43 oC) tested. The antimicrobial effect of TA was weaker than that of the OE, and TA did not show significant inhibitory effect at the end of storage period compared to BHA. Oregano essential oil treatment was more effective than the BHA and tannic acid in slowing down the growth of Stapylococcus aureus in cooked meat during storage at 10, 25, and 43 oC, but the OE + TA treatment was more effective than the EO alone. Based on these results it is concluded that EO has strong antimicrobial activities but its effect can be further improved when it is combined with TA. This study demonstrates that EO + TA combination could be a good natural antimicrobial treatment to suppress natural bacterial flora and food borne pathogens in ground chicken meat. Keywords: Oregano essential oil, tannic acid, aerobic plate count, pathogens, chicken meat.

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Introduction

Several foods including ground meats serve as a vehicle for transmission of enteric pathogenic bacteria to humans. The World Health Organization (WHO) reported that foodborne diseases are still highly responsible for the high levels of morbidity and mortality in the general population and especially in immunocompromised groups of people including infants and young children, the elderly, pregnant women, organ transplant patients, and patients undergoing chemotherapy (WHO, 2014). The Center for Disease Control and Prevention (CDC, 2011) estimated that approximately 1 in 6 Americans (or 48 million people) became ill, 128,000 were hospitalized, and 3,000 died of foodborne diseases in the U.S. annually (caused by known and unknown agents), and the recent cost estimation for the foodborne illness is around $ 51.0 billion to $ 77.7 billion each year (Scallan et al., 2011a; 2011b; Scharff, 2012). Although the 2011 estimation showed improvements from that of the 1999 (Mead et al., 1999), more efforts are needed to control the top known pathogens and determine the causes of foodborne illness (due to unknown agents), death without known causes, and economic cost estimation (Buzby and Roberts, 2009; Scallan et al., 2011a; 2011b; Scharff, 2012). In addition, the CDC indicated that reducing foodborne illness by 10% would keep about 5 million Americans from getting sick each year (CDC, 2014). The CDC considers that Salmonella and Staphylococcus aureus among the top five pathogens causing domestically acquired foodborne illnesses, Salmonella and E. coli O157 among the top five pathogens causing domestically acquired foodborne illnesses resulting in hospitalization, and Salmonella and Listeria monocytogenes among the top five pathogens causing domestically acquired foodborne illnesses resulting in death (CDC, 2011). Meat is an important part of our food and is an excellent medium in which many pathogens can grow. The contamination of meat by pathogens can occur before

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or after processing of the meat products (Jay, 2000), and through cross contamination between carcasses, handling, processing equipment, water, air, and food contact surface in the kitchen (Perez-Rodriguez et al., 2007). However the development of foodborne illness depends on the ability of pathogens to survive or grow in raw or cooked meat. For example, compared to cooked meat products, raw meat has a higher probability of being contaminated with Salmonella enterica, Listeria monocytogenes, and Escherichia coli and causing foodborne illness. Although each of these pathogens has their optimal conditions for survival, all can survive in fresh or ready-to-eat (RTE) poultry meat products. On the other hand, Staphylococcus aureus has higher ability to survive in cooked meat than in raw meat because their ability to compete with other microorganisms is very poor in raw meat products (Casman et al., 1963; Normanno et al., 2007). Poultry meat is the most popular meat consumed but can be easily contaminated by pathogens that cause food borne illness (Chouliara et al., 2007). Storage temperature and hygienic food handling are among the most important factors that can prevent contamination or suppress the growth of pathogens (Vernozy-Rozand et al., 2002; Solomakos et al., 2008). Most of the foodborne illnesses occur due to ineffective preservation methods, food contamination during processing, improper cooling of foods, and unhygienic practices of food handlers. Some pathogens such as Salmonella and Escherichia coli O157:H7 can be easily killed if proper cooking methods for the meat are used (Ingham et al., 2005) while others are resistant to heat treatment or produce toxins that cannot be destroyed by cooking. Many synthetic antimicrobial agents such as sodium lactate, acetate, benzoic acid etc. have been used to prevent or slow down the growth of pathogens in meat. Recently, the food industry has developed a greater interest in using natural antimicrobial agents in place of the synthetic ones to satisfy consumers who are concerned about the health effects of synthetic antimicrobial agents (Solomakos et al., 2008; Zhang et al., 2009; Davidson et al., 2013). Many spices, herbs and plant extracts have been studied in the food safety area

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because of their antimicrobial activity (Chung et al., 1998a; Horosova et al., 2006; Chouliara and Kontominas, 2006; Venkitanarayanan et al., 2013). Tannic acid and oregano oil exhibit antimicrobial activity because of their chemical structure and their properties (Scalbert, 1991; Botsoglou et al., 2003; Burt, 2004). Oregano essential oil contains high level of phenolic compounds such as carvacrol and thymol that are responsible for their antimicrobial activity (Beuchat, 1994; Sivropoulou et al., 1996). Tannic acid has been well studied and proven for its activity against several types of pathogens (Kim et al., 2010). The antimicrobial mechanisms of tannic acid are through their astringent effect, enzyme and substrate complex formation, and metal chelating activity (Chang, 1993, 1998b). However, no study has been conducted to determine the combined effects of these two natural antimicrobial agents on the growth of pathogens and the storage stability of ground chicken meat. A limitation of using these two natural antimicrobial agents together in meat is due to their effects on the flavor, odors, and their interactions with other food ingredients (Zucker, 1983; Chang 1998a; Burt, 2004). Thus, finding the optimal concentrations of these two natural antimicrobial agents to avoid changes in eating quality while maintaining the microbial safety of the meat are an important issue. The objectives of the present study were: 1) to evaluate the effect of oregano essential oil and tannic acid on the survival and growth of Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7, aerobic mesophilic bacteria and Enterobacteriaceae in raw ground chicken meat, and 2) to evaluate the effect of adding oregano oil and tannic acid on the survival and growth of Stapylococcus aureus, and aerobic mesophilic bacteria in cooked meat during storage at different temperatures.

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Materials and Methods

Sample preparation: Chicken breast meat was obtained from a local grocery store and ground twice a through a 10-mm plate then a 3-mm plate (Kitchen Aid, Inc., St. Joseph, MI). Five different treatments were prepared: 1) control (no antimicrobial agent), 2) 200 ppm oregano essential oil, 3) 10 ppm tannic acid, 4) 10 ppm tannic acid + 200 ppm oregano essential oil, and 5) 5 ppm butylatedhydroxyanisole (BHA). The amounts of oregano essential oil and tannic acid used were selected from the chemical studies to achieve the maximum antioxidant benefits of the additives. The oregano essential oil used was obtained from a certified company in Turkey (Healthy-Health Oil of Oregano, Turkey). The GC/MS analysis of the oregano essential oil indicated that 80.12% of the essential oil was carvacrol. BHA powder (0.1 g) and oregano essential oil (1.25 g) were dissolved in 10 ml of 100% ethanol, and then mixed with 50 ml mineral oil to make their stock solutions. The ethanol added was removed using a rotary evaporator (Model BUCH Rotavapor R-200) at (70 °C, 175 mbar vacuum pressure) before adding the stock to meat samples. Tannic acid powder (Sigma Chemical Co., St Louis, MO) was dissolved in de-ionized sterilized water before adding it to meat. Each treatment was added to the ground chicken meat and then mixed for 2 min in a bowl mixer (Model KSM 90; Kitchen Aid Inc., St. Joseph, MI, USA). All treatments were added with the same amounts of water and mineral oil to provide the same moisture and oil conditions. Samples of ground chicken meat (approximately 25 g each) were individually packaged in separate oxygen-permeable bags (polyethylene, 4 x 6.2 mil, Associated Bag Co., Milwaukee, WI), and used for the raw-meat study performed in the Microbial Food Safety Laboratory (MFSL) in the Department of Food Science and Human Nutrition. The same preparation method was used for cooked meat study, but the samples were packaged in oxygen impermeable vacuum

2 bags (O2 permeability, 9.3 mL O2/m /24 h at 0 °C, Koch, Koch, Kansas City, MO, USA). The patties were cooked in bag in a 90 °C water bath (Isotemp®, Fisher Scientific Inc., Pittsburgh, PA, USA) until the internal temperature of the meat

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reached 75 °C. After cooling to room temperature, the cooked meats were packed in insulated coolers and carried to MFSL for the cooked meat study. All meat samples were held in a walk-in cooler (4.4 ºC) and used in experiments within two hours after preparation.

Water activity and pH: The initial water activity (wa) of raw meat was measured using water activity meter (AQUA-LAB 4-TEV, Pullman, WA, USA). A 2-g portion of each non-inoculated raw ground meat sample was spread on the surface of a white plastic container. After the calibration of the instrument, the sample was inserted inside the meter and the water activity value was obtained and recorded. The water activity meter was calibrated and checked using an empty container, and AQUA-Lab Standard solution before use. The pH values of the ground chicken raw meat samples were determined using a pH meter (Accumet 925: Fisher Scientific, Fair Lawn, N.J., U.S.A) after homogenizing the 1.0-g samples with 9 ml de-ionzed distilled water (Sebranek et al., 2001).

Preparation of bacterial cultures: Five-strains each of Salmonella enterica, E. coli O157:H7, and Listeria monocytogenes, and four strains of Staphylococcus aureus were obtained from the Microbial Food Safety Laboratory at Iowa State University. Frozen stocks (in 10 % glycerol at -80 ºC) were thawed and separately transferred twice to 10 ml sterile tubes of tryptic soy broth (TSB; Difco, BD, Sparks, MD) before making the working cultures. Before the inoculation process each working culture (parent cells) was individually grown in 10 mL of tryptic soy broth (Difco laboratory, Detroit, MI) supplemented with 0.6 % yeast extract (TSBYE) at 35 ºC for 24h. These strains were adapted gradually in the TSBYE with different concentrations of added Naldixic acid (NA; antibiotic; M.W 232.24, 10 ug/ml, 30 ug/ml, and 50 ug/lm, Sigma-Aldrich, St. Louis, Missouri). Two consecutive transfers 24-h for each

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Naldixic acid resistant strain (NAR) cultures were prepared. For each pathogen, at the beginning of each study, 6 ml of each individual NAR adapted culture in TSBYE was aseptically transferred to a sterilized centrifuge tube to give a total 30 ml of strain mixture culture (NARC). Bacterial cells from each mixture of strains were harvested by centrifugation at 10,000 x g for 10 min at 4 ºC using a Sorvall Super T21 centrifuge (American Laboratory Trading, Inc., East Lyme, CT). The pelleted cells were re-suspended in a 30-ml sterilized saline (0.85 % NaCl), washed by vortexing, and then centrifuged again at the same speed and temperature conditions. After harvesting the cells from the second centrifugation, the harvested cells were suspended in fresh saline (0.85% w/v NaCl) and diluted (10-fold) using tubes of saline to obtain 106 CFU/ml in a suspension of washed cells used for inoculating the ground chicken meat.

Preparation and inoculation of meat samples: Samples of ground chicken meat were transported to the Microbial Food Safety Laboratory (Dept. of Food Science & Human Nutrition) for inoculation and microbial analysis. Each sample was inoculated with a mixture of five nalidixic-acid resistant serotypes of Salmonella enterica (S. Typhimurium, S. Newport, S. Kentucky, S. Oranienburg, and S. Montevideo), 5-strain mixture of Escherichia coli O157:H7 (ATCC 35150, ATCC 43894, ATCC 43895, WS 3062 and WS 3331) and 5-strain mixture of Listeria monocytogenes (Scott A, H7969, H7596, H7762 and H7962; all serotype 4b) to give an initial cell concentration of ~104 colony forming units (CFU)/g for each pathogen indiviually. All inoculated packages of ground chicken meat were loosely closed, manually massaged for 30 s from outside of the bag, and held at different storage times and temperature. At set intervals during storage, samples of raw meat were analyzed for Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes survivors. In addition, separate bags of non-inoculated ground meat were held at 4 and 10 °C and analyzed for aerobic plate counts and numbers of viable Enterobacteriaceae.

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The same inoculation preparation procedure was performed for the cooked meat using a 4-strain mixture of Staphylococcus aureus (ATCC 6538, ATCC 25923, ATCC 10390, and ATCC 29213) obtained from American Type Culture Collection. In addition, separate bags of non-inoculated ground cooked meat were held at different temperatures (10, 25, and 43 °C) and analyzed for aerobic plate counts.

Microbial analysis: Packages of ground chicken meat were aseptically opened, and two-25 g portions of meat were aseptically transferred into separate sterile filter-lined stomacher bags. Sterile 0.1% (w/v) peptone water (225 ml) was added to each bag. The resulting mixtures were homogenized for one minute in a laboratory stomacher blender operating at medium speed. Further 10-fold serial dilutions of meat samples were prepared in tubes of 0.1% peptone and 0.1 ml aliquots of diluted samples were surface plated on appropriate selective agar media to enumerate pathogenic bacteria. Sorbitol MacConkey agar (SMA), Modified Oxford (MOX) agar, Xylose lysine Deoxycholate XLD agar, and Baird-Parker Agar base were used to enumerate Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus, respectively. Inoculated agar plates were incubated at 35 °C and bacterial colonies were counted after 48 hours. The aerobic plate count (APC) was determined by surface plating aliquots (0.1 ml) of meat homogenate on tryptic soy agar (TSA), incubating the inoculated TSA plates at 35 °C, and counting bacterial colonies at 48 hours. The numbers of viable Enterobacteriaceae were determined using the agar overlay technique. Aliquots (0.1 ml or 1.0 ml) of meat homogenate were placed in a petri dish and molten TSA (48°C) was poured onto the dish to form the first layer. The TSA plate was held for 60 minutes at ambient temperature before pouring a layer of violet red bile agar (VRBA) over the TSA. The solidified TSA/VRBA plates were incubated at 35 °C and bacterial colonies were counted at 24 hours.

Statistical analysis: The experiment was a completely randomized design (CRD).

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Two separate samples per treatment per replication were analyzed over 2 replications of each independent experiment. The statistical analysis was performed using the SPSS (Chicago, IL, USA) package for Windows (Ver. 20.0). The mean values were compared using the one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests (p < 0.05). Mean values and standard error of the means (SEM) were reported at (P<0.05).

Results and Discussion

The aerobic plate count (APC) provides an estimate of the level of numbers of viable bacteria that can grow aerobically in a product. Obtaining these data may help evaluate good manufacturing practices for foods as well as determine potential sources of contamination (American public Health Association, 1984). In addition Enterobacteriaceae is a large family of rod-shaped Gram-negative, facultative bacteria, which includes some pathogens such as Salmonella, Yersinia, Escherichia coli, Klebsiella and Shigella. Most members of this family are a normal part of the gut flora and are found in the intestines of human and other animals. However, some are also found in water, soil, or parasites of a variety of different animals and plants. Enterobacter spp and Escherchia coli (members of the coliform group) are commonly used to indicate the level of sanitary quality of foods and water. Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus are considered important pathogens related to the microbial safety of meats (FSIS, 2014) in the US. The initial pH value of the ground chicken breast meat without additive was approximately 6.2 (Table 1), and was similar to that of meat samples containing the other treatments. Adding tannic acid or oregano oil to the meat did not change the pH values. In addition, the pH values for all treatments remained unchanged during storage time (data not shown). The initial water activity (aW) values (0.98, Table 1) were similar among all treatments, indicating that all the treated samples of meat have almost similar intrinsic conditions (with respect to pH and water activity) for

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microbial growth at the beginning of the study. For the raw meat samples, the storage temperatures of 4 ºC and 10 ºC were selected to represent a typical refrigeration temperature (4 ºC) and temperature abuse (10 ºC) of the meat in a faulty refrigeration unit. The total aerobic count of raw meat patties significantly (p < 0.05) increased during storage time in control group incubated at both 4 ºC and 10 ºC (Table 2). The initial APC (day 0) for the fresh ground chicken meat used for all treatments was almost similar (around 4.15 to 4.37 log10 cfu/g) indicating that the microbial quality of chicken meat was acceptable (Dawson et al., 1995). The differences between the treatments started to appear after 2 days of storage. The combination of oregano oil and tannic acid treatments resulted in lower total plate counts compared to the control and other treatments after two days. Meat samples with BHA also had lower APC than the control group over the storage time. Oregano essential oil treatment reduced the APC by 1.8 log compared with the control at Day 8; however, the combination of oregano oil and tannic acid showed the highest antimicrobial activity against the APC in raw meat samples (Table 2). The antimicrobial effect of combination treatment reduced the APC by 2.07 log unit compared with the control during storage at 10 oC (at Day 4). The ICMSF (1986) considered 7.0 log10 cfu/g the microbiological limit for microbial spoilage of fresh poultry meat. Therefore, the combination treatment was able to extend the shelf-life of the raw chicken meat by 2 days compared to the control (Tables 2). Chouliara et al. (2007) reported that oregano oil (0.1 %) extended the shelf-life of fresh chicken breast meat when stored at 4 oC. Skandamis and Nychas (2001) found that oregano essential oil (0.5% and 1%) delayed microbial growth and suppressed the final count of the spoilage micro-organisms in minced beef meat stored at 5 oC. The antimicrobial effect caused by adding oregano oil can be explained by the high level of carvacrol, thymol and the other polyphenolic structures (Sivropoulou et al., 1996). Tannic acid has high binding efficiency to iron which makes it like a siderophore to complex iron from the entire media affecting the iron availability to the microorganisms. The microorganisms living under aerobic conditions need iron for

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many biological functions, such as reducing ribonucleotide precursor of DNA, making heme compounds for the respiratory chain, and many essential processes for survival (Chung et al., 1998b). Many researchers believe that using combinations of natural antimicrobial plant extracts may increase their effectiveness through the synergistic effects (Pol and Smid, 1999; Lin et al., 2004; Brewer., 2011; Abd El-Kalek and Mohamed, 2012). Although, the combination of oregano oil and tannic acid showed better antimicrobial effects than using them alone, no synergistic antimicrobial effect of tannic acid and oregano oil was found in this study (Table 2). Similar results were found when tannic acid + oregano oil was added to the raw chicken meat that was later cooked. The initial number of APC was very low compared to the raw meat with around two log differences. Oregano oil, tannic acid + oregano oil combination, and BHA had the lowest initial APC numbers among the treatments and those numbers were significantly lower than that of the control and tannic acid treatments (Tables 5 & 6). These results strongly suggested that these preservatives, including the combination treatment, reduced the initial aerobic count in the meat during cooking. The APC for cooked meat incubated at 10 oC showed the highest bacterial count in the control group (around 8.31 Log10 CFU/g) at day 5 of storage. Tannic acid + oregano oil combination and oregano oil showed the strongest effect in suppressing the microbial growth at 10 oC. Tannic acid alone did not show significant effect at days 3 and 5 (Table 5). Also, tannic acid showed no antimicrobial effect at 0, 2, and 8 hour in cooked chicken meat held at 25 oC. These results indicate that tannic acid’s antimicrobial activity on the APC of the cooked meat patties was very low. All additive treatments showed significant antimicrobial effect in suppressing the growth of aerobic microorganisms at 43 oC after 6 hours of storage (Table 6). The same antimicrobial trend was found with oregano oil + tannic acid treatment on the total viable count of Enterobacteriaceae (ENT) in raw meat. The control group showed the highest ENT cell counts (8.74 CFU/g) after 8 days of

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storage at 4 oC, and reached to 7.60 CFU/g after storage at 10 oC for 4 days. The growth rate of Enterobacteriaceae was suppressed further when the oregano oil + tannic acid combination was used (Table 3). These results indicate the good potential of the combination treatment to suppress growth of Gram-negative, facultative bacteria, including pathogens in this group, in raw chicken during temperature abuse at 10 ºC. Among the pathogenic microorganisms, Escherichia coli O157:H7 was the most susceptible to oregano oil + tannic acid combination compared to the other treatments. After 6 days of incubation at 10 oC, E. coli O157:H7 with the oregano oil

+ tannic acid treatment showed the lowest log10 number (around 6.99 CFU/g) while the control had 8.51 CFU/g. No significant difference (P > 0.05) between tannic acid and BHA treatment during storage time was observed (Table 4). Oregano oil at 200 ppm showed higher effect in suppressing the growth of E. coli O157:H7 than the tannic acid and BHA treatments. The log10 difference between the oregano oil + tannic acid combination and the control was around 1.52 units at day 6. Oregano oil + tannic acid combination and oregano oil treatments showed the strongest antimicrobial effect against Salmonella. Adding a combination of oregano oil and tannic acid to raw ground chicken meat reduced the Log value by 1.5 unit compare to the control at day 6 of storage time (Table 4). Listeria monocytogenes was more affected by the oregano oil + tannic acid combination treatment than other treatments. The Listeria monocytogenes data showed that oregano essential oil has stronger antilisterial effect than tannic acid or BHA treatment. These results agree with those of Lin et al. (2004) who found that oregano suppressed the growth of Listeria monocytogenes in fish and other meats.

Oregano oil + tannic acid combination treatment showed 1.63 log10 lower than the control at Day 4, and 1.95 log10 lower than control at Day 8 of storage (Table 4). The oregano oil + tannic acid combination treatment did not show any synergistic effect against Enterobacteriaceae, Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes although the combination showed the highest

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antimicrobial effects among the treatments. The antimicrobial effect of oregano oil in suppressing the growth of E. coli O157:H7 is due to the high carvacrol content in the essential oil (Xu et al., 2008). Other researchers (Ultee et al., 1998; 2002; Di Pasqua et al., 2010) reported that oregano essential oil and its major component carvacrol changed the membrane potential of bacterial cell wall and disturbed the biological activities inside the cell. For the cooked meat, the temperatures of 10, 25, and 43 oC were all selected to represent temperature abuse conditions that allow the growth of Staphylococcus aureus in a faulty refrigeration unit (10 ºC), during inadvertent holding of cooked meat at ambient temperature (25 ºC) and during unsafe (43 ºC) hot-holding temperature that may often occur in foodservice operations. At all the temperature conditions used in the present study (10, 25, and 43 oC), Staphylococcus aureus in cooked meat was more sensitive to oregano oil + tannic acid combination compared to other treatments. The initial number of Staphylococcus aureus was the same for all treatments at the beginning of all studies. The oregano oil + tannic acid combination treatment in cooked meat samples resulted in numbers of viable Staphylococcus aureus that were 1.42 log10 lower than those of the control following 8 hours of exposure of the meat at 43 oC (Tables 7 & 8). The antimicrobial activity of oregano oil and tannic acid against Staphylococcus aureus growth in various foods is also reported by many research studies (Beuchat and Heaton, 1975; Akiyama et al., 2001; Ozcan, 2001).

Conclusion

Oregano oil at level 200 ppm showed a good antimicrobial activity in both raw and cooked meat. However, if oregano essential oil (200 ppm) and tannic acid (10 ppm) were combined, their antimicrobial effect could be better than using them singly. In terms of general food safety, the combination exhibited strong antimicrobial effects, but the effects were additive not synergistic. This combination could be a good antimicrobial treatment that can suppress most of the food borne pathogens as well as microorganisms that can grow aerobically in raw and cooked chicken breast meat.

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Table 1. Water activity and pH of raw chicken breast meat at 0 day.

Treatment Water activity (wa) pH value Control 0.986a 6.22a Tannic acid 0.987a 6.21a Oregano 0.991b 6.23a Combination 0.989ab 6.20a BHA 0.987a 6.24a SEM 0.001 0.061

a-fStatistically significant differences (p<0.05) between treatment. n = 4 for water activity and n = 3 for pH value Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 2. Total Plate Counts of raw chicken breast meat during storage at 4 °C and 10 °C. Storage time (days) Treatments 0 day 2 days 4 days 6 days 8 days SEM ------Log CFU/g meat ------Storage at 4°C Control 4.37aw 6.35bx 8.34dy 9.36cz 9.82dz 0.16 Tannic acid 4.51aw 5.31ax 7.42cy 7.99abz 8.37bz 0.13 Oregano 4.59av 5.46aw 7.20bx 8.04aby 8.34bz 0.05 Combination 4.58av 5.47aw 6.84ax 7.65ay 8.02az 0.05 BHA 4.39av 5.57aw 7.23bx 8.28by 8.89cz 0.08 SEM 0.09 0.15 0.02 0.14 0.07

Storage at 10 °C Control 4.15abx 7.20dy 9.35cz -1 - 0.04 Tannic acid 4.26bx 6.63cy 8.27bz - - 0.03 Oregano 4.15abx 6.42by 8.37bz - - 0.08 Combination 4.07ax 5.44ay 7.28az - - 0.11 BHA 4.18abx 6.66cy 8.24bz - - 0.01 SEM 0.04 0.06 0.09 1Not determined. n=4. a-eDifferent superscripts within a column (for a specified storage temperature) differ significantly (p < 0.05). x-zDifferent superscripts within a row (for a specified storage temperature) differ significantly (p < 0.05). Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 3. Numbers of viable Enterobacteriaceae in ground raw chicken breast meat stored at 4 °C and 10 °C. Storage time (days) Treatments 0 day 2 days 4 days 6 days 8 days SEM ------Log CFU/g meat ------Storage at 4 °C Control 4.21av 5.20bw 6.35cx 8.13cy 8.74dz 0.05 Tannic acid 4.14av 4.75aw 5.42bx 6.89by 7.79cz 0.07 Oregano 4.17av 4.70aw 5.49bx 6.72by 7.53bz 0.06 Combination 4.23av 4.59aw 4.86ax 6.20ay 7.10az 0.07 BHA 4.15av 4.71aw 5.50bx 6.73by 7.75cz 0.05 SEM 0.05 0.05 0.06 0.08 0.06

Storage at 10 °C Control 4.15ax 6.27cy 7.60dz -1 - 0.06 Tannic acid 4.33bx 5.34by 6.71cz - - 0.09 Oregano 4.22abx 5.08by 6.08bz - - 0.03 Combination 4.16ax 4.80ay 5.69az - - 0.05 BHA 4.15ax 5.24by 6.13bz - - 0.04 SEM 0.05 0.08 0.02 1Not determined. n=4. a-eDifferent superscripts within a column (for a specified storage temperature) differ significantly (p < 0.05). x-zDifferent superscripts within a row (for a specified storage temperature) differ significantly (p < 0.05). Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;

Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 4. Number of viable Escherichia coli O157:H7, and Salmonella enterica in ground raw chicken breast meat during storage at 10 °C. Storage (days) Treatments 0 day 2 days 4 days 6 days 8 days SEM ------Log CFU/g meat ------Escherichia coli Control 4.19aw 5.34cx 6.78cy 8.51dz -1 0.03 Tannic acid 4.15aw 5.26cx 6.43by 7.91cz - 0.12 Oregano 4.23ax 4.60abx 6.13aby 7.29bz - 0.12 Combination 4.04ax 4.21ax 6.00ay 6.99az - 0.06 BHA 4.09aw 5.01bcx 6.40by 7.93cz - 0.18 SEM 0.05 0.18 0.11 0.09

Listeria monocytogenes Control 4.21av 5.14cw 6.35dx 8.13dy 8.71dz 0.05 Tannic acid 4.15av 4.79bw 5.63cx 7.69cy 8.51cz 0.08 Oregano 4.17aw 4.59abw 5.12bx 6.14ay 6.86az 0.15 Combination 4.23aw 4.45aw 4.72ax 6.06ay 6.76az 0.09 BHA 4.15av 4.63abw 5.50cx 6.73by 7.68bz 0.05 SEM 0.06 0.07 0.12 0.12 0.04

Salmonella enterica Control 4.07aw 5.37cx 6.78cy 8.34dz - 0.06 Tannic acid 4.06aw 5.30cx 6.61bcy 7.67cz - 0.05 Oregano 4.18ax 4.60abx 5.90 ay 7.14bz - 0.17 Combination 4.03aw 4.21ax 5.75 ay 6.84az - 0.04 BHA 4.09aw 5.01bcx 6.30by 7.74cz - 0.17 SEM 0.06 0.17 0.12 0.07 1Not determined. n = 4. a-eDifferent superscripts within a column of the same meat differ significantly (p < 0.05). x-zDifferent superscripts within a row differ significantly (p < 0.05). Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 5. Aerobic plate count of cooked chicken meat patties during storage at 10 °C. Storage (days) Treatments 0 day 1 day 3 days 5 days SEM ------Log CFU/g meat ------

Control 2.58dw 4.29cx 7.54cy 8.31cz 0.04 Tannic acid 2.52dw 3.93abx 7.28cy 8.28cz 0.08 Oregano 2.01bw 4.16cx 5.70ay 6.33az 0.04 Combination 1.79aw 3.73ax 5.71ay 6.25az 0.03 BHA 2.30cw 4.12bcx 5.86by 6.71bz 0.03 SEM 0.04 0.07 0.03 0.05 1Not determined. n = 4. a-eDifferent superscripts within a column differ significantly (p < 0.05). x-zDifferent superscripts within a row differ significantly (p < 0.05). Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil;

Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 6. Aerobic plate counts of cooked meat patties held at 25 or 43 °C during storage. Storage (hrs) Treatments 0 hr 2 hr 4 hr 6 hr 8 hr SEM ------Log CFU/g meat ------Storage at 25 °C Control 2.60cv 3.46acw 4.51dx 5.49ey 6.88cz 0.04 Tannic acid 2.54cv 3.49cw 4.30cx 5.31dy 6.68cz 0.04 Oregano 2.32bv 2.89bw 3.62ax 4.32by 5.70bz 0.04 Combination 2.16av 2.62aw 3.59ax 4.07ay 5.26az 0.05 BHA 2.11av 2.61aw 3.77bx 4.94cy 5.58bz 0.09 SEM 0.03 0.05 0.04 0.04 0.09

Storage at 43 °C Control 2.60cv 3.88dw 5.11ex 6.16dy 8.32dz 0.03 Tannic acid 2.54cv 3.69cw 4.85dx 5.82cy 7.79cz 0.03 Oregano 2.31bv 2.89bw 3.67bx 4.65by 6.69bz 0.03 Combination 2.14av 2.68aw 3.48ax 4.31ay 6.46az 0.01 BHA 2.06av 2.86bw 3.79cx 4.67by 6.75bz 0.03 SEM 0.02 0.04 0.02 0.02 0.03 a-eDifferent superscripts within a column (for a specified storage temperature) differ significantly (p < 0.05). x-zDifferent superscripts within a row (for a specified storage temperature) differ significantly (p < 0.05). n = 4. Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 7. Numbers of viable Staphylococcus aureus in cooked ground chicken meat during storage at 10 °C. Storage (days) Treatments 0 day 1 day 3 day 5 day 7 day SEM ------Log CFU/g meat------Control 4.32av 5.05dw 6.77dx 7.48dy 8.39dz 0.06 Tannic acid 4.27av 4.64bw 5.45bx 6.70cy 7.30bz 0.02 Oregano 4.31aw 4.33aw 5.44bx 6.50by 7.13bz 0.07 Combination 4.24aw 4.28aw 5.30ax 6.00ay 6.63az 0.06 BHA 4.19av 4.93cw 5.69cx 6.68cy 7.33bz 0.04 SEM 0.07 0.02 0.02 0.05 0.08 a-eDifferent superscripts within a column of the same meat are differ significantly (p < 0.05). x-zDifferent superscripts within a row are differ significantly (p < 0.05). n = 4. Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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Table 8. Numbers of viable Staphylococcus aureus in cooked ground meat patties held at 25 or 43 °C. Storage (hrs) Treatments 0 hr 2 hr 4 hr 6 hr 8 hr SEM ------Log CFU/g meat ------Storage at 25 °C Control 4.32av 4.65cw 5.37dx 5.65dy 6.32cz 0.01 Tannic acid 4.33av 4.45abw 4.77bcx 5.15cy 5.79bz 0.03 Oregano 4.31aw 4.44abw 4.76bx 4.91by 5.77bz 0.04 Combination 4.24aw 4.43ax 4.60ax 4.78ay 5.31az 0.07 BHA 4.32aw 4.47bx 4.86cy 4.92by 5.81bz 0.02 SEM 0.07 0.01 0.03 0.01 0.04

Storage at 43 °C av bw dx cy dz Control 4.30 5.33 6.45 7.65 8.72 0.01 Tannic acid 4.27av 4.67aw 5.61bx 6.82by 7.46bz 0.02 Oregano 4.31av 4.62aw 5.60bx 6.77by 7.39abz 0.05 Combination 4.24av 4.62aw 5.39ax 6.48ay 7.30az 0.07 BHA 4.32av 4.68aw 5.75cx 6.81by 7.63cz 0.02 SEM 0.06 0.02 0.04 0.02 0.03 a-eDifferent superscripts within a column (for a specified storage time) are differ significantly (p < 0.05). x-zDifferent superscripts within a row are differ significantly (p < 0.05). n = 4. Treatments: Tannic acid, 10 ppm tannic acid; Oregano, 200 ppm oregano essential oil; Combination: 10 ppm tannic acid + 200 ppm oregano essential combination.

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CHAPTER 7 SUMMARY AND CONCLUISONS

Finding good combinations of natural antioxidants and antimicrobials from plant origins has been a hot topic in recent years. Researchers and food industry all share this approach because it reflects consumer preferences. Replacing synthetic antioxidants with natural ones is an excellent idea but requires further investigation. Several factors can affect the antioxidant and antimicrobial activity of these natural plant extracts in the meat system. For example the concentration and interaction of natural antioxidant or antimicrobial agents with other compounds, pro-oxidant, free radical, and oxygen availability in the meat system will affect their activity. So the objectives of the present study were: 1) to evaluate the antioxidant effect of oregano essential oil, tannic acid, and/or their combinations in ground chicken meat, 2) to evaluate the antimicrobial effect of oregano essential oil, tannic acid, and/or their combinations in ground chicken meat, and 3) to evaluate the sensory characteristics of combined oregano oil and tannic acid in ground chicken meat. Oregano essential oil, at levels 300 and 400 ppm, showed the highest antioxidant effect in ground chicken meat. Adding >100 ppm of oregano essential oil improved color stability of raw meat and decreased off-odor volatile of cooked meat, but their effects were much clearer in cooked than in raw meat. Over all, oregano essential oil could be used in place of synthetic antioxidant (e.g., BHA) to prevent quality deterioration in raw and cooked meat during storage. Tannic acid at 5 or 10 ppm was effective in maintaining meat color and retarding lipid and protein oxidation in raw ground meat. In addition, tannic acid at > 5 ppm effectively reduced lipid oxidation, protein oxidation, and off-odor-related volatiles in cooked ground breast during storage. Therefore, tannic acid could be a good candidate as a natural antioxidant to prevent oxidative changes and color changes in raw and cooked chicken breast meat during storage. Combination of tannic acid and oregano essential oil at level 200 ppm and 10

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ppm, respectively, showed positive effects on the TBARS values, total carbonyl, color stability, and off-odor volatile formation. The combination of 200 ppm oregano oil and 10 ppm tannic acid was a good replacement for the synthetic antioxidant in the ground chicken meat. In addition, combinations of tannic acid and oregano essential oil showed positive effects on the most sensory attributes of raw and cooked chicken meat. This combination has the highest over all acceptability score of both raw and cooked meat compared to the other treatments. Oregano oil at level 200 ppm showed a good antimicrobial activity for both raw and cooked meat. However, when oregano essential oil (200 ppm) and tannic acid (10 ppm) were combined, their antimicrobial effects were better than using them singly. In term of general food safety, the combination exhibited strong antimicrobial effects, but the effects were additive not synergistic. This combination showed a good antimicrobial treatment and suppressed most of the food borne pathogens as well as aerobic microorganisms in raw and cooked chicken breast meat. In addition, this combination showed higher score than other treatments for most of the sensory attributes used in this study. Over all the combination of tannic acid (10 ppm) and oregano essential oil (200 ppm) could be a good replacement for the synthetic antimicrobials and antioxidants currently used by the meat industry.