THE EFFECT OF STEAMA BLE BAG MICROWAVING AND STEAMABLE BAG DE SIGN ON NUTRITIONAL PRESE RVATION AND PHYSICAL QUALITY OF FROZEN BROCCOLI By Xueying Zhong A THESIS Submitted to Michigan State University i n partial fulfillment of the requiremen ts f or the degree of Packaging Master of Science 2014 ABSTRACT THE EFFECT OF STEAMA BLE BAG MICROWAVING AND STEAMABLE BAG DE SIGN ON NUTRITIONAL PRESE RVATION AND PHYSICAL QUALITY OF FROZEN BROCCOLI By Xueying Zhong T his study aims to ev aluate the effect of steamable bag microwaving and steamable bag design s on the nutritional value and physical properties of frozen broccoli. The results show that steamable bag microwaving performs better than traditional microwaving and is equal to steam er steaming in retaining ascorbic acid content and increasing antioxidant activity compared to thawed frozen broccoli. It lightness and yellow ness as compared to steamer steaming and tra ditional microwaving. These findings s upport that steamable bag microwaving is a cooking method that increases nutritional content, tenderizes at a quicker rate and produces minimal color change s in frozen broccoli, . M ost of the studied parameters are more affected by the shape of steamable bags than by the surface area. Frozen b roccoli cooked in more square - shaped steamable bag s retain s significantly higher ascorbic acid content , is less green and firmer than that c ooked in more rectangular - shaped steamable bag s . The smaller the surface area, the softer the broccoli became a fter cooking . Neither the shape nor the surface area of steamable bag s had an effect on the lightness or the antioxidant capacity of the broccoli . These results demonstrated the importance of controlling the surface area and shape of steamable bag s to optimize the qualities of frozen broccoli . iii ACKNOWLEDGEMENTS I would like to thank Dr. Almenar for her help and patience. I was exceptiona lly lucky s he was willing to work with me . Dr. Almenar is a true researcher and professor . I consider myself fortunate s he was my major professor, and I will always remember h er fondly. I would also like to thank Dr. Harte for h is guidance and supp ort. H is food packaging class was one of my favorite classes , and I will always be impressed by h is concern for h is students. I am also thankf ul for the assistance Dr. Dolan provided. His assistance helped me to overcome a big challenge of the stud y, and I am always be impressed by his language talent. Additiona lly, I would like to thank Dr. Siddiq, Dr. Bennink and Dr. Sogi for their support throughout my time of study. I am forever grateful for my parents. They have always supported me and encouraged me to ex cel in academic pursuits. I also appreciate their financial assistance during this time and when I am a master student. I would a lso like to thank my aunt for her continued support, kindness, and patience. iv TABLE OF CONTENTS LI ST OF TABLES ................................ ................................ ................................ ......................... vi LIST OF FIGURES ................................ ................................ ................................ ...................... vii 1 . Introduction ................................ ................................ ................................ ................................ .. 1 2 . Literature Review ................................ ................................ ................................ ......................... 3 2.1 Broccoli ................................ ................................ ................................ .............................. 3 2.1.1 Frozen Broccoli ................................ ................................ ................................ ....... 3 2.2 Nutritional Value ................................ ................................ ................................ ................ 4 2. 2.1 Ascorbic Acid ................................ ................................ ................................ .......... 4 2. 2.2 Antioxidant Capacity ................................ ................................ ............................... 5 2. 2.2.1 Hydrophilic Antioxidants ................................ ................................ ............. 5 2. 2.2.2 Lipophilic Antioxidants ................................ ................................ ............... 6 2. 2.2.3 Oxygen Radical Absorbance Capacity assay ................................ ............... 7 2. 3 Physical Quality ................................ ................................ ................................ ................. 7 2. 3. 1 Firmness ................................ ................................ ................................ ................... 7 2. 3.2 Color ................................ ................................ ................................ ........................ 8 2. 4 The Effect of Cooking Treatments on Broccoli ................................ ................................ . 9 3 . Effect of steamable bag microwaving versus steamer steaming and traditional microwaving on nutritional preservation and physical quality of frozen broccoli ................................ .................. 12 3.1 Materials ................................ ................................ ................................ ........................... 12 3.2 Methods ................................ ................................ ................................ ............................ 13 3 . 2.1 Packaging ................................ ................................ ................................ ............... 13 3 . 2.2 Treatments ................................ ................................ ................................ .............. 13 3 . 2.3 Ascorbic Acid ................................ ................................ ................................ ........ 1 5 3 . 2.4 Oxygen Radical Absorbance Capacity assay ................................ ......................... 1 7 3. 2. 5 Firmness ................................ ................................ ................................ ................. 1 9 3.2.6 Color ................................ ................................ ................................ ...................... 1 9 3.2.7 Moisture Content ................................ ................................ ................................ ... 22 3. 2. 8 Statistical Analysis ................................ ................................ ................................ . 22 3.3 Results and Discussions ................................ ................................ ................................ ... 22 3 . 3.1 Ascorbic Acid ................................ ................................ ................................ ........ 22 3 . 3.2 Antioxidant Capacity ................................ ................................ ............................. 2 4 3 . 3.3 Firmness ................................ ................................ ................................ ................. 2 6 3 . 3.4 Color ................................ ................................ ................................ ...................... 2 9 4 . Effect of steamable bag design on the nutritional and physical quality of cooked frozen broccoli ................................ ................................ ................................ ................................ .......... 32 4 .1 Materials ................................ ................................ ................................ ........................... 32 4 .2 Methods ................................ ................................ ................................ ............................ 33 4 . 2.1 Packaging ................................ ................................ ................................ ............... 33 v 4 . 2.2 Cooking Time ................................ ................................ ................................ ........ 33 4 . 2 . 3 Ascorbic Acid ................................ ................................ ................................ ........ 34 4 . 2 . 4 Oxygen Radical Absorbance Capacity assay ................................ ......................... 35 4. 2. 5 Color ................................ ................................ ................................ ...................... 36 4 .2.6 Firmness ................................ ................................ ................................ ................. 36 4 . 2 . 7 Moisture Content ................................ ................................ ................................ .... 37 4. 2. 8 Statistical Analysis ................................ ................................ ................................ . 37 4.3 Results and Discussions ................................ ................................ ................................ ... 3 7 4 . 3.1 Cooking Time ................................ ................................ ................................ ........ 3 7 4 . 3 . 2 Ascorbic Acid ................................ ................................ ................................ ........ 38 4 . 3 . 3 Antioxidant Capacity ................................ ................................ ............................. 42 4 . 3 . 4 Firmness ................................ ................................ ................................ ................. 44 4 . 3 . 5 Color ................................ ................................ ................................ ...................... 45 5. Conclusions ................................ ................................ ................................ ................................ 4 7 6. Future Work ................................ ................................ ................................ ............................... 4 9 BIBLIOGRAPHY ................................ ................................ ................................ .......................... 50 vi LIST OF TABLES Table 1. Color and F irmness of F rozen B roccoli after Steamable Bag Microwaving, Steamer Steaming and Traditional Microwaving as well as of Uncooked Thawed Frozen Broccoli ........ 2 8 Table 2. P ackage S pecification and C ooking T ime ................................ ................................ ...... 33 Table 3. Color and F irmness of Frozen Broccoli Cooked in Different Steamable Bags Designs 4 6 vii LIST OF FIGURES F i g u r e 1 . Sample size used to determine a scorbic a cid in uncooked broccoli and in broccoli cooked using steamer steaming, traditional microwavin g and steamable bag microwaving 16 F i g u r e 2. Sample size used to determine antioxidant capacity of uncooked broccoli and of broccoli cooked using steamer steaming, traditional microwaving and steamable bag microwaving ..18 F i g u r e 3. Sample size used to determine the firmness of uncooked broccoli and of broccoli cooked using steamer steaming, traditional microwaving and steamable bag microwaving 20 F i g u r e 4. Sample size used to determine the color of uncooked broccoli a nd of broccoli cooked using steamer steaming, traditional microwaving and steamable bag microwaving .21 Figure 5 . Ascorbic Acid Content of Frozen Broccoli after S teamable B ag M icrowaving, S teamer S teaming, and T raditional M icrowaving as well as of T ha wed F rozen B roccoli ......................... 24 Figure 6 . Antioxidant Capacity of Frozen Broccoli after S teamable B ag M icrowaving, S teamer S teaming, and T raditional M icrowaving as well as of T hawed F rozen B roccoli ......................... 2 6 Figure 7 . Ascorbic A cid C ontent of F rozen B roccoli cooked in D ifferent S teamable B ag D esigns in (a) D ry W eight B asis vs. (b) F resh W eight B asis ................................ ................................ ..... 41 Figure 8 . Antioxidant C apacity (ORAC) of F rozen B roccoli cooked in D ifferent S teamable B ag D esigns in (a) D ry W eight B asis vs. (b) F resh W eight B asis ................................ ....................... 43 1 1 . In troduction Numerous scientific evidence supports the relationship between the increase in the consumption of dietary antioxidants to the decrease in chronic illnesses (Bengtsson et al., 2006; Eberhardt , Kobira, Keck, Juvik & Jeffery, 2005; Wu et a l., 2004). V egetables are the major sources of dietary antioxidants ( Kurilich, Jeffery, Juvik, Wallig & Klein, 2002). C arotenoids, fat - soluble vitamins (such as tocopherol), water - soluble vitamins (such as Vitamin C), and many phenolic compounds together c ontribute to the total antioxidant capacity (Kalt , 2005; Wu et al., 2004). Consumption of frozen vegetables can not only fulfill the needs of health benefits as fresh vegetables but also provides consumers with bonus convenience such as time saving, easy s torage and longer shelf life. According to USDA report (2005), approximately 44 to 46.2 kg of frozen vegetables, not including frozen potatoes, has been consumed annually per person in the United State for the past 20 years. Broccoli is one of the most com monly consumed frozen vegetables because of its desired flavor and odor as well as high content in health - promoting compounds. Americans consumed approximately 1.23 kg of frozen broccol i per person during 2005. Frozen vegetables are typically cooked prior to consumption which the concentration of nutrients (i.e. ascorbic acid), their activity (antioxidant capacity) and sensory quality (i.e. color, firmness) may change through cooking practice s (Wu et al, 2004) . Although the effect of typical cooking methods (i.e. boiling, steaming and microwaving) on the quality of frozen vegetables has been widely investigated, the most popular cooking method for frozen vegetables, steamable bag microwaving is lack of study. Therefore, the objective s of this study are : (1) to investigate the effect of steamable bag microwaving on the nutritional value and physical properties of frozen broccoli compared to those thawed at room temperature (uncooked frozen broccoli); (2) to compare the effect of steamable bag microwaving versu s steamer steaming and traditional 2 microwaving on the nutritional value and physical properties of frozen broccoli; and (3) to investigate how the package design (shape and surface area) of a steamable bag containing a steam release valve affects the nutri tional value and physical properties of frozen broccoli during microwaving. 3 2 . Literature Review 2.1 Broccoli Broccoli is a member of the Brassicaceae family and belongs to the species Brassica oleracea (Wildman , 2001) , which are recommended to provide better health benefits against cancer and cardiovascular disease than many other vegetables according to recent epidemiological studies ( WCRF, 2007 ). Broccoli grows best in cool climates; therefore, in the United States of America t he majority of broccoli is grown in cool coastal areas of California generally during the winter and early spring. The Calabrese variety and the Italian variety are the two predominate types of broccoli. Calabrese is more common in the United States of Ame rica, and the Italian variety is more common in Great Britain and other regions in Europe (Wildman , 2001). Broccoli did not become a largely consumed vegetable in the United States until the ( Wildman , 2001 ) . 2.1.1 Frozen Broccoli Longer pre paration time is required for fresh vegetables which do not fit for current vegetables rapidly increased in recent years (Danesi & Bordoni, 2008). Frozen vegetable is a p roduct that has been undergone to different processes (e.g., blanching and quick freezing) . Balancing is a process of exposing vegetables or fruits to high temperatures for a short period. This process not only prolongs the shelf life of vegetables by inac tivating the enzymes responsible for browning (Nielsen, Larsen, & Poll, 2004) , such as polyphenoloxidase, lipoxygenase and peroxidase , but also improves both color and flavor. Broccoli is one of the most consumed frozen vegetables over the past 10 years in the United States (USDA , 2005) , 4 which is widely considered to contain high level of health benefit phytochemicals including glucosinolates, flavonoids, vitamins, and minerals (Cao, Sofic, & Prior, 1996; Plumb et al., 1996). The quality of frozen v egetables is defined in the United States Standards for Grades with color and texture as two of the common quality attributes assigned to broccoli. The color of s ince many of the natural colorants are either precursors to antioxidants or contain antioxidant ed rather than cooked to a very soft final product. Microwaving for approximately 6 - 8 min per pound or boiling for approximately 3 - 10 min per pound are recommended on most commercial packages of these vegetables sold today. 2.2 Nutritional Values 2.2.1 As corbic Acid Vitamin C, a water - soluble vitamin, which includes ascorbic acid and its oxidation product - dehydroascorbic acid (DHA), has many biological activities in human body. More than 85% of vitamin C in human diets is supplied by fruits and veg etables, such as citrus fruits, broccoli and tomatoes (Davey et al., 2000; Lee & Kader, 2000). Ascorbic acid is well known for its antioxidant activity, acting as a reducing agent to reverse oxidation in liquids. Biological function of ascorbic acid can be defined as an enzyme cofactor, a radical scavenger, and as a donor/acceptor in electron transport at the plasma membrane. Ascorbic acid is able to scavenge - tocopherol (Davey et al., 2000) . 5 The oxidation product of ascorbic acid, dehydroascorbic acid (DHA), is unstable at physiological pH and it is spontaneously and enzymatically converted to 2,3 - diketogulonic acid (Davey et al., 2000). 2.2.2 Antioxidant Capacity Antioxidants have gained lots of studies due to their potential effects in the prevention of chronic and degenerative diseases such as cancer and cardiovascular disease as well as aging (Diaz, Frei & Keaney, 1997; Ames, Shigenaga & Hagen, 1993; Ames, Gold & Willet, 1995; Young & Woodside, 2001). These health promotion effects may be related to components in the foods with antioxidant activity (Kaur & Kapoor, 2001). The ability of antioxidants to scavenge free radicals in the human body and thereby decrease the amount of free radic al damage to biological molecules like lipids and DNA may be one of their protective mechanisms. Since there are hundreds of antioxidant compounds in most foods, the total antioxidant capacity of a given food may be the integrated action from different com pounds instead of that from any single compound. The concept of total antioxidant capacity reflects the integrated and, if any, synergic effects of all the antioxidants. In order to evaluate the total antioxidant capacity of a given food and their health p romotion effects accurately, we need to consider the possible interaction of components in their contribution to antioxidant activity. In general, components in plants can be divided into two fractions, hydrophilic and lipophilic. 2.2.2.1 Hydrophilic Ant ioxidants Hydrophilic antioxidants are water - soluble antioxidants including vitamin C, phenolic compounds and others. Hydrophilic antioxidants are the main contributors to its total antioxidant 6 capacity in previous studies for broccoli. For exampl e, hydrophilic extracts are responsible for 80% to 95% of the total antioxidant capacity of fresh broccoli using the ORAC assay reported by Kurilich, Jeffery, Juvik, Wallig and Klein (2002). Similarly, Wu, Beecher, Holden, Haytowitz, Gebhardt and Prior (20 04) stated that hydrophilic antioxidants in Brassica vegetables provide more than 89% of the total antioxidant capacity while Roy, Juneja, Isobe and Tsushida (2009) found that 92% of the total antioxidant capacity of broccoli is provided by its hydrophilic extract. Most popular in vitro antioxidant measurement methods are designed primarily for hydrophilic components, and may not be suitable or adaptable for lipophilic measurements. 2.2.2.2 Lipophilic Antioxidants Lipophilic antioxidants are lipid - soluble antioxidants including carotenoids, vitamin E and others. In order to obtain a good measurement of total antioxidant capacity for a given food, Cano, Acosta and Arnao (2000) and Arnao, Cano and Acosta (2001) suggested that separating lipophilic co mponents from that of the hydrophilic components using similar chemical principles. FL methods were developed using a hydrophilic environment (Cao et al., 1993, 1995; Ou, Huan g, Hampsch - Woodill, Flanagan & Deemer, 2002). However, it has proven to be adaptable for lipophilic antioxidants as well. Recently, Huang, Ou, Hampsch - Woodill, Flanagan & Deemer (2002) developed a lipophilic ORAC FL measurement method that employed randomly methylated b - cyclodextrin (RMCD) as a solubility enhancer. This allowed for the measurement of the antioxidant capacity of lipophilic and hydrophilic components for a given sample separately, but based on the same peroxyl free radical. The ORAC FL method h as the advantage that it combines the inhibition degree and time of inhibition into one value. 7 2.2.2.3 Oxygen Radical Absorbance Capacity a ssay The use of different assays by research groups can result in reporting varying amounts of antioxidants f ound in various food products. Each assay typically employs a different free radical to use as the standard. The ability of the antioxidants to react with the free radical affects the antioxidant capacity. Oxygen radical absorbance capacity (ORAC) assay de pends on the free radical (AAPH generated) damage to fluorescein. The degree of the change of fluorescent intensity indicates the amount of radical damage. The presence of antioxidants results in the inhibition in the free radical damage to the fluorescent compound. This inhibition is observed as a preservation of the fluorescent signal calculated the area - under - curve (AUC) while the results are expressed as Trolox equivalents (Prior & Cao, 1999) . ORAC assay can be used for testing hydrophilic and lipophili c antioxidants. The area - under - curve (AUC) calculation combines both inhibition percentage at fixed time and the length of inhibition time of free radical action by an antioxidant into a single quantity that provides high specificity (Cao & Prior, 1998). W u et al. M Trolox equivalent/g when assayed used ORAC and cooked broccoli decreased in total M TE/g. 2.3 Physical Quality 2.3.1 Firmness Plant - based foods are subj ected to cooking or processing to increase their edibility and palatability, especially for frozen vegetables. Once people get used to the characteristics of a food, they expect to consume similar texture when they eat it again. Changes in nutrients are ex cluded because they are usually not apparent to the person eating the food but change in firmness will. Firmness of food may be considered as the combined effect of mechanical 8 properties and behavior perceived in the mouth, which is affected by both the ce ll wall and then cell contents (Brown, 1977). The turgor or the internal pressure of the living cell is considered as a very important aspect of the texture of raw fruits and vegetables. Maintenance of this pressure is a function of the integrity of the se mi - permeable membrane between the cell wall and the remainder of the cell contents (Brown, 1977). When the cell membrane has been damaged by cooking, water and soluble substances leak out of the cell and the rigidity of the tissues is lost. In fruits, sala d greens, leafy vegetables, and other thin - walled tissues, this loss of turgor pressure causes a major change in textural characteristics. Stems, roots, and seed pods contain specialized cells that support and protect the growing plant. These can provide f irmness or even crispness in the absence of turgor. Fruits that are heated in canning or in the preparation of a cooked dish, loss their crispness because the cell membranes are damaged to the extent that they are no longer able to retain the cellular flui d. In contrast to fruits, many vegetables have a crisp or firm texture after cooking because their cells have relatively thick walls. Although heating eliminates the contribution of turgor to the texture of vegetables, it causes only gradual softening of t he thick the degree of crispness may still be present. Therefore, since most of vegetables are cooked before consumption, cell turgor is not a factor in their t vegetable can be achieved by a suitable cooking method including proper temperature, time and pressure, etc. 2.3.2 Color Cooking or heat treatments have variable effect on pigments, such as chlorophyll, carotenoids and anthocyanins, which are responsible for the color of fruits and vegetables. 9 Chlorophyll is a green pigment found in the leaves and green stems of plants, which has different stabilities towards pressure and temperature. Butz et al. (2002) reported that a significant reduction in the chlorophyll content of broccoli juice at temperature higher than 50 . At a constant pressure level, the values of the degradation rate constants of chlorophylls increase with increasing temperature (Van Loey et al., 1998) whereas at constant elevated temperatures, pressure increase accelerates the degradation of chlorophyll. Turkmen, Poyrazoglu, Sari and Velioglu (2006) found that the loss of greenness in vegetables after cooking could be attributed to the degrad ation of chlorophyll along with the formation of pheophytins. However, the increase in green color intensity of broccoli at the early stage of blanching was related to cell disruption during blanching treatment which resulted in the leakage of chlorophyll into the intercellular space yielding a more intense bright green color on the vegetable surface (Oey, Lille, Loey & Hendrickx, 2008). Besides, structure and pigmentation of food interact with each other to affect both color and translucency/opaci ty. Firmness modification may result in changes in the nature and extent of internally scattered light and the distribution of surface reflectance, which in turn may produce changes of color appearance more than the effect of pigment concentration changes. 2.4 The Effect of Cooking Treatments on Broccoli Most vegetables, especially frozen samples, are commonly cooked before being consumed. It is known that cooking induces significant changes in chemical composition, affecting the bio - accessibili ty and the concentration of nutrients and health - promoting compounds such as vitamin C, carotenoids, and polyphenols (Pellegrini et al., 2010). 10 Domestic cooking methods, including high pressure, microwaving and boiling, reduced vitamin C (AA and DHAA) cont ent of broccoli between 20% and 46% except for steaming which no loss was found (Vallejo, Tomas - Barberan & Garcia - Viguera, 2002). Galgano, Favati, Caruso, Pietrafesa and Natella (2007) reported that boiling and steaming caused vitamin C losses of 34.2% and 22.4% in broccoli, respectively, while pressure cooking and microwaving conversely did not induce significantly loss of vitamin. Bernhardt and Schlich (2006) evaluated the influence of different domestic cooking method (i.e. boiling, stewing, steaming, pr essure steaming and microwave) on ascorbic acid content in fresh and frozen broccoli and found that all cooking methods caused small losses of ascorbic acid except boiling leaded to high losses. Zhang and Hamauzu (2004) reported that only 35% of antioxidan t capacity in broccoli was retained while up to 70% of ascorbic acid lost after boiling and microwaving for 5 minutes. It is noteworthy that the process used to microwave the vegetables in the study by Zhang and Hamauzu (2004) was essentially the same as b oiling since t he broccoli was plac ed in 200 m l of boiled water and then coo ked in the microwave oven . Both, microwaving and conventional cooking of broccoli with water, have been shown to decrease in antioxidant components and activity in broccoli (Gliszcz ynska - Swigo et al., 2006; Lopez - Berenguer, Carvajal, Moreno, & Garcia - Viguera, 2007). These findings indicated that loss of nutritional values due to nutrients being largely leached into the cooking water. On the contrary, Turkmen, Sari, and Vel ioglu ( 2005 ) found that the total antioxidant capacity of fresh broccoli increased up to 17% after cooking. This increase was the same during cooking by all boiling, steaming and microwaving. Wachtel - Galor, Wong and Benzie (2008) reported the increased ten dency of antioxidant capacity in broccoli after cooking while the antioxidant capacity of broccoli cooked by microwaving was about 30% lower than those cooked 11 by boiling but only approximately 30% - 50% compared to those under steaming. The enhanced antioxi dant capacity of broccoli after cooking may relate to the softening of matrix structure which improves the antioxidants extractability and formation of new antioxidant compounds such as Maillard - reaction products ( Turkmen, Sari, & Velioglu , 2005 ). Miglio, Chiavaro, Visconti, Fogliano and Pellegrini ( 2008 ) stated that cooking treatments including boiling, steaming and frying induced softening for broccoli. In their study, broccoli cooked by steaming and frying became less green ( - a* increased) while a signif icant increase in greenness was found in the boiled broccoli. The prolonged heating time in steaming may induce more chlorophyll degradation and then cause a decrease in greenness compared to the shorter cooking time used in boiling. Besides, oil and extre me high temperature in frying may change the light scattering and reflectance of green surfaces which leaded to loss of greenness. Pellegrini et al. (2010) claimed that fresh broccoli retain ed phytochemicals and total antioxidant capacity better than froze n broccoli , whereas frozen broccoli had color features more similar to raw samples because the blanching process inactivated the function of enzymes which limited the degradation of chlorophylls making the pigments more stable after cooking. 12 3. Eff ect of steamable bag microwaving versus steamer steaming and traditional microwaving on nutritional preservation and physical quality of frozen broccoli 3 . 1 Materials Frozen Broccoli. Individual quick freezing (IQF) broccoli (stems and florets) was purcha sed from a local marke t in 2.5 lbs bag 8 ºC. The frozen broccoli f rom the purchased bags was mixed together to obtain homogeneous samples. Chemicals. Meta - phosphoric acid (ACS reagent, 33.5~36.5%), 2, 6 - dichlorophenol sodium s alt hydrate (BioReagent), sodium bicarbonate (Analytical grade, 99.0%), 6 - methoxy - 2,5,7,8 - tetramethylchromane - 2 - - azobis(2 - amidino - propane) dihydrochloride (AAPH), sodium phosphate (mono and dibasic) were purchased from Sigma Aldrich (St. Louis, MO, USA). L - ascorbic acid (U.S.P. grade, 99.0% was purchased from Spectrum Chemical Manufacturing Corp. (New Brunswick, NJ, USA). Acetic acid (glacial, ACS reagent, 99.0%) was purchased from EMD Chemicals Inc. (Billerica, M A, USA). Sulfuric acid (ACS reagent, 96.4%) was purchased from J.T. Baker (Center Valley, PA, USA). Methanol (ACS reagent, 99.8%) was purchased from Macron Chemicals Inc. (Center Valley, PA, USA). Acetone (ACS reagent) was purchased from Jade Scientific I nc. (Westland, MI, USA). Distilled water was purchased from Meijer Distribution Inc. (Grand Rapids, MI, USA). Steamable bags. Steamable bags were composed of a high - barrier two - ply lamination film (Printpack Inc., Atlanta, GA, USA) and a steam - activ ated steam release valve (Avery Flexis valve, Avery Dennison, Pasadena, CA, USA). The laminated material consisted of polyethylene terephthalate (PET , out er layer ) and polypropylene (PP , in ner layer ) and had a total thickness was 5 mil . The dimension s of the steamable bags were 23 cm × 16 cm selected according to its capacity to contain 300 ± 3 g frozen broccoli. 13 Cookware. A T - fal steamer (VC133851, West Orange, NJ, U.S.A.), a Pyrex glass container with glass lid ( 2 quart, Greencastle, PA, U.S.A.) an d a Sharp microwave oven (1.4 cu. ft. 1100 watts , Sharp Model R410lk, Mahwah, NJ, USA) were purchased from a local retail store. 3 . 2 Methods 3 . 2 .1 Packaging Amounts of 300 ± 3 g frozen broccoli with an approx. equal amount of stems and floret s w ere packed into steamable bags. The transfer of product from its original packages was made in low lighting environment at 0 ºC. The bags were sealed using a thermal heat sealer (Model 24AS/1, Sencorp Systems Inc., Hyannis, MA, USA) for 1.5 seconds at a ja w pressure of 276 kPa and temperature of 199 ± 2 ºC. The seal integrity of the steamable bags was verified using a package leak test with the ARO Non - porous package tester (F099 - 1080, ARO Corporation, York, PA, USA). Brie f ly, a filled steamable bag was imm ersed in water inside the testing chamber , the chamber was closed , and then vacuum was drawn. The steamable bag was considered to maintain its integrity i f no bubble w as escaping from the package. The sealed packages were immediately stored in a freezer at 18 ºC. All product quality tests were done within one week to avoid variations caused by extended storage time. 3 . 2 .2 Treatments Un cook ed broccoli . Uncooked frozen broccoli was thawed to room temperature inside a corrugated box for 10 hours . The box wa s used to avoid light damage affecting the quality of broccoli during the thawing process . 14 Steamer steaming. 300 ± 3 g of frozen broccoli was steamed for 600 seconds using a T - fal steamer . Briefly, the broccoli was placed o n the plastic steaming rack abov e the steamer base containing boiling water and covered with the steamer lid . The cooking time was determined by using a T - type handheld flexible thermocouple probe ( Model 91100 - 40 , Cole - Parmer Instrument Co., Vernon Hills, IL, USA) . This was inserted into the broccoli floret ( stem towards - floret direction ) via the vent o f the steamer lid at time interval s between 480 and 720 seconds and then the temperature s were recorded after 2 seconds . The time needed for the broccoli to reach 74 ºC ( safe minimum cook ing temperature) was designated as cooking time. Traditional microwaving. 300 ± 3 g of frozen broccoli was microwaved for 330 seconds using a rectangular - shaped Pyrex glass container with a glass lid. Briefly, the broccoli together with 100 ml of distille d water at room temperature w as placed inside the glass container and then microwaved with the glass lid on . T he cooking time was determined by inserting the thermocouple probe into the broccoli floret as abovementioned immediately after microwaving at ti me intervals between 240 and 420 seconds , and then recording the temperature s after 2 seconds . The time needed for the broccoli t o reach 74 ºC was designated as the cooking time. The use of water was to prevent excessive water loss leading to serious colo r and firmness changes during microwaving , and an amount of 100 ml was used to avoid the Steamable bag microwaving. A steamable bag containing 300 ± 3 g of frozen broccoli was heated during 300 seconds in a Sharp microwav e oven. The cooking time was determined by opening the microwave oven after time intervals between 240 and 360 seconds, puncturing a small hole in the center of the steamable bag, inserting the thermocouple probe into the broccoli 15 floret as abovementioned and recording the temperatures after 2 seconds . The time needed for the broccoli to reach 74 ºC was designated as the cooking time. After each cooking process finished, some of the broccoli w as cooled rapidly on ice water slurry and then drained using a paper towel for ascorbic acid and antioxidant capacity analyses. Other broccoli samples were cooled to 50 ºC (mimicking consumption temperature) for firmness analyses , and to room temperature for color analyses. Temperatures were controlled by inse rting the above - mentioned thermocouple pro b e into the broccoli of the same batch used as the temperature control sample. 3 . 2 .3 Asco rbic A cid An amount of 100 g of uncooked or cooked frozen broccoli containing an approx. equal amount of stems and florets was blended with 100 ml of extraction solution (15 g meta - phosphoric acid: 40 ml acetic acid: 3.7 ml conc. sulfuric acid: 450 ml water) for 30 seconds using a food chopper (Model 72705, Hamilton Beach Brands Inc., Southern Pines, NC, USA). The homo genates were filtered using a nylon cloth and the resulting residues mixed with another 50 ml of extraction solution, blended for 30 seconds and filtered. The two filtrates were combined together and then centrifuged at 3500 rpm, 4 °C (Centrifuge 5804R, Ep pendorf, Germany) for 600 seconds. The supernatants were collected and titrated against a dye solution (50 mg 2, 6 - dichlorophenol Na salt, 42 mg NaHCO3 and 200 ml water) until a pink color persisted for 15 seconds ( AOAC method No. 967.21; AOAC , 2000 ). Each treatment was replicated 3 times. Three broccoli samples of 100 g each were analyzed per replication. The results from the nine samples of each treatment were averaged and expressed as mg ascorbic acid /100 g D.W . (dry weight basis) as shown in Diagram 1 . 16 F i g u r e 1 . Sample size used to determine a scorbic a cid in u ncooked broccoli and in broccoli cooked using s teamer s teaming, t raditional m icrowaving and s teamable b ag m icrowaving . A total of 9 results were averaged and expressed in units of mg ascorbic acid/100g D. W. Sample #1 - B Sample #1 - C Measurement #1 - A Replication # 2 Extraction #2 Sample # 2 - A Sample # 2 - B Sample # 2 - C Replication # 3 Extraction #3 Sample # 3 - A Sample # 3 - B Sample # 3 - C Extraction #1 Replication #1 Sample #1 - A Measurement #1 - C Measurement #1 - B Measurement #2 - A Measurement #2 - B Measurement #2 - C Measurement #3 - A Measurement # 3 - B Measurement #3 - C 17 3 . 2 .4 Oxygen R adical A bsorb ance C apacity assay Uncooked or cooked broccoli was homogenized in a high - speed blender, and a 5 g sample was mixed with 40 ml acidic methanol/water (50:50, v/v, pH 2). The mixture was placed in a water - bath shaker for 1hour and then centrifuged a t 10,000xg (Sorvall RC - 5B Refrigerated Superspeed Centrifuge, Du Pont Instruments, Wilmington, DE, USA) for 600 seconds. The supernatant was collected and the residues were extracted by adding to 40 ml acetone/water (70:30, v/v) followed by 1 hour shaking and then centrifuged at 10,000xg for 600 seconds. The two supernatants were combined and then acetone/water (70:30, v/v) was added to adjust the extracted solution volume to 80 ml. The ORAC assay was done following the analytical procedures of Huang, Ou, H amp sch - flourescein (20 nM) was added to a 96 - following solutions: blank (sodium phosphate buffer), Trolox (6 - methoxy - 2,5,7,8 - tetramethylchromane - 2 - carboxyl extracts were added to 10 ml sodium phosphate buffer). The mixture was incubated at 37 °C for 1800 seconds in Microplate Reader (Biotek Instruments, Winooski, VT, USA). After incubation, - azobis (2 - amidino - propane) dihydrochloride) was added to all the wells. Fluorescence was monitored using 485 nm excitation and 528 nm emissions at 120 seconds as (TE = Trolox Equivalent). Each treatment was replicated 3 times. Twelve broccoli samples of 5 g each were analyzed per replication. The results from the thirty - six samples of each treatment were averaged and expressed as µ M TE/g D . W. as shown in Diagram 2 . 18 F i g u r e 2. Sample size used to determine antioxidant capacity of u ncooked broccoli and of broccoli cooked using s teamer s teaming, t raditional m icrowaving and s teamable b ag m icrowaving . A total of 36 results were averaged and expressed in units of µM TE/g D.W. Replication #1 Extraction #1 Sample #1 - A Sample #1 - B ~ #1 - K Sample #1 - L Measurement #1 - A Measurement #1 - L Measurement #1 - B ~ #1 - K Replication # 2 Extraction #2 Sample # 2 - A Sample # 2 - L Measurement #2 - A Measurement #2 - L Replication # 3 Extraction #3 Sample # 3 - A Sample # 3 - L Measurement #3 - A Measurement #3 - L Sample #2 - B ~ #2 - K Measurement #2 - B ~ #2 - K Sample #3 - B ~ #3 - K Measurement #3 - B ~ #3 - K 19 3 . 2 .5 Firmness The firmness of uncooked and cooked broccoli stems was measured using TA.XTPlus texture analyzer (Stable Micro Systems, Godalming, UK) equipped with a 10 - blades Kramer shear cell since it produces simulated results similar to those from humans chewing food. Four stems of approx. similar size and amount (about 10 ± 0.2 g) were placed in the Kramer shear cell for evaluation. Maximum peak force was recorded at a shear press speed setting of 1.50 mm/s. In order to avoid temperature effect on the firmness of samples, all samples were placed on trays and stored in an oven (Fisher ISOTEMP, 200 series, Model 230F, Wood Dale, IL, USA) under controlled condition of 50 ºC. Each treatment was replicated 3 times and for each replication six samples consisting of four stems each were tested . The results from the eighteen samples of each treatment were averaged and expressed in units of kg - force/g broccoli as shown in Dia gram 3. 3 . 2 .6 Color The color of uncooked and cooked broccoli florets was measured using a Hunter LabScan XE colorimeter (LX17582, Reston, VA, USA) calibrated using standard black and white tiles. The color parameters values, L* (lightness, black = 0, white = 100), a*(redness > 0, greenness < 0), and b*(yellowness > 0, blue < 0), of the pieces were recorded. Results were also expressed by hue angle (h° = arctan (b*/a*), red = 0°, yellow = 90°, green = 180°, blue = 270°) and total color difference E . Florets were individually placed in the standard sample cup and duplicate readings were taken at room temperature . Each treatment was replicated 3 times and for each replication 6 florets were tested as shown in Diagram 4. 20 F i g u r e 3. Sample size used to determine the firmness of u ncooked broccoli and of broccoli cooked using s teamer s teaming, t raditional m icrowaving and s teamable b ag m icrowaving . Replication #1 Stems #1 - A A total of 18 results were averaged and expressed in units of kg - force/g broccoli Stems #1 - B ~ #1 - E Stems #1 - F Measurement #1 - A Measurement #1 - B ~ #1 - E Me asurement #1 - F Replication #2 Stems #2 - A Stems #2 - F Measurement #2 - A Measurement #2 - F Replication #3 Stems #3 - A Stems #3 - F Measurement #3 - A Measurement #3 - F Stems #2 - B ~ #2 - E Stems #3 - B ~ #3 - E Measurement #2 - B ~ #2 - E Measurement #3 - B ~ #3 - E 21 F i g u r e 4. Sample size used to determine the color of u ncook ed broccoli and of broccoli cooked using s teamer s teaming, t raditional m icrowaving and s teamable b ag m icrowaving . Replication #1 Floret #1 - A Floret #1 - B ~ #1 - E Measurement #1 - A A total of 36 results were averaged Floret #1 - F Measurement #1 - A A Measurement #1 - F Measurement #1 - FF Measurement #1 - B ~ #1 - E Measurement #1 - BB ~ #1 - EE Replication #2 Floret #2 - A Floret # 2 - B ~ # 2 - E Measurement #2 - A Floret #2 - F Measurement #2 - A A Meas urement #2 - F Measurement #2 - FF Measurement #2 - B ~ #2 - E Measurement #2 - BB ~ #2 - EE Replication # 3 Floret #3 - A Floret # 3 - B ~ # 3 - E Measurement #3 - A Floret #3 - F Measurement #3 - A A Measurement #3 - F Measurement #3 - FF Measurement #3 - B ~ #3 - E Measurement #3 - BB ~ #3 - EE 22 3 . 2 .7 Moisture Content The moisture contents of all analyzed samples (uncooked and cooked frozen broccoli) were determined by a moisture analyzer (MX - 50, AND Instruments Ltd., Abingdo, UK) and used to convert data from fresh weight basis to dry weight basis. Briefly, an amount of 5 - 6 g of homogenized broccoli was dried in the moisture analyzer at 105 °C using its standard drying program until reaching constant weight. Results were obtained in triplicate and averaged. A moisture content of 90.96%, 91.17%, 90.69% and 89.53% for uncooked, steamer steaming, traditional microwav ing and steamable bag microwaving, respectively , was obtained. 3 . 2 .8 Statistical Analysis One - compare the ascorbic acid, antioxidant capacity, firmness and color of uncooked and steamable bag microwaving cooked broccoli as well as the effect of steamable bag microwaving v ersus steamer steaming v ersus traditional microwaving on the abovementioned parameters of frozen broccoli. The significance level used was p 0.05. MINITAB® 16.1.1 Statistical Software (Minitab Inc., PA, US) was used for all statistical assessments. 3. 3 Results and Discussions 3. 3 .1 Ascorbic A cid Figure 1 shows the ascorbic acid content of frozen broccoli after steamable bag microwa ving, steamer steaming, and traditional microwaving as well as of thawed frozen broccoli. The latter was used to determine the ascorbic acid retained in broccoli after cooking because of the known n egative effect of heat on ascorbic acid preservation in ve getables 23 (Erdman & Klein , 1982) . The retained ascorbic acid was 90 % , 90 % and 84 % for the broccoli cooked by steamable bag microwaving, steamer steaming, and traditional microwaving retained, respectively. T he ascorbic acid content of frozen broccoli cooked by steamer steaming and steamable bag microwaving was the same ( 573 ± 16 mg/100g D.W. and 571 ± 17 mg/100g D.W. , respectively ) , and was significantly higher than that of frozen broccoli cooked by traditional microwaving (535 ± 18 mg/100g D.W.). The difference can be attributed to the leaching of ascorbic acid into water during traditional microwaving. Erdman and Klein (1982) reported that both, presen ce and amount of water , can significant ly affect ascorbic acid retention , with a larger quantit y of cooking water result ing in more loss of water soluble vitamins due to leaching. Miglio , Chiavaro, Visconti, Fogliano and Pellegrini (2008) also observed a loss of ascorbic acid in steamed broccoli while Vallejo , Tomas - Barberan and Garcia - Viguera (2002) and Gliszczynska - Swiglo et al. (2006) reported no effect of steamer steaming on ascorbic acid content. The difference between their results and our results can be attributed to the different methodologies used to extract and determine ascorbic acid. Sever al studies have also observed a higher loss of ascorbic acid in microwaved broccoli than in steamed broccoli ( Hudson, Dalal & Lachance, 1985; Vallejo, Tomas - Barberan & Garcia - Viguera, 2002; Pellegrini et al. 2010) . In summary , steamable bag microwaving per forms better than traditional microwaving and is equal to steamer steaming in retaining ascorbic acid content. However, steamable bag microwaving notably reduces cooking time compared to steamer steaming (300 and 600 seconds for steamable bag microwaving a nd steamer steaming, respectively) . 24 Figure 5 . Ascorbic Acid Content of Frozen Broccoli after S teamable B ag M icrowaving, S teamer S teaming, and T raditional M icrowaving as well as of T hawed F rozen B roccoli (different letters indicate significant difference s (p 0.05)). 3. 3 .2 Antioxidant Capacity H ydrophilic antioxidants have been reported to be the main contributors to the antioxidant capacity of broccoli (Kurilich, Jeffery, Juvik, Wallig & Klein, 2002 ; Wu, Beecher, Holden, Haytowitz, Gebhardt & Prior, 2004 ; Roy, Juneja, Isobe & Tsushida, 2009) and therefore, only these were extracted from thawed and cooked frozen broccoli and evaluated using oxygen radical absorbance capacity (ORAC) assay . Figure 2 shows the obtained ORAC values in a dry weight basis . The ORAC values of frozen broccoli upon s teamer steaming and steamable bag microwaving were significantly higher than those of thawed frozen broccoli . An increased antioxidant capacity in vegetables upon cooking has been attributed to cooking promoting the release of antioxidant compounds from the vegetable matrix and determining the 25 formation of n ew antioxidant compounds , such as Maillard - reaction products (Rechkemmer, 2007 ; Miglio, Chiavaro, Visconti, Fogliano & Pellegrini, 2008). The a ntioxidant capacity of frozen broccoli cooked by traditional microwaving was the same as that of thawed frozen br occoli, and 5 3 % and 48.0% lower than that of frozen broccoli cooked by steamer steaming and steamable bag microwaving , respectively . The different antioxidant capacity could in part be due to the major retention of ascorbic acid in broccoli cooked by steam er steaming and steamable bag microwaving compared to traditional microwaving (Figure 1). In addition, the leaching of other water - soluble nutrition al compounds such as glucosinolates into cooking water in traditional microwaving might have contributed to the lower antioxidant capacity of the microwaved frozen broccoli (Wachtel - Galor, Wong & Benzie, 2008 ; Miglio, Chiavaro, Visconti, Fogliano & Pellegrini, 2008 ) . Th is increased or maintained antioxidant capacity of the cooked frozen broccoli contras t s with the losses of antioxidant capacity of frozen broccoli during boiling, microwaving and steaming reported by P ellegrini et al. (2010) . But it is of note that different extraction solvents, procedures and analytical measurements might lead to results that are not easily compared (Perez - Jimenez et. al., 2008). However, Turkmen, Sari and Velioglu (2005) and Wachtel - Galor, Wong and Benzie (2008) observed an increased antioxidant capacity in boiled, microwaved and steamed fresh broccoli , which aligns with the results of this study . Our results partially disagree with the lower nutritional value attributed to processed vegetables compared to uncooked ones ( Zhang & Hamauzu, 2004; Danesi & Bordoni, 2008; Mazzeo et al., 2011) . According to this study, the final nutritional value of processed broccoli depends on the cooking treatment. In the case of steamer steaming and steamable bag 26 microwaving , some of the ascorbic acid of the frozen broccoli was lost during cooking but its antioxidant capacity was in creased. Figure 6 . Antioxidant Capacity of Frozen Broccoli after S teamable B ag M icrowaving, S teamer S teaming, and T raditional M icrowaving as well as of T hawed F rozen B roccoli (different letters indicate significant differences (p 0.05)). 3. 3 .3 Firmne ss T he firmness of frozen broccoli after steamable bag microwaving, steamer steaming, and traditional microwaving was determined and compared to that of thawed frozen broccoli to evaluate the effect of these cooking methods on broccoli tenderizati on . Table 1 shows that t he firmness of frozen broccoli after steamer steaming, traditional microwaving and steamable bag microwaving was significantly low er than that of thaw ed frozen broccoli, indicating the softening of the broccoli after all three cooking methods . By comparing the cooking methods, t raditional microwaving and steamer steaming softened the broccoli equally, and the softening 27 was less than that of the steamable bag microwaving . These results indicate the time - saving in tenderiz ing broccoli of steamable bag microwaving compared to traditional microwaving and steamer steaming ( 300, 330, 600 seconds for steamable bag microwaving , traditional microwaving , and steamer steaming , respectively ). In addition , steamable bag microwaving does not require cooking water to tenderiz e broccoli as traditional microwaving and steamer steaming do. Thus, steamable bag microwaving can soften broccol i faster and without the use of additional water , which upgrades microwave cooking to a higher level of efficacy and convenience . 28 Table 1. Color and Firmness of Frozen Broccoli after Steamable Bag Microwaving, S teamer Steaming and Traditio nal Microwaving as well as of Uncooked Thawed Frozen Broccoli Color 1 Firmness 2 (kg - force/g) L* a* b* Hue° E Uncooked 20.85 ± 2.18B 13.19 ± 1.39B 20.07 ± 2.79B 123.43 ± 1.68A 31.83 ± 3.60B 3.29 ± 0.36A Steamer Steaming 23.14 ± 2.50A 12.77 ± 1.01AB 23.43 ± 2.41A 118.66 ± 2.35C 35.35 ± 3.34A 2.31 ± 0.35B Traditional Microwaving 23.77 ± 2.04A 14 .17 ± 0.92C 23.29± 1.98A 121.34 ± 1.82B 36.19 ± 2.71A 2.61 ± 0.46B Steam Bag Microwaving 21.65 ± 1.16B 12.50 ± 0.88A 20.42 ± 1.66B 121.48 ± 1.27B 32.31 ± 1.69B 1.84 ± 0.36C ) 1 Values presented as mean ± SD (n = 18) 2 Values presented as mean ± SD (n = 18) 29 3. 3 .4 Color Cooked vegetables exhibit poor color quality in comparison with fresh ones ( Turkmen, Poyrazoglu, Sari & Velioglu, 2006) . Thus, the c olor of broccoli florets after steamable bag microwaving, steamer steaming and traditional microwaving was determined and compared to that of thawed frozen broccoli (Table 1). S teamer steaming and traditional microwaving significantly increased the L* value of the broccoli florets while steamable bag microwaving maintained the intense darkness of the broccoli florets compared to thawed frozen broccoli. The use of shortest cooking time in steamable bag microwaving might have caused this difference since less cell juice released by cell membrane deterioration replaced intercellular air ( Tijskens, Schijvens & Biekman, 2001) . In contrast to our results, Pellegrini et al. (2010) observed a decreased and maintained L* value in steamed and microwaved fro zen broccoli, respectively. The difference between their and our results can be attributed to the longer cooking times used for steaming and microwaving by Pellegrini et al. (2010) that might have increased cell membrane deterioration and consequently, mor e cell juice replacing intercellular air. Broccoli f lorets under steamer steaming and traditional microwaving became yellower ( increased + b* value) while those under steamable bag microwaving maintained yellowness (maintain ed + b* value) compared to thawe d broccoli florets. The use of shortest cooking time in steamable bag microwaving might have also caused this difference. A l oss of greenness ( increased - a* values ) was found in broccoli florets cooked by steamer steaming and steamable bag microwaving. Pel legrini et al. (2010) also found that steaming cause s loss of greenness in frozen broccoli . Loss of greenness in cooked broccoli has been attributed to chlorophyll pigment degradation along with the formation of pheophytins caused by the exchange of Mg +2 b y H + in the porphyrin ring of the chlorophyll ( Turkmen, Poyrazoglu, Sari & Velioglu, 2006) . In agreement, Pellegrini et al. 30 (2010 ) found that 29% chlorophylls broke down while 274% pheophytins were generated for frozen broccoli under steaming. In contrast to the aforementioned cooking methods , traditional microwaving yield ed greener ( decreased - a* ) broccoli florets . This greenness increase has been related to an alteration of surface reflecting properties and light penetration into the vegetable tissue caus ed by replacement in cells of air and other dissolved gases with cooking water (Miglio, Chiavaro, Visconti, Fogliano & Pellegrini, 2008 ; Turkmen, Poyrazoglu, Sari & Velioglu, 2006). Other factors could have contributed to this difference in greenness. T he shorter cooking time of traditional microwaving (330 seconds) compared to steamer steaming (600 seconds) could have reduced chlorophyll degradation and pheophytins formation. The different matrix structure (softness) between broccoli cooked u nder similar c ooking time s ( traditional microwaving (330 seconds) and steamable bag microwaving (300 seconds) ) could have been the reason for their different greenness since c hanges in matrix structure of vegetables have been reported to affect their color (Oey, Lille, Loey & Hendrickx, 2008) . In contrast to our results, Pellegrini et al. (2010) reported a 45% loss of greenness in frozen broccoli after microwaving. The difference between their and our results could be attributed to the longer cooking time used for microw aving by Pellegrini et al. (2010). The h ue angle of the broccoli florets decreased (shift toward yellow values) for all cooking methods, with steame d broccoli having the lowest h ue angle values. A decreased hue angle was observed for all cooking methods d ue to the combin ed effect s of a* and b* values since one or both changed but in a different way depending on the cooking method . The total color difference ( E) of the broccoli florets cooked by steamable bag microwaving was the same as that of the uncooked florets while an increased E value w as observed in the florets cooked by steamer steaming and traditional microwaving. Therefo re, steamable bag microwaving is a better cooking method compared to steamer steaming and 31 traditional microwaving in terms of minimiz ing color changes . This will most likely have an effect on since consumers prefer eating fres h - like vegetables . 32 4. Effect of steamable bag design on the nutritional and physical quality of cooked frozen broccoli 4 . 1 Materials Frozen broccoli. Individual quick freezing (IQF) broccoli (stems and florets) was purchased from a lo cal market in 2.5 lbs bag and stored in a freezer at 1 8 ºC. The frozen broccoli from the purchased bags was mixed together to obtain homogeneous samples. Chemicals. Meta - phosphoric acid (ACS reagent, 33.5 % ~ 36.5%) , 2, 6 - dichlorophenol sodium salt hydrate (BioReagent), sodium bicarbonate (Analytical grade, 99.0%), 6 - methoxy - 2,5,7,8 - tetramethylchromane - 2 - - azobis(2 - amidino - propane) dihydrochloride (AAPH), sodium phosphate (mono and dibasic) were purchased from Sigma A ldrich ( St. Louis, MO, US A ). L - ) was purchased from Spectrum Chemical Manufacturing Corp. ( New Brunswick, NJ, US A ). Acetic acid (glacial, ACS reagent, 99.0%) was purchased from EMD Chemicals Inc. ( Billerica, MA, US A ). Sul furic acid (ACS reagent, 96.4%) was purchased from J.T. Baker ( Center Valley, PA, US A ). Methanol (ACS reagent, 99.8%) was purchased from Macron Chemicals Inc. ( Center Valley, PA, US A ). Acetone (ACS reagent) was purchased from Jade Scientific Inc. ( Westland , MI, US A ). Steamable bags. S team able bags were composed of a high barrier two - ply lamination film (Printpack Inc., Atlanta, GA, US A Flexis valve, Avery Dennison, Pasadena, CA, US A ) with an approximat ely central location . The laminated material consisted of polyethy lene terephthalate (PET, outer laye r) and polypropylene (PP, inner layer), and had a total thickness was 5 mil. The material was shaped into bags differing in shape and surface area (length × width) as shown in Table 2 . The dimensions of the steam able bags were selected according to their capacity to contain the same amount of frozen 33 broccoli (330 ± 3 g). Different bag headspaces were obtaining by placing a same amount of broccoli in bags dif fering in dimensions. Table 2 . P ackage S pecification and C ooking T ime . Steamable Bag Type Length ( cm ) Width ( cm ) Surface Area ( cm 2 ) Cooking Time ( seconds ) SB - I 28.58 18.42 526.44 300 SB - II 22.23 18.42 409.48 315 SB - III 24.13 15.88 383 .18 300 * Time taken for broccoli to reach 74 °C at full microwave power . 4 . 2 Methods 4 . 2 .1 Packaging Amounts of 330 ± 3 g frozen broccoli with an approx. equal amount of stems and floret s were packed into the three steam able ba gs. The transfer of product from its original packages was made in low lighting environment at 0 ºC. The bags were then sealed using a thermal heat sealer (Model 24AS/1, Sencorp Systems Inc., Hyannis, MA, US A ) for 1.5 sec onds at a jaw pressure of 276 k Pa a nd temperature of 199 ± 2 ºC. The seal integrity of the steam able bags was verified using a package leak test with the ARO Non - porous package tester (F099 - 1080, ARO Corporation, York, PA, US A ). Briely, a filled steamable bag was immersed in water inside th e chamber , the chamber was closed and then va cuum was drawn . T he steamable bag was considered to maintain its integrity if no bubble w as escaping from the package. The sealed packages were immediately stored in a freezer at 18 ºC. All product quality test s were done within one week to avoid variations caused by extended storage time . 4 . 2 .2 Cooking Time Each of t he steamable bags contain ing frozen broccoli was heat ed up in a microwave oven ( 1.4 cu. ft. 1100 watts; Sharp Model R410lk, Mahwah, NJ, US A ) at different interval times 34 between 240 and 360 seconds. Then, the microwave oven was opened and a small hole in the center of the steamable bag was punctured . A T - type handheld flexible thermocouple probe ( Model 91100 - 40 , Cole - Parmer Instrument Co., Vernon Hills, IL, USA) was inserted via the puncture hole into the broccoli floret ( stem - towards - floret direction ) and the temperature was recorded after 2 seconds . The time needed for the broccoli to reach 74 ºC (safe minimum cooking temperature) was des ignated as the cooking time. Four packages of each type of steamable bag were used to confirm the cooking time. Results are presented in Table 2 . 4 . 2 . 3 Ascorbic A cid An amount of 100 g of steam able bag microwaved frozen broccoli containing an ap prox. equal amount of stems and florets was blended with 100 m l of extraction solution (15 g meta - phosphoric acid: 40 m l acetic acid: 3.7 m l conc. sulfuric acid: 450 m l water) for 30 seconds using a food chopper (Model 72705, Hamilton Beach BrandsInc., Sou thern Pines, NC, US A ). The homogenates were filtered using a nylon cloth and the resulting residues mixed with another 50 m l of extraction solution, blended for 30 seconds and filtered. The two filtrates were combined together and then centrifuged at 3500 rpm, 4 °C (Centrifuge 5804R, Eppendorf, Germany) for 10 minutes . The supernatants were collected and titrated against a dye solution (50 mg 2, 6 - dichlorophenol Na salt, 42 mg NaHCO 3 and 200 m l water) until a pink color persisted for 15 seconds (AOAC , No. 9 67.21, 2000 ). Three replicates from each package were evaluated, and the results were averaged and expressed as mg ascorbic acid/100g F . W . (fresh weight) and mg ascorbic acid /100 D . W. (dry weight) . Broccoli from a total of nine packages w as tested (three packages per type of steamable bag ). 35 4 . 2 . 4 Oxygen R a dical A bsorbance C apacity assay The cooked broccoli was removed from the steam able bag and homogenized in a high - speed blender . 5 g of the homogenized broccoli was mixed with 40 m l acidic methan ol/water (50:50, v/v, pH 2). The mixture was placed in a water - bath shaker for 1 hour and then centrifuged at 10,000x g (Sorvall RC - 5B Refrigerated Superspeed Centrifuge, Du Po n t Instruments, Wilmington, DE, US A ) for 10 minutes . The supernatant was collecte d and the residue s w ere extracted by adding to 40 m l acetone/water (70:30, v/v) followed by 1 hour shaking and then centrifuged at 10,000x g for 10 minutes . The two supernatants were combined and then acetone/water (70:30, v/v) was added to adjust the extra cted solution volume to 80 m l . The ORAC assay was done following the analytical procedures of Huang , Ou, Hampsch - Woodill, Flanagan and P rior (2002). Briefly, 150 l of flourescein (20 nM) was added to a 96 - wells black plate, followed by 2 5 l of each of the following solutions: blank (sodium phosphate buffer), Trolox (6 - methoxy - 2,5,7,8 - tetramethylchromane - 2 - carboxylic acid) standard and diluted sample l sample extracts were added to 10 m l sodium phosphate buffer). The mixture was incubated at 37 °C for 30 minutes in Microplate Reader (Biotek Instruments, Winooski, VT, l of - azobis (2 - amidino - propane) dihydrochloride) was added to all the wells. Fluorescence was monitored using 485 nm excitation and 528 nm emissions at 2 minutes intervals for 300 minutes in Microplate Reader. A Trolox standard (6.25, 12.5, 25, 50, ve and ORAC values were . W . . W . (TE = Trolox Equivalent). A total of nine packages were tested (three packages per type of steamab l e bag ). 36 4 . 2 . 5 Color The color of the frozen broccoli after steam able bag microw aving was measured using a Hunter LabScan XE colorimeter (LX17582, Reston, VA, US A ) calibrated using standard black and white tiles. Five florets of similar size from each package were analyzed immediately after microwaving. The floret pieces were individu ally placed in the standard sample cup and duplicate readings were taken per piece. The color parameter values , L*(lightness, black = 0, white = 100), a*(redness > 0, greenness < 0), and b*(yellowness > 0, blue < 0) , of the pieces were recorded. Results w ere also expressed by hue angle (h° = arctan (b*/a*), red = 0°, yellow = 90°, green = 180°, blue = 270°). Broccoli from a total of nine packages w as tested (three packages per type of steamab l e bag ). 4 . 2 . 6 Firmness The firmness of the frozen bro ccoli after microwaving the steam able bag s was measured using a TMS - TP Texture Press Analyzer (Model FTA - 300 Force Tr.; Food Technology Corp., Sterling, VA, US A ) , equipped with a 10 - blade Kramer shear cell (C - 332; 67 x 67 mm) , because it produces simulated results similar to those from humans chewing food. Four stems of approx. similar size and amount (about 10 ± 0.2 g) were placed in the Kramer shear cell for evaluation and a total of twenty pieces from each package were evaluated. In order to avoid a tem perature effect on the firmness of the samples, all samples were placed on trays and stored in an oven (Fisher ISOTEMP, 200 series, Model 230F, Wood Dale, IL, US A ) under controlled conditions of 50 ºC. Maximum force was recorded at a shear press speed sett ing of 0. 424 c m/s. The results were averaged and expressed in units of kg - force / g broccoli. Broccoli from a total of twelve packages was tested (four packages per type of steam able bag). 37 4 . 2 .7 Moisture Content The moisture content of frozen broc coli cooked in the microwav e steam able bags was determined using a moisture analyzer (MX - 50, AND Instruments Ltd., Abingdo , UK) with a standard drying program. An amount of 5 - 6 g of cooked homogenized broccoli (in triplicate) from each steam able bag was dr ied in the moisture analyzer at 105 °C until reaching constant weight. The average moisture content of broccoli from each type of steam able bag was then determined and used to convert the data from wet basis to dry basis. 4 . 2 .8 Statistical Analysis One - as used to evaluate the effect of the steam able bag design on the physical quality and nutritional content of ® 16.1.1 Statistical Software (Minitab Inc., PA, US) was used for all statistical assessments. 4.3 Results and Discussions 4. 3 .1 C ooking Time The time necessary for the frozen broccoli located in the center of each steam able bag to reach a temperature of 74 ºC (minimum cooking temperature recommended as safe) was determined and the results are presented in Table 2 . 300 sec onds was sufficient for the SB - I and SB - III to reach 74 ºC while 3 15 seconds were required for SB - II . These different cooking times were due to the different shape of the packages. Even though the SB - I and SB - III differed in microwavable surface area ( 526. 44 cm 2 vs. 383.18 cm 2 ) both had a rectangular shape and cooked the broccoli located in the center of the steam able bag 15 seconds faster than SB - II . The latter 38 had a microwavable surface area ( 409.48 cm 2 ) similar to that of SB - III ( 383.18 cm 2 ) but a more s quare shape. Therefore, the shape of a steam able bag significantly affect ed the time necessary for a specific temperature to be reached and , therefore, the speed at which a steam able bag can cook a frozen product. Since the temperature reached in all the s team able bags at the cooking times determined was the same, these cooking times were used for cooking the broccoli for the quality and nutritional evaluations. 4. 3 . 2 Ascorbic A cid Broccoli is known to be a significant source of vitamin C (Vallejo , Tomas - Barberan & Garcia - Viguera, 2002). L - ascorbic acid is the predominant form of vitamin C found in broccoli and other foods of plant origin (Erdman et al., 1982). It is a common practice to use ascorbic acid as an indicator of the effect of food proce ssing (heating) since vitamin C is considered one of the most labile vitamins. Thus, the effect of the different steam able bags on the loss of ascorbic acid in frozen broccoli during microwaving was determined and the results are presented in Figure 3 . Sig nificant differences in ascorbic acid content were fou nd between the broccoli cooked by different steam able bags. Broccoli from SB - II (square - shaped bag) had significantly higher ascorbic acid content in both fresh weight (F.W.) basis and dry weight (D.W. ) basis than broccoli from other steam able bags (rectangular - shaped bag). This was due to the lower heating environment reached inside the SB - II since this steam able bag was the one that cooked the broccoli the least as supported by the firmness results ( T able 3 ). Since the broccoli in SB - II was cooked longer and yet had the highest ascorbic acid content, cooking the broccoli for additional 15 sec onds did not affect its ascorbic acid content. Steam able bags are designed to control the amount of heat generat ed by releasing steam once a specific internal temperature in 39 the product is achieved. Comparing the remaining two steam able bags, the broccoli from SB - I had significantly higher ascorbic acid content than the broccoli from SB - III ( 73.2 0.5 mg /100g F.W. and 669.3 4.6 mg/100g D.W. vs. 70.7 0.9 mg/100g F.W. and 658.6 8.3 mg/100g D.W. for SB - I and SB - III, respectively ) . The higher loss of ascorbic acid in SB - III may be correlated to its smaller surface area, which cooked the broccoli the mos t as supported by the firmness results ( Table 3 ). The specific internal temperature necessary to activate the valve was achieved faster in SB - III than in SB - I due to its smaller headspace which p romoted faster hot air cycling which resulted in a higher amo unt of steam released from SB - III . Vitamin C is a water - soluble vitamin and therefore, it was lost with the loss of steam through the valve (leaching). This loss of vitamin C due to leaching is supported by the differences in vitamin C content bet ween fresh weight basis values and dry weight basis values. While the ascorbic acid content in fresh weight basis differed by 3.51% between the broccoli from SB - III and SB - I and by 2.34% between the broccoli from SB - I and SB - II , the ascorbic acid content i n dry weight basis differed by 1.62% between the broccoli from SB - III and SB - I and by 2.99% between the broccoli from SB - I and SB - II . This difference between the ascorbic acid content in fresh weight basis and in dry weight basis results from the loss of w ater through the valve since the water loss is included in the dry weight basis calculations but not in the fresh weight basis calculations. Therefore, the low ascorbic acid content in the frozen broccoli from SB - III resulted from a higher amount of water loss from this steam able bag due to its capability to reach a specific internal temperature and thus able to activate the valve to release the steam earlier than the other steam able bags. Loss of vitamin C associated to its water loss has previously been r eported for frozen broccoli. Pellegrini et al. (2010) reported that retention of ascorbic acid in frozen broccoli after cooking via microwaving without additional water, basket 40 steaming and boiling were 80%, 59% and 41%, respectively. However, these same authors found that fresh broccoli cooked by microwaving without additional water has almost complete loss of ascorbic acid while fresh broccoli cooked via basket steaming and boiling maintains 77% and 82% of its ascorbic acid. This different retention of a scorbic acid between frozen broccoli and fresh broccoli cooked using a microwave was attributed to the presence of ice on the surface of the frozen broccoli which prevented a large amount of water loss during microwaving (Erdman et al., 1982). Be sides the loss of water - soluble nutritional compounds, a significant evaporation of water from broccoli can result in flavor changes (Vallejo et al., 2003). Therefore, the retention of water in broccoli during cooking plays an important role in the preserv ation of its nutritional compounds and physical quality. Since microwav e steam able bags offer a cooking technology which protects the frozen food from large losses of water during cooking, these bags most likely minimize changes in flavor in addition to m aintain ing nutritional content. However, this minimal flavor change will depend on the design of the steam able bag system containing a valve. 41 Figure 7 . Ascorbic A cid C ontent of F rozen B roccoli cooked in D ifferent S teamable B ag D esigns in (a) D ry W eig ht B asis vs. (b) F resh W eight B asis ( n umbers between columns indicate ascorbic acid percentage differences and different letters indicate significant differences (p 0.05)) . 42 4 . 3 .3 Antioxidant Capacity Antioxidant capacity is used to evaluate the integrated and synergic effects of the several different antioxidants found in a food product (Danesi & Bordoni, 2008). In the case of broccoli, hydrophilic antio xidants are the main contributors to its total antioxidant capacity. Kurilich, Jeffery, Juvik, Wallig and Klein (2002) determined that hydrophilic extracts are responsible for 80% to 95% of the total antioxidant capacity of fresh broccoli using the ORAC as say. Similarly, Wu, Beecher, Holden, Haytowitz, Gebhardt and Prior (2004) stated that hydrophilic antioxidants in Brassica vegetables provide more than 89% of the total antioxidant capacity and Roy, Juneja, Isobe and Tsushida (2009) found that 92% of the t otal antioxidant capacity of broccoli is provided by its hydrophilic extract. Taking this into consideration, only the hydrophilic compounds of the broccoli were extracted and evaluated in this study. Figure 4 summarizes the ORAC values of the fr ozen broccoli cooked in the three microwav e steam able bags. These ORAC values are similar to the ORAC values reported for uncooked broccoli. Kurilich et al. (2002) determined that the antioxidant capacity of 8 genotypes of uncooked fresh broccoli ranged b etween 38.1 and 121.6 µM TE/g D . W . using the ORAC assay. Similarly, Ou, Huang, Hampsch - Woodill, Flanagan and Deemer (2002) reported ORAC values for uncooked fresh broccoli between 23 and 208 µM TE/g D . W. As observed in Figure 2 , the different designs of th e microwav e steam able bag did not have an effect on the antioxidant capacity of the frozen broccoli during microwaving. This means that the differences in cooking time between bags, temperature achieved inside the bags and/or steam released from the bags w ere not enough to produce a change in the antioxidant capacity of the cooked frozen broccoli. 43 Figure 8 . Antioxidant C apacity (ORAC) of F rozen B roccoli cooked in D ifferent S teamable B ag D esigns in (a) D ry W eight B asis vs. (b) F resh W eight B asis ( differ . 44 4. 3 . 4 Firmness The heating of vegetables is intended to tenderize the m for consumption ( Tijskens, Schijvens & Biekman, 2001 ) . Thus, the firmness of the cooked frozen broccoli was eval uated to determine the effect of the steam able bag design on its tenderization. Significant differences 0.05) between the shear force values of the frozen broccoli cooked in SB - I, SB - II and SB - III were found as shown in Table 3 . Frozen broccoli cooked in SB - I and SB - II was 27% and 1 3 % firmer , respectively, than that cooked in SB - III . The difference in firmness between the broccoli in SB - I and SB - III was due to the different surface area of the packages, 526.44 cm 2 and 383.18 cm 2 , respectively. The smal ler the surface area of the steam able bag , the softer the broccoli is after cooking. This result can be explained by the role of the surface area of the steam able bag in the loss of water from the broccoli. Under the same microwaving conditions (microwavin g power and cooking time), the amount of steam necessary to activate the valve was generated faster in SB - III than in SB - I due to its smaller headspace. Consequently, more steam was released from SB - III than from SB - I . This resulted in a higher loss of wat er for the frozen broccoli cooked in SB - III . Changes in the firmness of plants have been correlated to loss of water. The water content of plants has a direct effect on the turgor of their cells and the degree of cellular hydration is known to result in no ticeable changes in plant firmness (Sams, 1999 ; Jacobsson, Nielsen & Sjoholm, 2004). Therefore, a steam able bag like SB - III that allows a faster release of steam would tenderize the frozen products faster under the same microwaving conditions. The difference in firmness between broccoli in SB - II and SB - III resulted from a combined effect of surface area and shape of the steam able bag. SB - II and SB - III had very similar surface areas, 409.48 cm 2 and 383.18 cm 2 , respectively. This difference in sur face area of 26 cm 2 seems 45 not enough to cause a 1 3 % difference in broccoli firmness if compared with the 27% difference in broccoli firmness caused by a difference in area of about 143 cm 2 ( SB - I vs. SB - III ). Therefore, the different shapes of the steam able bags probably had an effect on tenderizing the frozen broccoli. The square - shaped steam able bag seems to provide a lower heating environment for the frozen broccoli during microwaving that leads to less tender broccoli. This agrees with the cooking time r esults which showed that more time is necessary for cooking the frozen broccoli in a square - shaped steam able bag than in a rectangular - shaped steam able bag. Therefore, there appears to be a relation between broccoli tenderization and steam able bag surfac e area and shape. Both of these parameters significantly affected the firmness of the cooked broccoli and would most likely affect consumer acceptance. This shows the importance of controlling the surface area and shape of the steam able bag. 4. 3 . 5 Color The color of frozen broccoli florets coo ked in the three studied microwav e steam able bags is presented in Tabl e 3 . No differences w as found betwee n the L* values of the florets cooked in the three different steam able bags. Zhong, Dolan and Almenar (2014) reported that the frozen broccoli cooked in steamable bag maintains its intense darkness (L* value) compared to thawed frozen broccoli. T he design of the steam able bags did affect other color parameters of the frozen broccoli after steam able bag mi crowaving . The florets cooked in SB - II were significantly 0.05) less green (less - a* value) than th ose cooked in SB - I and SB - III . No differences between t he green color of the florets cooked in SB - I and SB - III were observed. The reason for the frozen broccoli cooked in SB - II to be less green than those cooked in SB - I and SB - III is most likely due to the relative lower heating environment in the SB - II that resulted in 15 sec onds additional time 46 of cooking. Boekel (1999) and Turkmen, Poyrazoglu, Sari and Velioglu (2006) reported that the green color of vegetables is mainly related to chlorophyll pigment content and the loss of greenness is generally associated with the degradation of chlorophyll pigments and formation of pheophytins during heat processing . Therefore, the cooking of the frozen broccoli for 15 sec onds more in SB - II induced more chlorophyll degradation and pheophytin formation which led to an increased a* value . Broccoli florets cooked in SB - I became yellower (increased +b* value) than those cooked in SB - II and SB - III. This difference could be attributed to difference s in matrix structure (softness) between the broccoli florets , as supported by the firmness results, which chang ed the light penetration and surface - reflecting properties of the b roccoli florets ( Miglio, Chiavaro, Visconti, Fogliano & Pellegrini, 2008 ; Oey, Lille, Loey & Hendrickx, 2008 ). The hue angle of the florets cooked in SB - II shifted more towards yellow than that of the broccoli cooked in SB - I and SB - III . This low er hue angle could be attributed to the combin ed effect s of a* and b* values since both of them decreased and this did not happen in broccoli florets cooked in the other steamable bag design s . Therefore, the color results show that the design of the steama ble bag has a n important effect on color maintenance and consumers a cceptance of the cooked vegetables . Table 3 . Color and Firmness of Frozen Broccoli Cooked in Different Steamable Bags Designs Color 1 Firmness 2 (Kg - force/g) L* a* b* H ue° SB - 19.87 ± 1.20a 11.44 ± 0.94b 19.30 ± 1.91a 120.70 ± 1.70b 3.40 ± 0.39a SB - 19.78 ± 1.76a 10.22 ± 0.92a 18.42 ± 2.08ab 119.09 ± 1.95c 3.02 ± 0.34b SB - 20.06 ± 1.25a 11.31 ± 1.14b 17.97 ± 2.28b 122.28 ± 2.22a 2.68 ± 0.49c Means in rows follo 1 Values presented as mean ± SD (n = 15) 2 Values presented as mean ± SD (n = 20) 47 5 . Conclusion s This is the first time that the effect of steamable bag microwaving and the shape and surface area of a steamable bag on changes in nutritional content and physical properties of a frozen food product during cooking has been investigated. The nutritional content of frozen broccoli can be increased using steamable bag microwaving since this cooking method decreases ascorbic acid only slightly while it notably increases antioxidant capacity of cooked frozen broccoli compared to thawed frozen broccoli. Steamable bag microwaving also affects the physical properties of frozen broccoli since the broccoli is tenderized and changes in color but only in terms of greenness and not lightness or yellowness compared to thawed frozen broccoli. The nutritional content of frozen broccoli cooked by steamable bag microwaving is the same as that of frozen broccoli cooked by steamer steaming and higher than that of frozen broccoli cooked by traditional microwaving. Less color change and faster tenderization of frozen broccoli are obtained using steamable bag microwaving compared to steamer steaming or tradit ional microwaving. These findings show that steamable bag microwaving is a cooking method that increases nutritional content, tenderizes fast and produces minimal color changes in frozen Furthe rmore, b oth the shape and the surface area of a steamable bag (with a steam release valve) can significantly affect the nutritional content and physical properties of frozen broccoli. Frozen broccoli cooked in a more square - shaped steamable bag was signifi cantly less green and more yellow in color , less soft , and retained higher vitamin C content than frozen broccoli cooked in a more rectangular - shaped steamable bag. The lower heating environment created by the square shape of the steamable bag results in l ess loss of vitamin C and hardness due to less water loss from the steamable bag but a greater change in color due to the longer cooking time. 48 The smaller the surface area of the steamable bag the softer the broccoli became after cooking, independent of th e shape of the steamable bag. However, this trend was not observed for either color or vitamin C content. For steamable bags with a more rectangular shape, a smaller surface area results in less time needed to achieve an internal temperature at which the s team release valve opens and releases water. This results in softer broccoli and lower ascorbic acid retention, but does not affect the color of the broccoli. Neither the shape nor the surface area of the steamable bag had an effect on the lightness and an tioxidant content of the broccoli. It is concluded that the design of a steamable bag (with a steam release valve) can optimize the preservation of nutrients and the physical properties of cooked frozen food, such as frozen broccoli. 49 6 . Future W ork Only one type of steam release devices ( steam release valve ) and a constant location of the valve (in the center of the steamable bag), were studied. However , t he steam release valve location may affect its activated time which leads to new changes of nutrition value and physical quality of frozen broccoli after cooking. Moreover, different steam release devices, such as contaminated seal, mechanical score and perforations, have different influence s on the packaging hermeticity and generate d ifferent steaming environment inside the steamable bag. Therefore, future study can be conducted to better evaluate the performance of steamable bag microwaving and steamable bag designs. 50 BIBLIOGRAPHY 51 BIBLIOGRAPHY AO AC (2000). No. 967.21 Ascorbic acid in vitamin preparation and juices. Association of Gaithersburg, MD, USA. Ames, B. N., Gold, L. S., & Willet, W. C. ( 1995 ) . The causes and prevention of cancer. Proceedings of the National Academy of Sciences , 92, 5258 5265. Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Sciences , 90, 7915 7922. Arnao, M. B., Cano, A., & Acosta, M. (2001). The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chemistry , 73, 239 244. Andlauer, W., & Fürst, P. (2003). Non - nutritive bioactive food components of plants: Importance for nutri tion and health. International Journal for Vitamin and Nutrition Research, 73, Bengtsson , G . B . , Schöner , R . , Lombardo , E . , Schöner , J . , Borge , G . I . A . , & Bilger , W. ( 2006 ) . Chlorophyll fluorescence for non - destructive measurement of flavonoids in broccoli. Postharvest Biol ogy and Technol ogy , 39(3) , 291 - 29 8. Bernhardt, S., & Schlich, E. (2006). Impact of different cooking methods on food quality: Retention of lipophilic vitamins in fresh and frozen vegetables. Journal of Food Engineering, 77 , 327 3 33. Boekel, M. A. J. S. (1999). Testing of kinetic models: usefulness of the multiresponse approach as applied to chlorophyll degradation in foods. Food Research International, 32, 261 - 269. Brown, M. S. (1977). Texture of frozen fruits and vegetables. Journal of Texture Studies, 7, 391 - 404. Butz, P., Edenharder, R., Fernandez - Garcia, A., Fister, H., Merkel, C., & Tauscher, B. (2002). Changes in functional properties of vegetables induced by high pressure treatment. Food Research International, 35, 295 - 300. Cano, A., Acosta, M., & Arnao, M. B. ( 2000 ) . A method to measure antioxidant activity i n organic media: application to lipophilic vitamins. Redox Report , 5, 365 370. Cao, G., Alessio, H. M., & Cutler, R. G. ( 1993 ) . Oxygen - radical absorbance capacity assay for antioxidants. Free Radical Biology and Medicine , 14, 303 311. Cao , G., & Prior , R. L . (1998). Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry , 44 , 1309 - 1315 . 52 Cao, G., Sofic , E., & Prior, R. L. (1996). Antioxidant capacity of tea and common vegetables. Journal of Agricultural and Food Chemistry, 44(11), 3426 3431. Cao, G., Verdon, C. P., Wu, A. H. B., Wang, H., & Prior, R. L. ( 1995 ) . Automated ass ay of oxygen radical absorba nce capacity with the COBAS FARA II. Clinical Chemistry , 41, 1738 1744. Danesi, F., & Bordoni, A. (2008). Effect of home freezing and Italian style of cooking on antioxidant activity of edible vegetables. Journal of Food Science, 73, 109 112. Davey, M. W ., Montagu, M. V., Inze, D., Sanartin, M., Kanellis, A., Smirnoff, N., Benzie, I. J., Strain, J. J., Favell, D., & Fletcher, J. (2000). Plant L - ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. Journal of the Scienc e of Food and Agriculture, 80(7), 825 - 860. Diaz, M. N., Frei, B., & Keaney , Jr. J. F. ( 1997 ) . Antioxidants and a therosclerotic heart disease. New England Journal of Medicine , 337, 408 416 . Eberhardt , M . V . , Kobira , K . , Keck , A . , Juvik , J . A . , & Jeffery , E . H. ( 2005 ) . Correlation analyses of phytochemical composition, chemical, and cellular measures of antioxidant activity of broccoli ( Brassica oleracea L. var. italica ). J ournal of Agric ulture and Food Chem istry, 53(19) , 7421 - 74 31. Erdman, J. W., & Klein, B. P. (1982). Harvesting, processing and cooking influences on vitamin C in foods. In P. A. Seib, & B. M. Tolbert (Eds.), Ascorbic Acid: Chemistry, Metabolism, and Uses (pp. 499 - 532). Washington: American Chemical Society . Galgano, F., Favati, F., Carus o, M., Pietrafesa, A., & Natella, S. (2007). The influence of processing and preservation on the retention of health - promoting compounds in broccoli. Journal of Food Science , 72 (2), 130 135. Gliszczynska - Swiglo, A., Ciska, E., Pawlak - Lemanska K., Chmiele wski J., Borkowski T., & Tyrakowska B. (2006). Changes in the content of health - promoting compounds and antioxidant activity of broccoli after domestic processing. Food Additives and Contaminants, 23 (11), 1088 1098. Huang, D., Ou, B., Hampsch - Woodill, M. , Flanagan, J. A., & Prior, R. L. (2002). High - throughput assay of Oxygen Radical Absorbance Capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96 - well format. Journal of Agricultural and Food Chemi stry, 50 (16), 4437 4444. Hudson, D. E., Dalal, A., & Lachance, P. A. (1985). Retention of vitamins in fresh and frozen broccoli prepared by different cooking methods. Journal of Food Quality, 8, 45 50. 53 Jacobsson, A., Nielsen, T., & Sjoholm, I. (2004). Ef fects of type of packaging material on shelf - life of fresh broccoli by means of changes in weight, color and texture. European Food Research and Technology, 218, 157 - 163. Kalt , W. ( 2005 ) . Effects of production and processing factors on major fruit and veg etable antioxidants. J ournal of Food Sci ence, 70(1) , R11 - 1 9. Kaur, C., & Kapoor, H. C. (2001). Antioxidants in fruits and vegetables The millennium's health. International Journal of Food Science & Technology, 36, Kohlmeier, L., & Su, L. (1997 ). Cruciferous vegetable consumption and colorectal cancer risk: meta - analysis of the epidemiological evidence. The FASEB Journal, 11, 369. Kris - Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E., Hilpert, K. F., Griel, A. E., & E therton, T. D. (2002). Bioactive compounds in foods: Their role in the prevention of cardiovascular disease and cancer. Excerpta Medica, 30, 71S 88S. Kurilich, A. C., Jeffery, E. H., Juvik, J. A., Wallig, M. A., & Klein, B. P. (2002). Antioxidant capacity of different broccoli (Brassica oleracea) genotypes using the oxygen radical absorbance capacity (ORAC) assay. Journal of Agricultural and Food Chemistry, 50, 5053 5057. Lee, S. K., & Kader, A. A. (2000). Preharvest and postharvest factors influencing vi tamin C content of horticultural crops. Postharvest Biology and Technology, 20(3), 207 - 220. Lopez - Berenguer, C., Carvajal, M., Moreno, D. A., & Garcia - Viguera, C. (2007). Effects of microwave cooking conditions on bioactive compounds present in broccoli i nflorescences. Journal of Agriculture and Food Chemistry, 55(24), 10001 - 10007. Mabesa, L. B., & Baldwin, R. E. (1979). Ascorbic acid in peas cooked by microwaves. Journal of Food Science, 44, 932 933. gliano, V., & Pellegrini, N. (2011). Effect of two cooking procedures on phytochemical compounds, total antioxidant capacity and color of selected frozen vegetables. Food Chemistry, 128, 627 633. Miglio, C., Chiavaro, E., Visconti, A., Fogliano, V., & Pel legrini, N. (2008). Effects of different cooking methods on nutritional and physicochemical characteristics of selected vegetables. Journal of Agricultural and Food Chemistry, 56, 139 147. Ninfali, P., & Bacchiocca, M. (2003). Polyphenols and antioxidant capacity of vegetables under fresh and frozen conditions. Journal of Agricultural and Food Chemistry, 51, 2222 - 2226. Nielsen, G. S., Larsen, L. M., & Poll, L. (2004). Impact of blanching and packaging atmosphere on the formation of aroma compounds during long - term frozen storage of leek (Allium ampeloprasum Var. Bulga) slices. Journal of Agricultural and Food Chemistry, 52(15), 4844 4852. 54 Oey, I., Lille, M., Loey, A. V., & Hendrickx, M. (2008). Effect of high pressure processing on color, texture and flavo r of fruit - and vegetable - based food products: a review. Trends in Food Science & Technology, 19, 320 328. Ou, B., Huang, D., Hampsch - Woodill, M., Flanagan, J. A., & Deemer, E. K. (2002). Analysis of antioxidant activities of common vegetables employing o xygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: A comparative study. Journal of Agricultural and Food Chemistry, 50, 3122 - 3128. - Wollaston, V. (2001). Molecular and biochemica l characterization of postharvest senescence in broccoli. Plant Physiology, 125, 718 - 727. Pellegrini, N., Chiavaro, E., Gardana, C., Mazzeo, T., Contino, D., Gallo, M., Riso, P., Fogliano, V., & Porrini, M. (2010). Effect of different cooking methods on c olor, phytochemical concentration, and antioxidant capacity of raw and frozen brassica vegetables. Journal of Agricultural and Food Chemistry, 58, 4310 4321. Perez - Jimenez, J., Arranz, S., Tabernero, M., Diaz - Rubio, M. E., Serrano, J., Goni, I., & Saura - C alixto, F. (2008). Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results. Food Research International, 41, 274 285. Plumb, G. W., Lambert, N., Chambers, S. J., Wanigatun ga, S., Heaney, R. K., & Plumb, J. A. (1996). Are whole extracts and purified glucosinolates from cruciferous vegetables antioxidants . Free Radical Research, 25(1), 75 86. Price, K. R., Casuscelli, F., Colquhoun, I. J., & Rhodes, M. J. C. (1998). Composit ion and cooking. Journal of the Science of Food and Agriculture, 77, 468 472. Prior, R. L. & Cao , G . (1999). In vivo total antioxidant capacity: comparison of diffe rent analytical methods. Free Radical Biology & Medicine , 27, 1173 - 1187 . Rechkemmer, G. (2007). Nutritional aspects from thermal processing of food: Potential health benefits and risks. In G. Eisenbrand, K. H. Engel, W. Grunow, A. Hartwig, D. Knorr, & I. B. Knudsen, et al. (Eds.), Symposium. KGaA, Weinheim: Wiley - VCH Verlag GmbH & Co. Roy, M. K., Juneja, L. R., Isobe, S., & Tsushida, T. (2009). Steam processed broccoli ( Brassica oleracea ) has higher antioxidant activity in chemical and cellular assay syst ems. Food Chemistry, 441, 263 269. Sams, C. E. (1999). Preharvest factors affecting postharvest texture. Postharvest Biology and Technology, 15, 249 - 254. Tijskens, L. M. M., Schijvens, E. P. H. M., & Biekman, E. S. A. (2001). Modeling the change in colou r of broccoli and green beans during blanching. Innovative Food Science and Emerging 55 Technologies, 2, 303 313. Turkmen, N., Sari, F., & Velioglu, Y. S. (2005). The effect of cooking methods on total phenolics and antioxidant activity of selected green veg etables. Food Chemistry, 93, 713 718. Turkmen, N., Poyrazoglu, E. S., Sari, F., & Velioglu, Y. S. (2006). Effects of cooking methods on chlorophylls, pheophytins and color of selected green vegetables. International Journal of Food Science and Technology, 41, 281 288. Vallejo F, Tomas - Barberan F. A., & Garcia - Viguera, C. (2002). Glusinolates and vitamin C content in edible parts of broccoli florets after domestic cooking. European Food Research and Technology, 215, 310 316. Vallejo, F., Tomas - Barberan, F ., & Garcia - Viguera, C. (2003). Phenolic compound contents in edible parts of broccoli inflorescences after domestic cooking. Journal of the Science of Food and Agriculture, 83, 1511 - 1516. Van Loey, A., Ooms, V., Weemaes, C., Van de Broeck, I., Ludikhuyze , L., Denys, S., & Hendrickx, M. (1998). Thermal and pressure temperature degradation of chlorophyll in broccoli (Brassica oleracea L. italic ) juice: a kinetic study. Journal of Agriculture and Food Chemistry, 46 (12), 5289 - 5294. Wachtel - Galor , S., Wong , K. W., & Benzie , I. F. F. (2008). The effect of cooking on Brassica vegetables. Food Chemistry , 110, 706 710. Wattenberg, L. W. (1993). Inhibition of carcinogenesis by nonnutrient constituents of the diet. In K. W. Waldron, & I. T. Johnson (Eds.). Food and Cancer Prevention: Chemical and Biological Aspects, London: Royal Society of Chemistry, 12 24. WCRF , World Cancer Research Fund, & American Institute for Cancer Research (2007). Food, nutrition, physical activity and the prevention o f cancer: A globa l perspective. Washington, DC, USA: American Institute for Cancer Research. Wildman, R . E . C. ( 2001 ) . Handbook of Nutraceuticals and Functional Foods. Boca Rotan: CRC Press. 170 - 172. Wu, X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E. , & Prior, R. L. (2004). Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. Journal of Agriculture and Food Chemistry, 52, 4026 4037. Wu , X . L . , Liwei , G . , Holden , J . M ., Haytowitz , D . B . , Gebhardt , S . E . , & Prior , R . L. ( 2004 ) . Development of a database for total antioxidant capacity in foods: a preliminary study. J ournal of Food Compost Anal , 17 , 407 - 422. Young, I. S., & Woodside, J. V. ( 2001 ) . Antioxidant in health and disease. Journal of Clinical Pathology , 54, 176 186. 56 Zhang, D., & Hamauzu, Y. (2004). Phenolics, ascorbic acid, carotenoids and antioxidant activity of broccoli and their changes during conventional and microwave cooking. Food Chemistry, 88, 503 509. Zhong, X. Y., Dolan, K., & Almenar, E. (2014). Eff ect of steamable bag microwaving versus steamer steaming and traditional microwaving on nutritional preservation and physical properties of frozen broccoli. Food Chemistry.