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H-SS‘S. 93 mm: W 1121: 01mm m Jill Win milllln'rml 3 93 01572 2022 LIBRARY Michigan State University This is to certify that the' thesis entitled MASS TRANSFER OF a - TOCOPHEROL (VITAMIN E) FROM A.MULTI-LAYER LAMINATE STRUCTURE AND ITS INFLUENCE ON PRODUCT STABILITY Date presented by JU-FEN LIN has been accepted towards fulfillment of the requirements for MASTER PACKAGING degree in Major prof «- . June 12, 1996 0—7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE II RETURN BOX to remove thle checkout from your record. TO AVOID FINES return on or before dete due. ' DATE DUE DATE DUE DATE DUE MAGIC 2 ‘ APR 96 1999 mg 91999 *——'i;..94= I usu leAnMflrmetive Action/Emu Opportunity lnetltmon Wanna-9.1 _.——____h MASS TRANSFER OF a-TOCOPHEROL (VITAMIN E) FROM A MULTI- LAYER LAMINATE STRUCTURE AND ITS INFLUENCE ON PRODUCT STABILITY BY Ju-Fen Lin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE School of Packaging 1996 ABSTRACT MASS TRANSFER OF a-TOCOPHEROL (VITAMIN E) FROM A MULTI- LAYER LAMINATE STRUCTURE AND ITS INFLUENCE ON PRODUCT STABILITY BY Ju-Fen Lin The antioxidant, a-tocopherol (ATP) was incorporated into a coextruded laminate film consisting of a heat seal layer, a core layer of high density polyethylene (HDPE) impregnated with ATP, and an outer HDPE layer. The rate of loss of the antioxidant from the laminate film was determined as a function of time and temperature and was found to follow a first order rate expression, with an activation energy of 28.87 kcal/mole. The effectiveness of ATP and BHT impregnated laminate films to retard the oxidation of a packaged oat cereal product was also evaluated. The extent of oxidation was based on the level of hexanal in the product. Product packaged in pouches containing no antioxidant exhibited a significantly high level of lipid oxidation, as compared to the product packaged in the antioxidant impregnated laminate structures. Both antioxidants appeared to provide a similar level of effectiveness, in terms of retarding product oxidation. DEDICATION This thesis is dedicated to my parents and my sister, for their support, encouragement, patience, and love throughout this work. iii ACKNOWLEDGMENTS Dr. Jack Giacin, for his guidance, enthusiasm, and professional advice throughout my research and as my major advisor. Dr. Bruce Harete and Dr. Perry Ng, for serving on my committee members. Dr. John Culter, for sharing his expertise, wisdom, and serving on my committee members. General Mills Inc., for their help and cooperation. All my friends in School of Packaging, especially for Yueh- Yi Chang, for their help and friendship throughout this research study. iv TABLE OF CONTENTS LIST OF TABLES ......................................... vii LIST OF FIGURES ......................................... ix INTRODUCTION ............................................ 1 LITERATURE REVIEW ....................................... 4 General Review of Oxidation ........................ 4 Mechanism of Oxidation ............................. 5 Methods of Measurement Oxidation ................... 6 Peroxide Value (PV) ............................ 7 Thiobarbituric Acid Reactive Substances (TEARS) 8 Hexanal ....................................... 9 Composition of Oat Cereal .......................... 12 General Review of Antioxidants. .................... 14 Mechanism of Antioxidant ........................... 18 Characteristics of Hindered Phenolic Synthetic Antioxidants ..................................... 19 2-(1,1-Dimethylethyl)-1,4-Benzenediol (TBHQ) ....... 19 2-Tertiary-Butyl-4-Methoxy Phenol (BHA) ............ 20 3,5-Di-Tertiary-Butyl-4-Hydroxytoluene (BHT) .. 21 n-Propyl-3,4,S-Trihydroxybenzoate (PG) ........ 23 Characteristics of Natural Antioxidants ............ 25 Ascorbic Acid ................................. 25 Rosmarinus Officinalis ........................ ‘ 26 a-Tocohperol .................................. 28 Application of Antioxidant in Packaging Materials .. 31 Theory of Migration ................................ 41 Technology for Trapping Volatile ................... 46 Determination of Antioxidants ...................... 50 MATERIAL AND METHODS .................................... 52 Cereal Product ..................................... 52 Packaging Material ................................. 52 Film Storage Studies ............................... 55 Antioxidant Loss Rate Studies ...................... 55 High Pressure Liquid Chromatography (HPLC) Analysis Extraction Procedure .......................... 56 Percent Recovery of Antioxidant for Extraction Procedure ................................ 56 HPLC Analysis ............................ 57 Product Stability Studies .......................... 59 Hexanal Quantification ............................. 63 Apparatus for Trapping of Volatiles ........... 63 Hexanal Analysis Procedure .................... 68 Hexanal Calibration Curve Procedure ........... 70 Antioxidant Content of Cereal ...................... 71 Lipid Extraction .............................. 71 Antioxidant Content of Extracted Fat .......... 72 RESULT AND DISCUSSION ................................... 74 Thickness of Packaging Materials ................... 74 Loss of d-Tocohperol from HDPE Film ................ 75 % Recovery for Antioxidant ......................... 79 Relative Rate Lossed of Antioxidants ............... 87 Product Storage Studies ............................ 93 Retained Levels of Antioxidant in Packaging Sturctures ......................................... 98 Antioxidant Content of Cereal Product ............. 102 SUMMARY AND CONCLUSION ................................. 108 FUTURE STUDIES ......................................... 110 APPENDICES ............................................. 112 LIST OF REFERENCES ..................................... 123 vi 10. 11. 12. 13. 14. 15. LIST OF TABLES Antioxidants permitted in foods in the United States ............................................. 16 Average thickness for lamination film .............. 75 a-Tocopherol concentration (wt/wt%) from fresh roll at different time interval .................... 76 Loss of a-tocopherol from coextruded laminate film at 23°C determined by HPLC analysis ................... 80 Loss of a-tocopherol from coextruded laminate film at 30°C determined by HPLC analysis ................... 81 Loss of a-tocopherol from coextruded laminate film at 40°C determined by HPLC analysis ................... 82 Loss of a-tocopherol from coextruded laminate film at 50°C determined by HPLC analysis ................... 83 Rate constants for the loss of a-tocopherol from the coextruded laminate film sturcture ................. 84 The hexanal concentration of cereal product packaged in pouches fabricated from test coextruded laminate sturctures ......................................... 96 a-Tocopherol content in packaging pouches as a function of storage time .......................... 101 BHT content in packaged cereal product following storage at 23°C ................................... 107 a-Tocopherol calibration curve ................... 112 Hexanal data for the first calibration curve ...... 114 Hexanal data for the second calibration curve ..... 116 Thickness (um) measurements performed by optical microscopy ........................................ 118 vii 16. Thickness (pm) measurements performed by micrometer ........................................ 119 17. Percent recovery data and calculations ............ 120 viii 10. 11. 12. 13. 14. 15. 16. 17. LIST OF FIGURES The structure of TBHQ .............................. 20 The structure of BHA ............................... 21 The structure of BHT ............................... 22 The structure of PG ................................ 24 The structure of ascorbic acid ..................... 26 The structure of the rosemary antioxidant, rosmarinic acid? ................................... 27 Tocopherol structure ............................... 29 Cross section of three multi-layer structure ....... 53 The storage arrangement for cereal package ......... 61 Flow diagram of the test scheme .................... 62 Schematic diagram of dynamic purge and trap system 64 The apparatus with the sparging tube attached ...... '66 Photograph of dynamic purge and trap system ........ 67 Loss of ATP from the coextruded laminate film as a function of time/temperature ....................... 85 Arrhenius plot of loss rate constant versus temperature ........................................ 86 Relative percent loss of BHT and a-tocopherol from coextruded laminated film as a function of time (23°C) ............................................. 90 Relative percent loss of BHT and a-tocopherol from coextruded laminated film as a function of time (30°C) ............................................. 91 ix 18. 19. 20. 21. 22. 23. Relative percent loss of BHT and a-tocopherol from coextruded laminated film as a function of time (40°C) ............................................. 92 Hexanal concentration in cereal product packaged in pouches fabricated from test coextruded laminated structures ........................................ 97 Schematic of the mechanism of antioxidant activity of a polymeric packaging material ................. 106 a-Tocopherol calibration curve .................... 113 First hexanal calibration curve ................... 115 Second hexanal calibration curve .................. 117 INTRODUCTION Antioxidants have been used as an additive in food production for a number of years to prevent lipid oxidation. Antioxidants function by delaying the onset of oxidation by acting as free radical scavengers or metal chelating agents, and may be classified as either synthetic or natural. A recent innovation has emerged in the use of antioxidants in the manufacture of polymeric packaging materials, such as high density polyethylene (HDPE), to minimize the effect of oxidative degradation (Food Engineering, 1979). Antioxidants such as 3,5—di—tert-butyl-4-hydroxytoluene (BHT) have been incorporated within cereal packaging for a period of time (Charnas, 1992). As currently used, BHT not only stabilizes cereal liner films, but also functions as an antioxidant for time food product itself, inhibiting lipid oxidation and extending its shelf life. Due to the high level of unsaturated fatty acids in cereal grains, such products are subject to attack by oxygen, resulting in lipid oxidation. Impregnating packaging materials with an antioxidant will act to inhibit product oxidation. For example, Hoojjat et al.(1987) reported that 2 BHT impregnated HDPE film retarded the rate of oxidation of an oatmeal cereal. The proposed mechanism of antioxidant activity involves the following three-step process: (1) antioxidant diffusion through the polymer bulk phase; (2) evaporation of antioxidant from the surface of the packaging materials; and (3)subsequent antioxidant sorption onto the surface of the packaged product. BHT is widely used in food packaging. However, because of its low molecular weight, volatility and suspicion of being a cancer causing agent (Charnas, 1992), there has been considerable interest expressed in developing BHT alternatives (Monks, 1992). Recently, a-tocopherol was evaluated for the rather unconventional use as a polymer stabilizer for packaging film, when used at low concentrations, such as 100 ppm(wt/wt) (Urata, 1988; Laermer, 1990). Researchers (Laermer et al., 1993; Zambetti, 1995) have described its utility in the processing of polyolefins such as polyethylene and polypropylene, where the a—tocopherol functions to inhibit polymer oxidation during thermal processingu ‘Their studies showed polypropylene containing a-tocopherol had higher heat and color stability, when 3 compared to a polypropylene sample which had a commercial antioxidant added. a-Tocopherol also showed lower volatility and lower migration characteristics than other commercial antioxidants on the market. The present study focuses on determining the effect of temperature on the mass transfer properties of BHT and 0t- tocopherol from a multi—layer laminate film. The laminates consisted of an inner heat seal layer, a core layer of high density polyethylene impregnated with the antioxidant, and an outer HDPE layer. The specific objectives of this study included (1) To determine the rate of loss of a-tocopherol from the test laminate film as a function of time and temperature. (2) To evaluate the effectiveness of a-tocopherol and BHT impregnated laminates in inhibiting lipid oxidation of a cereal product via an evaporation-sorption mechanism. (3) Utilizing data obtained from the rate loss studies to develop a better understanding of the transfer mechanism of a—tocopherol from packaging film to product. LITERATURE REVIEW General review of oxidation Lipids become rancid as a result of oxidation, and rancidity is a major cause of food deterioration. Oxidative reactions occur as a result of such factors as the presence of oxygen or heavy metals, exposure to light and heat, the degree of product lipid unsaturation, product enzymes etc.. Oxidation of unsaturated fatty acids has been reported to be the primary factor in product rancidity (Buck, 1984). Oxidation is the result of the loss of electrons from a molecule or atom, and the corresponding reduction of the recipient component. Some fat- or oil- rich products, such as crackers and potato chips, are easily oxidized as a result of their large surface area. Product oxidation results in the development of rancid off-flavors, odors, discoloration of pigments, change in texture, loss of nutritional value, and even toxic oxidation products (Dziezak, 1986) . 5 Mechanism.of oxidation Oxidation of fats and oils is believed to occur as an autocatalytic reaction, which involves the following three steps: (1) initiation, when free radicals are produced. In: this step, the flavor and odor of the substance are slightly affected; (2) propagation, when free radicals react with oxygen to produce hydroperoxide radicals (ROOo) , which react further with other unsaturated hydrocarbons to regenerate the free radicals and yield hydroperoxides (ROOH); and (3) termination, which involves the combination of two radicals with the formation of stable products (Nawar, 1985; Dugan 1976; Paquette et al., 1985). The reaction sequence of the three-step oxidation process is as follows: activation Initiation : RH 4; Ro (free radicals) Propagation : lb + O2 4, ROO- ROO- + RH 47 ROOH + R- Termination : 1% + R- 4. RR R. + ROO- ___, ROOR ROO- + ROO- , ROOR + 02 6 RH refers to any unsaturated fatty acid, which has an active C-H group adjacent to a double bound. In the initiation stage, this hydrogen atom is easily removed from a carbon next to a double bound, when it has been activated. With light, lipoxygenases or a metal catalyst present, the unsaturated fatty acid, RH is easily activated. In the propagation step, oxygen quickly adds to the free radicals, forming an unstable peroxy free radical (ROOJ . 'Phis peroxy free radical abstracts a hydrogen atom producing a hydroperoxide (ROOH) . Hydroperoxides (ROOH) are the major initial reaction products from the reaction of a fatty acid with oxygen. At the termination stage, hydroperoxides readily decompose into various secondary byproducts such as aldehydes, ketones and hydrocarbons. These byproducts are the major source of rancid odor and flavors (Pokorny, 1971; Nawar, 1985). Method of Measurement Oxidation Sensory analysis provides a direct method to evaluate product oxidation. However, it is not a quantitative technique to determine the extent of product oxidation, and it lacks reproducibility. The following three methods are 7 the most popular being used to determine the extent of product oxidation. (1) Peroxide value (PV); (2) Thiobarbituric acid reactive substance (TEARS); and (3) Hexanal analysis. These three methods provide reproducibility, sensitivity and quantitativeness (Gray, 1978; Melton, 1983). Peroxide Value (PV) Hydroperoxides, commonly called peroxides, are the primary products of lipid oxidation. Therefore, measuring the peroxide concentration will determine the level of oxidation. NUmerous analytical. procedures for the measurement of the peroxide value are described (Dahle, 1962; Agbo, 1992; Van de Voort et al., 1994; Lrevi, et al., 1991). The American Oil Chemists’ Society (AOCS) official iodometric method Cd 8-53 is generally‘ used. Peroxide values can be neasured based on their ability to liberate iodine from jpotassium iodide, or to oxidize ferrous to ferric ions. However, the results of this method have been described as highly empirical and questionable. The results vary with details of the procedure used and the test is extremely' sensitive to ‘temperature changes (Gray, 1978). 8 The other errors associated with this method are: (1) iodide will be adsorbed at unsaturated bonds of the fatty material; and (2) the liberation of iodine from potassium iodide by oxygen present in the solution to be titrated (Mehlenbacher, 1960). Since the peroxides are likely to decompose to other secondary byproducts, it is xxx: a good choice for measurement of oxidation over long periods of time. Therefore, the peroxide value is applicable only at the early stages of oxidation. Thiobarbituric Acid Reactive Substances (TEARS) Kohn and Liversadge (1944) observed that a pink color was found. when animal tissue, which had. been incubated aerobically, reacted with 2-thiobarbituric acid (TBA). This pink color was formed from the oxidation product and TBA. The oxidation product has been reported to be the dicarbonyl compound, malonaldehyde, which is generated from the degradation of unsaturated fatty acids (Dahle et al., 1962). The TBA test originally had been used to measure the level of malonaldehyde in the product. When one mole of malonaldehyde reacts with two moles of TBA, this results in the formation of a pink chromagen. Absorbency of the pink 9 complex is monitored at 532 nm and the optical density recorded. This reaction has been used as a measure of the degree of lipid. oxidation” particularly' in ‘meat systems (Tarladgis et al., 1960). .An inherent problem associated with TEARS values is that other food components besides malonaldehyde, which are other oxidation products, can react with TBA to produce complexes that also absorb around 532 nm (Marcuse and Johansson, 1973). Therefore, TEARS values should be defined as resulting from compounds which react with. TBA" producing 21 pink. chromagan, rather than malonaldehyde exclusively. Also, the effect of other factors, such as acidity, heat and oxidizing agents on the TBA. reagent. will influence the accuracy' of TEARS (Gray, 1978; Melton, 1983). However, this method is still applicable to estimate the rancidity development of lipid oxidation after proper modification (Melton, 1983). Hexanal Hexanal is the major secondary product of the oxidation of linoleic acid. Since linoleic acid has been found in cereal grain products, soybean oil and other vegetable oils, the accumulation of hexanal has been used as an excellent 10 indicator of the degree of oxidation in food. products. Hexanal is derived from the 13—hydroperoxide of linoleic acid as follows (Dugan, 1976): of barley, Welch (1975) found differences between the species in composition and in their response to sowing date. He also found that the content and degree of unsaturation of the oat total fatty acid was affected by the sowing season. Although most cereals contain appreciable quantities of natural antioxidants, they may loss as high as 90% as a result of processing (Herting, 1969). Due to the high percentage of unsaturated fatty acids found in oat cereal, such oat grain products readily undergo autooxidation. General Review of Antioxidants The United States Food and Drug Administration (FDA) defines antioxidants as substances used to preserve food by retarding deterioration, rancidity or discoloration due to oxidation (21 Code of Federal Regulations (CFR) 170.3(o)(3)) (Dziezak, 1986). Antioxidants permitted for use as food additives are list in Table 1. Antioxidants have been used in the U.S. since 1947 to stabilize fats (Stuckey, 1972). In general, the total concentration of antioxidant must not exceed 0.02% by weight, based on the fat content of the food (Nawar, 1985). 15 The main antioxidants used for foods are monohydric or polyhydric phenols, with various ring substitutions. Antioxidants are generally classified as either synthetic or natural products. The four synthetic products, tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated. hydroxytoluene (BHT) and.jpropyl gallate (PG), are the antioxidants most widely used for food antioxidants. In the United States, they can be legally used, under the food additive regulations, at concentrations of not over‘ 200 ppm(wt/wt) of the fat content of food products (Dziezak, 1986). The natural antioxidants include tocopherols, ascorbic acid, lecithin, rosemary extract, gum guaiac, ascorbic acid, and others (Dougherty, 1988). 16 Table 1. Antioxidants permitted in foods in the United States (Source: Narwar, 1985) Primary antioxidants Synergists Tocopherols Gum guaiac Propyl gallate Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) 2,4,5—Trihydroxybutyrophenone (THBP) 4-Hydroxymethyl-2,6—di-tert- butylphenol tert-Butylhydroquinone (TBHO) Citric acid and isopropyl citrate Phosphoric acid Thiodipropionic acid and its didodecyl, dilauryl, and dioctadecyl esters Ascorbic acid and ascorbyl palmitate Tartaric acid Lecithin l7 Antioxidants can not reverse the oxidative process, or restore the food t1) its original quality; but they’ can interrupt the free-radical propagation. step» of oxidative reactions by contributing a hydrogen atom from the phenolic hydroxyl groups. They then become stable free radicals, which do not initiate nor propagate further oxidation of lipids (Sherwin, 1976). The effectiveness of an antioxidant is related to many factors, such as activation energy, rate constant, oxidation reaction potential and solubility properties. Its solubility' and ‘volatility affects accessibility to the peroxy radical sites and its permanence during storage or heating, respectively (Narwar, 1985). For maximum efficiency, primary antioxidants are often used in combination with other phenolic antioxidants or with a metal sequestering agent. Synergism occurs when a mixture of antioxidants perform more activity than the individual antioxidants, if used separately (Weng, 1993). Two kinds of synergism are recognized. One is the action of mixed free radical acceptors (Uri, 1961). The other involves the combined action of a free radical acceptor and a metal chelating agent (Narwar, 1985). 18 No single antioxidant is suitable for all food products. Antioxidants must be added to freshly produced oil or fat before the autooxidation reaction has been initiated (Dougherty, 1988; Dziezak, 1986; Coulter, 1988). It is also necessary to select a proper antioxidant to meet the needs of a particular food item. The choice of antioxidant is dependent upon the nature of the food product, processing conditions and the method by which it is added. Various methods have been described for the incorporation of antioxidants into a snack food, these include : (1) directly sprayed onto food; (2) treated packaging materials; (3) treated animal or vegetable fats; and (4) treated essential oils and flavors (Buck, 1984). Correctly applied antioxidants will help to maintain the product’s original freshness, flavor, odor and shelf-life. Mechanism of antioxidant Antioxidants have been used effectively and safely for many years to retard oxidative deterioration of foods. Antioxidants will inhibit or interfere with the formation of free radicals in food fats, terminating the oxidative reaction. in. its .initiating' step (Dougherty, 1988). The 19 mechanism by which the antioxidant (AH) acts to interfere with free radical formation is shown in the following sequence of reactions: 1% + AH 4;. RH + A- RO- + AH : ROH + A. ROOo-+.AH t ROOH + An lb + A. e RA Roe-+.Ae 11 ROA Antioxidants interrupt the autoxidation process by reacting with free fatty acid radicals or hydroperoxide radicals. The antioxidant free radicals are quite stable and will not further propagate the radical chain process (Everson et al., 1957). Characteristics of Hindered Phenolic Synthetic Antioxidants 2-(1,1-Dimethy1ethyl)-1,4-Benzenediol (TBHQ) 2-(1,1-Dimethylethyl)—1,4-benzenediol, commonly known as TBHQ. The molecular weight of TBHQ is 166.22. Its melting range is 126.5°C—128.5°C. It is a white to tan crystallive solid. TBHQ is soluble in oil, propylene glycol, ethanol and slightly soluble in water. It is effective in. providing oxidative stability to crude and 20 refined polyunsaturated oil, without encountering a problem of color. It is quite color stable even in the presence of metals. TBHQ exhibits good carry-through characteristics in the frying of potato chips. It was approved for use in the preservation of foodstuffs by FDA in 1972 (21 CFR 172.185) (Coulter, 1988). The structure of TBHQ is shown in Figure l (Coulter, 1988). C(CH3)3 HO OH (CH3)3 Figure 1. The structure of TBHQ 2-Tertiary-Eutyl-4-Methoxy Phenol (BHA) 2-Tertiary-butyl—4-methoxy phenol, commonly known as BHA. The molecular weight of BHA is 180. It has a boiling point of 264°C—270W2 and melting range at 48°C—55W2. It is a white solid which is soluble in fats, oils, propylene glycol, paraffin, and ethanol, but not soluble in water. BHA has a typical phenolic odor that may be noticeable if 21 the oil is subjected to high heat (Sims, 1972). The structure of BHA is shown in Figure 2 (Coulter, 1988). R1 HC) R2 10% R 1-H R2-C(CH 3)3 900/ R 1-c CH 3 3 OCH3 ° ( ) RZ-H Figure 2. The structure of BHA BHA has excellent carry-through activity after hot processing, therefore, BHA ie;aa popular antioxidant choice for dry breakfast cereal, animal fats and vegetable oils (Nawar, 1985). It is relatively effective when used in combination with other primary antioxidants. Because of its volatility, BHA has been incorporated as an additive to packaging material, from which it can migrate into food to prevent oxidation (Han et al., 1987). 3,S-Di-Tertiary-Eutyl-4-Hydroxytoluene (BHT) 3,5-Di-tertiary-butyl-4-hydroxytoluene (BHT) is the most prevalently used synthetic antioxidant. It has been used as an antioxidant in the food industry since 1970 for animal fat, dry breakfast cereals, and emulsion stabilizers 22 (Nawar, 1985; Dugan, 1976). The molecular weight of BHT is 220. It has a boiling point of 26532, and a melting point of 69.7%:. It is a white crystalline powdery solid, which is insoluble in water but is soluble in fats, oils and ethanol. The structure of BHT is as follows (Coulter, 1988): (CH3)3C C(CH3)3 CH3 Figure 3. Structure of BHT In accordance with good manufacturing practice (21 CFR 182.3173), BHT is listed as Generally Recognized As Safe (GRAS) for use in food, when the total content of antioxidants is not over 0.02% of the fat or oil content of the food (Coulter, 1988). In 1977, a proposal was made by the FDA to remove BHT from GRAS status and banning the substance from use in direct and indirect food packaging (Monks, 1992; Charnas, 1992). For the present, however, the official position of the FDA is that BHT still maintains 23 GRAS status (Monks, 1992). Because of the proposal, concerns had been raised regarding the safety and continued use of BHT in food packaging applications (Ho et al., 1994). Therefore, in order to meet consumer demands, it is necessary to find an antioxidant as a substitute for BHT. n-Propyl-3,4,S-Trihydroxybenzoate (PG) n-Propyl—3,4,5-trihydroxybenzoate, also known as PG, is the n-propyl ester of 3,4,5-trihydroxy-benzoic acid. The molecular weight of PG is 212. It begins to decompose when temperature is above 148°C. It has a melting range at 146°C—1480C. It is supplied as a solid white crystalline powder. It is soluble in propylene glycol and ethanol, slightly soluble in water, but not in fats or oils (Dziezak, 1986). PG is useful in inhibiting oxidation in oils and animal fats, meat products, including fresh and fresh frozen pork sausage, spices, and snacks. It was approved for food use by FDA in 1947. The FDA has also approved its use in chewing' gum, based. on levels not to (exceed 0.01% total antioxidant weight. PG is the most effective of the synthetic antioxidants in preventing oxidative rancidity. However, it can also be the most problematic due to possible 24 formation of black or purple colored complexes, when it comes in contact with metallic ions such as iron or copper. To prevent this problem, liquid blends are offered in a propylene glycol solution. combining jpropylgallate with. a cheating agent such as citric acid. The structure of PG is shown in Figure 4 (Coulter, 1988). HO OH COOC3H7 Figure 4. The structure of PG 2S Characteristics of Natural Antioxidants Ascorbic Acid Ascorbic acid, or vitamin C is a compound very widespread in natural. Ascorbic acid is constantly gaining importance as a natural food additive. It helps to improve the quality and increases the shelf-life of many food products (Dugan, 1976). In its removal of oxygen from air or food, ascorbic acid is oxidized to form dehydroascorbic acid, thereby asserting its antioxidant action (Narwar, 1985). Its ability to reduce components may be extended to antioxidants. For instance, ascorbic acid anxi its sodium salt, are hypothesized to regenerate phenolic antioxidants by contributing hydrogen atoms to phenoxyl radicals produced by lipid oxidation. By itself, ascorbic acid is freely water soluble and has hardly any antioxidant activity. It promotes the effectiveness of other antioxidants by combining as a synergist. Ascorbic acid has no restrictions on its usage levels and. has GRAS status for use as a chemical. preservative: (21 CFR 182.3013) (Dziezak, 1986). Ascorbic acid is a highly soluble substance that has both acidic and strong reducing properties. These qualities are attributable to its enediol structure which is conjugated 26 with the carbonyl group in a latone ring. The structure of ascorbic acid is as follows: 0 O H II << II = 0/ I HOCH HOCH CHOH iHOH Figure 5. The structure of ascorbic acid Rosmarinus Officinalis Rosmarinus Officinalis has been used for some time as an antioxidant in a number of foods (Liu et al., 1992; Pokrny, 1991). It is extracted from rosemary leaves by steam distillation. An alcoholic extraction of the leaves gives the crude rosemary extract, which can be directly used as a food grade antioxidant after evaporation of the alcohol (Yang, 1985). However, the extracts of rosemary have a strong odor and bitter taste and therefore, can not be used in most food products (Chang et al., 1977). To solve these problems, a nmdecular distillation followed by steam distillation was necessary. This gave an odorless and pale- 27 red/orange product of high activity (Loliger, 1983). Loliger (1983) studied the effectiveness of rosemary extract compared with BHT/BHA and the results indicated that the antioxidant activity from rosemary is about the same as BHT/BHA, at corresponding concentrations. Loliger also analyzed the rosmarinus Officinalis by a HPLC procedure and identified several of the compounds with very good antioxidant properties, such as rosmarinic acid, carnosol and carnosic acid. The structure of rosmarinic acid is shown in Figure 6. (DH COOH (Hi IHO (Hi Figure 6. The structure of the rosemary antioxidant, rosmarinic acid 28 a-Tocohperol a—TocopherolWitamin E) has recently been shown to have application as an alternative antioxidant for polymer processing (Laermer et al., 1993). It occurs naturally in vegetable oils. a-Tocopherol is a natural antioxidant which belongs to a class of compounds exhibiting vitamin E activity. At least seven types of tocopherol methyl substituted forms of tocol have been isolated. Tocopherols exhibit antioxidative properties, and have been shown to be effective in delaying oxidation in a variety of foods (Dugan, 1976). The antioxidative nature of tocopherols is attributed to their phenolic structure. Tocopherols differ in their antioxidant activity. In. general, antioxidant activity increases with decreasing vitamin E activity. The antioxidant activity of tocopherols increases in the following order: delta > gamma > beta > alpha. However, the relative activity of these compounds is significantly influenced In! temperature auxi light conditions (Sherwin, 1976). The structure of the respective tocopherols is shown in Figure 7. 29 PK) CH3 CH3 CH3 CH3 7 (3 CH5 5,7,8 - Trimethyl = d-tocohperol 5,8 - Dimethyl = B-tocopherol 7,8 - Dimethyl = y-tocopherol 8 - Methyl = 5-tocopherol Figure 7. Tocopherol structures The molecule weight of a-tocopherol is 430.72. It is a clear, viscous oily substances of pale yellow color, nearly odorless. It is insoluble in water, miscible at any ratio with vegetable oils, ethanol, ether, chloroform and acetone. IR: is heat stable and. not ‘volatile or steam distillable. INatural source tocopherols are regulated in the United States by the FDA in 21 CFR 182.3890 and by USDA at 9 CFR 318.7. In 1978 the FDA proposed to affirm GRAS status for 'mixed, concentrated tocopherols under 21 CFR 184.1894. Tocopherols have been approved for use as food additives leEi number of countries which include: Canada, 30 Japan, Korea, Australia and all members of the European Economic Community (Dougherty, 1988). The antioxidant effect of vitamin E in biological systems is well documented. It is believed that vitamin E functions in Vivo as a free radical scavenger, by being oxidized to a tocopheroxyl radical. The tocopheroxyl radical is easily formed due to its stabilization by the pyran ring oxygen (Burton, 1983). In the human diet, vitamin E acts to prevent biological damage associated with the effects of aging, chronic disease, and exposure to atmospheric pollution, and has been considered as an anticarcinogen. Epidemiological studies indicated that vitamin E, alone or in combination with other antioxidants can decrease the incidence of certain forms of cancer by quenching free radicals, or reacting with their products (Packer, 1992). a-Tocopherol has been shown to be effective in improving the oxidative stability of fats and is used at relatively low concentrations (100 to 300 ppm of fat weight) in a wide variety of food products to retard oxidative deterioration. (Dougherty, 1988; Coulter, 1988) 31 Application of Antioxidants in Packaging Materials Additives have been incorporated into food products and packaging materials for a number of years (Smith, 1993). Recently, consumer demands have lead to more interest towards reducing the levels of food additives in processed foods. However, elimination of additives such as antioxidants may reduce the shelf—life of the product. Therefore, manufactures have attempted.tx> reduce time total amount of antioxidant consumed with their products by incorporating less into the product, and more into the packaging material (Food Engineering, 1979). Since oxygen first attacks food at the surface, impregnating' packaging' materials with. an. antioxidant has long been used to protect the product from oxidative rancidity and thus prolong shelf life. The proposed mechanism of antioxidant activity involves the following 3 step process: (1) antioxidant diffusion through the polymer bulk phase; (2) evaporation of antioxidant from the surface of the packaging material; and (3) subsequent antioxidant sorption onto the surface of the packaged product. Antioxidant stabilizers have been incorporated into polyolefin resin to prevent degradation, improve heat 32 resistance and control color change. The basic steps associated with thermal degradation are similar to an oxidation reacticmn When. the polymer 'undergoes thermal degradation, the breaking of chemical bonds results in formation of reactive products such as free radicals and unstable hydroperoxides. Antioxidants, therefore, not only stabilize food packaging liner films during fabrication but as indicated above, due to their volatility and tendency to migrate, also become an antioxidant for the food product itself. various studies have been described dealing with methods of adding antioxidants indirectly, showing the positive utilization of the absorption phenomenon, in which the vapor from the antioxidants moves into the food. (Coulter, 1989). For example, Hoojjat et a1. (1987) demonstrated the effectiveness of a BHT impregnated film to retard lipid oxidation of a packaged oatmeal cereal, through the migration of antioxidant from the package to the product via an evaporation/sorption mechanism. There are a number of studies reported dealing with the migration characteristics and the stability of BHT in polyolefin films. Urata (1988) observed that when compared to vitamin E and Inganox®~1010 (Trade name of CGP-CIBA— 33 GEIGY Corporation) in polypropylene resin, BHT showed the greatest loss by heat deterioration at 100°C. Urata also reported that polypropylene (PP) resin, containing various concentrations of BHT as a stabilizer, was found to decrease in weight by heat deterioration and to be become brittle after 2 days heating at 130°C. When compared with vitamin E as a stabilizer, the higher the quantity of vitamin E added, the greater the number of days before the PP resin became brittle. Since BHT is somewhat volatile, even at ambient temperature, it readily migrates into food (Dziezak, 1986; Ho, 1994). Currently, BHT is added to plastic resins in concentrations of 3000 ppm(wt/wt) for breakfast cereal liner film (Monks, 1992). The BHT migrates to the cereal, acting as a preservative. Migration of BHT from polymer films has been widely studied. Terada and Naito (1989) reported that the higher the concentration of BHT in polyethylene (PE) film, the higher the BHT migration levels into a food model. Their studies showed that migration occurs when the product/package system are in contact with each other, or through the gaseous phase alone. They also found the levels of’ BHT' migrating' increased. with. time, but the level of 34 migration as a function of time varys significantly with the food. Till et a1. (1988) studied the migration of BHT from high density polyethylene (HDPE) to food and food simulates. They found that BHT migrated to fatty foods at a faster rate than to aqueous foods. The data also showed that temperature is a factor affecting the diffusion coefficient of BHT through HDPE. The higher the temperature, the higher the diffusion coefficient value. Therefore, the packaging material which has been impregnated with antioxidant must be stored properly before use. Figge et a1. (1976) studied the migration of BHT by a radio-tracer technique. They used several 14C-labeled plastic additives incorporated into resin samples which were compounded and molded into test sheets. The test sheets were then stored for various periods of time in contact with selected foods, at several different temperatures. They found that the amount of labeled additives migrating from HDPE is higher than from polyvinyl chloride (PVC). Bailey (1995) studied the mass transfer' of BHT' fremr a multi-layer lamination film and concluded that the rate of loss of BHT from the respective surface layers is controlled by surface evaporation, the 35 mass transfer coefficients and diffusion coefficients. Bailey also noted that the rate of loss of BHT from the heat seal layer' was greater than that from. the .HDPE surface layer. The (differences iJ1 antioxidant loss rates could affect the quality of a packaged product, due to the transfer rate of additive to the product. For many years, film manufactures used BHT as a stabilizer to prevent degradation. Recently, however, BHT has been suspected to be a cancer—causing agent (Charnas, 1992; Ho, 1994). As a result, many companies, such as Quantum Chemical Co., Mobil Chemical Co., and Exxon Chemical Co., have eliminated BHT from their resin and while the FDA has not taken any action with regard to the continued use of BHT, public concern has lead polyolefin producers and food manufactures to actively seek alternate antioxidants (Monk, 1992). Some costly, high molecular weight hindered phenols are being considered as alternative antioxidants by film manufactures (Monks, 1992). These antioxidants provide more permanent protection than BHT, while using the same additive level. They also diminished the color problem during processing. Therefore, most resin and additive suppliers 36 recommend high molecular weight hindered phenols as the best alternatives to BHT (Monk, 1992). In contrast, a study recently reported by the America Health Foundation (Monk, 1992) showed that BHT acts as an anticarcinogen, when ingested in small doses. In this study, rats were fed a potent liver carcinogen with 0 to 6000 ppm BHT. Data showed that rats eating all but the highest dosages of BHT developed less cancer than those who were denied the antioxidant (Monks, 1992). a-Tocopherol (ATP) or vitamin E is said to be unquestionably one of the safest, most environmentally acceptable additives that can be used for polymer stabilization. The use of vitamin E in the plastics area is relatively new and has been used as an alternative for BHT in food packaging since the mid-1980s (Urata, 1988; Ho, 1994). a—Tocopherol consists of an aromatic ring, which gives the molecule an enhanced redox potential and allows it to act as a extremely effective scavenger of free radicals. Also, the long side chain provides good solubility in the polymer (McMurrer, 1991). Based on its chemical structure, it is thought that a—tocopherol is 250 times more reactive toward peroxy radicals in styrene than BHT (Burton, 1986). 37 The properties of ATP make it effective in preventing polymer damage associated with the effect of long-term storage, processing temperature, and exposure to atmospheric pollutants. Laermer and Schuster (1990) observed the melt stability of polypropylene resin containing either ATP, BHT or IrganoxCFIOIO and found. ATP to be superior to the conventional antioxidants at very low concentrations, or in synergistic mixtures with other additives. When ATP was combined with synergistic additives, it also showed better color stability for PP. To provide the best processing and color stability, a secondary antioxidant, typically a phosphorous-based compound, is often incorporated to work synergistically with the primary phenolic antioxidants (McMurrer, 1991). Secondary antioxidants such as phosphites and phosphonites provided very important synergism for primary phenolic antioxidants. They must be hydrolytically stable to be blended and mixed thoroughly and homogeneously with the primary antioxidants. However, the commonly used phosphites are usually hydrolytically unstable and contribute to mixing/handing problems during transfer. They also occasionally form black specks during extrusion at high 38 temperature and for long residence time. A series of studies have been described which evaluated a “phosphite free” system to stabilize polyolefins. For example, Young et al. (1995) examined a “phosphite free” system based on a patented formulation. of tu-tocopherol, IrganoxCF1010, and other combinations of antioxidants, such as Irganox®h 1010/UltranoxCF626, and IrganoxCF1010/a-tocopherol. The data showed that using a-tocopherol alone was the most effective for HDPE and was found to improve film color and to reduce the formation of gels in films. For PP resin, the combination of a-tocopherol and IrganoxCfilolo offered excellent color and melt flow control. The results demonstrated that a-tocopherol can replace a phenolic antioxidant/phosphite combination as the primary antioxidant system, or replace the phosphite portion to achieve optimum performance. Therefore, the problems associated with phosphites can be eliminated or minimized. For overall performance, a “phosphite free” system offers balanced properties, as well as excellent economics and it is to be considered as the most cost effective system (Young et al., 1995). 39 Due to its excellent thermal stability, ATP will not begin to volatilize until 300°C. Its limited volatility reduces migration from the polymer matrix into food products (Smith, 1993). However, although ATP is very stable, it still has the potential to migrate to food products (Packaging, July 1992). The migration of ATP from a film to a series of food simulate systems was studies by Laermer et a1. (1993). The authors reported that 8.4% of the initial ATP additive present in HDPE film will migrate into heptane, 3.8% of the initial ATP will migrate to 50% ethanol, and no detectable level of migration was indicated with 8% ethanol and 4% acetic acid as the food simulant. Another study on the migration of ATP from drug packaging was carried out by Hoffmann-La Roche’s research group (Laermer et al., 1993). In these studies, strips of HDPE containing various additives were cut from the side wall of HDPE bottles and immersed in 100% ethanol in a sealed container. Data showed that only 5% of the initial ATP present in the HDPE bottle wall was extracted, as compared with 25% of BHT and 13% of Irganox®-1076 (Trade name of CGP—CIBA—GEIGY Corporation). It is beneficial to both the drug manufacturer and consumers to have less antioxidants extracted into a drug product. 4O “Odor and plastic taste” problems have challenged resin producers for years. During polymer processing, the heat and shear energy often causes bond-scission of polymer chains, leading to the formation of various low molecular weight compounds. Those compounds whidh are entrapped in the polymer matrix can evaporate in air as off—odor volatiles or migrate to food as off—taste compounds. The off-odors and off-taste can arise from a variety of materials such an; monomers, plasticizers, oligomers, solvents, etc.. Antioxidants have been used in plastic bottles to reduce their so called plastic taste. Laermer et al. (1993) and Ho (1994) have shown that HDPE incorporated with 100 ppm(wt/wt) ATP exhibited less taste and odor responses, when compared to 500 ppm(wt/wt) of IrganoxCF1076 or 250 ppm of BHT. This data also agreed with the results of Zambetti (1995), that ATP provided a 73%—83% reduction in aldehyde levels which are responsible for the unacceptable taste and odor of HDPE containers. ATP has been used as a flavor retainer by resin manufactures and food companies as well. A taste test carried out by Sensory Spectrum of Chatham (Laermer et al., 1993) demonstrated that any level of ATP incorporated into the HDPE liner structure retarded 41 the loss of flavor from a cereal product over the test period and temperature (37°C and 3 months). The development of gels in both blown film and cast film has been a problem to converters. Studies showed that the addition of ATP reduced the gel counts for both LLDPE and HDPE by 80%, when compared to the unstabilized resin. Thereby, reducing waste and allowing film to be processed at higher temperatures, with faster throughput. In nerketing, the cost of ATP is three times higher than other standard commercial antioxidants. However, it can be used at significantly lower dosages - 100 ppm for PE application and 250 ppm (or less) for PP. Because it is typically used at 1/4 to 1/5 the effect dosage of other antioxidants, ATP is more cost- effective tjuui conventional antioxidants (Laermer en: al., 1993) . Theory of Migration Migration is described as the transfer of material from plastic to food, under specified conditions. The migration of a plastic ingredient to food may take place through the headspace between the package and food. There are two types of migration. processes which have been defined in food 42 packaging, namely: global migration and specific migration. Global migration refers to the total transfer of all migrating species from plastic into packaged food. Specific migration refers to one or more identifiable species that is a constituent of the packaging material (Giacin, 1980). The rate of migration is affected by the following factors: (1) the solubility of additives in the polymer system; (2) the diffusion coefficient within the bulk of the polymer structure; and (3) the rate at which it volatilizes from the polymer surface (Calvert, 1979). Solubility of the additives has been recognized as an important factor :hi additive compatibility. Hawkins et al.(1969) demonstrated that the rates of loss of typical stabilizing additives are significant, relative to the lifetime of the polymer. If migration from the packaging material to a contained product is to occur, the migrant has to diffuse to the polymer surface, followed by dissolution of the migrant accumulated at the surface to the contact phase. Therefore, the loss rate of additives is determined by the rate of volatilization from the polymer surface and the diffusion coefficient within the bulk of the polymer. 43 A mathematical expression has been described by Crank (1975) for the additive loss from a film by surface evaporation with finite boundary conditions. According to this model, the total amount of additive leaving the polymer in time (t), is expressed as a fraction of the corresponding amount leaving at infinite time. Mt °° 2L2 exp(—B§T) 1’2; 2 2 2 oo [1:] Bn(Bn+L +1“) - 1 L4 ( ) where NR amount of additive leaving the polymer in time t amount of additive leaving the polymer at infinite time = Dt/l2 = la/D = half of film thickness, cm = time, second = diffusion coefficient of additive in polymer, cmz/sec a = mass transfer constant of additive from polymer, cm/sec Bu values are the positive roots of [intanfin = L 3 8 u UrTHt‘t-l Calvert and Billingham (1979) used Eq.(1) to describe the loss of additives from polymer film and sheet. In order to apply Eq.(1), they assumed that polymer degradation will proceed rapidly to sample failure, when the average concentration of additive falls to 10 percent of its initial 44 value, i.e., whenmt/M0° = 0.9. Eq.(2) is obtained when.Nh/Mm = 0.9, and n=1. 2L2 —exp(—[32T) _ BZ(BZ+L2+L) ’ ' (2) They plotted Eq.(2) for a variation of L value as a function of T and concluded that with high L values (thick film, rapid evaporation and low diffusion rate), the failure time is diffusion dominated and independent of CL The failure time is then given by Eq.(3) t = 0.87 12/D L > 10 (3) At the lower L value (thin film, slow evaporation and fast diffusion rate), Eq.(2) becomes a line of unit slope obeying Eq.(4) log L + log T = 0.383 (4) leading to the failure time given by Eq.(5) t = 2.42 l/Ot L < 0.6 (5) According to Eq,(3) and Eq.(5), the additives loss from a thick film is dominated by bulk phase diffusion and loss from a thin film is determined by surface evaporation. Diffusivity, (n: diffusion coefficient MD, is defined as the tendency of a substance to permeate through the polymer bulk phase. The driving force is dependent on a 45 concentration gradient, where the dissolved species diffuses from a high concentration to a low concentration area (Giacin, 1980). The rate of diffusion is defined by Fick’s first (6) and second (7) laws(Crosby, 1981): ldnl dc _____=_q)__ (6) A.dt dx where 1n = mass of the component transferred = time = migrant concentration = path of diffusion = diffusion constant = area of plane cross which diffusion occur 5 U N o n Fick’s second law is applied when the diffusion process is over an infinite surface (i.e., a sheet). dc dzc ——=I)——7 (7) dt dx In this expression, the diffusion coefficient is assumed to be constant and independent of the concentration. Therefore, the concentration of migration in the contact phase is a function of time, and product affinity. There are three different types of packaging materials/contact phase systems which can. be defined in terms of migrate diffusivity. Systenl I : non-migrating; system II : independently migration; and system III leaching. In system I, migration occurs only from the 46 packaging surface with near zero diffusion, therefore little if any migration takes place. In system II, migration take place and the diffusion coefficient is measurable under the test conditions and the specified contact time. In leaching, components of the contacting phase interact with the polymer and increase the diffusion coefficient of the migrating species through the polymer bulk phase (Crosby, 1981). Technique for Trapping Volatiles Techniques that have been used for trapping volatiles include solvent extraction, steam distillation, liquid— liquid extraction, simultaneous steam distillation-solvent extraction, and dynamic headspace or purge and trap procedures (Risch, 1989). Analytical. procedures for ‘the detection and identification of volatiles present in food products have been employed for many years. Dynamic headspace/gas chromatography has been recognized as one of the most effective and sensitive techniques for the analysis of volatile organic compounds (Vallejo-Cordoba, 1993). Headspace volatiles sampling technique can be achieved by: 47 (1) direct injection of volatiles into a gas chromatograph (GC) ,and (2) indirectly sorbing volatiles by sorption trap. Direct headspace sampling has been widely used. In 1971, Dupuy' et al. described. a the soxhlet extraction apparatus. iNo film was placed in the extraction thimble. After 24 hours of continuous operation, the extractant solution was recovered amd brought up to a volume of 100 ml using acetonitrile. The sample was filtered through 0.45pm size filter (HV4- 0.45um,4mm, Millipore), then transferred to a 4 ml vial for HPLC analysis. The area responses of three replicate injections of the extractant solution were averaged. The averages was divided by the area response set as a basis for 100% recovery, as described previously. HPLC Analysis Analysis of (Jr—tocopherol was carried out on a Waters Model 150-C ALC/GPC, with a Waters Data Module (Model 730) and a Waters 486 Tunable absorbance detector. The chromatographic conditions were as follows: 58 1) Column : Delta-Pak HPI C18 300 A (3.9mm x 150mm), Waters 2) Solvent : Pure Methanol (J.T.Baker Inc,99.9+% HPLC grade) 3) Flow rate : 1.0 ml/minute 4) Detection wavelength : 280nm 5) Amount injected : 10ul 6) Elution time : 3.65 minute Analysis for BHT also used the Waters HPLC system. The chromatographic conditions were as follows: 1) Column : Delta—Pak HPI C18 300A (3.9mm x 150mm) 2) Solvent : Methanol : Water = 85 : 15 3) Flow rate : 1.0 ml/minute 4) Detection wavelength : 280nm 5) Amount injected : 10 pl 6) Elution time : 3.7 minute The concentration of antioxidant in the test film was determined by comparison of detector response to a standard calibration curve which was constructed from standard solutions of known concentration. A standard solution of antioxidant was prepared by accurately weighing 0.01 gram of antioxidant, transferring the weighted sample to 51 100 ml volumetric flask and diluting with acetonitrile to volume. 59 The primary standard solution was then used to prepare a series of standard solutions of known concentration by a serial dilution. procedure. For construction of the calibration curve a 10 pl sample was withdrawn from the respective standard solutions and injected directly into the HPLC. The calibration data are shown in Appendix A. The concentration of antioxidant in the film was determined by substitution into the expression. AUX CF x Vtotal x 100 x Wt. antioxidant = injection polymer CF calibration factor (g/AU) AU average area units from integrator Vumm.= total extractant phase volume (100ml) Vnuumum = sample volume injected (1001) Wt.mflwmr = weight of polymer sample (9) Product Stability Studies Approximately 25 grams of oat cereal product were packaged in 4.5 inch by 5 inch test pouches. The pouches were fabricated from the three test films. The pouches were 6O sealed with an impulse sealer (Sencorp System Inc. Model No— 16TP). The heating time was 0.8 second, the cooling time was 1 second and the pressure was 35 psi. All test pouches were mounted on a wooden sampling rack in an environmentally controlled room. 13m: storage environment was monitored using a hygrometer. Temperature and relative humidity were maintained at 23%: i: 2°C and 50%RH i 2%. The storage arrangement is shown in Figure 9. Test pouches made from the respective film structures and filled with product were removed from storage every two weeks for the first two months of storage. After two months, filled pouches were removed monthly. Packaged product was sampled on a monthly basis for a period of eleven months. After sampling the pouches, the cereal product was removed from. the package and the following analysis performed on the product: (1)Hexanal analysis to determine the extent of lipid oxidation; and (2) the level of sorbed antioxidant present in.tflu3 cereal product. The pouches were also assayed to determinate the relative concentration of antioxidant retained as a function of the storage time. A flow diagram of the test scheme is shown in Figure 10. 61 The storage arrangement for cereal package Figure 9. 62 Impregnated Films 1 I Antioxidant Loss Cereal Package , Rate Studies 1 l Pouch Cereal 23%: 30 C 40 C 50 C l Antioxidant Loss Rate Studies 1 l Hexanal Analysis Antioxidant Content Figure 10. Flow diagram of the test scheme 63 Hexanal Quantification Apparatus for Trapping of Volatiles A dynamic gas purge and trap system was designed for headspace sampling of the cereal product. Six 250 ml Erlenmeyer flasks were modified to 29/42 standard taper male joints to which the dispersion tube assembly' of a gas washing bottle could be fitted (Stopper assemblies for Corning 31770 gas washing bottles, Fisher Scientific, Pittsburgh, PA). Modification of the dispersion tube assembly of the gas washing bottles and the Erlenmeyer flasks were performed by the Chemistry Department Glass Blowing Shop at MSU. A schematic diagram of the dynamic purge and trap system is shown in Figure 1d“ .As shown, a cylinder of compressed nitrogen was interfaced to a dispersing manifold which consisted of three flow meters and needle valves, all connections were through 1/8” O.D. copper tubing and. swagelok fitting. Fitmr meters were used to provide a continuous indication that a constant rate of flow of nitrogen was maintained to the individual purge and trap cells. Gas flow was regulated with NU PRO needle valves, type B-25G. 64 9C1) PC PC PC Re: regulator PC:purge cell R: rotormeter T: trapping tube WB: water bath Nitrogen Figure 11. Schematic diagram of dynamic purge and trap system 65 The trapping system was designed to ensure that the sample was continuously flushed with nitrogen gas and the desorbed volatiles conveyed to the trapping tube attached. The sorption trap was connected to the exit port of the dispersion head via swagelok adapters. The dispersion head exit port of 8 mm O.D. glass tubing was connected by a 5/16” swagelok nut and a series of reducing adapters to a 1/4” male swagelok fitting. The sorption trap was mounted to the dispersion head with a 1/4” thumb wheel swagelok fitting (Supelco Inc., Bellefonte, PA) for easy removal and could be fixed to both glass and metal desorption traps. Figures 12 and 13 show the trapping cell and the whole purge and trap system” The glass thermal desorption tubes-Carbotrap 300 (6mm I.D. x 4 mm I.D. x 11.5 cm) used in the present study were prepacked by Supelco Inc. (Bellefonte, PA). The trapping tubes were packed with 300 micrograms of Carbotrap C absorbent, 200 micrograms of Carbotrap E absorbent and 125 micrograms of Carbosieve S-III absorbent. 66 tube attached The apparatus with the sparging Figure 12 67 Figure 13. Photograph of Dynamic Purge and Trap System 68 Hexanal Analysis Procedure Individual packaged cereal product samples were removed from the environmental chamber as a function of storage time. The package was opened, and the product removed. Approximately 1.5 grams of cereal product were weighed and transferred to the modified Erlenmeyer flasks equipped with an inlet and outlet port and sparging tube as described. Nitrogen was flowed through the flask and the Carbotrap at a rate of approximately 25 cubic centimeters (cc) per minute. For the first hour, the flask was flushed at room temperature to remove oxygen from the system prior to heating. After one hour, the water bath (Blue M Constant Temperature Bath, Blue Island, IL) was turned on and allowed one hour to reach 55°C. The modified Erlenmeyer flasks were placed in the water bath and the system purged for 24 hours. Repetitive analyses of the cereal sample showed no additional hexanal detected. Each sample was analyzed in triplicate. After 24 hours trapping, the Carbotrap tubes containing trapped volatiles were transferred to the thermal desorption unit (Model 890, Dynathernl Analytical Instruments, Inc.) which was interfaced to a gas chromatograph for 69 quantification. The sorbed volatiles were desorbed by heating for 6 minutes at 340°C, with the valve and transfer line held at 230°C to maintain the desorbed compounds in the vapor phase, while being transferred to the gas chromatograph. Helium was used as a carrier gas through the thermal desorption unit at a flow rate of 7.5 ml./minute at 40 psi. After sample desorption, the sorbant tubes were conditioned at 340°C for 40 minute prior to re-use. Gas chromatographic (GC) analyses were carried out with a. Hewlett-Packard. 5890A. gas chromatograph, equipped. with dual flame ionization detectors (Avondale, PA, USA). The GC conditions were as follows: Column : Supelco, SPB5 (30m x 0.32nm, 10um) Helium carrier : 7.5 ml/minute H2 : 30 ml/minute Nitrogen : 30 ml/minute Air : 400 ml/minute Initial temperature : 40°C Initial time : 6 minute Rate : 5 degree/minute Final temperature : 200°C Final time : 10 minute 7O Hexanal was eluted at a retention time of 8.5 minutes. The concentration of hexanal in the cereal product was determined by substitution into the following equation ZHJX CF Conc. (Wt /Wt) = —— (9) Wt” total AU = average area units response from integrator CF = Calibration factor (gm/Au) Wt.umm.= Weight (gm) of cereal sample Hexanal Calibration Curve Procedure A standard solution of hexanal was prepared by accurately weighing 0.04 gram hexanal and transferring the weighed sample to a 100 ml volumetric flask and diluting with methanol to volume. The primary standard solution was then used to prepare a series of standard solutions of known concentration by a serial dilution procedure. To prepare a standard calibration curve to establish the linearity and sensitivity of the thermal desorption procedure, a 1.2111 sample was withdrawn from the respective standard solutions and the sample injected directly onto the Cbrbotrap tube. The Carbotrap tube was then transferred to the thermal 71 desorption unit. The GC conditions were as follows: programmed at an initial temperature of 40°C for six minutes, followed by heating at a rate of 5°C per minute to a final temperature of 200°C which was held for ten minutes. The calibration data are shown in Appendix B. Antioxidant content of Cereal Lipid extraction The cereal was extracted for total lipid using the method of Hoojjat (1987). A 40 gram sample of cereal was removed from the test pouches. A weighed quantity of the product was mixed in a blender (WARD Montgomery, 12 speed) at low speed with 50 ml chloroform and 100 ml methanol for 2 minutes. An additional 50 ml of chloroform was added and blended for 30 seconds. After 30 seconds, 50 ml of HPLC grade water was added and the solution blended another 30 seconds. The proportion of chloroform, methanol and water is 2:2:1, respectively. The mixture was filtered by vacuum filtration. After removing the cereal residue, the filtrate was transferred to a separatory funnel. The cflfltmoform layer (bottom layer) was collected and concentrated using a Buchi Rotavapor Model :12; 72 RE III series with a 50°C water bath and water aspirator pressure. After removing the chloroform, a yellow oily layer remained. This layer was then transferred to a screw capped vial and stored at —18°C for later analysis. Antioxidant Content of Extracted Fat Antioxidant content. of the cereal product following storage was determined using the method of Hoojjat (1987). A 0.5 gram sample of extracted oil was weighted into a 4 ml vial and 3 ml of acetonitrile was added. The mixture was shaken. by“ hand. for“ 30 second. and. allowed, to stand for approximately 15 minutes. After two phase separation, the top layer (acetonitrile) was collected, filtered and analyzed by HPLC for its antioxidant content (Analytical conditions were as previously described). The antioxidant content of the cereal product is determined by substitution into the following equation: Antioxidant content (g/g cereal) = AU X CF X Vtotal X Wt.total Vinjection X Wt.sample X Wt.cereal (10) CF calibration factor (g/AU) AU average area units from integrator 73 Vtotal = total solution phase volume (3ml) Vinjection = sample volume injected (lOul) Wt. total = total weight of extracted oil (9) Wt. sample = weight of oil sample assayed (~0.Sg) Wt. cereal = weight of cereal sample extracted (~40g) RESULTS AND DISCUSSION Thickness of Packaging Materials The optical microscopy procedure used to determine the thickness of the respective layers of the a-tocopherol impregnated laminate was found to give good resolution of the heat seal layer, but could not resolve the middle and outer HDPE layer. However, values of the total laminate thickness and the heat seal layer were obtained. Thickness measurements, were repeated five times, and the data obtained is presented in Appendix C. The averaged values obtained are summarized in Table 2. The total film thickness was also measured. by' a micrometer (Model 549 Micrometer, Testing machines, Inc. Amityville, NY) and the results are summarized in Table 2 for comparison. Ten measurements were recorded and are tabulated in Appendix D. The thickness values determined by Bailey (1995) for Film II are also summarized in Table 2. 74 75 Table 2. Average thickness for lamination film heat seal HDPE Total (m Total°° layer layer thickness thickness Film I 14.7 um 44.3 um 59.0 um 59.1 pm Film 11“” 10.16 pm 45.72 mm 61.9 mm 62 um (a) (b) (c) Source: Bailey (1995) Measured by microscopy Measured by micrometer As shown in Table 2, the total thickness of Film I measured by optical microscopy and by micrometer is 59pm and 59.1pm, respectively, providing good agreement between the two procedures. Loss of a-Tocopherol from Coextruded Laminate Film The relative loss of a-tocopherol from the test film, as a function of time and temperature, was determined by the HPLC procedure previously described. The initial concentration of a-tocopherol was determined at different time intervals, between receipt of the roll stock and termination of the studies, to insure a constant concentration. The results are summarized in Table 3. The test film was reported to contain 275 ppm(wt/wt) gm (1- 76 tocopherol per gm total film weight, according to the supplier (Bemis Company, Terre Haute, Indiana). The concentration level of a—tocopherol reported by the supplier is assumed to be the level added to the resin. Losses during' processing could. account for the somewhat lower a-tocopherol level determined by HPLC analysis. As shown, the level of a-tocopherol is reasonably constant over the course of the study. Table 3. a—Tocopherol concentration (wt/wt%) of laminate roll stock at different time intervals Storage 0 100 120 170 200 380 Avg. Day(a) wt/wtem 0.0238 0.022 0.023 0.0218 0.02 0.0195 0.0215 fa} (b) Time of storage from date of receipt Average of three replicate sample analysis, with an average standard deviation of i 5% The resulte; of monitoring’ the loss of a—tocopherol from the test film, at 23, 30, 40, and 50°C, by HPLC analysis, are summarized in Tables 4 through Table 7, respectively. The results are also presented graphically in Figure 14, where the relative percent a-tocopherol remaining in 77 the film is plotted as a function of time for the studies carried out at 23, 30, 40, and SOUS, respectively. According to Han et al. (1987), the rate of loss of an additive from the test film follows a first-order or pseudo first-order rate expression: ln(Ct/Co)= -kt (11) where Co is the initial concentration of a—tocopherol in the film and Ct is the concentration (wt/wt%) at any time (t); k is the loss rate constant, and t is the time interval. Graphical analysis (see Figure 14) indicated that 51 first order expression provided a good description of the rate loss of a-tocopherol from the lamination, up through greater than 85% loss. The rate constants determined from Equation (11) for the different temperatures are summarized in Table 8. As shown in Figure 14, a linear relationship was found for the plot of (Ck/Co)x100 versus time at the four different temperatures of test evaluated in.tflua rate loss studies. The following expressions were derived from a least squares fit of the rate loss data. 23°C (Ct/Co)x100 = 92.954 * exp(—0.0004t) (12) 30°C (Ct/Co)XI00 = 82.159 * exp(-0.0021t) (13) 78 40°C (Ct/Co)x100 129.46 * exp(-0.0068t) (14) ll 50°C (Ct/Co)x100 114.67 *exp(-0.0284t) (15) The R:2 values were 0.9914, 0.9069, 0.9177, and 0.9461 for 23, 30C, 40C, and 50°C, respectively. At 30°C, the relative percent loss of a-tocopherol from the test film was 83% after 720 hours, while a sample stored at 50°C showed loss of the same amount after 64 hours. The relationship between the rate loss constant and temperature is illustrated in Figure 15, where K is plotted as a function of temperature [1/T(°k)] . The activation energy for the loss of (Jr-tocopherol from the laminate structure was determined by solution of the Van’t Hoff-Arrhenius equation: k = ko exp(—E/RT) (15) where E is activation energy (kcal/mole) , R is the gas constant (1.98 cal/degree-mole) , k0 is the pre—experential constant, and T is temperature (°K) . As shown in Figure 15, the loss of a-tocopherol was found to follow the Arrhenius relationship. From the slope of the Arrhenius plot, the activation energy for the loss of ATP from the HDPE was determined to be 28.87 kcal/mole. Bailey (1995) determined 79 the activation energy for the loss of BHT from Film II, which is a similar multi—layer laminate film, to be 26 kcal/mole. Calvert and Billingham (1979) proposed that the loss of BHT and other simple low molecular weight additives from thin films is controlled by evaporation, while diffusion controls the loss of additive from thick films and bulk solids. The multi—layer lamination used in this study has a thickness of 59pm. It was therefore assumed that the rate of a-tocopherol loss is controlled by surface evaporation, since the sample was in film form. In analysis of the rate loss data, it was further assumed that the additive (i.e. a-tocopherol) had attained an equilibrium partition distribution between the respective layers of the laminate. % Recovery for Antioxidant The % recovery of (it-tocopherol carried through the soxhlet extraction/HPLC analysis procedure was determined to be 97%. The data and calculation are given in Appendix E. 80 Table 4. Loss of a-tocopherol (ATP) from coextruded laminate film at 23°C, determined by HPLC analysis (a) Time ATP Concentration Relative % ATP (hours) (Wt/Wt%) (Ct/Co)x100 0 0.022 100 672 0.014 62.4 1344 0.012 53.5 4368 0.003 14.3 5040 N/A N/A (a) Average of three replicate sample analyses, with an average standard deviation of i 5% 81 Table 5. Loss of a-tocopherol (ATP) from coextruded laminate film at 30°C, determined by HPLC analysis (a) Time ATP Concentration. Relative % ATP (hours) (wt/wt%) (Ct/Co)x100 0 0.0230 100 48 0.0180 77.7 96 0.0140 59.0 144 0.0150 65.70 192 0.0120 52.1 240 0.0130 56.7 288 0.0086 36.7 336 0.0090 38.6 384 0.0080 34.8 480 0.0065 28.1 528 0.0060 26.0 576 0.0050 21.0 624 0.0050 22.5 672 0.0075 30.8 720 0.0040 17.4 (a) Average of three replicate sample analyses, with an average standard deviation of i 5% 82 Table 6. Loss of a-tocopherol (ATP) from coextruded laminate film at 40°C, determined by HPLC analysis (a) Time ATP concentration Relative % ATP (hours) (wt/wt%) (Ct/Co)x100 0 0.0180 100.00 25 0.0177 97.8 49 0.0160 90.9 72 0.0158 87.2 96 0.0120 64.5 149 0.0100 57.4 198 0.0070 37.7 240 0.0062 34.4 312 0.0040 20.8 384 0.001 5.5 (3) Average of three replicate sample analyses, with an average standard deviation of i 5% 83 Table 7. Loss of a-tocopherol (ATP) from coextruded laminate film at 50°C, determined by HPLC analysis (a) Time ATP Concentration Relative % ATP (hours) (wt/wt%) (Ct/Co)x100 0 0.0283 . 100 8 0.0215 75.8 16 0.0212 74.7 24 0.0189 66.6 32 0.0142 50.1 40 0.0123 43.5 48 0.0108 38.1 56 0.0065 22.9 64 0.0049 17.4 72 0.0033 11.6 (it) Average of three replicate sample analyses, with an average standard deviation of i 5% 84 Table 8. Rate constants for the loss of ATP from the coextruded laminate film structure Temperature (°C) Loss Rate Constant k (hrq) 23 0.0004 30 0.0021 40 0.0068 50 0.0284 85 100 \\\ o \\ -————~- 3 \\\ . 50 0C \\ >4 l \\ C .40 0C 10 \ \\\ ‘30 0C " - ‘\ o° ‘\ .23 0C \u l" \ \\ ____ _ J 8 .( \ ' r, l x in Coextruded Laminate Film L L L l T 0 1300 2600 3900 5200 6500 Relative Concentration of alpha-tocopherol Storage Time (hours) Figure 14. Loss of ATP from the coextruded laminate film as a function of time/temperature 86 T ~ ~ 7444 01 A \\\ 3 \ I, 001 \\\\ ) \Q\\ I \. .\\\\\:\\ 0.001 . \ Rate Loss Constant 0.0001 0.0031 0.0031 0.0032 0.0032 0.0033 0.0033 0.0034 0.0034 l/T‘(°k*) J Figure 15. Arrhenius plot of loss rate constant versus temperature 87 Relative Rate Losses of Antioxidants The relationship between the rate of loss of BHT and a-tocopherol from the laminate structures evaluated in the present study is shown graphically in Figures 16, 17 and 18, where the relative percent antioxidant remaining in the film is plotted as a function of time for rate loss studies carried out at 23, 30 and 40°C, respectively. The rate loss data for BHT from Film II was reported by Bailey (1995). As can be seen, the a—tocopherol exhibits a significantly lower rate of loss, as compared to the loss rate of BHT from the lamination. The linear expression derived for the 0(- tocopherol rate loss at ambient temperature (23°C) gave a rate constant of 0.0004 (hr—1). When compared to the loss rate constant of BHT from the laminate at 23°C (k=0.0043 hrd), the rate of loss of BHT from the laminate was approximate 11 times greater than the rate of loss of a- tocopherol. At 30°C, the rate of loss of BHT from the laminate was approximately 10 times greater than the rate of loss of a—tocopherol, while the rate of loss of BHT at 40°C was also an order of magnitude greater than the rate of loss of a—tocopherol. 88 The lower rate of loss of a-tocopherol from the laminate may be attributed to a higher rate of diffusion of BHT through the laminate surface layers, or a difference in the rate of evaporation of the BHT from the respective surface layers, as well as to the equilibrium partition distribution of the antioxidants between the respective layers of the lamination. Antioxidant transport is related to the solubility of the additive within the respective layers of the lamination and the diffusion of the additive through it. Differences in either the solubility or diffusivity can affect the transmission characteristics of the respective antioxidants. The solubility difference depends primarily on the difference in the physico—chemical nature of the migrating species and the respective laminate layers and will be reflected in the partition distribution of the antioxidant between the laminate layers. On the other hand, differences in antioxidant diffusivity is determined largely by the size and shape of the nmflecule, and by the degree of aggregation among the diffusing species within the polymer layers. The fact that the initial concentration of the antioxidant BHT was significantly higher than the initial 89 level of a-tocopherol incorporated into the laminate may also be a contributing factor to the observed lower rate loss for a—tocopherol. 90 a 100 \ w-t ‘ -\‘\..\ ‘ u <5 Q ~ } ' .. (U . I... g b. \ 5 x E "6 e i O H .| -r-i -r-t ,, 1.) F14 + 0) . E U 0 )‘ \“‘\. Q4 (13 O l \ O C H t \ I 'r-i I I: E x l \\‘\ , o m 10 )_ “ ! 'H ’4 A0 “ f; 'U Q ( .Relative % of BHT (Film II) a) u ' B 'U U P '9 Relative 1: of ATP (Film I) c 5 V I a) :4) ), 2 x ) o 0) l U 8 1‘ l 3 t "-1 ) t; ( .—4 a) 1 m . 0 1000 2000 3000 4000 5000 Storage Time (hours) Figure 16. Relative percent loss of BHT and a-tocopherol from coextruded laminated films as a function of time (23°C) 91 1 O r ‘ \ \»t.\_ 1. .Relative % of BHT (Film II) (Ct/Co) x 100 O K .Relative % of ATP (Film I) Relative Concentration of Antioxidant in Coextruded Laminate Film l I l l | 0 100 200 300 400 500 600 700 800 Storage Time (hours) Figure 17. Relative percent loss of BHT and a-tocopherol from coextruded laminated films as a function of time (30°C) 92 = .5 100 H E 4.) :1»- I i n A “5. ! g )I .‘~\\ - 5 -H l. i X E ‘I l 3 '31 " l I i E m M \\ I : o q . | )m E 8 ) °!\ ; : o c H l \ ! l 'H ‘ “‘x\ ; ! s E x i E : o 6.1 10 t. i : "-1 .4 o i ( ; ‘J U a - ) ggg'g \: * ( .Relative % of BHT (Film II) . 4..) 'U U '. I ‘ s 5‘“ . 8 t: ( .Relative % of ATP (Film I) l C: K ) o o l : U 0 I E U l | m ) : > . .H ', D ) ‘° ) H g 1 k . . 0 100 200 300 . 400 1 Storage Time (hours) Li _A * _ __¥ _ k __ _ _ _ __¥m__ _-i Figure 18. Relative percent loss of BHT and a-tocopherol from coextruded laminated films as a function of time (40°C) 93 Product Storage Studies The results of hexanal analysis carried out on cereal product packaged in pouch I (a-tocopherol impregnated pouch structure), pouch II (BHT impregnated pouch structure), and pouch III (control pouch structure) are present in Table 9. The calibration factor applied for analyses carried out from week 0 through week 20 was based on the initial hexanal calibration curve constructed. The remaining calculations were all based on a calibration factor derived from a second hexanal calibration curve (see Appendix B). The relationship between the extent of lipid oxidation and the respective laminate package structures evaluated in the present study is shown graphically in Figure 19, where the hexanal concentration in the cereal product is plotted as a function of storage time. As shown, up through 20 weeks of storage the levels of hexanal detected in the cereal product packaged in the three test structures were very similar. Based on a one-way analysis of variance, by Tukey’s analysis from.C)txa 20 weeks, there was no statistically significant difference (p>0.05) between the levels of hexanal in the cereal product packaged in the three test pouches. After the 20th. week of storage, however, product packaged in 94 pouches without antioxidant exhibited a significantly higher level of hexanal (p<0.05), as compared to the product packaged in the antioxidant impregnated laminate structures. Over a 44 week storage period, there appeared to be no significant difference (p>0.05) between the a-tocopherol impregnated pouches and BHT impregnated pouches in terms of hexanal levels detected. inns statistical analysis carried out is presented in detail in Appendix F. Although the BHT and a-tocopherol have different characteristics such as volatility and stability, both appeared to provide similar effectiveness in terms of retarding lipid oxidation of the cereal product. Fritsch and Gale (1971) monitored the concentration of hexanal in oat, corn and wheat cereals stored at 37°C for 12 weeks. They determined a level of 5mg/kg (5ppm, wt/wt) to be indicative of significant deterioration in quality. As shown in Table 9, after 44 weeks, the cereal samples had not yet reached this level of hexanal concentration when held at 23°C and 50%RH. MCWeeny (1968) studied a series of reactions associated with food systems, which included: (1) the oxidation of unsaturated fats and oil; (2) oxidized flavor in milk fat; and (3) vitamin lose in fried foods. He 95 stated that the rate of a chemical reaction is affected by temperature and sometimes a negative rate of change of the reaction can occur when the substrate is involved in an alternate pathway. In the current study, the low concentrations of hexanal determined may be attributed to the low relative humidity and temperature conditions of the storage environment. In normal distribution, according to packaging scientists at General Mills Inc. (Culter, 1995), a cereal product has a shelf life of approximately nine months. In this study, the cereal product packaged in the antioxidant impregnated packaging structures showed decreasing levels of hexanal produced, as well as a reduction in the rate of oxidation, when compared to the product stored in laminate pouches to which no antioxidant was incorporated. These results provide supportive evidence for the effectiveness of the evaporation/sorption mechanism of antioxidant activity. Table 9. 96 packaged in pouches fabricated from test coextruded laminate structures Hexanal Concentration (pg/g cereal product) The hexanal concentration of cereal product Storage Time Pouch I m)M) Pouch II m>m> Pouch III W)M) (weeks) 0 0.141 0.141 0.141 2 0.163 0.171 0.164 4 0.147 0.182 0.168 6 0.165 0.187 0.190 8 0.172 0.162 0.180 12 0.180 0.201 0.162 16 0.202 0.164 0.191 20 0.206 0.182 0.234 28 0.249 0.188 0.382 36 0.265 0.296 0.460 44 0.289 0.324 0.552 (a) (b) (d (d) pouch I - a-tocopherol impregnated pouch structure pouch II - BHT impregnated pouch structure pouch III - control pouch structure Average of three replicate sample analyses, with an average standard deviation of i 5% 97 til +POUCh I (Tocopherol impregnated pouch) } - Pouch II (BHT impregnated pouch) I . pouch III (Control pouch) I,_. 0.5 rd ‘3 I rd >< I (D 5 I m 0.4 I \ I m . d I c I .2 0.3 _ ; 1(6) I )4 . . U 1| i c: I 9 I C: 0.2 . O I U l r-t (U I 8 I X 0.1 a) I :1: I 0 I . ¢ 0 10 20 30 40 50 ' Storage Time (weeks) Figure 19. Hexanal concentration in cereal product packaged in pouches fabricated from test coextruded laminated structures 98 Retained Levels of Antioxidant in Package Structures After removing the cereal product, the BHT and. a- tocopherol content of the pouch material was determined as a function of storage time. No BHT was found in the BHT impregnated laminate structure after 4 weeks storage at 23°C and 50%RH. Bailey (1995) carried out a series of studies monitoring the loss of BHT from the test Film II and found no detectable level of BHT after storage for 3.2 weeks at 23°C. These result were in agreement with the present finding. In earlier studies, Hoojjat et al. (1987) determined the rate of loss of BHT from HDPE at 21.5°C, and found approximately 80% of the initial quantity of BHT lost after 1 weeks storage. The results of the studies involving determination of the a-tocopherol levels lost from the package structure with time are summarized in Table 10. Comparing these results with those obtained from the laminate film (see Table 4) showed the rate of loss of a- tocopherol from the package to be somewhat less than that from the laminate film directly. In determining the rate of loss of antioxidant from the laminate film, the outer and inner surface layers are both exposed to the external environment, which acts as a infinite sink. However, when 99 the laminate film is fabricated to a package, the outer film surface is exposed to the external environment, while the heat seal layer is in contact with a finite environment. This may account for the observed differences in the losses of a-tocopherol. Loss of an additive, such as a-tocopherol or BHT, from the surface of a polymeric structure, like the laminate film studied, is determined by its solubility within the respective layers of the lamination and the diffusion coefficient of the additives in the polymer. Calvert and Billingham (1979) pointed out that BHT loss from thick films and bulk solids is determined by the rate of diffusion through the film, while antioxidant losses from thin films and fibers are controlled. by their ‘volatility from the surface. A number of studies have shown that BHT has high steam volatility (Dziezakm 1986; Figge, 1976; T111, 1982). Therefore, BHT is easily volatilized from the surface of the polymer into either the package headspace or to the external environment. Compared tx> BHT, a-tocopherol is rmxfli more stable while present in a polymer film. Its long side chain provides good solubility in the polymer and it does not begin to volatilize until temperatures reach 300°C 100 (McMurrer, 1991). Studies show that only 100 ppm(wt/wt) of a-tocopherol in. HDPE film are needed to enhance flavor retention and shelf life (Ho et al., 1994; Laermer, 1993). 101 Table 10. a—Tocopherol content in packaging pouches as a function of storage time Storage time a-tocopherol Relative % remaining concentration°fl in packaging pouches (weeks) (%wt/wt) 0 0.0238 100 4 0.0193 81.0 6 0.0192 80.7 8 0.0185 77.8 12 0.0124 51.9 16 0.0116 48.6 20 0.0115 48.6 28 0.0071 29.3 36 N/A(b) N/A(b) fa) Average of three replicate sample analysis, with an average standard deviation of i 5% m) N/A - Not available 102 Antioxidant Content of Cereal Product A schematic diagram of the mechanism by which antioxidant impregnated materials may control lipid oxidation is presented in Figure 20. As shown, the mechanism requires diffusion of antioxidant through the polymer bulk phase, evaporation of the antioxidant from the surface of the packaging material, diffusion of the antioxidant in air and, lastly, sorption of antioxidant onto the product surface. To establish the validity of this mechanism of antioxidant activity, storage studies were carried out as described and the product analyzed for the extent of lipid oxidation and for antioxidant content (i.e., extent of antioxidant sorption) as a function of time. The results of analysis for BHT levels in the cereal product packaged in the BHT impregnated laminate pouch are summarized in Table 11. As shown, after 4 week of storage, approximately 15% of the BHT initially present in the packaging film was transferred or sorbed by the product, while no detectable level of BHT was found to remain in the packaging structure. The results further show that the BHT concentration in the product decreased with increased storage time. :n: can 103 be assumed that the sorbed. BHT is ‘undergoing oxidative reactions as part of its antioxidant function, thus accounting for its decrease in concentration with time. The level of a—tocopherol present in the cereal product could not be determined, due to difficulties experienced with achieving chromatographic separation of a— tocopherol from the other compounds extracted from the cereal product. However, from the data shown in Table 10, it was assumed that some level of a-tocopherol migrated to the food product to provide antioxidant activity. Alternatively, the a-tocopherol incorporated in the laminate structure may act to retard lipid oxidation of the oat cereal product by inhibiting polymer surface catalyzed oxidative reactions. This assumes rapid diffusrmn of the a—tocopherol from the core layer, to the heat seal polymer surface. Precedent is found in the literature for surface catalyzed oxidation reactions involving polyolefins which are thermally processed at elevated temperatures resulting in their oxidation and the formation of hydroperoxide functionality on the polymer surface (Mannheim et al., 1987; Mannheim et al., 1988; Culter, 1992). 104 The migration of BHT has been widely studied by a number of investigators. Schmope et al. (1986) studied the migration of the antioxidants, BHT and Irganox®-1010 from LDPE to food and food-simulating liquids. They found BHT transferred into the food phase more rapidly than Irganox®— 1010. The rate of migration will also depend on the nature of the contact media and the polymeric contact phase. Figge et al. (1976) reported that BHT migration from PVC into various food products was much lower than from HDPE. Till et al. (1982) stated that BHT migration from HDPE to fatty foods occurs at a faster rate than to aqueous foods. There is little information available concerning the migration of a-tocopherol. Laermer et al. (1993) studied the migration of a-tocopherol from HDPE film to both heptane and a 50% aqueous ethanol solution by an immersion procedure and found that 8.4% of the initial (it-tocopherol additive present in the HDPE film migrated to the heptane while 3.8% transferred to the ethanol solution. Most cereal grains contain appreciable quantities of a—tocopherol (Herting, 1969; Slover, 1983; Slover, 1969). Manufacture of oatmeals resulted in relatively little or no decrease of vitamin E, but more extensive processing 105 increased losses to about 90% (Herting, 1969). In this study, the level of (IL-tocopherol initially present in the product was not determined for reasons stated above. 106 Outside I I Package Diffusion of Antioxidant Diffusion of I Through Polymer Structure Oxygen Polymer Film 17 Film Resistance I I Diffusion of Antioxidant in Air Head space V V l Antioxidant RH + 02—9 R 0 + 0 OH 02 L\\\ 1 r///J \/ \/ w + + + Product I.. I. I RH R00 0 Figure 20. Schematic of the mechanism of antioxidant activity of a polymeric packaging material 107 Table 11. BHT content in packaged cereal product following storage at 23°C Storage Quantity Relative percent (wt/wt) time distribution of BHT in (Weeks) (09) cereal product 0 4024.97“’ 100 4 597.36“) 14.84 6 408.81“) 10.16 8 362.41m) 9.00 12 357.44“) 8.88 16 223.00”) 5.54 28 119.96“) 2.98 fa) (b) Average of two replicate sample analysis Average of three replicate sample analysis SUMMARY AND CONCLUSIONS (it-Tocopherol loss from the multi-layer laminate structure was determined as a function of time and temperature. Rate loss studies were carried out at 23, 30, 40, 50°C, respectively. Graphical analysis indicated that a first order rate expression provided a good description of the rate of loss of the antioxidant from the laminate structure. The temperature dependency' of the transport process associated with the loss of antioxidant from the laminate film, over the temperature range studied, was found to follow the Arrhenius relationship. The determined activation energy value was 28.87 kcal/mole. The calculated loss rates for (at—tocopherol from the laminate film were compared with those reported for BHT from a similar multi- layer laminate structure and were found to be significantly lower. Based on the results of product storage studies, oat cereal product packaged in pouches containing no added antioxidant exhibited a signifiCantly high level of lipid oxidation, as compared to product packaged in the antioxidant impregnated laminate structures. Both antioxidants (BHT and a—tocopherol) were found to provide a 108 109 similar level of effectiveness, in terms of retarding product oxidation. The results of product analysis for the extent of cmidation and levels of transferred antioxidant provided supportive evidence for the evaporation/sorption mechanism of antioxidant activity. Knowledge of the rate of additive loss from a packaging structure can have significant practical applications in the packaging field, where the transfer of additives, such as antioxidants from the package to the product, plays a critical role in maintenance of product quality. The evaporation/sorption process provides a means of decreasing the levels of antioxidant present in the food stuff, and can avoid the direct incorporation of antioxidants into the food system" .Antioxidant transfer via.tflu3 evaporation/sorption mechanism offers economics advantages as well, since the antioxidant incorporated into the packaging material can both stabilize the packaging material during fabrication and retard product oxidation during distribution and storage. FUTURE STUDIES Future studies in this area may include determining the rate of loss of the antioxidant from the heat seal layer and outer HDPE layer, as well as determination of the diffusion coefficient of a-tocopherol through the respective laminate layers. Studies may also include determining the solubility of a-tocopherol in the respective polymer layers of the lamination. Such data will provide a better understanding of solubility and mobility characteristic of a-tocopherol in both the heat seal layer and HDPE layer and can provide a means of optimizing antioxidant transfer to a contained product, based on judicious selection of the package laminate structure and laminate layer in which the antioxidant is initially incorporated. Other areas of study may involve developing an appropriate analytical procedure to allow monitoring the extent of transfer of antioxidant (i.e. a-tocopherol) from the package to the contained product, and evaluating the effect of relative humidity and temperature on the oxidation of a food system stored in antioxidant impregnated laminate film structure. 110 111 The mass transfer characteristics of other commercial antioxidants, such as the Irganox series and their capacity to retard lipid oxidation also warrants investigation. APPENDICES 112 APPENDIX A Table 12. a-Tocopherol calibration curve Sample Grams of a- Area Response Tocopherol Injected 1a 1.0E-08 9251 lb 1.0E-08 9405 1c 1.0E—08 8673 Average 1.0E-08 9110 2a 2.0E-08 20654 2b 2.0E-08 20531 2c 2.0E—08 20930 Average 2.0E-08 20705 3a 3.0E-08 34364 3b 3.0E-08 33064 3c 3.0E-08 33870 Average 3.0E—08 33756 4a 4.0E-08 45620 4b 4.0E—08 44715 4c 4.0E—08 46183 Average 4.0E-08 45506 5a 5.0E-08 57035 5b 5.0E-08 56727 5c 5.0E-08 57877 Average 5.0E-08 57369 CF 8.24E-13 gm/AU 113 APPENDIX A 6E+04. 5E+04 4E+04 R? = 0.9997 3E+04 2E+04 Area Response 1E+04 OE+OO I L T; A T I 0E+00 1E-08 2E-08 3E-08 4E-08 5E-08 6E-08 Quantity of alpha-tocopherol (9) Figure 21. a-Tocopherol calibration curve 114 APPENDIX B Gas Chromatography Hexanal Calibration Data Table 13. Hexanal data for the first calibration curve (a) Grams of Hexanal Injected Area Response 1.20E—07 2101586 1.44E—07 2627925 1.68E-07 3235168 1.92E-07 3615613 2.16E-07 3832848 2.40E-07 4833968 2.64E-07 5260066 2.88E—07 5609180 3.12E-07 6538632 3.36E-07 6922237 (a) Average of three replicate sample analysis, with an average standard deviation of i 5% CF = 4.47E-14 gm/AU Area Response 115 APPENDIX B 8E+6 7E+6 6E+6_ 5E+6- 4E+6 3E+6 2E+6 1E+6 1 ii I l T I I 1.0E-07 1.5E-07 2.0E-07 2.5E—07 3.0E-07 3.5E-07 Quantity of Hexanal (9) Figure 22. First hexanal calibration curve 116 APPENDIX B Gas Chromatography Hexanal Calibration Data Table 14. Hexanal data for the second calibration curve (m Grams of Hexanal Injected Area Response 2.4E-08 476738 1.15E-07 2225511 1.88E—07 3571791 2.26E-07 4488571 3.07E-07 5652685 3.73E—07 6914579 (a) Average of three replicate sample analysis, with an average standard deviation of i 5% C.F = 4.34E-14 gm/AU ‘- 117 APPENDIX B 8E+06 7E+06 6E+06 5E+06e 4E+06 3E+06 Area Response I 2E+06 _ 1E+06 A I 0E+00 , T . 0.0E+00 1.0E-07 2.0E-07 3.0E-07 4.0E—07 I Quantity of Hexanal (g) Figure 23. Second hexanal calibration curve 118 APPENDIX C Table 15 Thickness(um) measurements performed by optical microscopy Sample Surlyn/EVA Total HDPE Total Film layer Thickness 1 15.9 44.0 59.9 2 14.5 43.2 57.7 3 12.6 47.2 59.8 4 17.2 42.1 59.3 5 13.4 45.1 58.5 Average 14.7 44.3 59.0 Standard Deviation i 1.86 i 1.95 i 0.93 119 APPENDIX D Table 16. Thickness(um) measurements performed by micrometer Sample Total Thickness 1 53.3 2 58.4 3 68.6 4 68.6 5 58.4 6 58.4 7 55.8 8 55.8 9 58.4 10 55.8 Average 59.1 Standard Deviation H- Ul .25 120 APPENDIX E Table 17. Percent recovery data and calculations Injection Initial Concentration of Concentration extract solution (gm/ml) (g/ml) 1 0.000144 0.000133 2 0.000131 0.000133 3 0.000136 0.000134 Average 0.000137 0.000133 Recovery 97.08% 121 APPENDIX F One-way Analysis of Variance (0-20 Weeks) Analysis of Variance on quantity Source DF SS MS F P Sample 2 0.00022 0.00011 0.10 0.905 Error 69 0.07738 0.00112 Total 71 0.07761 Individual 95% Cis For Mean Based on Pooled StDev Level N Mean StDev ----- + ------- + ------ +---- 1 24 0.16670 0.04347 ( ---------- * -------- ) 2 24 0.17079 0.02236 ( ---------- * --------- ) 3 24 0.16754 0.03122 ( ---------- * -------- ) ----- +-------+-—--—--+--- Pooled StDev = 0.03349 0.160 0.170 0 180 Tukey's Pairwise comparisons Family error rate = 0.0500 Individual error rate = 0.0193 Critical value = 3.39 122 APPENDIX F One-way Analysis of Variance (28-44 weeks) Analysis of Variance on quantity Source DF SS MS Sample 2 0.23090 0.11545 Error 24 0.09118 0.00380 Total 26 0.32208 Mean Level N Mean StDev 1 9 0.46467 0.07634 2 9 0.26933 0.06496 3 9 0.26767 0.03674 Pooled StDev = 0.06164 Tukey's Pairwise comparisons Family error rate = 0.0500 Individual error rate = 0.0198 Critical value = 3.53 F P 30.39 0.000 Individual 95% Cis For Based on Pooled StDev --+ ----- + ----- + ----- +--- (---*---) (---*---) (---*---> —-+ ----- + ----- + ----- +--- 0.24 0.32 0.4 0.48 LIST OF REFERENCES LIST OF REFERENCES Agbo, 0. 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