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The influence of fat on the deterioration of food aroma in model systems during storage
E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved
573
The Influence of Fat on the Deterioration of Food Aroma in Model Systems during Storage M. Chen and G.A. Reineccius Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108 USA Abstract Storage studies (30 and 45°C) determining the stability of selected aroma compounds (50 ppm) in the presence of simple microcrystalline cellulose (MCC)(with or without 5% vegetable oil and water activities of 0.11, 0.33, 0.53 and 0.76) were conducted. MCC was found to exhibit substantial aroma compound binding. Binding appeared to be strongly dependent on water activity with the binding being less at higher water activities and appeared to reach a maximum at an Aw of approximately 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. Furfurylmercaptan, pyrrole, 1-methylpyrrole, 2,4-hexadienal and ethyl-2mercaptoproprionate were found to be very unstable during storage (listed in decreasing order of loss). The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so rapid that the effect was small. The degree of increase in stability in the presence of oil was dependent upon the aroma compound studied.
1. INTRODUCTION There has been a relatively long term trend in the US to develop food products which have a reduced fat content. It is well documented in the literature that reduced fat foods lack the same sensory qualities as their full fat counterparts [1,2]. These differences in products may relate to any of several factors including flavor/food matrix interactions, flavor release, flavor formation (e.g. fried products) or textural changes [2-8]. Thus we have come to accept that reduced fat foods will generally be of lesser sensory quality than the full fat food. There are other flavor issues related to reduced fat foods that may be less obvious [1]. One relates to the quality of ingredients required in reduced fat versus the full fat products. We often find that a reduced fat food will have undesirable flavor notes associated with ingredients. Fat will mask off flavors and thus when fat is taken out of a food, the off flavors associated with ingredients are freely expressed. A low fat food may well have very perceptible off flavors using the same ingredients as were used in the formulation of an acceptable flavored full fat counterpart. A similar situation exists for the susceptibility of a food to off flavor development due to contamination from the environment (e.g. water, air or packaging sources). A low fat food is quite susceptible to off flavor development from these
574 sources relative to a full fat version of the product. This may translate to needs for improved quality packaging (less residual volatiles or better barriers to organic volatiles). The work to be reported in this paper addresses the issue of shelf-life of low fat versus full fat foods. Low fat foods typically have a shorter shelf-life than full fat foods. When a food changes in flavor during storage, it is seldom clear whether this change is due to the formation of undesirable flavors (e.g. oxidative rancidity) or the loss of desirable flavors. We know that some off flavors (e.g. lipid oxidation) are quite good at masking desirable flavor notes of a food. Also the question arises whether fat content has any influence on the stability of desirable aroma constituents in a food during storage. If the flavor of a food is more stable in the presence of fat, this might account for the stability differences one observes in low fat foods. This contribution is part of a larger study in which the stability of flavor compounds in the presence various food constituents (protein, sugar fat and microcrystalline cellulose) was monitored during storage [9]. We will present data and discuss the role of fat in stabilizing food aromas during storage.
2. MATERIALS AND METHODS 2.1. Materials Microcrystalline cellulose (MCC) (FMC Corp., Newark, DE.) and Crisco oil (Procter and Gamble, Cincinnati, OH) were used to make food model systems. Systems were prepared with distilled water purchased from the Glenwood Inglewood Company (Minneapolis, MN). Four salts were used to prepare saturated solutions for adjusting water activity: lithium chloride and magnesium chloride (Mallinckrodt Inc., Paris, KY); magnesium nitrate (Sigma Chemical Company, St. Louis, MO) and sodium chloride (E.M. Science, Gibbstown, NJ). Methylene chloride (Fisher Scientific; Fair Lawn, NJ) was used as a solvent to prepare the flavor solution. Sylon - CT silanizing reagent was obtained from Supelco Inc. (Bellefonte, PA), which was used to treat glass vials. Eleven flavor compounds and an internal standard were used in our studies: Isovaleraldehyde, 1-methylpyrrole, pyrrole, 2-methylthiophene, hexenal, nonane (internal standard), furfurylmercaptan, 2,4-hexadienal, diethyldisulfide, ethyl-3-mercapto-propionate, phenylacetaldehyde, and 2-ethyl-3,5(or3,6)-dimethylpyrazine. 2.2. Sample Preparation 2.2.1. Flavor Stock Solution A 5000 ppm stock solution of most flavor compounds was prepared by adding 0.250 g of each flavor compound to about 30 mL methlene chloride in a 50 mL volumetric flask. The solution was brought to 50 mL with methlene chloride. This yielded a concentration of 5000 ppm. Ethyl-3-mercaptopropinate, phenyl acetaldehyde and 2ethyl-3,5 (or 3,6) dimethylpyrazine were diluted to 15,000 ppm since these three compounds had a poor gas chromatographic response. The flavor stock solution was wrapped with aluminum foil to limit light exposure. 2.2.2. Saturated Salt Solution Preparation Salts were dissolved in distilled water in 1 L beakers according to the solubility limits given in the Handbook of Physics and Chemistry [10]. Solutions were heated and agitated constantly. Additional salt was added until undissolved salt crystals remained. Solutions were cooled to room temperature and transferred
575 into vacuum dessicators. The following salts were used to create the different water activities (77): LiCl (Aw = 0.11 at room temperature), MgCl (Aw = 0.33), Mg(N03)2 (Aw = 0.53) and NaCl (Aw = 0.76). 2.2.3. Model System Preparation Eight hundred mL of distilled water were added to 400 g MCC (or 380 g MCC plus 20 g Crisco oil) and thoroughly mixed by hand to yield a paste. The paste was frozen at -20°C first, then freeze dried using a freeze dryer (VIRTIS; Gardiner, NY) for 48 hr. Five g dried powder was weighed into 20 mL headspace sample vials (Chrom. Tech.; Apple Valley, MN). They were then placed in vacuum desiccators at 4 different water activities and allowed to reach equilibration for about 2 months. The model systems were then prepared by adding 50 |iL of stock flavor solution into each headspace vial which contained the MCC or MCC plus oil and sealed with a septum cap (Chrom. Tech., Apple Valley, MN). Thus, the concentration of flavor compounds in each vial was 50 ppm for most flavor compounds and 150 ppm for the 3 volatiles that had a poor gas chromatographic response. 2.2.4. Storage and Sampling. Samples were stored at 30 and 45°C in the dark and removed at 0, 2, 4, 7, 14, 21, 28, 36, 43, 50 and 90 days for headspace analysis. For the starting values, samples were held for 3 hr at 45°C or 12 hr at 30°C before analysis. This was based on preliminary experiments which indicated that the headspace concentration of volatiles reached equilibrium in approximately 3 hr and 12 hr at 45°C and 30°C, respectively. 2.3. Analysis 2.3.1. Static Headspace Analysis. Static headspace concentrations of the volatiles were measured using a Hewlett Packard headspace autosampler HSS19375A (Hewlett Packard; Little Falls Site, DE). Samples were held for 38 min at 45°C in the autosampler prior to analysis. One mL headspace was introduced into the GC. 2.3.2. Gas Chromatography. Headspace volatiles were separated and quantified using a Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard; Little Falls Site, DE) equipped with a flame ionization detector and GC Chem-Station (Hewlett-Packard, Avondale, PA). A DB-5 fused siUca capillary column, 30 m long, 0.32 mm i.d., 1.00 um film thickness (J&W Scientific; Rancho Cordoba, CA), was used to separate the model system volatiles. The carrier gas was helium (2.8 mL/min) at a head pressure of 12 psi. The GC oven was temperature programmed as follows: initial temperature, 40°C; initial time, 0 min; program rate 1, 5°C/min; final temperature, 80°C; final time 1, 5 min; program rate 2, 12°C/min; final temperature 2, 200°C; and final time 2 min. 2.3.3. Reproducibility. The reproducibility of analysis was determined by conducting the following experiment: 20 \xL of flavor stock solution was added to four 20 mL headspace vials containing 5 g MCC (or MCC plus 5% Crisco oil) at a water activity of 0.11. The GC peak areas were determined by GC-HS analysis under the described experimental conditions. The arithmetic means, standard deviations and the coefficient of variance values (%CV) were calculated. 2.4. Preliminary studies Preliminary studies were conducted to determine the equilibrium time required after adding flavor solutions to the model systems. Samples were analyzed at 0, 3, 6, 12, 24 and 48 hr at 30°C and 0, 3, 6, 12 hr. at 45°C. Peak area versus equilibration time were plotted at each temperature. Flavor compounds were also added to empty headspace vials without any food
576 matrix to determine if they interacted with each other or the glass vial wall. To check for interactions with the glass vials, the vials were treated with Sylon-CT silanizing reagent, flavor solution was then added and analyzed using the same experiment conditions. 2.5. Data presentation. The GC peak areas were determined for each flavor compound in all model systems. The assumption was that zero day values represented 100%, therefore, the peak areas of all the other days were compared to the zero day values and are presented as a % remaining.
3. RESULTS AND DISCUSSION 3.1. Aroma Stability during Equilibration The data that will be presented are based on the amount of flavoring in the headspace at what was considered "zero" time. The choice of "zero" time is not all that obvious as can be appreciated from the data in Figure 1 where we were attempting to determine the optimum equilibration time to use in analysis. Time 0 on the Figure corresponds to 38 min equilibration in the headspace sampler. The initial decrease in many of the aroma compounds appears to be related to their interaction with the MCC since their headspace concentrations decreased rapidly and then "stabilized" suggesting saturation of all binding sites. Based on these data, we chose to use 12 hr GC peak areas as the zero starting time at 30°C and 3 hr values as time zero for the 45°C samples (data not shown). 120 T ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL
FURFURYLMERCAPTAN 2,4-HEXADIENAL DIETHYLDISULFIDE ETHYL-3MERCAPTOPROPIONATE PHENYLACETALALDEHY DE 2-ET-3,5-DIMEPYRAZINE 2-ET-3,6-DIMEPYRAZINE
Figure 1. The influence of equilibration time in headspace sampler (30°C) on the concentration of headspace volatiles (MCC system).
577 We were concerned about the very rapid loss of volatiles in the MCC containing vials and wanted to determine if the volatile losses were due to interactions with the MCC or to possible interactions between the glass vials and aroma compounds or reactions between aroma compounds themselves. This prompted us to add the aroma compounds to empty glass vials (silanized and unsilanized) and monitor their losses during storage (Figure 2). This experiment pointed out that 38 min was not adequate for all compounds to reach equilibrium in the headspace. The amount of phenylacetaldehyde and ethyl-3-mercaptoproprionate reached equilibrium with the headspace some time between the initial sampling time and 3 hr sampling. Also as can be seen in Figure 2, most of the aroma compounds were quite stable in the empty vials (silanized or unsilanized). Only furfurylmercaptan, exhibited substantial losses during storage during this brief period. Thus it is assumed that the MCC was involved in hastening the loss of aroma compounds. ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL -FURFURYLMERCAPTAN -2,4-HEXADIENAL -DIETHYLDISULFIDE -ETHYL-3MERCAPTOPROPIONATE -PHENYLACETALALDEHY DE - 2-ET-3,5-DIMEPYRAZINE - 2-ET-3,6-DIMEPYRAZINE
Figure 2. The influence of equilibration time in headspace sampler (30C) on the concentration of headspace volatiles (empty glass vials - not silanized).
3.2. Effect of Oil Addition on Headspace Concentration of Aroma Compounds The addition of oil to the MCC resulted in decreased headspace concentrations of all of the volatiles used in this study except phenylacetaldehyde (Figure 3). While the method of analysis was reasonably reproducible (coefficients of variation (CV) ranged from 3 to 21% with most less than 10%), the largest CV was for phenylacetaldehyde. It is possible that the data point for this compound was in error since one would have anticipated that it also decreases in the oil containing system. The decrease in headspace concentration with the addition of oil is consistent with theory and not surprising.
578
MCC control MCC + 5% Oil
^O ^
.^ ^
A^ Jy
^
« * /
^^
rJ^
rJ^
Flavor Compounds Figure 3. The influence of including oil with MCC on the concentration of headspace volatiles (ISO-isovaleraldehyde; MP: 1-methylpyrrole; PYR: pyrrole; MT: 2-methylthiophene; HEX: hexanal; FFM: furfurylmercaptan; HD: 2,4-hexadienal; DD: diethyldisulfide; EMP: ethyl-3mercaptopropionate; PA: phenylacetaldehyde; EDP(3,5): 2-ethyl-3,5-dimethylpyrazine; EDP(3,6): 2-ethyl-3,6-dimethylpyrazine.
3.3. Effect of Oil Addition on the Stability of Aroma Compounds While we could have presented data on all of the aroma compounds studied, we will present only a sampling due to manuscript limitations (45C data only and selected aroma compounds). The reader is encouraged to obtain Ms. Chen's thesis [3] for greater detail. 3.3.1. Hexanal. Hexanal was lost from the headspace to a great extent during storage in the presence of MCC (Figure 4). There appears to be an effect of water activity in that hexanal was lost the least from the highest water activity (0.75) and the greatest at the lowest water activities (0.11 and 0.33). There appears to be no difference between the two lower water activities. Hexanal losses were greatly retarded in the MCC + oil system. Losses tended to follow what one might expect from typical oxidation reactions in that losses were lowest in the highest water activity system and increased with decreasing water activity until the monolayer (0.33) and then decreased once below the monolayer (0.11). 3.3.2. 2,4-Hexadienal. Losses of 2,4-hexadienal were very rapid with nearly complete loss from the headspace in 20 days. One could not discern any dependency on water activity and the effect of oil in the model system was positive but minimal (data not shovm).
579
100 90
T
MCC
-•-0.11 -^-0.33 -A-0.53 ^^0.76
80 3)70 = 60
150
|\
\/
30 20 10 0 1
\
Days
"
50
•—1 100
Days
50
100
Figure 4. The loss of hexanal at different water activities during storage (45°C, left Figure has no oil; right has oil).
3.3.3. Furfurylmercaptan. Furfurylmercaptan was one of the most labile compounds included in this study (Figure 5). It was nearly completely lost from the MCC sample headspace in 5 days storage at 45°C. The addition of oil to the system provided substantial stability. There is again an influence of water activity with the fiirfuryl mercaptan being most stable at the highest water activities. Losses were too rapid at either storage temperature to determine if they would have been lowest at the monolayer and increased again as is typical of oxidation reactions. 30 25
MCC 45*'C
?20 15
MCC + 5% Oil 45X
-0.11 •0.33 -0.53 -0.76
10 -t
0 l a t f fltf MM^tp Days
50
=M^ 100
Days
50
100
Figure 5. The loss offtirftirylmercaptanat different water activities during storage (45°C, left Figure has no oil; right has oil).
3.3.4. 2-Methylthiophene. While the data for the 0.53 Aw in the MCC system are somewhat erratic, the trend of continuous loss (nearly linear) during storage is evident (Figure 6). There appears to be increased losses with decreasing water activity. The presence of oil in the
580
120 J 100
MCC + 5% Oil
^ * * ^ " ' '
rv5S)>—^ • •
'E
1 *^v5
I 60 0)
—•—0.11 ^ -•-0.33 -A-0.53 -><-0.76
40 20
Days 50
+
100
Days
J
1
50
100
Figure 6. The loss of 2-methylthiophene at different water activities during storage (45°C, left Figure has no oil; right has oil).
system greatly reduced losses from the headspace although they followed similar loss patterns (linear with time). The effect of water activity is clearer in this system. 3.3.5. Diethyldisulfide. Diethyldisulfide (DEDS) losses during storage were somewhat different from compounds discussed to this point. Losses were nearly linear with time and water activity dependent in the MCC system (consistent with other compounds) but extremely stable in the oil containing system (Figure 7). It appears that there was some interaction with the MCC but once the binding sites on the MCC were satisfied, the DEDS was very stable.
140 n 120
MCC 45°C
dlOO !E
80
140 n -4—0.11 -•-0.33 -A-0.53 -^<-0.76
120 0)100 c ;E 80
ieo
1 60
^40
^
MCC + 5% Oil —•—0.11 45"'C -•-0.33 -A-0.53 -K-0.76
^^*MdSf^ ^ ^'m
20
Hi
40 20
1
0 Days
50
^ 100
0 Days
\
1
50
100
Figure 7. The loss of diethyldisulfide at different water activities during storage (45°C, left Figure has no oil, right has oil).
581 3.3.6. 2-Ethyl-3,5-(limethylpyrazine. Except for losses due to initial binding with the MCC, 2-ethyl-3,5-dimethylpyrazine was stable during storage in both model systems (Figure 8). Initial binding was influenced by water activity with a trend of less binding at the higher water activities, increased binding at lower water activities to 0.33 and then decreased binding at 0.11 water activity.
M C C + 5% 45°C
Days
50
100
Days
il —#—0.11 -•-0.33 —^k-0.53 ^«-0.76
100
Figure 8. The loss of 2-ethyl-3,5-dimethylpyrazine at different water activities during storage (45°C, left Figure has no oil, right has oil)
4. DISCUSSION We were rather surprised at the high level and general nature of aroma compound binding exhibited by MCC. We expected to use the MCC as an inert support for various food components and were disappointed to observe its activity in this respect. The binding appears to be strongly dependent on water activity with the binding being less at higher water activities and appears to reach a maximum at a water activity of ca. 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. We were surprised at the very rapid loss of furfurylmercaptan during storage. This bodes badly for the coffee industry since furfurylmercaptan is considered the character impact compound of coffee. Major losses were also observed for the two pyrroles (data not presented). Their loss rates were second only to furfiirylmercaptan. 2,4-hexadienal and ethyl2-mercaptopropionate losses were 3^^^ and 4^^ , respectively. Foods which derive a portion of their characteristic flavor from these compounds would not be expected to have a good shelflife. The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so still rapid that the effect was small. The increase in stability was dependent upon the aroma compound studied. It is clear that oil has a protective effect on the stability of aroma compounds in the headspace above foods. This may translate into an increase in the shelf-life of oil-containing foods depending upon the
582 contribution of oil to lipid oxidation and the masking effect of lipids on both desirable and undesirable aroma constituents.
5. REFERENCES 1
L.C. Hatchwell, In: Flavor-Food Interactions, ACS symposium series 633. R.C. McGorrin and J.V. Leland (eds), ACS, Washington DC, 1996, 15. 2 J. Bakker, In: Ingredient Interactions, Effects on Food Quality, A. Kumar and G. Gaonkar, (EDS), Marcel Dekker, Inc., New York, 1996, 411. 3 L.J. Farmer, D.S. Mottram, and F.B. Whitfield, J. Sci. Food Agric, Vol. 49 (1989) 347. 4 D.A. Forss, J. Agric Food Chem. no. 17 (1969) 681. 5 D.A. Forss, In: Progress In The Chemistry of Fats and Other Lipids. Vol. 13. R.T. Holman(ed), Pergamon Press, London, 1972,177. 6 B.M. King, and J. Solms, J. Agric. Food Chem. Vol. 27 (1979) 133. 7 J.E. Kinsella, In: Flavor Chemistry of Lipids in Foods. DE Min and T.H. Smouse (eds) American Oil Chem. Soc, Champaign. 1989, 376. 8 S. Shamil, L.J. Wyeth, and D. Kilcast, Food Qual. Pref, Vol. 3 (1991) 51. 9 M. Chen, The Stability of Aroma Compounds in the Presence of Food Components during Storage. M.S. Thesis, Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1997. 10. Handbook of Physics and Chemistry. CRC Press, Boca Raton, FL. 1995. 11 T.P. Labuza, Course titled: "Freezing and Dehydration of Food". Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1993, 81.
573
The Influence of Fat on the Deterioration of Food Aroma in Model Systems during Storage M. Chen and G.A. Reineccius Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108 USA Abstract Storage studies (30 and 45°C) determining the stability of selected aroma compounds (50 ppm) in the presence of simple microcrystalline cellulose (MCC)(with or without 5% vegetable oil and water activities of 0.11, 0.33, 0.53 and 0.76) were conducted. MCC was found to exhibit substantial aroma compound binding. Binding appeared to be strongly dependent on water activity with the binding being less at higher water activities and appeared to reach a maximum at an Aw of approximately 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. Furfurylmercaptan, pyrrole, 1-methylpyrrole, 2,4-hexadienal and ethyl-2mercaptoproprionate were found to be very unstable during storage (listed in decreasing order of loss). The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so rapid that the effect was small. The degree of increase in stability in the presence of oil was dependent upon the aroma compound studied.
1. INTRODUCTION There has been a relatively long term trend in the US to develop food products which have a reduced fat content. It is well documented in the literature that reduced fat foods lack the same sensory qualities as their full fat counterparts [1,2]. These differences in products may relate to any of several factors including flavor/food matrix interactions, flavor release, flavor formation (e.g. fried products) or textural changes [2-8]. Thus we have come to accept that reduced fat foods will generally be of lesser sensory quality than the full fat food. There are other flavor issues related to reduced fat foods that may be less obvious [1]. One relates to the quality of ingredients required in reduced fat versus the full fat products. We often find that a reduced fat food will have undesirable flavor notes associated with ingredients. Fat will mask off flavors and thus when fat is taken out of a food, the off flavors associated with ingredients are freely expressed. A low fat food may well have very perceptible off flavors using the same ingredients as were used in the formulation of an acceptable flavored full fat counterpart. A similar situation exists for the susceptibility of a food to off flavor development due to contamination from the environment (e.g. water, air or packaging sources). A low fat food is quite susceptible to off flavor development from these
574 sources relative to a full fat version of the product. This may translate to needs for improved quality packaging (less residual volatiles or better barriers to organic volatiles). The work to be reported in this paper addresses the issue of shelf-life of low fat versus full fat foods. Low fat foods typically have a shorter shelf-life than full fat foods. When a food changes in flavor during storage, it is seldom clear whether this change is due to the formation of undesirable flavors (e.g. oxidative rancidity) or the loss of desirable flavors. We know that some off flavors (e.g. lipid oxidation) are quite good at masking desirable flavor notes of a food. Also the question arises whether fat content has any influence on the stability of desirable aroma constituents in a food during storage. If the flavor of a food is more stable in the presence of fat, this might account for the stability differences one observes in low fat foods. This contribution is part of a larger study in which the stability of flavor compounds in the presence various food constituents (protein, sugar fat and microcrystalline cellulose) was monitored during storage [9]. We will present data and discuss the role of fat in stabilizing food aromas during storage.
2. MATERIALS AND METHODS 2.1. Materials Microcrystalline cellulose (MCC) (FMC Corp., Newark, DE.) and Crisco oil (Procter and Gamble, Cincinnati, OH) were used to make food model systems. Systems were prepared with distilled water purchased from the Glenwood Inglewood Company (Minneapolis, MN). Four salts were used to prepare saturated solutions for adjusting water activity: lithium chloride and magnesium chloride (Mallinckrodt Inc., Paris, KY); magnesium nitrate (Sigma Chemical Company, St. Louis, MO) and sodium chloride (E.M. Science, Gibbstown, NJ). Methylene chloride (Fisher Scientific; Fair Lawn, NJ) was used as a solvent to prepare the flavor solution. Sylon - CT silanizing reagent was obtained from Supelco Inc. (Bellefonte, PA), which was used to treat glass vials. Eleven flavor compounds and an internal standard were used in our studies: Isovaleraldehyde, 1-methylpyrrole, pyrrole, 2-methylthiophene, hexenal, nonane (internal standard), furfurylmercaptan, 2,4-hexadienal, diethyldisulfide, ethyl-3-mercapto-propionate, phenylacetaldehyde, and 2-ethyl-3,5(or3,6)-dimethylpyrazine. 2.2. Sample Preparation 2.2.1. Flavor Stock Solution A 5000 ppm stock solution of most flavor compounds was prepared by adding 0.250 g of each flavor compound to about 30 mL methlene chloride in a 50 mL volumetric flask. The solution was brought to 50 mL with methlene chloride. This yielded a concentration of 5000 ppm. Ethyl-3-mercaptopropinate, phenyl acetaldehyde and 2ethyl-3,5 (or 3,6) dimethylpyrazine were diluted to 15,000 ppm since these three compounds had a poor gas chromatographic response. The flavor stock solution was wrapped with aluminum foil to limit light exposure. 2.2.2. Saturated Salt Solution Preparation Salts were dissolved in distilled water in 1 L beakers according to the solubility limits given in the Handbook of Physics and Chemistry [10]. Solutions were heated and agitated constantly. Additional salt was added until undissolved salt crystals remained. Solutions were cooled to room temperature and transferred
575 into vacuum dessicators. The following salts were used to create the different water activities (77): LiCl (Aw = 0.11 at room temperature), MgCl (Aw = 0.33), Mg(N03)2 (Aw = 0.53) and NaCl (Aw = 0.76). 2.2.3. Model System Preparation Eight hundred mL of distilled water were added to 400 g MCC (or 380 g MCC plus 20 g Crisco oil) and thoroughly mixed by hand to yield a paste. The paste was frozen at -20°C first, then freeze dried using a freeze dryer (VIRTIS; Gardiner, NY) for 48 hr. Five g dried powder was weighed into 20 mL headspace sample vials (Chrom. Tech.; Apple Valley, MN). They were then placed in vacuum desiccators at 4 different water activities and allowed to reach equilibration for about 2 months. The model systems were then prepared by adding 50 |iL of stock flavor solution into each headspace vial which contained the MCC or MCC plus oil and sealed with a septum cap (Chrom. Tech., Apple Valley, MN). Thus, the concentration of flavor compounds in each vial was 50 ppm for most flavor compounds and 150 ppm for the 3 volatiles that had a poor gas chromatographic response. 2.2.4. Storage and Sampling. Samples were stored at 30 and 45°C in the dark and removed at 0, 2, 4, 7, 14, 21, 28, 36, 43, 50 and 90 days for headspace analysis. For the starting values, samples were held for 3 hr at 45°C or 12 hr at 30°C before analysis. This was based on preliminary experiments which indicated that the headspace concentration of volatiles reached equilibrium in approximately 3 hr and 12 hr at 45°C and 30°C, respectively. 2.3. Analysis 2.3.1. Static Headspace Analysis. Static headspace concentrations of the volatiles were measured using a Hewlett Packard headspace autosampler HSS19375A (Hewlett Packard; Little Falls Site, DE). Samples were held for 38 min at 45°C in the autosampler prior to analysis. One mL headspace was introduced into the GC. 2.3.2. Gas Chromatography. Headspace volatiles were separated and quantified using a Hewlett-Packard 5890A gas chromatograph (Hewlett-Packard; Little Falls Site, DE) equipped with a flame ionization detector and GC Chem-Station (Hewlett-Packard, Avondale, PA). A DB-5 fused siUca capillary column, 30 m long, 0.32 mm i.d., 1.00 um film thickness (J&W Scientific; Rancho Cordoba, CA), was used to separate the model system volatiles. The carrier gas was helium (2.8 mL/min) at a head pressure of 12 psi. The GC oven was temperature programmed as follows: initial temperature, 40°C; initial time, 0 min; program rate 1, 5°C/min; final temperature, 80°C; final time 1, 5 min; program rate 2, 12°C/min; final temperature 2, 200°C; and final time 2 min. 2.3.3. Reproducibility. The reproducibility of analysis was determined by conducting the following experiment: 20 \xL of flavor stock solution was added to four 20 mL headspace vials containing 5 g MCC (or MCC plus 5% Crisco oil) at a water activity of 0.11. The GC peak areas were determined by GC-HS analysis under the described experimental conditions. The arithmetic means, standard deviations and the coefficient of variance values (%CV) were calculated. 2.4. Preliminary studies Preliminary studies were conducted to determine the equilibrium time required after adding flavor solutions to the model systems. Samples were analyzed at 0, 3, 6, 12, 24 and 48 hr at 30°C and 0, 3, 6, 12 hr. at 45°C. Peak area versus equilibration time were plotted at each temperature. Flavor compounds were also added to empty headspace vials without any food
576 matrix to determine if they interacted with each other or the glass vial wall. To check for interactions with the glass vials, the vials were treated with Sylon-CT silanizing reagent, flavor solution was then added and analyzed using the same experiment conditions. 2.5. Data presentation. The GC peak areas were determined for each flavor compound in all model systems. The assumption was that zero day values represented 100%, therefore, the peak areas of all the other days were compared to the zero day values and are presented as a % remaining.
3. RESULTS AND DISCUSSION 3.1. Aroma Stability during Equilibration The data that will be presented are based on the amount of flavoring in the headspace at what was considered "zero" time. The choice of "zero" time is not all that obvious as can be appreciated from the data in Figure 1 where we were attempting to determine the optimum equilibration time to use in analysis. Time 0 on the Figure corresponds to 38 min equilibration in the headspace sampler. The initial decrease in many of the aroma compounds appears to be related to their interaction with the MCC since their headspace concentrations decreased rapidly and then "stabilized" suggesting saturation of all binding sites. Based on these data, we chose to use 12 hr GC peak areas as the zero starting time at 30°C and 3 hr values as time zero for the 45°C samples (data not shown). 120 T ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL
FURFURYLMERCAPTAN 2,4-HEXADIENAL DIETHYLDISULFIDE ETHYL-3MERCAPTOPROPIONATE PHENYLACETALALDEHY DE 2-ET-3,5-DIMEPYRAZINE 2-ET-3,6-DIMEPYRAZINE
Figure 1. The influence of equilibration time in headspace sampler (30°C) on the concentration of headspace volatiles (MCC system).
577 We were concerned about the very rapid loss of volatiles in the MCC containing vials and wanted to determine if the volatile losses were due to interactions with the MCC or to possible interactions between the glass vials and aroma compounds or reactions between aroma compounds themselves. This prompted us to add the aroma compounds to empty glass vials (silanized and unsilanized) and monitor their losses during storage (Figure 2). This experiment pointed out that 38 min was not adequate for all compounds to reach equilibrium in the headspace. The amount of phenylacetaldehyde and ethyl-3-mercaptoproprionate reached equilibrium with the headspace some time between the initial sampling time and 3 hr sampling. Also as can be seen in Figure 2, most of the aroma compounds were quite stable in the empty vials (silanized or unsilanized). Only furfurylmercaptan, exhibited substantial losses during storage during this brief period. Thus it is assumed that the MCC was involved in hastening the loss of aroma compounds. ISOVALERALDEHYDE 1-METHYLPYRROLE PYRROLE 2-METHYLTHIOPHENE HEXENAL -FURFURYLMERCAPTAN -2,4-HEXADIENAL -DIETHYLDISULFIDE -ETHYL-3MERCAPTOPROPIONATE -PHENYLACETALALDEHY DE - 2-ET-3,5-DIMEPYRAZINE - 2-ET-3,6-DIMEPYRAZINE
Figure 2. The influence of equilibration time in headspace sampler (30C) on the concentration of headspace volatiles (empty glass vials - not silanized).
3.2. Effect of Oil Addition on Headspace Concentration of Aroma Compounds The addition of oil to the MCC resulted in decreased headspace concentrations of all of the volatiles used in this study except phenylacetaldehyde (Figure 3). While the method of analysis was reasonably reproducible (coefficients of variation (CV) ranged from 3 to 21% with most less than 10%), the largest CV was for phenylacetaldehyde. It is possible that the data point for this compound was in error since one would have anticipated that it also decreases in the oil containing system. The decrease in headspace concentration with the addition of oil is consistent with theory and not surprising.
578
MCC control MCC + 5% Oil
^O ^
.^ ^
A^ Jy
^
« * /
^^
rJ^
rJ^
Flavor Compounds Figure 3. The influence of including oil with MCC on the concentration of headspace volatiles (ISO-isovaleraldehyde; MP: 1-methylpyrrole; PYR: pyrrole; MT: 2-methylthiophene; HEX: hexanal; FFM: furfurylmercaptan; HD: 2,4-hexadienal; DD: diethyldisulfide; EMP: ethyl-3mercaptopropionate; PA: phenylacetaldehyde; EDP(3,5): 2-ethyl-3,5-dimethylpyrazine; EDP(3,6): 2-ethyl-3,6-dimethylpyrazine.
3.3. Effect of Oil Addition on the Stability of Aroma Compounds While we could have presented data on all of the aroma compounds studied, we will present only a sampling due to manuscript limitations (45C data only and selected aroma compounds). The reader is encouraged to obtain Ms. Chen's thesis [3] for greater detail. 3.3.1. Hexanal. Hexanal was lost from the headspace to a great extent during storage in the presence of MCC (Figure 4). There appears to be an effect of water activity in that hexanal was lost the least from the highest water activity (0.75) and the greatest at the lowest water activities (0.11 and 0.33). There appears to be no difference between the two lower water activities. Hexanal losses were greatly retarded in the MCC + oil system. Losses tended to follow what one might expect from typical oxidation reactions in that losses were lowest in the highest water activity system and increased with decreasing water activity until the monolayer (0.33) and then decreased once below the monolayer (0.11). 3.3.2. 2,4-Hexadienal. Losses of 2,4-hexadienal were very rapid with nearly complete loss from the headspace in 20 days. One could not discern any dependency on water activity and the effect of oil in the model system was positive but minimal (data not shovm).
579
100 90
T
MCC
-•-0.11 -^-0.33 -A-0.53 ^^0.76
80 3)70 = 60
150
|\
\/
30 20 10 0 1
\
Days
"
50
•—1 100
Days
50
100
Figure 4. The loss of hexanal at different water activities during storage (45°C, left Figure has no oil; right has oil).
3.3.3. Furfurylmercaptan. Furfurylmercaptan was one of the most labile compounds included in this study (Figure 5). It was nearly completely lost from the MCC sample headspace in 5 days storage at 45°C. The addition of oil to the system provided substantial stability. There is again an influence of water activity with the fiirfuryl mercaptan being most stable at the highest water activities. Losses were too rapid at either storage temperature to determine if they would have been lowest at the monolayer and increased again as is typical of oxidation reactions. 30 25
MCC 45*'C
?20 15
MCC + 5% Oil 45X
-0.11 •0.33 -0.53 -0.76
10 -t
0 l a t f fltf MM^tp Days
50
=M^ 100
Days
50
100
Figure 5. The loss offtirftirylmercaptanat different water activities during storage (45°C, left Figure has no oil; right has oil).
3.3.4. 2-Methylthiophene. While the data for the 0.53 Aw in the MCC system are somewhat erratic, the trend of continuous loss (nearly linear) during storage is evident (Figure 6). There appears to be increased losses with decreasing water activity. The presence of oil in the
580
120 J 100
MCC + 5% Oil
^ * * ^ " ' '
rv5S)>—^ • •
'E
1 *^v5
I 60 0)
—•—0.11 ^ -•-0.33 -A-0.53 -><-0.76
40 20
Days 50
+
100
Days
J
1
50
100
Figure 6. The loss of 2-methylthiophene at different water activities during storage (45°C, left Figure has no oil; right has oil).
system greatly reduced losses from the headspace although they followed similar loss patterns (linear with time). The effect of water activity is clearer in this system. 3.3.5. Diethyldisulfide. Diethyldisulfide (DEDS) losses during storage were somewhat different from compounds discussed to this point. Losses were nearly linear with time and water activity dependent in the MCC system (consistent with other compounds) but extremely stable in the oil containing system (Figure 7). It appears that there was some interaction with the MCC but once the binding sites on the MCC were satisfied, the DEDS was very stable.
140 n 120
MCC 45°C
dlOO !E
80
140 n -4—0.11 -•-0.33 -A-0.53 -^<-0.76
120 0)100 c ;E 80
ieo
1 60
^40
^
MCC + 5% Oil —•—0.11 45"'C -•-0.33 -A-0.53 -K-0.76
^^*MdSf^ ^ ^'m
20
Hi
40 20
1
0 Days
50
^ 100
0 Days
\
1
50
100
Figure 7. The loss of diethyldisulfide at different water activities during storage (45°C, left Figure has no oil, right has oil).
581 3.3.6. 2-Ethyl-3,5-(limethylpyrazine. Except for losses due to initial binding with the MCC, 2-ethyl-3,5-dimethylpyrazine was stable during storage in both model systems (Figure 8). Initial binding was influenced by water activity with a trend of less binding at the higher water activities, increased binding at lower water activities to 0.33 and then decreased binding at 0.11 water activity.
M C C + 5% 45°C
Days
50
100
Days
il —#—0.11 -•-0.33 —^k-0.53 ^«-0.76
100
Figure 8. The loss of 2-ethyl-3,5-dimethylpyrazine at different water activities during storage (45°C, left Figure has no oil, right has oil)
4. DISCUSSION We were rather surprised at the high level and general nature of aroma compound binding exhibited by MCC. We expected to use the MCC as an inert support for various food components and were disappointed to observe its activity in this respect. The binding appears to be strongly dependent on water activity with the binding being less at higher water activities and appears to reach a maximum at a water activity of ca. 0.33. Aroma compounds varied greatly in terms of storage stability in the presence of our two model systems. We were surprised at the very rapid loss of furfurylmercaptan during storage. This bodes badly for the coffee industry since furfurylmercaptan is considered the character impact compound of coffee. Major losses were also observed for the two pyrroles (data not presented). Their loss rates were second only to furfiirylmercaptan. 2,4-hexadienal and ethyl2-mercaptopropionate losses were 3^^^ and 4^^ , respectively. Foods which derive a portion of their characteristic flavor from these compounds would not be expected to have a good shelflife. The inclusion of oil in the model systems invariably increased the stability of the aroma compounds studied although sometimes losses were so still rapid that the effect was small. The increase in stability was dependent upon the aroma compound studied. It is clear that oil has a protective effect on the stability of aroma compounds in the headspace above foods. This may translate into an increase in the shelf-life of oil-containing foods depending upon the
582 contribution of oil to lipid oxidation and the masking effect of lipids on both desirable and undesirable aroma constituents.
5. REFERENCES 1
L.C. Hatchwell, In: Flavor-Food Interactions, ACS symposium series 633. R.C. McGorrin and J.V. Leland (eds), ACS, Washington DC, 1996, 15. 2 J. Bakker, In: Ingredient Interactions, Effects on Food Quality, A. Kumar and G. Gaonkar, (EDS), Marcel Dekker, Inc., New York, 1996, 411. 3 L.J. Farmer, D.S. Mottram, and F.B. Whitfield, J. Sci. Food Agric, Vol. 49 (1989) 347. 4 D.A. Forss, J. Agric Food Chem. no. 17 (1969) 681. 5 D.A. Forss, In: Progress In The Chemistry of Fats and Other Lipids. Vol. 13. R.T. Holman(ed), Pergamon Press, London, 1972,177. 6 B.M. King, and J. Solms, J. Agric. Food Chem. Vol. 27 (1979) 133. 7 J.E. Kinsella, In: Flavor Chemistry of Lipids in Foods. DE Min and T.H. Smouse (eds) American Oil Chem. Soc, Champaign. 1989, 376. 8 S. Shamil, L.J. Wyeth, and D. Kilcast, Food Qual. Pref, Vol. 3 (1991) 51. 9 M. Chen, The Stability of Aroma Compounds in the Presence of Food Components during Storage. M.S. Thesis, Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1997. 10. Handbook of Physics and Chemistry. CRC Press, Boca Raton, FL. 1995. 11 T.P. Labuza, Course titled: "Freezing and Dehydration of Food". Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 1993, 81.