bk-2010-1059.ch024

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Chapter 24

Identification of Flavor-Active Volatiles in Soy Protein Isolate via Gas Chromatography Olfactometry Anthony J. Irwin,*,1 John D. Everard,2 and Robert J. Micketts1 1Solae, 2DuPont

4300 Duncan Ave., St. Louis, MO 63110, USA Crop Genetics, Wilmington, DE 19880-0353, USA *[email protected]

Stir bar sorption and dynamic headspace purge techniques were used to recover the flavor volatiles from soy protein isolate and defatted soy flakes. Thirty-seven compounds were found to be the most flavor-active volatiles present in soy isolate by combining the results of both techniques. The majority of the volatiles are products of lipid oxidation, combined with a smaller number of amino acid degradation products. None of the individual components has a soy isolate flavor which is concluded to be a composite of them all. The full complement of flavor-active volatiles identified in soy protein isolate is also present in the defatted flake and a portion of these are carried through the isolate manufacturing process into the final product. Additionally, a significant amount of lipid remains bound to the soy protein and its continuing oxidation acts as a source of flavor volatiles throughout the manufacturing process. The flavor of the final product appears to be a composite of these two sources of volatiles.

Introduction Soy products such as soy protein isolate provide a protein source that is less energy intensive and more environmentally friendly to produce than animal-based protein, while being at least as nutritious. However, its consumption by western consumers is limited by its characteristic flavor. These grainy, legumey off-flavors have been investigated and reported on for over 30 years. More than 300 volatile © 2010 American Chemical Society In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


organic compounds with a potential to impact the flavor have been identified in soy products (1). The identification and minimization of these constituents have been ongoing objectives for many soy protein producers. While hundreds of volatiles can be identified in many foods, it is usually a much smaller number that contributes most of the flavor. These compounds are best identified by Aroma Extract Dilution Analysis (AEDA) (2) and similar techniques relying on gas chromatography olfactometry. Boatright (3) used a vacuum distillation technique and AEDA to show that the most powerful odorants in soy protein isolate were, in order of flavor intensity, dimethyl trisulfide, E,E-2,4-decadienal, an unknown, 2-pentyl pyridine, E,E-2,4-nonadienal, hexanal, an unknown, and acetophenone. Kobayashi (4) coupled an unspecified sample preparation technique with AEDA and found that E,E-2,4-nonadienal and E,E-2,4-decadienal were the strongest odors present in soymilk, followed by hexanal, 2-pentyl furan, 1-octen-3-one, hexanol, E-2-nonenal, and E,Z-2,4-decadienal. Acree (5) isolated flavor volatile fractions from soymilk using Freon and ethyl acetate extraction and used GCO to demonstrate that E,E-2,4-decadienal, hexanal, beta damascenone, E-2-nonenal, E-4,5-epoxy-(E)-2-decenal, vanillin, E,Z-2,6-nonadienal, E,E-2,4-nonadienal, and E,Z-2,4-decadienal were the most powerful flavor volatiles present. Since most of the flavor–active volatiles were found to be lipid oxidation products, lipoxygenase-free soy varieties have been developed in an attempt to reduce the intensity of the characteristic flavor of soy products. In our experience, these soy varieties have not yielded isolates with improved flavor characteristics. Since no one isolation technique is likely to yield an extract that is totally representative of the volatile profile in the food being extracted, two techniques were selected to provide a more wide-ranging extraction of the flavor volatiles present in commercial and bench scale soy protein isolates. Firstly, stir bar absorptive technology (6) was selected because it is so convenient and because it was expected to recover the more hydrophobic, higher-boiling flavor volatiles. Secondly, the dynamic headspace purge and trap technique was selected because it was expected to preferentially isolate the more volatile flavors.

Materials and Methods Extraction of Soy Isolate Flavor Volatiles via Stir Bar Sorption Soy isolate (5g) was slowly added with stirring to a Waring blender containing deionized water (95ml). The slurry was stirred for 30 sec and then transferred to a sterile Nalgene container that was refrigerated overnight at 4C. Aliquots of slurry (20ml) were placed into 20mL Trace-Clean vials, fitted with Teflon-lined caps (VWR catalog #89093-834). A single Twister™ stir bar (10mm long X 0.5mm film thickness; Gerstel Inc., Baltimore, MD) was placed into each vial and the samples were stirred at 1700 rpm for 4 hr, at room temperature. At the end of the sampling period the slurries were discarded and the Twister™ bars were rinsed with seven exchanges of deionized water. The Twister™ bars were patted dry on Kimwipes, loaded into a Gerstel sample tubes and analyzed by GCMS. Because 390 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

of known losses of compounds from the volatile-loaded bars during dry storage, samples were prepared just prior to each olfactometry run.

Extraction of Defatted Soy Flake via Stir Bar Sorption


One Twister™ bar was stirred for 4 hr in a 7% aqueous slurry of commercial defatted flake from our production facility. The protein concentration of this slurry approximated that in a 5% soy isolate slurry.

GCMS and GCO Analysis The bars were loaded into a Gerstel TDS2 desorber unit that was operated in splitless/solvent vent mode. The bar was heated at a rate of 60C/min from 20C to 300C and then held for 3 min at 300C. The carrier gas flow during the desorption phase was 50ml/min. The desorbed volatiles were condensed at -150C in a CIS4 inlet fitted with a liner filled with deactivated quartz wool and subsequently volatilized on to the GC column by heating to 300C at a rate of 12C/sec. Gas chromatography was performed on a 50m 0.32mm Ultra 1 column with a 0.52micron film thickness mounted in an Agilent 6890. The initial oven temp of 40C was held for 6 min and the temperature was then increased at a rate of 5C/min until 270C was reached. The ramp rate was then increased to 20C/min until 325C was reached. Initial column flow rate was 2.4ml/min. The volatiles exiting from the column were split between the Agilent 5973 MSD and the Gerstel Odor Detection Port (ODP) in a 1:4 ratio by a split union linking a Restek Hydroguard FS 0.18mm ID capillary column (1.322m) to the ODP and a Restek Hydroguard FS 0.10 mm ID capillary column (1.222m) to the MSD. The pressure at the split union was set to 2.2psi. Air makeup gas flow to ODP was 10ml/min with humidification. Mass spectral data was acquired over a 10 – 330 mass range. Retention indices for compounds of interest were calculated from the retention times of ethyl ester standards according to equation 1.

where: RTi = Retention Time Index n = Carbon number of n-paraffin or ester (do not count carbons in ethyl group of ester) t i = Retention time of component (minutes). t n = Retention time of preceding standard (minutes). t n+1 = Retention time of next n-standard (minutes) In order to identify as many of the volatiles as possible, the total volatile load from eight stir bars was analyzed by GCMS analysis without splitting. Literature retention indices and, where available, authentic standards were used to confirm identifications. 391 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


GCO Analysis of Stir Bar Volatiles Aroma Extract Dilution Analysis (AEDA) was achieved by presenting 8X, 1X, 1/4, 1/16, and 1/64th of the amount volatiles collected on a single Twister™ bar to the GC column. This was accomplished either by loading multiple bars (for the 1X and 8X bar samples) or by splitting a portion of a single Twister™ bar extract during sample loading, so that only a defined portion (i.e., 1/4, 1/16, and 1/64th of the volatiles captured by a single bar) was presented to the column. The latter was achieved by pneumatically splitting portions of the sample, using the split/splitless capabilities of the Agilent/Gerstel 6890GC inlet, prior to the sample being introduced onto the chromatographic column. Preliminary experiments, in which defined ester mixtures were sequentially diluted (using the method described above) and quantified using the mass spectrometer, showed that proportional dilution using a combination of the number of bars loaded and the inlet pneumatics was effective and predictable. Each AEDA analysis was performed in duplicate by authors JE and AI. Odor descriptors for both sniffers were compared and only those odors which were reproduced are reported in the final tabulation. Odors were considered as reproduced if (i) both panelists detected the odor at the same retention time in the same sample, or (ii) one of the panelists detected the same odor at the same retention time in other dilutions of the same sample. Odors which were reproducibly detected during the 1X, 1/4, 1/16 and 1/64th bar experiments were thus detected at 1/8, 1/32, 1/128 and 1/512th respectively of the amount detected on eight bars. These odor compounds were assigned Flavor Dilution factors of eight, 32, 128 and 512 respectively.

Isolation of Flavor Volatiles via Dynamic Headspace Purge A headspace purge system was assembled as shown in Fig 1. The collection tube (Gerstel GC 09948) was packed with 290mg of 60/80 mesh Tenax-GR sorbent. Soy protein (5g) was stirred for 1 minute into 95ml deionized water in a Waring blender. The slurry was then sealed in an amber bottle and refrigerated overnight. A slurry sample (20g) was placed in the 50ml Erlenmeyer flask along with 7.5g sodium chloride and a magnetic stir bar. A teflon purge head adapter (SIS part 164372) was fitted to the Erlenmeyer flask, with a tube style purge head (SIS part 783009) fitted to the adapter. The sparging needle tip was positioned 3mm above the surface of the slurry. The assembly was then placed in a water jacketed beaker containing water at the level of the slurry in the flask and the water jacket was maintained at 45C. The nitrogen lines were attached and the slurry was stirred at 200rpm. The dry purge nitrogen stream was passed through the collection tube at 51ml/min to minimize water retention. The nitrogen flow rate through the sparge needle was 50ml/min. The collection tube was removed after 45 min purging.

392 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Dynamic headspace purge apparatus. GCO Analysis of Headspace Purge Volatiles The adsorbed volatiles were desorbed and analyzed by the same system described above. Dilutions of 1, ¼ and 1/16th of the total volatile load per collection tube were achieved as above via the pneumatic control system of the GC injector. Odors detected at ¼ and 1/16th dilutions were assigned FD factors of four and 16 respectively. For standard mass spectral analysis, the total load of desorbed volatiles from one Tenax trap was chromatographed and transferred from the GC column to the mass spectral detector without splitting. Retention time indices were calculated for the two different instrument configurations (with and without noseport) and enabled accurate alignment of the odors and mass spectra.

Results and Discussion GCO Analysis of Volatiles Recovered by Twister A standard commercial isolate Supro ® 500E was selected as the first candidate for AEDA GCO analysis. When its volatiles were trapped on eight Twister™ bars and chromatographed without any splitting, 46 volatiles were detected reproducibly by one or both of the sniffers. The intensity of many of the odors was very high and it allowed the total load to be diluted stepwise by a factor of 128 (i.e. 1/16th of the total load of eight bars), at which point 20 volatiles could still be detected. These constituted the most odor-active volatiles in Supro ® 500E. Many of these compounds were subsequently identified by matching their RTI and mass spectra with those of authentic standards. Many unknowns were present that either had spectra which were not matched with any spectra in 393 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


the NIST mass spectral library (version 2.0a), or were not present in sufficient quantity to generate a mass spectrum. The identities, RTIs, odor descriptors and flavor dilution (FD) factors of the volatiles detected in Supro ® 500E are listed in Table 1. Note that no odors with a retention index lower than that of hexanal were recovered by the stir bar sorption technique. Only when the instrument was configured without splitting for the noseport could volatiles be detected in this area of the mass spectral chromatogram. Supro ® XT219D, a commercial isolate that is slightly hydrolyzed by an endo protease enzyme to improve its functionality, was analyzed by the same technique. At a 1/16th dilution of the eight bars (FD factor of 128), 20 volatiles were detected and their identities coincided with those found in the Supro ® 500E. In order to identify the most flavor-active compounds within this group, the AEDA dilution was taken to 1/64th of the eight bar extract. At this dilution, only eight volatiles were reproducibly detected. These included an unknown at RTI 510, 1octen-3-one, an unknown at RTI 695, E-2-nonenal, E,E-2,4-nonadienal, E,Z-2,4decadienal, E,E-2,4-decadienal, and 2-butyl-2-octenal. These eight compounds with a FD factor of 512 are the most odor-active compounds in this particular soy isolate, and probably Supro ® 500E as well. The unidentified odor at RTI 510 is almost certainly due to an overlap of two compounds since its descriptors varied with the isolate type and with the dilution factor (see details in Table 1.) Judging by their odors, the two volatiles appear to be a sulfur-containing compound and an aldehyde. The presence of both compounds simultaneously at the odor port gave a cracker-type odor. A number of competitor commercial isolates, as well as isolates prepared in our bench scale pilot plant, were also analyzed by the same technique (results not shown). These were found to contain a similar range of volatiles as shown in Table 1, with similar FD factors. None of the individual volatiles recovered by the Twister™ bar technique had a characterizing soy isolate odor.

GCO Analysis of Volatiles Recovered by DHS The same Supro ® 500E-type isolate described above was also analyzed by using a DHS purge technique into a trap containing Tenax GR. Tenax TA was also evaluated as adsorbent and it was found to yield the same volatiles recovered by Tenax GR. The desorbed volatiles were split at 1:4 and 1:16 for AEDA analysis. The identities, RTIs, odor descriptors and flavor dilution (FD) factors of the volatiles detected in Supro ® 500E by DHS are listed in Table 2.



Table 1. All Odor Volatiles in Supro ® 500E, Supro ® XT219D and Defatted Soy Flake as Recovered by the Stir Bar Sorption Technique



Table 2. All Flavor-Active Volatiles Detected in Supro ® 500E by the DHS Technique



Unlike the volatile profile recovered by the Twister™ bars that did not contain any detectable volatiles with a retention time lower than hexanal, the DHS volatile profile covered the entire chromatogram. Since the recovery of volatiles by DHS and stir bar sorptive technology was expected to be both quantitatively and qualitatively different, there was no realistic means of quantitatively comparing the FD factors of the two procedures. Instead, dilution of the Tenax-trapped volatiles was only pursued until the twenty or so volatiles with the highest FD factor were present. Ultimately, 22 volatiles were detected when the volatiles split at a 1:16 ratio and these were considered to represent the most flavor-active volatiles as recovered by the headspace purge technique. These were then compared with the 20 most flavor- active volatiles found by the stir bar sorption technique, albeit with quantitatively different FD factors. The DHS technique recovered many odor-active volatiles that were not recovered by the Twister™ bar, and vice versa. As expected, the headspace purge procedure recovered much higher levels of lower molecular weight, lower boiling compounds such as acetaldehyde, diacetyl, 3-methyl butanal and pentanal that were not detectable as odors in the Twister™ bar volatiles. The polydimethylsiloxane coating of the bars seems to have a limited affinity for the low boiling volatiles known to be present, such that they are readily displaced by the more hydrophobic, higher boiling volatiles. The DHS technique also suggests that dimethyl trisulfide is an important contributor to soy isolate. The Twister™ bar recovered only a small amount of dimethyl trisulfide which did not persist during the AEDA. Whereas the Twister™ volatiles are overwhelmingly derived from lipid oxidation and convey papery and varnishy flavors, those recovered by the headspace purge convey a combination of caramel, grassy and sulfide notes in addition to a selection of the former flavors. None of the volatiles detected with the DHS technique had a characteristic soy isolate aroma. The Origins of Soy Isolate Flavor Volatiles The 20 volatiles with the highest FD factors from both techniques are combined in Table 3. Since none of these volatiles have an odor resembling soy isolate, it is concluded that the latter’s characteristic beany flavor is due to a combination of all of them. Many of the volatiles could not be identified because their spectra were not present in the mass spectral library or because there was insufficient compound to generate a mass spectrum. All of the unidentified volatiles with papery and varnishy odors are certainly aldehydes because their odors are very similar to the numerous saturated and unsaturated aliphatic aldehydes that were identified by comparison with authentic standards. All such aldehydes that occurred at retention times higher than pentanal are almost certainly arising from oxidative degradation of linoleic and linolenic fatty acids (7, 8). The other major source of aldehydes in food is the Strecker degradation of amino acids and none of the latter will yield aliphatic aldehydes with more than five carbons. With this assumption, 25 of the 37 flavor volatiles listed in Table 3 appear to be derived via lipid oxidation. Strecker degradation of amino acids accounts for the acetaldehyde, 3-methyl butanal and methional. 397 In Chemistry, Texture, and Flavor of Soy; Cadwallader, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Degradation of the methional is probably the source of the dimethyl trisulfide. Diacetyl may arise from both the Maillard reaction and microbial growth during the aqueous processing stages of the isolate manufacturing process. Soy protein isolate is prepared from defatted soy flakes via sequential aqueous extraction, centrifugation, precipitation at low pH, centrifugation, pasteurization, vacuum stripping, and spray drying. Given that most of the flavor volatiles in isolate were found to be derived via lipid oxidation, it was of interest to determine if they are being created during the manufacturing process from the flake, or if they are already present in the flake as a result of the soy oil extraction process. A sample of commercial defatted flake was therefore extracted as a 7% slurry with one Twister™ bar and the total volatile load was analyzed without splitting. A 7% slurry was used, as this concentration provided a similar soy protein level to the 5% slurries of soy isolate analyzed above. Of the 20 volatiles with the highest FD factor (via Twister™ bar), 19 were present in the defatted flake (see DFF in Table 1). While AEDA was not performed on the defatted flake, the intensity of these odors was overwhelmingly higher than those experienced when sniffing the volatiles recovered from the isolates with one Twister™ bar. Extraction of soy oil from the beans with hexane and the subsequent solvent removal steps thus lead to production and retention of lipid oxidation volatiles in the defatted flake. The extraction and washing steps in the current commercial isolate process are successful in lowering the concentration of most of these volatiles. For example, 1-octen-3-ol is present at very high concentrations in defatted flake and it provides a strong odor impact during Twister™ GCO. However, it was not detected (by odor) in the isolate by either volatile recovery technique. This behavior was not mimicked by the other volatiles, most of which retained sufficient concentration in the isolate to contribute to its characteristic flavor. For example, 1-octen-3-one remained as one of the most significant contributors to soy isolate flavor, despite having physical properties similar to 1-octen-3-ol. The latter should have led to removal of 1-octen-3-one in the washing and vacuumizing steps. The assumed removal of 1-octen-3-one in the washing/vacuumization steps and its probable subsequent regeneration in the pasteurization/drying steps can be traced to the residual fat in commercial defatted flake. Typically, the latter contains about 3% fat by acid hydrolysis, much of which is comprised of phospholipid. The isolate manufacturing process does not separate this residual fat from soy isolate, which consequently contains 4 – 5% fat by acid hydrolysis. This lipid accompanying the protein through the isolate manufacturing process appears to serve as a continuous source of lipid oxidation volatiles whose regeneration in thermal steps competes with their removal during washing/vacuumization. Overall, the net result of the isolate manufacturing process is a reduction of the volatile levels present in defatted flake, despite the continued presence of residual fat.



Table 3. Volatiles with the Highest FD Factors in Supro ® 500E Soy Protein Isolate as Detected via the DHS and Stir Bar Sorption Techniques

Conclusion The presence of the characterizing soy isolate flavors in the defatted flake strongly suggests that processing attempts to minimize the off-flavor of soy isolate during its manufacturing from the current raw material will be problematic. Efforts to produce a bland-tasting soy isolate should be focused either on genetically-modified beans with reduced polyunsaturated fatty acid content, or on conventional beans whose protein is separated from the oil in a novel way that minimizes exposure of the soy protein to oxidizing triglycerides and phospholipids.


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MacLeod, G.; Ames, J. Soy flavor and its improvement. CRC Crit. Rev. Food Sci. Nutr. 1988, 27, 219−400. Grosch, W. Determination of potent odourants in foods by aroma extract dilution analysis (AEDA) and calculation of odour activity values (OAVs). Flavour Fragrance J. 1994, 9, 147–158. Boatwright, W. L.; Lei, Q. Compounds Contributing to the beany odor of aqueous solutions of soy protein isolates. J. Food Sci. 1999, 64, 667–670. Kobayashi, A.; Tsuda, Y.; Hirata, N.; Kubota, K.; Kitamura, K. J. Agric. Food Chem. 1995, 43, 2449–2452. Feng, Y.; Acree, T. E.; Lavin E. H. Processing Modulation of Soymilk Flavor Chemistry. In Aroma Active Compounds in Food; ACS Symposium Series 794; Takeaka, G. R., Guntert, M., Engel, K.-H., Eds.; 2001; pp 251−264. Baltussen, E.; Sandra, P.; David, F.; Cramers, C. Stir bar sorptive extraction (SBSE), a novel extraction technique for aqueous samples: Theory and principles. J. Microcolumn Sep. 1999, 11, 737–747. Whitfield, F. B. Volatiles from interactions of Maillard reactions and lipids. Crit. Rev. Food Sci. Nutr. 1992, 31, 1–58. Grosch, W. Reactions of Hydroperoxides: Products of Low Molecular Weight. In Autoxidation of Unsaturated Lipids; Chan, H. W.-S., Ed.; Academic Press: London 1987; pp 95−139.


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