Thermally Generated Flavors

200 °C is primarily due to 5 compounds: diacetyl, 2-acetyl-1,4,5,6- tetrahydropyridine ... both model reactions as well as in actual food systems fl-...
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Chapter 7

Gas Chromatography—Olfactometry of Glucose—Proline Maillard Reaction Products

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Deborah D. Roberts and Terry E. Acree Department of Food Science and Technology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456

The aroma produced by the reaction between glucose and proline at 200 °C is primarily due to 5 compounds: diacetyl, 2-acetyl-1,4,5,6tetrahydropyridine, 2-acetyl-1-pyrroline, 2-acetyl-3,4,5,6tetrahydropyridine, and furaneol. Gas chromatography-olfactometry using the technique CharmAnalysis ™ produced chromatograms of odor-activity which, with gas chromatography - mass spectroscopy, allowed the identification of the odor-active compounds based on their retention index. At a lower reaction temperature, 180 °C, the minor odor-active compounds were not detected, thus altering the aroma profile. Maltoxazine, a major volatile, produced zero odor potency while the main volatile product of the reaction, 5-acetyl-2,31H-pyrrolizine, was found to contribute only 0.3 % to the total aroma. Maillard browning is one of the most complex and important odor and color producing reactions in food. In order to study the contribution of the Maillard reaction to flavor, the volatile products of a model reaction between a particular amino acid and monosaccharide are often identified. Several comprehensive books and review articles contain a vast amount of Maillard reaction literature, covering both model reactions as well as in actual food systems fl-41. More than 120 volatiles produced by monosaccharide-proline reactions have been characterized including twenty-two 2,3-dihydro-lH-pyrrolizines and twentyeight additional pyrrolidines, pyrrolines, piperidines, pyrroles, pyridinones, pyridines, pyrazines, and pentenimines [5-91. Specific glucose-proline reaction products include furfuryl alcohol, diacetyl, 2-acetyl-l,4,5,6-tetrahydropyridine, furaneol, 2-acetylpyridine, and the most abundant volatile, 5-acetyl-2,3-dihydro-lHpyrrolizine r7.10.111. The odors of many of these glucose-proline reaction products are caramel, bready, cracker-like, burnt, and maple. The presence of a volatile with odor, though, is not a measure of it's relative importance to Maillard browning aroma. The gas chromatography-olfactometry (GCO) techniques called CharmAnalysis [12] and aroma extraction dilution analysis (AEDA)[12] are odor bioassays in which the dilution to threshold approach quantitates each odor's potency. In this study, the CharmAnalysis procedure was applied to determine

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which of the many volatiles produced by a glucose-proline reaction are important to the aroma, based on odor potency.

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Materials and Methods Chemicals. Pure samples of the diacetyl, 2-acetyl-pyridine, and furaneol were obtained commercially (Aldrich, Milwaukee, Wis.) while 2-acetyl-l-pyrroline was a gift from Ron Buttery. The synthesis of 2-acetyl-l-pyrroline was reported by Buttery[14]. 2-Acetyl-1,4,5,6-tetrahydropyridine and it's tautomer, 2-acetyl- 3,4,5,6tetrahydropyridine were synthesized by heating sodium bisulfite, proline, and dihydroxyacetone, dissolving the product in water, adding NaOH, and extracting with pentane[15]. The pentane extract was analyzed by G C / M S employing the conditions described below and found to contain the two tautomers of 2-acetyltetrahydropyridine as the major products. Sample Preparation. Reaction of L-Proline and D-Glucose. Equimolar amounts (.02 mol) of D-glucose and L-proline dissolved in 100 mL of water were heated for 1 min at 180 C and 200 C in a closed reactor as described in "High temperature short time kinetics for the formation of Maillard products in the proline/glucose model system", Stahl, H.D. and Parliment, T.H., ibid. The resulting aqueous solutions were extracted sequentially with an equal volume of Freon and an equal volume of ethyl acetate. Both Freon and ethyl acetate phases were dried with anhydrous magnesium sulfate. 0

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Gas Chromatography-Olfactometry (GCO). The CharmAnalysis bioassay characterizes potent odor-active volatiles when humans sniffing the gas chromatographic effluent note the presence of an odor at a particular retention index (Kovats). In this study, one | i L samples were injected onto a G C O system made from a Hewlett Packard 5890 Gas Chromatograph (Datu Inc. Geneva, N Y ) . Two Hewlett Packard fused silica capillary columns were used: Carbowax 20M (12 m x 0.32 mm x 0.3 um film thickness) and OV101, a cross-linked methyl silicone (12m x 0.32mm x 0.52|im film thickness). The samples were injected at 35 ° C and after 3 min, the oven temperature was raised by 6 C/min to 225 C (200 C for Carbowax). The column was either connected to a flame ionization detector for determination of n-paraffin retention times in order to calculate retention indices or to an olfactometer. The olfactometer mixed the effluent into a stream of humidified air (20L/min). A person who was previously screened for olfactory ability with a set of 6 standard odors sniffed the effluent. Responses were recorded as the presence and quality of odor, and the time recorded on a Macintosh computer using specially designed software (DATU, Geneva, NY). The samples were diluted by factors of three and the analysis repeated until no more odor was observed (the detection threshold). The combination of these runs produced a charm chromatogram with odor potency defined as the area of the chromatographic peaks. This is in contrast to the dilution value, which is peak height. A l l charm values reported are the sum of the Freon and ethyl acetate fractions. Theoretically, charm is proportional to the ratio of the amount of odor compound to it's detection threshold[l£]. The compounds with high charm values were then identified by matching their mass spectra, retention indices on two substrates, and odor quality to authentic standards. Compounds with no authentic standard were labelled tentatively identified. 0

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Capillary Gas Chromatography / Mass Spectrometry (GC/MS). The characterization of the odor-active compounds was done by concentrating the original extract 270 fold and injecting lfiL on a Hewlett Packard 5890 Gas Chromatograph connected to a 5970 Series Mass Selective Detector, operated in electron impact at 70 ev. Two fused silica Hewlett Packard capillary columns were used: OV101, a methyl silicone, (25m x 0.2mm x 0.33n.m film thickness) and Carbowax 20M (25m x 0.32mm x 0.3^im film thickness). The samples were injected at 35 C and after 3 mins, the oven temperature was raised by 4 °C/min to 225 ° C (200 C for Carbowax 20M). In comparison to GC/MS, the rate of temperature increase for G C O was slower and the column was half as long to minimize charm chromatogram acquisition time. The mass spectrum of each odor-active compound was determined by using n-paraffin retention indices (RI) between G C O and GC/MS columns. 0

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Results and Discussion CharmAnalysis. The predominant odor qualities characterizing glucose-proline reaction products were burnt caramel, popcorn, and cotton candy. Nutty was previously used to describe the main odor in the glucose-proline reaction over a range of reaction temperatures [17]. Over 93% of the aroma potency in the glucoseproline reaction, as measured in charm units, can be explained by 4 compounds, and over 98% by 7 compounds, as seen in Table I. The two charm chromatograms made using different capillary columns are shown in Figure 1. The 200 C samples on Carbowax 20M yielded a charm chromatogram with more narrow retention indices and better separation of the odor active regions than on the OV 101 column. Thus, five main odor-active compounds (No. 1,2,3,5 and 6) were seen. The largest peak in the OV101 charm chromatogram, RI 1000-1100, was separated into 4 odor-active compounds on Carbowax 20M at RI 1332, 1539, 1619, and 1982. The compound with the largest odor potency was 2-acetyl-3,4,5,6tetrahydropyridine, which when synthesized, showed a retention index of 1110 on O V 101 and 1613 on Carbowax. A very similar mass spectrum to that of 2-acetyl3,4,5,6-tetrahydropyridine was seen at RI 1018 on OV101 and RI 1331 on Carbowax, due to the presence of the tautomer, 2-acetyl-l,4,5,6-tetrahydropyridine. These tautomers have very low odor thresholds, 1.6 |ig/L in water [18]. It has been reported that the tautomers are formed in concentrations up to 200 \ig/g in proline model experiments with reducing sugars, as well as detected in the low ng/g range in beer, malt, and bread[5]. A possible mechanism for their formation involving intermolecular condensation to form 5-substituted hexahydrocyclopenta-(b)pyridinones has been proposed [5]. 2-Acetyl-l-pyrroline is a high charm glucose-proline reaction product which has a very low threshold (0.1 jig/L in water) [19] and a popcorn aroma. In cooked, especially aromatic rice, wheat bread crust, and popcorn, 2-acetyl-l-pyrroline is a major contributor to the odor [19-211. Model experiments have shown the formation of 2-acetyl-l-pyrroline from an acetylation reaction between 1-pyrroline and 2-oxopropanal [22]. Furaneol (2,5-dimethyl-4-hydroxy-3(2H)-furanone) found in the ethyl acetate fraction has a very low odor threshold, 0.03 |ig/L[23], and is a character impact compound in the Maillard reaction. It is also a potent natural product contributing to the odor of fruit, especially the strawberry, pineapple, mango, raspberry, and grape [23-261. 0

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Table I. Odor-active Compounds in Proline/Glucose Reaction No Compound (type of identification)

Odor

1 Diacetyl (a,b,c)

Buttery

2

2-Acetyl-1pyrroline (a,b,c)

Popcorn

3

% Charm RI OV101

RI C20M

604

900

19

898

1257

2-acetyl-1,4,5,6Burnt tetrahydropyridine Caramel (a,b,c) 2-acetylpyridine Caramel (a,b,c)

12

1018

1332

1005

1539

5

2-acetyl-3,4,5,6Burnt tetrahydropyridine Caramel (a,b,c)

63

1110

1619

6

Furaneol (a,b,c)

3.9

1032

1982

7

5-acetyl-2,3lH-pyrrolizine (a,b)

0.3

1354

2033

4

Cotton Candy Medicinal

0.5*

0.5

Mass Spectra m/e (relative intensities) 86(M, 13), 44(4), 43(100), 42(12), 41(3) 111(M, 15), 83(29), 69(11), 68(13), 43(100), 42(26), 41(55) 125(M, 49), 95(25), 83(49), 82(56), 55(75) 54(59), 43(100), 41(24) 121(M,83), 120(20), 106(16), 93(58), 79(100), 78(93), 67(13), 52(33), 51(33), 50(32), 43(47) 125(M,74), 124(23), 93(15), 92(10), 83(9), 82(100), 80(13), 55(24), 54(60), 53(10), 52(11), 43(54) 128(M,60), 85(17), 57(58), 43(100) 149(46), 135(9), 134(100), 106(23), 79(18), 78(11), 77(15), 52(11), 51(15), 43(20)

99% a = mass spectral match , b = retention index match, c = standard match with odor * 12% of diacetyl recovered after extraction

Parliment et al.; Thermally Generated Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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2-Acetylpyridine is a minor odor compound in the glucose-proline reaction with a caramel aroma. It has been described as having a bread crust or popcorn odor and was isolated in peanuts, beer, and rye crust f 18.27-291. It's reported threshold is 19 ng/mL in water [!£]. The "buttery" smelling compound at RI 604 on O V 101 and 900 on Carbowax 20M is diacetyl (2,3-butanedione). It's reported threshold ranges from 2.6 ng/mL to 2.3 |ig/mL [18]. The extraction process used in this study showed a low recovery for diacetyl (12 % yield, Laurent, M . H . , Cornell University, unpublished data.). Diacetyl was present at higher levels in the original heated fraction than the extracted sample for CharmAnalysis. The analysis of headspace volatiles recovers more diacetyl than extraction. A study of the headspace volatiles of a glucoseglycine mixture heated at 95 °C for 2 hours showed diacetyl as the major volatileQQ]. The 0.5 % charm shown for diacetyl is an underestimate, and the value is more likely 4.2% charm. It's formation is at the early stage of the browning reaction and is assumed to be formed through fission of the C2 -C3 bond or C4-C5 bond of glucoseQQ]. It's presence is not limited to Maillard browning products, though, and is ubiquitous in dairy products from lactic acid bacteria fermentation [21]. 0

Temperature Effect. Heating at 200 C showed three of the same odor active regions (RI 1070, 937, 600 on OV101) found in the 180 ° C reaction but produced more minor odor-active compounds (Figure 2). The compounds 7 and 8 from the 200 C reaction were not detected in the 180 C reaction. Slightly different aroma profiles emerge from this 20 C difference in temperature. This is consistent with the Shigematsu variation theory of Maillard browning aroma formation where higher temperatures cause the amino acid to be degraded by pathways other than the Strecker degredation. Amines and ammonia are formed by decarboxylation and decarboxylation/deamination, thus providing further reactants to produce compounds such as pyridines [22]. In a study investigating the odors of Maillard browning and their change with reaction conditions, aromas became stronger, more unpleasant, aldehydic, and burnt with increasing temperature and time [17]. Charm values of the 200 ° C samples were indeed larger than for the 180 ° C samples, with 8 and 9 dilutions required to reach threshold for 200 C samples as compared to 5 and 7 dilutions for the 180 ° C samples. Several low charm aromas which appeared at 200° C were unpleasant: garbage, sweaty, and medicinal. 0

0

0

0

Comparison of Odor with Amount. The major volatile product in the glucoseproline reaction is 5-acetyl-2,3-lH-pyrrolizine [7JLQ]. Figure 3 shows the GC-FID chromatogram for the glucose-proline reaction with the pyrrolizine (No. 7) as the major product. The pyrrolizine's odor, though, makes up less than 1% of the total odor-activity as can be seen by the corresponding charm chromatogram. Maltoxazine (No. 9) is another major volatile, at RI 1622 on OV101 and RI 2428 on Carbowax, which does not exhibit an odor. In fact, most of the major volatile products have very little odor, while trace volatiles account for most of the odor potency.

Conclusion The key odor-active compounds in the glucose-proline reaction have been identified and are the trace, not the major volatile products of the reaction. Figure 4 shows the main compounds that compose 100% of the charm in the reaction sample. The

Parliment et al.; Thermally Generated Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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1. Buttery 2. Popcorn 3. Burnt Caramel 4. Caramel 5. Burnt Caramel 6. Cotton Candy 7. Medicinal 8. Fruity

(C 20M)

1000

1200

2 600

800

1400

4

1600

1800

2000

2400

(OVIOl) 8

5

" L

1000

2200

1600

1200

1800

Retention Index

Figure 1. Charm chromatograms of the volatile 200 ° C glucose-proline reaction products on OV101 and Carbowax 20M columns. Compound numbers refer to Table I. Y-axis measures dilution value and x-axis is n-paraffin retention index.

1. Buttery 2. Popcorn 3. Burnt Caramel 4. Caramel 5. Burnt Caramel 6. Cotton Candy 7. Medicinal 8. Fruity

800

1000

1200

1400

1600

1800

Retention Index (OV101)

Figure 2. Charm chromatograms of the volatile glucose-proline reaction products under different temperatures, 200 C and 180 C. Y-axis measures dilution value and x-axis is n-paraffin retention index. 0

0

Parliment et al.; Thermally Generated Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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7. ROBERTS & ACREE

GC-Olfactometry of Glucose-Proline Reaction

1. Buttery 2. Popcorn 3. Burnt Caramel 4. Caramel 5. Burnt Caramel 6. Cotton Candy 7. Medicinal 8. Fruity

600

800

1000

1200

1400

1600

1800

Retention Index (OV101)

Figure 3. Comparision between a Charm and FID chromatogram of the volatile glucose-proline reaction products at 200 C. Y-axis measures dilution value and x-axis is n-paraffin retention index. 0

4% (1)

4% (6)

Figure 4. Summary of the major odor-potent compounds in the glucose-proline reaction, with % Charm, 1: diacetyl, 2: 2-acetyl-l-pyrroline, 3: 2-acetyl1,4,5,6-tetrahydropyridine, 5: 2-acetyl-3,4,5,6-tetrahydropyridine, 6: furaneol.

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relation of percent charm to the odor quality in a mixture has yet to be determined. Based on charm values, one compound dominates the odor, that is 2-acetyl-3,4,5,6tetrahydropyridine. Two compounds, 2-acetyl-l-pyrroline and 2-acetyl-1,4,5,6tetrahydropyridine comtribute moderately to the aroma of the glucose-proline reaction. Furaneol and diacetyl contribute minor amounts to the aroma. Quantitating the levels of these potent flavor compounds can be used to optimize the production of food with the desired level of glucose-proline Maillard browning aroma. Acknowledgments

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This material is based upon work supported under a National Science Foundation Graduate Fellowship.

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(2) (3) (4)

(5)

(6) (7) (8) (9) (10) (11) (12) (13)

(14) (15)

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