Chapter 8
Effect of Amide Content on Thermal Generation of Maillard Flavor in Enzymatically Hydrolyzed Wheat Protein
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Qinyun Chen and Chi-Tang Ho Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick,NJ08903
Wheat gluten was hydrolyzed using fungal protease to obtain wheat gluten hydrolysate (WGH), and the resulting hydrolysate was deamidated to produce deamidated wheat gluten hydrolysate (DWGH). Acid-wheat gluten hydrolysate (AWGH) was prepared by hydrolyzing wheat gluten under strong acidic conditions. The three hydrolysates were reacted with glucose in closed systems and the volatile compounds that were generated were isolated and identified. About11times the amount of volatile compounds was generated in the DWGH-G system than was generated in the WGH-G system. By comparing the volatiles generated in these three systems, a clear trend was found that the deamidation reaction significantly increased the formation of aroma compounds.
Hydrolysis of proteins has been used to improve the quality of food for 100 years by adding the resulting mixture of amino acids and peptides. An older example is soy sauce, made by the action of mold enzymes in steamed soybeans and wheat. This enzymatic process causes the cleavage of proteins at various sites on the protein molecule and results in an increase of amino acids, oligopeptides or polypeptides. Although some short chain peptides or polypeptides resultingfromthe hydrolysis of meat or milk protein have been reported in food systems, the role of such hydrolysates as precursors in the generation of flavor compounds has not been investigated. Wheatflouris a major cereal that is rich in protein content. Therefore, enzymatically hydrolyzed wheat gluten would be a good substance to be used to improve theflavorquality of many foods. Hydrolyzed vegetable protein (HVP), traditionally produced by acid hydrolysis, has been used to produce various types offlavorsby the Maillard reaction in the food industry. Hydrochloric acid is commonly used in the production of protein hydrolysates because it works fast and yields a product with a highly accceptable savory profile (7). However, in recent years, there has been some concern about the safety of HVP due to the presence of chloropropanols in HVP. These chloropropanols are formed by the interaction of
0097-6156/96/0637-0088$15.00/0 © 1996 American Chemical Society
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Amide Content & Thermal Generation of Maillard Flavor
hydrochloric acid with glycerol-containing lipids and may have harmful effects on humans if present in high concentrations (7). Studies on the protein deamidation in food systems has recently been of great interest to the food industry. This is due to the fact that the deamidation reaction changes functional properties of protein such as solubility, emulsion property, and foaming ability (2). Deamidation is a reaction that results in the loss of the amide group of glutamine and asparagine residues in protein molecules, and further results in the release of ammonia. The ammonia releasedfromprotein deamidation will interact with other reactants such as reducing sugars when heated via the Maillard reaction possibly affecting both flavor and color attributes of the final product. With these points in mind, the major focus of this work was on the effect of amide content on the generation of aroma compounds via a thermal reaction using Wheat Gluten Hydrolysate (WGH), Deamidated Wheat Gluten Hydrolysate (DWGH), and Acid-Hydrolyzed Wheat Gluten (AWGH) as flavor precursors. MATERIALS AND METHODS Preparation of Wheat Gluten Hydrolysate (WGH) and Acid-Hydrolyzed Wheat Gluten (AWGH). Wheat gluten was purchasedfromSigma Chemical Company (MO). Fungal Protease 500,000 was purchasedfromGist-Brocades Food Ingredients, Inc. (PA). 10 g of wheat gluten and 1 g of fungal protease were weighed into a flask and 100 ml of distilled water was added. The flask was put in an incubator shaker (New Brunswick Scientific Company, Inc., NJ) at 180 RPM at 55°C for 2 hrs. The hydrolyzed mixture was thenfilteredto obtain thefiltrate.Thefiltratewas thenfreeze-driedto obtain the WGH. The degree of hydrolysis as measured by the ninhydrin method was 11.5%. It means that the major components in the WGH are oligopeptides. 3 g of wheat gluten was weighed into a reaction flask to which 100 ml of 6 N HC1 solution was added. Theflaskwas tightly capped and put in an oven at 130°C for 24 h. The reaction mixture wasfilteredto obtain the filtrate. The solvents of thefiltratewere evaporated with a rotary evaporator under vacuum The residue was dissolved with 90 ml of distilled water, and the pH was adjusted to 7 with 1 N sodium hydroxide solution and then diluted to 100 ml with distilled water. The resulting solution was then freeze-dried to obtain the AWGH. Preparation of Deamidated Wheat Gluten Hydrolysate (DWGH). 30 ml of hydrolyzed wheat gluten solution (WGH) was contained in a reaction bottle to which 120 ml of water was added. The pH of the solution was adjusted to 9. The bottle was capped and put in an oven at 110°C for 3 h to carry out the deamidation reaction. After deamidation, the bottle was cooled to room temperature. The pH of the deamidated solution was adjusted to 12, and then the solution was degassed overnight under vacuum in order to remove ammonia. The pH of the sample solution was then adjusted to 7 and the sample wasfreeze-driedto obtain the DWGH. Maillard Reaction and Volatile Extraction. Three equal amounts (3 g of wheat gluten) of WGH, DWGH, and AWGH, were separately reacted with 0.9 g of glucose at 155 °C in three reaction vessels for two hours. The volatile compounds were extracted
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with methylene chloride, and concentrated with a Kuderna-Danish concentrator to the appropriate concentrate of solutions. The three volatile solutions were analyzed by gas chromatography (GC) and GC-mass spectrometry. The volatile compounds were quantified by using dodecane as an external standard. RESULTS AND DISCUSSION In the WGH, DWGH, and AWGH model systems we identified and quantified 55, 51, and 53 volatile compounds, respectively (Table I). The major volatiles identified were furans, pyrazines, aldehydes, alcohols, ketones, pyrrolizines. These volatile compounds were mainly derivedfromStrecker degradation, sugar degradation, lipid degradation, and the further interactions of these degradation products. Strecker Aldehydes. The four Strecker aldehydes identified were 2-methylpropanal, 2methylbutanal, 3-methyIbutanal, and phenylacetaldehydes, which were derivedfromtheir corresponding free amino acids: valine, isoleucine, leucine and phenylalanine, respectively. Wheat gluten contains 6.1-8% leucine, 3.3-4.5% isoleucine, and 5.3-6.2% valine. Strecker aldehydes were the major volatile compounds generated in WGH-G, DWGH-G and AWGH-G model systems. The amount of Strecker aldehydes generated in the AWGH-G and DWGH-G systems were much higher thanfromWGH-G system Compared to the AWGH which contained mainlyfreeamino acids, DWGH consisted of mainly peptides and small amounts of free amino acids. However, the DWGH still generated a significant quantity of Strecker aldehydes when heated with glucose. These results are consistent with the observation of Rizzi (3). Rizzi heatedfructosewith different peptides, tripeptides, and mixtures of their corresponding amino acids. It was found that dipeptides containing valine and leucine produced significant amounts of the Strecker aldehydes, 2-methylpropanal and 3-methyIbutanal, despite the blocked amino group or carboxyl group. These experimental results indicated the Strecker degradation of peptides will take place in spite of their peptide bonding. Compared with 2-methylpropanal, 3-methylbutanal, and 2-methylbutanal, two other aldehydes, acetaldehyde and formaldehyde, were not detected in the model systems, but their corresponding substituted flavor compounds were identified. Acetaldehyde, having a low boiling point of 21°C, might either be too volatile to be detected or be very active and completely react with other components. Several heterocyclic compounds with ethylsubstituents were identified in the model systems. Alkylpyrazines. Besides the sugar-derived carbonyls, Strecker aldehydes, furans, and furanones were identified from the model systems (WGH-G, DWGH-G, and AWGH-G). The major volatile compounds identified were alkyl-substituted pyrazines. Table I lists seventeen alkyl-substituted pyrazines identified in the WGH-G, DWGH-G, and AWGH-G model systems. Compared with pyrazines generated from the WGH-G system, a significant amount of pyrazines were generated in the DWGH-G and AWGH-G systems. Although the nitrogen content of the wheat gluten hydrolysate was greater than the deamidated wheat gluten hydrolysate, there were 17 times more pyrazines generated in the DWGH-G system than the WGH-G system
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Amide Content & Thermal Generation of Maillard Flavor
Table I. Volatile Compounds Generated from the Thermal Reaction of WGH, DWGH and AWGH with Glucose Amount (ppm)
Compounds
Strecker Aldehydes 2-methylpropanal 3-methylbutanal 2-methyIbutanal phenylacetaldehyde total Alcohols 2-methylpropanol 1,2-propanediol 1,3-butanediol 2,3-butanediol cyclotene 2,3-dihydro-3,5-dihydroxy-6methyl-4H-pyran-4-one total Other Carbonvls 2,3-butanedione 2-butanone hydroxyacetone acetoin methyl propanoate hexanal 2-hydroxy-3-pentanone 4-chlorobutanoic acid 2-heptanone benzaldehyde acetophenone 2-phenylpropenal 4-phenyl-2-butanone 2-phenyl-2-butenal 5-methyl-2-phenyl-2-hexenal
DWGH-G
AWGH-G
9.8 141.2 39.1 1.3
277.1 2301.7 776.5 21.2
351.6 2912.0 1043.8 28.7
190.1
3355.3
4336.1
0.7 0.6 8.0 9.0 29.5
-
6.3
-
54.1
127.8
1.4 2.2 19.4 11.6
-
-
29.8 312.8 50.9
9.8 8.4
-
14.8 15.3 97.7
-
249.8
268.0
-
31.2 243.3 41.5 20.7
3.0 0.6 13.0 4.3 10.1 3.2 1.3
15.3 13.8 47.3
13.7 57.8
-
1.3 5.6
24.8 21.1 22.0
54.9
77.0
595.0
526.8
9.3 15.0 2.2
31.6 54.7 18.7
86.9 41.1 44.3
-
total Furans and Furanones 2-ftirfural 2-furfuryl alcohol 2-methyltetrahydrofuran-3-one
WGH-G
-
57.1 -
-
-
63.7 -
91
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Table I. Continued Amount (ppm)
Compounds WGH-G 2-acetylfiiran 5-methyl-2-furfural 5-hyaroxymethyl-2-furfural 2-phenylfuran dihydro-5-propyl-2(3H)-fiiranone total Pvrazines pyrazine methylpyrazine 2,5-dimethylpyrazine ethylpyrazine 2,3-dimethylpyrazine vinylpyrazine 2-methyl-5-ethylpyrazine trimethylpyrazine 2-methyl-5-ethylpyrazine 2-methyl-5-vinylpyrazine 2,5-dimethyl-3-ethylpyrazine 2,3-dimethyl-5-ethylpyrazine 2-methyl-5-propenylpyrazine 2-methyl-5-propylpyrazine 2-methyl-3-propylpyrazine 2,5-dimethyl-3-propylpyrazine 2,5-dimethyl-3-isobutylpyrazine
25.8 69.5 431.2 107.2
11.0 29.5 85.3 45.7 28.4
81.1
304.9
806.0
27.7 30.5 15.6 2.5 1.5 1.9
183.6 234.6 723.3 14.1 14.4 4.1 13.7 166.9 13.7
-
1.0 1.2
66.3 34.9
251.1 239.8 391.1 18.0 13.0 10.7 19.6 100.7 19.6 21.1 54.2 21.3 19.5 13.7 7.4 37.0 21.5
86.6
1543.6
1265.9
1.8 3.1 0.9 4.5 3.7 11.2 4.7 -
14.4
30.1
-
total
AWGH-G
0.9 2.6 35.8 6.4 8.9
4.7
total Other N-containing Compounds 2-acetylpyridine pyrrole-2-carboxaldehyde 2-acetylpyrrole 2,5-dioxo-3-methylpiperazine indole 5-acetyl-2,3-dihydro-1 H-pyrrolizine 5,6,7,8-tetrahydro-3-methylquinoline 5-propionyl-2,3-dihydro-1 H-pyrrolizine 5-acetyl-6-methyl-2,3-dihydro-1 H-pyrrolizine 2-acetyl-pyrido(3,4-d)imidazole
DWGH-G
-
61.0 -
9.1 9.1 -
-
19.6 -
141.6 26.9 8.4
-
24.6 24.0 -
-
-
1.3
35.3
339.9 74.1 52.5 47.1 40.7
31.2
236.2
633.0
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Amide Content & Thermal Generation of Maillard 93 Flavor
Table I. Continued Compounds
Amount (ppm) WGH-G
S-containing Compounds dimethyldisulfide thiophene-2-carboxaldehyde thiophene-3-carboxaldehyde 2-acetylthiophene 2-formyl-5-methylthiophene 2-acetylthiazole total
DWGH-G
AWGH-G
1.8 2.0 3.7 1.8 2.5 5.0
21.8 11.3
16.9 35.0
9.6
56.3
8.8
100.7
16.8
51.5
208.9
Furans and Furanones. There were eight furans and furanones identified in the WGHG, DWGH-G, and AWGH-G model systems. 5-Hycfroxymethyl-2-furfural, derived from the thermal degradation and caramelization of glucose in the Maillard reaction, was a dominant product for all model systems. The amount of cyclic oxygen-containing compounds generated in the AWGH-G system was much higher when compared to the other two systems. This may be due to the fact that amino acids catalyze the degradation of glucose, and that the AWGH contains the highest amount of amino acids. Pyrroles and Pyridines. In the three model systems, pyrroles were found in a minute amount, and 2-acetylpyrrole and pyrrole-2-carboxaldehyde were the only pyrroles detected. 2-Acetylpyridine was found in a minute quantity in the three model systems. The sensory properties of 2-acetylpyridine have been associated with popcorn, bread, cracker and coffee aromas (4). S-Containing Compounds. Four thiophene derivatives were identified in the WGH-G, DWGH-G, and AWGH-G systems; however two of them were not found in the DWGH and WGH-HC1 systems. This is probably because of a partial desulfuration reaction and the removal of hydrogen sulfide during either the deamination reaction or during acid hydrolysis. Thiazoles are somewhat unique among the Maillard products because they contain both a nitrogen and a sulfur atom in the same ring. Even though wheat gluten contains about 2% cysteine, only 2-acetylthiazole was identified in the model systems. This was, again, probably due to the lower abundance of hydrogen sulfide as a result of the deamidation reaction and acid-hydrolysis, which remove both ammonia and hydrogen sulfide. Dimethyldisulfide was the only non-cyclic sulfur compound identified in the WGH-G, DWGH-G, and AWGH-G model systems resulting from the condensation of the two molecules of methanethiol, derivedfrommethionine.
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Some Amino Acid-Specific Compounds. As listed in Table I, there were three pyrrolizines and one pyridoimidazole identified in the model systems. Pyrrolizines are a very important class of volatile compounds generated by the reaction of proline with reducing sugars. 2-Acetyl-pyrido(3,4-d)imidazole was another amino acid-specific Maillard reaction product identified in the model systems, which was derived from the reaction of histidine with dicarbonyls. Proline is the second most abundant amino acid (13-18%) contained in wheat gluten, and plays a very important role in flavor formation during food processing. A great deal of work has been carried out by Tressl et al. (5) on the volatile components generated in proline-specific Maillard reactions. The most abundant proline-specific Maillard reaction products are 2,3-dihydro-lH-pyrrolizines. The Maillard reaction of WGH-G, DWGH-G, and AWGH-G produced three 2,3dihydro-lH-pyrrolizines. The most abundant pyrrolizine was 5-acetyl-2,3-dihydro-lHpyrrolizine (5-ADHP). 5-Acetyl-2,3-dihydro-lH-pyrrolizine was identified in all three systems. 5-Propionyl-2,3-dihydro-l H-pyrrolizine was found in the DWGH-G and AWGH-G systems; while 5-acetyl-6-methyl-2,3-dihydro-1 H-pyrrolizine was only identified in the AWGH-G system It is very interesting to note that the amount of pyrrolizines generated were very different in each of the three model systems. The yields of pyrrolizines generated in the Maillard reactions were in this order: AWGH-G » DWGH-G » WGH-G. The main structural difference of proline between WGH, DWGH, and AWGH was that while the proline in the AWGH wasfreeform, most of the proline in WGH and DWGH existed as residues of peptides. Thefreeproline reacted with oc-dicarbonyls much faster than proline-containing peptides to form iminium ions which act as key compounds for proline specific compounds. The reason that only small amounts of pyrrolizines were generated in the DWGH-G and WGH-G systems was that most of proline-containing peptides had more than three residues which were sterically hindered to react with oc-dicarbonyls to form reactive intermediates. Histidine-specific Maillard reaction products have not been investigated to any appreciable extent, probably due to the fact that they do not contribute any characteristic flavors to cooked foods. Gi et al. (6) identified 2-acetyl-pyrido(3,4-d)imidazole and 2acetyl-pyrido(3,4-d)imidazole by the reaction of histidine with glucose at roasting and autoclaving conditions. While the wheat gluten contained relatively high amounts of histidine (up to 1.8-3.2%) DWGH contained 0.78% offreehistidine. It is interesting to find that the yields of 2-acetyl-pyrido(3,4-d)imidazole found in the DWGH-G and AWGH-G systems were 35.3 and 40.7 ppm, respectively. Again, the results indicate the fact that the peptides which contain histidine will react with glucose to form the peptide Schiff bases. The peptide Schiff bases undergo a rearrangement reaction to promote the hydrolysis of the peptide bonds leading to the formation offreehistidine. Histidine then undergoes further reactions leading to the formation of pyridoimidazoles. The yield of pyridoimidazole found in the WGH-G system was 1.3 ppm which was much less than that found in the DWGH-G and AWGH-G systems (35 & 40 ppm), respectively. Effects of Deamidation on Flavor Formation The deamidation reaction resulted in the release of ammonia from the residues of glutamine and asparagine in the wheat gluten hydrolysate. The ammonia released during
8. CHEN & HO
Amide Content & Thermal Generation of Maillard Flavor
the Maillard reaction will participate in the formation of heterocyclic compounds such as pyrazines, and also participate in pigment formation (7). The results listed in Table I demonstrate that the reaction of DWGH with glucose produces much greater amounts of volatile compounds than is produced by the interaction of WGH with glucose. About 17 times the amount of Strecker aldehydes, 8 times the amount of other carbonyls, 2.5 times the amount of hydrocarbons, 3.7 times the amount of furans, 18 times the amount of pyrazines, 4 times the amount of other N-containing compounds, and 3 times the amount of S-containing compounds were produced in the DWGH-G system than in the WGH-G system An examination of the difference in the amount of volatile compounds generated in the WGH-G and DWGH-G systems indicated that ammonia, a reactive species, played a very important role in the Maillard reaction. Shibamoto (8) found that ammonia participated in the formation of N-containing volatile compounds in the Maillard reaction by reacting ammonia and hydrogen sulfide with glucose. Shibamoto (9) reacted ammonia with rhamnose, and identified 65 volatile compounds which included pyrazines, pyrroles, and imidazoles. Another study was carried out by Hwang (70) to investigate the participation of amide groups of glutamine in the pyrazine formation by the Maillard reaction. The results indicated that the relative contribution of amide nitrogen to pyrazine formation was greater than the contribution of a-amino nitrogen. Other studies were carried out to demonstrate that ammonia reacted with sugars or sugar degradation products to form melanoidins. According to Kato (77) melanoidin is thought to consist mainly of a repeating aromatic moiety. Izzo (72) investigated the effects of residual amide content on the aroma generation and formation of brown color by comparing the reactions of deamidated and undeamidated wheat gluten with glucose in model systems. The experiments demonstrated that the deamidation reaction of wheat gluten greatly decreased the pigment formation in the Maillard reaction when the deamidation degree was higher than 20%. It was observed that the reacted mixture of WGH-G contained large amounts of dark colored products while the mixturefromthe reaction of DWGH with glucose contained only very small amounts of colored products. Investigation of this phenomena and the formation of volatile compoundsfromthe WGH-G and DWGH-G systems demonstrated that the deamidation reaction of wheat gluten hydrolysates would increase the formation of aroma compounds and decrease the formation of brown color. The fact that ammonia could enhance the generation of brown color and suppress pyrazine formation was investigated (72). It was thought to be attributed to the reactive nature of the chemistry of the ammonia molecule. The ammonia releasedfromthe amide groups of proteins or peptides during the Maillard reaction interacts with glucose leading to the formation of an unstable intermediate which when rearranged produces a primary amine. The primary amine, being quite reactive itself, then undergoes further reactions with another molecule of glucose or another carbonyl compound. The end result is a polymerization of the glucose in the early stages of the Maillard reaction resulting in a subsequent shift in chemical events away from flavor formation. The traditional mechanism for flavor formation could have been suppressed so that the net result was the formation of pigments with little formation of volatiles. An examination of the quantitative data of aroma formation in the heated WGH-G or DWGH-G samples provides a rather clear trend. Even though the ammonia released from
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the amides of proteins or peptides during the Maillard reaction could participate in both the volatiles formation and pigment formation by reacting with reducing sugars, it probably interacted with reducing sugars to generate colored polymers. Literature Cited 1. Nagodawithana, T. W. Savory Flavors. Esteekay Associates, Inc.: Milwaukee; 1995, pp. 401-434. 2. Hamada, J. S. CRC Crit. Rew. Food Sci. Nutri. 1994, 34, 283-292. 3. Rizzi, G. P. In Thermal Generation of Aroma; Parliment, T. H.; McGorrin, R. J.; Ho, C.-T., Eds. ACS Symp. Ser. 409; American Chemical Society: Washington, D. C. 1989, pp. 172-181. 4. Maga, J. A. J. Agric. Food Chem. 1981, 29, 895-898. 5. Tressl, R.; Helak, B.; Martin, N.; Kersten, E . In Thermal Generation of Aroma; Parliment, T. H.; McGorrin, R. J.; Ho, C.-T., Eds. ACS Symp. Ser. 409; American Chemical Society: Washington, D. C. 1989, pp. 156-171. 6. Gi, U.; Baltes, W. In Thermally Generated Flavors. Parliment, T. H.; Morello, M . J.; McGorrin, R. J., Eds. ACS Symp. Ser. 543; American Chemical Society: Washington, D. C. 1994, pp. 263-269. 7. Izzo, H.; Ho, C.-T. J. Agric. Food Chem. 1993, 41, 2364-2367. 8. Shibamoto, T. J. Agric. Food Chem. 1977, 25, 206-208. 9. Shibamoto, T.; Bernhard, R. A. J. Agric. Food Chem. 1978, 26, 183-187. 10. Hwang, H. I.; Hartman, T. G.; Rosen, R. T.; Ho, C.-T. J. Agric. Food Chem. 1993, 47, 2112-2115. 11. Kato, H.; Tsuchida, H. Prog. Food Nutr. Sci. 1981, 5, 147-156. 12. Izzo, H.; Ho, C.-T. J. Agric. Food Chem. 1991, 39, 2245-2248.
