J. Agric. Food Chem. 1998, 46, 5225−5231
5225
Comparison of the Aroma Characteristics of Acid-Hydrolyzed and Enzyme-Hydrolyzed Vegetable Proteins Produced from Soy Margit Dall Aaslyng,† J. Stephen Elmore,‡ and Donald S. Mottram*,‡ Chemistry Department, The Royal Veterinary and Agricultural University, Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, and Department of Food Science and Technology, The University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom
The aroma volatiles of an acidic hydrolyzed vegetable protein (HVP) and an enzymatic hydrolyzed vegetable protein (EVP) were compared by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GCO). Major differences were found between the two hydrolysates. Furans and furanones, pyrroles, and sulfur-containing compounds were mainly present in HVP, whereas alcohols (including phenols) and pyrazines were mainly found in EVP. These differences reflected the two production methods. The high temperature and low pH during the acid hydrolysis of HVP favored the production of furans and the decomposition of sulfur amino acids, resulting in the production of sulfur-containing volatile compounds. A pH above 5 during the enzymatic hydrolysis facilitated the production of pyrazines. The alcohols in EVP may be formed by the added enzymes or by enzymes naturally present in the soy grits. Significant differences in the odors between the hydrolysates were detected by GCO. Keywords: Acid-hydrolyzed vegetable protein; enzyme-hydrolyzed vegetable protein; Maillard reaction; aroma; volatiles INTRODUCTION
MATERIALS AND METHODS
Hydrolyzed vegetable protein (HVP) is a savory flavoring product produced by heating a protein source, such as soybean flour, wheat, or maize, with 4-6 M hydrochloric acid at 100-130 °C for 4-24 h (Manley et al., 1981; Dzanic et al., 1985; Weir, 1986). Enzymehydrolyzed vegetable protein (EVP), where the protein source is hydrolyzed by enzymes, is a newer alternative to HVP. Enzymatic hydrolysis is carried out at a pH between 5 and 7 at 50-55 °C for 10-24 h. Deactivation of the enzymes at 85 °C for 5 min is carried out after the hydrolysis (Pommer, 1995). The different production conditions result in two hydrolysates that are very different in color and flavor. The acidic hydrolysate is dark brown with a strong savory flavor, whereas the enzymatic hydrolysate is usually lighter in color and has a much less pronounced meaty or savory flavor (Weir, 1992). Very little has been published comparing the volatile composition of acid and enzymatic hydrolysates. Misharina et al. (1987) reported on the volatile compounds in HVP and EVP heated with xylose and cysteine. The composition and sensory properties of HVP and EVP preparations similar to those used in the present work have been reported recently (Aaslyng et al., 1998). Some of the major volatile components were also identified, but a detailed comparison of the volatile compositions of the two hydrolysates was not undertaken. This paper compares the volatile compositions of EVP and HVP and identifies the compounds that make significant contributions to the aromas of these hydrolysates.
Chemicals. Untoasted defatted soy grits (Unisoy, 49.6% protein) were obtained from Loders Crocklaan (Holland); Flavourzyme (from Aspergillus oryzae, freeze-dried preparation, 3872 LAPU/g) and Alcalase 2.4L (from Bacillus licheniformis, 2.4 AU/g) were obtained from Novo Nordisk A/S (Bagsvaerd, Denmark); 1,2-dichlorobenzene (99%, spectrophotometric grade) was obtained from Aldrich Chemical Co. (Gillingham, U.K.); n-hexane was obtained from BDH Laboratory Supplies (Poole, U.K.). Production of Hydrolysates. The acidic hydrolysate (HVP) was prepared by heating Unisoy 800 (100 g) with 240 mL of 4 M hydrochloric acid in a sealed glass bottle at 110 °C for 6 h (Aaslyng et al., 1998). After being cooled to room temperature, the mixture was neutralized to pH 6.5 with 4 M sodium hydroxide and centrifuged. The enzymatic hydrolysate (EVP) was produced using the proteolytic enzymes Flavourzyme and Alcalase. Unisoy 800 (150 g) was mixed with 825 g of water and pasteurized at 85 °C for 5 min. After cooling to 50 °C, the pH was adjusted to pH 7.0 with 4 M sodium hydroxide. Flavourzyme (0.75 g) and Alcalase (0.75 g) were added to the mixture, which was allowed to stand at 50 °C for 5 h, after which time the pH was adjusted to 5 with 4 M hydrochloric acid. Sodium chloride (14.5 g) and Flavourzyme (0.38 g) were added, and the hydrolysis continued without pH adjustment for 24 h at 50 °C. Both hydrolysates were freeze-dried and stored at -18 °C until analysis. Isolation of Volatiles. Sodium chloride was added to EVP so that its concentration was the same as in HVP (39%). A solution (100 mL) of each hydrolysate (15%) was then made up in water and placed in a 250-mL conical flask. Aroma isolates were collected on Tenax TA. The sample was held at 60 °C for 1 h while oxygen-free nitrogen, at 40 mL/min, swept the volatiles onto a glass-lined, stainless steel trap (105 mm × 3 mm i.d.) containing 85 mg of Tenax TA (Scientific Glass Engineering Ltd., Milton Keynes, U.K.). A standard (130 ng of 1,2-dichlorobenzene in 1 µL of n-hexane) was added to the trap at the end of the collection, and excess solvent and any water retained on the trap were removed by
* To whom correspondence should be addressed (e-mail
[email protected]; fax +44 118 931 0080). † The Royal Veterinary and Agricultural University. ‡ The University of Reading.
10.1021/jf9806816 CCC: $15.00 © 1998 American Chemical Society Published on Web 11/13/1998
5226 J. Agric. Food Chem., Vol. 46, No. 12, 1998
Aaslyng et al.
Table 1. Compounds Found in the Headspace Volatiles of Hydrolyzed Soy Protein Prepared Using Acid and Enzymatic Hydrolysis previously reported in LRIb
amount in headspacec HVP EVP
method of identificationd
soy and unfermented soy productse
HVP or EVP
3-methylbutanal 2-methylbutanal pentanal (E)-2-methyl-2-butenal 3-methyl-2-butenal hexanal heptanal (E)-2-heptenal benzaldehyde octanal phenylacetaldehyde (E)-2-octenal nonanal (E)-2-nonenal decanal
670 678 709 755 800 812 914 970 988 1016 1072 1076 1119 1178 1219
Aldehydes 1298 (47) 957 (209) 635 (44) 587 (154) nd 55 (24) 24 (1) 18 (4) 24 (3) 33 (6) 41 (1) 360 (84) 33 (2) 32 (6) nd 21 (4) 105 (4) 281 (43) 15 (0) 35 (5) 138 (23) 124 (35) nd 33 (10) 113 (5) 177 (36) nd 52 (9) 48 (1) 23 (5)
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI
X X X X
X f,g Xf Xe
X X X X X X X X X X
Xf,h
2,3-butanedione (diacetyl) 2-butanone 2-pentanone 3-pentanone 2,3-pentanedione 3-hydroxy-2-butanone (acetoin) 3-methyl-2-pentanone 2,3-hexanedione 2-methylcyclopentanone 5-methyl-2-hexanone 3-methylcyclopentanone 2-heptanone 2-methylcyclopent-2-en-1-one 2,3-octanedione 6-methyl-2,4-heptanedione 3-octen-2-one 3,5,5-trimethyl-2-cyclohexen-1-one (isophorone) acetophenone 2-dodecanone
613 614 698 707 710 717 762 796 859 862 868 900 922 995 1036 1053 1080 1093 1410
Ketones j j 25 (4) 8 (0) 188 (21) nd 3 (0) 25 (2) 3 (0) 5 (0) 3 (0) 31 (3) 23 (3) 15 (1) nd nd 55 (4) 42 (3) 86 (11)
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms + lri ms ms + lri MS + LRI MS + LRI MS + LRI
X X X
X
Xe,i
2-methyl-1-propanol 3-methyl-3-buten-1-ol 3-methyl-1-butanol 2-methyl-1-butanol 1-pentanol 1-hexanol 1-octen-3-ol phenol 2-ethyl-1-hexanol 1-octanol 2-methoxyphenol (guaiacol) 4-vinylphenol 1-decanol 2-methoxy-4-vinylphenol 1-undecanol 2-tert-butyl-4-methylphenol 1-dodecanol
644 743 750 753 780 880 991 1002 1038 1083 1114 1247 1285 1344 1387 1387 1487
MS + LRI ms + lri MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI msk MS + LRI msl + lri MS + LRI ms MS + LRI
X
Xf
X X X X X X
Xe,f Xf Xf Xe,f
X X X
Xe Xe,i
ethyl acetate 3-methylbutyl acetate methyl 4-oxopentanoate (methyl levulinate) phenylethyl acetate 4-vinylphenyl acetate 120, 91 (15), 91 (15), 43 (11), 121 (9), 162 (8), 65 (7)
626 884 1002 1279 1313
compounda
tetrahydro-3-methylfuran 2-ethylfuran dihydro-5-methyl-3[2H]furanone 2-furfural 5-methyl-2[3H]-furanone 2-acetylfuran 2-butyltetrahydrofuran 5-methyl-2-furfural
705 707 822 852 885 929 981 982
j j 9 (5) 1 (1) 68 (33) 54 (26) 27 (8) nd 1 (0) nd 1 (0) 39 (11) nd 31 (9) 38 (5) 30 (5) nd 5 (1) nd
Alcohols and Phenols 7 (0) 152 (42) nd 25 (6) 12 (0) 827 (222) 12 (1) 384 (97) 22 (3) 115 (30) 59 (6) 407 (114) 2 (0) 134 (32) 4 (1) 73 (29) 95 (10) 98 (36) nd 48 (17) 116 (9) 1968 (357) nd 58 (21) 52 (2) 6 (2) 1 (0) 218 (83) 95 (32) nd 50 (5) nd 1395 (123) 10 (4) Esters 1 (0) nd 71 (10) tr nd
28 (9) 81 (9) nd 36 (9) 31 (3)
Furans and Furanoids 54 (5) nd tr 22 (7) 29 (5) nd 391 (40) 24 (4) 105 (20) 2 (0) 1215 (137) 20 (3) 23 (1) nd 129 (14) 2 (0)
MS + LRI MS + LRI ms ms + lri se
ms MS + LRI ms MS + LRI ms MS + LRI ms MS + LRI
Xe,f,i Xe Xe,i
Xf Xe,f,i
X X X X X X
X
X X
Xe
X
X
Xe,f,i Xe Xe,f
X
Xe,f,i
X
Aroma Compounds from HVP and EVP
J. Agric. Food Chem., Vol. 46, No. 12, 1998 5227
Table 1. (Continued) previously reported in compounda
LRIb
2-pentylfuran 2-acetyl-5-methylfuran 3-acetyl-2,5-dimethylfuran an acetyldimethylfuran 123, 138 (38), 43 (15), 67 (8), 81 (8), 41 (7), 39 (6), 95 (6), 124 (6) 5-methyl-2-propionylfuran an acetyldimethylfuran 123, 138 (45), 43 (14), 67 (11), 124 (7), 39 (5) an acetyltrimethylfuran 137, 152 (49), 43 (32), 67 (11), 109 (11), 95 (9), 138 (8)
999 1054 1103 1144
amount in headspacec HVP EVP Furans and Furanoids nd 60 (26) 333 (36) 3 (0) 28 (2) nd 25 (3) nd
method of identificationd
soy and unfermented soy productse
MS + LRI MS + LRI MS + LRI se
X
1151 1177
219 (4) 46 (4)
2 (0) nd
MS + LRI se
1234
26 (3)
nd
se
2,5(and/or 6)-dimethylpyrazine ethylpyrazine trimethylpyrazine 2-methyl-5(and/or 6)-vinylpyrazine 2,5-dimethyl-3-ethylpyrazine a dimethylethylpyrazine tetramethylpyrazine ethyltrimethylpyrazine isopentyltrimethylpyrazine an MW 192 alkyldimethylpyrazine 122, 135 (19), 123 (12), 163 (5), 80 (5), 192 (3)
930 933 1020 1038 1094 1102 1105 1173 1406 1492
Pyrazines 6 (0) 230 (56) 15 (2) nd 24 (5) 481 (177) nd 15 (5) nd 51 (16) 34 (8) nd nd 395 (199) nd 182 (79) nd 91 (43) nd 4 (1)
MS + LRI MS + LRI MS + LRI ms MS + LRI se MS + LRI ms se se
2,5-dimethyl-1H-pyrrole an ethylmethylpyrrole 94, 109 (43), 93 (15), 108 (8), 95 (7), 106 (6) an ethyldimethylpyrrole 108, 123 (38), 93 (28), 106 (12), 107 (8), 94 (7), 109 (7), 122 (5)
937 1019
Pyrroles 444 (48) tr 85 (8) nd
ms se
1097
29 (3)
se
dimethyl disulfide 2-(methylthio)propanal 75, 104 (63), 47 (18), 49 (11) 1-(methylthio)-2-propanone tetrahydro-2-methylthiophene unknown, MW 113 3-(methylthio)propanal dimethyl trisulfide 2-acetylthiophene benzyl methyl sulfide dimethyl tetrasulfide benzothiazole toluene trimethyloxazole 1-dodecene 1-tetradecene unknown, MW 206
nd
Sulfur Containing 252 (14) 41 (16) tr nd
MS + LRI se
863 866 923 928 994 1116 1200 1260 1271
10 (1) 15 (1) 2 (0) 35 (1) 270 (9) 8 (1) 62 (2) 28 (3) 32 (3)
msm ms se MS + LRI ms + lri MS + LRI ms ms + lri MS + LRI
777 857 1195 1398 1494
Miscellaneous 5 (1) 18 (7) 2 (0) 79 (31) 52 (14) nd 64 (19) nd nd 2 (1)
756 825
tr nd nd 25 (8) 12 (5) nd nd nd 3 (0)
MS + LRI MS + LRI MS + LRI MS + LRI se
HVP or EVP
Xe
X X X X
Xe-g,i Xg,i Xf,i Xi Xe,f,i Xe Xe-g,i Xe-g
X
Xe-g
X X X
Xe,g Xe-g
X X
a Mass spectral data given for tentatively identified compounds, m/z (relative intensity). b Linear retention index on BPX-5 capillary column. c Amount in the headspace from 15 g of sample dissolved in 100 mL of water, reported as mean values of triplicate analyses with standard deviation in parentheses; nd,
