Changes in Aroma Concentrates during Storage - ACS Symposium

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6 ChangesinAroma Concentrates during Storage 1

1

2

1

G. R. Takeoka , M . Guentert , Sharon L. Smith , and W. Jennings 1

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Department of Food Science and Technology, University of California, Davis,CA95616 I B M Instruments Inc., P.O. Box 332, Danbury, CT 06810 Kiwifruit (Actinidia chinensis Planch.) concentrates were prepared by vacuum d i s t i l l a t i o n , followed by continuous l i q u i d - l i q u i d extraction and concentration of the extracts. The concentrates underwent various changes when stored at -10°C. The a r t i f a c t s produced were analyzed and characterized by c a p i l l a r y GC, GC/MS and GC/FTIR. Carboxylic acids, probably produced by f r e e - r a d i c a l oxidation, comprised the major portion of the a r t i f a c t s . Possible mechanisms of a r t i f a c t formation and methods to minimize their production are discussed. The formation of a r t i f a c t s during sample preparation has been addressed by various researchers (_1_~^)« A less frequently examined problem is the formation of a r t i f a c t s during storage of aroma concentrates. DeMets and Verzele (5) noted the decrease of myrcene and the production of new v o l a t i l e s , mainly a t t r i b u t a b l e to the degradation of b i t t e r acids, during the storage of hops. Badings (6) reported that storage at -40°C was required to keep a concentrate of cold-stored butter v o l a t i l e s unchanged for 12 hours. The influence of storage temperature was also observed by Kepner et a l . (7_) who found that while the amount of germacrene D in Douglas f i r o i l did not change for one year when stored at -20°C, J. disappeared within 6 weeks when the o i l was stored at 5°C. They postulated that the loss was not a thermal isomerization but rather interaction with another component in the o i l . The autoxidation of caryophyllene in Cannabis o i l has been reported by Paris ( 8 ) . Similarly, a marked decrease in caryophyllene l e v e l along with a corresponding increase in caryophyllene oxide l e v e l was observed in rough lemon (Citrus jambhiri Lush.) leaf o i l after storage at 9°C for 7 months (9). The i n s t a b i l i t y of the d i s t i l l e d o i l was attributed to the removal of nonvolatile natural antioxidants which are normally present in most cold-pressed c i t r u s o i l s . The formation of a r t i f a c t s in aroma concentrates is influenced by a number of factors including storage temperature, atmosphere, the presence of pro-oxidants and/or antioxidants, the concentration 0097-6156/86/0317-0065S06.00/0 © 1986 American Chemical Society Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

BIOGENERATION OF AROMAS

66

and s t a b i l i t y of the v o l a t i l e s present in the concentrate, and time. During our recent investigation of the v o l a t i l e constituents of k i w i f r u i t ( A c t i n i d i a chinensis Planch.) a rapid change in composition of the concentrated extracts during storage was observed. The purpose of this study was to i d e n t i f y the a r t i f a c t s formed during storage and to postulate mechanisms for their formation.


Experimental Sample Preparation. K i w i f r u i t of the major commercial variety, Hayward, was obtained l o c a l l y . The f r u i t was allowed to ripen at room temperature at which point the soluble solids content of the juice was 16-17%. After separation of the the skin, the pulp was gently blended in a Waring blender taking care not to fracture the seeds. The blended pulp was immediately subjected to vacuum steam distillation. Isolation of V o l a t i l e s . An aliquot of blended pulp (1.2 kg) was diluted with d i s t i l l e d water (700 mL) in a 3-L three-neck f l a s k and vacuum d i s t i l l e d (25-30°C/l mm Hg). D i s t i l l a t i o n continued for 2.5 to 3 h y i e l d i n g approximately 500 mL of d i s t i l l a t e which was collected in two l i q u i d nitrogen cooled traps. A t o t a l of 3.6 kg of f r u i t pulp was d i s t i l l e d in three batches. The d i s t i l l a t e s were combined and immediately frozen u n t i l use. The combined d i s t i l l a t e was extracted in 250 mL batches for 20 h with 60 mL t r i c h l o r o fluoromethane (Freon 11, b p 23.8°C) using a continuous l i q u i d l i q u i d extractor. The trichlorofluoromethane was d i s t i l l e d through a 120 χ 1.3 cm glass d i s t i l l a t i o n column, packed with Fenske helices, prior to use. Each extract was c a r e f u l l y concentrated to approximately 100 yL by d i s t i l l a t i o n of solvent using a Vigreux column (16 cm), and a maximum pot temperature of 30°C. Gas Chromatography. A Hewlett-Packard 5880A gas chromatograph with a FID, equipped with a 30 m χ 0.32 mm i d DB-WAX column ( d - 0.25 ym, bonded polyethylene g l y c o l phase, J&W S c i e n t i f i c ) was employed. The column temperature was programmed as follows: 30°C (2 min i s o thermal), to 38°C at l°C/min, then to 180°C at 2°C/min and held for 20 min. Hydrogen c a r r i e r gas was adjusted to an average l i n e a r v e l o c i t y of 49.7 cm/sec (30°C). The injector and detector were maintained at 225°C. A modified i n j e c t i o n s p l i t t e r (J&W Scien t i f i c ) was used at a s p l i t r a t i o of 1:30. f

Gas Chromatography-Mass Spectrometry. A Finnigan MAT 4500 series quadrupole gas chromatograph/mass spectrometer/data system equipped with the same c a p i l l a r y column and using the same temperature program described in the previous section was employed. Helium was used as the c a r r i e r gas at an average linear v e l o c i t y of 47.7 cm/sec (30°C). Injector temperature was 220°C, and the ion source temperature was 180°C. The outlet end of the fused s i l i c a column was inserted d i r e c t l y into the ion source block, which was maintained at approximately 180°C. Gas Chromatography-Fourier Transform Infrared Spectroscopy. An Instruments 9630 gas chromatograph was coupled with an

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

IBM IBM

6.

TAKEOKA ET AL.

Changes in Aroma Concentrates during Storage

67

Instruments IR-85 Fourier Transform infrared spectrometer, through an IBM GC-IR interface. The interface consisted of a gold-coated Pyrex light-pipe with potassium bromide windows. A scan rate of 6 scans/sec and a spectral resolution of 8 cm"** were used for data a c q u i s i t i o n . Samples were introduced into the system v i a s p l i t l e s s injections. A fused s i l i c a c a p i l l a r y column, 30 m χ 0.32 mm i d DB-WAX ( d = 1.0 ym), was employed with the outlet end connected d i r e c t l y to the GC-IR light-pipe entrance. Helium was used as the c a r r i e r gas at an average l i n e a r v e l o c i t y of 41.4 cm/sec (35°C). No make-up gas was employed in the system. The column temperature was programmed from 35°C to 180°C at 2°C/min. The GC-IR light-pipe assembly was maintained at 170°C.


f

Results and Discussion In our recent studies of k i w i f r u i t ( A c t i n i d i a chinensis Planch.) v o l a t i l e s (10), we noticed a rapid change in the composition of the concentrated extracts during storage in the freezer at -10°C. The a r t i f a c t s formed during storage were analyzed and characterized by c a p i l l a r y GC, GC/MS and GC/FTIR. To understand the s e n s i t i v i t y of the extracts to a r t i f a c t formation J. is informative to review the v o l a t i l e s in k i w i f r u i t . Quantitatively, peroxidation products of unsaturated fatty acids (11, 12), which include (E)-2-hexenal (77.87%), (E)-2-hexen-l-ol (6.80%), 1-hexanol (3.40%), hexanal (1.78%), (Z)-2-hexenal (0.87%), (E)-3-hexen-l-ol (0.32%) and (Z)-3-hexen-l-ol (0.17%), constitute over 90% of the t o t a l v o l a t i l e s . Other major constituents include the esters, methyl butanoate (2.54%) and ethyl butanoate (3.52%). The presence of large amounts of saturated and unsaturated aldehydes in the extract is noteworthy since they are quite susceptible to f r e e - r a d i c a l oxidation. We therefore expected that at least some of the a r t i f a c t s were the products of autoxidation. It has been suggested that autoxidation of saturated fatty acids and aldehydes occurs through a f r e e - r a d i c a l mechanism (13, 14). Supporting evidence of a r a d i c a l chain mechanism was provided by Palamand and Dieckmann (15) who studied the autoxidation of hexanal. The reaction involves peroxycarboxylic acid as an intermediate (16) and probably proceeds v i a the mechanism shown in Figure 1. Comparison of Fresh and Stored Samples. Figure 2 shows GC/FID chromatograms of a freshly prepared k i w i f r u i t extract (top), an extract which had been stored in the freezer f o r three months (middle) and an extract which had been stored in the freezer for four months (bottom). The a r t i f a c t s which developed during storage are numbered in the middle and bottom chromatograms. Table 1 l i s t s these a r t i f a c t s . As can be seen in Figure 2, there is a dramatic decrease in the levels of hexanal, (Z)-2-hexenal, (E)-2-hexenal, (E)-3-hexenol, (Z)-3-hexenol and (E)-2-hexenol upon storage. There is a corresponding increase in the l e v e l of a r t i f a c t s which generally elute at longer retention times than the native constituents. Mass spectral i d e n t i f i c a t i o n s were v e r i f i e d by comparison with Kovats retention indices of authentic reference standards.



0

0

II

II

->


R - C - H + X

R - C 0

0

II

II C

->

R

II 0-

+

0

->

C

II R - C - 0

OH

+

R

II

R - C - O - O H

0

0

0

II

II

R

0-0-

C

0

0

C - 0 -

+ HX

OH

II

I c I

C

+

R-C-

0

II 0 - 0 - C - R

H

:OH 1

©OH

II

/>

R - C - OH R - C - 0 - 0 H

©OH

II

R - C - OH

0

II C - R

2 R

OH

Figure 1. Possible mechanism for the oxidation of aldehydes to carboxylic acids.


6. TAKEOKA ET AL.

Changes in Aroma Concentrates during Storage

69

FTIR Studies. The identity of certain a r t i f a c t s was confirmed by FTIR spectral data. The application of GC/FTIR in flavor research has been demonstrated by Schreier et a l . (17). Examples of vapor phase IR spectra of selected a r t i f a c t s are shown in Figure 3. There is a s h i f t in the spectral bands of a l l molecules on a change of state. Therefore, IR spectra taken in the vapor phase w i l l d i f fer from those taken in the condensed phase. In general, stretch ing vibration bands move to higher wavenumbers in the vapor phase while deformation vibration bands move to lower wavenumbers (18). Spectrum A in Figure 3 shows bands t y p i c a l of an ester; the C=0 stretching absorption has been shifted to about 1751 cm" from the 1728 cm observed in the condensed phase while the C-0 stretching vibration occurs at about 1173 cm" . The other three spectra represent carboxylic acids. Carboxylic acids constitute the majority of the a r t i f a c t s formed. While carboxylic acids can exist either in the monomeric or dimeric form in the vapor state, higher temperatures favor the existance of monomers. It is evident from the sharp 0-H stretching band between 3580 and 3587 cm" that the acids exist as monomers. The normal saturated acids display a strong C=0 stretching absorption between 1778 and 1782 cm" with the C-0 stretching band occurring between 1142 and 1153 cm" . The t h i r d spectrum (C) displays bands at 1753, 1663 and 980 cm" , indicative of an unsaturated acid with a conjugated trans configuration. Comparison with a standard of (E)-2-hexenoic acid revealed an almost i d e n t i c a l spectrum. Another carboxylic acid (not shown) had absorption bands at 3587, 1763, 1645, 1138, 1111 and 818 cm suggesting an unsaturated acid with a conjugated c i s configuration. The spectrum was consistent with a standard of (Z)-2-hexenoic acid. The presence of the large amounts of carboxylic acids can lead to acid-catalyzed degradations and rearrangements of other constituents present in the extract.


1

-1

1

1

1

1

1

Formation of 2-Ethyl-2(5H)- Furanone. The presence of a r t i f a c t s with increased retention times suggests the formation of components of increased p o l a r i t y and/or the formation of higher molecular weight constituents from condensation or addition reactions. The acids, aldehydes and alcohols present can undergo oxidation to form γ- and δ-lactones (14, 15). The formation of the lactone, 5-ethyl2(5H)-furanone, probably occurs by the steps outlined in Figure 4. A plausible sequence would be reaction of 2-hexenoic acid to form a peroxy r a d i c a l at the γ-position followed by production of the hydroperoxide. Cleavage of the 0-0 bond with the subsequent addition of Η· could lead to 4-hydroxy-2-hexenoic acid. Intra molecular e s t e r i f i c a t i o n would then produce the i d e n t i f i e d lactone. Upon longer storage (see bottom chromatogram in Figure 2) a series of a r t i f a c t s with Kovats indices ranging from 2100 to 2400 developed. Mass spectral data suggested that many of these a r t i f a c t s are s t r u c t u r a l l y related (similar mass ions), possibly longer chain δ-lactones. We are unable to elucidate their structures at present. Conclusion The formation of a r t i f a c t s in aroma concentrates during storage is a potential problem in flavor research. Storage of concentrates


70



1

Figure 2. C a p i l l a r y gas chromâtograms of k i w i f r u i t v o l a t i l e s . Top to bottom: a freshly prepared extract; an extract which had been stored at -10°C for three months; an extract which had been stored at -10°C f o r four months. The peak numbers correspond to the numbers in Table 1.



0

c

c

f

d

e

c

1822 1846 1856 1924 1944 2078 2127 2176 2183 2200 2209 2226 2237 2250 2281 2287 2309 2383 2390

1710 1714

1161 1212 1439 1464 1517 1563 1592 1604 1637

Kovats index^ DB-WAX

116 100 114 114 114

112 102

200 88

tentatively identified by retention data only.

^Tentatively identified by mass spectral data ^Mass spectra of more than one component present. ^Retention

Identity also confirmed by GC/FTIR.

indices agree to within ± 2 units with reference compounds.

only,

c

^Mass spectrum and Kovats index are consistant with

41(98), 55(100), 60(62), 68(65), 69(53), 96(14), 99(14), 114(73) 41(33), 42(43), 55(35), 60(15), 68(34), 73(100), 99(39), 114(18) 39(5), 41(9), 55(40), 68(5), 81(2), 85(2), 97(100), 98(7) 57(54), 69(16), 83(14), 99(100), 113(13), 155(59), 171(5), 185(2) 43(59), 55(52), 62(31), 71(100), 73(29), 81(11), 109(10), 134(2) 55(41), 69(26), 81(27), 99(100), 141(58), 171(24), 185(10), 213(2) 43(24), 57(25), 61(100), 67(12), 85(16), 103(4), 121(14), 123(5) 57(95), 69(21), 99(100), 113(17), 155(78), 171(19), 185(8), 213(2) 57(100), 69(37), 81(84), 99(50), 171(46), 183(39), 185(20), 213(4) 57(77), 69(15), 81(24), 99(100), 145(5), 171(13), 185(4), 213(1) 55(39), 69(26), 81(28), 99(100), 141(58), 171(25), 185(11), 213(2) 57(100), 69(47), 81(94), 99(79), 145(17), 171(55), 185(25), 213(2) 57(63), 81(45), 97(53), 99(100), 145(10), 171(31), 185(14), 213(1) 57(60), 69(25), 81(24), 97(32), 99(100), 171(14), 185(4), 213(1) 43(59), 44(82), 55(98), 61(23), 73(100), 85(5), 91(6), 103(12) 55(20), 57(65), 69(14), 81(18), 99(100), 171(12), 185(4), 213(1)

42(5), 45(54), 46(6), 55(13), 56(13), 57(29), 73(65), 74(100) 39(13), 41(33), 43(44), 44(39), 45(14), 55(100), 57(34), 73(55) 43(74), 56(43), 61(12), 69(22), 71(18), 84(58), 99(68), 117(100) 41(16), 42(17), 43(14), 45(13), 55(7), 60(100), 73(37), 88(3) 39(13), 41(37), 43(50), 44(24), 45(10), 55(100), 57(23), 73(33) 41(100), 43(38), 45(30), 55(24), 57(84), 58(65), 59(73), 85(89) 39(4), 41(2), 54(3), 55(19), 57(8), 83(100), 84(14), 112(7) 41(13), 42(8), 43(10), 45(10), 55(9), 60(100), 73(46), 87(3) 39(10), 41(36), 43(50), 55(100), 57(18), 71(8), 73(18), 85(3) 41(20), 43(16), 55(13), 57(11), 60(100), 73(58), 87(20), 99(2) 39(39), 41(28), 45(26), 55(100), 60(15), 73(16), 82(18), 100(94)

39(8), 41(8), 55(5), 57(100), 68(4), 71(3), 86(7), 114(12) 41(33), 42(33), 43(34), 55(43), 56(100), 69(38), 84(13), 87(4) 41(100), 43(34), 45(22), 47(14), 55(62), 56(58), 60(9), 73(33)

114 130 74

mass spectral data, m/z (rel intensity)

MW

The peak numbers correspond to the numbers in Figure 2.

c

c

4-methyl-3-hexanone? hexyl formate unknown unknown propanoic acid unknown hexyl hexanoate butanoic a c i d unknown unknown 5-ethyl-2(5H)-furanone pentanoic a c i d unknown hexanoic a c i d 2-methyl-2-butenoic acid? (Z)-2-hexenoic acid » (E)-3-hexenoic a c i d (E)-2-hexenoic acid unknown 6-lactone? monoterpendiol? 6-lactone? unknown 6-lactone? 6-lactone? 6-lactone? 6-lactone? 6-lactone? 6-lactone? 6-lactone? unknown 6-lactone?

compound*

5

Artifacts formed during storage of kiwifruit extracts.

that of reference compounds.

a

1 2 3 4 5 6 6a 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

a

Peak no.

TABLE 1.



*0D0

0.2293

-0.0035L «iODO

Cû 0 . 1 1 3

< α ο

Changes in Aroma Concentrates during Storage - ACS Symposium

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