FALCONER et a1.-OFF-FLAVOUR I N DEHYDRATED CARROT 897

by a decrease in the level of acetic acid which is normally the predominant acid formed by the fermentation in the rumen of feeds high in cellulose. From this and earlier it appears that reductions in the digestion of cellulose by rumen micro-organisms can be brought about by lipid under a variety of dietary conditions. Increased formation of propionate in the presence of lipid also appears to be related to the digestion of the fibrous rather than the soluble consti- tuents of pasture species.

Acknowledgments The authors thank Dr. A. T. Johns for helpful discussion and Miss J. T. Barnett for assisting

with the analysis of long-chain fatty acids. These investigations were supported by a grant-in- aid from the University Grants Committee and one of the authors (J. A. R.) gratefuly acknow- ledges receipt of a Commonwealth Scholarship.

Massey University of Manawatu Palmerston North

New Zealand Received 6 April, 1964; amended manuscript 2 June, 1964

References 'Robertson, J. A,, & Hawke, J. C., J . Sci. Fd

Agric., 1964, 15, 274 Hawke, J. C., & Robertson, J. A., J . Sci. Fd

Agric., 1964, 15, 283 Brooks, C. C., Garner, G. B., Gehrke, C. W.,

Muhrer, M. E., & Pfander, W. H., J . Snimal sci., 1954, 13, 75s

* Brethour, J. R., Sirny, R. J.. & Tillman, X. D.,

6Davison, K. L., & Woods, W., J . A n i m a l Sci.,

Boda, J. M., & Johns, 4. T., Nature, Lond., 1962,

J . Aiaimal Sci., 1958, 17, 171

1960, 19, 54

193, I95 1 McDougall, E. I., Biochem. J . , 1948, 43, gg * Flatt, W. P., Warner, W. G., & Loosli, J. K.,

Cornell agric. Exp. Sta. iVemoir, 1959, No. 361

9 ' Official Methods of Analysis ', 9th edn, 1960 (Washington, D.C. : Ass. Off. Agric. Chemists)

lo Wilson, M. K., & Briggs, C. A. E., J . ufipl. B a d . , 1955, 18, 294

l1 Garton, G. A, Lough, A. K., & Viogue, E., J . gen.

l 2 Balch, C. C., &Johnson, V. W., Brit . J . Nutr., 1950,

l3 Miles, J. T., J . Dairy Sci., 1951, 34, 492 l4 Smith, P. H., Sweeney, H. C., Rooney, J. R., King,

K. W., & Moore, W. E. C., J . Dairy Sci., 1956,39, 598

l5 Chalmers, M. I., Abstr. 5th I n t . Congr. on Nutrition (Washington, D.C.), 1960

l6 Jayasinghe, J. R., Ceylon vet. J., 1961, 9, 135

Micvobiol., 1961, 25, 215

4, 389

CAROTENE OXIDATION AND OFF-FLAVOUR DEVELOP- MENT IN DEHYDRATED CARROT

B y M. E. FALCONER,' M. J. FISHWICK,? D. G. LAND? and E. R. SAYER*

Dehydrated carrot develops an unpleasant ' off '-flavour when stored in the presence of oxygen. The off-flavour has been described as ' having an odour similar to violets ' and i t was suggested to be due to the formation of p-ionone formed by oxidation of ,8-carotene.l This suggestion is supported by the fact that loss of colour, which is mainly due to the presence of b-carotene, accompanies the development of off-flavour. If the above suggestion is true it should be possible t o establish a direct relationship between loss of ,8-carotene and off-flavour, and this has now been done in a series of storage tests in which both carotene analyses and taste panel assessment for natural and off-flavour were carried out.

* Ministry of Agriculture, Fisheries & Food, Experimental Establishment, Greyhope Road, Aberdeen t Low Temperature Research Station, Downing Street, Cambridge

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898 FALCONER et al.-OFF-FLAVOUR I N DEHYDRATED CARROT

Experimental Preparation of material

Red-cored Chantenay carrots grown in N.E. Scotland were washed, scrubbed and diced on the day after harvest. The dice were blanched, dried by the accelerated freeze drying (A.F.D.) process2 and immediately canned under nitrogen and stored at -20". The dice were removed from the cans when required and finely powdered in a hammer mill in order to facilitate sampling and to eliminate texture differences which would interfere with taste panel assessment. Portions of the powder (120 g.) in 500-1111. flasks were exposed to oxygen and light (0.1 lux) at 18" ; flasks were periodically removed for analysis.

Taste panel analysis Each sample of the carrot powder was made into a puree with six times its weight of hot

water (50") for presentation to the trained taste panel for assessment of the following ratings : (I) Natural flavour on a scale of o to 4 based on a control sample taken straight from the can. The control was given the value 4. (2 ) Off-flavour on a scale of o to 4 based on a standard sample rated 2 on the scale shown in Table I, and on the control sample which was rated off-flavour 0.

Table I Offj'lavour scale for A.F.D. carrot

Off-flavour $4 Fully oxidised Off-flavour description rating carrot in mixture*

0 None-control sample I Slight-acceptable 5 Mild-unacceptable

1 0 Moderate-unacceptable 20 Strong and clinging-very distasteful

* The samples were made up as powders by mixing increasing amounts of well-oxidised (almost colourless) These samples were made up into individual flasks from one

Each flask was filled with nitrogen to prevent further oxidation and stored in darkness a t -zoo carrot with nitrogen-stored carrot as shown. batch. until required. One flask of each rating was used for each taste panel and any not utilised were discarded.

The panel, which consisted of seven members, was selected for their ability to discriminate between standard and off-flavour carrot samples in triangular tests. Training consisted of placing the standards in order of increasing off-flavour, followed by ratings of random standards (as unknowns) in terms of the two standards normally given to the panel with the unknown sample. No selective training in natural flavour assessment was given, as no sample of ' taste- less ' carrot powder, obtained by solvent extraction (e.g., with 80% methanol), was sufficiently free from solvent taint to be used as a diluent. Natural flavour was therefore rated in terms of that of the control sample.

The taste panel room was fitted with individual cubicles, the lighting of which could be varied to eliminate bias due to colour differences in samples. One unknown sample together with the two standards were presented to the panel a t each session, only one of which was held on any one day, and, in order to minimise the effect of diurnal rhythm, at the same time in the morning. Comments on the taste and aroma of the samples were always requested and noted after each session, partly to obtain any indica- tion of qualitative changes in flavour, and partly to maintain panel interest.

Soda water was provided as a palate cleanser.

Carotene analyses The amount of total carotenoid in a second sample of the carrot powder used for taste panel

assessment was estimated by a modification, suitable for dehydrated tissue, of a method described by G o o d ~ i n . ~ Two samples (6 g. in each) were first extracted with 80% aqueous methanol (4 x 30 ml.) to facilitate subsequent extraction of the carotenoids. The carrot residue was then exhaustively extracted with ether. Traces of carotene in the initial methanol extract were removed by extraction with ether after the addition of water (20%) to the extract. The combined ethereal extracts were washed with water, dried over anhydrous sodium sulphate and evaporated to dryness in vacuo at room temperature. The residue was dissolved in 250 ml.

J. Sci. Fd Agric., 1964, Vol. 15, December

FALCONER et a1.-OFF-FLAVOUR I N DEHYDRATED CARROT 899

of light petroleum (b.p. 40-60"), and suitably diluted for measurement of absorptivity a t 450 mp. Results were expressed as a percentage of the control values.

In pilot experiments attempts were made to separate and analyse the individual carotenoids present chromatographically. It was found, however, that losses of up to 10% occurred and this method of analysis was therefore abandoned. An indication of the changes in the individual carotenoids can be obtained by measuring changes in the shape of the absorption spectra of the extract a t the following wavelengths : 340, 350, 370, 400, 425, 435, 450, 466 and 478 mp.

Results and discussion

plotted against residual carotene measured by absorptivity a t 450 mp. equation relating off-flavour (t = taint) and residual carotene (c) is

The error of estimation with 95% probability is j-3-04 for t 2 and the correlation coefficient between t 2 and c2 is -094.

The curve indicates a close but non-linear relationship between carotene loss and the development of off-flavour. The off-flavour scale of course is not a linear function of the quantity of off-flavour substances in the standards, but when correction is made for this fact the plot between off-flavour rating and carotene loss is still curvilinear. As there are many other carotenoids in the carrot present in smaller quantities besides ,!I-carotene, a simple pattern of oxidation products is unlikely. Further work4 has demonstrated the presence of a- and ,!I-ionone in oxidised A.F.D. carrot, but they are by no means the only products of oxidation with violet- like aromas and can account for only part of the off-flavour of oxidised carrot. As oxidation proceeds, a bitter, soapy after-taste becomes apparent along with the violet-like aroma com- ponents. This occurs when about 50% of the carotene has been lost and probably accounts for the change in the slope of Fig. I from this point.

A change in the course of the loss of carotene can also be shown to occur a t the time when the bitter off-flavour appears by rate of reaction studies. If the reciprocal of the proportion a/(a - x ) of the initial amount (a) of carotene remaining (a - x) is plotted against time (T) , the points fall along two consecutive straight lines (Fig. 2) indicating a marked increase in the rate of reaction at about the thirteenth day. This is the time at which the taste panel began to remark on the presence of the bitter after-taste in the carrot samples. The graph

The results of three experiments are shown in Fig. I where mean off-flavour ratings are The statistically derived

t = (12.8 - 0.0013~~)~

0

X

0 1 I I I 0 1 . 2 3 4

OFF FLAVOUR RATING, f FIG. I .-Relationship between loss of carotene and off-jlavoaw development ilz accelerated freeze-dried

carrot stored in oxygen 0 Expt. I 0 Expt. z x Expt. 3

J. Sci. Fd Agric., 1964, Vol. 15, December

900 FALCONER et al.-OFF-FLAVOUR I N DEHYDRATED CARROT

oo 16

TIME fT1,days

FIG. 2.--Rate of reactioiis in loss of carotene in accelerated freeze-dried carrot a = initial weight of carotene x = amount of carotene reacted at time T

may be taken to indicate that the rate-limiting reactions causing loss of carotene are unimole- cular or, more likely, pseudounimolecular. Thus the development of off-flavour is not a simple process and it would be surprising if a direct relationship had been observed between carotene loss and off-flavour.

It is interesting that, whereas Tomkins et aZ.l stated that a slight off-flavour could be detected only when 20% of the carotene had been lost, under the present conditions off-flavour was detected by the panel when less than 5% had occurred.

The plot of change in absorbance at wavelengths other than 450 mp against off-flavour ratings showed no significant differences from Fig. I except at the three lowest wavelengths. The fall in absorbance at 340, 350 and 370 mp is delayed until off-flavour 3 is reached and then falls steeply towards zero at off-flavour 4. These changes suggest a small accumulation of an intermediate oxidation product absorbing in the region 340-370 mp. There is however no evidence to suggest accumulation of products of a stepwise oxidation of /?-carotene such as that obtained by osmium tetroxide and hydrogen peroxide,5 or accumulation of epoxides or furanoxides.6 The presence of traces of possible intermediates of these reactions has been demonstrated in A.F.D. carrot dice exposed to oxygen.'

The straight-line relationship between residual carotene (c) and natural flavour (f) in three experiments is shown in Fig. 3. The equation is

f = 0.027~ $. 0.15 The simple linear correlation coefficient is +0.94 and the error of estimation for the regres-

sion line at the 95% probability level is &o-68. As mentioned earlier the taste panel was not specifically trained to discriminate between different levels of natural carrot flavour and, as the particular batch of carrots used was rather low in natural flavour, the panel tended to downgrade all the samples rather than compare them with the low natural standard provided. Thus, even when little carotene oxidation had occurred, the maximum natural flavour rating was about 3. The results do indicate, however, that natural flavour components are lost as oxidation proceeds ; presumably these substances are themselves oxidised. The disappearance of such substances has since been confirmed by later work.*

Conclusion The precision with which natural or off-flavour can be predicted from the concentration

of p-carotene in carrot is quite high when one considers the complexity of the chemical processes

J. Sci. Fd Agric., 1964, Vol. 15, December

FALCONER et a].-OFF-FLAVOUR IhT DEHYDRATED CARROT 901

NATURAL FLAVOUR RATING, f

FIG. 3.-Relationshi@ betmeen loss of carotene and natural flavour in accelerated freeze-dried carrot

0 Expt. I 0 Expt. z x Expt. 3 stored in oxygen

involved, the general error usual in subjective measurements, and that three different taste panels were used over a period of 4 months. These experiments clearly demonstrate that some of the expected variability of such subjective assessments can be reduced under appropriate conditions. The investigation also clearly shows for the first time that, in A.F.D. carrot, loss of carotene by oxidation is closely related with development of off-flavour.

Acknowledgments The authors wish to thank Mr. J. M. Harries for statistical analyses, the various members

of the taste panel for their co-operation, and Mr. A. E. Joyce and Mr. T. C. Grey for helpful discussions.

This work was carried out as part of a research project supported by a U.S. Public Law 480 grant.

Ministry of Agriculture, Fisheries & Food Experimental Establishment

Greyhope Road, Aberdeen Received 22 May, 1964

References Tomkins, R. G., Mapson, L. W., Allen, R. J. L.,

Wager, H. G., & Barker, J., J . SOC. chew. Ind., 1944. 63, 225T

2 Gooding, E. G. B., Tucker, C. G., & Macdougall, D. B., Food Manuf., 1960, 35, 249

SGoodwin, T. W., ‘Modern Methods of Plant Analysis’ (eds. Paech and Tracey), Vol. 111, 1955. p. 280 (Berlin : Springer-Verlag)

4 Fishwick, 111. J., Land, D. G., & Ayers, J. E., in

5 Wendler, N. L., Rosenblum, C., & Tischler, M.,

6 Karrer, P., & Jucker, E., Helv. chiw. A d a , 1945.

Fishwick, M. J., Proc. 1st In t . Congr. Fa Sri.

preparation

J . Arner. chew. soc., 1950, 72, 234

28, 427

Technol. (London), 1962, in the press

J. Sci. Fd Agric., 1964, Vol. 15, December