Effect of Storage on Carotene of Plant Extracts A. R. KEMMERER’ AND G . .S. FRAPS Texas Agricultural Experiment Station, College Station, Texas
of 8-carotene, and neo-&carotene U, one fourth (6) to one third (3). Carotene is known to be lost in storage, but no information regarding the relative stability of the several isomers in storage is available. The experiments reported here were conducte$ to ascertain the relative changes during storage in the percentages of these isomers and other constituents of the crude carotene of plants and also their relative loss, during storage. I n the first experiment, samples of freshly dried ground grasses and dehydrated foods were stored a t room temperature in the dark. At the beginning of the experiment and from time to time thereafter, the crude carotene fractions of the samples were analyzed for carotenoid constituents by a method previously published (3). The sample is treated with alcoholic potassium hydroxide in a Waring Blendor. The crude carotene is extracted with petroleum ether, and the petroleum ether solution purified by shaking with 90% methanol. Since the coloring matter is not entirely carotene, it is termed “crude carotene”. All operations are carried out a t room temperature. The various carotenoids in the crude carotene solution are finally ascertained by chromatographic analysis on calcium hydroxide (3). With this procedure there is less than 10% loss of pigment. Table I gives the results. Impurity A consists of several pigments which do not have vitamin A potency. Pure carotene may be defined as the crude carotene minus impurity A , and consists of the vitamin A active pigments neo-&carotene U, &carotene, neo-8-carotene B, and, in the case of carrots, a-carotene.
During storage, up to 8 months, of thirteen samples of dried grasses and dried foods, the percentage of impurity A increased in the crude carotene fraction. The percentage of &carotene in the pure carotene fraction decreased in six samples and remained nearly constant or increased slightly in the other seven. The percentage of neo-@-caroteneU in the pure carotene increased in eleven of the samples, and that of neo-&carotene B decreased in ten. The quantity of impurity A increased in dehydrated carrots up to 14 months of storage and in beet greens up to 4 months of storage. In all other samples there was a decrease in the amount of impurity A . pCarotene, neo-@-carotene U, and neo-@-caroteneB decreased in all the samples except beet greens, in which there was a slight increase in neo-@-caroteneB‘during the second and fourth months of storage.
P
OLGAR and Zechmeister (6) showed that trans-&carotene
can exist in a number of other stereoisomeric forms. Kemmerer and Fraps (4) reported that two of these stereoisomeric forms, neo-&carotene U and neo-B-carotene B, are found in appreciable amounts in carote?e extracts of plants. Neo-8-carotene B (1,3) is reported to have one half the vitamin A activity 1
Present address, Arizona Agricultural Experiment Station, Tucson, Arili
TABLE I. EFFECTOF STORAGE ON P E R C E N T AOF G ~CONSTITUENTS IN CRUDEAND PURECAROTEXE
Sample Beet greens
Storage Time, Months
0 2 4 10
Eastern grama grass Buffalo grass
0
2 4 10 0 2 4 10
Bermuda grass
Dallis grass
Rhodes gram
Crab grass
0
2 4 10 0
2 4 10 0
2 4 10 0
2 4 10
Crude Carotene, P.P.M.
Impurity A in Crude Carotene,
Carotenes in Pure Carotene,
%
Neo-8 carotene U
8-Caro-
8.5 15 6 20.4 21.9
70 3 74.6 68 3 61.5
Neo-@carotene B
517 322 270 167
% 4.1 7.3 10.1 11.6
121.0 72.0 52.0 36.0
11.0 24.8 23.8 27.0
19.3 15.3 19.2 23.0
69.6 75.7 65.0 W3.0
11.7 9.0 15.8 9.0
79.0 45.0 28.0
60
13.0 23.9 18.7 19.6
19.4 31.4 20.6 26.6
65.7 55.7 66.4 67.5
14.9 12.9 13.0 5.9
94.0 52.0 32.0 17.2
11.2 17.5 20.3 27.8
18.8 20.7 24.7 26.6
69.8 67.0 61.5 68.4
11.4 12.3 13.8 5.0
65.0 18.5 9.3 4.4
14.2 35.4 40.3 57.3
18.6 22.3 27.1 30.9
71.7 71.3 60.4 61.7
9.7 6.4 12.5 , 7.4
117.0 74.0 51.0 34.8
11.3 15.6 15.7 20.5
16.6
15.8 18.7 25.6
71.0 67.7 70.0 64.5
12.4 16.5 11.3 10.0
90.0 47.0 34.0 18.6
11.2 23.9 24.6 31.0
16.0 15.9 21.3
72.5 71.0 66.6
11.6 13.1 12.1
...
tene
’
...
’
Storage
Sample Dehydrated carrots
21.2. 9.8 11.3 16.6
%
Carotenes i n Pure Carotene,
%
Neo-@ carotene U
0-Caro-
36.0a 34.25 28.5= 32.15 32.14
64.0 57.5 69.0 63.0 64.9
tene
Neo-@carotene B 0
862 765 650 ,650 675
3.0 6.2 6.2 9.1 7.5
Dehydrated alfalfa
0 2 6 10 14
213 173 128 97 65
14.9 20.0 22.1 22.5 26.1
25.1 23.0 29.0 29.4 33.1
57.4 61.4 56.0 56.0 55.0
17.5 15.6 15.0 14.6 11.9
Dehydrated bur clover
0 2 6 10 14
4.5 6.9 10.7 12.3 15.7
0.0 15.0 19.5 21.2 23.1
84.5 69.0 66.5 66.8 65.7
15.5 16.0 14.0 12.0 11.2
10.3 18.8 17.2 22.6
80.9 66.7 63.8 59.8
8.8 14.5 18.8 17.6
. 218.0 290.0 112 135 83 365.0
0
...
2 4 10
Turnip greens
0 2 4 to
Collards
0 4 10
a
457
Impurity A in Crude Carotene,
2 6 10 14
Swiss chard
...
Time MontLs 0
Crude Carotene, P.P.M
15.8 6.9 553.0
.
6.3 13.0 13.0 17.4
,
8.3 2.5 4.0 3.0
78.0 30.6
...
4.9 18.4 20.0 24.1
13.2 21.7 23.5 20.0
76.4 65.7 64.3 76.0
10.4 12.6 12.2 4.0
432 74.0 28.5
23.9 34.4 41.9
14.7 12.1
67.2 74.2 77.0
18.1 6.7 10 9
a-Carotene in oarrots.
19.1
I N D U S T R I A L AND E N G I N E E R I N G CHEMISTRY
4%
Crude caro-
Impurity
d 2.3. 0
terie
S w 5 a chard
365. 0
4 1110.
15.8
Deet greens 2 1110. 4 1110.
517.0 322.0 270.0 167.0
10 I l l O .
.4
? I : ,
2.1 1.0
2.: I.,
21 2 23.:) 27 2
-I2 4 4ii 7
5.0
I O mo.
Nro-,lwrw tei!i i-
4 x 2
37.4
19.4
Turnip grecii-
B-Curoiene 277.0 8.7 2.9
Nco-8- Crude call)carotrnc
tene R 29.6
..
2(i 0.9
!1S
104.0
28.8 27.6 24.2
38 48 68
387 0
63.2 7 J 0 .0
86 91
40 2 17.6
4 1110. I O nio. 862. 0
:mu c!
2 6 .0 47.5
2.14.0 174 0 190 0 201 i:
537.0 414 0 421 0 384.0 405.0
00.0 5%,i
9.2 6.6 3.7 2.5
10,: 2..
40.2
5.2
..
1.6
3.4 1.2
0 1
72 86 u3
94.0 52.0 32.0 17.2 79.0 60.0
10.5
15.8 8 9 D H 3 :j
58.3 28 7 15.7 8.4 45.4 25.6 24.3 15.2
117.0 74.0 51.0 35.0
13.2
763.0 650.0 660 0 675 0
40.0 60.0 50.0
?)allis grass 2'mo. 4 mo. 10 mo.
65.0 18.5 9.3 4.4
Hrrriiuda grass 2 1110.
lj.0
24.0 lY.0
,
carotene li
slight illcreme in the other w w n The percentage ol neo-B-camtcne U increased in elererr trf 111~. samples, and the prrcentagc of
neo-B-cttrotene B decreascd in tm. Apparently nco-B-cttrot cnc, 97 01 U i s more stable than t h o other !I9 (#i two carotenes. The a-ctirotcnc. i l l carrots (lwrc'wcd from 30.0 t o 32.1 "/o. 'L'lio tiatit n w e furtlicr csiiiniiied (Table 11) to asecrtuin if the chaiigcs in percentage w r e iluc t o an incrcasc in thr quantiiics of any of the carotcw:: or t o ~liffcrcnccsin t?ic ra 1 lic quantity of imp cxas:ctl only x i t h dchydratcdoarrots, and with beet grwns a f t e r storagt: for 2 or 4 months. ITT'hori iI ii2 iiorctl 10 morit.lis, tlicire H 7Y (5 R Rti 0 :: I i w of iiupurity A from ~ h hcc:1 c greens. An apparent, gain of 1 -1 11' iic~1-13-cnrotenc:T i occurrctl with 4 ti 32 fi7 87 tho beet grceris during 2 and 4 Inmiths of storage and v-i(,h tiiiifalo grass aftcr 2 mont,lii; of storage. I n the othcr test8 ihere wwe losses of d l pigmcnts in \lost, ol' t,hc increase in iwrcontages or impurity '1 in the crude carotetic and oT neo($-carotene U wcre due l o greater resistance t o destiuction than the other pigments poswssed, in yuairtitics present. In a second experiment a sample of dehydrated alfdfa Iraf nieel iviis divided into thrclc aliyuots. One aliquot was stored in >x refrigerator at 5 'C. and onr in :tn incubator at 37 a C.; the third waa stored in an incubator at 37" Ci. and stirred for 5 niinutc,s, twice ~ e e l d y .After the storage periods designated in Table 111,the conslituents of the crude carotene in t,he three aliquots were determined. The percentage of impurity A in all three was increased during storage. lIo~vcrpcrcentages of impurity A were found in the sample stored a t 5 C. than in the two samples stored at 37". The stirred saniple did n o t contain any greater percentages of impurity A than the unstirred samples. As in the first experiment, the carotenoid constituents werc calculated cxelusive of impurity A. There was an increase in the percentage of neo-&carotene U and a decrease in the percentage of 6-caroh i e in the pure carotene fraction of the two samples storcd in the incubator. The results on neo-8-carotene R were erratic but indicate no appreciable change in its percentage. Stirring did not affect' t'he percenta,ges of these pigments. I . .
!J6
349.4 223.0 166.0 91.0
8-Caroteue
Vol. 38, No. 4
,.
11.2
24.6 23 4 21.7
r
4 1110. IUmo.
tluffalo grass 2 mo. 4 mo. 10 n10.
;;;;
Kliodes grasp 2 no. 4 IIlO. 10 nio.
3ehydriLtcd alfalfa 2 11io. R 1110.
213.0 173.0 128.0 10 1110. 97.0 1.1 1110, 65 0 a = Cnivteile in car; ot&
0.P
9 1' 6.3
4.8 10.2 14.3
lS,4 14.4
;:E
17.2 $.3 8 1 7.;
11.6 8.0
7.2
31.8
4.i.C
34.(i 28 1
31 28 ,, 2% i
5
21.8 17.0
13 F
0.8 0.i
8.1
9.4
6.3 3 5 0.7
43 GI$
10.0 5.8 1.8 1 3
21 43 liR
74.0 42.3 30.0 17.9
12,G 10.3 4 R
3s
104.0 85.2 56.0 42.1 26.4
31.8
2.8 21.5 15.3
81'
58 70
10
The percentage of impurity A i t i the, crude carotene increased K i t h the length of the storage period. ror example, a t tlie end of 10 monihs the percentage of impurity A had increascd from 3.0 to 0.1 in dried carrots, 6.3 to 17.4 i t i Swiss chard, 4.1 t o 11.6 in beer greens, 4.9 t o 24.1 in turnip grecws, 11.2 t o 31.0 in crab grass,
Rnd 14.2 to 57.3 in Dallis grass, I n the pure carotene the percciitaye of &carotene decreased in six of the samples; it remaincd ncaily constant or exhibited a
TARLE 111. EFFECT OF STORAGCA Y D STIRRING ON Loss CAROTEXOID COSSTITI:IGKTS IN ALFALFA dtarage Conditions Kefrigerator, 5'C.
Timb Stored
0 1 mo. 2mo
3 mo.
4 mo. 8 moa
lncubator, ?i°C.
incu-
bator, 370 C., etiried
0
1 wk,
2wk. 4wk~ 2 mo. 3 mo. 4 mo. 6 mo. 8 moI
0
1 wk. 2 ak. 4 rrk. 2 mo. 3 mo. 4 mo. 6 mo. 8 mo.
Crude Carotene, P.P.N. 173 170 152 160 144 116
173 172 1.52 114 97 68 57 46 38
173 172 144 100 78 52 41
33 28
Impuriti A in Crude Carotene,
OF
Carotenes in Pure Carotene,
.
%
Ne=-caro-
Xeo-pcarotene U 16.5 18.3
8-Carotene 71.0 66.3
10 2 11.5
15.8 16.3 16.9-
68.8 71.7 62.5
15.4 12.0 20.6
7 8 12.6
16.5
71.0 70.5 74 5
12.5 12 2
6fi.3 70.2 61.6
11.1 17.4
Z' ,
7.e
9.6 , . .
ti.(! 1% 4
i!.? 1 1 . 6
18.5 19.4 22.0 27.3
7 8 9,3 11.3 14.3
20.9 21.8 21.8 23.5 28.3
...
17.3 18.3 18.6 18.7 21.0 23.3 20.5 23.6 16.5 18 3 17 ? 17.3 22.6. 23 8 24.8 27.2 25.2
...
tene
W
12.5 15.4
...
9.2 1S.l
65 2 69.0 62.3
11.5 20 5 14.1
71.0 72.2 71.5
12.5
68.7 67 2
64.5 62.6 56.7 58.3
9,5
10.8 16.0 10.2 12.7 12.6 16.1 16.5
1
L i m m ' r u m CITED
e.,
(1) Deuel, H. P., Jr., Johnston, Miserve, 22.. I'olgBr, 4., and Zeclimeister, L., Arch. Biochcm., 7, 247 (1945). (3) Deuel, H. F., Jr., Johnston, C.,Siamner, E., PolgLir, ,\., and Zechmeieter, I,., Ihid., 5, 107 (1944). (3) Kemrnerer, A . K.,and Frups, G. S., IND. ENO.C n ~ s i .AXAI,. , ED., 15, 714 (1943). (4) Kemmerer, A. It., and Fiaps, G . S . , J . Am. Chcm. Soc., 66, 306 (1944).
( 5 ) Kemmerer, A. It., and Fraps, G. S.,.J. B i d . Chem., 161, 197 (1945). (0) Polg&r, A, and Zeohmeist,er, I,., J . Am. Chern. Soc., 64, 18.56 (1942).
