Storage Stability of Carotenoids

P. G. CRANDALL, J. W. KESTERSON,

in Orange Oil
and S. DENNIS I

ABSTRACT
Citrus essential oils are. used as flavoring agents in foods and beverages. Recently a premium price was paid for oils with a high carotenoid content because they were also used as natural coloring agents. Commercially manufactured samples of Pineappleoils were concentrated 5, 10, and 25-fold, stored for 12 mo-at -18, 4, 26, and 32C and sampled periodically. The carotenoid content was measured spectrophotometrically with p-carotene as a standard. From an initialvalue of 7.2 mg of o-carotene/lOOg of oil, it decreased 6.6, 18.4, and 24.6% upon concentrating 5, 10, and 25-fold, respectively. The 25-fold sample lost 6% of its p-carotene at -18C and 90% at 32C duiing 12 mo of storage. There was a highly significant fit of the time, temperature and concentration data to a second order model.

INTRODUCTIOi
CAROTENOIDS are responsible for the characteristic orange to yellow color of most orange cultivars. About 70% of carotenoids are located in the plastids of the flavedo, the outer portion of the peel. There are widespread differences in the amount of color in the peel and juice among various citrus cultivars (Eilati, 1970). Out of the 300 carotenoids found in nature, a total of 115 have been reported in citrus. However, there is disagreement as to. how many of these carotenoids occur naturally, how many are artifacts formed during extraction and how they are synthesized (Stewart, 1980; Gross, 1977; Muller et al., 1980). Thus, the color of citrus oil is composed of a complex mixture of carotenoids and little information is available on the individual concentration levels. The major red to orange carotenoid is P-citraurin together with the orange colored /.?-cryptoxanthin they contribute the most to an orange color. The other carotenoid pigments, s including P-carotene, are discussed in recent reviews (Stewart, 1980; Gross, 1977). About 1.6 x lo7 MT of oranges were processed worldwide during the 1979-80 season (USDA, 1981a, b). From these fruit it was estimated that more than 4.5 x lo6 kg per year of citrus essential dils were produced, most of which was orange oil (Swisher and Swisher, 1977). The major producer of orange juice, Florida, processed 7.1 x lo6 MT of oranges during the 1979-80 season (Fla. Citrus Proc. Assoc., 1981). Light or poorly coloied juice is produced from Florida s early and midseason oranges and has to be blended with other more highly colored orange juices to increase its red-orange color. Color is important to the quality of citrus juice, accqunting, fbr 40 out of 100 grade points (USDA, 198 1) and color has been found to directly influence consumer preference (Taylor, 1965).
Author Crandall is with the Univ. of Florida, Institute 0; Food & Agricultu[al Sciences, Agricultural Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850. Author Kesterson is Professor Emeritus with the Univ. of Florida and consultant for Brown lnternaiional Corp., Covina, CA 91723. Author Dennis is with Plymouth Citrus Products Co-Op, Plymouth, FL

32768. 924-JOURNAL OF FOOD SCIENCE-Volume 48 (1983)

Numerous attempts have been made td extract color from citrus peel. This would be used as a colorant for poorly (colored citrus juice rather than import highly colored orange.juice concentrates from foreign countries for blending. There is also a substantial demand for natural colors for use in beverages and foods. Naturally occurring colorants have been a part of our diet for generations. Studies by Bagdon et al. (1960) showed low toxicity of pctirotene when administered either chronically or in single-dose. Most of the research to recover natural colorants used flavedo or peel as a starting material. It is a cheap raw material and contains the majority .of the carotetioids. Methods for recovering carotenoid pigments from peel have been published by Ting and Hendrickson (1969) and Wilson and Shaw (1977). Barron et al. (1973) has a patented method for centrifuging plastids from juice. Johnson et al. (1978) have a patented method that starts with orange essential oil, removers the d-limonene in an evaporator which also concentrates the carotenoids (folding the oil) and uses this material to increase the color of early season juice and beverages. Carotenoids in the flavedo are solubilized by the essential oil during normal commercial oil extraction because oils are a good solvent. Orange essential oils are widely used as flavoring agents-in beverages -and foods. Recently, a premium price was paid on-the carotenoid content of highly colored oils. These oils can act as flavoring and natural color enhancers.simultaneously. Much of the highly colored citrus oils are shipped to overseas~customer - a process that usually takes 2-3 mo after manufacturing the oil. So it is important to be able to economically store and transport oil to preser.ve its carotenoid content. In the transportation and storage of highly colored citrus oils, concentration and refrigeration have long been-used to preserve: the flavor citrus essential oils. Lund and Coleman (1977) reported that concentrating, or folding, essential oils increased their stability over that of single-strength oils, which have a higher d-limonene content. Swisher and Swisher (1977) reported an increase in the stability and greater flavor strength with a folded oil. Studies by Kesterson, on O2 uptake:_show that concentrated oils were less ed stable than single-strength oils; however, concentrated oils appeared to be more stable because their greater concentratibn of flavor compounds covered up more off-flavor development and concentrated oils may have a higher natural antioxidant, concentration. Kesterson et al. (197 1) found.citrus oil samples became lighter in color after absorbing l,,ZOO p*II of Oz. Blundstone et al. (1971) found little decrease in the P-carotene during processing and storage in citrus. Flores and Morse (1952) observed- that care-. tenqid pigments, in a model system (oil water em&ion), in were bleached white due to contact with air prior to developing terpeniness, the onset of off-flavors. Chichester and McFeeters (197 1) reviewed the stability of carotenoids in general. Haralampu and Karel (1981) measur ed the degradation kinetics for o-carotene under changing storage conditions. Sorensen et al. (198 1) developed a model for P-carotene degradation at temperatures

between 60-80C, while Onyewu (1981) looked at the thermal degradation products of P-carotene. This study was undertaken to determine the stability of carotenoids during: (1) concentration, used to reduce the volume of oil prior to shipment, (2) storage at temperatures above freezing, to reduce the storage energy consumption, and (3) interactions between temperature and time in storage. This should provide the data necessary to calculate the costs and benefits of concentration, storage temperatures, time of storage, and transportation of an oil to maintain its carotenoid content.

26C and 46% at 32C. It is possible to use these data to calculate the return on investing in refrigerated or frozen storage and transportation. Controlling temperature would probably be easier than appreciably decreasing the storage and transportation times. Effect of storage times The effects because of time of storage are shown in Fig. 2. It can be seen that there is a rapid initial loss in carotenoids then a slowing in this rate. This seems to be especially true for the more concentrated oils. There is not sufficient data to speculate if this change in rate is because of consumption of residual oxygen. Models In addition to looking at the data graphically to fit it to a second order model of the form: it is possible

MATERIALS & METHODS

SAMPLE OF ORANGE OILS were produced from Pineapple oranges using FMC extractors under commercial processing conditions by procedures described in Kesterson and Braddock (1976). The oil samples were pooled into a single batch and a single-strength sample was removed. Then a commercial wiped-film type oil evaporator (Luwa Corp., Charlotte, NC) was used to concentrate the samples. The oils were concentrated, or folded, 5, 10 and 25 times by a corresponding reduction in the volume of oil. Samples were overlaid with an atmosphere of N, gas, sealed in amber glass containers, then stored in the dark at four experimental storage temperatures. A commercial storage warehouse at 32C, room temperature at 26 refrigerated storage at 4 and the control samples C, C in frozen storage at -18 were used. Samples of single-strength oil C and 3-folded oils were analyzed initially, then at intervals of 1, 2, 3, 6, 9, and 12-mo storage. The carotenoid content was measured by diluting the sample in hexane and measuring the absorbance at 450 nm with a Beckman recording spectrophotometer (Fullerton, CA) according to Davies (1965). p-carotene (Hoffman-LaRoche Inc., NJ) was used as a standard. Color reflectance measurements were made using a Hunterlab D45D2 Citrus Colorimeter (Hunter Associates Laboratory Inc., Fairfax, VA). A second order model was fitted to the data for each concentration to determine if there were significant effects due to temperature, time or their interaction. A second order regression model which included concentration was also fitted to the data.

Y = PO + & Temp. + & Time + PI1 (Temp)2 + f122 (Time)2 + PI2 (Temp) (Time) where the temperature is in and time in months. Each C equation for an individual concentration is based on 24 measurements. There was a highly significant fit for the second order model for each concentration (Table 1). Substituting in the model for the lo-fold oil for 4 and 3 mo C we calculated a value for Y = 48.8 mg P-carotene/lOOg of oil and had an experimental value of 52.7 mg. The high R2 values indicate that transformations on the independent or dependent variables are unnecessary. The single-strength oil gave the .poorest fit and may fit a simpler model. The lofold data gave the best fit and all equations show &carotene decreasing with increasing time and-temperature. . A regression model using concentration (C) as fold increase, with the single strength concentration being 1, was also fitted to the data. The equation was: 5.8037 + 0.2887 - 0.0060 (Time)2 - 0.0273
I40 -

RESULTS & DISCUSSION

Effect of concentration Concentrating the cold-pressed orange oil in a commercial oil evaporator reduced the carotenoid content from the theoretical calculated amount. Expressed in terms of o-carotene, the single-strength sample initial contained the equivalent of 7.2 mg P-carotene/l OOg of oil. The theoretical p-carotene equivalents in 5, 10 and 25-fold oil samples should be 36.0, 72.0 and 180.0 mg/l OOg. The losses for 5, 10 and 25-fold samples were 6.6, 18.4 and 24.6%, respectively. Expressed as a loss of p-carotene per fold basis, the S-fold sample lost 0.48 mg of fi-carotene/lOOg of oil/each fold; 1.32 mg/fold for the 1 O-fold and 1.76 mg/fold for the 25-fold. This information enables a manufacturer of orange oil to calculate the additional revenue from the premium paid for carotenoids vs the extra transportation costs, handling larger volumes of oil in larger shipping containers. Effect of storage temperature The effect of storage temperature is shown in Fig. 1. Orange oils are normally stored under N2 or under minimum headspace at -18 C. These cold temperatures cause wax, which was dissolved in the oil during extraction, to precipitate. Then the oils are stored in refrigerated or unrefrigerated warehouses prior to shipments. In the 2-3 mo it normally takes between manufacture and receipt by the customer, a lot of deterioration can occur in an oil that is stored and shipped at 26 or higher. These data show that C after folding an oil lo-25 times, it is beneficial to ship this oil refrigerated or frozen to preserve its natural color. After 2 mo, the IO-fold oil retained 97% of its original o-carotene value at -18 C, 94% at 4C and only 48% at I

Temp + 5.6247 C - 1.0553 Time (Temp)2 - 0.0524 C2 t 0.0978 - 0.0884 (Temp) (Time) (Temp) C - 0.1157 (Time) C

-_I 0 0, 0 0 \ : al : + z 0 G

120-

100 -

80.

60 L

40-

20 -

5x OIL

IOX

I5 x

20 x

25 X

CONCENTRATION concentrations storage, 2 mo; storage, 2 mo.

Fig. l-Comparison of p-carotene content at four after 2 mo storage: original sample; o -18OC l 4 C storage, 2 mo; l 26C storage, 2 mo; A 32 C

Volume 48 (1983)--JOURNAL

OF FOOD SCIENCE-925

CAROTENOIDSSTABILITY

IN ORANGE OIL..

.
Interaction between storage temperature and time Fig. 3 shows the two concentrations at two temperatures for times up to 1 yr of storage. The constants for the model shown in Table 1 were used to generate the predicted values. The initial values were not used in developing the equations because they were not affected by storage temperature and time. Take as an example the IO-fold oil. After 3 mo the lo-fold stored at 26 was about equal to the 5-fold at C 4 C. The IO-fold oil stored 3 mo at 4 had a predicted C value Iof 48.8 mg which is about what the 25-fold oil stored 3 mo at 26OC was, 52 mg. The precision of the method was tested on four replicate samples of Pineapple orange oil after storage (Table 2). The coefficients of variation were 1.9% for the singlestrength oil, 1.3% for the 5-fold, 3.7% for lo-fold and 4.5% for 25-fold. Hunter color calorimeter values

The R2 value of 0.968 indicated a good fit and will give some indication of P-carotene content for higher concentrations and longer storage times. The final two terms in this equation -0.0273 (temp) C -0.1157 (time) C indicate that for a given concentration , the level of P-carotene C decreases with both increasing temperature and time. As the concentration or fold increases the size of these negative terms increases and the rate of decline is increased. The decline does not begin at a certain level of concentration, but is an interaction with temperature and time. This regression model or one like it developed for another oil can be useful in predicting the effect of storage time and temperature on the o-carotene content of an oil.
Table l-Regression equations for a second Concentration Term Constant Temperature Time (Temperature) (Time) 2 Temperature Time I32 Error mean square 1X 6.862 -0.03932 -0.1017 0.000309 0.000725 -0.002054 0.754 0.466 5x 31.45 -0.1764 -0.6096 -0.00279 0.02325 -0.03344 0.938 6.741 10X 57.89 -0.445 -2.602 -0.01075 0.1230 -0.0365 0.962 15.88 25X 118.3 -1.810 -5.651 -0.01088 0.2442 -0.03696 0.947 147.2 order storage model

2 x

The Hunter Citrus Colorimeter was used to measure the color of orange juice which served as a control model and the color of juice fortified with oil. This meter measures the color reflected light from a sample, the way our eyes of see color (Hunter, 1967). This meter is able to give a more accurate measure of the value of added color to a juice or beverage because it can measure the color of the finished product. The spectrophotometer has limited application to juice because of the cloud and interfering substances present in the juice. The Hunter citrus calorimeter measurements

Table .2-Precision of method on four concentrations of Pineapple orange oil Oil sample 1x 5X 10x 25x Pcarotene mg/lOO ml of oil 7.19 33.60 58.78 135.81

replicated

samples

of

four

60 _I 0 m 50
0 0

Standard deviation 0.14 0.45 2.23 6.12

,
E Q) c

40

; 30 L 0 0 Qk 20

, 0 -

40 t

IO

01

5 FOLD OF

IO ORANGE

25 OIL

I 0

4 TIME,

6 Months

IO

12

Fig. 2-Change in p-carotene content at 26C: 0 change from initial to the first month; q change from 1st to 2nd month; q change from 2nd to 3rd month; m change from 3rd to 6th month. 926-JOURNAL OF FOOD

Fig. S-interaction between time and temperature in the predicted degradation of p-carotene: * lo-fold oil, starting value; o lo-fold oil stored at 4OC; l IO-fold oil stored at 26OC; ** bfold oil, starting value; a dfold oil stored at 4C; X 5-fold oil stored at 26 C.

SCIENCE-Volume

48 (7983)

may be superior to a measurement of absorbance by a spectrophotometer which is currently used to fix the value of the color of an oil. Table 3 contains the color values for a Hamlin orange juice which is used as an example of a poorly colored juice. To this model juice, 0.035% by volume of lo-fold oil was added. This is the maximum amount of oil that could legally be added. It can be seen that the color number did not significantly change. So N rather than pay a premium on the basis of equivalent pcarotene content, the color increase in a model system should be better. The last column on Table 3 shows that 1 mg of pure P-carotene does increase the color number by about 1 point. So the Hunter can be used to evaluate the increase in color in the finished product similar to the eye evaluation. s

Table 3-Hunter color tified orange juice

Hamlin juice, orange control

difference

meter

measurements

on color

for-

OJ+l OJ + 0.035% by vol 1 O-fold oil

p-carotene/

mg

(OJ) x
CW s.d.

100 ml
x s.d.

x 26.6 71.9 34.9 27.6 25.7 9.1

s.d. 0.20 0.15 0.06 0.06 0.06 0.06

CY N Y X 2
a CR lent (Citrus color

28.4 69.8 35.0 23.1 21.7 8.3

red), score),

0.06 0.21 0.00 0.10 0.06 0.06

36.7 68.4 36.2 21 .3 20.8 8.0

or

0.10 0.25 0.06 0.06 0.12 0.06

Equiva-

CY (Citrus yellow), N Y, X, Z (CIE Tristimulus

(Color number values)

REFERENCES
Bagdon. R.E., Zbinden, G., and Studer, A. 1960. Chronic toxicity studies of p-carotene. Toxicol. Appl. Pharmacol. 2: 225. Barron, R.W., Fellers, P.J., and Huggart, R.L. 1973. Process for the production of naturally occurring coloring material from citrus fruit and the product thereof. U.S. Patent 3.725.083. Blunderstone, H.A.W., Woodman, J.S., and Adams. J.B. 1971. Canned citrus products. In The Biochemistry of Fruits and Their Products. Academic Press, New York. Chichester, C.O. and McFeeters, R. 1971. Pigment degeneration during processing and storage. In The Biochemistry of Fruits and Their Products. Academic Press, New York. Davies, B.H. 1965. Analysis of carotenoid pigments. In Chemistry and Biochemistry of Plant Pigments. Academic Press, New York. Eilati, SK. 1970. The changes in color of the ripening orange fruit as affected by endogenous and exogenous factors. Ph.D. thesis, The Hebrew University, Jerusalem. Cited in Gross, J. 1977. Citrus Science and Technology. AVI Publishing Co., Westport, CT. Flares. H. and Morse. R.E. 1952. Deterioration of oranee oil. Food Technol. 6(l): 6. Florida Citrus Processors Association. 1981. Statistical Summary 1979-80 season. FCP. Winter Haven. FL 33880. Gross, J. 1977. Carotenoid pigments in citrus. In Citrus Science and Technology. Vol. 1. AVI Publishing Co., Wesport, CT. Hamlampu, S. and Karel, M. 1981. An experimental method for the determination of degradation kinetics under dynamic storage conditions. Presented at the 41st Annual Meeting of the Institute of Food Technologists, June 7-10, Atlanta, GA. Hunter, R.S. 1967. Development of the citrus calorimeter. Food Technol. 21: 906. Johnson, J.D., Vi&, H.E., and Wait, D.M. 1978. Method for extracting carotenoid pigments from citrus oils. U.S. Patent 4.126.709. Kesterson, J.W. and Braddock, R.J. 1976. By-products and specialty products of Florida citrus. Bull. 784. Univ. of Fla. Agr. Expt. Sta. Kesterson, J.W., Hendrickson, R., and Braddock, R.J. 1971. Florida citrus oils. Bull. 749. Univ. of Fla. Agr. Expt. Sta. Lund. E.D. and Coleman. R.L. 1977. Concentrated oranze flavourinc powdered bases on methanol-treated starch. Int. Fla&rs & Food Additives 8(5): 193. Muller, R.K., Bernhard, K., Kienzle, F., Mayer, H., and Ruttimann, A. 1980. Some recent advances in the synthesis of natural carotenoids. Food Chem. 5: 15. Onyewu. P.N. 1981. Model system to analvze thermal degradation products of carotenoids. Presented at the 41st Annual Meeting of the Institute of Food Technoloeists. June 7-10. Atlanta. GA. Sorensen, J., Mishkin, M., Hsieh,-Y.. Kacyn, L, and Karel; M. 1981. Kinetic modeling of beta-carotene degradation at elevated temperatures. Presented at the 41st Annual Meeting of the Institute of Food Technologists, June 7-10, Atlanta, GA. Stewart, I. 1980. Color as related to quality in citrus. In Citrus Nutrition and Quality. American Chemical Society. Washington, DC. Swisher, H.E. and Swisher, L.H. 1977. Specialty citrus products. vol. 2, p. 290. AVI PubIn Citrus Science and Technology. lishing Co., Westport, CT. Taylor, E.A. 1965. Why improve frozen concentrated orange juice. Sixteenth Annual Citrus Processors Meeting. Univ. of Fla. Citrus Expt. Sm., Lake Alfred, FL 33850. Ting, S.V. and Hendrickson, R. 1969. Natural color enhancers orange peel carotenoids for orange juice products. Food Technol. 23(7): 87. U.S. Dept. of Agriculture, Foreign Agriculture Service. 1981a. Foreign Agriculture circular, Fresh and processed citrus fruits. FCF 4-81. U.S. Dept. of Agriculture, Food Safety & Quality Service, Fruit and Vegetable Quality Division, Processed Products Standardization and Inspection Branch. 1981b. United States standards for grades or orange juice. Effective April 1981. Wilson, C.W. and Shaw, P.E. 1977. Recovery of citrus pigments from processing waste - an overview. Proc. Int. Sot. Citriculture 3: 808.

M S received

6/12/82;

revised

l/3/83;

accepted

l/5/83.

Florida Agricultural The authors acknowledge the statistical analysis S. Longtin.

Experiment Stations the cooperation and the assistance

Journal Series No. 3888. of Dr. Frank G. Martin in of Ben F. Wood and Terry

TEMPERATURE

PRONLE

OF FRIED BACON.

From page 819

Pensabene. J.W.. Fiddler, W., Miller, A.H., and Phillips, J.G. 1980. Effect of preprocessing procedures for green bellies on N-nitrosopyrrolidine formation in bacon. J. Agric. Food Chem. 28: 966. Scanlan, R.A. 1975. N-Nitrosamines in foods. C.R.C. Critical Rev. Food Technol. 5: 363. Schroder, B.G. and Rust, P.E. 1974. Composition of pork bellies. 2. Compositional variation between and within animals and the relationshiD of various carcass measurements with the chemical components of the belly. J. Anim. Sci. 39: 1037. Sen, N.P., Iyengar. j.R., Donaldson. B.A.. and Panalaks, T. 1974. Effect of sodium nitrite concentration on the formation of nitrosopyrrolidine and dimethylnitrosamine in fried bacon. J. A&c. Food Chem. 22: 540. Wasserman, A.E., Pensabene, J.W., and Piotrowski, E.G. 1978. Nitrosamine formation in home-cooked bacon. J. Food Sci. 43: 276. Ms received 9/l/82; revised 12131182; accepted l/5/82. This study was supported in part by Grant No. IROlCA26576awarded by the National Cancer Institute, DHEW. Michigan Agricultural Experiment Station Journal Article 10558.

Hwang, L.S. and Rosen, J.D. 1976. Nitrosopyrrolidine formation in fried bacon. J. Agric. Food Chem. 24: 1152. Janzowski, C.. Eisenbrand, G.. and Preussmann, R. 1978. Occurrence of N-nitrosoamino acids in cured meat products and their effect on formation of N-nitrosamines during heating. Food Cosmet. Toxicol. 16: 343. Lee. M-L, Gray, J.I., and Pearson, A.M. 1983. Formation of Nnitrosopyrrolidine in fried bacon: model system studies. J. Food Sci. (submitted for publication). M&tram, D.S., Patterson, R.L.S., Edwards. R.A.. and Gough. T.A. 1977. The preferential formation of volatile N-nitrosamines in the fat of fried bacon. J. Sci. Food A&c. 28: 1025. Owens. J.L. and Kinast, O.E. 1980. An improved procedure for the determination of volatile N-nitrosamines in bacon grease by using the mineral oil distillation-thermal energy analyzer method. J. Agrlc. Food Chem. 28: 1262. Pensabene, J.W., Feinberg, J.I., Piotrowskl, E.G.. and Fiddler, W. 1979. Occurrence and determination of N-nitrosoproline and N-nitrosopyrrolidine in cured meat products. J. Food Sci. 44: 1700. Pensabene, J.W.. Fiddler, W., Gates. R.A., Fagan, J.C., and Wasserman, A.E. 1974. Effect of frying and other cooking conditions on nitrosopyrrolidine formation in bacon. J. Food Sci. 39: 314.

02

No.

Volume 48 (1983kJOlJRNAL

OF FOOD SCIENCE-927