Citrus Nutrition and Quality

18 Detection of Adulteration

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Visible and Ultraviolet Absorption and Fluorescence Excitation and Emission Characteristics of Florida Orange Juice and Orange Pulpwash DONALD R. PETRUS and NANCY A. DUNHAM Florida Department of Citrus, University of Florida, Agricultural Research and Education Center, Lake Alfred, FL 33850

Adulterations of citrus juices have evolved from simple dilutions with water, sugar and acids to highly sophisticated methods employing "tailor made" adulterants designed specifically to conceal adulteration. Orange pulpwash (water extraction of soluble fruit solids from orange pulp also referred to as WESOS or WEOS) as a substitution for or an adulterant of frozen concentrated orange juice (FCOJ) and single-strength orange juice (SSOJ) has become of great concern to the citrus industry and regulatory agencies, and an economic fraud on the consumer. Department of Citrus Rule 20-64.07 (3) states FCOJ packed in Florida and sold, shipped, or offered for sale or shipment in retail or institutional size containers shall not contain soluble solids recovered by aqueous extraction or washing of fruit pulp; in addition Rule 20-69.02 (1)(d) states that an imported product to be used in the production of FCOJ in Florida cannot contain soluble solids recovered by aqueous extraction or washing of fruit pulp (1). However, Federal Standard of Identity 146.146, for FCOJ, states "In its preparation, seeds (except embryonic seeds and small fragments of seeds that cannot be separated by good manufacturing practice) and excess pulp are removed, and a properly prepared water extract of the excess pulp so removed may be added" (2). The statement does not imply that a producer or packer may separately purchase orange pulpwash and add i t to FCOJ or orange juice from concentrate. In fact an official opinion (Feb. 4, 1980) from Joseph P. Hile, Associate Commissioner for Regulatory Affairs for the Food and Drug Administration, to the State of Florida Department of Citrus states "It is clear, both from the plain words of the standard and the background, that only the "pulpwash solids" (a properly prepared water extract) of the excess pulp removed from the particular batch of oranges used in preparing the frozen concentrated orange juice may be added back to that juice and that pulp from other sources cannot be used. The actual amount of added pulpwash solids would, of course, vary somewhat with the particular batch of orange juice being processed." The consensus of industry and professional personnel involved is that the product de0-8412-0595-7/80/47-143-423$05.00/0 © 1980 American Chemical Society


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s c r i b e d by Federal r e g u l a t i o n 146.146 would c o n t a i n an average of 6% and a t most 10% pulpwash. Therefore, anything greater than that amount should be considered an a d u l t e r a t e d product. To reveal the extent of the a d u l t e r a t i o n problem, three nat i o n a l surveys were conducted by the F l o r i d a Department of C i t r u s from June 1979 through February 1980. The samples (FCOJ and SSOJ packed o u t s i d e of F l o r i d a ) were obtained from the r e t a i l market by Department of C i t r u s f i e l d personnel and shipped to our l a b o r a t o r y f o r analyses. In the second survey, 26 of 55 FCOJ samples t e s t e d were i d e n t i f i e d a f t e r a n a l y s i s from c o n t a i n e r codes as packed i n F l o r i d a — n o n e had revealed any type of a d u l t e r a t i o n . Of the remaining 29 FCOJ samples, 86 percent i n d i c a t e d pulpwash a d u l t e r a t i o n i n amounts up to 60 percent. Twenty-five percent (6 of 25) of the SSOJ samples i n d i c a t e d pulpwash a d u l t e r a t i o n i n amounts to 40 percent. State of F l o r i d a C i t r u s F r u i t Laws (3j govern the i n s p e c t i o n , grading, c e r t i f i c a t i o n , s a l e and shipment w i t h i n or without the S t a t e of a l l c i t r u s f r u i t and the products thereof whether canned or concentrated. The laws a l s o govern the l i c e n s i n g or c e r t i f i c a t i o n of c i t r u s f r u i t i n s p e c t o r s by the United States Department of A g r i c u l t u r e . There are no r e q u i r e d i n s p e c t i o n s , grading or c e r t i f i c a t i o n of a product repacked by other d i s t r i b u t o r s out of the s t a t e of F l o r i d a . S c i e n t i s t s (4,j5,i6,7_) a t the U n i v e r s i t y of F l o r i d a A g r i c u l t u r a l Research and Education Center, Lake A l f r e d , F l a . conducted a comparison of equeous e x t r a c t s of orange pulp and r e c o n s t i t u t e d orange j u i c e concentrates. Results showed t h a t the chemical and p h y s i c a l c h a r a c t e r i s t i c s of water e x t r a c t s of orange pulp v a r i e d w i t h pulp washing procedures and v a r i e t y of f r u i t used. Also, f l a v o n o i d s and w a t e r - s o l u b l e p e c t i n were found i n greater q u a n t i t i e s i n pulpwash than i n t h e i r corresponding orange j u i c e s . Sawyer (8) conducted an i n v e s t i g a t i o n of the chemical composition of pulp e x t r a c t s , and f r e s h and concentrated orange j u i c e s . He concluded from r e s u l t s of chemical analyses t h a t a general comp o s i t i o n of a j u i c e may be determined, i n c l u d i n g type of a d u l t e r ation. L i f s h i t z et al_. (9) i n v e s t i g a t e d the p u r i t y of I s r a e l i c i t r u s j u i c e by a m u l t i v a r i a t e method. They s e l e c t e d 5 a n a l y t i c a l p a r a meters and concluded t h a t a d u l t e r a t i o n or d i l u t i o n of 15% or more was very l i k e l y to be detected at a 1% l e v e l of s i g n i f i c a n c e . However, a d u l t e r a t i o n w i t h orange pulpwash was not included i n t h e i r study. Schatzki and Vandercook (10) measured the chemical composit i o n ( t o t a l sugars, r e a c t i v e phenols, t o t a l amino a c i d s , a r g i n i n e and y-aminobutyric a c i d ) of concentrated orange j u i c e f o r manuf a c t u r e from A r i z o n a , C a l i f o r n i a , F l o r i d a and Mexico and orange pulpwash samples from F l o r i d a . They i n v e s t i g a t e d the use of nonparametric nearest neighbor c l a s s i f i c a t i o n techniques f o r the det e c t i o n of a d u l t e r a t i o n w i t h sugar, reducing sugars and c i t r i c acid. I t was concluded t h a t "sugared" pulpwash was o f t e n c l a s s i f i e d as pure concentrated orange j u i c e f o r manufacture.


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Vandercook and R o l l e (11) i n v e s t i g a t e d the u l t r a v i o l e t absorption c h a r a c t e r i s t i c s of alcoholic solutions of C a l i f o r n i a Arizona lemon j u i c e . They reported t h a t the r a t i o o f the absorbance o f 273-277 nm t o the absorbance o f 326-332 nm was e s s e n t i a l l y constant. From analyses o f the spectra obtained o f other f r u i t j u i c e s , they i n d i c a t e d i f one were added t o lemon j u i c e i t s p r e sence could be detected by a displacement o f t h i s r a t i o . Vandercook e t al_. (12) observed a s i g n i f i c a n t i n c r e a s e i n t o t a l p o l y phenolic absorbance a t 330 nm, o f lemon j u i c e , w i t h e x t r a c t i o n pressure. However, there was no s i g n i f i c a n t change i n the A/B absorbance r a t i o (273/326). Petrus and Dougherty (13) i n v e s t i g a t e d the combined v i s i b l e and u l t r a v i o l e t absorption c h a r a c t e r i s t i c s o f a l c o h o l i c s o l u t i o n s of F l o r i d a Hamlin, Pineapple and V a l e n c i a orange j u i c e s . S h o u l ders o r peaks were observed a t 465, 443 and 425 nm o f the v i s i b l e spectrum (due mainly t o the carotenoids present) and 325, 280 and 245 nm o f the u l t r a v i o l e t spectrum (due mainly to polyphenols, f l a v o n o i d s and a s c o r b i c a c i d , r e s p e c t i v e l y ) . Absorption o f d i l u t e d samples obeyed B e e r ' s law over the wavelength range o f i n t e r e s t . However, the 443/325 nm and 280/325 nm absorbance r a t i o s remained e s s e n t i a l l y the same as before d i l u t i o n . U l t r a v i o l e t a b s o r p t i o n increased and 443/325 nm absorbance r a t i o decreased w i t h i n c r e a s i n g f r u i t e x t r a c t o r pressure w h i l e only s l i g h t changes were observed i n the v i s i b l e a b s o r p t i o n . Spectra o f a l c o h o l i c s o l u t i o n s o f the rag and albedo components o f the orange v a r i e t i e s showed very l i t t l e v i s i b l e but very strong u l t r a v i o l e t absorbance. Whereas, s p e c t r a obtained o f j u i c e from j u i c e sacs revealed i t as the major c o n t r i b u t o r t o the v i s i b l e a b s o r p t i o n , and having a l s o strong u l t r a v i o l e t a b s o r p t i o n . Petrus and Dougherty (14) a l s o i n v e s t i g a t e d the s p e c t r a l c h a r a c t e r i s t i c s o f m i d - and l a t e season frozen orange concentrates and t h e i r corresponding f r o z e n orange pulpwash concentrates obtained from commercial processors. They concluded t h a t a l c o h o l i c s o l u t i o n s o f orange pulpwash samples were c h a r a c t e r i z e d by weak v i s i b l e and strong u l t r a v i o l e t absorpt i o n . They hypothesized weak v i s i b l e a b s o r p t i o n was due to the c o n c e n t r a t i o n o f parent orange j u i c e p r e s e n t , w h i l e the strong u l t r a v i o l e t a b s o r p t i o n was the r e s u l t o f parent orange j u i c e p r e sent plus i n c o r p o r a t i o n o f rag and pulp and t h e i r w a t e r - e x t r a c t e d s o l u b l e s o l i d s i n t o the product during t h e pulp washing process. Parent orange j u i c e c o n c e n t r a t i o n present i n the pulpwash product was estimated from the pulpwash to orange j u i c e absorbance r a t i o a t 443 nm. The purpose o f t h i s p r e s e n t a t i o n i s t o d i s c u s s the v i s i b l e and u l t r a v i o l e t a b s o r p t i o n and room temperature f l u o r e s c e n c e c h a r a c t e r i s t i c s o f a l c o h o l i c s o l u t i o n s o f F l o r i d a produced orange j u i c e and pulpwash samples, and to r e l a t e the c h a r a c t e r i s t i c s t o q u a l i t a t i v e d e t e c t i o n and q u a n t i t a t i v e approximation o f a d u l t e r a t i o n o f f r o z e n concentrated and s i n g l e - s t r e n g t h orance j u i c e s . Experimental d e t a i l s of our procedures may be found elsewhere (15).


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T y p i c a l v i s i b l e and u l t r a v i o l e t absorption spectra obtained f o r a l c o h o l i c s o l u t i o n s of F l o r i d a V a l e n c i a orange j u i c e s are shown i n F i g . 1 (please r e f e r to references (13, 14, i n press 15), f o r d i s c u s s i o n of a b s o r p t i o n wavelengths). Pineapple orange may be d i s t i n g u i s h e d from the V a l e n c i a orange v a r i e t y by g e n e r a l l y stronger absorbance at 245 nm ( a s c o r b i c a c i d being the main cont r i b u t o r at t h i s wavelength) and s l i g h t l y lower o v e r a l l v i s i b l e absorbance. V i s i b l e absorbance spectra obtained from the Hamlin v a r i e t y were s l i g h t l y l e s s than P i n e a p p l e , however, u l t r a v i o l e t absorbance s p e c t r a were s i m i l a r . To s i m p l i f y the p r e s e n t a t i o n only the above three v a r i e t i e s w i l l be discussed (the e f f e c t of v a r i e t y , m a t u r i t y , e x t r a c t o r type and s e t t i n g were i n v e s t i g a t e d during the 1971-72 (13J, 1972-73, and 1973-74 seasons, u n p u b l i s h ed d a t a ) . F i g . 2 shows t y p i c a l f l u o r e s c e n c e e x c i t a t i o n and emission spectra obtained from a l c o h o l i c s o l u t i o n s of F l o r i d a V a l e n c i a SSOJ's. Fluorescence spectra obtained from Pineapple and Hamlin orange j u i c e s were s i m i l a r to but of lower i n t e n s i t y than V a l e n c i a . E a r l y i n the Pineapple season the emission spectrum may appear as a f l a t apex (from about 310 to 333 nm). E a r l y season Hamlin j u i c e may produce an emission spectrum w i t h 310 nm as the maximum dropping to an i n f l e c t i o n a t 333 nm. As the f r u i t matured 333 nm became the emission maximum. Maximum e x c i t a t i o n and emission ( f o r the three v a r i e t i e s ) were a l s o evident a t 290-93 and 343 nm, r e s p e c t i v e l y , producing e x c i t a t i o n s p e c t r a s i m i l a r to F i g . 2 (shoulder a t 283 nm and s l i g h t i n f l e c t i o n changes a t 270 and 302 nm). V a l e n c i a v a r i e t y e x h i b i t e d g r e a t e r f l u o r e s c e n c e than Hamlin or Pineapple v a r i e t i e s . A s p e c t r a l ( v i s i b l e and u l t r a v i o l e t ) absorption curve comp a r i s o n of r e c o n s t i t u t e d , commercial F l o r i d a produced, FCOJ and orange pulpwash i s shown i n F i g . 3. The absorption shoulders and maxima were as p r e v i o u s l y d i s c u s s e d . I t i s evident t h a t orange pulpwash had a much weaker unresolved v i s i b l e absorption and very much s t r o n g e r , r e s o l v e d (at 280 nm) u l t r a v i o l e t absorption than orange j u i c e . I t i s a l s o evident t h a t the absorbance r a t i o a t 443/325 nm would be lower and the sum of absorbance (at 443, 325 and 280 nm) would be g r e a t e r f o r pulpwash than f o r orange j u i c e . Water e x t r a c t i o n of orange pulp and rag (during the pulp washing and f i n i s h i n g process) would tend to i n c o r p o r a t e more rag and pulp and t h e i r w a t e r - e x t r a c t a b l e s o l u b l e s o l i d s i n t o the product, thus i n c r e a s i n g the u l t r a v i o l e t and decreasing the v i s i b l e absorpt i o n (13,14). F i g . 4 shows the comparison of the f l u o r e s c e n t exc i t a t i o n and emission curves obtained from s o l u t i o n s of orange j u i c e and pulpwash. Maxima f o r orange j u i c e appeared a t 290 nm e x c i t a t i o n and 343 nm emission w i t h e x c i t a t i o n shoulders or i n f l e c t i o n s a t 270, 283, and 302 nm. Pulpwash revealed maxima a t 270 and 302 nm w i t h shoulders a t 283 and 290 nm e x c i t a t i o n and 353-55 nm emission. Pulpwash samples (not shown by F i g . 4) have a l s o shown strong e x c i t a t i o n maxima a t 270 and 302 nm w i t h a m i n i mum a t 280-85 nm. A l c o h o l i c s o l u t i o n s o f orange components ( r a g ,


18.

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Figure 1.

AND DUNHAM

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All

Visible and UV absorption spectra obtained from an alcoholic solution of Valencia orange juice

WAV E L Ε N GT H

Figure 2. Fluorescence excitation (283 nm maximum) and emission (333 nm maximum) spectra obtained from Valencia orange juice

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Figure 4. Comparison of fluorescence excitation and emission spectra of orange juice ( ) and orange pulpwash (·—·)

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albedo, j u i c e from j u i c e sacs) produced f l u o r e s c e n c e s p e c t r a s i m i l a r to FCOJ or SSOJ by the j u i c e component, w h i l e the rag and albedo components produced s p e c t r a s i m i l a r to pulpwash. Again pulp washing and f i n i s h i n g would tend to i n c o r p o r a t e more rag and pulp and t h e i r w a t e r - e x t r a c t a b l e s o l u b l e s o l i d s i n t o the product, thus an orange j u i c e sample e x h i b i t i n g f l u o r e s c e n c e e x c i t a t i o n peaks or pronounced shoulders a t 270 and 302 nm would i n d i c a t e the presence or use o f pulpwash. Table I l i s t s the e x c i t a t i o n and emission wavelengths u t i l i z e d f o r sample c h a r a c t e r i z a t i o n . I f an emission wavelength was absent f o r a sample under i n v e s t i gation the procedure was to set the emission monochromator at the d e s i r e d wavelength and scan the e x c i t a t i o n . Table I.

Fluorescence E x c i t a t i o n and Emission C h a r a c t e r i z a t i o n of Orange J u i c e and Pulpwash.

E x c i t a t i o n Emission

Range

E x c i t a t i o n Spectra Orange J u i c e

Pulpwash

(nm)

(nm)

Setting

340

423

0.1

Strong

Much stronger

230

310

0.03

Weak

Weaker

283

333

0.1

Strong Maximum Minimum

290

343

0.1

302

353

0.1

Inflection

Strong Maximum

270

333

0.1

Inflection

Shoulder or Maximum

"

"

Minimum or I n f l e c t i o n

F i g . 5 and 6 show the absorbance sum and r a t i o d i s t r i b u t i o n (normal curve constructed) o f F l o r i d a commercial packed FCOJ r e c o n s t i t u t e d to 12.80 B r i x . Sampling period was from 1972 through 1979. Absorbance sum of 416 samples showed a minimum of 2.002, maximum of 2.992, mean and standard d e v i a t i o n of 2.410 + 0.164, and c o e f f i c i e n t of v a r i a t i o n o f 6.8%. Absorbance r a t i o revealed a minimum of 0.092, maximum of 0.227, mean and standard d e v i a t i o n of 0.144 + 0.026, and c o e f f i c i e n t of v a r i a t i o n of 18%. F i g . 7 and 8 show the absorbance sum and r a t i o d i s t r i b u t i o n of F l o r i d a commercial packed f r o z e n orange pulpwash concentrate ( r e c o n s t i t u t e d or c a l c u l a t e d to 12.8° B r i x ) . Sampling p e r i o d was from 1971 through 1979. Absorbance sum o f 206 samples showed a minimum of 2.617, maximum of 4.992, mean and standard d e v i a t i o n of 3.781 + 0.473, and c o e f f i c i e n t o f v a r i a t i o n of 12.5%. Absorbance r a t i o showed a minimum of 0.017, maximum o f 0.112, mean and standard d e v i a t i o n of 0.048 + 0 . 0 2 0 , and c o e f f i c i e n t of v a r i a t i o n of 41.7%. Inspection o f the d i s t r i b u t i o n p l o t s and data revealed more v a r i a t i o n i n pulpwash than FCOJ, i n d i c a t i n g s t a n d a r d i z a t i o n of F l o r i d a FCOJ products. However, there are many processing v a r i ables i n orange pulp washing, such as number o f , and mechanical

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"A REL FREQ

+2.000

1-2.200

+2.400

1-2.600 »10~0

1-2.8Θ0

1-3.000

Figure 5. Distribution of absorbance sum (at 443, 325, and 280 nm) of Floridapacked FCOJ (N = 416; mean = 2.410; standard deviation = 0.164)

Ν

κ REL FREQ

2.100

Figure 6.

1-2.300

Distribution of absorbance ratio (at 443/325 nm) of Florida-packed FCOJ (N = 416; mean = 0.144; standard deviation = 0.026)

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Ν X REL FREQ 28 „. 13.6

+2. 5ΘΘ

1-2.980

+3.388

1-3.780 »Ι0 0

+4.100

+4.500

+4.908

Λ

Figure 7.

Ν

Distribution of absorbance sum of Florida-produced orange pulpwash (N = 206; mean = 3.781; standard deviation =

% REL FREQ

36.! 17.5 33

I

16.0

30.: 14.6 27

I 13. 1

24 £ 11 .7

+0 . 1 00

Figure 8.

+0 . 300

+0 . 500

+0 . 700 »10~-1

+0 . 900

+ 1 . I 00

Distribution of absorbance ratio of Florida-produced orange pulpwash (N = 206; mean = 0.048; standard deviation = 0.020)


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s e t t i n g of f i n i s h e r s , and i n c o r p o r a t i o n o f f r u i t core and c e n t r i fuge discharge i n t o the pulp washing process r e s u l t i n g i n p r o ducts of d i f f e r i n g s p e c t r a l c h a r a c t e r i s t i c s . A primary or s i n g l e stage f i n i s h e r produced pulpwash may resemble the e x c i t a t i o n curve shown i n F i g . 4, whereas pulpwash obtained from a m u l t i stage f i n i s h e r may show strong e x c i t a t i o n a t 270 and 302 nm w i t h a minimum a t 280-85 nm. The d i s t r i b u t i o n p l o t s a l s o reveal a s l i g h t overlapping r e g i o n . There were four FCOJ samples (1.0% o f t o t a l ) having an absorbance sum greater than 2.900 and seven pulpwash samples (3.3%) having a sum l e s s than 3.000. D i s t r i b u t i o n of absorbance r a t i o revealed more o v e r l a p than absorbance sum. There were 24 FCOJ samples (5.8%) having an absorbance r a t i o l e s s than 0.110 whereas pulpwash had 12 g r e a t e r than 0.090 (5.8%). There appear ed to be good s e p a r a t i o n of the general p o p u l a t i o n s . Model systems composed o f mixtures of r e c o n s t i t u t e d commer c i a l l y produced FCOJ and orange pulpwash were prepared, and t h e i r a l c o h o l i c s o l u t i o n s i n v e s t i g a t e d f o r c h a r a c t e r i s t i c v i s i b l e and u l t r a v i o l e t a b s o r p t i o n , and room temperature f l u o r e s c e n c e . Four models using 3 d i f f e r e n t FCOJ s and 4 d i f f e r e n t pulpwashes were i n v e s t i g a t e d from 100 percent orange j u i c e through 100 percent pulpwash. S t a t i s t i c a l a n a l y s i s o f the d a t a , (with absorbance sums a t 443, 325 and 280 nm, absorbance r a t i o s a t 443/325 nm and f l u o r e s c e n c e r a t i o s a t 302/284 nm) i n a l l cases showed each model system producing a c o e f f i c i e n t of determination r > 0.99. Mul t i p l e l i n e a r r e g r e s s i o n a n a l y s i s o f each system u t i l i z i n g the ab sorbance sum and r a t i o produced c o e f f i c i e n t s of determination R2 > 0.999. M u l t i p l e r e g r e s s i o n a n a l y s i s of the combined data obtained from the 4 models produced the e q u a t i o n : 1

2

y = 241.768 - 46.776( Σ ) + 19.043(ln

nm)

where y equals approximate percent orange j u i c e , and 100-y equals approximate percent pulpwash. This r e s u l t e d i n a high c o e f f i c i ent of determination R2 = 0.904, i n d i c a t i n g 90.4% of the v a r i a t i o n i n orange j u i c e content may be explained by the absorbance sum and n a t u r a l l o g of the r a t i o . The r e s u l t s approximated the orange j u i c e content f o r systems o f orange j u i c e a d u l t e r a t e d w i t h pulpwash, however, i t f a i l e d when the a d u l t e r a t i o n was f u r t h e r complicated by d i l u t i o n w i t h water. Since a d u l t e r a t i o n o f orange j u i c e w i t h pulpwash increased the absorbance sum ( l i n e a r function) i t was apparent t h a t f u r t h e r a d u l t e r a t i o n by d i l u t i o n w i t h water would decrease the sum ( l i n e a r f u n c t i o n ) . The a d u l t e r a t e d sample could then i n d i c a t e an absorbance sum i n the range found f o r orange j u i c e l e a d i n g to erroneous p r e d i c t i o n s o f orange j u i c e and pulpwash contents by m u l t i p l e r e g r e s s i o n equations. Since simple d i l u t i o n s of orange j u i c e r e s u l t e d i n e s s e n t i a l l y no change i n the absorbance r a t i o , and a d d i t i o n of pulpwash to orange j u i c e r e s u l t e d i n an absorbance r a t i o decrease (natural l o g f u n c t i o n ) , i t appeared l i k e l y a mathematical system could be e s t a b l i s h e d r e l a t i n g the sum and r a t i o to orange j u i c e and/or pulpwash contents and/or d i l u t i o n .

18.

PETRUS AND DUNHAM

Detection

of

Adulteration

433

The f o l l o w i n g equations were derived using the f o l l o w i n g constants: 1

=

A

v

Σ _ - Αν Σ . = 3.484 - 2.410 = 1.074 pw OJ

1

where Αν Σ ^ and Αν Σ . were average absorbance sums f o r p u l p wash ( a t 11.8° B r i x ) and orange j u i c e ( a t 12.8° B r i x ) respec tively. Citrus Nutrition and Quality Downloaded from pubs.acs.org by EAST CAROLINA UNIV on 03/16/16. For personal use only.

Q

y = In Av R . - In Av R = In 0.144 - In 0.048 = 1.099 2 oj pw where In Av R . and In Av R were n a t u r a l l o g o f average absorboj pw ance r a t i o s f o r orange j u i c e and pulpwash r e s p e c t i v e l y . Then x = in R

u n k

- i n Av R

- In R

p w

u n R

- In 0.048

3

where In R , was the n a t u r a l l o g absorbance r a t i o o f the unknown sample. The average absorbance sum c o r r e c t i o n f a c t o r u

a =

n

K

-

x

y

x

1.099

and absorbance c o r r e c t i o n value b = Z(a) = 1.074(a)

5

and the c o r r e c t e d average absorbance sum c = A v £ - b pw R

unk

= 3.484 - b

6

At t h i s p o i n t i t may be explained t h a t i f χ = 0 (when In ^ ) ' 0» t h e r e f o r e b = 0 and the c o r r e c t e d

=

n A

v

R

t

i e n

a

=

p w

absorbance sum c = Αν Σ

- 0 = 3.484 and the product would conpw t a i n pulpwash i n pure or d i l u t e d form. However, i f χ = 1.099 (when In R = In R .) then a = 1, u n k

t h e r e f o r e b = 1.074 and the c o r r e c t e d absorbance sum c = 3.484 1.074 = 2.410 which i s equal t o the average absorbance sum f o r orange j u i c e and the product would i n d i c a t e orange j u i c e i n pure or d i l u t e d form. % Τ CM =

£

"

n

c

k

χ 100

7

where % TCM i s the approximation o f t o t a l c i t r u s m a t e r i a l present i n the unknown sample and Σ i s the unknown sample absorbance sum. u

n

k

CITRUS

434

NUTRITION

AND

QUALITY

Then

% TCM(a) = % OJ

8

and % TCM - % OJ = % PW

9

and Citrus Nutrition and Quality Downloaded from pubs.acs.org by EAST CAROLINA UNIV on 03/16/16. For personal use only.

100% - % TCM = % D i l u t i o n

10

The f o l l o w i n g cases i l l u s t r a t e use of the above equations: Case 1. I f the r e s u l t s i n d i c a t e a combination of orange j u i c e , pulpwash and d i l u t i o n , then equation 8, 9 and 10 are reported as c a l c u l a t e d and product f o r m u l a t i o n equals 100 percent. Case 2. I f r e s u l t s i n d i c a t e only orange j u i c e and pulpwash, then r e s u l t s of equations 8 and 9 are set p r o p o r t i o n a l to 100% as % 0 J : % TCM = x: 100 and % PW: % TCM = x: 100 r e s p e c t i v e l y . Solved f o r x, product f o r m u l a t i o n equals 100 percent. Case 3. I f r e s u l t s i n d i c a t e orange j u i c e , or orange j u i c e w i t h d i l u t i o n , then equations 7 and 10 are reported as c a l c u l a t e d and product f o r m u l a t i o n equals 100 percent. Case 4. I f r e s u l t s i n d i c a t e only pulpwash or pulpwash w i t h d i l u t i o n , then equations 7 and 10 are reported as c a l c u l a t e d and product f o r m u l a t i o n equals 100 percent. The equations were performed on the data obtained from the 416 FCOJ samples p r e v i o u s l y d i s c u s s e d . Q u a l i t a t i v e l y the absorpt i o n and fluorescence s p e c t r a d i d not i n d i c a t e the presence of orange pulpwash. Therefore, the j u i c e s were examples of Case 3, and orange j u i c e content reported as % TCM (equation 7 ) . Statist i c a l a n a l y s i s of the r e s u l t s showed a mean of 99.6% TCM and standard d e v i a t i o n of 8.5. To f u r t h e r v e r i f y the equations, 29 unknown samples (4 s e t s ) composed of various combinations of orange j u i c e s , and/or p u l p washes and/or product d i l u t i o n s were prepared by others f o r a n a l ysis. C a l c u l a t e d values were c o r r e l a t e d w i t h a c t u a l values and gave the f o l l o w i n g c o e f f i c i e n t s of d e t e r m i n a t i o n : orange j u i c e content r2 = 0.942; pulpwash content r2 = 0.981; and d i l u t i o n r2 = 0.937. Even w i t h s i g n i f i c a n t c o e f f i c i e n t s o f d e t e r m i n a t i o n , i t must be stressed t h a t the equations provide only approximat i o n s of sample f o r m u l a t i o n s . The constants used i n the equat i o n s were based on r e s u l t s obtained from commercial F l o r i d a


18.

PETRUS AND DUNHAM

Detection

of

Adulteration

435

c i t r u s products and could vary w i t h other c i t r u s producing r e gions. The f o l l o w i n g examples were s e l e c t e d from the aforementioned n a t i o n a l surveys conducted by the F l o r i d a Department of C i t r u s . The examples were packed o u t s i d e F l o r i d a and obtained from the r e t a i l market. They w i l l serve to i l l u s t r a t e both q u a l i t a t i v e l y and s e m i - q u a n t i t a t i v e l y the information which may be obtained from the spectra and equations. V i s i b l e and u l t r a v i o l e t absorption and fluorescence s p e c t r a , obtained from a l c o h o l i c s o l u t i o n s of a commercially packed (out o f the State o f F l o r i d a ) FCOJ, are presented i n F i g . 9, 10, 11 and 12. Q u a l i t a t i v e l y , F i g . 9 r e v e a l s a l a c k o f r e s o l u t i o n i n the v i s i b l e absorption region and a well resolved peak a t 280 nm. Comparison with F i g . 1 and 3 shows i t s absorption c h a r a c t e r i s t i c s to be more s i m i l a r to those o f orange pulpwash i n F i g . 3, i n d i c a t ing a d u l t e r a t i o n by pulpwash a d d i t i o n . Fluorescence e x c i t a t i o n spectra ( F i g . 10, 11) reveal w e l l defined peaks a t 270-75 nm, and F i g . 12 a shoulder a t 270-75 nm. The spectra appear deformed when compared to F i g . 2 and 4 of pure orange j u i c e . However, charac t e r i s t i c s are s i m i l a r to those obtained from pulpwash and very s i m i l a r to those obtained from prepared model systems. Qualita t i v e l y both v i s i b l e and u l t r a v i o l e t a b s o r p t i o n , and room tempera ture fluorescence i n d i c a t e the presence of pulpwash i n the FCOJ sample. The spectra are complementary. Absorption a l s o d i d not i n d i c a t e f u r t h e r a d u l t e r a t i o n by d i l u t i o n which would have been denoted by weaker o v e r a l l absorption and a s h i f t a t 227 nm to s h o r t e r wavelength. The sum of absorption a t 443, 325 and 280 nm i s 0.098 + 1.040 + 1.622 = 2.760 absorbance u n i t s . F l o r i d a State s t a t u t e 20-64.07(1)(a) r e q u i r e s FCOJ to be 44.8° B r i x which r e c o n s t i t u t e d to 12.8° B r i x (16) and Federal standards, Section 52.2582(a), r e q u i r e 4 1 . 8 ° B r i x , r e c o n s t i t u t e d to not l e s s than 11.8° B r i x (17). Therefore, the sum o f absorbance i s m u l t i p l i e d by the r a t i o o f 12.8 to 11.8° B r i x , w i t h a c o r r e c t e d sum o f 2.995. The sample absorbance r a t i o a t 443/325 nm i s 0.098/1.040 which i s equal to 0.094. I f the sum and natural l o g r a t i o values are sub s t i t u t e d i n t o the r e g r e s s i o n equation: y = 241.768 - 46.776( Σ ) + 19.043(ln ^ | nm) the sample was found to c o n t a i n approximately 57% orange j u i c e and 43% pulpwash (100-57 = 4 3 ) . I f the sum and natural l o g r a t i o are used w i t h equations 1 through 10, the sample was found to c o n t a i n approximately 65% orange j u i c e and 41% pulpwash (106% TCM) and there was no i n d i c a t i o n o f f u r t h e r d i l u t i o n . The sample c o n s i s t e d o f only orange j u i c e and pulpwash, and i s a Case 2 example. T h e r e f o r e , s o l v i n g the proportions (65:106 = x:100 and 41:106 = x:100) r e s u l t e d i n a product f o r m u l a t i o n o f approximately 61% orange j u i c e and 39% pulpwash. The values are comparable to those obtained using the r e g r e s s i o n equation. V i s i b l e and u l t r a v i o l e t absorption and room temperature f l u orescence e x c i t a t i o n and emission spectra obtained from a l c o h o l i c

436

CITRUS

NUTRITION

A N D QUALITY

1^ CM CM


I

WAVELENGTH,nm Figure 9.

Visible and UV absorption spectra obtained from a commercially packed (outside Florida) FCOJ

18.

PETRUS

AND DUNHAM

in in to h- ω en CM CM CJ

Detection

of

Adulteration

437

ο ιο


I e, I

>-

UJ

>

-

Figure 16. Fluorescence excitation (302 nm) and emission (353 nm) spectra obtained from a commercially packed SSOJ

WAVELENGTH,nm


18.

PETRUS A N DDUNHAM

Detection

of

Adulteration

441

have been concerned mainly w i t h d i l u t i o n s o f the product followed by f o r t i f i c a t i o n w i t h c e r t a i n inexpensive chemicals or elements to d i s g u i s e the a d u l t e r a t i o n . However, simple d i l u t i o n o f orange j u i c e s a d u l t e r a t e d w i t h orange pulpwash would r e s u l t i n chemical and elemental p r o f i l e s s i m i l a r to orange j u i c e and thus confuse these types o f analyses. M u l t i p l e r e g r e s s i o n analyses o f the absorbance data produced an equation ( u t i l i z i n g the absorbance sum and r a t i o ) f o r the e s t i mation o f orange j u i c e and pulpwash present i n a product. The equation worked w e l l f o r orange j u i c e s a d u l t e r a t e d w i t h pulpwash t h a t were not d i l u t e d . Therefore, a s e r i e s o f equations was de r i v e d , using model systems and absorbance d a t a , to approximate orange j u i c e and/or pulpwash content and/or d i l u t i o n o f an orange j u i c e sample. The constants used were obtained from analyses o f commercial F l o r i d a c i t r u s products and may vary w i t h other c i t r u s producing r e g i o n s . ACKNOWLEDGMENT A s s i s t a n c e from the f o l l o w i n g t e c h n i c a l persons i n preparing and a n a l y z i n g the numerous samples and t a b u l a t i n g the data has been g r e a t l y a p p r e c i a t e d ; John Glennon, G a i l H a l l , E l a i n e South, Pam B i r k y , and Betty Murphy.

Literature Cited 1. Official Rules Affecting the Florida Citrus Industry Pursuant to Chapter 601, Florida Statutes. 2. Code of Federal Regulations, Food and Drugs Parts 100 to 199, Revised April 1, 1977. 3. State of Florida Citrus Fruit Laws--Chapter 601, Florida Statutes, 1979. 4. H i l l , E. C.; Patrick, R. Proc. Fla. State Hort. Soc., 1959, 72, 233. 5. Huggart, R. L.; Olsen, R. W.; Wenzel, F. W.; Barron, R. W.; Ezell, G. H. Proc. Fla. State Hort. Soc., 1959, 72, 221. 6. Rouse, A. H.; Atkins, C. D.; Moore, E. L. Proc. Fla. State Hort. Soc., 1959, 72, 227. 7. Wenzel, F. W. Proc. Fla. State Hort. Soc., 1959, 72, 235. 8. Sawyer, R. J. Sci. Fd. Agric., 1963, 14, 302. 9. Lifshitz, Α.; Stepak, Y.; Brown, M. B. J . Assoc. Off. Anal. Chem., 1974, 57, 1169. 10. Schatzki, T. F.; Vandercook, C. E. J . Assoc. Off. Anal. Chem., 1978,61,911. 11. Vandercook, C. E.; Rolle, L. A. J . Assoc. Off. Anal. Chem., 1963, 46, 359. 12. Vandercook, C. E.; Rolle, L. Α.; Postlmayr, H. L.; Utterberg, R. A. J . Food Sci., 1966, 31, 58. 13. Petrus, D. R.; Dougherty, M. H. J . Food Sci., 1973, 38, 659.


442

CITRUS

NUTRITION

AND

QUALITY

14. Petrus, D. R.; Dougherty, M. H. J. Food Sci., 1973, 38, 913. 15. Petrus, D. R.; Attaway, J. A. J. Assoc. Off. Anal. Chem., in press, 1980. 16. Official Rules Affecting the Florida Citrus Industry Pursuant to Chapter 601, Florida Statutes, Section 20-64.07(1)(a). 17. United States Standards for Grades of Frozen Concentrated Orange Juice Effective Feb. 17, 1976, Section 52.1582(a) Without Sweetener. United States Department of Agriculture, Agricultural Marketing Service, Fruit Safety and Quality Service, Processed Products Standardization and Inspection Branch, Washington, D.C. RECEIVED July

14, 1980.

Citrus Nutrition and Quality

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