Protein Functionality in Foods - American Chemical Society

2

Color

Downloaded by COLUMBIA UNIV on February 15, 2015 | http://pubs.acs.org Publication Date: March 6, 1981 | doi: 10.1021/bk-1981-0147.ch002

F. A. BLOUIN, Z. M. ZARINS, and J. P. CHERRY Southern Regional Research Center, Science and Education Administration, Agricultural Research, U.S. Department of Agriculture, P.O. Box 19687, New Orleans, LA 70179

The first encounter with any food product is usually visual. Immediately, a judgment is made on the appearance of the product. The individual making the evaluation is probably not consciously aware of the complex factors that influence this judgment. Color is usually the major criterion used to evaluate quality, and past experience plays a role in this evaluation. For example, the color of fresh fruit indicates ripeness and the time the fruit is most likely to taste best. Off-colors in cheese or meats are associated with poor flavor quality. Generally, we expect each food product to have a certain color. Deviations from this expected color can result in rejection of a product even when this color does not adversely influence the flavor or nutritional value of the food. If plant-protein products are to attain widespread utilization in food applications, the color they impart to the product must be considered an important factor in consumer acceptability. Cottonseed flour is a plant protein product which can be used in foods to improve nutritional and functional properties. However, in some applications, cottonseed flour causes a serious color problem. Biscuits prepared with 20% cottonseed flour are yellow brown (Figure 1). Liquid-cyclone-processed (LCP) cottonseed flour is made from glanded seeds by liquid centrifugation in hexane to remove the pigment glands containing the toxic gossypol pigment (1_). LCP flour produced a much darker color in biscuits than hexane-defatted flour made from glandless cottonseeds. Undesirable colors in raw and processed foods have often been minimized by 1) altering their processing to include solvent extraction steps to remove pigments, 2) adding antioxidants or bleaching reagents to reduce color changes, or 3) including additives to mask undesirable color. These approaches to the color problem in cottonseed flours have been generally unsuccessful (2^, 3_) • The objective of the present research is to isolate and identify the pigments responsible for the color problem that occurs when cottonseed flours are used as food ingredients. This basic approach has led to a better understanding of the This chapter not subject to U.S. copyright. Published 1981 American Chemical Society

In Protein Functionality in Foods; Cherry, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

22

PROTEIN FUNCTIONALITY

IN FOODS

problem. The methods and techniques developed i n t h i s study should a l s o be a p p l i c a b l e to d e f i n i n g the c o l o r problem of other p l a n t - p r o t e i n m a t e r i a l s , i n c l u d i n g sunflower and a l f a l f a l e a f proteins.


Experimental Procedures Cottonseed f l o u r s and methods used to f r a c t i o n a t e , i s o l a t e , and i d e n t i f y pigments i n these f l o u r s are f u l l y described by B l o u i n and Cherry (4) and B l o u i n , et a l . ( 5 ) . Sunflower and soy f l o u r s were obtained from the Food P r o t e i n R&D Center, Texas A&M U n i v e r s i t y , College S t a t i o n , Tex.; a l f a l f a l e a f p r o t e i n from the Western Regional Research Center, U.S. Department of A g r i c u l t u r e , Berkeley, C a l i f . ; and peanut f l o u r from Gold K i s t , Inc., L i t h o n i a , Ga. B i s c u i t s were prepared from p l a n t - p r o t e i n f l o u r s based on 20% replacement f o r wheat f l o u r . In b i s c u i t s prepared with f r a c t i o n s i s o l a t e d from cottonseed f l o u r s , the q u a n t i t i e s used were c a l c u l a t e d using the percentages these f r a c t i o n s represented of the o r i g i n a l f l o u r , e.g., the s a l t s o l u t i o n s o l u b l e f r a c t i o n of LCP f l o u r was 43% of the o r i g i n a l f l o u r and 8.6%, (20 x 0.43) replacement f o r wheat was used to prepare the b i s c u i t . Solvent systems used f o r t h i n l a y e r chromatography were 1) n-butanol:acetic acid:water (4:1:5 upper phase), 2) a c e t i c a c i d : water (15:85), 3) e t h y l acetate:pyridine:water (12:5:4), and 4) chloroform:acetic acid:water (50:45:5). S i l i c a gel plates were used f o r chromatography of f l a v o n o i d aglycones and c e l l u l o s e p l a t e s for a l l other components. Aluminum c h l o r i d e was used for d e t e c t i o n (under long UV l i g h t ) of f l a v o n o i d s , a n i l i n e phthalate f o r sugars, ninhydrin for amino acids and iodine for other components. C e l l u l o s e t h i c k l a y e r plates were developed with solvents 1 or 2. Column chromatography of f l o u r extracts was c a r r i e d out on Sephadex G-15 with water as the eluent, Sephadex LH-20 with 50% methanol as the eluent f o r f l a v o n o i d s , and DMF as the eluent for gossypol components. Flavonoid i d e n t i f i c a t i o n methods were those of Mabry et a l . (6). Color Measurements In any study of food c o l o r , one of the basic problems i s measuring c o l o r and r e p o r t i n g the r e s u l t s i n a r e a l i s t i c and understandable manner. Color i s not j u s t a p h y s i c a l property of the object. Color i s a sensory property dependent on both p h y s i c a l and p s y c h o l o g i c a l f a c t o r s r e l a t e d to the object, the conditions of observation, and the i n d i v i d u a l making the observation. Because of the complexity and great economic importance of the c o l o r phenomena, a vast " c o l o r science l i t e r a t u r e that deals with measuring and s p e c i f y i n g c o l o r has evolved during the 11



2.

BLOUIN ET AL.

Color

23

past 20-30 years (7_, 8_). I f the incident l i g h t and the observer are removed as v a r i a b l e s by using appropriate i n t e r n a t i o n a l l y accepted standards, then the problem of c o l o r measurement and s p e c i f i c a t i o n i s reduced to measuring the l i g h t - r e f l e c t i n g or l i g h t - t r a n s m i t t i n g p r o p e r t i e s of the colored object. To define the c o l o r of an object q u a n t i t a t i v e l y , three fundamental q u a n t i t i e s must be s p e c i f i e d . They are: 1) hue or s p e c t r a l c o l o r , which i d e n t i f i e s the object as red, green, yellow, blue, or an intermediate c o l o r between these, 2) s a t u r a t i o n or p u r i t y , which i s the strength or i n t e n s i t y of the hue, and 3) l i g h t n e s s or luminance, which i s the amount of l i g h t r e f l e c t e d or transmitted from the o b j e c t . Numerous v i s u a l and instrumental methods and c o l o r scales have been devised to measure and express these three q u a n t i t i e s . In the work with cottonseed f l o u r s , we used the Hunterlab c o l o r meter D25D2A and expressed these measurements as Hunter L, a, b c o l o r values. These are coordinates of the three-dimensi o n a l opponent-color space shown i n F i g u r e 2. The L value measures l i g h t n e s s , or the amount of l i g h t r e f l e c t e d or transmitted by the o b j e c t . The a and b values are the c h r o m a t i c i t y coordinates from which information about hue and s a t u r a t i o n can be obtained. The a value measures redness when plus and greenness when minus. The b value measures yellowness when plus and blueness when minus. Comparison of c o l o r measurements on wheat f l o u r and s i x d i f f e r e n t p l a n t - p r o t e i n products with the v i s u a l appearance of these f l o u r s shows that the L, a, b scale does y i e l d meaningful values ( F i g u r e 3). The L value f o r the a l f a l f a l e a f p r o t e i n (L • 75.5) shows that t h i s product i s much darker than the wheat f l o u r (L = 89.5) and somewhat darker than the other p l a n t p r o t e i n m a t e r i a l s (L = 79.5 to 88.2). The a values do not show any s i g n i f i c a n t v a r i a t i o n s (a = +1.0 to -1.7), i n d i c a t i n g the absence of s i g n i f i c a n t red or green c o l o r a t i o n i n the f l o u r s . A l l of the p l a n t - p r o t e i n f l o u r s gave p o s i t i v e b values (b * 7.6 to 13.8), i n d i c a t i n g some yellow c o l o r a t i o n i n these products. This comparison of wheat and p l a n t - p r o t e i n products as dry powders or f l o u r s also i l l u s t r a t e s another important p o i n t : the c o l o r of the dry f l o u r s does not a c c u r a t e l y r e f l e c t the magnitude of the c o l o r problem observed when these f l o u r s are used i n food products. C o l o r measurements made on wheat and LCP cottonseed f l o u r as dry powders, aqueous pastes, and a l k a l i n e pastes are shown i n F i g u r e 4. The L and b values f o r aqueous and a l k a l i n e (pH 10) pastes of LCP f l o u r , when compared to the values f o r wheat f l o u r pastes, more adequately r e f l e c t the magnitude of the c o l o r problem of the cottonseed f l o u r . The aqueous paste i s a dark yellow brown, and the darkness and yellowness are even more pronounced i n the a l k a l i n e paste.


PROTEIN FUNCTIONALITY IN FOODS


24

Figure 1. Biscuits containing 100% wheat and 20% LCP and glandless cottonseed flours

L=IOO

L = 0

Figure 2.

L,a,b

color

solid



BLOUIN ET AL.

Color

25

Figure 3.

90}-

Plant protein

flours

WHEAT FLOUR O COTTONSEED FLOUR V 9 DRY FLOUR

80 h

70h

i

ftn 6

T

50H

-

X

AQUEOUS PASTE

\ A

4 0

~

io ^ io b VALUE (yellowness)

ALKALINE PASTE

' flours

Journal of Food Science

F/gwre 4. Hunter L and b values of dry (O), aqueous pastes (X), and alkaline pastes (A) of LCP cottonseed and wheat flours (4)


26

PROTEIN F U N C T I O N A L I T Y IN FOODS

B i s c u i t s as a Model System


The best method f o r e v a l u a t i o n of the c o l o r problem i s to prepare a food product c o n t a i n i n g the p r o t e i n f l o u r . Biscuits were r o u t i n e l y used as the model food system. F i g u r e 5 i l l u s t r a t e s the c o l o r of b i s c u i t s prepared with 100% wheat f l o u r and with 20% p l a n t - p r o t e i n products. The c o l o r of the b i s c u i t s prepared with soybean and peanut f l o u r s shows that these i n g r e d i ents do not cause a serious c o l o r problem. However, sunflower, a l f a l f a l e a f , and cottonseed f l o u r s do produce a d i s c o l o r a t i o n i n t h i s model food system. The L and b values g e n e r a l l y r e f l e c t this visual evaluation. Plant

Phenols

Most b r i g h t l y c o l o r e d plant parts contain anthocyanin, carotenoid, or c h l o r o p h y l l pigments. These pigments, however, do not seem to play a s i g n i f i c a n t r o l e i n the c o l o r problem associated with products c o n t a i n i n g o i l s e e d - p r o t e i n . P l a n t phenols, on the other hand, seem to be the most important c o n t r i butors to the c o l o r problem of these p r o t e i n m a t e r i a l s . Phenols are substrates f o r enzymatic and nonenzymatic browning r e a c t i o n s . Oxidative r e a c t i o n s of phenol q u i t e o f t e n lead to attachment of these compounds to p r o t e i n and polysaccharide components of the p l a n t - p r o t e i n products. The r o l e of chlorogenic acid as the source of the c o l o r problem i n sunflower-seed-protein products has been reported (j), 12> cottonseed f l o u r s (4_, 5), two types of phenolic compounds involved i n the d i s c o l o r a t i o n problem are: gossypol and f l a v o n o l s . I

n

Cottonseed F l o u r F r a c t i o n a t i o n LCP cottonseed f l o u r was separated into four f r a c t i o n s by methods developed f o r p r o t e i n i s o l a t i o n ( 4 ) . The f l o u r was f i r s t e x t r a c t e d with water to y i e l d a water-soluble and a waterinsoluble fraction. The water-soluble f r a c t i o n was d i a l y z e d i n c e l l u l o s e tubing with a molecular weight c u t o f f of 3,500 to give low-molecular-weight (L) and high-molecular-weight (H) f r a c t i o n s . The w a t e r - i n s o l u b l e part of the f l o u r was e x t r a c t e d with 10% sodium c h l o r i d e s o l u t i o n to produce a s o l u b l e (S) and an insoluble (I) f r a c t i o n . B i s c u i t s c o n t a i n i n g these f r a c t i o n s i n the percentages that were present i n the o r i g i n a l f l o u r were prepared (Figure 6). The c o l o r of the b i s c u i t s showed that the two major p r o t e i n f r a c t i o n s , the high-molecular-weight water s o l u b l e s (H) and the s a l t - s o l u t i o n s o l u b l e s ( S ) , d i d not c o n t a i n the pigments r e s p o n s i b l e f o r the c o l o r problem. The pigments r e s p o n s i b l e f o r the yellow c o l o r a t i o n i n b i s c u i t s were present i n the low-molecular-weight water-soluble f r a c t i o n ( L ) . P i g ments causing the brown c o l o r i n b i s c u i t s were present i n the water and s a l t - s o l u t i o n i n s o l u b l e f r a c t i o n ( I ) .


Color


B L O U I N ET A L .

Figure 5.

Biscuits containing

100%

wheat and 20%

plant-protein

products



Figure

6.

Biscuits

containing 100% wheat flour, 20% LCP cottonseed flour, 4.5% L 2.5% H fraction, 8.5% S fraction, and 4.5% I fraction (4)


fraction,

Journal of Food Science

H

•5 > r

H

z o

a

O H M

oo

2.

B L O U I N ET A L .


Role of

Color

29

Flavonoid8

The low-molecular-weight water-soluble f r a c t i o n of LCP f l o u r was found by t h i n l a y e r chromatographic methods to contain seve r a l f l a v o n o i d components. To e s t a b l i s h the r o l e of flavonoids in the production of yellow c o l o r i n b i s c u i t s , these components were extracted from LCP and glandless cottonseed f l o u r s with 85% aqueous i s o p r o p y l a l c o h o l (which i s a b e t t e r solvent f o r f l a v o noids than water). Before removal of the f l a v o n o i d s , the f l o u r s had been treated with petroleum ether to e x t r a c t r e s i d u a l l i p i d s that could i n t e r f e r e with f l a v o n o i d i s o l a t i o n . E x t r a c t i o n of the r e s i d u a l l i p i d s did not s i g n i f i c a n t l y a l t e r the c o l o r of b i s c u i t s prepared with the extracted f l o u r s (Figure 7). B i s c u i t s prepared with LCP and glandless cottonseed f l o u r s a f t e r e x t r a c t i o n with aqueous i s o p r o p y l a l c o h o l to remove flavonoids were brown; those prepared with the a l c o h o l i c e x t r a c t s ( i n quant i t i e s equivalent to that present i n the o r i g i n a l f l o u r ) were yellow (Figure 8). Thin layer chromatography (TLC) of the aqueous i s o p r o p y l a l c o h o l e x t r a c t s showed that i n a d d i t i o n to flavonoids considerable amounts of other components were i n the e x t r a c t s . Consequently, the a l c o h o l i c e x t r a c t s were next separated on a Sephadex G-15 column, with water as the eluent, i n t o nonflavonoid (95%) and f l a v o n o i d (5%) f r a c t i o n s . Figure 9 shows two-dimens i o n a l TLC p l a t e s of the o r i g i n a l e x t r a c t s and the nonflavonoid and f l a v o n o i d f r a c t i o n s of these e x t r a c t s from both LCP and glandless f l o u r s . The c h a r a c t e r i s t i c yellow flourescent spots observed under u l t r a v i o l e t l i g h t (long) i n d i c a t e the presence of at l e a s t s i x major flavonoids i n the o r i g i n a l e x t r a c t s and the f l a v o n o i d f r a c t i o n s . Because the concentrations used on the p l a t e s for the o r i g i n a l e x t r a c t s and for the nonflavonoid f r a c tions were the same, the absence of the yellow f l o u r e s c e n t spots f o r the nonflavonoid f r a c t i o n s i n d i c a t e s that the Sephadex column f r a c t i o n a t i o n was e f f e c t i v e i n separating the flavonoids from the nonflavonoid components. B i s c u i t s prepared with the nonflavonoid f r a c t i o n s of the a l c o h o l i c e x t r a c t s from LCP and glandless f l o u r s were tan and near white, r e s p e c t i v e l y (Figure 10). B i s c u i t s containing the f l a v o n o i d f r a c t i o n s were yellow. This very c l e a r l y e s t a b l i s h e s that the yellow c o l o r i n b i s c u i t s prepared with cottonseed f l o u r s i s caused by the flavonoids i n these f l o u r s . This f i n d i n g i s a l s o supported by the observation that b i s c u i t s containing the commercially a v a i l a b l e f l a v o n o i d , r u t i n , have the same yellow hue and s a t u r a t i o n as the b i s c u i t s containing the cottonseed f l a v o noids (Figure 11). Rutin i s a f l a v o n o l glycoside with q u e r c e t i n as the aglycone and with a disaccharide of glucose and rhamnose attached to the 3-hydroxyl p o s i t i o n of the aglycone. I t i s , i n f a c t , one of the components found in the cottonseed f l o u r s . Seven major flavonoids have been i s o l a t e d from LCP f l o u r and t e n t a t i v e l y i d e n t i f i e d (Table I ) .



PROTEIN FUNCTIONALITY I N FOODS

Journal of Food Science Figure 7. Biscuits containing 100% wheat flour, 20% LCP and glandless cottonseed flour, and 20% LCP and glandless cottonseed flour after extraction with petroleum ether (5)

Journal of Food Science Figure 8. Biscuits containing 18.0% LCP and 18.5% glandless cottonseed flours after extraction with 85% aqueous isopropyl alcohol and extracts isolated from these flours, 1.8% and 1.5%, respectively (5)



2.

BLOUIN E T AL.

31

Color

Journal of Food Science Figure 9. Two-dimensional cellulose TLC of flavonoids in aqueous isopropyl alcohol extracts, nonflavonoid fractions of extracts, and flavonoid fractions of extracts of LCP and glandless cottonseed flours (5)

Journal of Food Science Figure (0.1%

10. Biscuits containing the nonflavonoid (1.7% and 1.4%) and flavonoid and 0.1%) fractions of the aqueous isopropyl alcohol extracts of LCP and glandless cottonseed flours, respectively (5)



1.2.3 1.2.4 1,2

q u e r c e t i n 3 - 0 - g l u c o s i d e or i s o q u e r c e t r i n (3-D-glucoside)

kaempferoll/ 3-0-neohesperidoside (2-0-a-L-rhamnosyl-3-D-glucoside)

kaempferol

6

3

2/quercetin

O

H

1/ kaempferol

1/For number designations see F i g . 9 .

3-0-glucoglucoside

4/

1,2,3

quercetin 3-0-rutinoside or r u t i n (6-0-a-L-rhamnosyl-3-D-glucoside)

5A

4

1,2

quercetin 3-0-robinoside (6-0-a-L-rhamnosyl-3-D-galactosi de)

(1) U l t r a v i o l e t - v i s i b l e d i a g n o s t i c spectral a n a l y s i s . (2) H y d r o l y s i s and TLC o f aglycones and sugars. (3) Chromatographic m o b i l i t y w i t h standards. (4) NMR s p e c t r a .

1,2

5B

3-0-glucoglucoside

quercetin

2

1,2,4

CHARACTERIZATION METHOD!/

g u e r c e t i n l / 3-0-neohesperidoside (2-0-a-L-rhamnosyl-3-D-glucoside)

T e n t a t i v e I d e n t i f i c a t i o n o f Cottonseed Flavonoids TENTATIVE IDENTIFICATION

1

DESIGNATION!/

Table I .



2.

BLOUIN ET AL.

Color

33

The f l a v o n o i d mixture obtained by f r a c t i o n a t i o n on the Sephadex G-15 column was f u r t h e r separated on a Sephadex LH-20 column, with 50% methanol as the e l u e n t , into one f r a c t i o n cont a i n i n g f l a v o n o i d s 1, 3, and several minor f l a v o n o i d s , and i n t o a second f r a c t i o n c o n t a i n i n g f l a v o n o i d s 2 and 4 to 6 (see Figure 9 f o r number d e s i g n a t i o n s ) . These mixtures were then separated on t h i c k layer c e l l u l o s e p l a t e s into i n d i v i d u a l f l a v o n o i d s . They were rechromatographed on t h i c k layer plates u n t i l they appeared to be pure by two-dimensional TLC. As a f i n a l p u r i f i c a t i o n step, they were again chromatographed on a Sephadex LH-20 column. T h i s f i n a l step y i e l d e d two components, designated A and B, from f l a v o n o i d 5. D i a g n o s t i c u l t r a v i o l e t - v i s i b l e s p e c t r a l a n a l y s i s i n d i c a t e d that f l a v o n o i d s 1, 2, 5A, 5B, and 6 were 3-0-glycosides of q u e r c e t i n and that f l a v o n o i d s 3 and 4 were 3-0-glycosides of kaempferol. A c i d h y d r o l y s i s of the f l a v o n o i d s followed by TLC of the aglycones substantiated the above assignments. TLC of the hydrolyzed sugar components showed that f l a v o n o i d s 2, 4, and 6 contained only glucose, f l a v o n o i d s 1, 3, and 5A contained glucose and rhamnose and f l a v o n o i d 5B contained galactose and rhamnose. Chromatography with known f l a v o n o i d s , r u t i n , and i s o q u e r c e t r i n , i n d i c a t e d the i d e n t i t y of f l a v o n o i d s 5A and 6. The chemical s h i f t of the H-l rhamnosyl protons i n the NMR spectra of f l a v o noids 1 and 3 i n d i c a t e d the d i s a c c h a r i d e s i n these compounds are l i n k e d a-(l+2) and are thus neophesperidosides. F l a v o n o i d 5B because of i t s composition and chromatographic s i m i l a r i t y with r u t i n i s i d e n t i f i e d as a q u e r c e t i n r o b i n o s i d e c o n t a i n i n g a rhamnosyl g a l a c t o s i d e l i n k e d a-d+6). The linkage of the glucose d i s a c c h a r i d e u n i t s i n f l a v o n o i d s 3 and 4 has not yet been established. The above i d e n t i f i c a t i o n s agree with the work of P r a t t and Wender (12, 13) on cottonseed f l a v o n o i d s . The Role of Bound Gossypol Pigments Gossypol i s the major pigment i n glanded v a r i e t i e s of cottonseed. I t i s a binaphthyl terpenoid and i s t o x i c to monog a s t r i c animals (14). In the seed, t h i s pigment i s contained i n s t r u c t u r e s r e f e r r e d to as pigment glands. The l i q u i d cyclone process (1_) separates these glands from the p r o t e i n f l o u r . Rupture of some of the glands during processing frees the gossypol- allowing i t to react with the cottonseed p r o t e i n s . A t y p i c a l e d i b l e LCP f l o u r contains less than 0.045% free gossypol and 0.26% bound gossypol (15). One of the aldehyde groups of the gossypol i s b e l i e v e d to react with the primary amine of the l y s i n e u n i t s of the c o t t o n seed p r o t e i n s to form the S c h i f f base or imine type compound as shown at the top of Figure 12 (16, 17). An a n a l y t i c a l method f o r determination of bound gossypol, developed by Pons et a l . (18), involves treatment of the cottonseed f l o u r with 3-amino1-propanol and a c e t i c acid i n N, N-dimethylformamide (DMF)



34


Journal of Food Science Figure

11.

Biscuits containing flavonoid fractions of LCP and glandless seed flours and rutin (5)

.NHL

CH$ I NH,

CH ' (CH ) 2

4

N=CH OH H0HO-

cotton-

OH CHO OH OH

f

CH -CH CH -OH 2

I

R

N=CH OH

(CH ) 2

NH,

4

HO HO

2

OH CHO OH OH

Journal of Food Science Figure

12.

Reaction

of protein-bound gossypol with 3-amino-l-propanol presence of acetic acid in DMF solution (4)


in the


2.

BLOUIN ET AL.

Color

35

(Figure 12). Under these c o n d i t i o n s , the gossypol and g o s s y p o l - l i k e pigments become bound to the aminopropanol as the S c h i f f base of t h i s low-molecular-weight compound. The gossypolaminopropanol d e r i v a t i v e can then be washed from the f l o u r and measured spectrophotometrically. T h i s aminopropanol treatment for removal of bound gossypol appeared to be an e x c e l l e n t means to t e s t the r o l e of bound gossypol i n the c o l o r problem. The water and s a l t - s o l u t i o n i n s o l u b l e f r a c t i o n (I) of LCP f l o u r that contained the brown c o l o r causing pigments was treated for 15 min at 80°C with 2% 3-amino-l-propanol and 2% a c e t i c a c i d i n DMF s o l u t i o n . The aminopropanol e x t r a c t was f i l t e r e d o f f , and the f l o u r residue was washed and d r i e d . A b i s c u i t prepared with the treated f l o u r f r a c t i o n showed that most of the brown c o l o r was removed by the aminopropanol treatment (Figure 13). This r e s u l t suggests that bound gossypol pigments play a major r o l e i n the c o l o r problem i n LCP f l o u r . In order to t e s t t h i s idea f u r t h e r , the e x t r a c t of the aminopropanol treated f l o u r was f r a c t i o n a t e d on a Sephadex LH-20 column with DMF as the eluent. The e x t r a c t before f r a c t i o n a t i o n gave an u l t r a v i o l e t spectrum s i m i l a r to that of standard gossvDol treated with aminopropanol (Figure 14, curves B and A, respectively). Three colored f r a c t i o n s were obtained by column chromatography of the e x t r a c t . The spectrum of the second band e l u t e d (Figure 15, curve 2), was almost i d e n t i c a l to the spectrum obtained from the gossypol-aminopropanol d e r i v a t i v e (Figure 14, curve A). The f i r s t and l a s t bands eluted from the column exhibi t e d the spectra i n curves 1 and 3 of Figure 15, which are s i m i l a r to the gossypol-aminopropanol curve; however, they do d i f f e r considerably i n the 350-450 nm region. These data i n d i cate that bound gossypol and at l e a s t two bound g o s s y p o l - l i k e pigments are responsible f o r the brown c o l o r a t i o n i n b i s c u i t s prepared with LCP cottonseed f l o u r . Other Color Components Glandless cottonseed f l o u r a l s o caused yellow-brown c o l o r i n b i s c u i t s , and a s i m i l a r c o l o r d i s t r i b u t i o n was obtained when b i s c u i t s were prepared from f r a c t i o n s of glandless f l o u r (Figure 16). The i n t e n s i t y of the brown c o l o r was much l e s s i n the b i s c u i t prepared with the water and s a l t - s o l u t i o n i n s o l u b l e f r a c t i o n of glandless f l o u r than that prepared with the i n s o l u b l e f r a c t i o n of LCP f l o u r (Figure 6). The brown c o l o r i n t h i s f r a c t i o n of glandless f l o u r was not a f f e c t e d by the aminopropanol treatment, which removed brown c o l o r from the LCP f l o u r . A n a l y s i s of t h i s f l o u r i n d i c a t e d the absence of any detectable q u a n t i t i e s of f r e e or bound gossypol. The brown c o l o r i n glandless f l o u r i s believed to be due to phenolic compounds of another type. The brown pigment containing f r a c t i o n i s composed mainly of i n s o l u b l e




Figure 13. Biscuits containing the water and salt-solution insoluble LCP cottonseed flour before and after treatment with aminopropanol

300

400

500

fraction of (4.5%)

600

WAVELENGTH,nm

Journal of Food Science Figure 14. UV spectra of (A) gossypol-amino reaction mixture from aminopropanol treatment of water and salt-solution insoluble cottonseed flour (4)

and (B) extract fraction of LCP



BLOUIN ET A L .

Color

300

400 500 WAVELENGTH,nm

600

Journal of Food Science Figure 15. UV spectra of Fraction extract of the water and salt-solution

Figure

16. 6.0%

1, 2, and 3 separated from the aminopropanol insoluble fraction of LCP cottonseed flour (A)

Biscuits containing 100% wheat flour, 20% glandless cottonseed flour, L fraction, 1.5% H fraction, 8.0% S fraction, and 4.0% I fraction


38

protein functionality in foods

proteins and carbohydrates. If the remaining proteins are removed by treatment with sodium dodecyl sulfate and 2-mercaptoethanol, most of the brown color remains with the insoluble carbohydrate residue. These results suggest that the brown color causing components in glandless cottonseed flour are either bound to the polysaccharides (hemicellulose or cellulose) or are insoluble polymers such as tannins or lignins.


Conclusions When cottonseed flours and some other plant-protein products are used in food applications, there is a serious discoloration problem. Plant phenolic constituents are the major contributors to this problem. When cottonseed flours are used in food products, a yellow coloration is caused by cottonseed flavonoids. The seven major flavonoids in these flours were isolated and identified as 3-0-glycosides of kaempferol and quercetin. The marked brown discoloration observed when LCP glanded cottonseed flour is used in a food product is caused by bound gossypol and at least two bound gossypol-like pigments. The brown color observed when glandless cottonseed flour is used in food is believed to be due to other phenolic constituents that are either insoluble polymers or are bound to the insoluble plant polysaccharides. Color in food products ranks second in importance to taste in relation to consumer acceptability of a product. Discoloration problems caused by plant-protein products must be solved if these products are to be accepted. Isolation and identification of the pigments producing color is an important step in solving this problem and the methods developed in the studies presented in this chapter with cottonseed flours are applicable to color problems caused by other plant-protein products. Acknowledgment Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. Literature Cited 1. 2. 3. 4.

Gardner H. K., J r . ; Hron, R. J., Sr.; Vix, H. L. E. Cereal Chem., 1976, 53, 549. Kadan, R. S.; Ziegler, G. M., Jr.; Spadaro, J . J . Cereal Chem., 1978, 55, 919. Kim, M. K.; Calvin, B. M.; Lawhon, J . T. Cereal Sci. Today, 1971, 16, 216. Blouin, F. A.; Cherry, J . P. J . Food Sci., 1980, 45, 953.


2. 5. 6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

blouin et al.

Color

39

Blouin, F. A,; Zarins, Z. M.; Cherry, J . P. J . Food Sci., 1981, in press. Mabry, T. J.; Markham, K. R.; Thomas, M. B. "The Systematic Identification of Flavonoids"; Springer-Verlag: N.Y., 1970; p. 354. Judd, D. B.; Wyszecki, G., Eds. "Color in Business, Science, and Industry"; 2nd ed., Wiley and Sons, Inc.: N.Y., 1963; 500. Francis, F. J.; Clydesdale, F. M., Eds. "Food Colorimetry: Theory and Applications"; Avi Publ. Co., Inc.: Westport, Conn., 1975; p. 477. Sabir, M. A.; Sosulski, F. W.: Finlayson, A. J . J . Agric. Food Chem., 1974, 22, 575. Sodini, G.; Canella, M. J . Agric. Food Chem., 1977, 25, 822. Guilleux, F. Rev. Fr. Corps Gras, 1976, 23, 11. Pratt, C.; Wender . H. J . Am. Oil Chem. Soc., 1959, 36, 392. Pratt, C.; Wender, S. H. J . Am. Oil Chem. Soc., 1961, 38, 403. Berardi, L. C.; Goldblatt, L. A. Gossypol, In: "Toxic Constituents of Plant Foodstuffs"; Ed. I. E. Liener; Academic Press: N.Y., 1969; pp. 211-266. Gastrock, E. A.; D'Aquin, E. L., Eaves, P. H.; Cross, D. E. Cereal Sci. Today, 1969, 14, 8. Baliga, B. P.; Lyman, C. M. J . Am. Oil Chem. Soc., 1957, 34, 21. Conkerton, E. J.; Frampton, V. L. Arch. Biochem. Biophys., 1959, 81, 130. Pons, W. A., J r . ; Pittman, R. A.; Hoffpauir, C. L. J . Am. Oil Chem. Soc. 1958, 35, 93.

RECEIVED September 5, 1980.


Protein Functionality in Foods - American Chemical Society

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