Discoloration of Proteins by Binding with Phenolic Compounds - ACS

4 Discoloration of Proteins by Binding with Phenolic Compounds F. A. BLOUIN, Z. M. ZARINS, and J. P. CHERRY

1

United States Department of Agriculture, Southern Regional Research Center, Agricultural Research Service, New Orleans, LA 70179

Phenolic compounds that occur in plant materials have very diverse chemical structures. Some are simple benzene deriva t i v e s , l i k e the phenolic acids, p-hydroxybenzoic acid and caffeic acid (Figure 1). Others contain more complex ring systems such as the flavonoid quercetin and the terpenoid gossypol (Figure 1). Still others are extremely complex in structure. The vegetable tannin, procyanidin, i s thought to be an oligomer of flavan-3-ol units (Figure 1). Lignin i s a network type polymer composed of substituted cinnamyl alcohols (Figure 1). Phenolic compounds are found in a l l plant tissues. G r i f f i t h s (1), for example, found gentisic acid in the wood, bark, roots, stems, leaves, flowers, pod w a l l , cotyledons and testa of the cacao tree (Table I). Leucoanthocyanins and epicatechin were also present in most of the parts of this particular plant. The concentration of phenolics in plant materials can be quite high. Lignin represents 20-30% of the dry weight of the woody stems of trees. The dry matter of tea leaves is 40% polyphenols of the tannin type. Chlorogenic acid, one of the com monly occurring phenolic esters, is present in fruits at about a 0.1% level whereas in sunflower seed i t s concentration can be as high as 2-3%. In the natural state, the low molecular weight phenolic compounds are generally bound to carbohydrate moieties. For example, one of the commonly occurring flavonoids is rutin (Figure 2). It is a quercetin 3-0-rutinoside; a disaccharide of glucose and rhamnose is bound to the 3-hydroxyl of the C-ring of the quercetin. Even the polymeric polyphenol, 1ignin, i s covalently bound to the hemicellulose components of the cell walls. Phenols sometime occur in the free state but usually in storage tissues such as seeds, in dead tissues such as the heartwood of trees, or in diseased plant tissues. 1

Current address: United States Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118. This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

Figure 1.

p-Hydroxybenzoic acid Quercetin

OAr(Lig)

Gossypol

Structures of various types of phenolic compounds found in plant materials.

Caffeic acid

+

+ ++ +

+++ +

+++

+++

+

+

+

+

++

+

Cotyledon

Testa

+

+

++

+

+++

+++

++

++

+

+++

++

+

++++

+

++

+

+

++

+

+

Unhydrolysed extract Leuco(-)-Epianthocyanin catechin

+

,

+++

Pod wall

Flower

Old leaf

Young leaf

Green stem

Roots

++

Bark

Gentisic acid

++

p-Coumaric acid

Heart wood

Caffeic acid

++

Quercetin

Hydrolysed extract

Compound in

j

++++

++

++

Cyanidin glycoside

~

Distribution of Flavonoid Compounds in the Tissues of the Cacao Tree (Treobroma cacao) ( 1 ) ·

Sap wood

Plant part

Table I .

FOOD P R O T E I N

OH

OH Figure 2.

Structure of rutin.

DETERIORATION

4.

BLOWN ET AL.

Discoloration of Proteins

71

Discoloration When plant tissues are disrupted by grinding, heating or blending with other ingredients such as occurs in food processing, the phenolic components usually undergo enzymatic and nonenzymatic reactions that can cause undesirable discoloration in the processed food. Figure 3 i l l u s t r â t e s the effect of several types of plant protein products on the color of biscuits (2). Soybean and peanut flours used at the 20% replacement level do not cause a color problem. Sunflower, a l f a l f a leaf and cottonseed flours, on the other hand, cause a marked discoloration of the b i s c u i t s . In the case of the sunflower seed f l o u r , this discoloration is believed to be due to chlorogenic acid. The coloration caused by the a l f a l f a leaf protein product has been attributed mainly to chlorophyll pigments (3) but polyphenol s are also thought to contribute to the discoloration (4J. Work at the Southern Regional Research Center (SRRC) has shown that flavonoids, gossypol and possibly other phenolic compounds contribute to the color observed when cottonseed flours are used in foods (2). Interactions There are two major types of interactions between proteins and phenolic compounds (5). F i r s t , phenols combine reversibly with proteins by hydrogen bonding. Second, they interact i r r e v e r s i b l y by oxidation and condensation-type reactions. Infrared spectral data indicate that hydrogen bonding between phenols and N-substituted amides is one of the strongest types of H-bonds {§_). Research on col lagen, vegetable tannins and synthetic polymers (j>) has established the importance of the peptide linkage in the formation of Η-bonded complexes between tannins and proteins. The interaction is probably between the peptide oxygen and the hydrogen of the phenol. Condensed tannins are bound to proteins almost independent of pH below pH 7.0 to 8.0. This bonding is thought to involve only un-ionized phenolic hydroxyl groups. Hydrolyzable tannins, on the other hand, are strongly bound to proteins at pH 3.0 to 4.0 but the binding decreases above pH 5.0. In this case, the stronger Η-bonding has been attributed to the un-ionized carboxyl groups and the weaker Η-bonding to the un-ionized hydroxyl groups of the tannins. Oxidation of phenols is the f i r s t step in the irreversible type protein-phenol interaction. This oxidation can be enzymatic (as i l l u s t r a t e d in Figure 4) or nonenzymatic. The primary reac tion products are qui nones which are themselves very reactive compounds. These qui nones can then react directly with protein groups or can f i r s t undergo polymerization and/or oxidation-reduc tion reactions to produce secondary reaction products which can form covalent bonds with the proteins. The work of Mason and Peterson (7j has shown that sulfhydryl and N-terminal ami no groups are the most reactive. The epsilon-amino group of lysine also

FOOD PROTEIN

DETERIORATION

ure 3. Biscuits containing 100% wheat and 20% plant-protein products. (Reproduced from Ref. 2. Copyright 1981, American Chemical Society.)

Figure 4.

SULFHYDRYL. C-AMINO

N - T E R M I N A L AMINO, β

REACTIONS

POLYMERIZATION

POLYPHENOLOXIDASE

• [o]

BONDING

OH

OH

TO PROTEIN GROUPS.

COVALENT

I

Reaction sequences involved in covalent bonding of phenols to proteins.

REACTIONS

REDUCTION

OXIDATION-

0-8ENZ0QUIN0NE

CATECHOL

74

FOOD PROTEIN DETERIORATION

reacts with qui nones but more slowly. These qui none-type reactions are most commonly cited as causing discoloration problems in plant food products. Cottonseed Flours One of our interests at SRRC is the use of high protein cottonseed flours as edible food products. Glanded cottonseeds contain high concentrations of the phenolic compound gossypol, enclosed in pigment glands. This compound i s toxic to humans but an edible flour can be made by removal of the glands in a process developed at SRRC called the Liquid Cyclone Process (8). Glandless cottonseeds do not contain gossypol and an edible flour is prepared from this material by simple hexane extraction methods. Biscuits prepared using 20% cottonseed flours as a replacement for wheat flour are yellow-brown in color (Figure 3 ) . The biscuit prepared with glanded flour is much darker in color than that containing glandless f l o u r . Our research work was to isolate and identify the pigments responsible for this color problem. Experimental. Cottonseed flour was prepared from glanded seeds at SRRC by the Liquid Cyclone Process of Gardner, et a l . (8). The glanded flour had a proximate composition of 57.3% protein, 32.1% carbohydrate, 1.6% l i p i d , 7.7% ash and 2.3% crude f i b e r . The glandless cottonseed flour was obtained from the Plains Cooperative Oil M i l l , Lubbock, Texas and had a proximate compositon of 56.6% protein, 29.9% carbohydrate, 3.7% l i p i d , 7.5% ash and 2.3% crude f i b e r . Analysis of glanded flour indicated 0.04% free gossypol and 0.15% total gossypol. Free and total gossypol analyses of glandless flour indicated that i t s gossypol content was below the detectable level s of the methods. Extraction with petroleum ether, chloroform, 85% aqueous isopropyl alcohol, water and 10% sodium chloride were conducted at room temperature with s t i r r i n g at a solid to solvent ratio of 1:10. Extraction times were 1 hr for the water and salt solution systems and 24 hr for the other solvents. Solids were separated from extracts either by f i l t r a t i o n or centrifugation and residues were washed with a volume of sol vent equal to the original vol ume used. Insoluble residues were dried in a i r (to remove petroleum ether), in vacuum oven (chloroform) or by freeze drying after removal of alcohol by rotary evaporation or salt by d i a l y s i s . Solvents were removed from the extracts by rotary evaporation and/or freeze drying and salt by d i a l y s i s . Pepsin digestions were conducted on the salt insoluble residues at 37°C for 24 hr at pH 2 using 1:100 ratio of enzyme to substrate. Cellulase treatments were at 45°C for 24 hr at pH 4.5 with an enzyme ratio of 1:50. Digestions with amylase were run at 75°C for 4 hr at pH 6.5 with an enzyme ratio of 1:25.

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Discoloration

of

Proteins

75

Insoluble residues were separated from digests by centrifugation, washed with water, neutralized and freeze dried. Digests were also neutralized and freeze dried. Insoluble amylase residues were treated with di methylsulfoxide (DMSO) with s t i r r i n g for 1 hr at 160°C. The insoluble residue was collected on a f i l t e r , washed with 50% methanol , dispersed in water, neutralized and freeze dried. The DMSO extract was poured into a large volume of 50% methanol and stirred for about 1 hr. The precipitated sol ids were separated from the solution by centrifugation, washed with 50% methanol, dispersed in water, neutralized and freeze dried (fraction A ) . The non-precipitable fraction was isolated by rotary evaporation of the methanol-water-DMSO solution (fraction B). Wheat flour biscuits were prepared with 20.0 g wheat or composite f l o u r , 1.0 g baking powder, 0.5 g s a l t , 4.5 g shortening, and 15.0 g f l u i d milk. Biscuits were prepared from plant-protein flours based on 20% replacement for wheat f l o u r . In biscuits prepared with fractions isolated from cottonseed f l o u r s , the quantities used were calculated using the percentages these fractions represented of the original f l o u r , e.g. the salt solution soluble fraction of glanded flour was 35.7% of the original flour and 7.1 % (20 χ .357) replacement for wheat was used to prepare the b i s c u i t . Other methods and procedures are f u l l y described by Blouin et a l . (9,10). Sol vent Extraction Steps. Glandless and glanded cottonseed flours were subjected to a series of five mild extraction steps. Biscuits were prepared with the extracted flours and in most cases with the extracts (Figure 5). The quantity of cottonseed material used in preparation of a biscuit was proportional to the amount that the fraction represented in the original f l o u r . These percentages of the orginal flour are shown in Figure 5 in parentheses. In this way, the preparation of biscuits was used as a guide to determine i f the pigments responsible for the color had been modified or lost and to establish in which fractions they occurred. Figure 5 i l l u s t r a t e s that extraction of glandless flour with petroleum ether and chloroform does not alter the color of b i s c u i t s . After extraction with aqueous alcohol , the yel1ow color is present in the biscuit containing the extract and the brown color is present in the biscuit containing the insoluble flour residue. We were able to establish that the yel1ow is caused by flavonoids present in the flours ( £ ) . Seven flavonol compounds were isolated and identified as glycosides of quercetin and kempf e r o l . No strong interactions between these phenolic components and the cottonseed proteins were evident since they were readily removed by simple room temperature extraction. The pigments responsible for the brown color, however, appear to be strongly bound to the more insoluble flour components. They remain in the


Figure 5. Biscuits containing glandless cottonseed flour fractions isolated in five solvent extraction steps. Values in parentheses represent the amount of cottonseed material used in preparation of the biscuit.

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77

insoluble flour fractions after extractions with water and 10% sodium chloride solution. At this point in the isolation proce dure, the brown color causing pigments are contained in a fraction which represents 29% of the orginal f l o u r . Figure 6 shows biscuits prepared from glanded flour frac tions obtained by the same five extraction steps. The yellow color due to the flavonoids is in the biscuit containing the aqueous alcohol extract and the brown coloration remains in the insoluble fraction through the salt solution extraction step. Our i n i t i a l assumption about the more intense brown color in glanded flour was that i t was caused by gossypol bound to the cottonseed proteins. The original flour contained 0.1% bound gossypol measured by an analytical method developed by W. Pons and co-workers at SRRC (1JJ. This analytical method assumes gossypol is bound as a Schiff base to the epsilon-amino groups of the lysine in proteins (Figure 7). Although this bonding is not of the type previously described for phenol-quinone-proteiη systems, i t is a type quite common in biological systems (12). In this analytical method, the cottonseed flour is treated with 3-amino-l-propanol and acetic acid in dimethylformamide (DMF) s o l u t i o n . Under these conditions, i t is assumed that the gossypol becomes bound to the aminopropanol as the Schiff base of this low molecular weight com pound. This gossypol-aminopropanol derivative can then be washed from the flour and the gossypol content measured spectrophoto metrically. This reaction appeared to be a good means to test the role of bound gossypol in the color problem. The flour residues after extraction with salt solution were treated with 2% aminopropanol and 10% acetic acid in DMF solution for 15 min at 90°C. The treated fractions were washed, dried and used in biscuits (Figure 8 ) . Most of the brown color was removed from the glanded flour fraction by this treatment. The color of the biscuit prepared with the glandless flour fraction was not altered by this aminopropanol treatment. These results suggest that bound gossypol is responsible for the intense brown color observed in the glanded cottonseed flour-containing b i s c u i t . Enzyme Digestion Steps. To further concentrate and isolate the brown pigments in glandless and glanded flours, the salt solution insoluble residues were subjected to a series of four sequential enzyme treatments. Biscuits prepared from the soluble and insoluble fractions of glandless flour are shown in Figure 9. Pepsin digestion solubilized about 50% of the salt solution insoluble residue. Treatment of the pepsin residue with a eel 1ulase enzyme, which also contained hemicellulase a c t i v i t y , removed an additional 50-60% from the insoluble f r a c t i o n . This was followed by a second pepsin digestion which removed 20-30% from the eellulase residue. Treatment with amylase solubilized an additional 20-25%. At each step of these treatments, biscuit preparation showed that the brown color-causing pigments remained


78

Figure 6.

Biscuits containing glanded cottonseed flour fractions isolated in five solvent extraction steps.

BLOUIN ET AL.

Discoloration

of

Proteins

CHJT

CH -CH -OH 2

2

NH*

OH CHO

CH C0 H/DMF 5

2

CH-CH£-CH£-OH OH CHO

Figure 7. Reaction of protein-bound gossypol with 3-amino-l-propanol in the presence of acetic acid in DMF solution. (Reproduced, with permission, from Ref. 10. Copyright 1980, Institute of Food Technologists.)


Figure 8. Biscuits containing the salt solution insoluble fractions of glanded (top) and glandless (bottom) cottonseed flour before and after treatment with aminopropanol.

4.

BLOUiN ET AL.

Figure 9.

Discoloration

of

Proteins

Biscuits containing glandless cottonseed flour fractions isolated in four enzyme digestion steps.

81

82


in the insoluble fractions. The amylase residue represented 2% of the original flour. Glanded cottonseed flour responded in an identical manner when subjected to the same four enzyme treat ments. Biscuits prepared with these fractions are shown in Figure 10. The last insoluble fraction in this case was 2.6% of the original f l o u r . Pimethyl sulfoxide Treatment Step. The last step in the iso lation procedure was to treat the amylase residues from glanded and glandless flours with DMSO at 160°C for 1 h r . This treatment solubilized 70 to 75% of the residues. For both f l o u r s , the DMSO solution was f i l t e r e d off to isolate the insoluble f r a c t i o n . A second fraction was isolated by precipitation on mixing of the DMSO solution with a large volume of 50% methanol (soluble frac tion A ) . A third fraction was obtained by removal of the solvents on a rotary evaporator (soluble fraction B). Biscuits prepared from these DMSO treated fractions, i l l u s trated in Figure 11, showed some coloration i n a l l six b i s c u i t s . However, a marked difference in color d i s t r i b u t i o n is now evident between the two types of f l o u r . For the glanded f l o u r , the intense brown color is clearly present in the DMSO soluble frac tion A. With the glandless f l o u r , the major proportion of the brown pigments is in the insoluble f r a c t i o n . Also, a brown color of- similar hue and intensity is observed in the biscuit containing the insoluble fraction of glanded cottonseed f l o u r . This indicated that the pigments responsible for the brown color in glandless flour are also present in glanded f l o u r . Chemical analyses indicated that these insoluble fractions are about 40% carbohydrate (presumably polysaccharide), 30% protein and 5% f a t . As yet, we have no experimental evidence that the brown color in these insoluble fractions is caused by phenolic compounds. Because of the extremely insoluble nature of these residues, how ever, we suspect that lignins or condensed tannins are present. The DMSO soluble fraction A of glanded flour is about 70% protein. It also contains about 7% carbohydrates and 8% f a t . The fact that this f r a c t i o n , which contains the intense brown color characteristic of glanded flour, is mainly protein, is in agreement with the assumption that gossypol bound to proteins is responsible for this discoloration. The DMSO soluble fraction A of glandless flour is about 50% protein. This lower proportion of protein i s due to a higher fat content (18%). This high 1i pi d content and the amino acid pro f i l e data suggests that the DMSO soluble A fractions are mainly membrane type lipoproteins (13). The DMSO soluble Β fractions appear to be mixtures of pro t e i n s , 35 to 50%, and carbohydrates, 14 to 19%. They cause the least amount of brown color in b i s c u i t s . The infrared spectra of these six fractions, shown in Figure 12, mainly confirm the chemical data previously described. The spectra of the DMSO insoluble residues are identical for both

4.

BLOUIN ET AL.

Figure 10.

Discoloration

of

Proteins

Biscuits containing glanded cottonseed flour fractions isolated in four enzyme digestion steps.

83

84


Figure 11. Biscuits containing glanded and glandless flour fractions isolated in DMSO treatment step. Precipitable DMSO soluble fraction is Fraction A and nonprecipitable DMSO soluble fraction is Fraction Β in text.

4.

BLOWN ET AL.

Discoloration

4000 30002500 ι ι t

85

of Proteins

2 0 0 0 160014001200 ι i i ι

1000900 ι i

800 ι

700 cm" ι

1

DMSO INSOLUBLE FRACTION

DMSO SOLUBLE FRACTION A

DMSO S O L U B L E FRACTION Β

ι 1 1 1 ι microns 2.5 3.0 3.5 4.0 4.5 Figure 12.

1 5

1 6

1 7

1 8

ι 9

1 10

1 II

1 12

1 13

1 14

1 — r 15 16

IR spectra of DMSO treated fractions of glanded (A) and glandless (B) cottonseed flours.

86


f l o u r s . The -OH, -CH and -C-0- absorption regions indicate a pre dominance of carbohydrate components. For the DMSO soluble A fractions, the -NH and amide I and II absorption regions indicate a predominance of protein components. The -CH absorption region confirms the presence of greater amounts of l i p i d s in the gland less f l o u r . There i s , however, no indication of the presence of the intense brown color causing components in the spectrum of the DMSO soluble fraction A of the glanded f l o u r . The spectra of the DMSO soluble Β fractions again confirm the presence of protein and carbohydrate mixtures in these fractions. There are some d i f ferences between these two spectra which are unexplainable at this time. The u l t r a v i o l e t - v i s i b l e spectra of the DMSO soluble fractions, shown in Figure 13, do indicate significant differences between the two types of flours. The concentrations used were 0.2 mg/ml for the four flour fractions and 0.02 mg/ml for the gossypol spectra. The glanded flour fractions exhibited s i g n i f i cantly higher UV-visible absorption than the glandless flour fractions. Even more significant, both of the glanded fractions exhibited absorption in the 375-400 nm region similar to the 379 nm maximum exhibited by free gossypol. These data again suggest that gossypol bound to protein is the cause of the intense brown color in glanded flour-containing b i s c u i t s . Sunflower Seed Flour Chlorogenic acid (Figure 14) i s the phenolic compound, which according to several research groups (14-17), is responsible for the discoloration problem observed when sunflower proteins are used in food systems. The concentration of phenolic compounds in sunflower seed flour is 3.0 to 3.5% (18). Chlorogenic acid and caffeic acid constitute about 70% of these phenolics (18). As an interesting comparison with cottonseed flour studies, the research of M. A. Sabir and co-workers (14,15) on the chlorogenic acidprotein interactions in sunflower is reviewed below. Sabir et a l . (14) extracted proteins from sunflower seed flours with 2.5% sodium chloride at pH 7. They fractionated the salt-extractable proteins on a Sephadex G-200 column and isolated five protein fractions (Figure 15). When the undialyzed extracts (Figure 15a) were used, peak V showed far greater absorption at 280 nm than the high molecular weight fractions. Peak V also exhibited a UV-visible maximum at 328 nm which was not observed in the spectra of the other fractions. If the salt extractable proteins were dialyzed before fractionation, the elution curve shown in Figure 15b was obtained. Peak V exhibited, in this case, an absorption at 280 nm more in proportion to the other peaks. This fraction s t i l l exhibited a second absorption maximum at 328 nm. Chlorogenic acid gives a UV-visible maximum at 328 nm and other phenolic acids and esters absorb between 310 and 335 nm. Presumably, phenolic components were present in fraction V, some


ε c

LU

U

ι

i

m

ELUTION

m

VOLUME

Figure 15. Fractionation of undialyzed (top) and dialyzed (bottom) salt-extractable sunflower proteins on a Sephadex G-200 column with neutral salt solution. (Repro duced from Ref. 14. Copyright 1973, American Chemical Society.)

4.

BLOUIN ET AL.


89

of which were removed by d i a l y s i s . The phenolics that remain in fraction V after dialysis are strongly bound to this protein fraction. Sabir and coworkers (15) refractionated fraction V on a Sephadex G-200 column with neutral salt solution and 7 M urea. They reasoned that a strong hydrogen-bonding medium such as 7 M urea would dissociate any phenolics that were hydrogen bonded to the proteins but would not influence any phenolic compounds covalently bound. As shown in Figure 16, they isolated two subfractions, VI and V2. Fraction VI exhibited a maximum at 280 nm characteristic of proteins but no absorption in the 328 nm region (Figure 17). This subfraction represented 68% of the proteins in fraction V. Fraction V2, on the other hand, showed absorption maxima at 280 and 328 nm. Since 7 M urea did not dissociate these bonds, these researchers concluded that chlorogenic acid was covalently bonded to this low molecular weight protein fraction. This fraction represented 4% of the original s a l t extractable proteins. Conclusions Current research on sunflower and cottonseed flours indicates that phenolic compounds contribute to discoloration problems when these plant protein products are used in food systems. In the case of cottonseed flours, flavonoids cause a yel1ow coloration but these phenolics do not interact to any significant extent with the proteins. The phenolic compound gossypol in glanded cottonseed flour is believed to be covalently bonded to membrane type lipoproteins and cause an intense brown color in food products containing this f l o u r . Both glanded and glandless cottonseed flours contain pigments causing 1ight brown color in foods. These pigments are bound to or are part of the most insoluble flour components. Lignin or condensed tannins may be the moieties responsible for this light brown c o l o r . Sunflower seed protein products also cause marked discoloration in food products. Experimental evidence obtained by Sabir et a l . (14, 15) indicates that strong hydrogen bonding and covalent bonding of chlorogenic acid to low molecular weight sunflower proteins occurs and that these interactions are probably responsible for the discoloration problem. If plant protein products are to be used in food systems, discoloration problems must be solved. A knowledge of the nature of the bonding between the color-causing components and the proteins should lead to development of better methods for prevention and elimi nation of these problems.

90


Vt f ι V™ ι 1— Ο 120 140 160 180 2 0 0 2 2 0 ELUTION V O L U M E ( m l ) f

l

Figure 16. Refractionation of salt-extractable sunflower protein Fraction V on a Sephadex G-200 column with neutral salt solution and 7 M urea. Key: O-O, 280 nm; and Δ - Δ , 328 nm. (Reproduced from Ref. 15. Copyright 1974, Ameri can Chemical Society.)

1.0] • ! 0.8

U

yj

ο

ζ 0.6 < m oc ο £ 0.4 CD

1

< 0.2h

; 0.0

340

300 260 220

WAVE LENGTH(nm) Figure 17. UV spectra of Subfractions F , and V of salt-extractable sunflower proteins. (Reproduced from Réf. 15. Copyright 1974, American Chemical Society.) t

4.

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91

Acknowledgement Names of companies or commercial products are given solely for the purpose of providing specific information; t h e i r mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

G r i f f i n s , L . Biochem. J., 1958, 70, 120. Blouin, F. A.; Zarins, Ζ. M . ; Cherry, J. P. Color, In: "Protein Functionality in Foods"; Cherry, J. P . , E d . ; Amer. Chem. Soc. Symposium Series 147: Washington, D. C . , 1981; p. 21. Kohler, G. O.; Knuckles, Β. E. Food Technol., 1977, 31(5), 191. Lahiry, N. L.; Satterlee, L. D . ; Hsu, H. M . ; Wallace, G. W. J. Food Sci., 1977, 42, 83. Loomis, W. D.; B a t t a i l e , J. Phytochem., 1966, 5, 423. F l e t t , M. St. C. J. Soc. Dyers Colourists, 1957, 68, 59. Mason, H. S.; Peterson, E. W. Biochim. Biophys. Acta, 1965, 111, 134. Gardner, H. K . , Jr.; Hron, R. J., S r . ; V i x , H. L . E. Cereal Chem., 1976, 53, 549. Blouin, F. A . ; Zarins, Ζ. M . ; Cherry, J. P. J . Food Sci., 1981, 46, 266. Blouin, F. Α.; Cherry, J. P. J. Food Sci., 1980, 45, 953. Pons, W. A., Jr.; Pittman, R. A.; Hoffpauir, C. L . J. Am. Oil Chem. Soc., 1958, 35, 93. Singleton, V. L . Common Plant Phenols Other Than Anthocyanins, Contributions to Coloration and Discoloration, In: "The Chemistry of Plant Pigments"; Chichester, C. A . , Ed; Academic Press: Ν. Υ., 1972; p . 162. Gurr, M. I., James, A. T. "Lipid Biochemistry: An Introduction"; Cornell Univ. Press: Ithaca, Ν. Y., 1972; p. 192. Sabir, M. A.; Sosulski, F. W.; MacKenzie. S. L . J. Agr. Food Chem., 1973, 21, 988. Sabir, M. A.; Sosulski, F. W.; Finlayson, A. J. J. Agr. Food Chem., 1974, 22, 575. Sodini, G . ; Canella, M. J. Agric Food Chem., 1977, 25, 822. Cheftel, C . ; Cuq, J. L.; Pronansal, M . ; Besancon, P. Rev. Francaise des corps Gras, 1976, 23, 9. Sabir, Μ. Α.; Sosulski, F. W.; Kernan, J. A. J. Agr. Food Chem., 1974, 22, 572.

RECEIVED July

16, 1982.

Discoloration of Proteins by Binding with Phenolic Compounds - ACS

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