TEXAS AGRICULTURAL EXPEHIMENT STATION H. D. LEWIS. Director. College Station, Texas 1141a» 748 Nature, Causes and Correction of Discoloration of Canned Blackeye and Purple Hull Peas ne 195.2 The TEXAS AGRICULTURAL AND MECHANICAL COLLEGE SYSTEM GIBB GILCHRIST, Chancellor [Blank Page in Original Bulletin] DIGEST Properly canned blackeye or purple hull peas are a fav- orite food with many people in the Southern States. How- ever, due to a brown or black liquor discoloration, the canned produ-ct has met with some sales resistance. Most of the discoloration is caused by the anthocyanin pigment of the seed coat. The discoloration can be controlled by the use of 3 per- cent ammonium alum or aluminum sulfate as a blanching solution, or by the use of a high-temperature and short-time sterilization process, or by combining these two methods. Unless pressurized cooling facilities are available, the use of a high-temperature and short-time process is not recommended. a A desirable side-effect derived f.rom the use of aluminum salts in the blanch solution is the reduction of bo-th the vis- cosity of the liquor and gelling in the can. Though ammonium alum is used extensively in the manu- facture of pickles from cucumbers and also is used as an aid in purifying drinking‘ water, any one anticipating the use of ammonium alum or aluminum sulfate to control discoloration should first clear the process with the U. S. Food an-d Drug Administration. CONTENTS Digest ' , a Introduction Review of Literature Procedure, Results and Discussion .. , Relation of Globulin t0 Discoloration Association of Anthocyanin with Discoloration Acknowledgments >-7 . . Bibliography s 1 s s , 1 1 s s s s . , , . . s . . s . . . , _ , s . _ . . , _. 22 7 ............ _.23 BULLETIN 748 JUNE 1952 Nature, Causesand Correction of Discoloration of Canned Blac/(eye and Purple Hull Peas W. H. Culver and R. F. Cain* BLACKEYE PEAS can be grown in nearly all parts of Texas. The area generally spoken of as “East Texas” is especially well adapted to the growing of this crop and a number of canneries are located in that part of the State. Large acre- ages of blackeyes are grown and packed in the Lower Rio Grande Valley. Some peas also are produced in the vicinity of Lubbock. The blackeye pea canning industry is centered mainly in Texas. Seventy-nine percent of all blackeyes canned in the United States in 1949 were packed in this State. Approxi- mately 1,500,000 cases of blackeye and purple hull peas were packed in Texas during 1948. (10,11) . In spite of the desirable characteristics of blackeye and purple hull peas, the canning of these products has not ex- panded as much as normally would be expected. The three main reasons for this are: uneven maturation of the pods, which makes mechanical harvesting impractical; the tendency of canned peas to have a viscous liquor that sometimes forms a gel; and the very dark, undesirable appearance of most can- ned blackeye and purple hull peas. This bulletin is concerned with the last named characteristic of the peas. Brown or black discoloration of peas is undesirable from several standpoints. Of primary concern is the fact that the consumer not familiar with Southern peas (edible varieties of the cowpea, Vigna sinensis), is likely to consider the badly discolored product as spoiled and discard it. Home economists long have recognized the fact that eye appeal is very import- ant. It is sometimes the factor which determines whether the product is accepted or rejected. Discolored peas certainly lack eye appeal. The darkening of the canned peas masks the green color of the fresh green-shelled product. On the basis *Formerly research assistant and associate professor, Department of Horticulture. 6 BULLETIN 748, TEXAS AGRICULTURAL EXPERIMENT STATION of color alone, canned green peas frequently are indistinguish- able from canned dry peas. Presence of discolored peas in the can is also undesirable - from the standpoint of U. S. grade. Typical examples of commercially canned peas have a very viscous dark liquor and the peas themselves may be dis- colored so badly that the discoloration extends through the cotyledon. REVIEW OF LITERATURE Only brief mention has been made in the literature of discoloration in canned Southern peas. Sherman (24) ob- served that the severity of darkening was proportional to the amount of seed-coat coloration. Cain and Brittingham (4) reported that blackeye and purple hull peas canned in plain tin were subject to more severe discoloration than were those canned in “C” or “R” enamel cans. The C enamel was slightly more desirable. Cream peas were not influenced as greatly by the container coating. The darkening of pigmented seedcoats, which can be decreased through the use of the proper container, is secondary to the more severe darkening of the product. The literature revealed that morphology and pigmentation of the seedcoat of Southern peas are associated with the dis- coloration of the canned product. Therefore, a portion of this review is devoted to the work of Albert Mann on the seedcoat of cowpeas. Mann (17) published a treatise on the morphology and pigmentation of the seedcoat of cowpeas. The seedcoat is composed of three distinct layers of cells, the outer palisade layer, the middle “hour glass” layer and the inner “basal- color” layer. He stated that there are two types of pigment in the seedcoat of cowpeas. They are anthocyanins and a melanin-like compound. The anthocyanin is responsible for the dark colors in the seedcoat. It occurs only in the palisade layer and sometimes only in some of the cells of that layer. In blackeye peas, it occurs only in the cells immediately around the hilum. The melanin-like substance occurs mostly in the basal-color layer but sometimes is found in the palisade cells. The anthocyanin in cowpeas consists of two phases or types. One is an acid-reacting anthocyanin, ranging in color DISCOLORATION OF CANNED PEAS 7 from a decided rose red to a strong purple. The other, an alkaline-reacting anthocyanin, is uniformly a deep indigo blue but in mass it often appears dead black. Gortner (5) found that the melanin in sheep’s wool is in- soluble in neutral or acidic solutions, but that it is soluble in alkalies; therefore, it is concluded that, at the pH normally "encountered in canned blackeye and purple hull peas, the melanin-like substance probably is insoluble and is not an im- portant factor in discoloration. Many excellent reviews have been published on the prop- erties and chemistry of anthocyanins (13, 14, 15, 21, 22, 23, 25). A complete discussion of properties and nature of these plant pigments is beyond the scope of this report. However, it is pertinent to point out that anthocyanins are the gluco- sides of anthocyanidins, according to Bancroft and Ratzler (1). They found that anthocyanins can be prepared by the reduc- tion of the corresponding flavinols or the oxidation of leuco- anthocyanins. In mltro this can be accomplished only with strong reagents, although these reactions probably proceed in vivo with the assistance of enzymes. Leuco-anthocyanins are almost always found in plant tissues where anthocyanins are present (16). PROCEDURE, RESULTS AND DISCUSSION Griswold (9) observed that neutral lead acetate precipi- tated the anthocyanin pigment in Montmorency cherries. Therefore, it was deemed advisable to ascertain the effect of this salt on the appearance of dry blackeye peas which were soaked, then canned. Two samples were soaked 8 hours at room temperature, one in water and the other in 2 percent neutral lead acetate solution. The soaking solutions were discarded, then the peas were packed in cans and covered with hot water. The cans were heated for 35 minutes at 240°F. When opened for examination, the peas treated with lead acetate Were free from discoloration. The liquor was slightly turbid but it was not discolored. The eyes of the peas were black and well defined. The peas soaked in water were normal- ly discolored. 8 BULLETIN 748, TEXAS AGRICULTURAL EXPERIMENT STATION Relation 0f Globulin to Discoloration Since lead acetate precipitated both water soluble protein (globulin) and anthocyanin, the relation of the globulin to dis- coloration was determined. About 4.5 pounds of dry peas were ground in a hand- operated food chopper. The resulting meal was treated twice with petroleum ether to extract the fat. After the meal was dried at room temperature, six 300-gram samples were weigh- ed and extracted with various solutions. These solutions were distilled water, 0.5 N sodium chloride, 1.0 N sodium chloride, 2.0 N sodium chloride, distilled water extraction followed by the addition of enough sodium chloride to the filtered extract to make it approximately 1.0 N, and normal sodium chloride extraction followed by precipitation -of the protein by the ad- dition of ammonium sulfate. The precipitate was separated and the liquid of the last solution was treated as a sample. Different concentrations of sodium chloride were used because the solubility of globulins varies with the concentra- tion of neutral chlorides of monovalent metals, according to Gortner (7). The samples of meal were placed in large flasks and cov- ered with 1.5 liters of the extraction solution. They were shaken thoroughly several times during the 2-hour period of extraction. After separation by centrifuging followed by fil- tering, the solid material was discarded. Several test tubes were filled with portions of each ex- tract, plugged with cotton and autoclaved at 248°F. for 20 minutes. Portions of the extracts of samples 1, 2, 3, 4 and 5 were analyzed for nitrogen content to determine the amounts of protein extracted. Sample 6 was not analyzed because of the addition of ammonium sulfate. Sample 3 was accidentally destroyed. There was a noticeable difference in the behavior and ap- pearance of the samples. In every case, where salt was pres- ent, coagulation of the protein occurred during the autoclav- ing process. The precipitates of all samples extracted with salt solutions were lighter in color, even several weeks after autoclaving, than was the sample extracted with water to which salt had been added later (sample 5). The extract that contained no salt did not coagulate (sample 1). The super- natant liquids in all the samples that precipitated were uni- form, light straw-yellow and clear. The protein precipitated DISCOLORATION OF CANNED PEAS 9 from sample 6 darkened within a few hours after autoclaving. However, the darkening in all cases was slight and was not comparable with that of darkened canned peas. The protein contents of the extracts are shown in Table 1. Apparently, the substances extracted by water and dilute salt solutions are not the ones directly responsible for the dis- coloration of canned blackeye and purple hull peas. Further- more, the protein normally extracted "by these solvents is co- agulated by heating in the presence of salt. This is not direct- ly related to discoloration, but, as will be shown later, is sig- nificant. The quantity of globulin extracted varied with the concentration -of sodium chloride, in accordance with the find- ing of Gortner (7). Association of Anthocyanin with Discoloration Results of the preceding experiments indicated that an- th-ocyanin is the principal component of peas associated with the discoloration of the canned product. To obtain more concrete evidence of this, the following experiment was con- ducted. F-our samples were prepared in the following manner: One hundred grams of dry blackeye peas were ground and placed in a flask. Another 100-gram sample of dry blackeye peas, from which the eyes had been removed, was ground and placed in a flask. A 100-gram portion of ground dry cream peas was placed in a flask along with the eyes from the blackeyes in sample 2. One hundred grams of ground dry cream peas were placed in a fourth flask. Table 1. Protein content of extracts of blackeye peas Sample Solvent Percent protein (N X 6.25) 1 Water 12.79 2 0.5 N sodium chloride 17.90 4 2.0 N sodium chloride 14.61 5 Waterl 10.41 1The slight difference between samples 1 and 5 is probably due, in part, to the dilution of the extract of sample 5 with salt. 10 BULLETIN 748, TEXAS AGRICULTURAL EXPERIMENT STATION One hundred and fifty cubic centimeters of distilled water were added to each flask. The flasks were plugged with cotton and then were heated in the autoclave at 15 Pounds pressure for 2O minutes. The ground whole blackeyes were dark colored, and ground eyeless blackeyes were almost White. The creams with the blackeye were almost as dark as the whole ground black- eyes. This with the observations made in previous experi- ments, definitely established that the eye pigment is associa- ted with the discoloration of canned blackeye peas. To study the distribution and behavior of the pigment in greater detail, a microscopic examination was made of some badly discolored commercially canned blackeye peas. Samples of liquor and of the peas were examined. The effect of adding dilute hydrochloric acid or ammonium hydroxide to the sample on the slide was noted, in addition to the gross morphology of the solid particles. Many, but not all, of the starch granules were brown in color. The liquid portion was almost colorless. Many of the particles of solid material other than starch were tinted a brown or black color. The introduction of hydrochloric acid or ammonium hydroxide had no effect on the color. From these observations, it is concluded that during the canning process, the pigment was adsorbed on or otherwise attached to the insoluble solids in the pea. The anthocyanin lost its property of changing from red in acid to green in alkaline solution. To study the behavior of the eye pigment in greater de- tail, approximately 4.5 pounds of peas were coarsely ground. The meal was placed in a pan. When the pan was filled with water, most of the eyes floated and were skimmed off. The eyes thus obtained were extracted with 2 percent formic acid solution by placing them in a Waring Blendor with the solvent and blending for about 10 minutes. The mixture was allowed to stand for several hours. Subsequent centrifug- ing and filtering separated the solid material from the liquid. This crude extract was then shaken with isobutyl alcohol and the alcoholic layer was separated from the aqueous layer. When the pink alcoholic solution of starch, protein and anthocyanin was treated with ammonium hydroxide, the starch and protein coagulated and formed an amorphous mass. The anthocyanin turned to a blue-green color and was immediately adsorbed on or otherwise attached to the coagu- DISCOLORATION OF CANNED PEAS ll lum. All attempts t0 dislodge the pigment were unsuccessful, except refluxing the mass with 6 normal hydrochloric acid for about 30 minutes. Since anthocyanins can be decolorized only by drastic oxidation or reduction, the control of discoloration seemed to be limited to precipitation of the pigment in situ or otherwise preventing its disperson. Actual precipitation of the pigment seemed to be out of the question because this can be done only with such toxic reagents as lead acetate. Therefore, the search for a remedy for the undesirable darkening of canned peas was confined largely to a study of treatments and of materials that will inhibit the dispersion of the pigment from its natural location in the eye. The ideal treatment would be one in which the starch and protein are coagulated and a suitable environment created so that the union of the pigment with the coagulated particles takes place before any dispersion occurs. This effect would be obtained more easily by adding salts to the blanching solu- tion. Therefore, an experiment was conducted to determine the relative effectiveness of several salts and the temperature at which coagulation occurs. Dry blackeye peas were ground and extracted with water in the same manner as in the preceding experiments. Fifteen cubic centimeter portions of the extract were brought to a concentration of 3.3 percent of various salts by the addition of‘ five cubic centimeters of 10 percent solution of the salt. Chemicals used were none (control), urea, sodium chloride, magnesium sulfate and aluminum sulfate. Five cubic centimeters of water were added to sample 1. The tube-s were immersed in a water bath equipped with a mechanical stirring device. A thermometer was inserted in a test tube containing 2O cubic centimeters of water. The temperature of the bath was raised slowly at a fairly uniform rate and the temperature at which coagulation occurred was noted. After the preliminary treatment, all the tubes were plugged with cotto-n and autoclaved. The results and observa- tions are shown in Table 2. There was a striking difference between the coagulating ability of the inorganic salts. This is in agreement with the Hardy-Schulze rule that “the precipitating power of an elec- trolyte depends upon the valence of the ion whose charge is opposite to that on the colloidal particle.” From this it was concluded that the particles precipitated from the pea extract were negatively charged. Gortner (6) stated that “the in- 12 BULLETIN 748, TEXAS AGRICULTURAL EXPERIMENT STATION Table 2. Temperature of coagulation of pea globulin in the presence of various salts Temperature of Normality - Appearance of precipitate Salt of salt coagulatlon one week after autoclaving (deg. F.) None (Control) 0 1851 Urea — 144 Almost black sodium Very slight darkening com- chloride 057 161468 pared with overall appear- ance of control. Slfifgarégslum 026 132 Very slight darkening Aluminum Uniform straw-colored gel; sulfate 0.27 821’- weeping 1Only very slight flocculation which disappeared after autoclaving. 2Room temperature. _ fluence of valence is not an arithmetical 1 :2:3 ratio but more nearly a geometrical progression 1: >< : X2.” The results of this experiment agree with Gortner’s principle. It is realize-d that these conclusions would be somewhat less open to question if sodium sulfate had been included in the series. However, the primary object of this experiment was not to determine the charge of the particles but to de- termine the coagulating ability of various salts which could be used in the blanch solution in commercial practice. The charge on the particles is of minor significance since antho- cyanins are amphoteric and are readily adsorbed on either positively or negatively charged particles, provided the pH is correct. In view of the foregoing observations, an experiment was designed to determine the effect of sodium chloride, mag- nesium sulfate and aluminum sulfate, when used in the blanch- ing solution, on the appearance of the canned peas. Urea was omitted because of the black precipitate formed in its presence. A sample of fresh green-shelled purple hull peas was blanched in each of the following solutions: water, 2 percent chloride, 2 percent magnesium sulfate, 0.5 percent aluminum sulfate and 0.25 percent aluminum sulfate. A blanch temper- ature of 185 to 190° F. was used. The peas were immersed in the blanch solution for 4 minutes. They were then washed and canned in the usual manner. Twelve days later, there were no differences in any of the samples except those blanched in 0.5 percent aluminum DISCOLORATION OF CANNED PEAS 13 sulfate. Those peas were darkened slightly less than the others. Since the coagulation of the starch and protein stopped the further spread of the anthocyanin, it was assumed that the lapse of time between sealing the cans and processing them was a factor in the discoloration of the peas. To ascertain whether this was correct, small lots of peas Were blanched 4 minutes at 185° F. After cooling them by washing in cold water, each lot was placed in a can, covered with boiling water, sealed, and placed in the autoclave. The time Was noted when the boiling water was added. One sample Was canned at each 5-minute interval. When the last can was placed in the autoclave, it was closed and brought to 240° F. as rapidly as possible. Thus, samples were obtaine-d which had been held various lengths of time prior to beginning the sterilization process. No difference was noted between the samples of peas. Those held 27 minutes appeared the same as those held only 2 minutes prior to the time that the retort reached 240° F. Therefore, the interval between sealing and processing is not critical. Since coagulation of the starch and protein early in the canning procedure is desirable, it was assumed that the use of a higher temperature during the sterilization process would be advantageous in bringing about the desired effect more rapidly. In fact, this had already been found to be the case by Blair and Ayers (2) with English peas. They recom- mended a process time and temperature of 7 minutes at 260° F. With these facts in mind, two lots of peas were packed. One lot was processed 7 minutes at 260° F. and the other for 30 minutes at 240° F. The peas cooked at the higher temperature were very much greener and less discolored than the peas cooked at 240° F. The liquor of the former, however, was much darker. The findings of the preceding experiments Were com- bined in designing an experiment to determine the interaction of salts in the blanch and different processing times and temperatures. A sample of fresh green-shelled purple hull peas was blanched 4 minutes at 185° F. in 4 percent magnesium sulfate and another in 1 percent aluminum sulfate and water. They were then placed in cans, covered with Water and sealed. 14 BULLETIN 748, TEXAS AGRICULTURAL EXPERIMENT STATION Some of the cans from each treatment were cooked in each of the following sterilization processes: 53 minutes at 235° F., 11 minutes at 260° F. and 35 minutes at 240° F. Ex- amination re-vealed that the use of aluminum sulfate reduced discoloration, With only one exception, the peas cooked at the lower temperatures were inferior to those cooked at the higher temperatures. The use of magnesium sulfate increased the toughness of the peas so much that this compound was dis- carded. Table 3 indicates the treatments in their relative order to desirability of the basis of color. This experiment was repeated on blackeye peas with al- most identical results. Subsequent experiments conducted in a similar manner showed that ammonium alum (Al (NH4) (SO4)2.12 H2O) is only slightly less effective than aluminum sulfate. This dif- ference probably is due to the fact that ammonium alum con- tains approximately 8.09 percent of the metal. A beneficial side effect was noted when aluminum salts were used in the blanching solution. The viscosity of the liquor and the severity of gelling were reduced markedly by the aluminum treatments. No attempts were made to obtain quantitative data on this effect. The Blair and Ayres process for canning English peas (2) involves a pre-processing soak containing calcium hydroxide, a cover brine containing magnesium hydroxide and the pre- viously mentioned high-temperature and short-time steriliza- Table 3. Relative desirability of various blanching solutions and sterilization processes (lieesiiiiiiiity Blanchmg solutwn . Time