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" [13.1. 04'031‘ . 0 ‘ ‘ _. . .0: I - s 5 .4 .¢.. . l ’ y- . ... .,.‘.'°." . . 0 5'. ‘_ . v . .. _ V "“"'l"‘°"f‘"""“"' .AO-¢ .7 . - .-. l 0 U - o ' ' . - ‘.O '* - ‘ "".l‘.’—I° "‘ {-‘W U 5 'l' O ' "Ofirnw.’.-- Lift: 4 5-. 101‘. “.3 O [J LIBRARY ‘5 Michigan Stage: University ABSTRACT EFFECTS OF GAMMA RADIATION ON WATER HOLDING CAPACITY AND NITROGEN SOLUBILITY OF ISOLATED SOYBEAN PROTEIN BY Hiroyasu Toriumi This study was initiated to investigate and to improve the water holding capacity of soybean protein for use in vegetable protein foods. The protein used (93% protein content) was obtained by Soxhlet extraction with ethyl ether of cracked soybeans followed by alkaline water extraction and acid precipitation. Final drying was by freeze dehydration. In this method, the protein was prepared without the use of high temperature in order to minimize denaturation. Two methods, the filter paper method and the centri- fugal method, for water holding capacity of the protein were investigated. Both methods gave identical values. The filter paper method, however, was preferred because it is easier to use. The protein was irradiated in dry and wet forms and the water holding capacity was measured. Irradiation of the dry form did not increase the water holding capacity. In the wet form, at high doses (10 and Hiroyasu Toriumi 20 Mrad) increased water holding capacity occurred. This increase, however, was not retained when the protein was dried subsequent to irradiation. Irradiation of both wet and dry protein decreased the soluble nitrogen at pH 7 at doses of 1.5 and 10 Mrad. EFFECTS OF GAMMA RADIATION ON WATER HOLDING CAPACITY AND NITROGEN SOLUBILITY OF ISOLATED SOYBEAN PROTEIN BY Hiroyasu Toriumi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1970 ACKNOWLEDGMENTS The author wishes to express his deep appreciation to his grandfather, the late Saihei Toriumi, for his hearty affection and financial assistance to enable him to complete the study. The author's appreciation is extended to Miss Hildegard Czeskleba for encouraging him in a kind and sincere manner during this period of study. Thanks are expressed to Dr. W. M. Urbain for serving as academic advisor, to Dr. L. E. Dawson for the extensive use of laboratory equipment, and to Dr. D. E. Ullrey for serving as committee member. Thanks are given also to Mr. George Giddings for his help with the irradiation of the protein, and to the Department of Food Science at Michigan State Uni- versity for financial assistance. ii ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION . LITERATURE REVIEW Isolation of Nature of Water Holding Capacity Determination of Water Holding Capacity Effect of Radiation of Proteins PROCEDURES . Preparation of Isolated Soy Protein Determination of Water Holding Capacity Determination of Soluble Nitrogen Index Nitrogen Determination Fat and Ash Determination Moisture, TABLE OF CONTENTS Soybean Proteins RESULTS AND DISCUSSION Preparation of Isolated Soy Protein Water Holding Capacity of Soy Protein Effect of Gamma Radiation on Nitrogen Solubility . SUMMARY . . LITERATURE CITED iii Page ii 16 16 20 21 22 22 23 23 26 32 39 41 LIST OF TABLES Table Page 1. Analysis of Isolated Soybean Protein Extracted by Batch Method with Hexane-Ethanol Mixture . . 25 2. Analysis of Isolated Soybean Protein Extracted in Soxhlet Apparatus with Ethyl Ether . . . . 26 3. Water Holding Capacity (%) of Protein Irradiated in Dry Form, and Measured at Indicated pH's . . 30 iv LIST OF FIGURES Figure Page 1. Relation between pH and Water Holding Capacity (%) of Non-Irradiated Soybean Protein- Centrifuge Method . . . . . . . . . . 27 2. Relation between pH and Water Holding Capacity (%) of Non-Irradiated Soybean Protein-Filter Paper Method . . . . . . . . . . . . 28 3. Relation between pH and Water Holding Capacity (%) of Protein Irradiated in Wet Form . . . . 31 4. Water Holding Capacity (%) of the Isolated Protein Sample that was Irradiated in Wet Form at the Doses of 10 and 20 Mrad and Freeze Dried . . . . . . . . . . . . 33 5. Soluble Nitrogen Index of Isolated Soy Protein Without Irradiation . . . . . . . . . . 35 6. Soluble Nitrogen Index of Isolated Soy Protein with Dry Irradiation . . . . . . . . . 36 7. Soluble Nitrogen Index of Isolated Soybean Protein with Wet Irradiation . . . . . . . 37 INTRODUCTION Simulated meats from vegetable proteins are being developed in the growing realization that there is an economic advantage in the future to utilize directly protein-rich vegetable sources for human foods. There are, however, some problems with such products before this can be done. These problems include the following: 1. texture, 2. absorption of water and fat, 3. stability during storage, 4. nutritional value, and 5. ability to carry flavor and color. These problems are primarily related to the carbohydrate and protein portions of these foods, and to a smaller extent, to the lipid. Certain problems are related to the physical structure secured by the processing utilized in manufacturing these foods. The texture of meats and meat products are related in part to the interaction between water and the protein constituents. The structure of meat largely is due to the protein fibers of the animal muscle, whose properties in turn depend upon highly hydrated proteins. The simulated meats from vegetable proteins require similar characteristics. Simulated meats can be produced as follows: The proteins of 95 to 98% purity of any of several vegetable materials are dispersed in somewhat alkaline water, and the resulting dope is extruded through spinnerets into an acid coagulating-bath where fiber formation occurs. This process takes advantage of the fact that the vege- table protein is more soluble at alkaline pH's and has a minimum solubility at the isoelectric point. For soy protein, the isoelectric point is approximately pH 4.6. It can be seen, therefore, that a spun protein-food can be made by this process only at near the isoelectric point, where the solubility of the protein is minimal. At the isoelectric point, however, vegetable proteins have minimum water holding capacity (WHC). Muscle pro- teins such as myosin, actin and others have quite high WHC as present in meat. Vegetable proteins have greater WHC at higher pH's but at such pH's the protein is quite soluble, and fibers cannot be formed. One way to improve the low WHC of the fibers would be to use a highly hydrated binder such as a starch gel, agar, gelatin and so on. Such substances, however, do not give a meat-like structure to the product.but one more of a cereal type. Another way to overcome the low WHC of the fibers is to alter the normal properties of the protein, making up the fibers. This may be accomplished in at least two ways: (1) to increase the cross linking of protein with ionizing radiation and in that way to build up a network of protein so as to increase water holding capacity; and (2) by irradiation, to reduce the solubility of the pro- tein at pH's greater than the normal isoelectric point and, through use of higher pH's in the coagulating bath to secure improved water holding capacity. (Some cross- linking also could be involved.) It was the purpose of this study to investigate the above two methods for improving the WHC of proteins suit- able for making Spun protein foods. This study was limited to soy protein. LITERATURE REVIEW Isolation of Soybean Proteins The first published work on the isolation of soy- bean protein is that of Meissl and Bocker in 1883. These authors recognized three proteins, one of which they called casein. It was extractable by potassium hydroxide and comprised 30% of the soybean (moisture 10%) and more than 90% of the total protein content. The second protein fraction they termed albumin, because it was coagulable by heating the whey obtained from the precipitation of the casein. They recognized its dif- ference from common albumin (ovalbumin) but likened it to pea albumin, and said it was possibly derived from the casein. The third fraction, comprising 7% of the soybean, was insoluble on repeated extraction, and was designated insoluble casein. In 1898, Osborne and Campbell, gave the name glycinin to the globulin-like protein extracted from the soybean by 10% sodium chloride solution. In 1921 and 1923 the most extensive early work in this field is that of Satow, who investigated the separation and properties of soybean protein with a view to its industrial application. In addition to preparing glycinin by the procedure of Osborne, gt_al., he separated protein by extraction with water and various alkalies, acids or salts. In 1920, Muramatsu characterized the water- soluble portion as containing 84 parts globulin, 5.36 parts albumin, 4.36 parts proteose, and 6.03 parts non- protein nitrogen. In 1928 Tadokoro and Yoshimura desig— nated the water-extracted dialysis-precipitated protein as glycinin A. They obtained glycinin B by extracting the residual meal with 10% sodium chloride. Glutelin was obtained from the residual from the second extraction by using 0.2% sodium hydroxide. Legumelin was derived from the filtrate containing glycinin A by precipitation with ammonium sulfate. The order of increasing content of free amino nitrogen was glycinin, glutelin, legumelin. In 1931 among other properties given for glycinins A and B, glutelin and legumelin by Ivanov are amino acid content, nitrogen distribution, composition of silver salts, presence of free amino groups and behavior toward aqueous sodium hydroxide or sodium chloride. In 1932 Jones and Csonka separated five fractions from a 10% sodium chloride extract of soybean meal by adding ammon- ium sulfate at specific degrees of partial saturation. The fraction precipitated at 55% saturation of ammonium sulfate most nearly resembled Osborne, g£_31., glycinin. It had an isoelectric point of pH 5.2 and was not coag- ulable even at boiling water temperature. In 1945 Evans and St. John determined the percentage protein present in soybean meal as albumin (soluble in water), globulin (soluble in 5% potassium chloride), prolamin (soluble in 70% ethanol), glutelin (soluble in 0.2% potassium hydroxide), and as residual protein. The foregoing discussion illustrates the state of confusion brought about by the failure to characterize unequivocably any particular protein species of the soy- bean. Glycinin would appear to be the fraction most nearly approaching a state of chemical individuality, but many differences in its properties are reported by different investigators. Another complicating factor is the observation of Csonka and Jones (1933) that the glycinin fractions from different varieties of soybean differ not only in amount, but also in nitrogen content and amino acid composition. In 1945 Vickery, however, applied modern methods to a known variety of soybeans to isolate a protein most nearly like the glycinin described by Osborne and Camp- Zbell (1898). Although 91% of the nitrogen could be brought into solution by extracting the meal in a Waring Blendor twith 6% sodium chloride (adjusted to pH 7.1 to 7.5 by loarium hydroxide) to insure complete cell wall rupture, the suspension was turbid even after removing the insoluble portion. It was not possible to clarify it further by centrifugation or filtration. Vickery (1945) states that an essential prerequisite to the preparation of a protein fraction free from non-protein contaminants is complete clarification of its solution. He achieved such clarification by the use of a saturated sodium chloride extraction of the meal. In 1946, Smiley and Smith preferred the use of nitrogen content as the most convenient criterion for the purity of soybean protein fractions from a single source, in the absence of crystallinity or other dis- tinctive physical properties. This preference was based on the belief that variation in nitrogen content of the main protein in soybean is due to non-protein impurities. They found that a higher nitrogen content of separated protein was obtained when the meal had been defatted with ethanol, even when alkaline extraction and acid precipitation were used instead of Vickery's procedure. The recent literature specifically relating to preparation and properties of the isolate includes that of Smith, gt_al. (1938). About 92% of the protein of oil-free soybean meal can be extracted with distilled water at a pH of about 6.6. Contrary to the behavior of most vegetable proteins, low concentrations of neutral salts reduce the dispersion of the protein. For example, 0.1 N sodium chloride in water lowers the dispersion from 92 to 45%, and 0.0175 N calcium or magnesium chloride lowers the dispersion of nitrogen components to 21%. This cation effect is overcome by increasing the con- centration of the salt or by raising the pH of the system. Most industrial sources of water would be too high in salt concentration for extracting protein in good yield, and alkali must be used to overcome the salt effect. To obtain a high yield of protein, the meal is extracted by adding water and adjusting to about pH 9.0. The insoluble residue is removed in a centrifuge and the protein precipitated with acid in the pH range of 4.6 to 4.1. Protein isolated by this procedure is a mixture of globulins and glutelins. In laboratory-scale operation as much as 84% of the total protein of the meal is extracted. Thus, with dehulled, oil-free meal containing 50% protein, the yield will be 42% based on the weight of the meal. In large-scale processing, however, the yield will be much lower, owing partly to the lower water-to- meal ratio which is necessary in commercial Operations and partly to loss of protein through chemical degradation into a more soluble form which is not precipitated upon acidification. A yield of 30% is considered good commer- cial practice. Nature of Water Holding Capacity The water holding capacity of meat has been the subject of considerable study. For example, Hamm (1960) reviewed the biochemistry of meat hydration in which the WHC of muscle tissue was reported to concern mainly actin and myosin of the complex actomyosin. Though there are no studies reported on the water holding capacity of soybean protein, some basic studies of water holding characteristics of proteins were reported. In 1940 Sponsler, gt_gl., reported that the hydrophilic groups responsible for the fast binding of water by protein are of two types. One type includes the polar groups of the protein side chains, such as the carboxyl-, amino-, hydroxyl-, and sulfhydryl- groups. The other type is made up of undis- sociated carbonyl- and imido- groups of the peptide bonds in which the binding of water is due to the dipolar char- acter of water. The water molecule is a dipole because of the unsymmetrical space distribution of the negative charge of oxygen and positive charge of hydrogen. This circumstance leads to attraction and association with polar groups in the protein. For example, the water mole- cule is bound by hydrogen bond (H), probably as follows: 10 / O- H '\ OH é——C') — H Carboxyl group —-C\ OH or --C H 1. o—H/ O-QH—O —H H /.H Amino and imidazol groups /N—H(—O -N H [\H H- 0—H Carbonyl groups C — O -—9 H— O —H Guanidino groups H H H H l I I l H—N N—-)H ll Olcott and Frankel-Conrat (1946) found that the activity of proteins for binding water is diminished by blocking polar groups by certain reagents, and proved in that manner that polar groups are at least partly respon- sible for the water binding. With the same technique, they showed that carboxyl groups play a less important role in hydration than do amino groups (Seehof, gt_31., 1953). At a very low relative humidity (about 5%) one molecule of water is bound by two amino groups. When the rela- tive humidity is increased to 60%, one amino group binds 2-1/2 m01ecules of water. At that point, the ability of amino groups to bind water is exhausted. According to Pauling (1945) the initial phase of binding of water by proteins consists in the binding of one water molecule by one polar group. The affinity for water of the different polar group varies. There- fore, water attaches first to the most active groups, and then to the less active. Whereas Pauling supposed that peptide groups do not play any role in binding water, Mellon, g£_al., (1947, 1949) found that the -CO-NH- group takes part in hydration. At 60% relative humidity, peptide groups appear to be responsible for about 45% of the vapor—phase water absorption by casein and 70% of the absorption by zein. According to the hypothesis of Klotz (1958), non- polar groups can also have some influence on hydration, Ibut are not concerned in the true binding of water. 12 Determination of Water Holding Capacity Ultrafiltration was used by Lloyd and Moran (1933) for determining bound water in gelatin. This technique might be primarily applicable for homogenates of tissues, but has the disadvantage of requiring a long time to carry out the determination.‘ At first, pressing meat between two plates was used only for a qualitative judgement of wetness (Schonberg, 1937). Pusch (1950) screw-pressed pieces of muscle between two steel plates and measured the volume of juice squeezed out. Grau (1952) also used the pressure method for the estimation of the quality of frankfurters. In his method the meat was pressed in a sieve-ended cylinder by a moving piston and the juice was weighed. The water holding capacity of cooked meat was determined by press methods, particularly for study of the correla- tion between a subjective impression of "juiceness" and an objective test using "pressometers." The press method was transformed to a quantitative technique by using filter paper. The more loosely the water bound, the better it is absorbed by filter paper. Grau and Hamm (1953, 1957) developed a quantitative method for determining the WHC of meat--a combination of the press technique and the filter paper techni- que. The method of Gran and Hamm was modified by several authors by applying a constant pressure or ‘weighing the filter paper before and after pressing. 13 Mohler and Kiermeir (1953) used a sedimentation method for ground meat which was suspended in a calibrated cylinder. This method was used for studying the influ- ence of certain salts on the swelling of meat. The advantages of this method are its simplicity and usability for serial-type experiments. Wierbicki (1957) put a meat sample on a fritted glass disk in a special centrifuge glass tube and sepa- rated the loose water from tissue by centrifuging the juice at 1000 r.p.m. into a smaller graduated section of the tube. Effect of Radiation of Proteins The principal changes in proteins produced by ionizing radiations are explained by Bacq, as follows: 1. Main Chain Scission This is related to a reduction in molecular weight. Since proteins are made up of a large number of identical or similar repeating units, there is an equal probability that a break is produced at almost every unit along the molecule. If all the molecules are of uniform molecular weight to start with, then radiation-induced breaks will product a non-uniform (or polydisperse) product. If the polymers are polydisperse at the beginning, then degrada— tion by radiation does not change the character of the distribution but only the average molecular weight. l4 2. Crosslinking This can be of two types. If two different molecules are joined together, this is inter-molecular crosslinking. As this process proceeds, more and more molecules are joined together. This is crosslinking of an insoluble gel network. Initially the network is so loose that the gel may be difficult to detect especially with polymers of high molecular weight. A certain number of crosslinks have to be formed before a sufficiently large network is formed to give a gel and consequently there will be a minimum radiation dose before any gel can be detected. When a large network is formed of molecules, they are no longer soluble, but only swell in solvents which dis- solved the starting material. If crosslinks can then be formed between different groups in the same molecule, this is intramolecular crosslinking. The effect of such intramolecular cross- linkins is to pull the molecule together so that it occupies a smaller volume in solution and this brings with it a reduction in viscosity without any change in molecular weight. In such randomly coiled long molecules intramolecular crossing will predominate, if the cross- linking reaction is carried out in very dilute solution; at higher concentrations the reaction will be predomi- nantly between different molecules and give rise to gel networks. 15 3. Disruption of Secondary Structure of Macromolecules Proteins in their native state are maintained in rigid steric configurations by secondary valency forces and do not assume in solution purely random configura- tions, as do most synthetic polymers. The main chains are constrained in fixed configurations by hydrogen bonds. Radiation disrupts this secondary folding. This type of effect has so far only been encountered in proteins. PROCEDURES Preparation of Isolated Soy Protein 1. Batch Method with Hexane-Ethanol Mixture Approximately 100g of raw soybeans (var. Harosoy 63) produced in Michigan, were cracked in a Waring Blendor for 30 seconds and passed through a lOO-mesh screen. The crushed full-fat soybeans were then extracted with hexane—absolute-ethanol azeotropic mixture (79:21). A ratio of three parts by weight of solvent to one of beans was employed. The mixture was stirred for six hours without heating. Stirring was stopped and the mixture allowed to stand for one hour. The supernatant containing some suspended solids was removed and centrifuged at 6000 r.p.m. for 20 minutes in a Sorvall Superspeed RC2-B centrifuge with a GSA Rotor. The clear oil-containing solvent thus obtained, was discarded. The solids were added to those obtained by decanting the original mixture. The combined solids were extracted five times with the hexane—ethanol mixture for one hour periods in order to get essentially oil-free residues. The solids so obtained, 16 17 were extracted with water with five times the weight of the solids. The mixture was adjusted to pH 8 with sodium hydroxide. The solution obtained after one hour of stir- ring contained the soluble protein, carbohydrate and mineral constituents. These were separated from the insoluble material by centrifuging at a speed of 6000 r.p.m. for 20 minutes. After removing the supernatant, the residue was again diSpersed in the alkaline solution of pH 8, and another protein-containing supernatant was obtained in the same way. These two portions of super- natant were combined and acidified to pH 4.6 with hydro- chloric acid. This precipitated the globulin fraction. The resulting curd containing liquid was then centrifuged to obtain the insoluble protein, and the supernatant portion was discarded. This curd was washed by dispers- ing it again with water at pH 8.0 obtained with sodium hydroxide and precipitated again at pH 4.6 in the same manner. This washing procedure was repeated three times. The purified protein so obtained was freeze-dried. The freeze-dried product was converted to a particulate form by placing it in a Waring Blendor for 10 seconds. Product so obtained and when passed through a 100 mesh screen was used in some of the studies reported below. 18 Raw Soybeans crack (30 sec. in Waring blendor) Oil Extraction (79:21 Hexane:Ethanol mixture for 6 hours solvent + oil residue discard wash 5 oil extraction times by solvent J F 1 residue oil + solvent discard water extraction adjust to pH 8 with NaOH 1 hour centrifuge (6000 rpm) f I residue ____4 supernatant water . adjust to pH 4.6 extraction at with HCl pH 8 I I . centrifuge centrifuge f 1 l l residue supernatant supernatant protein ppt I discard water extraction at disperse and pH 8 ppt 5 times centrifuge freeze drying residue supernatant discard isolated soy protein l9 2. Method with Ethyl Ether, Using Soxhlet Apparatus Approximately 100g of raw soybeans (var. Harosoy 63) produced in Michigan, were cracked in a Waring Blendor for 30 seconds and passed through a loo-mesh screen. The' cracked full-fat soybeans were extracted with ethyl ether by Soxhlet apparatus for 30 hours. The defatted soybeans were treated in the same way as with the batch method in order to secure isolated soybean protein. crack (30 sec. in Haring blendor) Oil extrac io (with ether by Soxhlet) I 1 solvent and oil discard residue water extraction adjust to pH 8 with NaOH stir 1 hour centrifuge (6000 rpm) 1 residue F'—_'_—_T$supernatant water , adjust to pH 4.6 egtgaction at with gel P I centrifuge centrifuge I 1 p I I 1 supernatant protein ppt residue supernatant discard disperse and ppt Sltimes water extraction at freeze dry pH 8 l isolated soy centrifuge protein residue supernatant discard 20 Determination of Water Holding Capacity 1. Water Holdipg Capacity bprilter Paper Method The filter paper method by Sherman (1961) for the determination of percentage water retention was used. Two grams of isolated soy protein were weighed into a tared centrifuge tube (50 ml capacity) and 20 ml of distilled water added. After thorough mixing with a glass rod, the tube was stOppered and mixture kept for three hours at room temperature. The stopper was removed and the tube was centrifuged at 3000 r.p.m. by Lourdes Model AX centrifuge for 20 minutes. The supernatant fluid was decanted through a presoaked and drained filter paper rest- ing in a glass funnel into a 10 ml graduated bylinder that could be read to within 0.05 ml. The mixture was drained into the filter paper for 10 minutes, and the amount of liquid passing the filter was measured. The water holding capa- city of the sample expressed as percentage water retention per gram of soybean protein was calculated as follows: Percentage of WHC = vol (ml) fluid added minus vol (ml) fluid not absorbed x weight (g) of protein 100 3. Water Holdipg Capacity py Centrifuge Method The procedure used for determining WHC in this present study was a modification of the method reported by Wierbicki, gp_al. (1957). A specially constructed tube was used for this determination. Overall length was 180 mm; the top section was 30 mm in diameter, 100 mm in length 21 and the bottom section was 18 mm in diameter, 80 mm in length. The quantity of expressed water was determined in the graduated bottom section calibrated in 0.1 ml division. A medium coarse fritted glass disc was placed at the intersection of the large and small diameter sec- tions of the tube. One gram of the isolated soybean protein and 10 m1 of buffered solution which was adjusted to a specified pH with hydrocloric acid or with sodium hydroxide, were added and mixed well in upper portion of the tube. After 30 minutes, the mixture was centrifuged for 20 min at 1000 r.p.m. by International Refrigerated Centrifuge Model PR-6. The water holding capacity was calculated from the following relationship: Percentage of WHC = vol (m1) of fluid added minus m1 fluid not absorbed wt (9) of protein x 100 Determination of Soluble Nitrogen Index The method is a modification of the method for the dispersible nitrogen in soybean products by A.O.C.S. (1946) Tentative Method Ba 11-65. Two grams of the soybean pro- tein were weighed into a 400 m1 beaker. A small portion (of 200 m1 of distilled water was added and the protein 'thoroughly dispersed with a stirring rod. The remainder of the 200 ml of water was added and the mixture stirred. The EDH of the protein-water mixture was adjusted to selected pH's (either with hydrochloric acid or sodium hydroxide. This solu- ‘tion was stirred by a mechanical stirrer for 120 minutes at 22 room temperature, and transferred quantitatively to a 250 m1 volumetric flask and additional water added to make 250 ml. After a few minutes about 40 ml were drained into a 50 m1 centrifuge tube, and centrifuged for 10 min- utes at 1500 r.p.m. Then the supernatant was decanted through a funnel containing a plug of glass wool and collected in the 100 ml beaker. Twenty-five m1 of the clear liquid were pipetted into a Kjeldahl flask. The standard procedure for Kjeldahl nitrogen determination was followed. Soluble nitrogen index is expressed as follows: water soluble nitrOgen 100 % Soluble Nitrogen Index = total nitrogen Nitrogen Determination All nitrogen determinations were made by the micro- Kjeldahl method outlined by the American Instrument Company (1961). Nitrogen values were determined in duplicate. Moisture, Fat and Ash Determination The methods of the A.O.A.C. (1965) were applied for moisture (13.070), fat (13.074) and ash (13.071) deter- minations. RESULTS AND DISCUSSION Preparation of Isolated Soy Protein It was reported by Honig, et a1., (1969) that dehulled soybean flakes, defatted with pentane- hexane, could be further extracted with an azeotropic mixture of hexane and ethanol in order to remove free and bound lipids including phospholipids, sterols and trigly— cerides. In their procedure, however, lipids in soybeans were extracted in a Soxhlet which is designed to do a continuous operation by raising the solvent mixture to a boiling point. Generally five physical factors affecting the extraction of protein from soybean meal are important: (1) size of meal particle; (2) solvent-meal ratio; (3) temperature; (4) time; and (5) separation of meal residue from extractant. The size of meal particles is a very important factor and loo-mesh or finer meal is needed for maximum extraction. In this study, the soybeans were cracked in a Waring Blendor for 30 seconds and passed through a lOO-mesh sieve. 23 24 During the cracking in the Blendor, heat is gener- ated. Since such heat can cause denaturation of the protein, a limit of 30 seconds for the Operating of the Blendor was employed. With this limitation the soybeans were heated only slightly above room temperature. Although batch-type of extraction is not used in the industry because of the small throughput per man hour and the outlay of equipment necessary to handle moderately large tonnage, it was applied in this study when the hexane-ethanol solvent was used. With batch- type extraction it was possible to keep the extraction temperature close to room temperature and to cause a mini- mal heat denaturation of the protein. Using the convenient Soxhlet operation method with this solvent would require quite high temperature (above 70°C). This would cause significant denaturation of the protein. In the batch operation employed, the solvent and soybean meal were mixed in a polyethylene beaker and stirred for six hours. After settling, the liquid por- tion was decanted from the solid. The oil-extracted residues were washed five times with the solvent mixture in order to reduce the oil content further. Finally, the residue was freed of solvent by air drying at room temperature. The results of analysis of the extracted meal are shown in Table 1. Although a relatively high percentage of protein was obtained (89.1%), it was not adequate. Isolated soy protein is defined as not less 25 Table 1.--Analysis of Isolated Soybean Protein Extracted by Batch Method with Hexane-Ethanol Mixture. Moisture 2.5% Protein (N x 6.25) 89.8% Ash 4.1% Fat 3.1% than 90% protein. It was realized that this method of extraction led to a quite high fat content (3.1%), being higher than commercial products by a factor of about ten. It was concluded, therefore, that this batch-type method was not suitable, since too much oil remained in the meal. It is conceivable that this resulted from the low temperature during extraction. Because of this failure of the solvent used with the batch system, a second system was employed. This was the continuous extraction method, employing the Soxhlet apparatus with anhydrous ethyl ether as the solvent. Although ethyl ether is not a practical com- mercial solvent for the extraction, it is a most effec- tive solvent for lipid extraction and, for the purpose of this study, is attractive because it boils at only 35°C. This temperature is not likely to cause denat- uration of the protein. As shown in Table 2, the iso- lated soy protein was obtained with a higher percentage protein content (93.2%) and with very low-percentage fat content (0.2%). The Soxhlet extraction method, with 26 ethyl ether as the solvent, therefore, was used to pre- pare isolated soybean protein for this study. TABLE 2.--Analysis of Isolated Soybean Protein Extracted in Soxhlet Apparatus with Ethyl Ether. Moisture 2.2% Protein (N x 6.25) 93.2% Ash 4.2% Fat 0.2% Water Holding Capacity of Soerrotein Two methods were employed for measuring the WHC of soy protein. Both methods, filter paper method and centrifuge method, yielded quite similar results for the WHC of non-irradiated soy protein. The influence of pH on the WHC of non-irradiated soy protein is shown in Figures 1 and 2. It is clear that WHC is dependent on» pH value. Practically, however, the filter paper method is easier to use than the centrifuge method. In the centri- fuge method, a fritted glass disc was placed between the upper portion and the bottom portion of the glass tube. This disc, however, does not hold its position prOperly during the centrifugation, and moves so that the sample in the upper part of the tube leaks into the graduated bottom part. Paraffin can be used to seal the fritted 27 .coaumz wasMfluucmoulswwuoum somehow connecmnancoz mo Amy muflommso mcacaom wwum3 can an swm3umn c0wumammnn.a musmwm mm s as m l w ms 1 '- 1 d dim -«V - d» ‘ 1.ooH L.oom ..oom Tcow L room loom :oon (%) Katoedeo Burpton :equ 28 .oonums momma Hmuafimllcwmuoum cmmnmom coumwcmuancoz mo Amy muwommmo mcflcaom Hmumz was an cmm3umn cowumammll.~ gunman an Ad ad m m h o m e m N H LP P P 4 d d an- d? 1:- q)- 1|- qu- J.oS 1 room Luoom irooe com 1 room room I (g) Kiroedeo burpton 1932M 29 glass disc to the glass tube. This, however, was incon- venient. The curves in Figures 1 and 2 give a good picture of the relative electrochemical conditions of the protein with changing pH. When pH increases or decreases from the isoelectric point (4.6), the protein net charge increases, and consequently attraction of water to the peptide chains increases. Therefore, the protein hydra- tion increases. .On the other hand, at the isoelectric point, the protein net charge is zero, and hydration bonds between the peptide chains caused a tightening of the protein network. Therefore, the WHC at the isoelectric pH is at a minimum. Since the filter paper method was convenient and effective, this method was used for the determination of WHC in this study.’ Two different methods by which a chemical change affecting WHC can be brought about by ionizing radiations were considered possible in this study. One is direct action in which the molecule undergoing change itself becomes ionized or excited by the passage through it of an electron or other energy particle. The other is indirect action in which the molecule in question does not itself directly absorb the energy but receives it by transfer from another molecule or molecular fragment. In this study, when soy protein is irradiated dry, the action or the soy protein is essentially direct. The data on WHC of the soy protein irradiated in dry form I are shown in Table 3. It is clear that the WHC is 30 quite independent of the irradiation doses, and in respective pH ranges, it is similar to the WHC of non- irradiated soy protein. TABLE 3.--Water Holding Capacity (%) of Protein Irradiated in Dry Form, and Measured at Indicated pH's. Dose - rad a k k k k pH 5 50 100 500 1 M 5 M 10M 20M . 330 340 330 335 340 320 340 330 .5 300 320 310 320 320 310 300 310 380 365 386 370 370 370 360 370 365 380 370 380 380 350 365 370 605 600 585 590 590 595 585 585 600 605 580 590 595 600 595 605 QOU'IubU When soy protein is irradiated in water, the action of the radiation can be due to both direct and indirect effects. The WHC of the protein in the wet form was determined over a pH range of 2 to 11, and for irra- diation doses from 0.5 to 20 Mrad. The curves given in Figure 3 for 0.5, 1 and 5 Mrad, are all similar, showing the minimum water absorption at near the isolectric point and higher values at higher pH ranges. For 10 and 20 Mrad, the curves are quite different. The WHC's at these doses are significantly greater, particularly above pH 7. Above pH 7, the WHC's reach the value of about 1000%, and all water added was absorbed in the protein. Water Holding Capacity (%) 1000 f—ut + +4 900-. ll ' I W 800j_ 7004 600., sooc 400.L 300 ~L ‘A‘ 0—0 1/2 Mrad C>———-c) 1 Mrad 20° “ H 5 Mrad D—U 10 Mrad 100-1 H 20 Mrad r l _‘L I i 1 ‘r 2 4 5 6 7 8 9 1o 11 pH Figure 3.--Relation between pH and Water Holding Capacity (%) of Protein Irradiated in Wet Form. 32 At this state, gels were characteristically formed. It is conceivable that the crosslinkings responsible for the aggregation of the proteins, were set up by this rather large amount of gamma radiation. It is clear that irradiation of wet form is quite effective in increasing WHC of the protein. This could be due to the change of chemical bonds in the protein or to simple physical change in the structure in which water is absorbed, as in a sponge, without being chemically bonded. In an effort to determine which of these explana- tions might be correct, proteins which had been irradiated at 10 and 20 Mrad, were freeze-dried, ground, and passed through 100 mesh sieve. The WHC was again measured. The curves for the WHC values over the pH range 2 to 11 are given in Figure 4. They are essentially identical with. the one given in Figure 2 for the protein which was not irradiated. It is believed, therefore, that the increase of WHC observed for the irradiation in the wet form with 10 and 20 Mrad resulted in water held in the structures without true chemical bonding. Effect of GammaRadiation on'Nitrogen’Solubility One of the chemical and physical changes produced by ionizing radiation is an alteration of solubility of protein. The purpose of a determination of soluble Water Holding Capacity (%) 700 l 600' 500 » 400C 300 y 33 O———O 10 Mrad [3-—-—-£3 20 Mrad 200 w 100 « 2 4 5 6 7 8 9 10 11 PH Figure 4.--Water Holding Capacity (%) of the Isolated Protein Sample that was Irradiated in Wet Form at the Doses of 10 and 20 Mrad and Freeze Dried. 34 nitrogen of the soy protein in this study was to find the amount of the decrease at various pH's and radiation. doses. It was hoped that the irradiated protein would display a reduced solubility at a higher pH and at the same time would have a WHC greater than non-irradiated protein. In Figure 5, the soluble nitrogen index of non- irradiated soy protein is shown for the pH range 1 to 11. In accord with theory, minimum solubility lies around isoelectric point. On both the acid and alkaline sides of the isoelectric point, the solubility is quite high. As seen in Figure 6, the solubility of soy protein which was irradiated in the dry form, decreases, giving the minimal solubility near the isoelectric point. At alka- line pH's, higher irradiation doses give lower solubili- ties but at acid pH's irradiation clearly did not affect the solubility. In Figure 7 data for the solubility of the protein which was irradiated in wet form, are shown. Quite different curves from those of the previous two graphs (Figures 5 and 6) were obtained. Again the higher doses decrease solubility. The most characteristic change of the solubility with wet irradiation, however, is that the solubilities around pH 7 are very low even at l, 5 and 10 Mrads. This result is interesting when it is considered with the results shown in Figures 2, 3 and 4. In Figure 3, the WHC of the doses 1 and 5 Mrads 100 . Soluble Nitrogen Index (%) 90‘ 80» 70 O‘ O U! 0 Jr 05 O d: ‘P 20“ 1Q. Figure 5.—-Soluble Nitrogen Index of Isolated Soy Protein 35 3 4 5 5 Without Irradiation. PH ~10 1b 9 Soluble Nitrogen Index (%) 36 100 .r. 904' . ‘_ .A A; 4‘ 801)- . I . I I" """"*. 0.5 Mrad O———o 1Mrad Ak—-—-—-i§ 5 Mrad >CP—---4J 10 Mrad f 6 7 E '9 10 Figure 6.--Soluble Nitrogen Index of Isolated Soy Protein with Dry Irradiation. Soluble Nitrogen Index (%) 37 lOO'f ‘F----{D 1/2 Mrad 90)” C¥-—--4D 1 Mrad Ar 1 A 5 Mrad 80" H 10 Mrad 70-- 60-- 504)- 400 30 ' L O 20 1‘ . 10" ‘ x O/ \'L\ ‘7 If D ‘Igl. l‘....i:=‘n!'l L J Q ‘ 1 1 L l ' D f I r T f l 3 4 5 6 7 8 9 10 pH Figure 7.--Soluble Nitrogen Index of Isolated Soybean Protein with Wet Irradiation. 38 is given as approximately 500% and 10 Mrads about 825. In Figure 4, the WHC of the protein which was irradiated in a wet form and dehydrated again, is about 530% at pH 7. In Figure 2, the water holding capacity of unirra- diated protein is shown about 625% at pH 7. From these results, one may conclude that if the protein is irradiated in wet form at about pH 7, the soluble nitrogen will be low and the WHC high. This could result in an improved spun protein food. S UMMARY In order to obtain a soy protein isolate with minimum denaturation, two methods of oil extraction were evaluated. The first, a batch method, in which hexane-ethanol mixture was used at room temperature did not remove sufficient oil, as evidenced by the analysis, which showed 3.1% oil and 89.1% protein. The second method, in which ethyl ether was used in a Soxhlet apparatus yielded a product analyz- ing 0.2% oil and 93.2% protein. This method was employed in this study. In the determination of water holding capacity (WHC), two methods were tested. Both methods yielded similar results. The filter paper method was more convenient than the centrifuge method and was used for this reason. The minimal values of WHC were observed near the isoelec- tric point. Gamma radiation was applied to soy protein isolate, both dry and dispersed in water. Irradiation in the dry form did not change the WHC, whereas irradia- tion in the wet form caused some changes. At relatively high doses only (10 and 20 Mrad) irradiation in the wet form increased the WHC substantially, eSpecially at the 39 40 higher pH's. Freeze drying of the irradiated protein restored the WHC to the values before irradiation. From this, it is concluded that the radiation-induced increase in WHC involved only absorption of water in the physical structure and was not due to significant chemical altera- tion of the protein. Changes in the soluble nitrogen index due to irradiation of the protein both dry and dis- persed in water were measured. Only irradiation in the wet form produced an increase. LITERATURE CITED Anon. A.O.A.C. 1965. Official Method of Analysis, Assoc. Off. Agr. Chemists, 10th, p. 203, Washington, D. C. Anon. A.O.C.S. 1946. Official and Tentative Methods Nitrogen Solubility Index, Ba 11-65, Am. Oil Chemists Soc., Chicago, Illinois. Bacq, Z. M. 1961. Effect of Radiation on Macromolecules. In Fundamentals of Radiobiology. 157-212. Pergamon Press, New York. Csonka, F. A. and Jones, D. B. 1933. Differences in the amino acid content of the chief protein (glycinin) from seeds of several varieties of soybean. Agr. Research, 46:51-55. Evans, R. J. and St. John, J. L. 1945. Estimation of the relative nutritive value of vegetable proteins by two chemical methods. J. Nutrition, 30:209-217. Grau, R. 1952. Die Beeinflussung der Wasserbindung von Fleisch durch Zusatz von Salzen. Fleischurtschaft 4, 83. Quoted in Biochemistry of Meat Hydration by Reiner Hamm, 1960, in Advances in Food Research. Vol. 10:366. Academic Press, New York. Grau, R. and Hamm, R. 1953. Eine einfoche Methode zur Bestimmung der Wasserbindung im Muskel. Natur- wissenschaften 40, 29. Quoted in Biochemistry of Meat Hydration by Reiner Hamm, 1960, in Advances in Food Research. Vol. 10:366. Academic Press, New York. Grau, R. and Hamm, R. 1957. Uber das Wasserbindungs vermogen des Saugetiermuskels. ll. Mitt. Uber die Bestimmung der Wassergindung des Muskels. Z Lebensm Untersuch. U. Forsch 105, 446. Quoted in Biochemistry of Meat Hydration by Reiner Hamm, 1960, in Advances in Food Research. Vol. 10:366. Academic Press, New York. Hamm, R. 1960. Biochemistry of Meat Hydration. Advances in Food Research. Vol. 10, 355-443. Academic Press, New York. 41 42 Honig, D. H.; Sessa, D. J.; Hoffmann, R. L.; and Rackis, J. J. 1969. Lipids of defatted soybean flakes: Extraction and characterization. Food Tech. 23, 95-99. Ivanov, N. N. 1931. Tsentral Nauch. Issledovatel. Biokhim. Inst. Pishchevoi Vkusovoi Prom., Narkomsnaba S.S.S.R., Separate, p. 36. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vol. 1, 291. Inter- science Publishers, Inc., New York. Jones, D. B. and Csonka, F. A. 1932. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vol. 1, 291. Interscience Publishers, Inc., New York. Klotz, I. 1958. Protein hydration and behavior. Science 128:815. Lloyd, D. J. and Moran, T. 1933. Bound water of gelatin gels. Nature 132:515. Meissl, E. and Bocker, F. 1883. Monatsh., 4, 349-368; Sitzber, Akad. Wiss. Wien, Mathnaturw Klasse, Abt. l, 87, 372-391. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vol. 1, p. 275. Mellon, E. F.: Korn, A. H.; and Hoover, S. R. 1949. Water absorption of proteins. IV. Effect of physical structure. J. Am. Chem. Soc. 71:2761. Mohler, K. and Kiermeir, F. 1958. Die Wirkung anoganischer phosphate auf tierisches Eiweiss II. Mitt. Der Einfluss auf die Quelling von Fleischeiweiss. Z. Lebensm Untersuch u Forsch 96, 90. Quoted in Bio- chemistry of Meat Hydration by Reiner Hamm, 1960, in Advances in Food Research. Vol. 10:364. Academic Press, New York. Muramatsu, S. 1920. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vol. 1:291. Inter- science Publishers, Inc., New York. Olcott, H. S. and Fraenkel-Conrat, H. 1946. Water resist- ance of proteins. Ind. Eng. Chem. 38:104. Osborne, T. B. and Campbell, G. F. 1898. J. Am. Chem. Soc. 20:419-428. Pauling, L. 1945. The absorption of water by proteins. J. Am. Chem. Soc. 67:555. 43 Pusch, I. 1950. Vergleichende Untersuchurger zur Bestimmung der Wasserigkeit und des Ausblutungsgrades in der Fleischuntersuchung. Lebensmitteltierarzt 1, 82. Quoted in Biochemistry of Meat Hydration by Reiner Hamm in Advances in Food Research. Vol. 10. 365. Academic Press, New York. Satow, S. 1921, 1923. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vol. 1:289. Inter- science Publishers, Inc., New York. Schonberg, F. 1937. Die Fleisspapierprobe, ein einfaches Hilfsmittel zur Feststellung des Ausblutungs und Wasserigkeitsgrades im Fleisch, insbesondere bei der Beurteilung von not- und Krankschlachtungen. Berlin tierarztl Wochschr 33, 510. Quoted in Biochemistry of Meat Hydration by Reiner Hamm in Advances in Food Research. Vol. 10:366. Academic Press, New York. Seehof, M.; Keilin, B.; and Benson, S. W. 1953. The surface area of proteins. V. The mechanism of water absorption. J. Am. Chem. Soc. 75, 2427. Sherman, P. 1961. The water binding capacity of fresh pork I. Food Tech. 15:79-86. Smiley, W. G. and Smith, A. K. 1946. Preparation and nitrogen content of soybean protein. Cereal Chem. 23:288-296. Smith, A. K. and Circle, S. J. 1938. Peptization of soybean protein. Ind. Eng. Chem. Vol. 30:1414- 1418. Smith, A. K.: Circle, S. J.; and Brother, G. H. 1938. Peptization of soybean protein. The effect of neutral salts on the quantity of nitrogenous con- stituents extracted from oil free meal. J. Am. Chem. Soc., 60:1316-1320. Sponsler, O. L.; Bath, J. D.; and Ellis, J. W. 1940. Waterbound to gelatin as shown by molecular struc- ture studies. J. Phys. Chem. 44:996. Tadokoro, T. and Yoshimura, K. 1928. Quoted in Markley, K. S. 1950. Soybeans and soybean products. Vo1. 1:291. Interscience Publishers, Inc., New York. Vickery, H. B. 1945. The proteins of plants. Physiol. Revs. 25:347-376. 44 Wierbicki, E.’ Kunkle, L. B.; and Deatherage, F. E. 1957. Changes in the water holding capacity and cationic shifts during the heating and freezing and thawing of meat as revealed by a simple centrifugal method for measuring shrinkage. Food Tech. Feb:69-73. ”71111111111111 (11111311113111ES