SOYBEAN PROTEINS

Legumes are widely grown to be utilized directly as foods for utilization of their oil and for utilization of the oil and proteins. Table 1 gives the composition of some common legumes. This data indicates that peanuts provide the greatest percentage of oil while soybeans contain the greatest amount of protein.

Until very recently in the U.S., soybeans were grown for the value of their oil and the protein was considered as a by product of much less economic importance. The current price of soy protein and the fact that soybeans contain twice as much protein as fat has made these components of about equal economic importance.

Types of Soy Products

Full-fat soy flours are made from dehulled whole soybeans. The dehulled beans are steam treated to inactivate the anti-nutritional factors and to denature the enzyme lipoxygenase. These products are very similar in composition to whole soybeans as the hull comprises only about 10% of the total bean. The composition of a typical full fat flour is given in Table 2. Removal of the soybean lipid by either mechanical means or by solvent extraction yields defatted soy flour. The protein content of this flour must be at least 50% of a dry weight basis. This flour serves as the starting point for further processed soy products.

Further purification of the soy protein yields the formation of soy protein concentrates which by definition have a dry weight protein content of at least 70%. Defatted soy flour is used as a starting material and the soluble carbohydrate material is removed. The soluble sugars (mostly sucrose, raffinose and stachyose) can be removed by one of three procedures. The defatted soy flour can be washed with dilute acid at pH 4.5. This is the isoelectric point of the main soy protein and they are insoluble at this pH if the salt content is low. Another procedure is to soak the flour in a mixture of alcohol and water that contains enough alcohol to prevent the proteins from becoming soluble but enough water to remove the carbohydrate. In the final procedure, the soy proteins are denatured with steam to make them insoluble and the soluble carbohydrate is then removed by a water wash.

The composition of the concentrate produced by all these procedures is about the same and a typical one is presented in Table 2. Use of alcohol or heat causes extensive loss of protein solubility in the finished products.

To produce soy protein isolates which by definition must contain greater than 90% protein on a dry basis, defatted flakes are utilized as a starting material. The soluble protein is removed by extraction with dilute alkali (pH 7-9) at moderate temperatures. The insoluble carbohydrate (fiber) and some proteins are left behind. The proteins are then recovered from the soluble carbohydrates and ash by isoelectric precipitation at pH 4.5. The precipitated proteins are called soy curd and the supernatant fraction is called whey. The curd is then washed, sometimes neutralized, and then dried. Table 2 contains a typical composition for a soy protein isolate.

Classification of Soy Proteins

Most soy proteins are insoluble in water at their isoelectric point, but are solublized in the presence of salt. The addition of NaCl to 0.7N will make most of the soy proteins soluble at their isoelectric points. This behavior is typical of proteins that have classically been classified as globulins and thus we frequently refer to the soy proteins as globulins.

Studies of soy proteins by analytical ultracentrifugation in a phosphate buffer of pH 7.6 with an ionic strength of 0.5 containing 0.01M mercaptoethanol has revealed the presence of four Schlieren peaks. These peaks have approximate Svedberg coefficients of 2S, 7s, 11S and 15S. These fractions are not homogeneous but rather are mixtures of proteins. They have been studied to differing degrees and have been characterized.

2S Proteins

The 2S fraction has been reported to contain from 8 to 22% of the extractable soybean protein and consists of a number of enzymes. The fraction has been assigned an average molecular weight of 26, 000, but as we will see, it is composed of a number of proteins with a variety of molecular weights.

Probably the most studied protein of the 2S fraction are the trypsin inhibitors. The smaller of these inhibitors has been termed the Bowman-Birk inhibitor. It consists of 71 amino acid residues and has a molecular weight of 7,861. Its primary sequence is known and is shown in Figure 1. While the protein has a low molecular weight, it has been shown to form dimers and trimers in solution which probably explains its association with the other 2S protein.

This molecule is a highly symmetrical protein composed of a number of tight rings. These rings are held together by the presence of 7 disulfide bonds. While soy proteins as a whole are low in sulfur-containing amino acids, this inhibitor has 14 of its 71 amino acids composed of cysteine. The trypsin inhibiting site is the bond between lysine -16 and serine -17. While trypsin would normally cleave a Lys-Ser bond, it appears that the ridged ring structure in which this bond exists prevents the cleavage from occurring. Thus, trypsin binds to the molecule, but can not cleave the bond and thus is not readily released to cleave other molecules.

At the opposite end of the molecule, leucine 43 and serine 44 make a bond that can interact with chymotrypsin, but for similar reasons to those presented for the Lys-Ser bond is not cleaved. This protein, while named the Bowman-Birk trypsin inhibitor, can also inhibit the activity of chymotrypsin.

The high number of disulfide bonds in the small protein molecule make it a very rigid structure and tends to inhibit thermal unfolding of the molecule. The gain in conformational entropy upon complete denaturation of the molecule is also considerably lowered by the presence of the seven disulfide bonds making this protein very resistant to denaturation.

The other main trypsin inhibitor of soybean is the Kunitz inhibitor. This is a much larger molecule being composed of 181 amino acids with a molecular weight of about 21,500. This protein contains two disulfide bridges and is not as rigid a molecule as is the Bowman-Birk inhibitor. Its primary sequence is presented in Figure 2.

Arginine 63 and isoleucine 64 comprise the bond at the active site of the inhibitor. It appears that trypsin can actually cleave this bond, but that the enzyme can not be released from the inhibitor once contact is made. Figure 3 shows the complex formed between the Kunitz inhibitor and porcine trypsin at a resolution of 5A. In the area of contact between the two molecules, the Arg-Ile bond contacts the active site of the trypsin molecule. The x-ray diffraction patterns of the contact area of the inhibitor-protein complex shows that about 300 atomic contacts exists between the two proteins. Apparently, these contacts are strong enough to resist interaction with water and render the trypsin catalytically inactive. While the feeding of non-heat treated soy proteins to animals has been demonstrated to have a detrimental affect on growth due to the trypsin inhibitor, they can be inactivated almost completely by commercial heat treatment.

7S Fraction

The 7S fraction of soy protein comprises about 35% of the soluble protein. It contains some enzymes, a number of hemagglutinins and a protein known as the 7S globulin. The enzyme of greatest commercial significance in this fraction is lipoxygenase. A protein having a molecular weight of about 105,000. This enzyme causes the formation of hydroperoxides by the addition of oxygen to the double bonds that occur in linoleic and linolinic acid. Breakdown of these hydroperoxides can lead to further lipid oxidation and the creation of off-flavors in the soy protein. Much of the typically objectionable, beany flavor often associated with soy protein is thought to arrive from the oxidative deterioration of lipids that are tightly complexed with protein molecules.

For most uses of soy proteins, the minimal heat treatment required will be designed to inactivate this enzyme. In some applications, however, low-heat soy proteins are added that contain active lipoxygenase. These particular applications often require that a moderate amount of oxidation be allowed to take place. This oxidation can cause the destruction of pigments and thus a bleaching of the products. It can also cause the oxidation of free sulfhydryl groups to form disulfide bonds. Both of these functions can be of importance in the preparation of freshly ground flour for the baking industry.

The 7S fraction also contains a group of molecules called hemagglutinins. These carbohydrate containing proteins are thought to be made up of four possibly non-identical subunits, each having a molecular weight of 30,000 to yield complete molecular weights of 120,000. These proteins have the ability to cause the aggregation of red blood cells in vitro and hence, their name. In some foods, hemagglutinins have been shown to lower the utilization of proteins in vitro, but they seem to have no affect on the protein quality of soybean proteins.

The majority of the 7S protein consists of the 7S globulins also called conglycinin. This protein makes up about 85% of the 7S fraction and consists of from 4 to 6 different components and contains about 6% carbohydrate. The molecular weights of the various 7S fractions range from 140,000 to about 170,000. At low ionic strengths, the 7S globulins form dimers with molecular weight of about 280,000 to 350,000 and a sedimentation coefficient of 9S.

Recently, the 7s globulins have been shown to consist of three subunit proteins labelled a, a1 and b. The a1 and a subunits have molecular weights of 57,000 and can be separated by ion exchange chromatography. The b subunit has a molecular weight of 42,000. This data suggests that native 7s globulins are composed of trimers of their subunits. Six possible combinations of three subunits, i.e: aaa, aaa1, aab, aa1b, abb, and a1bb would explain the presence of six fractions of 7S globulins and would have the correct molecular weights. This scheme is shown diagrammatically in Figure 4.

At concentrations below 7%, total protein, soy protein are extremely heat stable. The 7s proteins show a thermal transition at 77C by use of differential scanning calorimetry. The proteins do not, however, precipitate from solutions after such heat treatments and are stable toward heat treatments in excess of 100C. As the ionic strength of the heating medium increases, the 7S globulin became more heat sensitive.

11S Fraction

The 11S fraction comprises from 31 to 52% of the soluble soy protein with about 85% of the total being, the 11S globulin. The 11S globulin is also called glycinin and it's reported molecular weight range from about 320,000 to 360,000.

Glycinin is the major component of a fraction of proteins recovered from soybeans by cold precipitation. This cryoprecipitation is characteristic of the 11S globulin. When the 11S globulin is completely dissociated, fractions having molecular weights of 34,800 and 19,600 are recovered. The larger species have isoelectric points of from 4.75 to 5.4 and are called the acidic subunits. The smaller components have isoelectric focusing results indicate that the three acidic subunits have isoelectric points of 4.75, 5.15 and 5.40. The basic subunits were also separated into three groups having isoelectric points of 8.0, 8.25 and 8.5. The acidic subunits have been shown to contain three proteins with differing N-terminal amino acids, eg., Leucine, Isoleucine and Phenylalanine. The basic subunits all contain a terminal glycine.

It appears that the intact 11S globulin is a dodecimer compound of six basic and six acidic subunits. Under proper conditions, the 11S can be fractionated into units of ~ 7S. These 7S units appear to be composed of three acidic and three basic subunits. The 11S protein can also be broken to yield 3S subunits which appear to be dimers consisting of one acidic and one basic subunit each. The bond between the acidic and basic subunits might be a disulfide linkage. Reduction of this bond yields individual subunits of about 2S which are individual acidic and basic subunits.

This data has been interpreted to mean that the native 11S globulin is composed of two 6-numbered rings stacked one on top of the other as shown in Figure 5. Each ring is composed of three acid and three basic subunits. It has been suggested that one each of the three types of acidic and basic units comprise a given 6-numbered ring.

When isolated 11S proteins are heated to 80C, a portion of the protein precipitates. Examination of the precipitate shows that it consists almost exclusively of basic subunits. The acidic subunits remain soluble after this heat treatment. When whole soybean protein is given a similar heat treatment, no precipitation occurs. Recent work has shown that the addition of isolated 7S globulin to the 11S fraction will prevent the precipitation of the basic subunits.

This heat stability can be of benefit when it is desirable to obtain proteins that can be processed at high temperatures and maintain their solubility. In other cases where partial gelation of the proteins is desired, this heat stability can be a detriment.

15S Protein

The final fraction of the extractable soy protein is the 15S fraction. This material comprises about 5% of the total extractable protein. It is only poorly characterized and is thought to be composed of polymers of the other soy proteins.

TABLE 1. Proximate compositions of various legumes.

Table 2. Proximate composition of soy products.

Figure 1. Primary sequence of the Bowman-Birk trypsin inhibitor.

Bowman-Birk in Rasmol is here The inhibitor complexed with trypsin is presented here.

Figure 2. Primary sequence of the Kunitz trypsin inhibitor.

The Rasmol version of the structure is here.

Figure 3. Complex formation between trypsin and the Kunitz trypsin inhinitor.

Figure 4. Model of b-conglycinin, the major 7s soybean globulin.

Physicochemical properties of isomers of b-conglycinin:

Subunit Molecular Weights:

a = 57,000

a¢ = 57,000

b = 42,000

Figure 4. Model of b-conglycinin, the major 7s soybean globulin.

Basic subunits = B and acidic subunits = A

Physicochemical properties of Glycinin

N - Terminals

Leu = 2

Ile = 2

Phe = 2

Gly = 6

Molecular weight

Acid subunit = 34,800

Basic subunit = 19,600

Total Glycinin = 326,000

Figure 5. Model for glycinin, the major soybean 11S globulin.