Food or edible material: processes – compositions – and products – Products per se – or processes of preparing or treating... – Stabilizing or preserving agent or emulsifier other than...
Reexamination Certificate
2001-08-24
2003-10-28
Carlson, Karen Cochrane (Department: 1653)
Food or edible material: processes, compositions, and products
Products per se, or processes of preparing or treating...
Stabilizing or preserving agent or emulsifier other than...
Reexamination Certificate
active
06638562
ABSTRACT:
This application is a 371 of PCT/JP00/02051 filed Mar. 30, 2000, which claims priority to Japan 898,34/1999 filed Mar. 30, 1999.
FIELD OF THE INVENTION
The present invention relates to a method for fractionating and a process for producing a 7S globulin-rich fraction and an 11S globulin-rich fraction from a soybean protein-containing solution.
BACKGROUND ART
Soybean storage protein is precipitated at about pH 4.5 and can be relatively easily separated from components other than the protein. This is referred to as isolated soybean protein and, in many cases, soybean protein in this form is utilized in the food industry. The protein is further divided into 2S, 7S, 11S and 15S globulins according to sedimentation constants in ultracentrifugation analysis. Among them, 7S globulin and 11S globulin are predominant constituent protein components of the globulin fractions (note: 7S globulin and 11S globulin are classification names in a sedimentation method and substantially correspond to &bgr;-conglycinin and glycinin according to immunological nomenclature, respectively), and both of them have specific different properties such as viscosity, coagulability, surface activity, etc. Then, factionation of 7S globulin and 11S globulin makes it possible to utilize properties of respective protein components, and it is expected to expand industrial utilization of proteins.
7S Globulin and 11S globulin are composed of several subunits. 7S Globulin is composed of three subunits, i.e., &agr;, &agr;′ and &bgr; subunits. 11S Globulin is composed of several subunits each of which is a pair of an acidic polypeptide (A) and a basic polypeptide (B). The molecular weights and charge states of 7S globulin and 11S globulin are very similar to each other. In particular, both globulins are diversified due to combinations of subunits, and properties thereof range to some extent to thereby overlap each other. Then, for fractionating both globulins effectively, a certain essential difference should be found out.
Known fractionation methods are as follows. That is, a method utilizing a difference in isoelectric point: extraction is carried out by adjusting pH to about the isoelectric point of 11S globulin and only 7S globulin is extracted (JP 55-124457 A); a method utilizing a difference in reactivity with calcium: a small a-mount of a calcium salt is added upon extraction to extract a 7S globulin-rich fraction (JP 48-56843 A); a method utilizing a difference in solubility at a certain pH and ionic strength: an insoluble fraction is removed in the presence of sodium chloride or potassium chloride at pH 1.2 to 4.0 to prepare 7S protein (JP 49-31843 A), or a slurry precipitated at an isoelectric point is adjusted to pH 5.0 to 5.6 and its molar concentration of sodium chloride is adjusted to 0.01 to 2 M to separate 7S and 11S fractions (JP 58-36345 A); and a method utilizing a cold-precipitation phenomenon and a reducing agent, etc.: this utilizes a phenomenon that the solubility of 11S globulin is lowered at a low temperature (referred to as cryo-precipitation phenomenon), and a soybean protein raw material is treated in the presence of a sulfurous acid compound, glutathione compound or cysteine compound in an aqueous system at pH 6.5 or higher, followed by adjusting pH to 5.5 to 7.0 and a temperature to 20° C. or lower to fractionate into a 7S globulin-rich fraction and an 11S globulin-rich fraction (JP 61-187755 A).
These known fractionation methods skillfully utilize a difference in solubility between 7S globulin and 11S globulin due to pH, ionic strength, the presence of a certain salt, temperature, etc. However, there are such problems that these known fractionation methods are unsuitable for an industrially applicable fractionation method because clear fractionation of both globulins cannot be achieved due to overlap of their properties as mentioned above or, even if clear fractionation can be achieved to some extent, these methods are still those for the level of experimental use. Thus, problems still remain in practice. For example, in the method of JP 61-187755 A, cryo-precipitation phenomenon highly depends on a temperature and it is necessary to cool a reaction mixture to about 5° C., which results in such a practical problem that a large amount of a sulfurous acid compound, etc. should be added to separate fractions with an industrially available low centrifugal force, as well as which results in such a problem of fractionation precision that a little amount of 11S globulin is contaminated in a soluble fraction.
Then, it has been desired to develop a fractionation method which can simply and efficiently produce a 7S globulin-rich fraction and an 11S globulin-rich fraction in an industrial scale with minimizing contamination of the soluble fraction with the insoluble fraction or vice versa.
On the other hand, phytic acid is an organic phosphate compound (myo-inositol hexakis-phosphate: 6 phosphoric acid groups are attached to inositol) which is predominantly present in plant seeds in the form of its calcium salt, magnesium salt and potassium salt. Soybeans contain about 1% of phosphorus most of which is present in the form of phytin. Since phytic acid forms a slightly soluble compound by being attached to a nutritiously important mineral component (calcium, magnesium, iron, zinc, etc.) through a chelate bond, it is pointed out that phytic acid lowers absorption of these trace minerals in a living body. In addition, phytic acid tends to form a complex with a protein and a multivalent metal cation and, normally, soybean protein contains 1 to 3% by weight of phytic acid based on the weight of the protein. An activity of decomposing phytic acid used herein refers to the activity for liberating phosphoric acid from phytic acid. A representative enzyme having such an activity is phytase.
Utilization of phytase which has an activity of decomposing phytic acid of soybean protein is divided into that for removing phytic acid which is considered to be an inhibitor of mineral absorption and that for recovering a protein having a high solubility under acidic conditions (pH 5 or lower). Examples of the former include those described in JP 49-7300 A, JP 50-130800 A and JP 4-503002 A. Examples of the latter include a method for isolating a soluble protein fraction from a phytic acid-containing protein material which comprises adding phytase to an aqueous suspension of a phytin-containing soybean protein material to decompose phytin, adjusting the suspension to pH about 4.6 to form an insoluble precipitate, collecting a protein solution, adjusting the protein solution to pH about 5.0 to 5.4 to precipitate a protein fraction and collecting the precipitated protein fraction (JP 48-18450 A); and a method for collecting a protein from a vegetable protein raw material which comprises washing the vegetable protein raw material with water at an isoelectric point, digesting the washed vegetable protein raw material with acidic phytase, and separating a soluble protein-containing liquid extract from an insoluble digested residue to collect the protein (JP 51-125300 A).
Phytase is reacted under the following conditions (extracted from Examples of each publication). JP 49-7300 A: endogenous phytase, pH 5, 65° C., 9.3 hours; JP 50-130800 A: wheat phytase, pH 5.5, 45° C., 16 hours; JP 4-503002 A: microorganism phytase, pH 5.0, 40° C., 4 hours; JP 48-18450 A: wheat phytase, pH 6, 50-55° C., 24 hours; and JP 51-125300 A: microorganism phytase, pH 2.8, 50° C., 10 hours.
Since bacteria generally tend to grow at pH 5 or higher, normally, a solution containing soybean protein is liable to be putrefied unless it is subjected to sterilization treatment such as heat sterilization, etc. In addition, since a protein is liable to be denatured with an acid by treatment under strong acidic conditions such as at pH 2.8 for a long period of time, such treatment adversely affects fractionation of 7S globulin and 11S globulin. Further, in the method of JP 51-125300 A, the dissolved fraction is fractionated from “okara (insoluble residue f
Kohno Mitsutaka
Kugimiya Wataru
Saitoh Tsutomu
Tsumura Kazunobu
Carlson Karen Cochrane
Fuji Oil Company Limited
Snedden Sheridan K
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