Abstract:
Hydrothermal processes are provided for preparing hybrid proteins containing altered SS/SH bonds, thereby yielding hybrid proteins having enhanced functional properties. The processes involve steam treatment of an aqueous protein-containing slurry containing at least two different proteins in a jet cooker ( 16 ) or similar device in order to heat shock and thereby alter the conformation of some of the proteins, followed by relatively rapid cooling to cause formation of the desired hybrids. Plant and animal proteins may be processed, and the starting slurry can be pH-modified and/or supplemented with one or more additional ingredients (e.g., salts, phosphates, fatty acids, polysaccharides, alcohols, aromatic compounds). The hybrid proteins are useful as food ingredients (e.g., solubility, wetability, dispersibility, foaming, emulsification, viscosity, gelation or thickening agents).

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is broadly concerned with processes for the production of hybrid proteins formed by the interprotein and/or intraprotein rearrangement of SS/SH bonds in a plurality of different starting proteins, in order to obtain hybrid proteins having desired functional characteristics. More particularly, the invention is concerned with such processes and the resultant hybrid proteins wherein an aqueous, protein-containing slurry comprising at least two different proteins is hydrothermally treated in a jet cooker or similar device in the presence of steam, in order to alter the conformation of at least some of the proteins. The treated slurry is then cooled to cause the formation of hybirds proteins, which are recovered by spray drying or similar methods. 
     2. Description of the Prior Art 
     Proteins are essentially composed of linear chains of amino acid residues linked together by peptide bonds which join the nitrogen atoms of amino groups to the carbon atoms of preceding carboxyl groups. All amino acids have identical backbone structure and differ only in their side chains. The physiochemical properties of amino acid residue side chains and the sequence of these residues are the dominant factors in determining the structure and function of proteins. Protein molecules also vary widely in size, e.g., enzymes may vary in size from about 13 kDa up to several thousand kDa. 
     The structure of proteins is recognized at four distinct levels of importance. The most basic level is the primary structure, i.e., the sequence of amino acid residues in the chain. The secondary structure of proteins relates to the conformation of amino acid residues which are relatively close to one another in the chain. Three conformations are known: α-helix, β-pleated sheet and aperiodic (also known as random coil). The tertiary structure of proteins refers to the spatial structure thereof, resulting from hydrophobic and electrostatic forces, and disulfide bridges between aliphatic and aromatic side chains of the protein. Hydrophobic interactions are the major forces responsible for tertiary structure. The fourth and last protein structure is quaternary structure. This essentially describes the nature of the assemblage of protein subunits to form a massive aggregated molecule. 
     The properties of food and proteinaceous feed ingredients may be placed in two categories, namely nutritional and functional properties. Functional properties are defined as those properties of a food or food ingredient that affect its utilization, or influence the behavior of the food or food system during processing, handling, storage, preparation and consumption. For a given protein to perform well in a food system, it should normally possess multiple functionalities. For example, egg white possesses multiple functionalities including foaming, emulsifying, heat setting, and binding/adhesion. The functional properties of any protein are basically related to its physiochemical and structural properties including size, shape, amino acid composition and sequence, net charge, charge distribution, hydrophobicity/hydrophilicity ratio, and the secondary, tertiary and quaternary structural arrangements. 
     Efforts have been made in the past to modify or rearrange proteins in order to alter the functional properties thereof. For example, European Patent No. 782825 describes a method of rendering whey protein more hydrophobic in order to improve its gelling properties. Commercially available whey protein concentrate was heated to 75° C. along with sodium or magnesium caseinate, giving the resultant protein an increase in hydrophobicity. Lasztity et al.,  Narung,  42:210 (1998) studied wheat germ protein systems modified with urea to disassociate quaternary structures, β-mercaptoethanol to reduce SS bonds and aeration to reoxidize SH groups to SS bonds. This treatment altered the surface protein properties of the wheat germ protein. 
     The dissertation of Ballegu,  Effect of Hydrothermal Process on Functional Properties of Wheat Gluten Isolate  (2001), describes hydrothermal processing of wheat gluten isolate using a jet cooker. HPLC profiles of the recovered protein samples revealed polymerization of gliadin molecules through aggregation and/or crosslinking to give glutenin or glutenin-like molecule; the extent of polymerization was found to depend upon the process severity. The viscosity of the hydrothermally processed wheat gluten isolate was found to be higher than that of the control, regardless of processing conditions. 
     Other references include: Cosio et al.,  J. Dairy Sci.,  83:1933 (2000); Apichartsrangkoon,  Food Sci.,  67:653 (2002); U.S. Pat. Nos. 4,038,431, 4,500,454, 3,754,926, 5,100,679, 5,068,117, 4,036,996, 3,965,268, 4,038,432, 4,062,987, and 4,650,856; and Japanese Patents Nos. 356021568, 362146659, 361227739 and 360030645. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to the formation of hybrid proteins from plural, different starting proteins. Broadly speaking, the method of the invention involves providing an aqueous, protein-containing slurry including at least two different proteins therein. This slurry is introduced together with steam into a pressurized injection zone, where the proteins are treated under conditions of heat and pressure and for a time sufficient to alter the conformation of at least some of the proteins. Following such hydrothermal treating, the treated slurry is cooled and the hybrid proteins are recovered. 
     Preferably, the aqueous starting slurry should have a solids content of no more than about 35% by weight, and the conditions within the pressurized injection zone should be selected so that a temperature of from about 100-350° F. and a pressure of from about 10-150 psi are maintained. The residence time of the slurry within the injection zone should be on the order of 10 seconds to 2 ½ minutes. The cooling step is preferably carried out over a short period of time (about 10-60 seconds) to achieve a temperature of from about 50-150° F.; cooling may be accomplished by exposure to the atmosphere and/or by supplemental cooling. The dried hybrid protein products should have a moisture content of from about 3-10% by weight, wet basis. 
     Hybrid proteins in accordance with the invention find particular utility in food systems, serving as solubility, wetability, dispersibility, foaming, emulsification, viscosity, gelation or thickening agents, depending upon the specific properties of the hybrid proteins. The processes of the invention can be tailored to enhance particular properties of the starting proteins. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic illustration of a suitable processing apparatus in accordance with the invention; 
         FIG. 2  is a schematic representation of a preferred type of jet cooker used in the process of the invention; and 
         FIG. 3  is a schematic representation illustrating a mechanism for the production of hybrid proteins using the process of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     A suitable apparatus  10  for carrying out the process of the invention is schematically illustrated in  FIG. 1 . Broadly speaking, the apparatus  10  includes a steam injection assembly  12 , a slurry preparation and injection assembly  14 , a jet cooker  16 , and a recovery assembly  18 . 
     The steam assembly  10  includes an inlet valve  20  with an inline separator  22  and filter  24  leading to electronically controlled valve  26 , the output of the latter leading to the steam inlet  28  of jet cooker  16 . The assembly  14  includes a slurry feed tank  30  together with a preheat tank  36 ; the latter has product line  38  directed to product pump  40 . The outlet of the pump  40  leads to the slurry inlet  42  of cooker  16 . 
     The jet cooker  16  is further illustrated in  FIG. 2  and includes a main body  44  having steam inlet  28  and slurry inlet  42  coupled thereto, as well as a processed slurry output line  46 . Internally, the body  44  presents a converging passageway  48  leading to the output line  46 . An adjustable valve member  50  is disposed within passageway  48  and is axially shiftable therein by means of rotatable adjustment wheel  52 . It will be observed that the member  50  presents a conical wall  54  which generally mates with the adjacent defining wall surfaces of the body  44 . As will be readily appreciated, the body  50  may be adjusted to provide a greater or lesser clearance between the conical wall  54  and the adjacent main body wall surfaces. This in effect creates a restricted pressurized injection zone  56  within the confines of the body  44 . 
     The recovery assembly  18  includes a product conveying line  58  equipped with appropriate valving, and leading to a three-way diversion valve  60 . One output leg  62  of the valve  60  leads to flash chamber  64  permitting flash of steam to the atmosphere with consequent cooling. The slurry output from chamber  64  is directed to a heated kettle  66  coupled to product collection tank  68 . The recovered slurry within tank  68  is then passed via line  70  to a conventional spray dryer. The opposite leg  72  from valve  66  passes to plate-type heat exchanger  74 , operated using conventional cooling system  76 . The output  78  from exchanger  74  may pass to kettle  66  or directly to tank  68 . As will be readily appreciated, the assembly  18  thus allows the user the option of cooling solely by exposure to ambient atmosphere, or with supplemental cooling via exchanger  74  prior to drying. 
     In use, the apparatus  10  functions to treat protein slurries so as to create hybrid proteins having desired functional characteristics. As explained above, in broad terms the method of the invention involves providing an aqueous, protein-containing slurry made up of at least two different proteins; this slurry is introduced along with steam into a pressurized injection zone, and the proteins are treated therein under conditions to alter the conformation of at least some of the proteins. Thereafter, the treated slurry is cooled and hybrid proteins are recovered. 
     The incoming slurry can have a solids content of up to about 50% by weight, but more preferably it is dilute and should have a solids content of up to about 35% by weight and still more preferably from about 0.5-20% by weight. The total protein content of the starting slurry is generally in the range of from about 3.5-45% by weight, and more preferably from about 10-45% by weight. 
     A wide variety of proteins may be used in the invention, but advantageously the selected proteins should themselves be concentrated, i.e., the protein-bearing materials used should have a protein content of at least about 65% by weight, more preferably from about 70-90% by weight. In terms of soy protein for example, either soy concentrate (typically around 75% by weight soy protein) or soy isolate (typically about 90% by weight soy protein) should be used in lieu of lower protein concentration products such as soy flour or meal. Virtually any combination of proteins may be employed, i.e., the proteins may be selected from the group consisting of plant and animal proteins. Exemplary plant proteins are selected from the group consisting of soy, wheat, oat, rice, peanut, cotton seed, corn, sorghum, fruits, and mixtures thereof, whereas, suitable animal proteins are selected from the group consisting of beef, poultry, pork, milk, whey, eggs, and mixtures thereof. It should also be understood that the starting proteins may be native proteins or may be modified by any known means such as chemical or enzymatic modification. To give but one example, deamidated gluten may be used in the invention along with another protein such as corn zein. 
     In many cases, it is desirable to alter the pH of the starting protein slurry to a pH approximating the average isoelectric point of proteins within the slurry. In practice, pH levels of from about 2-9, and more preferably from about 3.5-7.5 are useful; in many cases, a pH modification to a level of from about 2-4 or from about 7-9 are effective. 
     If desired, the slurry may also be supplemented with additional ingredients designed to achieve further or different protein hybridization. Thus, the slurry may include one or more additional ingredients such as those selected from the group consisting of sulfur-containing compounds such as bisulfites or SO 2  (20-200 ppm), oxygen (20-200 ppm), alkali metal and/or alkaline earth metal salts (e.g., chlorides, bromides, or carbonates, about 0.01-2% by weight), phosphates (poly and pyrophosphates, 0.01-2% by weight), C12-C22 fatty acids (0.01-2% by weight), polysaccharides (e.g., xanthan gum, 0.1-2% by weight), C1-C4 aliphatic alcohols or aromatic compounds (e.g., toluene, 0.1-10% by weight). The foregoing additional ingredient levels of use are approximate, and are based upon the total weight of protein in the slurry taken as 100% by weight. 
     The processing conditions within jet cooker  16  are selected so as to alter the conformation of at least some of the proteins within the starting slurry. Thus, temperature conditions of from about 100-350° F. should be maintained within zone  56 , more preferably from about 250-350° F. Pressure conditions in the zone  56  are typically maintained at a level of from about 10-150 psi, more preferably from about 60-135 psi. Retention time within the zone  56  should be from about 10 seconds to 2 ½ minutes, and more preferably from about 25-125 seconds. 
     The treated slurry exiting jet cooker  16  via output line  46  is normally cooled in order to assist in the formation of hybrid proteins. It is preferred that the treated slurry be cooled to a temperature of from about 50-150° F., and more preferably from about 75-125° F. Moreover, such cooling should be done over a relatively short period of time usually from about 10-60 seconds and more preferably from about 15-40 seconds. In some instances sufficient cooling may be obtained simply by flashing the product to the atmosphere. 
     The treated slurry, whether cooled or not, is advantageously dried to permit recovery of the hybrid proteins. A variety of techniques may be used for drying, but most efficient drying is carried out in a conventional spray dryer. The moisture content of the final recovered hybrid proteins should be from about 3-10% by weight, or more preferably from about 4-7% by weight, wet basis. 
     Although not wishing to be bound by any theory, it is believed that hybrid proteins are formed in the process of the invention by the combination of heat shock effected in the jet cooker  16 , followed by cooling.  FIG. 3  schematically illustrates an exemplary process wherein wheat gluten and egg proteins are co-processed in a jet cooker. In the jet cooker, the protein heat shock effectively alters the conformation of the starting protein molecules. Thereafter, upon release to atmospheric pressure with or without cooling, the heat shocked proteins reform by the rearrangement of SS/SH bonds. This SS/SH bond rearrangement may occur interprotein or intraprotein or both as shown in  FIG. 3 , so that the hybrid protein molecules are different from the starting proteins owing to changes in gross amino acid composition, and/or the quantity of disulfide bonds or thiol groups present. Thus, the hybrid proteins have different charge densities (domains), which correspondingly alters the hydrophobic and hydrophilic properties thereof. The overall hybrid protein hydrophobicity and hydrophilicity, along with rearrangement of disulfide bonds therein, essentially decides the status of the secondary, tertiary and quaternary protein structures which in turn influences the functionality of the hybrid proteins in food systems for example. Moreover, these alterations in the hybrid proteins will impact upon their molecular surface related properties (solubility, wetability, dispersibility, foaming and emulsification), and hydrodynamic properties (viscosity, gelation, thickening).