JP2002515723A - Bioelastomer suitable as food additive - Google Patents

Abstract

  (57) [Summary] The present invention is a method of improving the texture of a food, comprising incorporating a bioelastic polypeptide into the food or a precursor of the food in an amount sufficient to increase the elasticity of the food, A method is provided wherein the bioelastic polypeptide has a tetrapeptide or pentapeptide repeat unit or a mixture thereof, and wherein the repeat unit is in a conformation having a β-turn. The invention also provides a method for binding a food precursor, comprising adding a bioelastic polypeptide in an amount sufficient to bind the food precursor, A method is also provided wherein the stick polypeptide has a tetrapeptide or pentapeptide repeat unit or a mixture thereof, and wherein said repeat unit is in a conformation having a β-turn. The invention further relates to bioelastic polypeptides having tetrapeptide or pentapeptide repeat units or a mixture thereof, wherein said repeat units are in a conformation having a β-turn, and suitable for human or animal consumption. A food additive comprising a novel edible material is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION Bioelastomer suitable as food additive                                   Introduction Technical field   The present invention relates to substances suitable for improving the texture of food products.background   Flour prepared from wheat always has a special place in baking and cooking. ing. Fine flour breads and cakes can be made from flour, From a crushed preparation of cereal grains (often called another name, for example corn meal) Cannot be made. Protein elasticity determines the functionality of flour and dough It is important in that. The proteins involved are the main storage proteins, e.g. Luteinine, which accounts for about 10% of the dry weight of the grain, moistens and kneads the flour When the dough is applied, a continuous network structure called gluten is formed. This net The eye structure traps the carbon dioxide produced by the yeast during the fermentation of the dough, It swells to form a lightly porous aggregate structure. Therefore, fermented bread is essentially To support other flour components (of which the most important component is starch) Protein form.   The ability of wheat gluten protein to trap carbon dioxide and form foam , Depending on a combination of two physical properties, elasticity and extensibility. Insufficient dough Very elastic (weak and too low modulus) or excessively elastic (strong and Sometimes too bad) A good balance of these properties is important is there. Gluten elasticity is also important for other wheat applications, including noodle and pasta production. You.   Wheat gluten consists of more than 50 species that fall into two groups that exist in approximately equal amounts. A complex mixture of the above individual proteins. Gliadin has strong non-covalent bonding (Mainly through hydrogen bonding and hydrophobic interaction), contributing to gluten extensibility. It is a monomer protein to offer. In contrast, glutenin is an interchain disulfide bond High M stabilized by_r(About 1-10 × 10⁶Da) Is it a subunit that forms a polymer? Consisting of These polymers seem to be the main determinant of gluten elasticity, The exact molecular basis for is unknown. However, it may be relevant Two notable features are the number and distribution of disulfide bonds, and gluteninsa. The presence of a β-helix-like structure within one group of buunits.   These proteins are called the high molecular weight (HMW) subunits of glutenin. , Somewhat more detailed because allelic variation in its composition is associated with differences in wheat baking quality [Shewry et al. (1992) J. Am. Cereal Sci. 15: 105-120]. Individual tabs Proteins range in length from 627 to 827 residues (M_rDiffers from about 67,500 to 88,100) Flanked by non-repetitive N-terminal (84-104 residues) and C-terminal (42 residues) regions Center repeat region. Repeat regions are nonapeptides (common GYYPTSP or Are based on LQQ), hexapeptide (PGQGQQ) and tripeptide (GQQ) motifs. Tandem scattered repetitive nucleotide sequences, based on repeated β-turns, It appears to form a helical structure [Miles et al. (1991) Proc. Natl. Acad. Sci. US A 88: 68-71]. Their helical structure has been proposed to be inherently elastic (D Tatham et al. (1985) Cer. Chem. 62: 405- 412; Tatham et al. (1984) FEBS Lett. 177: 205-208] But the mechanism is It is clearly different [Belton et al. Cereal Sci. 19: 115-121]. Non-anti The reversible N- and C-terminal regions are spherical and form crosslinks via disulfide bonds. I think it will.   Elastin is high alanine and lysine containing alternating high glycine hydrophobic sequences. Consisting of a single protein containing a contiguous array of potential cross-linking sequences. Is known For a full-length bovine sequence, the most prominent hydrophobic sequence, from both a length and a composition standpoint, is Including Ripentapeptide (PPP) and Including Polyhexapeptide (PHP) Things. Elastin also contains several tetrapeptide (TP) units. The inventor As a result of a study performed by one of them, when crosslinked, polypentape The peptide is found to be elastic, and its polyhexapeptide is inelastic It was found that the chains were aligned and entangled during elastic development (interloc king). Elastin polypentapeptide and Both polytetrapeptides undergo entropy elasticity and strength when subjected to inverse temperature transition To form a mechanical structure containing regular β-turns. It has also been discovered that this is an application-dependent elastomer.   More importantly, a certain length of cross-linked PPP, PTP and its analogs Elastic activity at different temperatures ranging up to about 75 ° C depending on several controllable variables To show elastomeric force. Furthermore, their crosslinked elastomers Exhibits near maximum elastic activity in a relatively narrow temperature range. Therefore, various moles Modified with hexameric repeat units along with the amount of constituent pentamers and tetramers By synthesizing a bioelastomer material having such units, By selecting a specific solvent that supports the first viscoelastic phase, Strictly the temperature at which the tomer exhibits elastic activity Can be controlled.   In general, the step of raising the temperature to produce the elastic state is different from typical rubber, Create regular, non-random structures using hydrophobic intramolecular interactions as characteristic components The reverse temperature transition that results. This regular structure creates a space between spiral turns. Having a β-turn as a probe, providing a hydrophobic bond between the turns of the helix and water containing loose peptide segments with peptide segments hanging between the β-turns It is proposed to be a β-helix, which is a ricks structure. Those various bikes The elastic activity of an oelastomer develops as its regular structure develops. In addition, loss of the regular structure due to high temperature denaturation results in reduced elastic activity. Monomer -By selecting different amino acids for different positions of the unit, and The cross-linking method used to make the product (eg, chemical, photochemical, enzymatic) , Radiation irradiation method), by a wide range of different water composition, Hydrophobic, in almost any desired morphology and porosity, and variable degree of crosslinking Can prepare those polymers.   These bioelastomer polypeptides were obtained from one of our labs. It is a relatively new discovery that has taken place, It has been disclosed. For example, U.S. Pat. Numerous tetrapeptides and peptides that can be used to make The non-peptidic repeating units are described. Special bioelastic polymers U.S. Pat.Nos. 4,132,746; 4,187,852; 4,589,882 and 4,870. , 055. U.S. Pat.No. 5,064,430 teaches Polynona A peptide bioelastomer is disclosed. Bioelastic polymers Prepared for another purpose Pepti but can also contain bioelastomer polymers in the final polymer Also disclosed in related patents directed to polymers containing repeating units . U.S. Pat. Nos. 4,605,413, 4,976,734 and 4,693,718; / Bioelastomers Containing Pentapeptide Units ”(“ Bioelastomer Containin g Tetra / Pentapeptide Units "), U.S. Patent No. 4,898,926, entitled the invention; “Temperature-related force and structure evolution of elastin polytetrapeptide” (“Te mperature Correlated Force and Structure Development of Elastin Polytetr U.S. Pat. No. 4,783,523 entitled "Apeptide"); Constructed from bioelastomer with modulatable temperature transition for interconversion of mechanical work Reversible Mechanochemical Engines Com ” prised of Bioelastomers Capable of Modulable Temperature Transitions for  the Interconversion of Chemical and Mechani-cal Work ”) U.S. Patent Nos. 5,032,271, 5,085,055 and 5,255,518; "Elastomeric Composite Mate" US Patent No. 4,500,700 entitled "rial Comprising a Polypeptide") Specification; and US Patent No. 5,250,516. Numerous other bioelastics Materials and methods of using them are described in pending U.S. patent applications, including: "Elastomers suitable for preventing adhesion of biological materials Litetrapeptide matrix ”(“ Elastomeric Polytetrapeptide Matrices Suitable for Preventing Adhesion of Biological Materials ”) U.S. Application No. 184,873 filed April 22, 1988; U.S. application filed October 16, 1992 Application No. 07 / 962,608; U.S. Application No. 08 / 187,441, filed January 24, 1994; Filed May 20, 1994 in the United States Application No. 08 / 246,874. These patents and patent applications are And / or their components and their preparation are described in detail, All are incorporated herein by reference. This information can be used to prepare the compositions of the present invention. And use as well as in the method of the invention.   Wheat varieties are screened for favorable properties for food processing (cereal tampering Are primarily storage proteins that are stored for germination), Properties such as elasticity and extensibility that are optimal for the production of noodles, noodles and cakes Not screened during evolution. Recent developments in routine wheat transformation methods [Vasil (1992) Bio / Technology 10.667-675; Weeks et al. (1993) Plant Physiology 102: 1077-1084] takes into account the manipulation of wheat proteins by genetic engineering. Farming To improve the texture of breads, cakes and other spices made from food Milling dough, especially other economically valuable cereals that are less commonly used, e.g. Improve the quality of mills made from corn, oats, rye and millet The need still remains.Related literature Summary of the Invention   The present invention relates to a method for producing a food precursor or food precursor in an amount sufficient to increase the elasticity of the food. Food texture by incorporating bioelastic polymers into the material Provide a way to improve security.   The present invention provides bioprecursors for food precursors in an amount sufficient to bind the food precursor. Method for binding food precursors by adding elastic polymer I will provide a.   The invention also relates to a tetrapeptide or pentapeptide repeat unit or a A bioelastic polymer comprising a mixture, and a human or animal Provide a formulation consisting of edible ingredients suitable for cost. The formulations vary in structure and vary Can provide different properties and can be used in sheet, gel, foam or powder Available in a variety of physical forms such as Has the potential.                             BRIEF DESCRIPTION OF THE FIGURES   BRIEF DESCRIPTION OF THE DRAWINGS The present invention is described in detail below with reference to certain embodiments, taken together with the drawings that form a part hereof. A better understanding may be had by reference to the description.   Figure 1 is (GVGVP)_n1 is a schematic diagram of the gene cloning step.   A. 1. Neighboring outer BamH1 and inner PflM1 restriction endonuclease recognition sites (GVGVP)_TenFor synthetic genes. The gene and plasmid pUC118 were each Cleavage with H1, mixed with ligase, the plasmid into which the gene was inserted Was recyclized. Plasmid containing the cloned gene was Amplified in coli I let you. 2. Cleavage of large-scale plasmid preparation with PflMI and free (GVGVP)_TenPurify the fragment Was. 3. Concatenated gene was prepared by self-ligating the purified PflM1 fragment . 4. Add the adapter oligonucleotide to the ligation reaction and add (GVGVP)_TenHeredity Ends containing restriction sites necessary for cloning offspring in various expression plasmids Sequence provided. Genes are referred to in multiples of 10, but adapter oligonucleotides Since the tide gives an additional pentamer sequence, it ends up [(10)_n] +1.   B. Illustration of the process described in A.   Figure 2 shows (GVGVP) of tobacco_Ten1 shows the sequence of a gene.   FIG. 3 is a schematic diagram of the plasmid pHD203-GUS-EPSPS (EN / polyA).                             DETAILED DESCRIPTION OF THE INVENTION   The introduction of protein-based elastic polymers into food or food precursors is To provide the desired properties of texture and consistency, or Use it to take away and replace unwanted properties such as fat and calories. Can be. Protein-based elastic polymers are not only edible, Those having different physical forms can also be manufactured. For example, they are Hui May be an alternative to cream or used to make meringue Can be squeezed out of the aerosol can as a foam. They are like Can be used in pastry layers and coatings. As foams, foams, malleable materials such as gums, sheets and viscoelastic substrates Dope them with a flavor so that they can be (See U.S. Patent Application No. 07 / 962,608 filed October 16, 1992. Teru. This specification is incorporated herein by reference). Protein The adhesive properties of the base elastic polymer allow the polymer to adhere to other processed crops. It can be used to act as a binder.   Now the availability of these protein-based elastic polymers in food is Clearly, expansion to countless uses is not hard to imagine. With the present invention , In terms of production and disposal, and in terms of nutrition, which is protein instead of fat, It becomes environmentally friendly. In addition, bioproduction of protein-based elastic polymers is sufficient. Low cost is particularly important for products that have properties that are less desirable in other ways. When used in combination with economically viable crops that will give Provide inexpensive resources for consumables.   Bioelastic polypeptides have been previously characterized and have It has been described in a number of patents and patent applications. Those materials are monomers Tetrape, which individually acts as an elastomer unit, in all polypeptides containing Contains either peptide, pentapeptide or nonapeptide monomers. This The elasticity of the monomeric units of the dynamics that hang during the β-turn Protein secondary structure (ie, its Likely due to the presence of a series of β-turns in the peptide chain conformation). It is. The β-turn is characterized by a hydrogen bonded 10 atom ring of the formula:In this formula, R₁~ R_FiveRepresents a side group of each amino acid residue. The 10-atom ring is the first The carbonyl oxygen of the amino acid, the amino hydrogen of the fourth amino acid, and the second and third amino acids Consists of intervening skeletal atoms of the amino acids of the eye. In this monomer unit as shown the chain Remaining skeletal atoms (the remaining amino acid at the fourth, the fifth amino acid, and the next The first part of the first amino acid of the tantameric unit) hangs between adjacent β-turns Make up the bridging segment. Elastomers of similar construction or other lengths It is also present in peptide units. Other peptide structures, such as β-barrels, Elastic polymers can be made elastic. Bioelastici ty) extends the polymer Imparts an internal damping of the dynamics of the chain when it is Provided by a structure that is free to rotate or vibrate around the mating. This Reduces the degree of freedom obtained in the extended state.   This β-turn-containing structure is described in the above-mentioned prior patents and patent application specifications. As such, it need not be described again in detail here. To preserve elasticity Multiple β-turns with intervening hanging bridge segments are retained Substantial mutations in amino acids at various positions in the repeat unit are possible . Except as imposed by the process of making those polymers, they are used in the present invention. It appears that there is no upper limit on the molecular weight of the polymer to be obtained. Up to about 250 pentamer units A polymer containing E. coli was obtained using recombinant DNA technology. It is synthesized in E. coli. Typical At least 5, preferably at least 10, more preferably at least Also contains 20 tetrapeptide or pentapeptide monomers, and To squeeze foam from an aerosol can because it increases the viscosity at Contains usually less than 1000, usually less than 500, such units, but is used for fabric production. Can be of any length, and the maximum length that can be obtained is Would be preferable. In addition, these monomer units can be used to Polypeptides scattered throughout longer polypeptides, including peptide segments Can be prepared. For example, to increase the modulus or special Hard segments can be included for cross-linking to achieve a perfect alignment . Lysine or tryptic digestion to improve the digestibility of the polymer Protease cleavage sites such as arginine or pepsin Can be introduced. The invention relates to other molecules, such as proteins or peptides. Do, lipid, charcoal Bioelastomer interspersed between segments of hydrates or other organic compounds Polymers having-units can also be used.   The upper limit on the number and type of substituents that can be attached to the polymer is Bullets that fold / assemble correctly to obtain a β-helical structural element in a relaxed state Is affected by the ability of the conductive polymer. Polymers related to monomer residue side chain positions The positions of the substituents in the middle are heavy as long as the β-turn is not formed in the relaxed state. It is not necessary. Preferred positions for various peptides are incorporated by reference. Taught in patents in this field and pending patent applications from the inventor's lab It is as it is.   Prototype Poly (Val¹-Pro^Two-Gly^Three-Val^Four-Gly^Five[Poly (VPGVG) in the present specification ] And Poly (Val¹-Pro^Two-Gly^Three-Gly^Four) Molecules and countless analogs These bioelastomer materials, which include, form a viscoelastic phase when mixed with water. The phase, when cross-linked, results in a soft, compliant, elastic matrix. Crosslinking of the polymer solution to form an elastic matrix can be achieved by various crosslinking methods, For example, it can be performed using a chemical method, an enzymatic method, or a radiation irradiation method. Rice No. 4,589,882 (incorporated herein by reference) Teaches enzymatic cross-linking by synthesizing block polymers with cross-linkable units ing. Applying the teachings of this patent to recombinant bioelastic polypeptides be able to. No. 4,589,882 describes synthetic polymers. Bioelastic with the required ratio of lysine to glutamic acid residues Recombinant technology is used to generate vectors encoding the polypeptide. This In this case, the enzymatic cross-linking method involves the action of lysyl oxidase, which is an extracellular enzyme. It progresses more in vivo. Alternatively, cross-linking in vivo can result in glutenin binding. Next that seems to be It can occur via a disulfide bridge in a similar manner. In this case, the bio Elastic polypeptides can form interchain disulfide bonds with each other. Engineered to include a possible cysteine residue.   In general, the choice of the sequence of amino acids in a particular monomer unit and the The selection of the required ratio determines (or investigates) the properties of the known bioelastomer. Made similar but different bioelastomers, and cited patents and As described in the patent application, the transition temperature (T_tExperimental) starting with measuring) Can be achieved by law. However, preferably, the amino acid residues (natural The transition temperature without experimentation using the relative hydrophobicity table (either natural or modified) calculate. For example, Y. Nozaki & C. Tanford, J. Biol. Chem. (1971) 246: 2211- 2217 or H.B. Bull & K. Breese, Archives Biochem. Biophys. (1974) 161: 6 65-670, as well as particularly useful compilations of hydrophobicity data, are described by D.C. Urry (1993) Ang ewandte Chemie Omt. Ed. Engl. See 32: 819. For example, polymer objects The average hydrophobicity of each amino acid residue in the mer unit is summed, and the result is By comparing to the sum obtained for the polymer with the transfer temperature, or_t Hydrophobic scale based on US patent application Ser. No. 08 / 187,441 filed Jan. 24, 1994 The sum of the transition temperatures from Table 1) and dividing it by By obtaining the average value, a rough estimate of the transition temperature can be obtained.   To determine the transition temperature of a series of related polymers with only one component changed Thus, a more accurate value can be calculated for any given polymer. An example For example, a variable amount of VPGXG monomer (e.g., 2 %, 4% and 8% X, where X is any selected amino acid residue) Containing polymer Can be prepared and tested for transition temperature. This test is simply The rimer is prepared in uncrosslinked form, the polymer is dissolved in water and the polymer from solution And raising the temperature of the solution until "turbidity", which indicates precipitation of the solution, appears. Turn When the transfer temperature is plotted against the ratio of VPGXG monomer in the polymer, a straight line is obtained. The ratio of VPGXG monomer obtained and required from this graph for another desired temperature Rate (within the limits indicated by 0-100% VPGXG monomer) directly Obtainable. This technique should be able to estimate the total hydrophobicity as described above When combined with can obtain any desired transition temperature in the region of liquid water it can. It should be understood that the position of "X" is chosen only as an example in the above description X can be at any position in the repeating unit and It can be in multiple locations as long as the overall properties are maintained. Elastomer alone The choice of position and the individual amino acids that synthesize the resulting polypeptide can be The resulting structure is described, for example, in U.S. Pat.Nos. 4,474,851 and 5,064,430. Elastomer formation having the features described herein, especially including β-turn structures Unlimited, as far as it can go. Thus, the amino acid sequence of the elastomer unit is obtained. Is selected to provide the desired properties to the food product to be prepared.   The synthesis of bioelastomer repeat units is not tedious; Can be easily achieved. Synthetic bioelastomers are patented and patented by the inventor. It can be produced and crosslinked by the method described in the patent application . In that case, the bioelastomer polypeptide is a food precursor during the production of food. Is added to Bioelastomer polypeptides increase the elasticity of foods Will be added in a sufficient amount. Usually 1% to 99.5% by weight depending on the application About 10% for the production of dough, and whipped cream Nearly 100% will be added to the system.   An alternative to organic synthesis of protein-based polymers is to use trendy recombinant DNA This is a biosynthetic approach using technology. Using this approach, the desired pepti A gene encoding the coding sequence is created and artificially inserted into the host organism, and then The host organism produces the peptide. The host can be a prokaryote, such as a bacterium, It can be a eukaryote, such as a yeast, a plant. Using recombinant DNA, given Synthetic genes encoding multiple repeat units of the selected peptide sequence. And self-polymerize those synthetic genes to create longer coding sequences, Long chain protein-based polymers can be produced. In a suitable host organism Manipulating genetic information (ie, DNA sequences) in preparation for their efficient expression in Molecular biology techniques known in the art are used [eg, Sambrook et al., Molecular Clon. ing: A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York (1989); D eguchi et al. (1993) Mat. Res. Soc. Symp. Proc., 292: 205-210; Capello, J. (199 2) Review Protein Engi-neering Biomaterial, Curr. Opin. Struct. Biol., 2 : 582-586; McPherson et al. (1992); Perbal, B. (1988) A Practical Guide to Mo. Aucular, F. lecular Cloning, 2nd edition, John Wiley & Sons NY; M. (1989) Curr ent Protocols in Molecular Biology, Volumes 1 & 2, John Wiley & Sons NY Teru]]. The main tools that make this possible are well known in the art, for example Includes enzymes that cleave, ligate, replicate and otherwise modify oligonucleotides . In addition, a vector that enables this information to be introduced into the host organism in a form suitable for expression. Tar is also known in the art. When the host is a microorganism, the protein produced is Large quantities can be purified from cultures grown in fermenters and produced synthetically Method similar to bioelastomer Can be added to the food precursor. Specific examples of production of poly-VPGVG include: McPherson et al., "Production and P," a publication from one of our labs. urification of a Recombinant Elastomeric Polypeptide, G- (VPGVG)₁₉-VPGV, from Escherichia coli ", Biotechnol. Prog., 1992: 347-352, and" Bio Overproduction of elastic polypeptides "(" Hyperexpression of Bio-elastic Polypeptides ") and a" BioElastic "Simple Method for the Purification of Bioelas tic Polymers ") (both filed on the same date as the present application) ). They are useful for gene production and purification of the material of the invention. Can be used as a guide for and incorporated herein by reference. .   Certain crops have been engineered by techniques involving plasmid-mediated DNA insertion into the chromosome. Or random impact of plant cell lines using DNA-coated microprojectiles It can be transformed by bombardment. The latter method uses chloroplasts and nuclear genomes. It is used for transformation. Multiple copies of the chloroplast genome are Chloroplasts for gene amplification and protein overproduction This is advantageous for targeting the last. Exotic tans produced by plants Protein may involve the production of storage or intracellular proteins, More recent studies have identified suitable leader sequences for secretion of transgenic proteins. I'm using   The produced polypeptide can be obtained by techniques known in the art [eg, Scopes, R.K. (1987) ) Protein Purification, Springer Verlag, NY; McPherson et al. (1992)] Can be isolated and described above for proteins expressed by microorganisms. Use the same as be able to. Alternatively, a host selected for processing into food, for example, wheat, Express polypeptides in corn, oats, rye, millet and legumes Can be done. Methods for transforming plants are well known in the art [Shimam oto, K. (1989) Nature 338: 274-277; Datta, S.K. Other (1990) Bio / Technolo gy 8: 736-740; Cristov, P .; Et al. (1991) Bio / Technology 9: 957-962; Gordon-Kamm (1990) The Plant Cell 2: 603-618; Fromm, M.E. et al. (1990) Bio / Techn. Vasil, V. et al. (1992) Bio / Technology 10: 667-674; Weeks, J. et al. T. et al. (1993) Plant Physiol. 102: 1077-1084; Somers, D.A. Other (1992) Bio / Tec hnology 10: 1589-1594; Bower, R .; And Birch, R.G. (1992) Plant J. 2: 409- 416; Kung, S. and Wu, R. (1993) Transgenic Plants Engineering and Utili zation 1: 382; Daniell, H. (1993) Methods in Enzymol. 217: 536-556].   Artificial protein-based polymers, such as elastomeric poly (VPGVG) [Poly (GVGVP), poly (GVPGV) or other permutations are equivalently described. Bioproduction can take place in bacteria or in plants. In addition, the following are advantageous: (i) Codons suitable for the codon preference of the chosen expression host Synthesize genes with multiple tandem copies of peptide coding units using (Ii) that the synthetic gene can be easily cloned into a vector plasmid and Should be transferable as a cassette to the expression vector of (iii) synthesis The gene is then ligated to the desired polypeptide using the appropriate transcriptional and translational regulatory signals for the expression host. Expressed in a peptide product, preferably (iv) a form in which the polypeptide allows for purification. And (v) the presence of foreign amino acids relative to the VPGVG repeat sequence. To be avoided or removed after expression.   Briefly, for example, a gene encoding 10 repeat units of an elastomeric peptide Is made using synthetic oligonucleotides and cloned into an expression vector. To run. Genes for bioelastomer polypeptides may contain additional oligonucleotides Certain additional repeats using amide and PCR (eg, US Pat. No. 4,683,202) It can be further extended to units, allowing 10-mer genes to overlap. This technology is Insert 3 'of the unit gene (10 mer in this case) to insert each column addition of the unit. It relies on the presence of an appropriate restriction enzyme recognition site at the end. This module Use the approach to build continuous genes, increasing the size of each additional unit Can be Essential issues in generating genes with multiple repeated copies Is one of E. Possible recombination and deletion in E. coli. Genetics using repetitive sequences The offspring can be prepared using an appropriate host strain (for example, recA^-) When used in poly (GVPV) No problem for G).   Alternatively, the “concatemer” route is used to produce high molecular weight polymer proteins. It is possible. One is a double chain, each encoding a bioelastomer peptide unit Involves the use of oligonucleotide monomers, which are linked through sticky ends Ligated to form a multimeric sequence (concatemer) encoding the polypeptide It is a method. Another route combines a modular approach with the concatemer route. In combination, having a terminal restriction enzyme site that when cleaved leaves a nonpalindrical cohesive end, When a gene encoding a peptide unit multiple repeat sequence (for example, 10 mer) is created, It is a route called. High molecular weight polymer by linking those gene monomers Multimers that encode can be produced. This alternative is Easier and faster than high-molecular-weight genes Function.   According to the broadest aspect, the invention relates to a method for producing foodstuffs in an amount sufficient to increase the elasticity of the foodstuff. Or by incorporating bioelastogenic polypeptides into food precursors Food additives and methods for improving food texture are provided. The present invention Biodegradation of food precursors in amounts sufficient to bind them. Also provides a way to bind food by adding stick polypeptide I do.   A food additive as used herein is an edible material suitable for human or animal consumption. Ingredients such as synthetic or natural flavors such as peppermint, lemon or basil Leek essence, and sweeteners such as saccharin, aspartame or sugar Can be formed into various physical forms, with the optional inclusion of It can be used with those precursors.   The method of the present invention increases the elasticity and extensibility of a food or its precursor. This improves the texture of the food. For example, wheat in dough production The quality of its elongation and elasticity when making dough by mixing with water (these are mainly Is a property provided by the protein of the present invention). Their quality is general In addition, by examining the baking quality, for example, SDS precipitation It has been measured by measuring velocity [Moonen et al. (1982) Euphytica 31: 6 77-690]. Standard and micro baking tests are described, for example, in Smak, C. (1972) Cer. Chem. 49554-560 or Meppelink (1981) Getrelde, Mehl und Performed as described by Brot 35: 107-109. Alternatively, the viscoelasticity of the dough Can be measured directly.   Improved dough viscosity then results in improved food texture, for example, Make foods that are less likely to be crumbling or disintegrating. This It is a mill that cannot currently be processed into high quality dough, for example Suitable for milling from corn, corn and oats.   In this case, the bioelastomer can be added directly to the mill or Introduce their specific properties (ie, elasticity and extensibility) into the flour obtained from the product Can be expressed by transgenic plants. VPGVG pair Motifs or, for example, VPGVG, IPGVG and VPAVG Intrinsically elastic subunits based on combinations produce the preferred elastic module I can do it. Baking is polymer T_tSo that it takes place at a higher temperature It is only important that the repeating unit is selected for the first time.   Such subunits are converted to natural proteins by interchain disulphide bonds, For example, incorporation into gluten is augmented which is optimal for bread making and other applications Will result in elasticity and improved quality.   Furthermore, by incorporating polyhexapeptide units into the bioelastomer, Structure, eg (pentapeptide)_n(Hexapeptide)_n(Pentapeptide)_nStructure Construction can be provided. For example, (GVGVP)_n(GVGIP)_n(GFG VGP)_nOffers a soft polymer that can be used as a chewing gum base. Offer. The ratio of repeating units may be different for the flavor used, which may further soften the gum. Will often depend. However, the ratio will be known to those of skill in the art in light of the teachings of the present application. It can be determined more easily.   The bioelastomer is formed according to the teachings of the inventor's patent application and patent specification. It can also be machined so as to have adhesion. Their properties bind food precursors Used for food production, in which case about 1% to 99.5% by weight of the food precursor % By weight of bioelastomer Is present, preferably about 10% to 90% by weight, more preferably about 30% to 70% by weight. % Is present.                                  Example Example 1 Expression of genetic constructs encoding bioelastic polymers   Using a synthetic oligonucleotide having the sequence depicted in FIG. 1 (GVGVP )₁₂₀Was prepared. The oligonucleotide was ligated with BamH1 (G'GATCC) and PflM1 (CC AGGCGTTGG) flanked by sequences containing restriction endonuclease recognition sites. this The nucleic acid was inserted into plasmid pUC118 and Used to transform E. coli Was. After isolation of the amplified plasmid, the sequence of the gene insert was Analysis [Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Sp ring Harbor, New York (1989)].   This 10-mer gene is then converted to a high molecular weight (GVGVP)_nLong chain encoding It was used as a module unit for producing a gene. Plastic containing 10-mer gene Sumids were prepared and digested with PflM1. Then PflM 1 (GVGVP)_TenPurify gene fragments Then [(GVGVP)_Ten]_nIn subsequent ligation reactions to make Used. It also allows for subsequent cloning of linked gene fragments For this purpose, separate adapter oligonucleotides with unique restriction sites Added to the ligation reaction. These adapter oligonucleotides have high molecular weight It was added in a ratio that would be preferable for recovery of the "Temers". This step is schematically illustrated in FIG. 1B. expressed.   For recovery and cloning of individual length concatamer genes, the ligation mix is used. The mixture was digested with BamH1, then electrophoresed through an agarose slab gel to give various Different molecular weight sizes were fractionated. Sections corresponding to various size ranges are then removed from the gel. Cut, collect DNA, And cloned into plasmid pUC118 [Urry et al., “Elastic and Protei n-based Polymers: Potential for Industrial Uses ”(Am. Chem. Soc.) Div. P olym. Mat., Sci. & Engr., “Industrial Biotechnological Polymers,” Washi ngton, D.C., 1994]. Insert the gene insert in this plasmid with the restriction endonuclease. Agaro adjacent to concatamer “ladder” analyzed by ase digestion The size was accurately fractionated by gel electrophoresis. Native protein-based poly Marker (GVGVP) for expression in E. coli₁₂₀Conca to code The timmer gene is used as a gene fusion behind the sequence encoding the six-fold histidine. It was subcloned from pUC118 into the expression vector pQE-32 (Quiagen, Inc.). Expression using this plasmid is compatible with proteins with an amino-terminal polyhistidine fusion. Quality, specifically MRGSH₆GIQTM- (GVGVP)_nProduced. This fusion component Ability to affinity purify proteins by metal chelate chromatography Empower. Several different sized concatemer genes in pQE-32 vector Subcloned and E. coli. E. coli. Poly (GVGV) produced P) The polymer is affinity purified from bacterial cells and reversed phase chromatograph Of phenylisothiocyanate (PITC) derivatives of amino acids separated by Amino acid analysis shows that glycine and valine required for poly (GVGVP) at the expected ratio Proven to have phosphorus and proline. This allows high molecular weight Protein based polymers such as (GVGVP)₂₅₀Create a gene that encodes And E. Efficient expression in E. coli host organisms Is established. Using a gene fusion and purification approach, Purify the protein to high purity and also prove that it is the desired product It was possible.Purification of expression products   Affinity-purified MRGSH₆GIQTM- (GVGVP)_nUse to raise or lower the temperature The protein-based polymer precipitates reversibly (ie, Coacervate). When entering or leaving this solution, the liquid becomes cloudy It can then be determined visually by observing the transparency.   Using this property, (GVGVP)_n(Where n = 40, 140 and 250) The histidine fusion protein consisting of was purified. First, a protein-based polymer E. Dissolve the cooled (on ice) suspension of E. coli cells by sonication. The components were dispersed. At low temperatures, poly (GVGVP) is in a spread soluble state It remains and insoluble cell debris can be removed by centrifugation. Still cooled As it was, the cell lysate was centrifuged at high speed to remove insoluble cell debris. Then recovered Warm the supernatant to 37 ° C to form visible aggregates from the protein-based polymer At which time it was separated from the soluble fraction by centrifugation. 37 ℃ it When heated above the transition temperature of the poly (GVGVP) species, the poly (GVGVP) Form a new phase that takes into account selective removal by the mind. Wash this operation again When repeated as purification, analysis was performed by SDS-polyacrylamide gel electrophoresis. Sometimes resulted in a product that was as pure as affinity purified material. like this As a result, more than 90% of bacterial endotoxins were removed.Example 2 E. FIG. Expression and purification of native poly (GVGVP) _{120 in} E. coli   E. FIG. To achieve expression of native protein-based polymers in E. coli , (GVGVP)₁₂₀Concatemer gene encoding pUC described in Example 1 The expression vector from 118 as an Nco1-BamH1 fragment And subcloned into the vector pET-11d (Novagen, Inc.). Previously described High-level protein expression from this plasmid without an amino-terminal affinity component Was done. The protein was also described in Example 1 using the temperature-induced aggregation method. Purified efficiently as follows.Example 3 An elastic protein-based polymer, poly (GVGV), in tobacco chloroplast Expression of P)   Coding poly (GVGVP) protein in the range of 150 tandem pentapeptide units Synthetic oligonucleotide encoding 10 repeat units of GVGVP And PCR for expression in tobacco systems. Figure 2 shows the maximum code degeneracy Figure 2 shows the sequence of a gene comprising codons optimal for tobacco while maintaining. The gene Describes two oligonucleotides, each representing slightly more than half of the gene. We make using. The oligonucleotides have a complementary 25 base overlap ( (Dotted underline in the drawing), which is extended by PCR to form a full-length double-stranded sequence I do. Digest the PCR product with BamH1 for sequence confirmation and Inserted into Rasmid pUC119. The fragment is excised from pUC119 using PflM1 and the desired Self-ligate, including adapter fragments, to end multimerization over a range of sizes To form a concatemer.   Essentially described by Daniell [Methods Enzymol. 217 (1993) 536-556]. As described above, the synthetic gene was used in chloroplasts of cultured tobacco cells using Gene Gun. Is introduced.³⁵Continuously transforming cells in MS saline medium in the presence of S-methionine After culturing, a mini-bead beader [Daniell et al. (1993) Nucleic Acids Res. 21: 1 503-1504] to isolate chloroplast. Chloroplast in hypertonic buffer To obtain soluble chloroplast protein You. Purify the bioelastomer essentially as described in Example 1. You.Example 4 Expression of a protein-based plastic polymer, poly (AVGVP), from tobacco   Encodes a range of poly (AVGVP) in the range of 150 repeat units for expression in tobacco Different approaches are used to generate the genes. In detail, each is (AVGV P) Codon suitable for tobacco, representing one chain of a double-stranded unit encoding a monomer Two 15 base degenerate oligonucleotides with selection, 5'-CCNGCNGTNGGNGTN-3 'and Synthesize 5'-CNGGNACNCCNACNG-3 '(where = G, A, T or C). These two Strands leave 4-5 base overlapping ends that are complementary when they anneal, leaving 5 It is an offset so as to allow the connection between the 'and 3' ends. (GVGV P)_TenAs described above for the gene, the annealed oligonucleotide, Links catemers through their complementary termini, forming long-chain multimers, Form a timmer. Bioelastomer polypeptide expression and purification Essentially the same as above, but using the transition temperature of poly (AVGVP).Example 5 Expression of poly (VPGVG) after stable chloroplast or nuclear transformation   Plasmid pHD203-GUS-EPSPS (EN / polyA) (see Fig. 3) contains the aroA gene (gly Encoding an EPSP synthase that confers resistance to fosate) and 35S promoter of CaMV flanked at the 3 'end by a polyA fragment to stabilize the transcript Contains a motor / enhancer element. G- (VP fused with gst coding sequence GVG)₁₉-VPGV The (20-mer) coding sequence can be modified by using an adapter or E. coli. Coli DNA Filling in the concave 3 'end using the Klenow fragment of polymerase I Thus, it is inserted at the BglII site in pHD203-GUS-EPSPS- (EN / polyA). gst- Bombarding an EPSPS vector containing the EG20 mercoding sequence into tobacco cultured cells, and Stable expression was achieved by growing them in the presence of glyphosate. It is. The coding sequence is tobacco chloroplast to achieve high frequency transformation. Inserted into the region of the genome between rbcL and ORF 52 [Svab and Maliga (1993) Proc . Natl. Acad. Sci. USA 90: 913-917].   Transgenic tobacco plants expressing polymers inside chloroblasts By shooting leaves from aseptically grown plants with chloroplast vectors can get. Selection and regeneration of tobacco chloroplast transgenic plants Optimal conditions for this are well known in the art [Svab et al. (1990) Proc. Natl. Acad. Sci. U SA 87: 8526-8530; Staub and Maliga (1992) The Plant Cell 4: 39-45; Staub And Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917].Molecular and biochemical analysis of chloroplast transformation   Chloroplast DNA from transgenic plants by methods known in the art Is isolated. Examine ethidium bromide stained gels of ctDNA preparations digested with restriction enzymes To detect additional ctDNA fragments. Chloroplastec into the tobacco ct genome Insertion of the EPSPS / gst-EG20 mer fragment from the transgenic plant Introduce additional restriction sites into the system. Digest ctDNA with restriction enzymes and use agarose Separate by electrophoresis on a gel and blot on a nylon membrane. EPSPS Alternatively, a fragment containing gst or polymer coding sequence Use as a probe. All transges Genetic system with the EPSPS-gst-polymer coding sequence in the tobacco chloroplast genome. Test for the presence of The chloroplast extract was prepared as described in Example 3. And purify the protein.Example 6 Stable nuclear expression of protein-based polymers in transgenic tobacco   Synthetic gst-G- (VPGVG)₁₉-VPGV gene cassette (Mc Pherson et al.) As follows Insert into the pKYLX vector [Schardl et al. (1987) Gene 61: 1-11]. The cassette Was modified to change the Xho1 / Nco1 adapter (5′-TCGA GCCATGG-3 '/ 3'-CGGTACC-5') to incorporate a new ATG codon, and And transfer it into pKLYX7.1 as an Xho1 / EcoR1 fragment. Xba1 site replaced by EcoR1 site PKYLX7.1 [Daniell et al. (1986) Proc. Natl. Acad. Sci. USA 83: 248-2 55), a derivative of pKLYX7.2, synthesized gst-G- (VPGVG)₁₉-Contains VPGV cassette Use to   Young fully opened tobacco leaf [Nicotian tabacum (Nicotiana tabacum ) Cv KY 14] was collected from an 8-week-old plant, and the surface was washed with 10% chlorox. Sterilize for 0 minutes, then for 3 minutes in 70% alcohol, and wash three times with sterile distilled water You. Agrobacterium tumefaciens (Agrobacterium tumefaciens)But Mediated leaf transformation and stem regeneration are described in Horsch et al. (1985) Science 227: 1229-123. Perform as described by 1. Briefly, Svab et al. [Proc. Natl. Acad . Sci. USA (1990) 87: 8526-8530]. Putative transformation on MS medium containing mycin and 500 mg / l mefoxin Choose a body. Transfer kanamycin resistant stems to rooting medium. About 50 kanamycin resistant Sexual plantlets are selected for analysis. Control plants were transformed using only pKYLX7.2 Exchange.   Putative transformants were confirmed by Southern hybridization and NP TII phosphotransferase activity and gst-G- (VPGVG)₁₉-VPGV protein (McP herson et al.) assay for production. Field experiments with selected transformants For the production of homozygous individuals to be used as first ancestors of seeds to be used in select. About 1 acre (about 4047 square meters) of each type of transgenic Grow cigarettes. Cultivate plants transformed only with vectors and untransformed plants The small plot serves as a growth control or other control. Seedlings They were bred in the greenhouse and transplanted in the test plot. Cell extraction as described in Example 1 Prepare the product (Scopes et al.) And purify the polymer.   All publications and patent applications mentioned in this specification are It was noted that the patent application was specifically and individually incorporated herein by reference. Are incorporated herein by reference to the same extent as   Having described the invention well, it departs from the spirit or scope of the appended claims. It will be apparent to those skilled in the art that many changes and modifications can be made without .

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 C12N 15/00 A (81) Designated country EP (AT, BE, CH, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN , TD, TG), AP (KE, LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU , AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK , TJ, TM, TT, UA, UG, UZ, VN (71) Applicant University of Bristol United Kingdom, Bristol BS 189 A.F. (No address), Department of Agricultural Science, Long Ashton Research Station (72) Inventor Early, Dan Wright. United States, Alabama 35216, Birmingham, Vestavia Road 2423 (72) Inventor Shawley, Peter Earl. United Kingdom, Bristol BBs 66 Edabru, Cozam Blow 73 (72) Inventor Cali , Yuma Ezwara Prasad, United States, Pennsylvania 19446-4363 Lansdale, Winfield coat 1005

Claims (1)

[Claims]   1. a method of improving the texture of a food, which increases the elasticity of the food The food or precursor of the food in a sufficient amount to Incorporating a repeptide, wherein said bioelastic polypeptide is Is a tetrapeptide, pentapeptide or nonapeptide repeating unit or And wherein the repeating unit comprises a β-turn. A method characterized by being present in an application.   2. The bioelastic polypeptide is used during production of the food product during the production of the precursor. The method of claim 1, wherein the method is added to the quality.   3. The bioelastic polypeptide is a recombinant protein Item 1. The method of Item 1.   4. The method of claim 3, wherein said recombinant protein further comprises glutenin.   5. The recombinant protein is expressed in a host selected for processing into the food. 4. The method of claim 3, wherein   6. said host is from wheat, corn, oats, rye, millet and beans 6. The method of claim 5, wherein the method is selected from the group consisting of:   7. A method for binding a food precursor, said binding a food precursor. Including adding a bioelastic polypeptide in an amount sufficient to Wherein the bioelastic polypeptide is a tetrapeptide or pen Or a mixture thereof, wherein the repeating unit comprises β A method characterized by being in a conformation having turns.   8. Food additives, including tetrapeptide or pentapeptide repeat units. Or a mixture thereof, wherein the repeating unit is β- Polypeptides in a Conformation with a Chain And food additives comprising edible materials suitable for human or animal consumption.   9. The method wherein the bioelastic polypeptide is used as a foam Item 8. The food additive according to Item 8.   Ten. 9. The food of claim 8, wherein said bioelastic polypeptide is a malleable material. Goods additives.   11． 9. The food additive of claim 8, wherein the bioelastic polypeptide is a gel. Addictive.   12． 9. The food of claim 8, wherein said bioelastic polypeptide is a sheet. Additive.   13. Wherein the bioelastic polypeptide is substantially cross-linked. Item 8. The food additive according to Item 8.   14. The bioelastic polypeptide is substantially uncrosslinked. 8 food additives.   15． The bioelastic polypeptide further comprises a hexapeptide repeat unit 9. The food additive of claim 8, comprising a food additive.   16． Whether the edible material is saccharin, sugar, peppermint and aspartame 9. The food additive of claim 8, wherein the food additive is selected from the group consisting of: