WO2009036493A1

WO2009036493A1 - Wheat gluten modified for food application

Info

Publication number: WO2009036493A1
Application number: PCT/AU2008/001369
Authority: WO
Inventors: Robert John Pearce; Amy Lee Barrie
Original assignee: Shoalhaven Starches Pty Ltd
Priority date: 2007-09-19
Filing date: 2008-09-16
Publication date: 2009-03-26
Also published as: AU2008301212B2; AU2008301212A1

Abstract

There is a process for producing a modified wheat gluten for incorporation into a protein enriched food. The process includes the step of adding a protease enzyme to said wheat gluten in aqueous dispersion under conditions of predetermined pH, ionic strength, temperature and lime whereby selective peptide bond cleavage is obtained to give a degree of hydrolysis for said gluten in a range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons being less than 15% of total proteinaceous material, a content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material and a glass transition temperature in a range of about 50 C to about 59 C.

Description

Wheat gluten modified for food application

Field of the invention

The present invention relates to wheat gluten ingredients that have been enzymaticalr) -modified to render them suited for incorporation in extruded and expanded foods and to such foods with consequent enhanced nutrition due to incorporation of such enzymatically-modified wheat gluten ingredients.

Background to the invention

The provision of foods providing adequate nutrition for children and adults is necessary for good health and well being. Foods are selected by consumers for qualities including taste, appearance, convenience, texture, mouthfeel, satisfaction and enjoyment in addition to the nutrition. US Patent 6,899,905 reports tasty, ready-to-eat nutritionally balanced food compositions prepared by selecting fat and fiber ingredients with minerals and cofactors in formulated foods with low water activity in a variety of prepared food forms. US Patent 5,894,027 describes breakfast cereal products coated with a cold-water-soluble coating made from milk solids or high protein, vitamins and mineral supplement powders adhering to the surface of the cereal. It eliminates the need for liquid milk and requires only water to generate similar taste, color and sensation at consumption.

Consumers may select a satisfactory mix of fresh foods and prepared foods for the preparation of main meals to provide adequate nutrition. Children and adults may select foods between main meals often referred to as snack foods. Advisedly these snack foods may be items of fresh foods including fresh fruits and vegetables containing low levels of fat and salt and assessed as having a low glycaemic index as defined in "Glycemic index of foods: A physiological basis for carbohydrate exchange'. (Jenkins, Thomas, Wolever, Taylor, Barker, Fielden, Baldwin, Bowling, Newman, Jenkins & Goff, 1981) Alternatively, foods manufactured for flavour, convenience and enjoyment may be selected that may contain levels of fat, salt, sugar and starch considered to be excessive for healthy living and likely to cause consumers to be overweight as reported in 'The Australian Diabetes, Obesity and Lifestyle Study'. (2005) The provision of a high concentration of protein in snack products has been described in US Patent 6,051,492 wherein the protein, particularly soy protein, is provided in a textured form with fruit or savoury flavour and a semi-chewy or crisp texture. By contrast, US Patent 4,126,705 describes a process for making a dehydrated protein snack food from protein material such as raw meat, poultry, fish etc.

US Patent 4,183,966 describes a method for manufacturing a high protein snack food from cheese whey and yeast mixed with a starch base from potato or corn starch and other ingredients to make dough that can be extruded into pieces that can be fried.

US Patent 4,212,892 describes a method of making a high protein snack food from a protein-rich gel derived from fish or soybean mixed with starch or flour to make a mass that can be extruded in the form of chips.

US Patent 4,124,727 describes the preparation of nutritionally balanced protein snack foods prepared from legume seeds. After combining with cereal grain flours and water, a dough is formed that can be extruded into sheets cut in pieces and fried.

High protein snack foods are also described in Canadian Patent 1021994 in which a puffed fried snack food contains protein and gelled starch derived from non-oilseed legumes, for example; peas, faka beans, white pea beans and kidney beans. Cereal flour for example from wheat corn or rice and vegetable protein concentrate may also be included. The final protein content is normally 12 to 30% and the starch content is 40 to 70%. For reasons of improved palatability, generation of flavour, provision of nutritional availability and improved functionality for incorporation into foods, proteins may be modified by enzymatic processes.

US Patent 3,753,728 describes a process for the incorporation of soy protein at levels greater than 20% into a ready-to-eat breakfast cereal. Soy protein is made more palatable by subjecting it to partial hydrolysis using a mixture of proteases, one of which is papain.

Enzymatic hydrolysis of proteins is well known to generate flavour in products. Such flavour may be considered detrimental for the use of the product in foods and a process such as described in US Patent 4,482,574 may be used to minimise such flavour development. By contrast extensive proteolytic digestion may result in flavours of a savoury nature deemed desirable as seasonings and flavorants. Proteins from animal meats have been hydrolysed with enzymes for flavour as seasonings is described in Korean Patent No KR20060083452. US Patent 6,803,062 describes a process in which a hydrolysed protein can be used as seasoning that is first substantially sterilised by heating under acidic conditions before treatment with proteolytic enzyme derived from a microorganism. For further flavour enhancement US Patent 6,036,983 describes a method of obtaining protein hydroly sates in which the level of free or peptide bound glutamic acid is increased by subjecting the substrate to a deamidation process in addition to a proteolytic process using specific enzymes.

Combining protein hydroly sates may facilitate the provision of a desired nutritional composition of amino acids as described in US Patent 6,420,133, wherein a mixture of different oilseed flours is used as substrate for successive enzymatic reactions to get a final product having optimum composition of amino acids. The hydrolysate so obtained has molecular weight in the range 2500+/- 1000 to 10,000+/-l,500 Daltons. US Patent 5,520,935 describes a process for the preparation of a pea protein hydrolysate with very high purity and with desirable organoleptic properties that is fully soluble and low in phytate. Degree of hydrolysis was in the range 15- 35% and the final hydrolysate was prepared from the process stream permeating an ultrafiltration membrane with molecular weight cut-off above 5000 Daltons.

US Patent 7,112,424 provides a process for the recover} of protein from soy flour in high concentration by means of preparation of a protein hydrolysate from soy flour using a sequential hydrolysis first with fungal protease followed by papain achieving up to 45% degree of hydrolysis and obtaining solubility of 95- 98%.

By enzymaticalrj -modifying vegetable glycoprotein isolates with an acid protease such as pepsin, the glycoproteins are functionally- modified for use as an egg albumin replacer or whip stabilising agent. The enzymatic alteration described in US Patent 4,409,248 produces a glycoprotein with different physical and functional properties from the precursor source materials. The enzyme modified glycoproteins are capable of forming white opaque heat-set gels similar to those of egg albumin.

US Patent 6,171,621 describes the preparation of a liquid protein hydrolysate food which may be a hypoallergenic infant food in which a protein substrate solution is sterilised by ultra-heat processing and into which a limited amount of protease enzyme is introduced that previously was sterilised by microfiltration. The hy drolysis is allowed to progress until the entire enzyme is consumed. This method overcomes deterioration of quality in the form of impaired coloration or taste, instability of emulsions or decreased nutritional value.

US Patent 5,486,461 describes the conversion of essentially insoluble rennet casein to a fully soluble hydrolysate using three defined proteolytic enzymes to achieve a degree of hydrory sis of between 15 and 35% and having a number average molecular weight in the range 400-650 Daltons. Wheat protein has a unique amino acid composition and structure which in its natural intact form provides for its widespread use in bakery applications. The high content of glutamine can readily be converted to glutamic acid and is utilised in wheat protein hydrolysates for generating savoury flavour. US Patent 6,036,980 discloses a process in which glutamine-rich proteins including cereal proteins and wheat gluten are enzyme hydrorysed and the hydrolysate added to the feedstock for glutamic acid fermentation.

More recently increased glutamine intake has been shown to improve recovery from physical stress and various food formulations have been supplemented with either the free amino acid, L-glutamine, or by use of wheat protein hydrolysates generated by use of protease enzymes on wheat gluten. Such wheat protein hydrolysates have identical glutamine bioavailability but allow foodstuffs incorporating such hydrolysates to be heat processed (Barr, Magliano, Zimmet, Polkinghorne, Atkins, Dunstan, Murray & Shaw, 2006). US Patent 6,864,230 describes such a process, product and application.

Wheat protein is also well recognised for its role in eliciting the autoimmune condition known as coeliac disease. Enzymatic hydrolysis to fragment the protein to an extent that it might not elicit such condition has been described in US Patent 6,692,933 which discloses a method for producing a glutamine-rich gluten-free preparation from wheat gluten. A fully soluble product is obtained with a degree of hydrolysis up to 30%.

Protein hyroh/sates are also used in many surface-active formulations by virtue of their dispersing properties and their ability" to influence the dermatological compatibility of anionic surfactants by interaction with the protein molecules of the skin. US Patent 5,945,299 discloses a method which overcomes previously encountered difficulties of discoloration and instability in storage. In the preferred embodiment of the invention, wheat gluten is modified sequentially with proteinases first at pH 2 - 5, then at pH 8-10. Finally the resulting hydrolysate is treated with peptidases at pH 6-7. The outcome is a wheat protein hydrolysate most preferably- in the range 2,000 - 5,000 Daltons which after filtration processes results in a clear solution which shows particularly high stability in solutioα

In common with all the above disclosures of inventions of wheat protein hydrolysates, is the high degree of hydrolysis and high level of solubility of the product. By contrast the object of the present invention is to provide for a wheat gluten enzymaticaHy-modified only to the extent that is required for the purpose of suitability as an ingredient in protein-rich extruded and expanded foods.

Processes for preparing expanded and extruded snack food products have been described, for example by Matz (1984). Early manufactured snack foods were essentially based on fried potato starch dough pieces to achieve the crispness and flavour of fried thin potato slices, as described in US Patent 3,997,684.

Variously such snack foods may be made from dried starch pieces in which the starch is wholly or partially gelatinised and dried during passage through a hot double roller dryer and the resulting film is cut in to pieces and fried as described in US Patent 4,140,803

Alternatively, wet dough containing fully gelatinised starch at 30 to 85% solids may be cut into pieces and fried as described in US Patents 3,297,450; 3,451,822 and 3,539,356.

Furthermore wet dough may be prepared containing a mixture of gelatinised and ungelatinised starch at 30 to 70 solids as described in US patent 3,997,684. With such dough when cut into pieces and fried, the expansion is only about 1.6 times the original volume compared to over three times expansion by the processes described in the previous categories. Processes and products have been described that result in snack food products with improved nutrition by incorporation of various fibre ingredients such as bran. Furthermore, grain materials, meals and flours from a variety of sources may be used as in US Patents 2,701,200; 3,656,966 and 4,526,800 to produce palatable snack food products with a fibre content useful in the diet.

Traditional Mexican food specialities, such as tortillas and tacos, made from corn have given rise to various corn-based foods. Traditionally com kernels are steeped in hot lime solution to soften and allow removal of the outer hull, the remainder being worked into a plastic dough of about 50% solids known as masa. Thin pieces of such dough may be formed by extrusion and fried as in US Patents 2,002,053; 3,083,103 and 3,278,311. As a result of the initial high moisture the fried products are hard and brittle and take up high fat content, 35- 40%. Processes have been described to reduce the moisture content before frying as in US Patents 2,905,559; 3,690,895. As a result,.fat content may be reduced to 20-25%. In order to overcome uneven expansion, masa can be dried and the masa flour later rehydrated to form a dough for extrusion or sheeting, as described in US Patent 4,623,548. In order to produce corn-based fried snacks with uniform porosity and expansion and lower fat content a dough may be prepared from a mixture of dry solids incorporating both raw or partially gelatinised, low water absorption flour and pre-gelatinised high water absorption flour as also described in US Patent 4,623,548. A raw starch component comprising one or more ungelatinised starches should also be included. The combination of the three components in the dough at the time of frying is critical to the process described in US Patent 4,769,253.

The ingredients used to make corn chips, potato chips or expanded foods contain little or no gluten and consist essentially of starch but without gluten hydrated starch or gelatinous composition does not form a workable or sheetable dough. US Patents 4,834,996 and 5,104,673 describe a process in which heating and mixing are performed continuously in a cooker extruder which imparts a dough- like consistency that can be sheeted while hot An embodiment of the invention includes the use of a gluten-containing starch ingredient including wheat flour in a mixture with non-gluten containing ingredient.

Objects of the invention

It is an object of the present invention to provide novel protein-rich food ingredients derived from wheat gluten wherein the physical and chemical properties of the wheat gluten have been modified to render the derived ingredients particularly suited to applications in extruded and expanded foods.

Another object of the present invention is to provide a novel process for the preparation of novel protein-rich food ingredients from wheat gluten involving the use of protease enzymes to modify the structure of the wheat proteins to an extent that the product is particularly suited to applications in extruded and expanded foods.

A further object of the present invention is to provide food products that are beneficial to consumers due to the improved nutritional value of such foods having an elevated content of protein and consequent reduced content of other major ingredients including fats and carbohydrates achieved by incorporation of said novel protein-rich food ingredients of the present invention

Description of the invention

In accordance with a first aspect of the invention a wheat gluten is disclosed for incorporation into an extruded or expanded food, the structure of the wheat gluten being modified by the addition of a protease enzyme whereby said wheat gluten shows a degree of hydrolysis in the range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons, being less than 15% of total proteinaceous material, content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material, and a glass transition temperature in the range of about 50⁰C to about 59°C. Preferabry said degree of hydrolysis is in the range of about 2% to about 3%.

In accordance with a second aspect of the invention a process is disclosed for producing a modified wheat gluten for incorporation into an extruded or expanded food, said process including the step of adding a protease enzyme to said wheat gluten in aqueous dispersion under suitable conditions of pH, ionic strength and temperature whereby selective peptide bond cleavage is obtained to give a degree of hydrolysis for said gluten in the range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons, being less than 15% of total proteinaceous material, content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material, and a glass transition temperature in the range of about 50⁰C to about 59°C.

Preferably said degree of hydrolysis is in the range of about 2% to about 3%.

In accordance with a third aspect of the invention an extruded or expanded food is disclosed which includes as an ingredient thereof a modified wheat gluten having a degree of hydrolysis in the range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons, being less than 15% of total proteinaceous material, content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material, and a glass transition temperature in the range of about 50⁰C to about 59°C.

Preferably said degree of hydrolysis is in the range of about 2% to about 3%.

In accordance with a fourth aspect of the invention an application of a modified wheat gluten as an ingredient of an extruded or expanded food is disclosed, said modified wheat gluten having a degree of hydrolysis in the range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons being less than 15% of total proteinaceous material, content of proteinaceous material having molecular weight less than 15,000 Daltons, being less than 50% of total proteinaceous material, and a glass transition temperature in the range of about 50⁰C and to about 59°C.

Preferably said degree of hydrolysis is in the range of about 2% to about 3%.

According to the present invention there is provided an enz>τnaticallj -modified wheat gluten composition possessing improved properties of cohesion, extension and melting that render the said composition particularly suited to incorporation at an elevated level for the manufacture of protein-rich extruded and expanded foods.

The novel protein-rich ingredient of the present invention is derived from vital wheat gluten that is well known and characterised as a complex protein system constituted by a number of defined proteins having molecular weights in the range 5,000 to 200,000 Daltons. The complex protein system is further characterised by intermolecular cross-links resulting in macro-structures that provide the well known physical attributes of vital wheat gluten in an aqueous medium. For the purpose of analysis of the complex protein system the well- known technique of PolyAcrylamide Gel Electrophoresesis (PAGE) operating in the presence of reducing agents to break all intermolecular cross links is used to characterise the molecular weights of the multiple defined proteins of wheat gluten. (Wrigley, Robinson & Williams, 1981)

When a protease enzyme is added to wheat gluten in aqueous dispersion under conditions of pH, ionic strength and temperature suited to the particular protease, one or more of the peptide bonds that linearly link the constituent amino acids of the protein is cleaved. If the cleaved peptide bond is the first or last in the polypeptide structure that is the protein by a so called exo-protease or exo- peptidase then the molecular weight of the protein is reduced by just the molecular weight of the amino acid. If the cleaved peptide bond is at any other position on the polypeptide by a so called endo-protease or endo-peptidase, then two fragments are generated of sizes determined by the position of the cleaved peptide bond. Multiple peptide bond cleavage on the multiple proteins of wheat gluten may result in multiple structures having molecular weights different from the parent defined proteins. In general, the more extensive the extent of peptide bond cleavage referred to as Degree of Hydrolysis, the smaller and more diverse in size are the structures derived from such treatment Application of the techniques of PAGE, High Performance Liquid Chromatography (HPLQ and Mass Spectrometry (MS) allows the Degree of Hydrolysis to be estimated and visually represented.

Furthermore and additionally, chemical analysis is used to determine the Degree of Hydrolysis. Several well known methods are described for the determination of the increase in amino acids appearing at the ends of polypeptide structures so obtained by cleavage of proteins by protease enzymes. The OPA method is fully described by Frisher, Meisel and Schlimme, 1988. Thus for an intact protein molecule having only one N-terminal amino acid and one C-teπninal amino acid the degree of hydrolysis is zero. If said protein is hydrorysed to the extent that all peptide bonds are cleaved and the protein is converted to only the constituent amino acids then the Degree of Hydrolysis is 100%. Enzyme-modified wheat gluten of the present invention is characterised by a degree of hydrolysis in the range 1 to 4% and preferably in the range 2 to 3%.

For the purpose of the present invention it has been discovered that certain commercially-available protease enzymes applied under defined conditions can be used to advantageously modify the physical properties of wheat gluten through a limited extent of and selective peptide bond cleavage that may be considered to be a low Degree of Hydrolysis in the range 1 and 4% preferably in the range 2 to 3% of all peptide bonds. Application of the technique of PAGE to wheat gluten so treated shows that most of the largest protein subunits categorised as high molecular weight glutenins in for example Wrigley, C.W., Robinson, P.J. and Williams, W.T. (1981) have been degraded to smaller structures in enzyme-modified wheat gluten. Similarly some of the medium molecular weight glutenins and gliadins are apparently no longer recognisable as intact units with reference to the subunits of the parent wheat gluten but additional structures in the molecular weight range 3,000 to 30,000 Daltons are identified. Additional smaller structures in the molecular weight range 3,000 to 10,000 Daltons not present in the parent gluten are also identified. Use of the technique of PAGE in conjunction with further physical and chemical tests and tests for suitability of enzyme-modified wheat gluten in certain food applications shows that products that are insufficiently modified by an enzyme treatment or excessively modified by an enzyme treatment can be identified. For the purpose of the present invention products having greater than 15% of the protein units in the higher molecular weight range 50,000 to 200,000 Daltons relative to then- content in the parent wheat gluten as determined by PAGE are inadequately modified. For the purpose of the present invention products having less than 30% of the protein units in the medium molecular weight range, 15,000 to 50,000 Daltons, and more than 50% in the lower molecular weight range, 3,000 to 15,000 Daltons, relative to their content in the parent wheat gluten as determined by PAGE are excessively modified.

Furthermore and additionally, use of Differential Scanning Calorimetry (DSC) allows the melting properties of materials to be demonstrated and in the instance of the complex material that is natural wheat gluten the method can be used to determine an apparent transition enthalpy and a glass transition temperature which is indicative of the temperature at which the material melts. Natural wheat gluten shows a glass transition temperature in the range 61 to 75°C dependent on the heating rate when determined using DSC according to the method of Lawton and Wu (1993). Enz>maticall> -modified wheat gluten of the present invention shows a glass transition temperature in the range 45 to 60⁰C, preferably in the range 50 to 60°C. Use of DSC characterises the enzymatically-modified wheat gluten as having a glass transition temperature lower than that of natural wheat gluten and comparable to that of wheat starch.

Furthermore and additionally, use of Dynamic Mechanical Analysis (DMA) allows the melting profile of materials to be demonstrated and in the instance of the complex material that is natural wheat gluten the method can be used to determine a glass transition temperature which is indicative of the temperature at which the material melts. Natural wheat gluten shows a glass transition temperature in the range 30 to 35°C when determined using DMA according to the method of Zhang, Hoobin, Burgar and Do (2006). Enzymaticalry-modified wheat gluten of the present invention shows a glass transition temperature in the range 15 to 30 ⁰C. Use of DMA characterises the enzymaticalry-modified wheat gluten as having a glass transition temperature lower than that of natural wheat gluten and comparable to that of wheat starch.

The process of the present invention for the preparation of enzyme-modified wheat gluten particularly- suited to use in extruded and expanded foods requires the treatment of wheat gluten in aqueous dispersion with selected protease enzymes under suitable selected reaction conditions.

Wheat gluten can be obtained in a wet form directly from a process well known for its separation from an aqueous dispersion of wheat flour such as described by Cornell and Hoveling (1998). In said wet form from which as much free water as can be removed by physical pressing typically the total solids content of the said wet gluten is in the range 30 to 35% preferably 31 to 33% w/w and the protein content in the range 70-85% determined by the Kjeldahl or Dumas methods using the Nitrogen Conversion Factor for wheat gluten of 5.7.

Alternatively , wheat gluten can be obtained in dried form having a protein content determined by the same method on the basis of dry solids in the range 70 to 85%. In this instance dried wheat gluten is dispersed in water in the concentration range 0 to 50% and preferably in the range 20 to 35% and most preferably in the range 28 to 33%

To said aqueous dispersion of wheat gluten a quantity of selected protease enzymes in aqueous dispersion is added with vigorous mixing to ensure uniform treatment of the wheat gluten by the protease enzymes. It will be observe that the highly cohesive and viscous wheat gluten dispersion becomes less cohesive and less viscous. After an appropriate period of time a treatment is applied to terminate the proteolytic activity of the protease enzymes.

Alternatively, wherein dried gluten is to be used, the selected protease enzymes may be dispersed wholly or partially in the water prior to the addition of the dried gluten such process being undertaken in either a batch or continuous mixing operation. The presence of protease enzyme from the initial contact of gluten and water facilitates the dispersion of the gluten and by almost immediate commencement of proteolytic breakdown of the wheat gluten protein the tendency to form a cohesive agglomerate is reduced. The remainder of any of the selected protease enzymes may be added at a later stage particularly in a batch process to provide greater uniformity to the degree of hydrolysis.

Protease enzymes are available in commercial quantities derived from animal, plant, fungal and bacterial sources. Most such enzymes demonstrate particular selectivities or preferences for cleavage of peptide bonds between certain amino acids in proteins and polypeptides. Some commercially available protease enzyme products contain a mixture of protease enzymes with different specificities. Some commercially available protease enzyme products contain both endo-peptidases and exo-peptidases. The determination of which protease enzymes to use in the process for the production of enzyme modified wheat glutens of this invention is, therefore, not prescribed specifically but exemplified through non-exclusive identification of preferred protease enzymes that having been evaluated and compared to numerous protease enzyme products are recommended non-exclusively for use by their ability to modify wheat gluten to achieve the physical properties for enzyme modified wheat gluten of this invention.

Protease enzymes from such said sources may show a magnitude of proteolytic activity that is determined by the physical and chemical conditions of the aqueous environment. In general, increasing the temperature results in an increase in proteolytic activity up to a temperature at which said proteases may be de-activated due to denaturatiσn of their intrinsic protein structure. In general, proteins including enzymes are more soluble and hence reactive in the presence of low concentrations of ions in aqueous solution, that is, at low ionic strength. This has the effect of minimising electrostatic interactions and prevents charge- mediated flocculation. However, excessive ionic concentration has the effect of enhancing entropically driven interactions between protein molecules again resulting in insolubility. The ionic strength of the dispersion during proteolysis should preferably be in the range of 0.01 to 0.5 and more preferably in the range of 0.01 to 0.1. Some protease enzymes are proteorytically most active over a narrow and specific pH range; other protease enzymes may be active over a broad pH range. Wheat gluten occurs naturally in aqueous dispersion at about pH 6. Protease enzymes that show a high level of proteolytic activity at this pH may be preferable for the process of this invention but not exclusively as the pH of the said aqueous dispersion of wheat gluten can be changed by the addition of acid or alkali to be more suited to a particular protease enzyme product Selection of the reaction conditions for enzyme modification of wheat gluten in the process of this invention is not prescribed but may be determined according to the protease enzymes selected for the purpose of achieving enzyme modified wheat gluten of this invention. In conjunction with said preferred protease enzymes for the process of this invention, reaction conditions are recommended non-exclusively for use and exemplified for their suitability to achieve enzyme modified wheat gluten of this invention.

The extent of enzyme modification of wheat gluten is determined by the period of contact time between the active protease enzyme and the wheat gluten in addition to the selection of a certain or preferred protease enzyme product and the selected or preferred aqueous reaction conditions. For the process of the present invention, the period of time for contact between active protease enzyme and wheat gluten is not prescribed but may be determined and is exemplified for its suitability to achieve enzyme modified wheat gluten of this invention according to the protease enzymes selected for the purpose of achieving enzyme modified wheat gluten of this invention in conjunction with reaction conditions that are recommended non-exclusively for use. In a continuous process the contact time may preferably be a number of minutes; in a batch process the contact time may preferably be a number of hours.

The process of the invention requires that the contact time between the active protease enzyme and wheat gluten is limited according to the requirement to achieve enzyme modified wheat gluten of this invention. This might be achieved by a process that would separate the protease enzymes from the wheat gluten and its enzyme-modified derivatives but is preferably achieved by inactivating the said protease enzymes. Any process that may disrupt the intrinsic structures of active enzymes may be suited to inactivation of the selected protease enzymes in contact with the wheat gluten. This might include modern techniques such as application of ultra-high pressure or ultra-sonics but may preferably be achieved by simpler and cheaper technologies including thermal denaturation, acid-shock and alkaline-shock according to the sensitivity of the selected protease enzymes to such said technologies.

Enzyme-modified wheat glutens of the present invention are exemplified in which either thermal denaturation or acid-shock technology has been used to inactivate the selected protease enzymes. The selection of either of these technologies for the process of this invention as preferable may depend on the application for the product and the severity of the inactivation conditions. The use of high temperature particularly greater than 80°C may adversely affect the colour and flavour of the enzyme-modified wheat gluten if an extended period of time at this temperature is necessary, however, ultra-high-temperature (UHT) conditions applied for a very short time may be a preferred technology. The use of acid-shock technology may involve a substantial reduction of the pH by addition of an acid while maintaining a moderate temperature for a period of time followed by restoration of the initial pH by addition of an alkali. Under these conditions the intrinsic structure of the selected protease enzymes may be irreversibly changed and the activity of the enzymes negated. The use of this technology for inactivation of the selected protease enzymes results in the formation of a salt that may be desirable or undesirable in the enzyme modified wheat gluten of this invention for a particular application. Additionally, acid deamidation of glutamine and asparagine amino acid components of the enzyme modified wheat gluten may occur if the pH is sufficiently low, the temperature sufficiently high and the exposure time sufficiently long. This may adversely affect the physical properties of the enzyme-modified wheat gluten. The advantage of the acid-shock technology is that the use of only moderate temperature eliminates the potential to adversely affect the colour and flavour of the enzyme modified wheat gluten of this invention.

The resulting enzyme-modified wheat gluten of the present invention is finally dried using one of a number of well known commercial drying technologies. Owing to the somewhat viscous and cohesive nature of the enzyme-modified wheat gluten in the final aqueous dispersion certain drying technologies may be preferred. An attrition drier or ring drier as often employed for drying wheat gluten may be preferable.

Enzyme-modified wheat gluten of the present invention provides for a food ingredient that may be used to manufacture of foods having an elevated content of protein and consequent reduced content of other major ingredients including fats and carbohydrates. Furthermore, incorporation of the enzyme-modified wheat gluten of the present invention as an ingredient in said novel foods enables desirable colour and flavour to be achieved and facilitates desirable texture, shape and form characteristics to be generated in said novel foods.

Protein-enriched expanded foods are manufactured using facilities such as single- or twin-screw extruders by incorporating a quantity of enzyme-modified wheat gluten of the present invention into a mix of predominantly starch- containing ingredients as used in the manufacture of many less nutritious expanded food types concomitantly displacing an equivalent quantity of said predominantly starch ingredients. The extent to which the predominantly starch- containing ingredients are displaced is not prescribed as it will depend on the desired nature, form and nutritional composition of the extruded expanded product. Expanded food products have been manufactured containing 40% protein by incorporating enzyme-modified wheat gluten of this invention. In the instance that the dry ingredient mix containing enzyme-modified wheat gluten of this invention is introduced into an extruder with or without pre-hydration according to the desired product and the capability' of the extruder it is introduced in the same manner as introduction of the dry mix not containing the protein-rich ingredient. Such extruder is operated with essentially the same screw configuration and with essentially the same operating conditions as for extrusion of a predominantly starch-containing ingredient mix not containing the enzyme modified wheat gluten of the present invention. Because the enzyme-modified wheat gluten of the present invention melts under pressure and shear at a temperature equivalent to that of starch then the enzyme-modified wheat gluten and starch melt together and form a near uniform plastic material that when expressed through a die and allowed to freely expand and cool forms a crisp expanded form equivalent to that produced when a predominantly' starch- containing mix is extruded and expanded. The configuration of the die is not prescribed nor is the resulting form of the extruded protein-enriched food. Novel expanded foods enriched with protein by incorporating enzyme-modified wheat gluten of the present invention can thus be made in a variety of shapes, sizes, forms and protein contents as is perceived to be desirable for consumers. Expanded foods of this type may be variously coloured and flavoured by addition of suitable colorants or flavorants prior to expansion or after the expanded food form is manufactured as is perceived to be desirable for consumers.

Protein-enriched, sheeted (extruded in the form of a thin sheet) and fried or baked foods including potato-chip-like, corn-chip-like and tortilla-like products are manufactured by incorporating a quantity of enzyme-modified wheat gluten of the present invention into a mix of predominantly starch-containing ingredients as used in the manufacture of many less nutritious sheeted and fried or baked food types concomitantly displacing an equivalent quantity of said predominantly starch ingredients. The extent to which the predominantly starch- containing ingredients are displaced is not prescribed as it will depend on the desired nature, form and nutritional composition of the sheeted and fried or baked product. Sheeted and fried food products have been manufactured containing 30% protein by incorporating enzyme-modified wheat gluten of this invention. The dry ingredient mix containing enzyme-modified wheat gluten of this invention is formed into a dough by addition of water and use of a mixer. The said dough is introduced into a sheeter according to the desired product, and cut into shapes and fried or baked in the same manner as applied to a dough not containing the enzyme-modified wheat gluten ingredient of the present invention.

The sheeter is operated in essentially the same configuration and with essentially the same operating conditions as for sheeting of a predominantly starch- containing ingredient mix not containing the enzyme-modified wheat gluten of the present invention. The enzyme-modified wheat gluten of the present invention has sufficient elasticity to assist the formation of a cohesive dough but is weak enough to allow sheeting without contraction, so subsequent cutting and shaping can be achieved as for compositions not containing the enzyme-modified wheat gluten of the present invention.

The configuration of the sheeter is not prescribed nor is the resulting shape or form of the resulting protein-enriched food. The mode of cooking is not prescribed but baking or frying to cook the starch are both possible. Novel sheeted and cooked foods enriched with protein by incorporating enzyme- modified wheat gluten of the present invention can thus be made in a variety of shapes, sizes, forms, compositions and protein contents as is perceived to be desirable for consumers. Sheeted and cooked foods of this type may be variously coloured and flavoured by addition of suitable colorants or flavorants prior to sheeting and cooking or after the sheeted and cooked food form is manufactured as is perceived to be desirable for consumers.

The invention will now be further described in the following examples where reference is made to the following accompanying figures: Figure 1 is a SDS-PAGE gel of wheat gluten and enzyme-modified wheat gluten products analysed relative to calibrant proteins of known molecular weight

Figure 2 shows percentage of protein in each band relative to total protein in all bands in each lane for wheat gluten and enzyme-treated wheat gluten products; Lane assignment as in Figure 1

Figure 3 shows band identification and molecular weight assignment for wheat gluten and enzyme-treated wheat gluten products; Lane assignment as in figure 1.

Figure 4 shows Mass Spectrum of enzyme-modified wheat protein products identified as Sample 3 prepared as in Example 2 and Sample 6 prepared as in Example 5 showing overlapping regions of the Mass ranges 4982 to 7491 and 6244 to 9290 mass units

Figure 5 shows Mass Spectrum of enzyme-modified wheat protein products identified as Sample 3 prepared as in Example 2 and Sample 6 prepared as in Example 5 showing overlapping regions of the Mass ranges 8753 tol0899 and 10477 to 13599 mass units

Figure 6 shows Mass Spectrum of enzyme-modified wheat protein products identified as Sample 3 prepared as in Example 2 and Sample 6 prepared as in Example 5 showing overlapping regions of the Mass ranges 12736 tol9189 mass units

Figure 7 shows Glass transition temperature and heat capacity determined by Differential Scanning Calorimetry for (i) wheat gluten and enzyme-modified wheat gluten products,(ii) Sample 3, (ϋi) Sample 5 and (iv) Sample 6. Figure 8 shows Storage modulus and tan δ values determined by Dynamic Mechanical Analysis obtained as a function of temperature for (#1) Wheat gluten, (#2) Sample 3 and (#3) Sample 5.

Examples

Example 1 - Preparation of enzyme-modified wheat gluten using the protease, Neutrase 0.8L

Water at a temperature of 55^σC was placed in a temperature-controlled vessel and stirred continuously. To this water Neutrase 0.8L (Novozymes) was added sufficient to result in a concentration of 0.05% relative to the amount of dry vital gluten to be used. Dry vital wheat gluten was added over a five minute period to achieve a final mix of 30% solids (w/w). This mixture was stirred continuously at a temperature of 55^DC for a period of two hours. After this time the pH of the mixture was adjusted to a value of 3.0 by adding sufficient hydrochloric acid (16% w/w) and stirred at 55°C for a further thirty minutes. The pH of the mixture was then adjusted to pH 6.5 using sodium hydroxide solution (15% w/w). The resulting enzyme-modified wheat gluten product was then freeze dried and identified herein as Sample 2. The product composition and Degree of Hydrolysis are shown in Table 1

Table 1 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 2

Product Analysis Content (%)

Moisture 4.5

Protein N x 6.25 82

Ash 1.5

Degree of hydrolysis 2.59

Example 2 - Preparation of enzyme-modified wheat gluten using the protease, Multifect Neutral Water at a temperature of 55°C was placed in a temperature-controlled vessel and stirred continuously. To this water Multifect Neutral (Genencor) was added sufficient to result in a concentration of 0.05% relative to the amount of dry vital gluten to be used. Dry vital wheat gluten was added over a five minute period to achieve a final mix of 30% solids (w/w). This mixture was stirred continuously at a temperature of 55°C for a period of two hours. After this time the temperature was rapidly increased to 80⁰C and after 2 minutes at 8O⁰C, the temperature was cooled to less than 50⁰C. The resulting enzyme-modified wheat gluten product was then freeze dried and identified herein as Sample 3. The product composition and Degree of Hydrolysis are shown in Table 2

Table 2 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 3

Product Analysis Content (%

Moisture 4.2

Protein N x 6.25 82

Ash 0.5

Degree of hydrolysis 2.45

Example 3 - Commercial production of enzyme-modified wheat gluten using the protease, Multifect Neutral

Fresh, wet vital wheat gluten at about 40°C was obtained directly from a starch and gluten manufacturing plant The wet gluten together with continuous introduction of the appropriate quantity of Multifect Neutral, as in Example 2, was pumped through a continuous high shear mixer and into a steam jacketed stirred vessel to increase the temperature of the mix to 55°C. This mixture was stirred continuously at a temperature of 55°C for a period of two hours. After this time the mixture was pumped through a tube-in-tube heat exchanger to increase the temperature to 80⁰C, then through a jacketed tube with 2 minute retention and then through a second tube-in-tube heat exchanger to cool the mix to about 50⁰C. The resulting enzyme-modified wheat gluten product at about 28% w/w solids was then dried through a ring drier by introducing the wet feed into the recycle blender and identified herein as Sample 4. The product composition and Degree of Hydrolysis are shown in Table 3

Table 3 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 4

Product analysis Content (%

Moisture 4

Protein N x 6.25 82

Ash 0.5

Degree of hydrolysis 2.27

Example 4 - Preparation of enzyme-modified wheat gluten using the protease, Multifect Neutral

Water at a temperature of 60°C was placed in a temperature-controlled vessel and stirred continuously. To this water Multifect Neutral (Genencor) was added sufficient to result in a concentration of 0.2% relative to the amount of dry vital gluten to be used. Dry vital wheat gluten was added over a five minute period to achieve a final mix of 30% solids (w/w). This mixture was stirred continuously at a temperature of 60°C for a period of two hours. After this time the pH of the mixture was adjusted to a value of 3.0 by adding sufficient hydrochloric acid (16% w/w) and stirred at 60⁰C for a further thirty minutes. The pH of the mixture was then adjusted to pH 6.5 using sodium hydroxide solution (15% w/w) and identified herein as Sample 5. The product composition and Degree of Hydrolysis are shown in Table 4

Table 4 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 5

Product analysis Content %

Moisture 4

Protein N x 6.25 85

Ash 1.5

Degree of hydrolysis 2.96

Example 5 - Preparation of enzyme-modified wheat gluten using the protease, Multifect Neutral Water at a temperature of 55⁰C was placed in a temperature-controlled vessel and stirred continuously. To this water, Multifect Neutral was added sufficient to result in a concentration of 0.5% relative to the amount of dry vital gluten to be used. Dr>- vital wheat gluten was added over a five minute period to achieve a final mix of 30% solids (w/w). This mixture was stirred continuously at a temperature of 55^DC for a period of two hours. After this time the temperature was rapidly increased to 80°C and after 2 minutes at 80⁰C, the temperature was cooled to less than 50⁰C. The resulting enzyme-modified wheat gluten product was then freeze dried and identified herein as Sample 6. The product composition and Degree of Hydrolysis are shown in Table 5

Table 5 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 6

Product analysis Content

Moisture 4

Protein N x 6.25 82

Ash 0.5

Degree of hydrolysis 4.86

Example 6 — Preparation of enzyme-modified wheat gluten nsing the protease, Bromelain 110

Water at a temperature of 55°C was placed in a temperature-controlled vessel and stirred continuously. To this water Bromelain 110 ( Genencor) was added sufficient to result in a concentration of 0.1% relative to the amount of dry vital gluten to be used. Dry vital wheat gluten was added over a five minute period to achieve a final mix of 30% solids (w/w). This mixture was stirred continuously at a temperature of 55°C for a period of two hours. After this time the temperature was rapidly increased to 80⁰C and after 2 minutes at 80°C, the temperature was cooled to less than 50⁰C. The resulting enzyme-modified wheat gluten product was then freeze dried and identified herein as Sample 7. The product composition and Degree of Hydrolysis is shown in Table 6 Table 6 Composition and Degree of Hydrolysis of Enzyme-modified wheat gluten product, Sample 7

Product analysis Content (%

Moisture 4

Protein N x 6.25 82

Ash 0.5

Degree of hydrolysis 1.71

Example 7 - Characterisation of Enzyme-Modified Wheat Gluten products by SDS-PAGE analysis

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with process variations to define and optimise the product and process were analysed by PAGE essentially according to the detailed method of Kasarda, Woodard and Adalsteins (1998) using reducing conditions.

Samples for analysis were prepared by dispersing lOmg of sample in 1.OmL of sample buffer containing 2% SDS, 10% glycerol, 5OmM ditbiothreitol, 62.5mM Tris(hydroxymethyl) aminomethane-HCl and 0.05% bromophenol blue as marker dye at pH 8.5. Samples were heated to 8O⁰C for 5 minutes and analysed on a polyacrylamide gradient (4 to 12%) gel. 9 microL of each sample was layered onto the polyacrylamide gel and run at 120V for 90 minutes. Gels were stained in Coomassie Brilliant Blue R250 without trichloroacetic acid fixation and destained with 30% methanol overnight. Standard protein calibration mixture was run on each gel to enable molecular weights of protein fragments to be determined.

The gel was photographed and scanned for band intensity along each sample slot. The intensity and relative migration distance data for each band was compared to that obtained from the protein calibration mixture using BIORAD Image Analysis Software, Quantity One Version 4.4 The results shown in Figures 1 to 3 and Table 7 indicate the extent to which the wheat gluten has been fragmented according the proportion of each sample in a particular size range.

Table 7 Distribution of molecular size in proteins and peptides constituting wheat gluten and enzyme-modified wheat gluten products as assessed by SDS-PAGE

Percent of Bands in Lane

Sample 50- 20OkD 30-5OkD 15-3OkD 3- 15 kD

Wheat gluten 23.5 41.3 14.8 20.4

Sample 3 10.6 31.3 20.5 39.7

Sample 5 2.8 31.4 19.4 46.2

Sample 6 0 0 21.5 78.5

Sample 7 15.1 49.8 12.6 22.3

Example 8 — Characterisation of Enzyme-Modified Wheat Gluten products by estimation of Degree of Hydrolysis

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with process variations to define and optimise the product and process were analysed for Degree of Hydrolysis according to the detailed OPA method as described by Frisher et al. (1988).

Samples were solubilised at 125mg mL^"1 in 12.5mM borate buffer at pH 8.5 containing 2% (w/v) SDS. 50μL of this solution was mixed with ImL of a reagent (50ml 0.1M sodium borate buffer pH 9.3, 1.25mL 20% (w/v) SDS solution, lOOmg of N, N-dimethyl-2-mercaptoethylammonium chloride (DMMAC) and 40mg OPA dissolved in ImL methanol). The reaction mixture was allowed to stand for 2 minutes before measuring its absorbance at 340 nm. The number of amino groups was determined with reference to a L-Leucine standard curve between 0.5 - 5 mM. The Degree of Hydrolysis was calculated by- comparing the increase in amino groups between gluten and the enzyme- modified gluten samples using the equation:

DH = [(α - n,) /n_τ] x l00 in which:

DH is the Degree of Hydrolysis α is the number of free amino groups measured in the enzyme-modified wheat gluten samples n, is the number of amino groups in native gluten and n_τ is the total number of amino groups in native gluten after total hydrolysis with 6M HCl for 24 hours.

Table 8 shows that enzyme-modified wheat gluten products suited to extruded and expanded foods have been modified to a Degree of Hydrolysis within the range 2 - 3%. Such products having DOH below or above this range were not suited to such applications.

Table 8 Degree of Hydrolysis determined for wheat gluten and enzyme- modified wheat gluten products

Sample Degree of Hydrolysis

Wheat gluten 0

Sample 2 2.59

Sample 3 2.45

Sample 4 2.27

Sample 5 2.96

Sample 6 4.86

Sample 7 1.71

Example 9 - Characterisation of Enzyme-Modified Wheat Gluten products by Mass Spectrometry

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with process variations to define and optimise the product and process were analysed by Mass Spectrometry essentially according to the method developed by Applied BioSystems

Samples were prepared for analysis at about 7mg /mL by dispersing in 45% acetonitrile containing 0.1% formic acid. For mass spectrometry the matrix was lOmicrogram / mL of sinnapinic acid in 30% acetonitrile containing 0.1% formic acid. The sample to matrix ratio was 1:1. Samples were analyses in a Voyager STR mass spectrometer ( Applied Biosy stems) over a Mass /Charge( m/z) range from 4829 to 21927 mass units.

Figures 4,5 and 6 show sequential and overlapping portions of the mass spectrum achieved for each of Samples 3 and 6. It is apparent that the peaks representing peptides of different mass/ charge ratio (m/z) can be categorised broadly into three Categories: (i) peaks substantially present in Sample 3 representing a product with low Degree of Hydrolysis but not significantly present in Sample 6 representing a product with a higher Degree of Hydrolysis, (ii) peaks approximately equivalently represented in Samples 3 and 6 and (iii) peaks substantially represented in Sample 6 but not significantly represented in Sample 3.

The results obtained from the mass spectra for Samples 3 and 6 show that an enzyme- modified wheat protein product suited to extruded and expanded foods with a Degree of Hydrolysis within the range 2 - 3% can be clearly identified by the presence of peaks representing peptides in Category (i). It is rational to infer that these peptides are produced in the early stages of enzyme modification up to a Degree of Hydrolysis in the range 2-3% and are subsequently fragmented further with increased degree of hydrolysis. Table 9 provides a list of mass/ charge ratios for peaks representing peptides that are most distinctly of this type and which therefore most distinctly identify an enzyme-modified wheat protein product suited to extruded and expanded foods.

Table 9 Mass Spectrometry data identifying peptides most distinctly present in products suited to extruded and expanded foods application after enzymatic modification of wheat gluten.

Mass/ charge ratio Extent of presence in Extent of presence in

(m/z) Sample 3 Sample 6

17,424 ***** *

16,076 *** *

15,490 *** 12,713 ♦♦♦ *

12,348 ♦♦♦♦♦

9,873 **** *

9,024 *♦♦ *

8,592 *♦ ♦

5,835 *** **

Size of peak

(% chart scale)

0-20

21-40

41-60

61-80

***** 81-100

It is apparent that mass spectrometry provides a more detailed and precise evaluation of the peptide entities resulting from enzyme modification of wheat gluten and can be taken as complimentary to the data provided by SDS-PAGE as in Example 7. While SDS-PAGE provides a direct "fingerprint" representation of the apparent molecular weights of the peptides present in a product sample, mass spectrometry identifies a sequence of peptides according to the mass/ charge ratio and consequently cannot be directly compared to results from SDS-PAGE since the charge value for each peak is not known.

Example 10 - Characterisation of Enzyme-Modified Wheat Gluten products by Differential Scanning Calorimetry

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with ingredient and process variations to define and optimise the product and process were analysed by Differential Scanning Calorimetry (DSC) essentially according to John W Lawton and Y. Victor Wu (1993). Samples were measured as obtained on a differential scanning calorimeter PerkinElmer DSC Pyris 1. The measurement was made on 3-5 mg samples at a scan rate of 10 °C/min. The glass temperature (Tg) was recorded as the midpoint of the heat capacity change during the transition at the first scans. The results in Table 10 and Figure 7 show that enzyme-modification of wheat gluten resulted in significant decrease in glass transition temperature, Tg, and a significant corresponding decrease in heat capacity. Extensively modified wheat gluten, Sample 6, showed a high heat capacity and glass transition temperature indicative of greater molecular mobility resulting from greater protein fragmentation.

Table 10 Physical properties of wheat gluten and enzyme-modified wheat gluten products determined by Differential Scanning Calorimetry

Sample Tg (⁰Q ΔCp (J/g°C)

Wheat gluten 63.5 0.196

Sample 3 53.9 0.189

Sample 5 50.6 0.135

Sample 6 57.6 0.276

Example 11 -Characterisation of Enzyme-Modified Wheat Gluten products by Dynamic Mechanical Analysis

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with ingredient and process variations to define and optimise the product and process were analysed by Dynamic Mechanical Analysis (DMA) according to Zhang et al. (2006).

Samples were prepared as sheets with water (15%) as plasticiser and adjusting the pH to 4.0 with acetic acid. Each sample with a designed formulation was mixed with a high speed mixer for 1 minute and left overnight to equilibrate. The sample was then compression-moulded at 130°C for 5 minutes using a heating press with a pressure of 12 ton. The sample size was 145mm x 145mm with a thickness of lmm ± 0. lmm. DMA testing was performed on samples after conditioning at 50% relative humidity using a Perkin-Elmer PYRIS Diamond DMA in dual cantilevers bending mode at a frequency of IHz. The temperature range was -100 to 150⁰C with a heating rate of 2 ^oC/min. The storage modulus (E % the loss modulus (E") and tan δ ( E" /E ' ) were recorded as a function of tempεrature. Results obtained for wheat gluten (#1), Sample 3 (#2) and Sample 5 (#3) are shown in Figure 9

The results in Table 11 and Figure 8 show that enzyme-modification of wheat gluten resulted in significant decrease in the storage modulus, E', and a significant increase in tan δ. Extensively modified wheat gluten, Sample 6, was too brittle to handle when pressed into a sheet

Table 11 Rheological properties of wheat gluten and enzyme-modified wheat gluten products determined by Dynamic Mechanical Analysis

Sample E' (GPa, tan δ storage Maximum at modulus at 120⁰C

22°Q

Wheat gluten 1.05 0.28

Sample 3 0.88 0.47

Sample 5 0.69 0.58

Sample 6 nd nd

Example 12 -Application of Enzyme-Modified Wheat Gluten products in a formulated potato chip-type food

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with ingredient and process variations to define and optimise the product and process were analysed for sheeting properties and suitability for application in a cooked sheeted food as in a formulated potato chip-type food according to the formulations shown in Table 12 and the detailed method below. The composition of finished products is shown in Table 13

Table 12 Formulation of potato chip-type food Ingredients Standard Standard Product Product product (g) product (%) including including

Enzyme- Enzyme- modified modified wheat gluten wheat gluten

(g) (%)

Potato 930 23.25 930 23.25 granules

Modified 1274 31.84 634 15.85 potato starch

GMS 16 0.4 16 0.4

Salt 38 0.96 38 0.95

Potato flakes 682 17.05 682 17.05

Water 1060 26.5 1060 26.5

Gempro LH 0 0 640 16.0

Samples were prepared in a high shear mixer by first adding dry ingredients and mixing for 10 seconds. Water was then added and mixed for 30 seconds. Enzyme-modified wheat gluten was included in the range 0-20% w/w. The resulting dough was fed into a 3 roll sheeter with a roil diameter of 110mm, front roll speed of 6 rpm, back roll speed of 2 rpm and a roll gap of 0.65mm. Sheeted dough was cut into oval shaped pieces of approximately 8cm x 5 cm and fried in a batch fryer at 180°C for 15-20 seconds. Chip thickness was 0.55-0.80mm.

Table 13 Composition of finished potato chip-type product

Composition Standard product Product including enzyme-modified wheat gluten

Moisture 4.8 4.6 * ( Formatted Table

Protein 3.2 16.9

Fat 35.5 27.9

Carbohydrate 52.2 45.7

Fibre 1.7 1.6

Ash 2.6 3.3

Table 14 shows that satisfactory sheeting behaviour was achieved at least up to 16% w/w inclusion of enzyme-modified wheat gluten in a potato chip-type food formulation with some advantage due to low level cohesion increasing with protein content. The pieces of the sheet, when deep fried, cooked to an attractive appearance with colour intensity increasing with included protein content. The flavour at all levels of enzyme-modified wheat gluten was pleasant and typical of a deep fried potato chip-type food, however, a mild cereal flavour note increased with increasing protein content. The fat uptake by the potato chip-type food decreased with increased protein content

Table 14 Processing and product quality' assessment of potato chip-like food containing levels of enzyme-modified wheat gluten

Enzyme Modified Sheeting behaviour Colour Flavour/

Wheat Gluten Mouthfeel

Inclusion

0% Satisfactory, brittle Light yellow Bland, fried

Crisp

4% Satisfactory, less Light Yellow brittle

8% Satisfactory, even Light-medium More flavour less brittle yellow Crisp

12% Satisfactory, Medium-dark

Becoming cohesive jellow

16% Satisfactory, more Dark yellow-light Even more cohesive brown flavour

Crisp

Example 13 -Application of Enzyme-Modified Wheat Gluten products in a formulated corn-based, tortilla-type food

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with ingredient and process variations to define and optimise the product and process were analysed for sheeting properties and suitability for application in a cooked sheeted food as in a corn-based tortilla-type food according to the formulation shown in Table 15 and the detailed method below. The compositions of finished products are shown in Table 16.

Table 15 Formulation of corn-based tortilla-type food

Ingredients Standard Standard Product Product product (g) product (%) including including

Enzyme- Enzyme- modified modified wheat gluten wheat gluten

(g) (%)

Corn masa 2900 72.5 2260 62.95 flour

GMS 25 0.63 25 0.7

Salt 15 0.38 15 0.42

Water 1060 26.5 0 0

Gempro LH 0 0 640 17.83

Vinegar 0 0 650 18.11

Samples were prepared in a high shear mixer by first adding dry ingredients and mixing for 10 seconds. Water was then added and mixed for 30 seconds. Enzyme-modified wheat gluten was included in the range 0-20% w/vv. The resulting dough was fed into a 3 roll sheeter with a roll diameter of 110mm, front roll speed of 6 rpm, back roll speed of 2 rpm and a roll gap of 0.65mm. Sheeted dough was cut into oval shaped pieces of approximately 8cm x 5cm and fried in a batch fryer at 180°C for 15-20 seconds. Chip thickness was 0.55-0.80mm

Table 16 Composition of finished corn-based tortilla-type product

Composition Standard product Product including enzyme-modified wheat gluten

Moisture 8.2 7.2

Protein 5.6 19.6

Fat 25.9 22.8

Carbohydrate 58.1 47.4

Fibre 0.45 0.41

Ash 1.69 2.58

Table 17 shows that satisfactory sheeting behaviour was achieved at least up to 16% w Av inclusion of enzyme-modified wheat gluten in a corn-based tortilla- type food formulation with some advantage due to low level cohesion increasing with protein content The pieces of the sheet, when deep fried, cooked to an attractive appearance with colour intensity increasing with included protein content. The flavour at all levels of enzyme-modified wheat gluten was pleasant and typical of a deep fried corn-based tortilla-type food, however, a mild wheat flavour note increased with increasing protein content The fat uptake by the corn-based tortilla-type food decreased with increased protein content.

Table 17 Processing and product quality assessment of corn-based tortilla- typε product containing levels of enzyme-modified wheat gluten

Enzyme Modified Sheeting behaviour Colour Flavour/

Wheat Gluten Mouthfeel

Inclusion

0% Satisfactory, brittle Light yellow Bland

Crisp

4% Satisfactory, less Light yellow brittle

8% Satisfactory', even Light-medium yellow More less brittle flavour

Crisp

12% Satisfactory, Medium-dark yellow

Becoming cohesive

16% Satisfactory, more Dark yellow-light Even more cohesive brown flavour

Example 14 —Application of Enzyme-Modified Wheat Gluten products in an extruded, expanded food type of ready-to-eat products

Enzyme-modified wheat gluten samples prepared as in Examples 1 to 6 and with ingredient and process variations to define and optimise the product and process were analysed for extrusion properties in an expanded, extruded food as in a breakfast cereal food or a snack food according to the formulations shown in Table 18 and the detailed method below. Compositions of finished products is shown in Table 19.

Ingredients were dry blended in a ribbon blender and included enzyme-modified wheat gluten in the range of 0-20% w/w. Water was then added to the dry ingredients and mixed for approximately 1 minute. The blend was then extruded through a pilot-size Werner and Pfleiderer C-37, Fully Intermeshed Twin Screw Cooking Extruder using the following design and operating details: Extruder Specifications:

Screw Diameter - 37.0mm

Total Screw Length - 590.0mm, 220.0mm (Working

Length)

Maximum Screw Speed - 400rpm (6.67Hz)

Die Geometry:

Hole Diameter - 4.00mm

Number of holes - 2

Process Data:

Estimated Processing Rate - 8.5kg/h

Table 18 Formulation for extruded, expanded cereal-based ready-to-eat products

Ingredients Standard Standard Product Product product (kg) product (%) including including

Enzyme- Enzyme- modified modified wheat gluten wheat gluten

(g) (%)

Wheat flour 8.0 20 8 16

Maize flour 16.0 40 16 32

Rice flour 12.0 30 12 24

Oat flour 2.0 5 2.0 4

Sugar 2.0 5 2.0 4

Gempro LH 0 0 10 20

Table 19 Composition of extruded, expanded cereal-based ready-to-eat products

Composition Standard product Product including enzyme-modified wheat gluten

Moisture 6.0 6.0

Protein 8.9 24.3

Fat 1.3 1.7

Carbohydrate 81.5 64.9

Fibre 1.4 1.7

Ash 1.1 1.3

The results in Table 20 show that enzyme-modified wheat gluten may be included in a typical starch-rich formulation used in manufacturing some extruded, expanded breakfast cereal food and snack food types. Direct replacemeπt of predominantly starch ingredient with enzyme-modified wheat gluten at least up to 15% w/w resulted in satisfactory products with only small increases in product diameter and expansion ratio.

Table 20 Processing and product quality assessment of extruded, expanded cereal-based ready-to-eat products containing levels of enzyme- modified wheat gluten

Enzyme-modified Product Expansion Colour/Flavour Product texture Wheat Gluten level Diameter Ratio Development

0% 11.5 8.27 Sweet flavour, Crisp, pale yellow non-uniform bubbles

5% 12.5 9.77 Less sweet, Crisp Pale yellow

10% 13.5 11.39 Even less sweet, Crisp Pale yellow

15% 14.0 12.25 Not so sweet, Crisp, more bland finer more

Pale yellow uniform bubbles

References Cited [Referenced by] U.S. Patent Documents

2002053 May., 1935 Doolin

2701200 Feb., 1955 Huber

2905559 Sep., 1959 Anderson et al.

3083103 Mar., 1963 Anderson et al.

3278311 Oct., 1966 Brown et al.

3297450 Jan., 1967 Loska

3451822 Jun., 1969 Fast et al.

3539356 Nov., 1970 Benson et al.

3656966 Apr., 1972 Ball et al.

3690895 Sep., 1972 Amadon et al.

3753728 Aug., 1973 Bedenk et al.

3997684 Dec, 1976 Willard

4124727 Nov., 1978 Rockland et al.

4126705 Nov., 1978 Hait

4140803 Feb., 1979 Panchuk et al.

4183966 Jan., 1980 Mickle, deceased, et al.

4212892 JuI., 1980 Chahine et al.

4409248 Oct, 1983 Lehnhardt et al.

4482574 Nov., 1984 Lee

4526800 JuI., 1985 Howard

4623548 Nov., 1986 Willard

4769253 Sep., 1988 Willard

4834996 May, 1989 Fazzolare et al.

5104673 Apr., 1992 Fazzolare et al.

5486461 Jan., 1996 Nielsen

5520935 May, 1996 Eriksen et al.

5894027 Apr., 1999 Kazemzadeh

5945299 Aug., 1999 von Kries et al.

6036980 Mar., 2000 Beck et al.

6036983 Mar., 2000 Nielsen

6051492 Apr., 2000 Park et al.

6171621 Jan., 2001 Braun et al.

6420133 JuI., 2002 Cheruppanpullil et al.

6692933 Feb., 2004 Merrell et al.

6803062 OcL, 2004 Yamamoto et al.

6864230 Mar., 2005 Ostrom

6899905 May, 2005 Prosise et al.

7112424 Sep., 2006 Rao et al.

Foreign Patent Documents

20060083452 JuI., 2006 Masamichi et al. KR

1021994 Dec., 1977 Panchuk et al. CA Other References

Barr, E.L.M., Magliano, DJ., Zimmet, P.Z., Polkinghome, K.R., Atkins, R. C, Dunstan, D. W., Murray, S. G. and Shaw, J.E. (2006) The Australian Diabetes, Obesity and Lifestyle Study, AusDiab 2005. International Diabetes Institute, Melbourne.

Beynon, R. and Bond, JS (ed), (2001) Proteolytic Enzymes, 2^nd ed. Oxford University Press, New York.

Boza, J.J., Dangin, M., Moennoz, D., Montignon, F., Vuichoud, J., Jarret, A., Gremaud, G., Oquey-Araymon, S., Courtois, D., Woupeyi, A., Finot, P.A. and Ballere, O. (2001) Free and protein bound glutamine have identical splanchnic extraction in healthy human volunteers. Am J. Physiol Gastrointest. Liver Physiol. 281, G267-74

Jenkins, D. J., Thomas, M.S., Wolever, T.M., Taylor, R.H., Barker, H., Fielden, H., Baldwin, J.M., Bowling, A.C., Newman, H.C., Jenkins, AL. and Goff, D.V. (1981). Glycemic index of foods: A physiological basis for carbohydrate exchange. American Journal of Clinical Nutrition, 34(3), 362-366

Frisher, H., Meisel, H. and Schlimme, E. (1988) OPA method modified b use of N,N-dimethyl-2-mercaptoethylammonium chloride as thiol components. Fresenius Zeitschrifi Anyafytical Chemistry, 330, 631-633

Kasarda, D.D., Woodard, K.M. and Adalsteins, AE. (1998) Resolution of High Molecular Weight Glutenin Subunits by a new SDS-PAGE System Incorporationg a Neutral pH Buffer. Cereal Chemistry,^') '5(1), 70-71

Matz, S. A (1984) Snack Food Technology, 2^nd ed. The AVI Pub!. Co., Westport, pp. 144-149

Owusu-Apenten, R.K. (2002) Food Protein Analysis. Quantitative Effects on Processing. Marcel Dekker, Inc., New York.

Wrigley, C. W., Robinson, P.J. and Williams, W.T. (1981) Association between electrophoretic patterns of gliadin proteins and quality characteristics of wheat cultivators. J. Sci. Food Agric. 32:433-442

Zhang, X., Hoobin, P., Burgar, I. and Do, M.D. (2006) Chemical modification of Wheat protein-based natural polymers: cross-linking Effect on mechanical properties and phase Structures. J. Agric. Food Chem. 54: 9858-9865

Cornell, HJ. and Hoveling, AW. (1998) The Wet Milling of Wheat Flour in Wheat Chemistry and Utilisation publ. Technomic Publishing Co. Inc., Lancaster, Pennsylvania, USA

Lawton, J. W. and Wu, Y. V. (1993) Thermal Behaviour of Annealed Acetic Acid-soluble Wheat Gluten. Cereal Chem. 70(4): 471-475

Claims

The claims :-

1. A process of producing a modified wheat gluten for incorporation into a protein enriched food, said process including the step of adding a protease enzyme to said wheat gluten in aqueous dispersion under conditions of predetermined pH, ionic strength, temperature and time whereby selective peptide bond cleavage is obtained to give a degree of hydrolysis for said gluten in a range of about 1% to about 4%, a content of proteinaceous material having molecular weight greater than 50,000 Daltons being less than 15% of total proteinaceous material, a content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material and a glass transition temperature in a range of about 50 degrees C to about 59 degrees C.

2. The process as claimed in claim 1 wherein said degree of hydrolysis is in a range of about 2% to about 3%.

3. The process as claimed in claim 1 wherein said wheat gluten is in a wet form in which total solids content is in a range of about 30% to about 35% w/w and the protein content is in a range of about 70% to about 85%.

4. The process as claimed in claim 1 wherein said wheat gluten is in a dry form and has a protein content in a range of about 70% to about 85% and said process includes the step of dispersing said protease enzyme wholly or partially in water prior to the addition of said dried gluten in either a batch or a continuous mixing operation.

5. The process as claimed in claim 1 wherein said wheat gluten is in a dry form and has a protein content in a range of about 70% to about 85% and said process also includes the steps of dispersing said wheat gluten in water in a concentration range up to about 50% and then adding a quantity of said protease enzyme in aqueous dispersion.

6. The process as claimed in claim 1 and including the step of deactivating said protease enzyme after said predetermined time by thermal denaturation, acid-shock or alkaline-shock.

7. A wheat gluten for incorporation into a protein enriched food, the structure of said wheat gluten being modified in accordance with the process as claimed in claim 1 whereby said wheat gluten shows a degree of hydrolysis in a range of about 1% to about 4%, a content of proteinaceous material having a molecular weight greater than 50,000 Daltons being less than 15% of total proteinaceous material, a content of proteinaceous material having molecular weight less than 15,000 Daltons being less than 50% of total proteinaceous material and a glass transition temperature in a range of about 50 degrees C to about 59 degrees C.

8. The wheat gluten as claimed in claim 7 wherein said degree of hydrolysis is in a range of about 2% to about 3%.

9. A protein-enriched food which includes as an ingredient thereof a modified wheat gluten as claimed in claim 7.

10. The protein-enriched food as claimed in claim 9 wherein said food is expanded or extruded.

11. The protein-enriched food as claimed in claim 9 wherein said food is a potato chip-type food which has been extruded in the form of a sheet and then fried or baked.

12. The protein-enriched food as claimed in claim 9 wherein said food is a com based tortilla-type food which has been extruded in the form of a sheet and then fried or baked.

13. The protein-enriched food as claimed in claim 9 wherein said food is a breakfast cereal food or a snack food which has been extruded or expanded.

14. A process of producing a modified wheat gluten for incorporation into a protein-enriched food, said process being substantially as described herein with reference to the examples.

15. A wheat gluten for incorporation into a protein-enriched food, said wheat gluten being modified in a manner substantially as described herein with reference to the examples.

16. A protein-enriched food substantially as described herein with reference to the examples.