Wheat-based emulsifying agent

Abstract

Provided herein is a wheat-based emulsifying agent comprising a modified wheat protein component in a concentration of at least about 50% by weight on a dry basis, relative to the total weight of the wheat-based emulsifying agent. The modified wheat protein component preferably has a high molecular weight protein fraction of no greater than about 45%, a low molecular weight protein fraction of at least about 15%, and a gliadin protein fraction of at least about 35%. Also provided herein are methods of making a modified wheat protein composition, methods of preparing baked goods comprising a modified wheat protein composition, and baked goods prepared using such methods.

Description

WHEAT-BASED EMULSIFYING AGENTTECHNICAL FIELD

[0001] Provided herein are wheat-based emulsifying agents that are useful, for example, in the preparation of baked goods. Also provided herein are methods of making wheat-based emulsifying agents, methods of preparing baked goods that comprise wheat-based emulsifying agents, and baked goods prepared using such methods.BACKGROUND ART

[0002] Emulsifying agents are food additives commonly used in baking to stabilize, or “emulsify,’' mixtures of water and oil. Typical emulsifying agents are surfactants that comprise a polar moiety that interfaces with the aqueous phase, and a non-polar moiety that interfaces with the oil phase, thereby enabling the two phases to mix and form a stable or semi-stable emulsion.

[0003] Eggs, and particularly egg yolks, are traditional emulsifying agents that have been used in baking recipes for centuries. Lecithin has also been traditionally used as an emulsifying agent. Chemical emulsifying agents, such as monoglycerides, lactic acid esters, propylene glycol esters, polyglycerol esters, and polysorbates, first began to see widespread use in the 1930s and have now become commonplace in processed commercial baked goods.

[0004] In recent years, there has been an increasing interest in “plant-based” foods, which do not incorporate any ingredients derived from animal sources. Traditional emulsifying agents used in baking, particularly eggs and dairy proteins, are often derived from animal sources and may be viewed by consumers as unsuitable for use in plant-based baked goods.

[0005] Consumer interest in “clean label” food products has also been rapidly rising in recent years. While precise definitions can vary, the term “clean label” generally encompasses food products made exclusively from natural ingredients that are familiar to most consumers. Stated another way, “clean label” food products are generally free of artificial ingredients and synthetic chemicals. Products meeting these criteria are viewed favorably by consumers and are often able to command a premium price in the marketplace.

[0006] It is therefore desirable to provide a plant-based emulsifying agent that can be used to prepare plant-based baked goods. Ideally, a plant-based emulsifying agent would be capable of providing the same, or at least similar functionality as traditional, animal-based emulsifying agents, and would be suitable for use in plant-based recipes as well as traditional recipes that may incorporate animal products. Additionally, a plant-based emulsifying agent would ideally be suitable for use in “clean label” food products.DISCLOSURE OF INVENTION

[0007] In one aspect, provided herein is a method of making a wheat-based emulsifying agent. The method may comprise any one or more of the following steps: (1) providing a reaction mixture comprising a starting material; (2) a proteolysis step wherein the reaction mixture is contacted with a protease; (3) a lipolysis step wherein the reaction mixture is contacted with a lipase; (4) a supplemental enzyme treatment step wherein the reaction mixture is contacted with an enzyme; and (5) an enzy me deactivation step wherein one or more enzymes present in the reaction mixture are substantially deactivated. For example, the supplemental enzy me treatment step may comprise contacting the reaction mixture with a glutaminase, a xylanase, or a combination thereof. The method may optionally comprise one or more additional steps, including but not limited to drying steps and enzyme deactivation steps, as described in further detail below.

[0008] Also provided herein is a wheat-based emulsifying agent. The wheat-based emulsifying agent may be prepared, for example, using a method as provided herein.

[0009] The wheat based emulsifying agent may have one or more desirable properties as described in further detail below. For example, the wheat-based emulsify ing agent may have wheat protein content of at least about 50% by weight on a dry' basis (i.e., excluding water content). The wheat-based emulsifying agent may comprise a modified wheat protein component having an average molecular weight of less than about 150 kDa. For example, the wheat-based emulsifying agent may comprise a modified wheat protein component having an average molecular weight of from about 30 kDa to about 150 kDa. The wheat-based emulsifying agent may have a fat content of at least about 0.5% by weight on a dry' basis. For example, the wheat-based emulsify ing agent may have a fat content of from about 2% by weight to about 8% by weight on a dry basis. The fat content the fat content of the wheat-based emulsifying agent may be enriched in monoglycendes and diglycerides as compared to the naturally occurring fat present in wheat flour.

[0010] For example, provided herein is a wheat-based emulsify ing agent comprising a modified wheat protein component in a concentration of at least about 50% by weight on a dry' basis, relative to the total weight of the wheat-based emulsifying agent. The modified wheat protein component preferably has a high molecular weight protein fraction of no greater than about 45%, a low molecular weight protein fraction of at least about 15%, and a gliadin protein fraction of at least about 35%.

[0011] Also provided herein are methods of preparing baked goods that comprise wheatbased emulsifying agents as described herein, and baked goods prepared using such methods.

[0012] Other objects and features will be in part apparent and in part pointed out hereinafter.BEST MODE FOR CARRYING OUT THE INVENTION

[0013] Provided herein are methods of preparing wheat-based emulsifying agents that are suitable for use in preparing baked goods. Also provided herein are methods of preparing baked goods comprising a wheat-based emulsifying agent, and baked goods prepared using such methods.

[0014] Several advantages may be provided by a wheat-based emulsifying agent as described herein. Wheat protein is readily available and relatively inexpensive, particularly compared to animal-based ingredients, such as eggs and dairy products, that are traditionally used as emulsifying agents in baking applications. Accordingly, the wheat-based emulsifying agent described herein may be produced consistently, at a relatively low cost, and without exposure to the price instability and supply chain issues that have plagued animal-based ingredients in recent years.

[0015] The wheat-based emulsifying agent provided herein may be capable of providing similar functionality as traditional, animal-based emulsifying agents.

[0016] The wheat-based emulsifying agent provided herein is suitable for use in plantbased recipes as well as traditional recipes that may incorporate animal products. Additionally, the wheat-based emulsifying agent provided herein is suitable for use in ‘‘clean label” food products. The provided wheat-based emulsifying agents are particularly suitable for use in baked goods — because they contain a relatively high percentage of wheat protein, they are able to maintain the gluten network that provides the desirable texture in many bakery products.Methods of Making a Wheat-Based Emulsifying Agent

[0017] For example, provided herein is a method of making a wheat-based emulsifying agent. The method may comprise any one or more of the following steps: (1) providing a reaction mixture comprising a starting material; (2) a proteolysis step wherein the reaction mixture is contacted with a protease; (3) a lipolysis step wherein the reaction mixture is contacted with a lipase; (4) a supplemental enzy me treatment step wherein the reaction mixture is contacted with an enzyme; and (5) an enzyme deactivation step wherein one or more enzymes present in the reaction mixture are substantially deactivated.

[0018] The method may further comprise one or more dilution steps, drying steps, starch addition steps, or other steps as described in further detail below'.

[0019] The proteolysis, lipolysis, and supplemental enzyme treatment step(s) may be conducted simultaneously (i.e., in a single stage), or separately (i.e., in multiple stages). Inembodiments where the method comprises multiple stages, the proteolysis, lipolysis, and supplemental enzyme treatment step(s) may be conducted in any order or combination.

[0020] As used herein, the term “reaction mixture” refers initially to the starting material, which may optionally be present as a mixture with water and / or other components as described below. With respect to subsequent method steps, the term “reaction mixture” refers to the product of the preceding step.

[0021] Starting Material

[0022] The method provided herein utilizes a starting material comprising a source of wheat protein. Generally, the starting material can comprise any source of wheat protein known in the art. Non-limiting examples of suitable wheat protein sources include wet wheat gluten, vital wheat gluten, whole wheat flour, and wheat protein isolate.

[0023] The starting material may be provided as a dry composition, such as a wheat protein isolate powder, a vital wheat gluten powder, or a mixture thereof. Alternatively, the starting material may be provided as a wet composition wherein a source of wheat protein (e.g., wheat protein isolate, vital wheat gluten, or a combination thereof) is combined with water. The compositions and percentages provided below are provided on a dry basis, that is, without taking into account any water present in the starting material.

[0024] The starting material preferably has a wheat protein content of at least about 50% by weight. For example, the starting material may have a wheat protein content of at least about 60% by weight, at least about 65% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight.

[0025] The starting material preferably comprises a small amount of fat. For example, the staring material may have a fat content of at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, or at least about 5% by weight. Typically, the starting material has a fat content of no greater than about 15% by weight, for example, no greater than about 10% by weight, no greater than about 8% by weight, no greater than about 6% by weight, or no greater than about 5% by weight. The fat content of the starting material may fall within a range defined by any two values listed above. As non-limiting examples, the starting material may have a fat content of from about 0.5% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 3% by weight to about 8% by weight, or from about 4% by weight to about 6% by weight.

[0026] Wheat protein sources suitable for use as the starting material will typically comprise some amount of naturally occurring fat. For example, vital wheat gluten typicallycomprises about 5% fat by weight. Commercially available wheat protein isolate typically comprises from about 1% to about 5% fat by weight.

[0027] The starting material may comprise, consist essentially of, or consist of vital wheat gluten. For example, the starting material may comprise, consist essentially of, or consist of vital wheat gluten having a wheat protein content of at least about 60% by weight. For example, the vital wheat gluten may have a wheat protein content of at least about 65%, at least about 70%, or at least about 75% by weight. Conversely, vital wheat gluten typically has a wheat protein content of at most about 95%, at most about 90%, at most about 85%, or at most about 80% by weight. The protein content of the vital wheat gluten may fall within a range defined by any two values listed above. As non-limiting examples, the vital wheat gluten may have a wheat protein content of from about 60% by weight to about 90% by weight, from about 65% by weight to about 85% by weight, or from about 70% by weight to about 80% by weight.

[0028] The vital wheat gluten may have a fat content of at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, or at least about 5% by weight. The vital wheat gluten preferably has a fat content of no greater than about 10% by weight, for example, no greater than about 8% by weight, or no greater than about 6% by weight. The fat content of the vital wheat gluten may fall within a range defined by any two values listed above. As non-limiting examples, the vital wheat gluten may have a fat content of from about 0.5% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, or from about 4% by weight to about 6% by weight.

[0029] The starting material may comprise, consist essentially of, or consist of a wheat protein isolate. For example, the starting material may comprise, consist essentially of. or consist of wheat protein isolate having a wheat protein content of at least about 80% by weight. For example, the wheat protein isolate may have a wheat protein content of at least about 85% by weight, or at least about 90% by weight. Conversely, wheat protein isolate typically has a wheat protein content of at most about 99%, at most about 98%, at most about 97%. at most about 96%, at most about 95%, or at most about 90% by weight. The protein content of the wheat protein isolate may fall within a range defined by any two values listed above. As non-limiting examples, the wheat protein isolate may have a wheat protein content of from about 80% by weight to about 99% by weight, from about 85% by weight to about 98% by weight, or from about 90% by weight to about 97% by weight.

[0030] The wheat protein isolate may have a fat content of at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, or at least about 5% by weight. The wheat protein isolate preferably has a fatcontent of no greater than about 10% by weight, for example, no greater than about 8% by weight, or no greater than about 6% by weight. The fat content of the wheat protein isolate may fall within a range defined by any two values listed above. As non-limiting examples, the wheat protein isolate may have a fat content of from about 0.5% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, or from about 4% by weight to about 6% by weight.

[0031] Proteolysis Step

[0032] The method may comprise a proteolysis step wherein a reaction mixture is contacted with a protease.

[0033] The protease may comprise any protease known in the art. Non-limiting examples include serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases.

[0034] As a specific example, the protease may comprise an aminopeptidase. For example, the protease may comprise a dipeptidyl-aminopeptidase.

[0035] The protease may comprise a naturally occurring protease derived from a plant. For example, the protease may comprise one or more enzymes naturally occurring in pineapple (for example, bromelain). The protease may comprise one or more enzymes naturally occurring in papaya (for example, papain). The protease may comprise one or more enzymes naturally occurring in kiwifruit (for example, actinidin).

[0036] The protease may comprise a naturally occurring protease derived from fungi. For example, the protease may comprise a naturally occurring protease derived from Aspergillus oryzae.

[0037] The protease can be utilized in any available form. For example, the methods described herein may comprise using a fruit comprising the protease ( blended or pureed pineapple, papaya, or kiwi fruit), using a natural fruit extract comprising the protease, or using a commercially or industrially produced protease.

[0038] Without being bound to a particular theory, it is believed that the protease acts to reduce the average molecular weight of the wheat protein present in the starting material. Wheat proteins consist of glutenins and gliadins. Glutenins are protein aggregates divided into high- molecular-weight (HMW-GS, about 70 - 140 kDa) and low-molecular-weight (LMW-GS, about 30-50 kDa) subunits. Gliadin accounts for the remaining 40-50% of the total seed storage proteins of wheat and is a heterogeneous mixture of monomeric protein having an average molecular weight of 30-78 kDa. A proteolysis step as defined herein modifies the wheat protein by shifting the proportion of larger molecular weight proteins to lower molecular weight proteins. Forexample, modified wheat proteins produced using a proteolysis step as defined herein will exhibit a lower percentage of polymeric protein (glutenin), and an increased percentage of lower molecular weight proteins (albumin and globulins). In some cases, the overall average molecular weight of the modified wheat protein is close to that of naturally occurring gliadin (e g,, from about 30 kDa to about 78 kDa).

[0039] Accordingly, in preferred embodiments, the proteolysis step produces a reaction mixture comprising a modified wheat protein having a reduced molecular weight as compared to the starting material. For example, the proteolysis step preferably produces a reaction mixture comprising a modified wheat protein having an average molecular weight of between about 30 kDa and about 150 kDa.

[0040] For example, the proteolysis step preferably produces a reaction mixture comprising a modified wheat protein having an average molecular weight of less than about 150 kDa, less than about 140 kDa, less than about 130 kDa. less than about 120 kDa, less than about 110 kDa. less than about 100 kDa, less than about 90 kDa, less than about 80 kDa. or less than about 70 kDa. Conversely, the proteolysis step preferably produces a reaction mixture comprising a modified wheat protein having an average molecular weight of at least about 10 kDa, at least about 20 kDa, at least about 25 kDa, or at least about 30 kDa. The average molecular weight of the modified wheat protein may fall within a range bounded by any two values listed above. For example, the modified wheat protein may have an average molecular weight of from about 20 kDa to about 80 kDa, or from about 25 kDa to about 45 kDa.

[0041] The proteolysis step preferably produces a reaction mixture comprising a modified wheat protein having a reduced proportion of glutenins as compared to the starting material. As used herein, the “high molecular weight protein fraction” refers to the fraction of the total wheat protein content that is attributable to high-molecular-weight glutenins and / or low-molecular- weight glutenins on a weight basis. For example, the modified wheat protein preferably has a high molecular weight protein fraction that is less than about 98%, less than about 96%, less than about 94%, less than about 92%, less than about 90%, or less than about 88% of the high molecular weight protein fraction of the starting material. Conversely, the modified wheat protein preferably has a high molecular weight protein fraction that is at least about 70%, at least about 75%, at least about 80%, or at least about 85% of the high molecular weight protein fraction of the starting material. The modified wheat protein may have a high molecular weight protein fraction that falls within a range bounded by any two values listed above. As non-limiting examples, the modified wheat protein may have a high molecular weight protein fraction of from about 80% to about 92%,or from about 85% to about 88% of the high molecular weight protein fraction of the starting material.

[0042] The proteolysis step preferably produces a reaction mixture comprising a modified wheat protein having an increased proportion of albumin and globulins as compared to the starting material. As used herein, the “low molecular weight protein fraction’7refers to the fraction of the total wheat protein content that is attributable to albumin and / or globulins on a weight basis. For example, the modified wheat protein preferably has a low molecular weight protein fraction that is at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, or at least about 130% of the low molecular weight protein fraction of the starting material. Conversely, the modified wheat protein preferably has a low molecular weight protein fraction that is at most about 200%, at most about 175%, at most about 150%, at most about 140%, or at most about 135% of the low molecular weight protein fraction of the starting material. The modified wheat protein may have a low molecular weight protein fraction that falls within a range bounded by any two values listed above. As non-limiting examples, the modified wheat protein may have a low molecular weight protein fraction of from about 120% to about 150%, or from about 125% to about 140% of the low molecular weight protein fraction of the starting material.

[0043] The proteolysis step can be conducted under increased temperature (i.e., a temperature in excess of room temperature). For example, the proteolysis step can be conducted at a temperature of at least about 40 °C (104 °F), at least about 50 °C (122 °F), or at least about 55 °C (131 °F). In some embodiments, the proteolysis step is conducted at a temperature of from about 45 °C (113 °F) to about 80 °C (176 °F), from about 55 °C (131 °F) to about 70 °C (158 °F), from about 50 °C (122 °F) to about 65 °C (149 °F), or from about 55 °C (131 °F) to about 60 °C (160 °F).

[0044] The proteolysis step is preferably conducted for a time sufficient for the reaction between the one or more enzymes and the wheat protein to proceed substantially to completion. For example, the proteolysis step may be conducted for a period of at least about 15 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 1 hour. In some embodiments, the proteolysis step is conducted for a period of from about 30 minutes to about 4 hours, from about 45 minutes to about 3 hours, or from about 1 hour to about 2 hours.

[0045] In some embodiments, a reaction mixture comprising the one or more enzy mes and the wheat protein composition is mixed during at least a portion of the proteolysis step. In preferred embodiments, the reaction mixture is mixed for substantially the full duration of the proteolysis step.

[0046] The proteolysis step can be carried under acidic conditions, wherein the pH of the reaction mixture is less than 7. For example, the proteolysis step can comprise maintaining the pH of the reaction mixture within a range of from about 3 to about 7. In preferred embodiments, the proteolysis step comprises maintaining the pH of the reaction mixture within a range of from about 5 to about 7, or from about 5.5 to about 6.5. In other embodiments, the proteolysis step comprises maintaining the pH of the reaction mixture within a range of, for example, from about 3 to about 5, or from about 3.5 to about 4.5.

[0047] Lipolysis Step

[0048] The method may comprise a lipolysis step wherein a reaction mixture is contacted with a lipase.

[0049] The lipase may comprise any lipase known in the art. The lipase may be used in any available form. Non-limiting examples of suitable lipases include phospholipases, triacyl glycerol lipases, and mixtures thereof.

[0050] Without being bound to a particular theory, it is believed that the lipase acts to convert triglycerides present in the starting material into free monoglycerides and diglycerides. It is believed that the free monoglycerides and diglycerides contribute to the emulsifying properties of the w heat-based emulsifying agent described herein.

[0051] Preferably, the lipolysis step comprises contacting the reaction mixture with a lipase for a time sufficient to convert at least about 5% of the triglycerides present in the starting material into monoglycerides and diglycerides. For example, the lipolysis step may comprise converting at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%. at least about 40%, or at least about 50% of the triglycerides present in the starting material into monoglycerides and diglycerides.

[0052] The lipolysis step may be conducted before, after, or concurrently with a proteolysis step as discussed above. For example, the method may comprise conducting a proteolysis step followed by a lipolysis step. Alternatively, the method may comprise conducting a proteolysis step and a lipolysis step simultaneously.

[0053] The lipolysis step can be conducted under increased temperature (i.e., atemperature in excess of room temperature). For example, the lipolysis step can be conducted at a temperature of at least about 40 °C (104 °F), at least about 50 °C (122 °F), or at least about 55 °C (131 °F). Conversely, the lipolysis step is typically conducted at atemperature of no greater than about 90 °C (194 °F), no greater than about 80 °C (176 °F), no greater than about 70 °C (158 °F), no greater than about 65 °C (149 °F), no greater than about 60 °C (140 °F), or no greater than about 55 °C(131 °F). The lipolysis step may be conducted within a temperature range bounded by any two values listed above. As non-limiting examples, the lipolysis step may be conducted at a temperature of from about 45 °C (113 °F) to about 80 °C (176 °F), from about 55 °C (131 °F) to about 70 °C (158 °F), from about 50 °C (122 °F) to about 65 °C (149 °F), or from about 55 °C (131 °F) to about 60 °C (140 °F).

[0054] The lipolysis step is preferably conducted for a time sufficient for the reaction between the one or more enzymes and the wheat protein to proceed substantially to completion. For example, the lipolysis step may be conducted for a period of at least about 15 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 1 hour. In some embodiments, the lipolysis step is conducted for a period of from about 30 minutes to about 4 hours, from about 45 minutes to about 3 hours, or from about 1 hour to about 2 hours.

[0055] In some embodiments, a reaction mixture comprising the one or more enzy mes and the wheat protein composition is mixed during at least a portion of the lipolysis step. In preferred embodiments, the reaction mixture is mixed for substantially the full duration of the lipolysis step.

[0056] The lipolysis step can be carried under acidic conditions, wherein the pH of the reaction mixture is less than 7. For example, the lipolysis step can be carried under acidic conditions wherein the pH of the reaction mixture is less than about 6.5, less than about 6, less than about 5.5, less than about 5, or less than about 4.5. Conversely, the pH of the reaction mixture is typically at least about 3, at least about 3.5, at least about 4, at least about 4.5, or at least about 5. The pH of the reaction mixture may fall within a range defined by any two values listed above. As a non-limiting example, the lipolysis step can comprise maintaining the pH of the reaction mixture within a range of from about 3 to about 7. In preferred embodiments, the lipolysis step compnses maintaining the pH of the reaction mixture within a range of from about 5 to about 7, or from about 5.5 to about 6.5. In other embodiments, the lipolysis step comprises maintaining the pH of the reaction mixture within a range of, for example, from about 3 to about 5, or from about 3.5 to about 4.5.

[0057] Supplemental Enzyme Treatment Step

[0058] The method may comprise a supplemental enzyme treatment step wherein a reaction mixture is contacted with an enzyme.

[0059] In most cases, conducting a proteolysis step and a lipolysis step on a suitable starting material, as generally described above, will result in a wheat-based emulsifying agent that is suitable for use in many food applications. In situations where a greater emulsification strengthis desirable, however, an additional enzyme treatment step can be conducted further process the wheat protein and provide the desired functionality.

[0060] As a non-limiting example, the supplemental enzyme treatment step may comprise contacting the reaction mixture with a glutaminase, transglutaminase, a xylanase, a P-glucanase, or a combination thereof.

[0061] For example, the supplemental enzyme treatment step may comprise contacting the reaction mixture with a transglutaminase. Without being bound to a particular theory, it has been discovered that an additional enzyme treatment step utilizing a transglutaminase may result in a wheat-based emulsifying agent having increased emulsification strength.

[0062] As a further example, the supplemental enzyme treatment step may comprise contacting the reaction mixture with a glutaminase.

[0063] As a further example, the supplemental enzyme treatment step may comprise contacting the reaction mixture with a xylanase.

[0064] As a further example, the supplemental enzyme treatment step may comprise contacting the reaction mixture with a P-glucanase.

[0065] The supplemental enzyme treatment step may be conducted before, after, or concurrently with a proteolysis step and / or a lipolysis step as discussed above. For example, the method may comprise a supplemental enzyme treatment step that is conducted subsequent to a lipolysis step and a proteolysis step. Alternatively, the method may comprise a supplemental enzyme treatment step that is conducted concurrently with a lipolysis step and / or a proteolysis step.

[0066] The supplemental enzyme treatment step can be conducted under increased temperature (i. e.. a temperature in excess of room temperature). For example, the supplemental enzyme treatment step can be conducted at a temperature of at least about 40 °C (104 °F), at least about 50 °C (122 °F), or at least about 55 °C (131 °F). In some embodiments, the supplemental enzyme treatment step is conducted at a temperature of from about 45 °C (113 °F) to about 80 °C (176 °F), from about 55 °C (131 °F) to about 70 °C (158 °F), from about 50 °C (122 °F) to about 65 °C (149 °F), or from about 55 °C (131 °F) to about 60 °C (140 °F).

[0067] The supplemental enzyme treatment step is preferably conducted for a time sufficient for the reaction between the one or more enzymes and the wheat protein to proceed substantially to completion. For example, the supplemental enzyme treatment step may be conducted for a period of at least about 15 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 1 hour. In some embodiments, the supplemental enz me treatment stepis conducted for a period of from about 30 minutes to about 4 hours, from about 45 minutes to about 3 hours, or from about 1 hour to about 2 hours.

[0068] In some embodiments, a reaction mixture comprising the one or more enzymes and the wheat protein composition is mixed during at least a portion of the supplemental enzyme treatment step. In preferred embodiments, the reaction mixture is mixed for substantially the full duration of the supplemental enzyme treatment step.

[0069] The supplemental enzy me treatment step can be carried under acidic conditions, wherein the pH of the reaction mixture is less than 7. For example, the supplemental enzy me treatment step can be carried under acidic conditions wherein the pH of the reaction mixture is less than about 6.5, less than about 6, less than about 5.5, less than about 5, or less than about 4.5. Conversely, the pH of the reaction mixture is typically at least about 3, at least about 3.5, at least about 4, at least about 4.5, or at least about 5. The pH of the reaction mixture may fall within a range defined by any two values listed above. As a non-limiting example, the supplemental enzyme treatment step can comprise maintaining the pH of the reaction mixture within a range of from about 3 to about 7. In preferred embodiments, the supplemental enzyme treatment step comprises maintaining the pH of the reaction mixture within a range of from about 5 to about 7, or from about 5.5 to about 6.5. In other embodiments, the supplemental enzyme treatment step comprises maintaining the pH of the reaction mixture within a range of, for example, from about 3 to about 5, or from about 3.5 to about 4.5.

[0070] Dilution Step

[0071] The methods may optionally comprise one or more dilution steps in which a reaction mixture, or a component thereof, is mixed with w ater.

[0072] Particularly where the starting material has a low moisture content, diluting the starting material and / or one or more of the enzyme components in water can enable the enzymes to more readily react with the starting material. For example, the method may comprise a dilution step in which the starting material is diluted with w ater. As a further example, the method may comprise a dilution step in which one or more enzymes are diluted with water.

[0073] Water added during a dilution step may be removed using a subsequent drying step, as explained in further detail below.

[0074] Enzyme Deactivation Step

[0075] The method may comprise an enzyme deactivation step wherein one or more enzymes present in a reaction mixture are substantially deactivated.

[0076] For example, the deactivation step can comprise heating the reaction mixture comprising the primary enzyme, the optional secondary enzyme, and the wheat protein to a temperature sufficient to deactivate the primary and secondary7enzymes. In some embodiments, the deactivation step comprises heating the reaction mixture to a temperature of at least about 65 °C (149 °F), at least about 70 °C (158 °F), at least about 75 °C (167 °F), at least about 80 °C (176 °F), at least about 85 °C (185 °F), or at least about 90 °C (194 °F).

[0077] The deactivation step is preferably conducted for a duration sufficient to ensure that substantially all of the primary enzyme and optional secondary enzyme is deactivated. For example, the deactivation step can be conducted for a period of at least about 2 minutes, at least about 5 minutes, or at least about 10 minutes.

[0078] Drying Step

[0079] The methods described herein can further comprise a drying step wherein a reaction mixture is dried to remove excess water.

[0080] In some embodiments, the method comprises an enzyme deactivation step as discussed above that is effective to dry the reaction mixture, for example by exposing the reaction mixture to an increased temperature for a sufficient duration to substantially remove the water content. Accordingly, in these embodiments, the enzyme deactivation step effectively serves as the drying step, and no further drying is required.

[0081] In other embodiments, the reaction mixture can be dried by any means known to those skilled in the art. Non-limiting examples of suitable drying methods include spray drying, freeze drying, and convective hot air drying, for example using a ring dryer, an oven dryer, or a flash dryer.

[0082] For example, the drying step may comprise freeze drying the reaction mixture, thereby providing a wheat-based emulsifying agent suitable for use in the preparation of baked goods.

[0083] As a further example, the dry ing step may comprise drying the reaction mixture using a ring dryer, thereby providing a wheat-based emulsifying agent suitable for use in the preparation of baked goods.

[0084] Starch Addition Step

[0085] The method may comprise a starch addition step wherein a starch is added to the reaction mixture. The starch may be, for example, wheat starch. The starch addition step may be conducted before, concurrently with, or after any7of the proteolysis, lipolysis, or supplemental enzyme treatment steps described above.

[0086] For example, the method may comprise a starch addition step wherein starch is added in an amount sufficient to comprise at least about 1%, at least about 2%, at least about 5%, at least about 10%, or at least about 20% by weight of the reaction mixture.

[0087] Wheat-Based Emulsifying Agent

[0088] Also provided herein is a wheat-based emulsifying agent that is useful, for example, in the preparation of baked goods. The wheat-based emulsifying agent may be prepared, for example, using a method as described herein.

[0089] The wheat-based emulsifying agent preferably comprises a modified wheat protein component in a concentration of at least about 50% by weight on a dry basis (i. e. , excluding water content) relative to the total weight of the wheat-based emulsifying agent. For example, the wheatbased emulsifying agent may comprise the modified wheat protein component in a concentration of at least about 60% by weight, at least about 65% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight on a dry basis. Conversely, the wheatbased emulsifying agent typically comprises the modified wheat protein component in a concentration of at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, or at most about 90% by weight. The wheat-based emulsifying agent may comprise the modified wheat protein component in a concentration that falls within a range defined by any two values listed above. As non-limiting examples, the wheat-based emulsifying agent may compnse the modified wheat protein component in a concentration of from about 80% by weight to about 99% by weight, from about 85% by weight to about 98% by weight, or from about 90% by weight to about 97% by weight on a dry basis, relative to the total weight of the wheat-based emulsifying agent.

[0090] The wheat-based emulsifying agent preferably comprises a modified wheat protein component having a high molecular weight protein fraction of no greater than about 45%. For example, the modified wheat protein component may have a high molecular weight protein fraction of no greater than about 44%, no greater than about 43%, no greater than about 42%, no greater than about 41%, or no greater than about 40%. Conversely, the modified wheat protein component preferably has a high molecular weight protein fraction of at least about 30%, at least about 32%, at least about 34%, at least about 36%, or at least about 38%. The modified wheat protein component may have a high molecular weight protein fraction that falls within a range bounded by any two values listed above. As non-limiting examples, the modified wheat protein component may have a high molecular weight protein fraction that falls within a range of from about 30% to about 45%, from about 36% to about 43%, or about 38% to about 42%.

[0091] The wheat-based emulsifying agent preferably comprises a modified wheat protein component having a low molecular weight protein fraction of at least about 15%. For example, the modified wheat protein component may have a low molecular weight protein fraction of at least about 16%, at least about 16.5%. at least about 17%, not at least about 17.5%, at least about 18%, or at least about 18.5%. Conversely, the modified wheat protein component preferably has a low molecular weight protein fraction of no greater than about 30%, no greater than about 28%, no greater than about 26%, no greater than about 24%, no greater than about 22%, or no greater than about 20%. The modified wheat protein component may have a low molecular weight protein fraction that falls within a range bounded by any two values listed above. As non-limiting examples, the modified wheat protein component may have a low molecular weight protein fraction that falls within a range of from about 15% to about 30%, from about 16% to about 24%, or about 17% to about 22%.

[0092] The wheat-based emulsifying agent preferably comprises a modified wheat protein component having a gliadin protein fraction of at least about 35%. As used herein, the “gliadin protein fraction” refers to the fraction of the total wheat protein content that is attributable to gliadin on a weight basis. For example, the modified wheat protein component may have a gliadin protein fraction of at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, or at least about 41%.

[0093] In some embodiments, the wheat-based emulsifying agent comprises a modified wheat protein component having an average molecular weight of less than about 80 kDa, less than about 70 kDa, less than about 60 kDa, less than about 55 kDa, less than about 50 kDa, or less than about 45 kDa. The wheat-based emulsifying agent may comprise a modified wheat protein component having an average molecular weight of at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, or at least about 25 kDa. The average molecular weight of the modified wheat protein component may fall within a range bounded by any two values listed above. For example, the modified wheat protein component may have an average molecular weight of from about 20 kDa to about 80 kDa, or from about 25 kDa to about 45 kDa.

[0094] The wheat-based emulsifying agent may have a fat content of at least about 0.5% by weight, at least about 1% by weight, at least about 2% by weight, at least about 3% by weight, at least about 4% by weight, or at least about 5% by weight on a dry basis. Typically, the wheatbased emulsifying agent has a fat content of no greater than about 15% by weight, for example, no greater than about 10% by weight, no greater than about 8% by weight, no greater than about 6% by weight, or no greater than about 5% by weight on a dry basis. The fat content of the wheatbased emulsify ing agent may fall within a range defined by any of the values listed above. As non-limiting examples, the wheat-based emulsifying agent may have a fat content of from about 0.5% by weight to about 10% by weight, from about 2% by weight to about 8% by weight, from about 3% by weight to about 8% by weight, or from about 4% by weight to about 6% by weight on a dry basis.

[0095] In some embodiments, the fat content of the wheat-based emulsifying agent is enriched in monoglycerides, and diglycerides as compared to the naturally occurring fat present in wheat flour. For example, monoglycerides and diglycerides may comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the fat content of the wheat-based emulsifying agent.Preparation of Baked Goods Comprising the Wheat-Based Emulsifying Agent

[0096] Also provided herein are methods of preparing baked goods comprising a wheatbased emulsifying agent, and baked goods prepared using such methods.

[0097] Generally, the wheat-based emulsifying agent provided herein may be used as a replacement for conventional emulsifiers such as eggs, egg yolks, and lecithin, as well as chemical emulsifiers such as monoglycerides, lactic acid esters, propylene glycol esters, polyglycerol esters, and polysorbates. In general, the wheat-based emulsify ing agent may be used in rates and amounts similar to those used for conventional emulsifiers.EXAMPLES

[0098] The following non-hmiting examples are provided to further illustrate the present disclosure. The examples should, of course, be understood to be merely illustrative of only certain embodiments and not to constitute limitations upon the scope of the invention which is defined by the claims that are appended at the end of this description.

[0099] General Procedure and Components

[0100] The commercial products listed below were used as indicated in the following examples:

[0101] GemPro® HPG (“HPG”) is a natural wheat protein isolate available from Manildra USA.

[0102] Lipopan® Xtra is a phospholipase available from Novozymes.

[0103] Lipopan* 50 is a phospholipase available from Novozymes.

[0104] GemStar® 100 is a highly purified unmodified wheat starch available from Manildra USA.

[0105] Ficin 100 is a biocatalyst from the latex of l-icus species available from Enzyme Development.

[0106] Panzea BG is a standardized to 235 NXU / grams xylanase available from Novozymes.

[0107] Ban® 480L is an alpha-amylase available from Novozymes.

[0108] Rohalase® is a liquid enzy me preparation with beta-glucanase activity and xylanase side activity available from AB Enzy mes.

[0109] PG 500 is a protein glutaminase available from Amano.

[0110] Example 1

[0111] An emulsify ing agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C(135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM using a T- bar mixer attachment.

[0112] To the beaker was added 0.28125 grams of a bromelain enzyme. Next, 0.1406 grams of a lipase (Lipopan® Xtra) was added and allowed to mix for 5 minutes.

[0113] To the beaker was added 281.25 grams HPG in 20 gram increments over 27 minutes while mixed at an RPM of up to 1200. The timer was started at the first addition of HPG. The mixing was kept at up to 1200 RPM to ensure consistent mixing once the first 20 grams of HPG was added. The resulting mixture was thick as the last portion of HPG was mixed in. Next, after all the HPG was added, the RPM was adjusted to 600-1200 RPM to maintain a uniform mix. a uniform mix as the mixture was mixed, the RPM was decreased to 800 RPM.

[0114] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0115] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) to deactivate the enzyme. The temperature was increased over 25 minutes. Once the temperature was consistently 80 °C (176 °F) it was maintained for 10 minutes. The pH was measured after the deactivation was completed. The resulting mixture was a very7thin liquid. Finally, the resulting mixture was frozen and dried.

[0116] Example 2

[0117] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath.To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM using a T bar attachment.

[0118] To the beaker was added 0.5625 grams of a bromelain enzy me. Next, 0. 1406 grams of a lipase (Lipopan® Xtra) was added and allowed to mix for 5 minutes.

[0119] To the beaker was added 281.25 grams HPG in 20 gram increments over 30 minutes while mixed at an RPM of up to 1000. The timer was started at the first addition of HPG. 20 grams of the HPG was added every' 3 minutes until the entirety7of the amount was added. The mixing was kept at up to 1200 RPM to ensure consistent mixing once the first 20 grams of HPG was added. The resulting mixture was more fluid than Example 1 and water was separating out of the gluten as it was breaking down. Next, after all the HPG was added, the RPM was adjusted to 800 RPM to maintain a uniform mix. As the mixture was mixed, the mixture was already thinned out and broken down. A layer of w ater separated on the top of the mixture.

[0120] The resulting mixture w as maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0121] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was a very thin liquid, very7homogenous, and had a pH of 6.06. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0122] Example 3

[0123] An emulsify ing agent w as prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker w as added 656.25 grams w ater. The added water was stirred at 600 RPM using the T bar attachment.

[0124] To the beaker was added 0.5625 grams of a bromelain enzyme. Next, 0. 1406 grams of a lipase (Lipopan* Xtra) was added. Next, 0.703 grams of a transglutaminase was added and allow ed to mix for 5 minutes.

[0125] To the beaker was added 281.25 grams HPG slowly and uniformly over 28 minutes. The timer was started at the first addition of HPG. The mixing was kept at 600 RPM or enough to consistent mixing. The resulting mixture thick at first but mostly was thinned out by the time theentirety of the HPG was added. Next, after all the HPG was added, the RPM was adjusted to 1100 RPM a uniform mix. The RPM was adjusted to 800 RPM after the mixture thinned out.

[0126] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0127] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.98. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0128] Example 4

[0129] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM using a T bar attachment.

[0130] To the beaker was added 0.5625 grams of a protease (MT). Next, 0.1406 grams of a lipase (Lipopan® Xtra) was added and allowed to mix for 5 minutes.

[0131] To the beaker was added 281.25 grams HPG slowly and uniformly over 28 minutes. The timer was started at the first addition of HPG. The mixing was kept at 600 RPM or enough to consistent mixing. Next, after all the HPG was added, the RPM was adjusted to 500 RPM. a uniform mixin the mixture, the gluten was mostly a cohesive ball with little to no breakdown. The ball was so cohesive that a mixing speed of 500 RPM did not create a funnel, but simply spun the ball.

[0132] The resulting mixture was maintained at a temperature of 57.2 °C (135 ) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added. The resulting mixture was still in the form of a thick ball.

[0133] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour. The resulting mixture was still a thick cohesive ball showing little signs of breakdow n. Another 0.5625 grams of protein was added, and the reaction was allowed to progress 1 more hour. 5 minutes after the protease addition, the gluten broke down and was smooth rather than a ball. The mixture was run for a total time of 2 hours and 15 minutes prior to deactivation. Next, the temperature of the water bath was raised to 80 °C (176 °F) and wasmaintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.92. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0134] Example 5

[0135] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0136] To the beaker was added 0.5625 grams of a protease (MT). Next, 0.1406 grams of a lipase (Lipopan® Xtra) was added. Next. 0.703 grams transglutaminase was added and allowed to mix for 5 minutes.

[0137] To the beaker was added 281.25 grams HPGin 20 gram increments over 30 minutes while mixed. The timer was started at the first addition of HPG. 20 grams of the HPG was added every 3 minutes until the entirety of the amount was added. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. During mixing there was heavy water separation while adding the HPG. A big ball of gluten formed in the T bar attachment. Next, after all the HPG was added, the RPM was adjusted to up to 1600 RPM to maintain a uniform mix. Maintaining a funnel was difficult. As more HPG was added, the HPG piled on top and did not incorporate while water separated from the mixture. An additional 0.5625 grams of protease was added to help break down the gluten. The reaction mixture w as in the form of a large ball.

[0138] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG as added. At this point the mixture was broken down but had heavy water separation on top. Next, the pH was recorded 30 minutes after the HPG was added.

[0139] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture thinned out considerably with a thin layer of separated water around the edge, was smooth, had a pH of 5.96. Next, half of the resulting mixture w as frozen and dried, and the other half was run through a spray drier.

[0140] Example 6

[0141] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0142] To the beaker was added 0.5625 grams of a bromelain enzyme. Next, 0. 1406 grams of a lipase (Lipopan® Xtra) was added. Next, 0.703 grams transglutaminase was added. Next, 0.5625g of GemStar®100 was added and allowed to mix for 5 minutes.

[0143] To the beaker was added 281.25 grams HPG in 20 gram increments over 26 minutes. The timer was started at the first addition of HPG. 20 grams of the HPG was added every 3 minutes until the entirety of the amount was added. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. The gluten went in easily and started breaking down after about 20 minutes. Next, after all the HPG was added, the RPM was adjusted to 1300 RPM to maintain a uniform mix.

[0144] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0145] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.99. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0146] Example 7

[0147] An emulsify ing agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0148] To the beaker was added 0.225 grams of a bromelain enzyme. Next, 0. 1406 grams of a lipase (Lipopan® Xtra) was added and allowed to mix for 5 minutes.

[0149] To the beaker was added 281.25 grams HPG in 20 gram increments over 28 minutes. The timer was started at the first addition of HPG. 20 grams of the HPG was added every 3 minutes until the entirety of the amount was added. The mixing was kept at 600 RPM or enoughto consistent mixing once the first 20 grams of HPG was added. N ext, after all the HPG was added, the RPM was adjusted to 1300 RPM to maintain a uniform mix. The mixture was very thick and massed around the spindle. The mixture thinned out after 15 minutes of final HPG addition. Rpm was maintained between 1300 and 1000 for the first 55 minutes.

[0150] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0151] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thicker but pourable, and less pourable as it cooled, and had a pH of 6.06. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0152] Example 8

[0153] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM.

[0154] To the beaker was added 0.2813 grams of a bromelain enzyme and allowed to mix for 5 minutes.

[0155] To the beaker was added 281.25 grams HPG in 20 gram increments over 30 minutes. The timer was started at the first addition of HPG. 20 grams of the HPG was added every73 minutes until the entirety of the amount was added. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. The resulting mixture was very gloopy at the end. The gluten went in smooth and even for the first half of the addition. Next, the RPM was increased to 1300 RPM to maintain a uniform mix. The mixture appeared like the gluten is spinning in the mixture rather than breaking down. After all the HPG was added, 0. 1406 grams of an esterase lipase was added. The mixture was thick and gloopy after 25 minutes. After the full 75-minute reaction time, the mixture was still thick and gloopy. As a result, the time for reaction was extended until the breakdown was visible.

[0156] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0157] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 2 hours. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was still very thick but pourable and had a pH of 6.0. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0158] Example 9

[0159] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0160] To the beaker was added 0.2813 grams of a bromelain enzy me. Next, 0.1406 grams of a lipase (Lipopan® Xtra) was added and allowed to mix for 5 minutes.

[0161] To the beaker was added 281.25 grams HPG in 20 gram increments over 28 minutes along with 28.125 grams of blended GemStar® 100. The timer was started at the first addition of HPG. 20 grams of the HPG was added every73 minutes until the entirety7of the amount was added. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. The HPG incorporated easily as the RPM increased to 1600 RPM. The mixture became thicker as more was added before decreasing the mixing continued. The mixture contained a noticeable white color in the water. The RPM was adjusted to 1000 RPM 10 minutes after the final HPG and starch addition. The mixture thinned out but was still slightly thicker and appeared like a textured paint. The mixture was pourable and had no water separation. Next, after all the HPG was added, the RPM was adjusted to 600-1200 RPM as needed to maintain a uniform mix.

[0162] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0163] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture thinned out as it heated up, was easily pourable, and had a pH of 5.96. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0164] Example 10

[0165] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM.

[0166] To the beaker was added 0.5625 grams of a protease (MT) and allowed to mix for 5 minutes.

[0167] To the beaker was added 281.25 grams HPG in 20 grams amounts time. The timer was started at the first addition of HPG. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. The first half of the amount of HPG was added over 20 minutes. The RPM was increased with even' addition of HPG as needed to maintain a funnel. After half of the HPG was added, the remaining half was added over 45 minutes with the RPM maintained at 2000 RPM. In the mixture, the gluten went in, but became much thicker and started climbing the spindle and flicking off to the side of the container. The RPM was decreased to 1300 RPM to keep the moisture moving, but there is no free water. The resulting mixture appeared like a dough ball around a dough hook.

[0168] After all the HPG was added, 0.5626 grams lipase (Lipopan® Xtra) was also added, and the RPM was adjusted to 1000 RPM to maintain a uniform mix.

[0169] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0170] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was still thick going into deactivation and had a final pH of 6.01. Finally, the resulting mixture was frozen and dried.

[0171] Example 11

[0172] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 46. 1 °C (115 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0173] To the beaker was added 0.5626 grams of a protease (MT) and allowed to mix for 5 minutes.

[0174] To the beaker was added 281.25 grams HPG in 20 gram increments over time. The timer was started at the first addition of HPG. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. After 70% of the total HPG was incorporated, the mixture turned very light and airy and foamy, tripling in size in the container. The spindle could not properly spin the mixture and instead was whipping the inside. Mixing speeds of 700-1500 RPM were used to try to get the mixture to spin, but with no success. Paddle mixing was attempted but was also unsuccessful. Next, mixing using a T bar was tried again, and the temperature was increased to 57.2 °C (135 °F) and allowed to mix at 600 RPM for 30 minutes. Afterward, the mixture shrunk back down to size, more water became available, and the mixture formed a cohesive gluten ball. The remaining HPG was added over 10 minutes at 1000 RPM. The resulting mixture w as very thick and gloopy at this point.

[0175] After 30 minutes of additional mixing, the gluten was still not completely broken down. The temperature was increased to 71.1 °C (160 °F). The mixture was still very thick, and the T bar stopped spinning after 15 minutes. Next, the mixture was mixed using a paddle bar. The paddle bar broke down some of the gluten but started to shake the container. Next, the mixture was mixed using a T bar and mixing at 1200 RPM. This resulted in a resulting mixture where gluten was climbing the spindle. After 15 minutes, the gluten had thinned out into a smooth mixture. The RPM was decreased to 875 RPM. The total reaction time up until this point was 2.75 hours. 0.5626 grams lipase (Lipopan81Xtra) was added to the mixture and the mixture was allow ed to stir for 1 hour at a range of 600-1200 rpms as needed to maintain a funnel.

[0176] The resulting mixture w as maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG w as added. Next, the pH was recorded 30 minutes after the HPG was added.

[0177] The resulting mixture was raised to a temperature of 80 °C (176 °F) and allowed to deactivate for 10 minutes. The pH was measured after the deactivation was completed. The resulting mixture was very thick and had a pH of 5.98. Next, half of the resulting mixture was frozen and dried, and the other half was run through a spray drier.

[0178] Example 12

[0179] An emulsifying agent w as prepared using the following experimental procedure. A water bath was heated to a temperature of 46. 1 °C (115 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0180] To the beaker was added 0.9846 grams of a protease (MT) and allowed to mix for 5 minutes.

[0181] To the beaker was added 281.25 grams HPG in 20 gram increments over 1 hour. The timer was started at the first addition of HPG. The mixing was kept at 600 RPM or enough to consistent mixing once the first 20 grams of HPG was added. Half of the HPG was added over the first 20 minutes, and the rest of the HPG was added 20 grams at a time over the next 40 minutes. The mixture started out thick but was able to keep a funnel as the HPG was added. This mixture was thinner than Examples 10 and 11 but still semi thick. Next, the RPM was increased to 2000 RPM. Next, after all the HPG was added, the RPM was adjusted to 1700 RPM to maintain a uniform mix. The mixture was allowed to continue breaking down for an additional hour.

[0182] Two hours after the initial HPG addition, 0.5626 grams lipase (Lipopan® Xtra) was added to the mixture and mixed at 600-1200 rpms. The mixture was allowed to react for 1 additional hour, for a total reaction time of 3 hours.

[0183] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0184] Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.96. Finally, the resulting mixture was frozen and dried.

[0185] Example 13

[0186] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 46. 1 °C (115 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 RPM using a T bar attachment.

[0187] To the beaker was added 0.7736 grams of a protease (MT) and allowed to mix for 5 minutes.

[0188] To the beaker was added 281.25 grams HPG in 20 gram increments over time. The timer was started at the first addition of HPG. The first 50% was added over 20 minutes and stirred at a speed of up to 1000 RPM. The remaining 50% of the HPG was added over the next 40 minutes with a mixing speed of up to 1900 RPM. At the end of the addition, the resulting mixture was thick and climbing the spindle but still able to mix. The mixture was thinner than previous examplesthat utilized 0.5626 grams of protease, but thicker than previous examples that used 0.9846 grams of protease.

[0189] Next, about 70 minutes after the first gluten addition, 0.5626 grams of lipase (Lipopan® Xtra) was added and mixed at 600-1200 RPM. The mixture was still thick at this point, but gluten breakdown was visible and the HPG became more homogenous.

[0190] After the lipase was added, the mixture was allowed to mix at 1600 RPM for 1 hour. 10 minutes after the lipase addition, the thickness was dissipating, and the mixture had more flow.

[0191] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG w as added. Next, the pH was recorded 30 minutes after the HPG was added.

[0192] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for a total of 2 hours and 10 minutes before deactivation. Next, the temperature of the water bath was raised over 20 minutes to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was pourable, but had some creaminess to it, and had a pH of 5.98. Finally, the resulting mixture w as frozen and dried.

[0193] Example 14

[0194] An emulsifying agent w as prepared using the following experimental procedure. A w ater bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0195] To the beaker was added 0.7876 grams of a protease (MT). Next, 1.4065 grams of a lipase (Lipopan* Xtra) was added.

[0196] To the beaker was added 281.25 grams HPG in 20 gram increments over 50 minutes. The timer was started at the first addition of HPG. About half of the HPG was able to be mixed in within the first 20 minutes, with the remaining half added in over another 30 minutes. Once the first 20 grams of HPG was added, the mixture was mixed at 600-1700 RPM as needed to maintain a consistent funnel. The resulting mixture was very foamy, and was significantly foamier where it was pushed to the outer edges while spinning / mixing.

[0197] After the HPG addition, the mixture was allowed to continue mixing for 10 minutes at 1700 RPM. Next, after 10 minutes, the RPM was lowered to 1000 RPM. The mixture was still very foamy with the foam covering the top of the mixture.

[0198] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0199] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 50 minutes after the start of the HPG addition. The mixture was still very foamy with a layer on top. The mixture was much foamier than in other examples. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 46.1 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture comprised protein that was broken down to thinner consistency and no foam was present in the mixture after deactivation. The resulting mixture had a pH of 5.86. Finally, the resulting mixture was frozen and dried.

[0200] Example 15

[0201] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0202] To the beaker was added 0.4219 grams of abromelain enzyme. Next, 0.5626 grams of a lipase (Lipopan® Xtra) was added.

[0203] To the beaker was added 281.25 grams HPG in 20 gram increments over 35 minutes. The timer was started at the first addition of HPG. The HPG incorporated well and did not become too thick to spin / mix. Some HPG floated on the top as it was mixing but was slowly incorporated. The mixing was maintained at 600-1300 RPM as needed to ensure consistent mixing after the first 20 grams of HPG were added. Next, after all the HPG was added, the RPM was adjusted to 600-1200 RPM to maintain a uniform mix. Five minutes after the final gluten addition, the HPG had broken down into a thinner consistency with some residual layer of water floating on top. The mixture contained minor to little foam in the water.

[0204] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added. The mixture continued breaking down into an easy pourable consistency with no chunky bits.

[0205] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes from first HPG addition while mixed at 1000 RPM. Next, the temperature of the water bath was raised to 80 °C (176 °F) over 20 minutes and wasmaintained at 80 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.94. Finally, the resulting mixture was frozen and then freeze dried.

[0206] Example 16

[0207] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0208] To the beaker was added 0.4219 grams of abromelain enzyme. Next, 1.4065 grams of a lipase (Lipopan® Xtra) was added.

[0209] To the beaker was added 281.25 grams HPG in 20 gram increments over 35 minutes. The timer was started at the first addition of HPG. The mixing was kept at 600-1300 RPM or enough to consistent mixing once the first 20 grams of HPG was added and as the HPG was continuously added. HPG addition became more difficult as it was added, due to foam formation and HPG floating to the top of the mixture. After the last amounts of HPG were added the resulting mixture broke down to liquid. The water of the resulting mixture appeared a little foamy and pushing towards the edges. After all the HPG was added, the RPM was adjusted to 1100 RPM to maintain a uniform mix.

[0210] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0211] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes after HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) over 20 minutes and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture comprises a protein liquid but appeared grainy. The resulting mixture had a pH of 5.89. Finally, the resulting mixture was frozen and freeze dried.

[0212] Example 17

[0213] An emulsify ing agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0214] To the beaker was added 0.2812 grams of a bromelain enzyme. Next, 1.4065 grams of a lipase (Eipopan® Xtra) was added.

[0215] While it was mixing, 281.25 grams Vital wheat gluten (VWG) was added to the beaker in increments of 20 grams. The gluten was added over the course of 32 minutes from the first addition. The VWG incorporated easier than the HPG and without as much conglomeration. The mixture only had to mix at a speed of up to 1000 RPM to maintain a uniform mix. The resulting mixture smoothed out quickly, more quickly than HPG examples.

[0216] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the VWG was added. Next, the pH was recorded 30 minutes after the VWG was added.

[0217] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes after the first VWG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was liquid but had a slightly grainy appearance and had a pH of 5.92. Finally, the resulting mixture was frozen and dried.

[0218] Example 18

[0219] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0220] To the beaker was added 0.2812 grams of a bromelain enzyme. Next, 1.4065 grams of a lipase (Lipopan® Xtra) was added.

[0221] Next, a blended mixture of 281.25 grams HPG and 28.125 grams GemStar® 100 was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition. This mixture incorporated similarly to Example 17. The mixture only had to mix at a speed of up to 1000 RPM to maintain a uniform mix. The resulting mixture smoothed out quickly, more quickly than other HPG examples.

[0222] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0223] The resulting mixture was maintained at a temperature of 57.2 °C (135 ° and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature ofthe water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 15 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was liquid but had a slightly grainy appearance and had a pH of 5.74. Finally, the resulting mixture was frozen and freeze-dried.

[0224] Example 19

[0225] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0226] To the beaker was added 0.2812 grams of a bromelain enzyme. Next, 0.14065 grams of an esterase lipase was added.

[0227] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 35 minutes from the first addition. The resulting mixture comprised foamy water towards the end of HPG addition (with 30 grams of HPG left to be added). As HPG was added, the RPM was increased to 1000 RPM. HPG addition became more difficult to incorporate as it was added due to HPG floating on the top of the mixture for a few moments. Once spinning, the HPG was incorporated. After all the HPG was added, the RPM was adjusted to 850 RPM to maintain a uniform mix.

[0228] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0229] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture had a pH of 5.92. Finally, the resulting mixture was frozen and dried.

[0230] Example 20

[0231] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0232] To the beaker was added 0.225 grams of a bromelain enzyme, 0.14065 grams of Ficin 100, and 1.406 grams of a lipase (Lipopan* Xtra).

[0233] While it was mixing. 225 grams HPG and 56.25 grams chickpea flour was added to the beaker in increments of 20 grams. The HPG and chickpea flour was added over the course of 30 minutes from the first addition. The gluten mixed in easily and made a smooth but very thick textured mixture. The resulting mixture had the texture of thin oatmeal. Next, after all the gluten was added, the RPM was adjusted to 1100 RPM to maintain a uniform mix.

[0234] The resulting mixture was maintained at a temperature of 60 °C (122 °F) and the pH was recorded 10 minutes after the HPG was added. 45 minutes after the first addition of gluten, the resulting mixture was gloopier and stringier, with a layer of foam floating on the top of the mixture. Next, the pH was recorded 30 minutes after the HPG was added. 75 minutes after the first addition of gluten, the resulting mixture had a thick layer of foam on the top and the gluten was still stringy. Half of the mixture was removed and deactivated at 75 °C (167 °F) for 10 minutes on a hot plate while the remaining mixture was allowed to spin / mix and continue to break down. The first deactivated mixture cooked on the hot plate as the temperature was raised which resulted in a mixture that was a chunky paste.

[0235] The second half of the mixture was maintained at a temperature of 60 °C (122 °F) and allowed to react for 2 hours and 30 minutes after the first gluten addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) and was maintained at 80 °C (176 °F) for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thinned out more but still was stringy and slightly foamy. As the temperature increased, the mixture thinned out. The final pH of the mixture was 5.89. Finally, the resulting mixture was frozen and dried.

[0236] Example 21

[0237] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F). A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 RPM using a T bar attachment.

[0238] To the beaker was added 0. 1124 grams of a bromelain enzyme, 1. 124 grams of an esterase lipase, and 0. 1124 grams of Ban® 480L.

[0239] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 600 RPM or enough to get the HPG to incorporate. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0240] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0241] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 176 °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thick, appeared foamy, and had some separation. Finally, the resulting mixture was frozen and freeze-dried.

[0242] Example 22

[0243] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F) °F. A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0244] To the beaker was added 0.281 grams of a bromelain enzyme. Next, 1.4065 grams of a lipase (Lipopan® 50) was added.

[0245] Next, 281.25 grams HPG was added to the beaker in increments of 20 grams each. The HPG was added over the course of 30 minutes from the first addition. The HPG went in very smoothly at a RPM of 800 RPM. After all the HPG was added, the mixture was stirred at 800 RPM to maintain a uniform mix.

[0246] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added. The resulting mixture was slightly foamy around the spindle.

[0247] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was very smooth and had thinned out even more as it heated up. The resulting mixture was very homogenous after deactivation and had a pH of 5.96. Finally, the resulting mixture was frozen and freeze-dried.

[0248] Example 23

[0249] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F) °F. A beaker was placed in the waterbath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0250] To the beaker was added 0.281 grams of a bromelain enzy me, 1.4065 grams of an esterase lipase, and 1.4065 grams of a transglutaminase.

[0251] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The gluten was added over the course of 27 minutes from the first addition mixing at 850 RPM. The resulting mixture was able to funnel but was thick and chunky. After all the HPG was added, the mixture was stirred at 1050 RPM to maintain a uniform mix.

[0252] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0253] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture looked smooth and thinned out as it heated. Upon pouring into the pans, there was still some slightly chunky, unbroken HPG at the bottom and the thinned-out portion had floated to the top of the mixture. The mixture had a thin layer of water on the bottom of the pan and had HPG residue down around the pan. The resulting mixture had a pH of 5.97. Finally, the resulting mixture was frozen and freeze-dried.

[0254] Example 24

[0255] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0256] To the beaker was added 0.2248 grams of a bromelain enzyme. Next, 1.4065 grams of an esterase lipase.

[0257] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed in at 900 RPM. The HPG incorporated easily and went in smooth. After all the HPG was added, the mixture was stirred at 900 RPM to maintain a uniform mix.

[0258] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0259] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture was slightly chunky, separated from the water, not homogenous, and had a pH of 5.94. Finally, the resulting mixture was frozen and dried.

[0260] Example 25

[0261] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F). A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0262] To the beaker was added 0.281 grams of a bromelain enzyme, 1.4065 grams of an esterase lipase, and 1.4065 grams of Panzea BG.

[0263] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The gluten was added over the course of 60 minutes from the first addition, mixing at 900 RPM. The HPG went in smoothly, but the mixture got slightly chunky towards the end of the HPG addition. After all the HPG was added, the mixture thinned out quickly in about 10 minutes. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0264] The resulting mixture was maintained at a temperature of 50 °C (122 °F) and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0265] The resulting mixture was maintained at a temperature of 50 °C (122 °F) and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was very' smooth, homogenous, poured smoothly into pans, and had a pH of 5.92. Finally, the resulting mixture was frozen and freeze-dried.

[0266] Example 26

[0267] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 50 °C (122 °F). A beaker was placed in the water bath.To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0268] To the beaker was added 0.281 grams of a bromelain enzy me, 1.4065 grams of an esterase lipase, and 1.4065 grams of PG 500.

[0269] While it was mixing, 281.25 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 24 minutes from the first addition, mixing at 1100 RPM. The HPG was added in very smoothly and the resulting mixture had no conglomeration and broke down almost immediately into a smooth consistency. After all the HPG was added, the mixture was stirred at 600-1200 RPM as needed to maintain a uniform mix.

[0270] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added. The resulting mixture turned grayer as it reacted, and the RPM was reduced to 600 RPM because the mixture was very fluid.

[0271] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was very thin, homogenous, very fluid, was mixed at 300 RPM. Finally, the resulting mixture w as frozen and freeze-dried.

[0272] Example 27

[0273] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 656.25 grams water. The added water was stirred at 600 using a T bar attachment.

[0274] To the beaker w as added 0.2248 grams of a bromelain enzyme, 1.4065 grams of an esterase lipase, and 1.4065 grams of PG 500.

[0275] While it was mixing, 281.25 grams HPG w as added to the beaker in increments of 20 grams. The HPG was added over the course of 26 minutes from the first addition and mixed at 900 RPM. The resulting mixture had no clumping and no climbing of the spindle. After all the HPG was added, the mixture was stirred at 600 RPM to maintain a uniform mix. the resulting mixture had HPG that went in smooth and incorporated nicely. The resulting mixture had no significant chunks.

[0276] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0277] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed. The resulting mixture was thinned out, easily pourable, and had a pH of 5.99. Finally, the resulting mixture was frozen and freeze-dried.

[0278] Example 28

[0279] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0280] To the beaker was added 0.1 124 grams of a bromelain enzy me, 1.25 grams of an esterase lipase, and 0.70325 grams of PG 500.

[0281] While it was mixing, 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 24 minutes from the first addition and mixed at 300 RPM, then increased to 550 RPM and as more HPG was added. The resulting mixture was thick at the beginning of the addition of the HPG due to lower water and spin, and it was harder to incorporate the HPG. The mixture started to thin out after 20 minutes after first addition of HPG. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix. Next the RPM speed was adjusted to 300 RPM.

[0282] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0283] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was smooth, fluid, pourable, and had a pH of 5.94. Finally, the resulting mixture was frozen and freeze-dried.

[0284] Example 29

[0285] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0286] To the beaker was added 0.1124 grams of a bromelain enzyme, 0.70325 grams of an esterase lipase, and 0.42195 grams of PG 500.

[0287] While it was mixing, 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 25 minutes from the first addition and mixed at 600 RPM or fast enough to make a consistent mixture. The resulting mixture incorporated smoothly and broke down quickly. After all the HPG was added, the mixture was stirred at 600 RPM to maintain a uniform mix.

[0288] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was reduced to 300 RPM for the remainder of the reaction. Next, the pH was recorded 30 minutes after the HPG was added.

[0289] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzy me. The pH w as measured after the deactivation was completed. The resulting mixture was smooth, pourable, and had a pH of 5.99. Finally, the resulting mixture was frozen and freeze-dried.

[0290] Example 30

[0291] An emulsify ing agent w as prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0292] To the beaker was added 0. 1124 grams of a bromelain enzyme, 1. 124 grams of an esterase lipase, and 0.42195 grams of PG 500.

[0293] While it w as mixing, 140.625 grams HPG w as added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 592 RPM. The resulting mixture had more starch in the water and slightly milky appearance around the edges. The HPG did not break down as fast as other examples and still had a thinnertexture 10 minutes after the final addition of HPG. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0294] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was decreased to 300 RPM. The mixture was still slightly thicker with a layer of starch around the edges. Next, the pH was recorded 30 minutes after the HPG was added.

[0295] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thinned out but still very slightly thick, had a starch layer in the water around the edge, and had a pH of 5.91. Finally, the resulting mixture was frozen and freeze-dried.

[0296] Example 31

[0297] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0298] To the beaker was added 0. 1124 grams of a bromelain enzyme, 1. 124 grams of an esterase lipase. 0.42195 grams of PG 500. and 0.3518 grams of a transglutaminase.

[0299] While it was mixing, 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 600 RPM. The resulting mixture broke down easily and started being smooth shortly after the final HPG addition. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0300] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was decreased to 300 RPM about 10 minutes after the final HPG addition. Next, the pH was recorded 30 minutes after the HPG was added.

[0301] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed.The resulting mixture was thin, easily pourable, and had a pH of 5.94. Finally, the resulting mixture was frozen and freeze-dried.

[0302] Example 32

[0303] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0304] To the beaker was added 0.1124 grams of a bromelain enzy me, 1.124 grams of an esterase lipase, 0.42195 grams of PG 500, and 0.1124 grams of Ban® 480L, and 0.1124 grams Rohalase®.

[0305] While it was mixing, 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 600 RPM. The resulting mixture started out slightly chunky but thinned out very quickly as more HPG was added. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0306] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was decreased to 300 RPM about 10 minutes after the final HPG addition. Next, the pH was recorded 30 minutes after the HPG was added.

[0307] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thin, easily pourable, and had a pH of 5.97. Finally, the resulting mixture was frozen and freeze-dried.

[0308] Example 33

[0309] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0310] To the beaker was added 0. 1124 grams of a bromelain enzy me, 0.3513 grams of an esterase lipase, and 0.42195 grams of PG 500.

[0311] While it was mixing. 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 600 RPM or an RPM that is enough to make a consistent mixture. The resulting mixture went in smooth but had a foamy top. The foamy top settled as time went on. After all the HPG w as added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0312] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG w as added.

[0313] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was thinned out but still had a starch ring around the top of the mixture and had a pH of 6.0. Finally, the resulting mixture was frozen and freeze-dried.

[0314] Example 34

[0315] An emulsifying agent w as prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0316] To the beaker was added 0.1124 grams of a bromelain enzyme, 1.124 grams of an esterase lipase, and 0.2109 grams of PG 500.

[0317] While it was mixing, 140.625 grams HPG w-as added to the beaker in increments of 20 grams. The HPG was added over the course of 25 minutes from the first addition and mixed at 600 RPM. The resulting mixture w-as less broken down and slightly separated. After all the HPG w as added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0318] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was decreased to 300 RPM about 10 minutes after the final HPG addition. Next, the pH was recorded 30 minutes after the HPG w as added.

[0319] The resulting mixture w as maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzy me. The pH was measured after the deactivation was completed.The resulting mixture was still slightly thick but pourable, noticeably not as thin as previous examples, and had a pH of 5.91. Finally, the resulting mixture was frozen and freeze-dried.

[0320] Example 35

[0321] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0322] To the beaker was added 0.1124 grams of a bromelain enzy me, 1.124 grams of an esterase lipase, 0.1756 grams of a transglutaminase, and 0.4218 grams of PG 500.

[0323] While it was mixing. 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 30 minutes from the first addition and mixed at 800 RPM to get the HPG to incorporate. The resulting mixture was foamy as it mixed, was slightly more thick than previous examples, and broke down about 10 minutes after all the HPG was added. After all the HPG was added, the mixture was stirred at 600-1200 RPM to maintain a uniform mix.

[0324] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the RPM was decreased to 600 RPM about 10 minutes after the final HPG addition. Next, the pH was recorded 30 minutes after the HPG was added.

[0325] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was slightly thick but still pourable and had a pH of 5.94. Finally, the resulting mixture was frozen and freeze-dried.

[0326] Example 36

[0327] An emulsifying agent was prepared using the following experimental procedure. A water bath was heated to a temperature of 57.2 °C (135 °F) °F. A beaker was placed in the water bath. To the beaker was added 328.125 grams water. The added water was stirred at 600 using a T bar attachment.

[0328] To the beaker was added 0. 1406 grams of a bromelain enzy me, 0.1406 grams of an esterase lipase, and 0. 1406 grams of PG 500.

[0329] While it was mixing. 140.625 grams HPG was added to the beaker in increments of 20 grams. The HPG was added over the course of 25 minutes from the first addition and mixed at 600 RPM. The resulting mixture started off very thick and somewhat stringy' as the reaction progressed. After all the HPG was added, the resulting mixture was very thick, and was stirred at 600-1200 RPM as needed to maintain a uniform mix.

[0330] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and the pH was recorded 10 minutes after the HPG was added. Next, the pH was recorded 30 minutes after the HPG was added.

[0331] The resulting mixture was maintained at a temperature of 57.2 °C (135 °F) °F and allowed to react for 1 hour and 15 minutes after the first HPG addition. Next, the temperature of the water bath was raised to 80 °C (176 °F) °F and was maintained at 80 °C (176 °F) °F for 10 minutes to deactivate the enzyme. The pH was measured after the deactivation was completed. The resulting mixture was a thicker mixture but still pourable, not as thin as other examples, and had a pH of 5.91. Finally, the resulting mixture was frozen and freeze-dried.

[0332] Example 37: Mixograph Measurements

[0333] Each of the wheat-based emulsifying agents prepared in Examples 1-36 was evaluated on a mixograph. The resulting midline data is summarized in the table below.a75 minute reaction timeb2.5 hour reaction time

[0334] Example 38: Evaluation of Emulsifying Properties

[0335] The following procedure was used to evaluate the emulsifying properties of the wheat-based emulsifying agents prepared in Examples 1-36.

[0336] First, a dilute test solution was prepared by adding 198 grams distilled water into beaker, followed by 2 grams of the wheat-based emulsifying agent.

[0337] Using a Silverson L5M-A Laboratory Mixer, the mixer head was lowered until the top of the mixer head was even with the top of the water. The beaker remained stationary. The test solution was then mixed at 3000 RPM for 2 minutes.

[0338] Oil was then added to the test solution while the solution was mixed at 3800 RPM. In each case, approximately 565 grams oil was added over a period of 2 minutes. Following addition of the oil, the mixer head was raised to the middle of the mixture. The beaker remained stationary. The mixture was then mixed for an additional 2 minutes at 3800 RPM.

[0339] The initial appearance of the test mixture was noted, and the volume of any nonemulsified aqueous fraction was recorded. Where indicated in the table below, the test mixture was then reevaluated after 12 hours, and again the following day (after approximately 24 hours).

[0340] Observations regarding the homogeneity and stability of each test mixture are summarized in the table below.

[0341] Example 39: Faty Acid Composition

[0342] The faty acid compositions of a wheal-based emulsifying agent prepared by contacting wheat protein isolate with a protease and a lipase (Sample A) and a wheat-based emulsifying agent prepared by contacting wheat protein isolate with a protease, a lipase, and a protein glutaminase (Sample B) were evaluated. The fatty acid compositions of GernPro HPG was also evaluated for purposes of comparison. The compositional data are presented in the tables below.

[0343] Example 40: Protein Content Analysis

[0344] The protein content of a wheat-based emulsifying agent prepared using the procedure described in Example 7 was analyzed and compared with the protein content of the HPG starting material.

[0345] For each sample, the wheat total protein was extracted for SE-HPLC as described by Gupta and Macritchie (1993) with minor modifications (Ohm et al., 2009; Malalgoda et al., 2018). The SDS- extractable fraction was obtained by adding 1 rnL of extraction buffer to 10 mg ± 0.05 (adjusted to 14% moisture content) of the flour sample. The extraction buffer was 1 mL of 0.5 % SDS and 0.05M sodium phosphate buffer (pH 6.9). Wheat protein sample was suspended in extraction buffer followed by stirring for 5 min at 2,000 rpm using a Thermomixer C with a SmartBlock 2.0 mL. Then the mixture was centrifuged at 20,000 x g for 10 min, and the supernatant which is the extractable protein fraction was filtered through a 0.45 gm PVDF membrane filter. The sample was immediately heated at 80 °C for 2 minutes in a water bath and then cooled at room temperature as for the SDS-extractable fraction following the filtration. In order to solubilize SDS unextractable proteins, the residue was resuspended in 1 mL of the same extraction buffer and sonicated in a probe-type sonicator for 30 s at 10 W power setting. The sonicated mixture was also centrifuged as described for the unextractable fraction, then the supernatant was filtered, and heat treated at 80 °C for 2 min in a water bath as for the SDS- unextractable fraction.

[0346] SE-HPLC was performed using an Agilent 1100 Series (Agilent Technologies, Santa Clara, CA). The SDS-extractable and SDS-unextractable protein fractions were performed individually on a narrow-bore size exclusion column with a precolumn in-line filter assembly anda guard cartridge. The SE-HPLC settings were as follows: the injection volume was 10 LIL and the elution solution was 50 % acetonitrile (ACN) and 50 % aqueous trifluoroacetic acid (TFA) solution (0.1% v / v). The flow rate was 0.5 mL / min. The detection of solutes was done at UV 214 nm absorbance using Agilent 1200 Photodiode Array Detector. These experiments were performed in duplicate.

[0347] The SE-HPLC profiles were divided into four fractions (F1-F4) for both SDS extractable and unextractable fractions. The retention time intervals are Fl (3.5-4.7 min), F2 (4.7- 5.2 min), F3 (5.2-5.8 min), and F4 (5.8-7.4 min). Primary components constituting individual fractions were high molecular weight (HMW) glutenin polymers for Fl; low molecular weight (LMW) glutenin polymers for F2; gliadin and for F3; and albumin and globulins proteins for F4.

[0348] The results of the analysis are presented in the table below.

[0349] The protein content of the wheat-based emulsifying agent comprised a significantly smaller high molecular weight protein fraction (categories Fl and F2) and a significantly larger low molecular weight protein fraction (category F4) as compared to the starting material. Specifically, the high molecular weight protein fraction of the wheat-based emulsifying agent was 86.22% of the high molecular weight protein fraction of the starting material. Conversely, the low molecular weight protein fraction of the wheat-based emulsifying agent was 131.78% of the low molecular weight protein fraction of the starting material.

[0350] In view of the above, it will be seen that the several objects of the disclosure are achieved and other advantageous results attained.

[0351] It will be understood that certain features and sub combinations are of utility and may be employed without reference to other features and sub combinations. This is contemplated by and is within the scope of the present disclosure and the following claims. As various changes could be made in the provided products and methods without departing from the scope of the disclosure, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

[0352] The embodiments described above are presented by way of example only and are not intended to limit the concepts and principles of the present invention. Thus, there has been shown and described several embodiments of a novel invention. As is evident from the foregoing description, certain aspects of the present disclosure are not limited by the particular details of theexamples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. When introducing elements of the present disclosure or the preferred embodiment(s) thereof, the articles “a”, “an”, “the”, and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive, mean that there may be additional elements other than the listed elements, and are used in the sense of “optional” or “may include” and not as “required.” Many changes, modifications, variations and other uses and applications of the disclosed embodiments will, however, become apparent to those skilled in the art after considering the specification. All such changes, modifications, variations and other uses and applications which do not depart from the spirit and scope of the disclosure are deemed to be covered by the invention(s) described herein, which are limited only by the claims which follow.

Claims

CLAIMSWhat is claimed is:

1. A method of making a wheat-based emulsifying agent, the method comprising:(1) providing a reaction mixture comprising a starting material having a wheat protein content of at least about 50% by weight;(2) a proteolysis step wherein the reaction mixture is contacted with a protease, thereby producing a modified wheat protein having a decreased high molecular weight protein fraction and an increased low molecular weight protein fraction as compared to the starting material;(3) a lipolysis step wherein the reaction mixture is contacted with a lipase, thereby converting at least about 10% of the triglycerides present in the starting material into monoglycerides and diglycerides; and(4) an enzyme deactivation step wherein one or more enzy mes present in the reaction mixture are substantially deactivated.

2. The method of claim 1 wherein the staring material has a fat content of at least about 0.5% by weight.

3. The method of claim 1 wherein the starting material is selected from the group consisting of wet wheat gluten, vital wheat gluten, whole wheat flour, and wheat protein isolate.

4. The method of claim 1 wherein the protease is selected from the group consisting of serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases.

5. The method of claim 4 wherein the protease comprises an aminopeptidase.

6. The method of claim 5 wherein the protease comprises a dipeptidyl-aminopeptidase.

7. The method of claim 1 wherein the protease is selected from the group consisting of bromelain, papain, and actinidin.

8. The method of claim 1 wherein the proteolysis step produces a reaction mixture comprising a modified wheat protein having an average molecular weight of from about 20 kDa to about 80 kDa.

9. The method of claim 1 wherein the proteolysis step produces a reaction mixture comprising a modified wheat protein having a high molecular weight protein fraction that is less than about 92% of the high molecular weight protein fraction of the starting material.

10. The method of claim 1 wherein the proteolysis step produces a reaction mixture comprising a modified wheat protein having a low molecular weight protein fraction that is at least about 120% of the high molecular weight protein fraction of the starting material.

11. The method of claim 1 wherein the proteolysis step is conducted at a temperature of from about 45 °C to about 80 °C for a period of at least about 15 minutes; and wherein the proteolysis step is conducted under acidic conditions wherein the pH of the reaction mixture is within a range of from about 3 to about 7.

12. The method of claim 1 wherein the lipase is selected from the group consisting of phospholipases, triacyl glycerol lipases, and mixtures thereof.

13. The method of claim 1 wherein the lipolysis step converts at least about 20% of the triglycerides present in the starting material into monoglycerides and diglycerides.

14. The method of claim 1 wherein the lipolysis step is conducted at a temperature of from about 45 °C to about 80 °C for a period of at least about 15 minutes; and wherein the lipolysis step is conducted under acidic conditions wherein the pH of the reaction mixture is within a range of from about 3 to about 7.

15. The method of claim 1 further comprising a supplemental enzyme treatment step wherein the reaction mixture is contacted with a glutaminase, transglutaminase, a xylanase, a - glucanase, or a combination thereof.

16. The method of claim 15 wherein the supplemental enzyme treatment step comprises contacting the reaction mixture with a transglutaminase.

17. The method of claim 16 wherein the supplemental enzyme treatment step is conducted at a temperature of from about 45 °C to about 80 °C for a period of at least about 15 minutes; and wherein the supplemental enzyme treatment step is conducted under acidic conditions wherein the pH of the reaction mixture is within a range of from about 3 to about 7.

18. The method of claim 16 wherein the proteolysis step, the lipolysis step, and the supplemental enzyme treatment step are conducted concurrently.

19. A wheat-based emulsifying agent prepared by the method of any one of claims 1 to 18.

20. A wheat-based emulsifying agent comprising a modified wheat protein component in a concentration of at least about 50% by weight on a dry basis, relative to the total weight of the wheat-based emulsifying agent; wherein said modified wheat protein component has a high molecular weight protein fraction of no greater than about 45%, a low molecular weight protein fraction of at least about 15%, and a gliadin protein fraction of at least about 35%.

21. The wheat-based emulsifying agent of claim 20 wherein said modified wheat protein component has an average molecular weight of from about 20 kDa to about 80 kDa.

22. The wheat-based emulsifying agent of claim 20 wherein said modified wheat protein component has a high molecular weight protein fraction of from about 30% to about 45%.

23. The wheat-based emulsifying agent of claim 20 wherein said modified wheat protein component has have a low molecular weight protein fraction of from about 15% to about 30%.

24. The wheat-based emulsifying agent of claim 20 comprising said modified wheat protein component in a concentration of from about 80% by weight to about 99% by weight.

25. The wheat-based emulsifying agent of claim 20 having a fat content of at least about 0.5% by weight.

26. The wheat-based emulsifying agent of claim 25 wherein monoglycerides and diglycerides may comprise at least about 80% of said fat content.

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