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Title:
INCREASING THE FREE PHENOLIC ACID IN GRAIN-BASED FOODS
Document Type and Number:
WIPO Patent Application WO/2015/048727
Kind Code:
A1
Abstract:
Disclosed herein are methods for increasing the free phenolic acid content of grain-based food products, such as powders, breads, cereals, and animal feed. Methods disclosed herein comprise pretreating bran, including aleurone and/or pericarp, with at least one enzyme, adding the pretreated bran to a preferment composition, and allowing the preferment composition to ferment. In further embodiments of the disclosure, additional ingredients may be added to the fermented composition, and the fermented composition may be allowed to further ferment in a second fermentation step. Yet further embodiments relate to products and baked goods prepared from the pretreated bran.

Inventors:
AIMUTIS, Ronald (3126 Rodeo Drive NE, Blaine, Minnesota, 55449, US)
CASPER, Jeffrey L. (4705 East 37th Street, Minneapolis, Minnesota, 55406, US)
FINNNIE, Sean M. (315 N 5th St, Manhattan, Kansas, 66502, US)
CAMPAYO, Vicenta Garcia (17101 Creek Ridge Pass, Minnetonka, Minnesota, 55345, US)
Application Number:
US2014/058261
Publication Date:
April 02, 2015
Filing Date:
September 30, 2014
Export Citation:
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Assignee:
ARDENT MILLS, LLC (1875 Lawrence St, Denver, Colorado, 80202, US)
AIMUTIS, Ronald (3126 Rodeo Drive NE, Blaine, Minnesota, 55449, US)
CASPER, Jeffrey L. (4705 East 37th Street, Minneapolis, Minnesota, 55406, US)
FINNNIE, Sean M. (315 N 5th St, Manhattan, Kansas, 66502, US)
CAMPAYO, Vicenta Garcia (17101 Creek Ridge Pass, Minnetonka, Minnesota, 55345, US)
International Classes:
A21D2/38; A23L7/10; A23L33/00
Domestic Patent References:
2013-02-21
Foreign References:
US20070184175A12007-08-09
Other References:
SAULNIER, L ET AL.: "Thermal and enzymatic treatments for the release of free ferulic acid from maize bran.", CARBOHYDRATE POLYMERS., vol. 45, 2001, pages 269 - 275
Attorney, Agent or Firm:
LEFORT, Brian et al. (Faegre Baker Daniels LLP, 2200 Wells Fargo Center90 S. Seventh Stree, Minneapolis Minnesota, 55402, US)
Download PDF:
Claims:
WHAT IS CLAI ED IS:

1 . A process for increasing the free phenolic acid content in bran comprising: pretreating bran with at least one enzyme;

subsequently adding the pretreated bran to a preferment composition; and allowing the preferment composition to ferment to form a fermented composition.

2. The process according to claim 1 , wherein the bran comprises aleurone.

3. The process according to claim 1 , further comprising:

adding additional ingredients to the fermented composition, and

allowing the fermented composition to ferment in a second fermenting

step.

4. The process according to claim 1 , further comprising baking, extruding, pelletizing, pulverizing, and/or drying the fermented composition.

5. The process according to claim 3, further comprising baking the fermented composition after the second fermenting step.

8. The process according to claim 1 , wherein the at least one enzyme is chosen from xylanase and ferulic acid esterase.

7. The process according to claim 1 , wherein the bran is pretreated with the at least one enzyme for at least about 10 minutes before adding the pretreated bran to the preferment composition.

8. The process according to claim 1 , wherein the bran is pretreated with the at least one enzyme for at least about 2 hours before adding the pretreated bran to the preferment composition.

9. The process according to claim 1 , wherein the bran is pretreated at temperature ranging from about 40°C to about 85°C.

10. The process according to claim 1 , wherein the preferment composition is allowed to ferment for at least about 15 minutes.

1 1 . The process according to claim 1 , wherein the preferment composition is allowed to ferment for at least about 2 hours.

12. The process according to claim 1 , wherein the preferment composition comprises at least one of yeast and lactic acid bacteria.

13. A composition comprising free phenolic acid prepared from a process comprising:

pretreating bran with at least one enzyme;

subsequently adding the pretreated bran to a preferment composition; and allowing the preferment composition to ferment to form a fermented composition.

14. The composition according to claim 13, wherein the process further comprises:

adding additional ingredients to the fermented composition, and

allowing the fermented composition to ferment in a second fermenting step.

15. The composition according to claim 13, wherein the bran comprises aleurone.

16. The composition according to claim 13, wherein the at least one enzyme is chosen from xyianase and feruiic acid esterase.

17. The composition according to claim 13, wherein the bran is pretreated with the at least one enzyme for at least about 10 minutes before adding the pretreated bran to the preferment composition

18. The composition according to claim 13, wherein the bran is pretreated at temperature ranging from about 40°C to about 85°C.

19. The composition according to claim 13, wherein the preferment

composition comprises at least one of yeast and lactic acid bacteria.

20. A grain-based food product comprising free phenolic acid prepared from a process comprising:

pretreating bran with at least one enzyme;

subsequently adding the pretreated bran to a preferment composition; allowing the preferment composition to ferment to form a fermented composition; and

optionally baking, extruding, pel!etizing, pulverizing, and/or drying the fermented composition.

21 . The grain-based food product according to claim 20, wherein the baked good is bread.

Description:
INCREASING THE FREE PHENOLIC ACID IN GRAIN-BASED FOODS

Technical Field

[001 ] The present disclosure relates to novel methods for increasing the free phenolic acid content of grain-based food products, such as powders (for example, germ, bran, or aleurone fraction), breads, cereals, and animal feed. Various

embodiments according to the disclosure comprise the steps of pretreating bran, including aleurone and/or pericarp, with at least one enzyme, adding the pretreated bran to a preferment composition, and allowing the preferment composition to ferment. In further embodiments of the disclosure, additional ingredients may be added to the fermented composition, and the fermented composition may be allowed to further ferment in a second fermentation step. Yet further embodiments relate to dough products and grain-based food products prepared from the pretreated bran.

Background

[002] For certain food products, it is desirable to have a product that is high in whole grains. This is because in flour and food products produced using flour, whole grains are desirable for their improved nutritional benefits. Whole grains have been shown to impart nutritional benefits such as, for example, improved cardiovascular health, improved bowel health, anti-carcinogenic effects, and anti-inflammatory effects.

[003] In cereal grains, the majority of desirable "whole grain" nutritional components are concentrated in the bran portion of the grain, which comprises pericarp, seed coat (or testa), hyaline layer, and aleurone. Bran Is a source of several essential vitamins and minerals, including, for example, vitamin B8, niacin, vitamin E, potassium, magnesium, calcium, iron, and zinc. Bran is also a source of many antioxidants and phytochemicals.

[004] !t has been shown that within the bran, it is the aleurone component of bran that is responsible for the majority of the nutritional benefits of whole wheat, as the aleurone layer is more nutritionally dense than other components. For example, the aleurone layer of bran is a rich source of vitamins, minerals, sterols, essential fatty acids, nutrient fibers, high-quality protein and bioactive substances, such as phenolic compounds, lignan, betaine, and phyfate. Aleurone contains a high concentration of fiber, and the fiber in aleurone may improve digestion in the large intestine as well as slow resorption in the small intestine and bind undesired substances. In so doing, aleurone may contribute to detoxification and a long-lasting feeling of satisfaction.

[005] The aleurone layer is also a concentrated source of both "free" and "bound" forms of phenolic acid compounds, which are believed to have antioxidant and anti-inflammatory properties. The free versus bound status of phenolic acid compounds results in differences in health benefits and properties. Indeed, whether a phenolic acid compound is bound or free will impact which tissues of the body will receive the antioxidant and/or anti-inflammatory benefits of the phenolic acid compound.

[008] For example, bound phenolic acid compounds cannot be absorbed in the upper digestive tract, but may be released in the lower digestive tract by, for example, bacterial feru!ic acid esterases and further metabolized or absorbed by the endothelial ceils of the gut. Free phenolic compounds, however, are readily absorbed from the upper gastrointestinal tract into the blood, where these free phenolic acid compounds may confer antioxidant and anti-inflammatory properties to many tissue types in the body. Accordingly, it is desirable to increase the amount of free phenolic acid

compounds in cereal grains, and particularly the bran or aleurone portion of cereal grains, in order to impart increased nutritional benefits to food products comprising whole grains, such as powders, breads, cereals, and animal feed,

SUMMARY

[007] Disclosed herein are novel methods for increasing the free phenolic content of grain-based food products, such as powders, breads, cereals, and animal feed by increasing the free phenolic acid (such as ferulic acid) content of bran, and in particular the aleurone component, in cereal grains. In certain embodiments, the free phenolic acid content of bran, including aleurone and/or pericarp, is increased by pretreating bran with at least one enzyme, adding the pretreated bran to a preferment composition, and allowing the preferment composition to ferment to form a fermented composition. In further embodiments of the disclosure, additional ingredients may be added to the fermented composition, and the fermented composition may be allowed to further ferment in a second fermentation step. In certain embodiments the fermented composition may be, for example, a fermented dough composition or a fermented batter composition.

[008] Further disclosed herein are dough products and grain-based food goods comprising free phenolic acid prepared by pretreating bran with at least one enzyme, adding the pretreated bran to a preferment dough composition, and allowing the preferment dough composition to ferment to form a fermented dough composition, and, in the case of grain-based food products goods, baking, extruding, pel!etizing, pulverizing, and/or drying the fermented dough composition.

[009] Both the foregoing general summary and the following detailed description are exemplary only and are not restrictive of the disclosure.

B HEF DESCRIPTION OF THE FIGURES

[010] Figure 1 is a graph showing the impact of solid state enzyme treatment over time on the free trans-ferulic acid content of aleurone, bran, and pericarp.

[01 1 ] Figure 2 is a graph showing the impact of pellitizing on free trans-ferulic acid content, wherein "untreated" indicates no enzymes were added, "2hr" indicates the material was enzyme treated for 2 hours, "4hr" indicates the material was enzyme treated for 4 hours, "low temp" indicates the material was enzyme treated at a low temperature, and "high temp" indicates the material was enzyme treated at a high temperature.

[012] Figure 3 is a graph showing the percent increase in free trans-ferulic acid content in pellets comprising enzyme-treated material compared to pellets comprising untreated material under the same conditions. In the graph, "2hr" and !, 4hr" indicates the material was enzyme treated for either 2 hours or 4 hours, respectively, and "low temp" and "high temp" indicted the material was enzyme treated at a low temperature or a high temperature, respectively.

[013] Figure 4 is a graph showing the free trans-ferulic acid content of pre- exfruded cereal premix and finished cereals made with untreated and enzyme-treated wheat bran and aleurone. In the graph, "2hr" and "4hr" indicate the material was enzyme treated for either 2 hours or 4 hours, respectively. [014] Figure 5 is a graph showing the effect of fermentation time, enzyme pretreatment, and direct enzyme addition during bread making on the free trans-ferulic acid content of bread made with whole wheat, bran, and aieurone. In the graph, "Ohr", "2hr", and "4hr" indicate the material was not pretrea ed with the enzyme, the material was enzyme pretreated for 2 hours, or the material was enzyme pretreated for 4 hours, respectively.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[015] Disclosed herein are processes for increasing the free phenolic acid content of grain-based food products, such as powders (including, for example, germ and bran), breads, cereals, and animal feed. According to certain processes disclosed herein, the free phenolic acid content of bran or aieurone enriched bran fraction may be increased by pretreating the bran, which may comprise aieurone and/or pericarp, with at least one enzyme, adding the pretreated bran to a preferment composition, and allowing the preferment composition, also referred to herein as the sponge, to ferment, or rest. During prefermentation, the yeast or lactic acid bacteria in the preferment composition will ferment and the other ingredients may develop. It is contemplated that in certain embodiments, the preferment composition may be dried, and the dried composition subsequently may be added to a fermented composition or consumed directed, for example as a nutritional supplement.

[018] The resultant fermented composition is referred to herein as unproofed. In certain embodiments disclosed herein, the fermented composition may be a fermented dough composition, !n other exemplary embodiments, the fermented composition may be a fermented batter composition.

[017] According to certain exemplary embodiments of the disclosure, the fermentation may be carried out by any organism or combination of organisms recognized in the art. For example, in certain embodiments, the fermentation may be a yeast fermentation. In certain exemplary embodiments, the fermentation may be a bacterial fermentation, such as a lactic acid fermentation. One skilled in the art would be capable of choosing the appropriate fermentation organism to arrive at the desired fermentation composition. For example, a lactic acid fermentation may be used to acquire a fermented bread dough composition, such as a sourdough or non-sourdough bread composition. It is recognized that the fermentation organism may be able to ferment fibers in the composition and may have specific enzyme activities, such as esterase activity.

[018] In further embodiments of the disclosure, after fermenting, additional ingredients may be added to the fermented composition, and the fermented composition may be allowed to further ferment in a second fermentation step, known as proofing.

[019] As used herein, the term "bran" refers to the outer layers of cereal grain, comprising both aleurone and pericarp. Aleurone is botanical!y part of the endosperm, but is separated from the endosperm with the bran during milling and therefore may be considered part of the bran. Bran is an integral part of whole grains, and may be present in and may be milled from any cereal grain, including for example rice, corn, wheat, oats, barley, and millet. Bran contains ferulic acid, which is a phenolic acid compound found in plant ceil walls. As used herein, the term bran may include aleurone.

[020] As used herein, the term "aleurone" means the outermost layer or layers of endosperm in cereal grains, such as wheat. The aleurone surrounds the endosperm and may be separated from the wheat germ and starchy endosperm by standard milling processes known in the art. Aleurone may also comprise low levels of starch and pericarp, and represents a major portion of whole grain's nutritional benefits. It is believed that aleurone is more accessible to enzymatic conversion by certain enzymes, such as xylanase and feruiic acid esterase, than bran as a whole, and therefore the aleurone portion of bran, may, in certain embodiments, result in greater quantities of free phenolic acid compounds after enzyme pretreatment.

[021 ] The bran used in accordance with the embodiments disclosed herein may be obtained in a conventional manner in a grain mill or by any other method known in the art. The aleurone used in accordance with the embodiments disclosed herein may be isolated from bran and further processed using physical, such as mechanical- abrasive and biological-enzymatic, methods known in the art, \n certain embodiments disclosed herein, the aleurone used may be obtained from any cereal grain comprising aleurone, including for example rice, rye, corn, wheat, oafs, barley, and millet.

[022] Enzyme pretreatment of bran has shown potential in increasing the content of free phenolic acid compounds. Although not wishing to be bound by theory, it is believed that enzymatic pretreatment in combination with standard food processes, such as providing shear and heat, may amplify the quantity of free phenolic acid compounds in the composition and/or the finished food or feed material. As disclosed herein, in certain embodiments, the free phenolic acid content of a fermented composition may be increased by preteating bran with at ieast one enzyme, adding the pretreated bran to a preferment composition, and allowing the preferment composition to ferment. It is noted that the sequence of events, wherein first the bran is pretreated with at Ieast one enzyme, followed by subsequently adding the pretreated bran to a preferment composition yields superior results (e.g., increased phenolic acid content) as compared to simultaneously adding at Ieast one enzyme and bran to a preferment composition or adding at Ieast one enzyme to a preferment composition followed by the addition of bran.

[023] According to certain exemplary embodiments, the free phenolic acid content of a fermented dough composition may be increased by preteating bran with at Ieast one enzyme, adding the pretreated bran to a preferment dough composition, and allowing the preferment dough composition to ferment.

[024] In certain embodiments, first pretreating bran with at Ieast one enzyme, subsequently adding the pretreated bran to a preferment composition and then ailowing the preferment composition to ferment, in the order recited, results in an increased amount of free phenolic acid compounds as compared to adding untreated bran and at Ieast one enzyme to a preferment composition and allowing the preferment composition to ferment. In certain other embodiments disclosed herein, pretreating bran with at ieast one enzyme, adding the pretreated bran to a preferment composition and allowing the preferment composition to ferment results in an increased amount of free phenolic acid compounds as compared to pretreating bran with at Ieast one enzyme and adding the pretreated bran to a preferment composition without ailowing the preferment composition to ferment.

[025] In certain embodiments disclosed herein, the bran is treated with the at least one enzyme for at least about 15 minutes before the enzyme-treated bran is added to the preferment composition. In certain other exemplary embodiments, the bran is treated with the at least one enzyme for at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, or at least about 4 hours before the enzyme-treated bran is added to the preferment composition.

[028] According to certain embodiments, the at least one enzyme for

pretreatment may be any enzyme capable of increasing the free phenolic acid content of bran. For example, the at least one enzyme may be chosen from protease, xylanase, β-glucanase, celiulase, phytase, and feruiic acid esterase.

[027] !n certain exemplary embodiments disclosed herein, the pretreatment step may be performed at a temperature at which the at least one enzyme is active. One skilled in the art would be able to discern an appropriate temperature range for the activity of the at least one enzyme. In certain embodiments, the bran may be pretreated at a temperature ranging from about 40°C (104°F) to about 74 °C (185°F). !n certain exemplary embodiments, the bran may be pretreated at a temperature ranging from about 45°C to about 60°C, or about 50°C to about 55°C.

[028] In addition to the at least one enzyme, water may also be added to the bran during the pretreatment. In certain embodiments, water is added to yield a moisture content of the pretreatment mixture comprising bran, water, and at least one enzyme ranging from about 20% to about 70%. In certain exemplary embodiments, the bran may be pretreaied to have a final moisture content ranging from about 30% to about 80%, such as about 35% to about 55%, In certain embodiments, water may be added to the bran at a temperature that is higher than the desired pretreatment temperature, such that when the water is mixed with the bran and the at least one enzyme, the final temperature of the mixture is the desired pretreatment temperature. For example, in certain embodiments, the water may be added to the bran at a temperature of at least about 45 °C, at least about 55°C, at least about 65°C, and at least about 74°C.

[029] According to certain embodiments, phenolic acid compounds include any known free or bound phenolic acid compounds. In certain embodiments, phenolic acid compounds may include, for example, p-coumaric acid, sinapic acid, caffeic acid, and feruiic acid, such as trans-ferulic acid.

[030] As disclosed herein, the preferment composition may include any ingredients known in the art for producing baked goods, such as, for example, flour, yeast, water, salt, and sugar. One skilled in the art could readily adjust the amounts and types of ingredients in the preferment composition in order to arrive at the desired baked good. When adding the pretreated bran to the preferment composition, it is contemplated that the pretreated bran may be added in any order, including together with the other ingredients, before the addition of other ingredients, or after the addition of other ingredients.

[031 ] !n certain exemplary embodiments, the preferment composition may be allowed to ferment for at least about 10 minutes, such as at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, or at least about 4 hours. In certain embodiments, after the preferment composition has fermented for the desired time, the composition may be baked extruded, peiietized, pulverized, and/or dried.

[032] In certain embodiments, in lieu of baking the fermented composition, any additional ingredients known to those in the art may be added, and the fermented composition subsequently may be allowed to further ferment in a second fermentation step. The second fermentation step may comprise steps know to those in the art of baking. One skilled in the art would readily recognize appropriate times, temperatures, and conditions in order to arrive at the desired fermented composition. After the second fermentation step, the composition may then be baked to form a baked good, such as bread.

[033] According to certain embodiments, the enzyme pretreated bran may be used to make dough and dough products; batter, baked products, such as bread, pastries, muffins; beverages such as beer and non-alcoholic beverages, and the like, by any method known to those of skill in the art. It is contemplated that any grain may be used in the processes described herein, such as wheat, barley, rye, oats, hulled varieties, hull-less varities, etc. In at least one embodiment, the grain is wheat, and the wheat may be chosen from any wheat variety, including red wheat, white wheat, soft wheat, hard wheat, spring wheat and winter wheat.

[034] Unless otherwise indicated, ail numbers used in the specification and claims are to be understood as being modified in ail instances by the term "about," whether or not so stated. It shouid also be understood that the precise numerical values used in the specification and claims form additional embodiments of the disclosure, as do all ranges and subranges within any specified endpoints. Efforts have been made to ensure the accuracy of the numerical values disclosed in the

Examples. Any measured numerical value, however, can inherently contain certain errors resulting from the standard deviation found in its respective measuring technique.

[035] As used herein the use of "the," "a," or "an" means "at least one," and should not be limited to "only one" unless explicitly indicated to the contrary.

[038] It is to be understood that both the foregoing description and the following examples are exemplary and explanatory only and are not intended to be restrictive. In addition, it will be noted that where steps are disclosed, the steps need not be

performed in that order unless explicitly stated.

[037] The accompanying figures, which are incorporated in and constitute a part of this specification, are not intended to be restrictive, but rather illustrate embodiments of the disclosure.

[038] Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure.

Ό39] The following examples are not intended to be limiting of the disclosure.

[040] Parent hammermiiled bran, aieurone, and aleurone by-product

(hereinafter "pericarp") was obtained from an aieurone production line processing hard red winter wheat bran. [041 ] Enzyme treatment was performed using a combination of the foliowing two commercially available enzymes: (1 ) ferulic acid esterase Depo! 740L (from

BioCatalysts Ltd., Wales, UK); and (2) xylanase 8-200 (from Enzyme Development Corp., New York, NY). It is noted that ferulic acid esterase (Fase) has a working pH ranging from about 4 to about 8 and an optimal temperature ranging from about 40°C to about 85°C.

[042] The solid state enzyme treatment of the bran, aleurone and pericarp was conducted by adding 500ppm each of xylanase and Fase, on a substrate weight basis, to water to provide a final moisture content of approximately 30%. The water was added very slowly while mixing in a Hobart N-50 mixer outfitted with a paddle, and mixed on 2 nd speed until uniform, approximately 5 minutes. After this time, the paddle and bowl were scraped and the material was mixed for an additional 5 minutes on 2 nd speed. The product was then transferred to plastic containers outfitted with lids and placed in a humidified temperature controlled chamber set at 48°C (1 18°F). Samples were pulled at the desired time and the enzyme mixture was inactivated by heating the material to 82°C (180°F).

[043] The bran, aleurone, and pericarp were compared prior to enzyme treatment, 1 hour after enzyme treatment, 2 hours after enzyme treatment, 3 hours after enzyme treatment, and 4 hours after enzyme treatment. Figure 1 illustrates the impact of the solid state enzyme treatment over time on the free ferulic acid content in the bran, aleurone, and pericarp. It is shown that the bran, aleurone and pericarp all contained similar amounts of free ferulic acid prior to enzyme treatment. As the enzyme treatment progressed, however, it is shown that the aleurone and pericarp contained similar amounts of free feruiic acid with similar increasing rates of evolution of the free ferulic acid. Although the free feruiic acid content increased over time in the bran, the rate of evolution was less. See Figure 1 . This experiment demonstrates that the enzyme mixture was effective at increasing the free phenolic acid content of bran, aieurone, and pericarp.

[044] The process detailed above in Example 1 was scaled to produce 15 kg of material to cover the following three application experiments: animal feed pellets, cerea and bread. For this process, an 80qt Hobart planetary mixer was used to blend in the enzymes and water to reach approximately 30% moisture. The water was heated to 74°C (185°F) to produce a finished temperature of approximately 60°C (140°F) in the materials so that the material would be close to incubation temperature. After incubation with the enzyme mixture for the given period of time, the treated materials were spread on trays and heated to 82°C (180°F) to inactivate the enzyme mixture. After inactivation, the moisture content was determined so that the solids added from each materiai could be properiy standardized for each application. Table 1 shows the percent moisture and percent solids of the enzyme-treated and untreated bran and aieurone.

Table 1

(4hrs) bran

2a. Animall Feed Pellets

[045] For this study, equine feed pellets containing about 16% of the tested materials (bran, aleurone, pericarp) at various treatment times (enzyme addition for 0, 2, and 4 hours) and at two temperatures (low and high) were produced to evaluate the impact of pellet processing (peptization) on the free phenolic acid content of the finished material.

[046] A standard equine pellet formula was utilized to measure the free phenolic acid content in pellets produced using untreated wheat bran, untreated aleurone, bran treated with enzymes for 2 and 4 hours, and aleurone treated with enzymes for 2 and 4 hours. Each formula was pellefized at two temperature targets: low (60-63°C) and high (77-79°C). The formula in Table 2 below was used as the base formula, and 2 nd clear flour was used for the standard pellet. The 2 nd clear flour was replaced with the material being evaluated at the same percent inclusion, adjusting for the moisture to keep the % solids added to the pellet formula constant.

Table 2

Mono-dical phos 0.08 0.20

Salt 1 .37 3.42

Maxi bond 0.1 1 0.27

Total 100.00 250.00

[047] Pellets were produced using a Buhler pilot pellet mill (Buhler AG, Uzwil Switzerland). During pel!etizing, approximately 3-6% moisture was added via steam, most of which was removed during the cooling process.

Table 3

[048] Figure 2 illustrates the results of the peptization on the free trans-ferulic acid content of the pellets. It is shown that the free trans-feru!ic acid content is greatest in the pellets containing aleurone that had been enzyme treated for 4 hours, and second greatest in the pellets containing aleurone that had been enzyme treated for 2 hours.

[049] Figure 3 shows the percent increase in free trans-ferulic acid in pellets with enzyme treated materials as compared to pellets produced from untreated materials under the same conditions. As shown in Figure 3, the percent increase in free trans-ferulic acid was greatest in pellets containing aleurone that had been enzyme treated for 4 hours, and second greatest in pellets containing aleurone that had been enzyme treated for 2 hours.

2b. Cereal

[050] A direct expanded corn puff type cereal (having a formula approximating a Kix ® type cereal) was prepared, containing 20% of the tested materials (wheat bran, aleurone, and pericarp) at various treatment times (enzyme addition for 0, 2, and 4 hours) for analytical purposes.

[051 ] Seven cereal premixes were created with untreated pericarp, treated and untreated aleurone, and treated and untreated wheat bran. All mixes were then extruded to see if the free phenolic acid content increased. The pericarp was the only product that was not treated and was used to start up the twin screw, but a sample was collected to see if the twin screw processing did make an effect. Wheat bran and aleurone enzyme treated for 2 hours and 4 hours were extruded. Samples of the pre- extruded premix and the processed cereal were collected and were evaluated to see if the twin screw process was additive or synergistic to increasing the bioavailability of the phenolics in both the untreated and treated material.

[052] For the pericarp formula, an Anderson Dahlen Ribbon Blender (801b size) was used. For the other formulae, a Leland Southwest Double Action Paddle Mixer (501b size) was used. The minor ingredients (ingredients weighing less than one pound) were weighed out in the lab. The major ingredients were weighed out and added to the mixer, and then the minor ingredients were added to the mixer. The materials were blended for 7 to 10 minutes and then placed in a lined drum for future use.

[053] For the extrusion process, the extruder used was a Baker Perkins MPF24 (24mm diameter; 25:1 L/D). For the drying procedure, the dryer used was an Aeroglide Tray dryer. The puffs were dried with the dryer temperature set to 1 10°C with a residence time in the dryer of 4 minutes.

[054] Tables 4-8 below show the formulae used for the control cereal puffs, the cereal puffs with wheat bran, and the cereal puffs with aieurone, respectively. Table 7 lists the moisture content and density of the untreated pericarp, untreated aieurone, aieurone that has been enzyme treated for 2 hours and 4 hours, untreated wheat bran, and wheat bran that has been enzyme treated for 2 hours and 4 hours.

Table 4 - Control cereal puffs

Total 100 80.0 36,320.0

Table 5 - Puffs with Wheat Bran

Table 7

(4hrs)

[055] Figure 4 shows the free trans-ferulic acid content of pre~extruded cereal prernix and finished cereals made with untreated and enzyme treated (2 and 4 hours) wheat bran and aleurone. As shown in Figure 4, the greatest concentration of free trans-ferulic acid was bran and aleurone that had been treated for 2 hours and 4 hours, with the free trans-ferulic acid content increasing with increased treatment time.

[058] Breads were prepared using standard formulas and methods to determine the impact of enzymatic pretreatment, fermentation, and baking on the free phenolic acid content in bread applications, it was also desired to compare the pretreated materials to the addition of enzymes directly to dough compositions comprising untreated materials; therefore, enzymes at the same substrate basis were added directly to dough compositions for this purpose.

[057] A whole wheat bread formula was used as a controi reference for both straight dough (no prefermentation) and sponge-in-dough (prefermentation) processes, both of which are common in the baking industry. The whole wheat reference was also treated with a direct addition of enzymes at the same level, bran content basis, as in the enzyme treatment process. This would provide an understanding of the efficacy of whole wheat flour with enzymes.

[058] Figure 5 shows the effect of fermentation time (2 and 4 hours), enzyme pretreatment (untreated versus 4 hour pretreatment), and direct enzyme addition during bread making on the free irans-ferulic acid content of bread made with whole wheat, bran, and aieurone.

[059] As shown in Figure 5, there is an increase in the free transferu!ic acid content when comparing (1 ) bread made from adding untreated aieurone to a preferment dough composition with no enzymes to (2) bread made from adding untreated aieurone to a preferment dough composition with an enzyme mixture.

Moreover, the free-transferuiic acid content increased significantly when comparing (1 ) bread made from adding untreated aieurone to a preferment dough composition together with an enzyme mixture to (2) bread made from adding enzyme pretreated aieurone to a preferment dough composition when the preferment dough composition is allowed to ferment. It is this sequential process of enzyme pretreatment, followed by fermentation, that provided the greatest liberation of free phenoiics compounds using standard food processes.