CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/586,400, filed November 15, 2017, which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Malted grains are used to make many foods and beverages for humans and animals. Most importantly, malted barley is used in the brewing industry to make beer.

Millions of tons of barley and other cereal grains are malted every year for use in beer, foods, and beverages.

SUMMARY OF THE INVENTION

[0003] Described herein are methods for producing malt. In one aspect, the malt produced by the methods can be used to make a beer having reduced stale flavor attributes, i.e., a more stable beer. In one aspect, the malt produced by the methods can be used to make a beer and/or a malt extract having an enhanced color, e.g., a beer with an enhanced red, yellow, or orange color.

[0004] In one aspect, the method for producing a malt kernel, comprises: selecting a cereal grain kernel for malting, wherein the kernel has a protein content of at least 12.0%, steeping the kernel, wherein the moisture content of the kernel is at least 43% at the end of steeping, germinating the kernel to form a green malt kernel, wherein the temperature during germination is about l5°C or less, the germination step is at least 4 days in length, and the moisture content of the green malt kernel at the end of germination is at least 42%, and kilning the green malt kernel to form a malt kernel.

[0005] In some embodiments, the cereal grain kernel selected has a protein content of at least about 12.5%. In some embodiments, the cereal grain kernel selected has a protein content in the range of 12.0 to 13.0%. In some embodiments, the moisture content of the kernel is at least 45% at the end of steeping. In some embodiments, the steeping step comprises alternating periods of immersing the kernel in water (wet period) followed by removing the kernel from water (dry period). In some embodiments, the steeping step comprises at least 3 wet periods and at least 3 dry periods. In some embodiments, the ratio of the time of each dry period to the time of each wet period is in the range of 0.5 to 1.5. In some embodiments, the germination step is at least 5 days. In some embodiments, the moisture content of the green malt kernel at the end of germination is at least 45%. In some embodiments, the germination step further comprises heating the kernel to about 20°C for at least 4 hours prior to kilning. In some embodiments, the kilning step is performed by maintaining the green malt kernel at about 43 to 60 °C for 24 to 72 h. In some embodiments, at least a portion of the kilning step is performed by maintaining the green malt kernel at about 43 to 49°C.

[0006] In some embodiments, the malt kernel produced has a high content of

chromophore precursors. In some embodiments, the chromophore precursors comprise 4- hydroxy-5-methylfuran-3(2H)-one and/or 5-hydroxymethyl-2-furanaldehyde. In some embodiments, the chromophore precursors are quinizolate and homoquinizolate. In some embodiments, at least some of the chromophore precursors are precursors of the chromophore (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-met hylfuran-3(2H)-one, (E)-4- hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-methylfur an-3(2H)-one, or a mixture thereof. In some embodiments, the chromophore precursors comprise one or more amino acids. In some embodiments, the amino acid is tryptophan. In some embodiments, the amino acids comprise histidine, leucine, and arginine. In some embodiments, the chromophore precursors comprise one or more bound chromophore precursors. In some embodiments, the bound chromophore precursor is bound HMF.

[0007] In some embodiments, the cereal grain is barley. In some embodiments, the barley is 2-row. In some embodiments, the barley is 6-row.

[0008] In some embodiments, the kilning step comprises a curing phase. In some embodiments, the green malt kernel is maintained at less than about 65 °C during the curing phase. In some embodiments, the green malt kernel is heated to 70°C or greater during the curing phase. In some embodiments, the temperature delta of air-in versus air-out during the curing phase is at least 20°C. In some embodiments, the temperature delta of air-in versus air- out during the curing phase is at least 30°C. In some embodiments, the temperature of the air-out is at least 80°C. In some embodiments, the green malt temp is increased to 45 °C prior to kilning.

[0009] In one aspect, the disclosure relates to a malt composition comprising a malt produced according to the above embodiments of the malting method, or any combination of such embodiments.

[0010] In one aspect, the method of brewing a red beer, comprises: obtaining a malted barley kernel according to any of the malting methods above; milling the malted barley kernel to form a milled malt, mashing the milled barley to provide a mash wort; separating husks from the mash wort to provide a brew wort; boiling the brew wort to provide a brew of the desired color; adding yeast to the brew to provide a brew mixture; and fermenting the brew mixture to provide a beer, wherein the beer has a yellow, orange, and/or red color.

[001 1 ] In some embodiments, the one or more cereal grains and/or malted cereal grains are combined with malted barley kernel or milled barley prior to mashing. In some

embodiments, the beer is shelf stable. In some embodiments, the beer comprises a lesser amount of Strecker aldehydes than a beer brewed with a different malt. In some embodiments, the beer comprises a lesser amount of decanal than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of Perlolyrine than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of Flazin than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of (E) and/or (Z)-4-hydroxy-2-( 5-( hydroxymethyl )furan-2-yl methylene )-5-methylfuran-3(2//)-one than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of bound Perlolyrine than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of bound Flazin than a beer brewed with a different malt. In some embodiments, the beer comprises a larger amount of bound (E) and/or (Z)-4-hydroxy-2-(5- (hydroxymethyl)furan-2-ylmethylene)-5-methylfuran-3(2H)-one than a beer brewed with a different malt.

[0012] In one aspect, this disclosure also relates to malt extracts and methods of producing malt extracts. In some embodiments, the method for producing a malt extract, comprises:

extracting a malt produced according to any of the methods above with water to form a malt extract.

[0013] It is also to be understood that the elements or aspects of any embodiment of the processes, methods, or compositions described above can be applied to any other embodiment, as would be understood by a person skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [0015] Figure 1 is a diagram of a formation pathway leading to 4-hydroxy-2-(5- (hydroxymethyl)furan-2-ylmethylene)-5-methylfuran-3(2H)-one (1) from the Amadori-products of hexoses (la; Rl = -CH20H) and pentoses (lb; Rl = -H).

[0016] Figure 2 is a RP-HPLC chromatogram (l = 360 nm) of the ethyl acetate extractables isolated from barley malt.

[0017] Figure 3 is an excerpt of the H,H-COSY NMR chromatogram (400 MHz, DMSO- d6, 298 K) of (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-met hylfuran-3(2H)- one showing the 3J couplings of H-C(3) and H-C(4) as well as H-C(l) and HO-C(l).

[0018] Figure 4 is a set of HPLC-MS/MS chromatograms showing the quantitative analysis of (£)/(Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)- 5-methylfuran-3(2H)- one (la/lb) in barley malt and beer A) reference compound and ECHO standard B) barley malt sample C) beer sample using the ECHO technique. The peak of the ECHO standard is labeled with an“e” the peak of the target compounds with an“a” for la and with a“b” for lb.

[0019] Figure 5 is a diagram showing the possible formation of Perlolyrine from maltose/maltodextrin reacting with the amino acid tryptophan.

DETAILED DESCRIPTION

[0020] It is to be understood that the figures and descriptions of the present invention provided herein have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating other elements found in the related field(s) of art. Those of ordinary skill in the art would recognize that other elements or steps may be desirable or required in implementing the present invention. However, because such elements or steps are well known in the art or do not facilitate a better understanding of the present invention, a discussion of such elements or steps is not provided herein.

[0021 ] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 7 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 6, from 2 to 5, from 3 to 5, etc., as well as individual numbers within that range, for example, 1, 2, 3, 3.6, 4, 5, 5.8, 6, 7, and any whole and partial increments in between. This applies regardless of the breadth of the range. Methods for Producing Malt and Malt Compositions Produced Therefrom

[0022] This disclosure provides improved methods for producing malt. In one aspect, the malt produced by these methods can be used to make beer with extended stability, beer having a unique or enhanced flavor profile, and/or beer having a unique or enhanced color profile. In one aspect, the malt is produced using germination conditions and/or kilning conditions which provide a malt having a different chemical profile than a malt made using methods currently known in the art. In one aspect, the malt can be used to produce a beer with enhanced red, orange, and/or yellow color.

[0023] It has been unexpectedly found that red and yellow-orange chromophores are enhanced in beer brewed with malt produced using extended low temperature and high moisture germination, and/or low temperature, extended kilning conditions. Such germination and kilning conditions in the malting process are not currently used in methods for producing color malts such as Crystal, Caramel, Munich, Vienna, Amber, Melanoiden, Victory, Biscuit and others. These malts typically have colors of greater than 22 Standard Reference Method (SRM), and are used in making beer with reddish/orange, amber, and brown colors.

[0024] It has also been surprisingly found that malts produced using the process conditions described herein are typically pale in color, with low SRM, for example in the range of 1-20, yet these malts can be used to produce beer exhibiting significantly high color. The malts of the present invention allow for greater control and diversification of beer color when added with typical mash grain formulas. These malts can also be used to formulate new mash grain recipes to produce beer with unique and novel colors and flavors.

[0025] In one aspect, the malting process can produce a malt with a higher concentration of chromophore precursors than other malting processes. When using such a malt in a beer brewing process, these precursors can react to form chromophores during the brewing process.

In one aspect, the conditions of the beer brewing process can be modified to maximize the formation of chromophores. It has been surprisingly found that by minimizing the formation of chromophores in the malting process, and instead optimizing the malting process conditions to form chromophore precursors instead, the malt produced by such a malting process can be used to make a beer with improved red color and/or improved stability. Accordingly, in one aspect, the conditions of the beer brewing process can also be optimized for the formation of chromophores. In one aspect, the germination step includes using cooler and/or wetter conditions than other methods for producing a malt. These milder germination conditions can provide a malt with a higher content of chromophore precursors and a lower content of chromophores than other malts.

[0026] However, in some embodiments, it may be desirable to produce a higher concentration of chromophores in the malt itself. Accordingly, in one aspect, the malting process can be used to produce a malt with a higher concentration of chromophores than other malting processes.

[0027] In one aspect, the chromophore produced by the malting and/or brewing process described herein is the compound (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5- methylfuran-3(2H)-one and/or its (E)-isomer (the compound is also referred to herein as “compound 1,”“colorant 1,” or“chromophore 1,” with reference to“la” designating the Z isomer and“lb” designating the E isomer). In one aspect, the malting process described herein produces precursor molecules that can react to form (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan- 2-ylmethylene)-5-methylfuran-3(2H)-one and/or its (E)-isomer. In some embodiments, the precursor molecules include 4-hydroxy- 5 -methylfuran- 3 (2H) -one and 5-hydroxymethyl-2- furanaldehyde.

[0028] In some embodiments, the chromophore can be any desirable chromophore produced via a Maillard reaction, i.e., reactions between reducing carbohydrates and amino compounds. While it has been surprisingly found that (£)/(Z)-4-hydroxy-2-(5- (hydroxymethyl)furan-2-ylmethylene)-5-methylfuran-3(2H)-one can be the only significant chromophore exhibiting UV-Vis absorption above 360 nm produced in the malting and/or brewing process, this disclosure is not meant to be limited to malting and/or brewing processes in which this compound is the only chromophore.

[0029] In one aspect, the chromophore produced by the malting and/or brewing process is Perlolyrine. In one aspect, the chromophore produced by the malting and/or brewing process is Flazin. Both Perlolyrine and Flazin are associated with yellow color. Perlolyrine and Flazin can be formed from glucose, maltose, maltodextrin or high molecular weight carbohydrates during the malt and/or beer brewing process. During the malting or wort boiling process, glucose can form hydroxymethylfurfural (HMF), and react with tryptophan or its degradation product tryptamine to form Perlolyrine or Flazin. The reducing end of maltose, maltodextrin or any high molecular weight carbohydrate is also able to form“bound” HMF at the reducing end of the oligo- or polysaccharide, and this reaction product is also suited to form“bound” Perlolyrine or Flazin by the reaction with tryptophan, which could be released from the high molecular weight fraction of the beer, e.g., by acid hydrolyses (see, e.g., Figure 5). [0030] In some embodiments, the malting and/or brewing process can form Perlolyrine and/or Flazin in amounts which result in the malt or beer having an increased yellow color compared to other malt or beer. In some embodiments, the malting and/or brewing process can form (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-met hylfuran-3(2H)-one and/or its (E)-isomer in amounts which result in the malt or beer having an increased red or orange color compared to other malt or beer.

[0031] In one aspect, the present disclosure also relates to increasing the formation of bound chromophore precursors and/or bound chromophores in a malt or beer. A“bound” chromophore or chromophore precursor refers to a compound formed by a reaction of an amino acid with HMF or a similar compound which is bound to an oligo- or poly-saccharide. For example, an oligo- or poly-saccharide can be substituted for the di-saccharide in the reaction shown in Figure 5. The Perlolyrine can remain bound to the oligo- or poly-saccharide if not subjected to acid hydrolysis. Such bound chromophores can also be used to impart red, orange, and/or yellow colors to a malt and/or beer. Accordingly, in some embodiments, the

chromophore produced by the malting and/or brewing process is a bound chromophore. In some embodiments, the bound chromophore is bound Perlolyrine. In some embodiments, the bound chromophore is bound Flazin. In some embodiments, the bound chromophore is bound (E) or (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-met hylfuran-3(2H)-one. In some embodiments, the chromophore precursors produced by the malting process are bound chromophore precursors, for example, HMF or a similar compound capable of reacting with an amino acid to form a bound chromophore.

[0032] This disclosure also relates to malt compositions. In some embodiments, the malt compositions are pale in color, yet are capable of producing a beer with significant color, especially beer with a high degree of red, orange, and/or yellow color. In some embodiments, the malt compositions are capable of producing a more stable beer than other malts. In some embodiments, the malt compositions are capable of producing a beer with less stale attributes than other malts. In some embodiments, the malt compositions have a higher concentration of (£j/(Z)-4-hydroxy-2-(5-( hydroxymethyl )furan-2-yl methylene )-5-methylfuran-3(2//)-one than other malt compositions known in the art. In some embodiments, the malt compositions have a higher concentration of (£ ^' )/(Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylen e)-5- methyl furan-3(2//)-one precursors than other malt compositions known in the art. In some embodiments, the malt compositions have a higher concentration of chromophores exhibiting

UV-Vis absorption above 360 nm than other malt compositions known in the art. In some embodiments, the malt compositions have a higher concentration of chromophore precursors that can react to form chromophores exhibiting UV-Vis absorption above 360 nm than other malt compositions known in the art. In some embodiments, the malt compositions have a higher concentration of Perlolyrine and/or Flazin precursors than other malt compositions known in the art. In some embodiments, the malt compositions have a higher concentration of certain amino acids which are more likely to act as chromophore precursors. In some such embodiments, the amino acids include arginine, leucine, and/or histidine. In some such embodiments, the amino acids include phenylalanine and tyrosine. In some such embodiments, the amino acids include tryptophan.

[0033] The general malting process is well known in the art. The general malting process steps and conditions described below are not meant to be comprehensive, and some elements of a general malting process may be omitted which are not necessary for an understanding of how to perform the malting methods or produce the malts of the present invention.

[0034] Many cereal grains can be malted including, but not limited to, barley, wheat, buckwheat, rye, maize, rice, and oats. Cereal grains can be malted to modify their kernel structure, composition and enzyme content. The general malting process is described below for barley, but it is to be understood that other types of cereal grains can be used in a malting process.

[0035] The process of malting barley consists primarily of three stages: steeping, germination, and kilning. Prior to steeping, barley kernels can go through a selection process and/or processing steps to prepare them for the malting process. For example, because different sized barley kernels absorb moisture at different rates, it can be desirable to process uniform kernel sizes to improve product uniformity and quality. Therefore, processing steps can include separating barley kernels into similar sizes prior to steeping. Other processing steps such as cleaning of barley kernels can also be performed. The selection process can include selecting barley kernels for a variety of different attributes based on the desired qualities of the final malted barley. Process conditions and equipment also are considered. Once the appropriate kernels are selected and prepared, these kernels are then steeped.

[0036] Steeping refers to the immersion of barley kernels in water to increase the moisture content of the kernels. The barley kernels are immersed in water which may or may not be aerated. Typically, steeping can include a series of water immersions. These water immersions can be separated by periods of air rest under ventilation. During steeping, respiration of the barley begins, and heat and gases are given off although no significant growth takes place. Properly hydrating the barley to target moisture levels can be accomplished by manipulation of immersion times, air rest time, and immersion water temperature among other infrastructure and process recipe means. When steeping is completed, the embryo is swollen with moisture and is generally visible. Tips of the barley rootlets are generally just appearing. This visible swelling of the embryo and emergence of the rootlets is referred to as“chitting.”

[0037] After steeping, the barley kernels are typically transferred to germination compartments to undergo the germination process. Germination refers to a period of controlled growth and modification of the kernels. Modification of barley kernels is well known in the art to encompass cell wall degradation in the starchy endosperm, creation of soluble proteins and free amino nitrogen, and synthesis of desirable enzymes.

[0038] Germination broadly involves subjecting the steeped barley kernels to appropriate conditions of temperature, moisture, and airflow for a time sufficient for the starchy interior portion of the barley kernel (the endosperm mass) to be made more friable and modified by cell wall degradation, and growth of the embryo facilitated. Growth typically begins slowly on the first day of germination, and accelerates during the second day. Most germinations are 4-day processes, though 3-day processes are also known as well as germination processes which can last 5 or more days.

[0039] During germination, the barley kernel completes chitting and rootlets grow outwardly from the embryo of the kernel. The acrospire (also known as“first leaf’) also starts to grow from the embryo at the base of the kernel and grows under the hull toward the top end of the kernel. Growth of the acrospire in germination is a key parameter monitored by the commercial maltster. Typically, it is desired for well-modified malt to have the acrospire reaching ¾ to 100% of the kernel length by the end of germination. Malt products desiring a lower degree of modification may be ½ to ¾ or less than 100% the length of the kernel. Malt products requiring an extremely high degree of modification may be well in excess of 100% of the kernel, and may even have acrospires past the end of the kernel by 100% or more the length of the kernel.

[0040] Germination compartments generally use a slotted screen false floor allowing the compartment to receive a continuous humidified and temperature controlled airflow and allowing excess moisture to drain through the bed. Germination compartments are equipped with turning machines, or some means for turning the germinating barley kernels to minimize temperature differences between the top and bottom of the germination bed and to prevent rootlets from growing together and matting. Large air handling fans are used to transfer fresh air, recirculation air, and/or any blend of fresh and recirculation air through water spray humidification and temperature controlled chambers, and force the air through the germinating grain. The barley kernels, during growth, give off considerable heat and carbon dioxide. It is important to near continuously pass temperature controlled humidified air through the germination compartment to remove the carbon dioxide and heat produced by grain respiration, as well as to slow the rate of moisture loss, provide a means of controlling the germination compartment temperature, and control overall rate of barley growth.

[0041] Turning machines are typically equipped with a spray bar capable of delivering water to the growing barley. Other means of applying water to the barley are also possible. Generally, the germinating barley loses approximately 1% moisture per day during germination. The spray bars can be used to add back this lost moisture, and increase moisture level if desired, to the germinating barley through a metered watering.

[0042] Generally, sufficient water is applied to the germination compartment to wet the germinating barley, while allowing a minimal amount to leak out the bottom of the germination compartment. The germinating barley is ready to water when it is dry of surface moisture from steeping or a previous watering. Typically, after 24 hours from the start of the germination stage, the germinating barley is ready for its first watering. Depending on the initial moisture level of the barley kernels out of the steeping stage, the process goals for degree of modification, or customer specification, watering may occur every 8-12 hours after the initial watering. A second, third, fourth, or more, watering can be used. It is understood that different conditions and parameters can be used in the germination stage of the malting process depending on the barley conditions, the variety of barley starting material, the desired attributes of the malt output, and the size, type, or other physical attributes and limitations of the particular germination compartment used.

[0043] Barley respiration, rootlet growth, and acrospires are considered typical examples of malting loss. In the case of respiration loss, the barley releases carbon dioxide as a byproduct of metabolic activity which consumes grain mass. In the case of rootlet growth, rootlets are formed consuming grain mass and subsequently cleaned off following kilning. Acrospires in excess of the kernel length or that grow outside of the husks are also cleaned off following kilning, resulting in a loss of saleable malt mass. Though acrospires in excess of kernel length and rootlets retain minimal value in residual feed streams, they are a significant loss to the malting process. [0044] After the barley kernels have been modified to the desired degree, the grains are subjected to the kilning stage. Kilning refers to the controlled drying of the germinated barley. In the kilning process the germinated barley (green malt) is heated in a kiln to reduce its moisture content and stop further growth. Kilning is typically comprised of 3 phases: wither, pre cure/post wither, and cure. Green malt is typically transferred to a kiln immediately following germination. Most commercial kilns are slotted floor false bottom allowing air to pass through the green malt. Kilns usually have a means to minimally level the green malt bed for efficient drying, and may utilize turning machines as in germination to mix, turn, or level the green malt bed. Kilning is essentially a process of performing a regulated removal of water from the green malt.

[0045] The first main phase of kilning is the wither phase. In this phase the green malt at the end of germination is subjected to moderate temperature and high-volume airflow. During the wither phase, germination continues during the initial period when the grain still contains high levels of moisture, but growth and modification slows and stops once the rootlets are “withered”, with removal of all surface moisture as well as the more easily removed moisture in the embryo area of the grain.

[0046] In the second phase of kilning, the pre-cure/post wither phase, moisture level in the grain is further reduced, and the grain appears dry to the touch. Applied temperature is increased and airflow is typically decreased in this phase.

[0047] In the third phase of kilning, the cure phase, the temperature is increased to the maximum set point for the process, kiln, or the desired malt outcome. The cure phase typically drives off undesirable volatiles, and reduces final product moisture content to a

microbiologically, food safe level of approximately 4%. Temperatures used and time applied vary widely among commercial maltsters, and among product lines being produced.

[0048] After kilning is complete, the kernels are screened during which time the bulk of rootlets and in some cases acrospires are separated from the kernels. The separated rootlets, acrospires, as well as grain respiration which occurs during malting, represent a loss in the malting process, the so called“malting loss.” The amount of malted barley remaining after completion of the malting process and removal of rootlets, acrospires, and other undesirable materials is referred to as the malt yield. The conventional malting process is well known in the art and, for example, is described in D. E. Briggs, Malts and Malting, Springer (1998); D. E. Briggs, J. S. Hough, R. Stevens, and T. W. Young, Malting and Brewing Science, Volume i,

Malt and Sweet Wort, Springer Verlag (1981); A. W. MacGregor and R. S. Bhatty, eds., Barley: Chemistry and Technology, American Association of Cereal Chemists (1996), all of which are incorporated by reference herein in their entirety.

[0049] Two principal types of cultivated barley are used in the malting process, 2-row and 6-row. Cultivated barley can further be broken into fall planted winter barley, and spring planted spring barley. Each of these types of barley has several varieties which are used in the malting industry. A malt barley variety refers to a variety of barley typically cultivated and developed from a barley breeding program. Barley variety development will typically utilize germ plasm collections to develop varietal traits most beneficial to desired malt quality, and desired agronomical characteristics. Examples of barley varieties include Sebastian, Moravian, Copeland, Tipple, Metcalfe, Tradition, Scarlett, Barke and Stellar. This list is not exclusive as there are hundreds of barley varieties and more are constantly being created.

[0050] In one aspect, the malting process of the present invention relates to germination and/or kilning conditions during the malting process which result in higher levels of hydrolysis and greater amounts of monosaccharides such as hexoses and/or pentoses; di-, oligo-, and poly saccharides; and amino acids, peptides, and protein precursors, than malting processes known in the art. In one aspect, the malting process also relates to kernel selection, steeping conditions, and/or kilning conditions which result in higher levels of hydrolysis and chromophore or chromophore precursors than malting processes known in the art.

[0051 ] As described herein, the malting process of the present invention produces greater amounts of chromophore precursors than other malting processes. In one aspect, the

chromophore precursors are mono-, di-, oligo-, and/or poly-saccharides (“saccharide chromophore precursors”). In one aspect, the chromophore precursors are amino acids. As described herein, certain chromophores associated with red, orange, and/or yellow color are produced when saccharide chromophore precursors react with amino acid chromophore precursors. In one aspect, the malting conditions of the present invention can produce a malt with increased amounts of saccharide chromophore precursors and/or amino acid chromophore precursors. In one aspect, the malting conditions increase the amount of saccharide chromophore precursors and/or amino acid chromophore precursors while substantially preventing reactions between these two types of precursors. Therefore, the resulting malt contains higher amounts of these chromophore precursors and lower amounts of chromophores than other malts. It is to be understood that the chromophore precursors can then react to form chromophores during the beer brewing process instead of during the malting process. [0052] In one aspect, both types of chromophore precursors are produced in increased amounts in a single malt. In one aspect, only one type of precursor is produced in an increased amount in a single malt. For example, the malting conditions can be selected to drive increased production of saccharide chromophore precursors in one malt without a substantial increase in amino acid chromophore precursors in the same malt. Similarly, the malting conditions can be selected to drive increased production of amino acid chromophore precursors in one malt without a substantial increase in saccharide chromophore precursors in the same malt. In one aspect, malts with different chromophore precursor compositions can be combined in the grist for beer brewing. For example, a malt rich in saccharide chromophore precursors can be blended with a different malt which is rich in amino acid chromophore precursors. A beer brewed with such a blend can also be used to produce a beer with increased red, orange, and/or yellow color.

[0053] In some embodiments, the malting process of the present invention includes the steps of: selecting a cereal grain kernel for malting, steeping the kernel, germinating the kernel to form a green malt kernel, and kilning the green malt kernel to form a malted kernel. In some embodiments, the process further includes a curing step, which can also be considered as part of the kilning step. Provided below are embodiments for each of these steps. It is to be understood that each embodiment listed for a given step can be combined with any of the embodiments for any of the other steps, as would be understood by a person skilled in the art. For example, a malting process can have a kernel selection step that includes selecting a kernel having a protein content of at least 12.5% and steeping step wherein the final moisture content is at least 43%, or the malting process can instead have the same kernel selection step with different steeping conditions, for example a final moisture content of at least 45%.

[0054] Throughout this disclosure, the description of the malting process and the claims may recite selecting“a kernel,” i.e., a single kernel. However, it is to be understood that the processes and methods of the present invention relate to processing significant quantities of grain kernels simultaneously. Accordingly, process steps referring to a single kernel in the description and/or claims are also meant to cover simultaneously processing multiple kernels in a similar way, as is done in a typical industrial grain malting process. For example, a claim referring to“selecting a kernel, wherein the kernel has a protein content of at least 12.5%” should also be interpreted as“selecting a plurality of kernels, wherein each kernel, on average, has a protein content of at least 12.5%.” Kernel Selection

[0055] In one aspect, the malting process includes a kernel selection step. Kernels can be selected for the malting process based on one or more characteristics, including the protein content of the kernel, the size of the kernel, and/or other aspects of the composition of the kernel, such as gibberellic acid content.

[0056] In one aspect, the kernel selected is a kernel containing gluten. The gluten present in the kernel can be a source of amino acids during the malting and/or brewing process. As described elsewhere herein, these amino acids can react with other compounds, such as hexoses or pentoses, to form chromophores and/or chromophore precursors. Non- limiting examples of gluten-containing kernels useful for the methods described herein include 6-row barley, 2-row barley, wheat and wheat varieties such as spelt, and rye.

[0057] In some embodiments, the kernel is selected for having a high protein content. In some embodiments, the kernel selected has a protein content of at least 12.5%. In some embodiments, the kernel selected has a protein content of at least 11.0, 11.5%, 12.0%, 13.0%, or 13.5%. In some embodiments, the kernel selected has a protein content in the range of 12.0 to

13.0%. In some embodiments, the kernel selected has a protein content in the range of 11.0 to

13.0%. In some embodiments, the kernel selected has a protein content in the range of 10.0 to

13.0%.

[0058] In some embodiments, the kernel is selected based on size. In some embodiments, the kernel selected is a small-sized kernel, i.e., a kernel having a size that is less than average for a particular grain. In some embodiments, the kernel selected is an average-sized kernel. As the kernel size increases, the amount of protein can increase depending on the type of grain.

Accordingly, choosing a larger kernel size can increase the amount of proteolysis that occurs during malting, which can result in an increased content of chromophore precursors in the final malt.

[0059] In some embodiments, the kernel selected contains gibberellic acid. In some embodiments, the kernel is selected based on the kernel having a higher than average gibberellic acid content for a particular type of grain.

Steeping

[0060] In one aspect, the malting process includes a steeping step. The conditions of the steeping step can include the number of immersions, i.e., the number of times the kernel is immersed in water or otherwise contacted with water after being removed from contact with water; the dry/wet ratio, i.e., the ratio of time the kernel is out of water versus in water; the total time of water immersion of the kernel; and/or the percent moisture at the end of the steeping step.

[0061 ] In some embodiments, the steeping step can include at least 2 immersions, at least 3 immersions, or at least 4 immersions. In some embodiments, the dry/wet ratio is in the range of 0.5 to 1.5. In some embodiments, the dry/wet ratio is in the range of 1.0 to 1.5. In some embodiments, the dry/wet ratio is in the range of 0.5 to 2.0. In some embodiments, the dry/wet ratio is in the range of 1.0 to 2.0. In some embodiments, the dry/wet ratio is about 1.5.

[0062] In some embodiments, the moisture content of the kernel is at least 42% at the end of steeping. In some embodiments, the moisture content of the kernel is at least 43% at the end of steeping. In some embodiments, the moisture content of the kernel is at least 44% at the end of steeping. In some embodiments, the moisture content of the kernel is at least 45% at the end of steeping. In some embodiments, the moisture content of the kernel is at least 46% at the end of steeping. In some embodiments, the moisture content of the kernel is in the range of 43 to 50% at the end of steeping. In some embodiments, the moisture content of the kernel is in the range of 42 to 48% at the end of steeping. In some embodiments, the moisture content of the kernel is in the range of 44 to 46% at the end of steeping. In some embodiments, the moisture content of the kernel is in the range of 45 to 50% at the end of steeping.

Germination

[0063] In one aspect, the malting process includes a germination step. The conditions of the germination step can include the total length of the germination step; the temperature of the air contacting the kernel at the start of germination; the temperature of the air contacting the kernel at the end of germination; the addition of gibberellic acid to the kernel; the fan or air flow conditions at any point during germination; and/or the level of moisture of the kernel maintained during germination. In some embodiments, the germination step is cooler and/or wetter than germination conditions currently used.

[0064] In some embodiments, the length of the germination step is at least 4 days, at least 5 days, or at least 6 days. In some embodiments, the length of the germination step is 4 to 6 days. In some embodiments, the length of the germination step is 5 to 6 days.

[0065] In some embodiments, the temperature (of the air contacting the kernel) at the start of the germination step is relatively cool. In some embodiments, the temperature is about l5°C or less at the start of germination. In some embodiments, the temperature is about 10 to l5°C at the start of germination. In some embodiments, the temperature is about 20°C or less at the start of germination. In some embodiments, the temperature is about 10 to 20 °C at the start of germination.

[0066] In some embodiments, the temperature at the start of germination is maintained throughout the entire germination step. In some embodiments, the temperature is increased at or near the end of germination to be greater than the temperature at the start of germination, e.g., to increase proteolysis. In some embodiments, the temperature is about 20°C during the final portion of the germination. In some embodiments, the temperature is about 15 to 20°C during the final portion of the germination. In some embodiments, the temperature is about 20 to 25 °C during the final portion of the germination. In some embodiments, the temperature during the final portion of the germination is at least about 5°C higher than the temperature at the start of germination. In some embodiments, the temperature during the final portion of the germination is about 5 to l0°C higher than the temperature at the start of germination. In some embodiments, the temperature during the final portion of the germination is at least about l0°C, l5°C, 20°C,

25 °C, or 30°C higher than the temperature at the start of germination. In some embodiments, the temperature during the final portion of the germination is about 10 to 20 °C, 15 to 25 °C, or 20 to 30°C higher than the temperature at the start of germination.“The final portion of the germination” refers to part of the germination process immediately prior to heating the kernel for kilning. In some embodiments, the final portion of the germination can be the last 2 h, 4, 6 h, 8 h, 12 h, 16 h, 24 h, 36 h, or 48 h of the germination step.

[0067] In some embodiments, the flow of air contacting the kernel can be reduced or shut off at some point(s) of the germination step. In some embodiments, the air flow is shut off for at least 6 hours prior to the end of germination. In some embodiments, the air flow is shut off for at least 4 hours of the last 24 hours prior to the end of germination. In some embodiments, the air flow is shut off for at least 6 hours of the last 24 hours prior to the end of germination. In some embodiments, the air flow is shut off for at least 8 hours of the last 24 hours prior to the end of germination. In some embodiments, the air flow is shut off for at least 12 hours of the last 24 hours prior to the end of germination.

[0068] In some embodiments, the level of moisture of the kernel is maintained at a minimum level or within a range during germination, e.g., by continuously or regularly adding water to the kernel. In some embodiments, the moisture content of the kernel is maintained at a level of 42% or greater, 43% or greater, 44% or greater, 45% or greater, or 46% or greater during germination. In some embodiments, the moisture content of the kernel is maintained in the range of 42% to 50%, 43% to 50%, 44% to 50%, 45% to 50%, 46 to 50%, 43% to 48%, or 44% to 46% during germination.

[0069] In some embodiments, gibberellic acid is added to the kernel during the germination step. In some embodiments, the amount of gibberellic acid added is an amount suitable for decreasing the time for the kernel to sprout.

Kilning

[0070] In one aspect, the malting process includes a kilning step. The conditions of the kilning step can include the time the kernel is heated or maintained at the kilning temperature, and the temperature of the kernel after heating. The kilning step can also include a curing step or phase. The curing step or phase may be referred to as a separate step in some portions of this disclosure. However, the curing step can be considered as part of the kilning step. The conditions of the curing phase can include the time of the curing phase; the temperature of the curing phase; and the air flow conditions (including the temperature of air-in v. air-out, and the presence or absence of air flow/contact with the kernel).

[0071] In some embodiments, the kilning step is performed by maintaining the green malt kernel at about 43 to 60°C (about 110 to 140°F). In some embodiments, the kilning step is performed by maintaining the kernel at about 32 to 60 °C. In some embodiments, the kilning step is performed by maintaining the kernel at about 37 to 60°C. In some embodiments, the kilning step is performed by maintaining the kernel at about 40 to 65 °C. In some embodiments, the kilning step is performed by maintaining the kernel at about 40 to 70°C. In some embodiments, the kilning step is performed by maintaining the kernel at about 43 to 65°C. In some

embodiments, the kilning step is performed by maintaining the kernel at about 43 to 68°C. In some embodiments, the kilning step is performed by maintaining the kernel at less than about 60°C. In some embodiments, the kilning step is performed by maintaining the kernel at less than about 65 °C. In some embodiments, the kilning step is performed by maintaining the kernel at less than about 68°C. In some embodiments, the kilning step is performed by maintaining the kernel at less than about 70°C. In some embodiments, the time of kilning is at least 24 h, 48 h, or 72 h. In some embodiments, the time of kilning is about 24 to 72 h. In some embodiments, the kilning step is performed by maintaining the green malt kernel at about 43 to 60 °C for 24 to 72 h. In some embodiments, the air in versus air out temperature delta for the kilning phase is about 10°C or less. In some embodiments, the air in versus air out temperature delta for the kilning phase is about 20°C or less. In some embodiments, the air in versus air out temperature delta for the curing phase is about 30°C or less.

[0072] In some embodiments, the kilning step includes a curing phase. In some embodiments, the curing phase includes heating the kernel to a higher temperature than the initial part of the kilning step, e.g., a higher temperature than the values listed in the previous paragraph. In some embodiments, the air contacting the kernel is heated to greater than 68°C for the curing phase. In some embodiments, the air contacting the kernel is heated to greater than 70°C for the curing phase. In some embodiments, the air contacting the kernel is heated to at least 80°C for the curing phase. In some embodiments, the air in versus air out temperature delta for the curing phase is at least 20°C. In some embodiments, the air in versus air out temperature delta for the curing phase is at least 30°C.

[0073] In some embodiments, the final moisture of the malt at the end of the kilning step is less than 2%. In some embodiments, the final moisture of the malt at the end of the kilning step is less than 3%. In some embodiments, the final moisture of the malt at the end of the kilning step is less than 4%. In some embodiments, the final moisture of the malt at the end of the kilning step is less than 5%.

[0074] It is to be understood that any condition, embodiment, or aspect of the process steps described above can be combined with any other condition, embodiment, or aspect of a process step. Further, as would be understood by a person skilled in the art, different types of cereal grains can require different malt process conditions to produce the same relative qualities in the final malt. The following malt process conditions are exemplary conditions for certain types of grain. These examples are non-limiting, and it is to be understood that these types of grain can be used in a malt process with one or more changes to the process parameters, such as other options for process conditions described herein, to produce a malt having increased chromophore precursors and/or other qualities of the malts of the present invention.

Methods for Brewing Beer

[0075] In one aspect, the present invention relates to methods for brewing beer using a malt produced by the malting process described herein. In some embodiments, the malts produced by the methods of the present invention are relatively pale malts that can be used to produce beer with a high degree of color, especially color in the red-orange spectrum. This surprising effect is due to the production of a malt with a high content of chromophore precursors instead of the chromophores themselves. The relatively mild malting conditions can be used to make a pale malt that can be used to make a red, orange, or yellow beer. Further, the malt has other benefits besides increased color, such as increased stability of the beer and improved taste due to a decrease in compounds associated with stale flavors.

[0076] When beer becomes stale, it can have a lower hedonic rating that has been associated with an increase in,“stale”,“cabbagy”,“musty”,“metallic”,“s our”, and“catty” attributes (Techakriengkrai et ah, Journal of the Institute of Brewing, 2006. 112(1): p. 28-35). Several volatiles have been associated with stale flavor of beer, including increased levels of 2- furfural, Acetal, 5-hydroxymethyl-2-furfural, t,t-2,4-hexadienal, dihydro-5-pentyl-2(3H)- furanone (P-nonalactone), t,t-2,4-decadienal, l-heptanol, and decreased levels of furfuryl acetate, ethyl hexanoate, l-heptyl acetate, and ethyl octanoate (Foster, et ak, Journal of the American Society of Brewing Chemists, 2001. 59(4): p. 201-210).

[0077] 5-Hydroxymethyl furfural (5-HMF) and furfural were reported to be good index compounds for beer staling (Shimizu, et ak, Journal of the American Society of Brewing Chemists, 2001. 59(2): p. 51-58). Two main sources of these stale volatiles are oxidative and Maillard reactions during the malting process (Npddekter, T.V. and M.L. Andersen, Journal of the American Society of Brewing Chemists, 2007. 65(1): p. 15-20). The Strecker aldehydes, and series of aldehydes derived from the decarboxylation of amino acids produced in the part of the Maillard reaction called the Strecker reaction, were reported to be main influence on the formation of stale flavors. And, the thiobarbituric acid index (TBI) in malt, un-boiled wort and fresh beer was found to correlate highly with the sum of Strecker aldehydes and the tasting results of the aged beer (Gastl, et ak, Monatsschrift fur Brauwissenschaft, 2006. 59(9-10): p. 163-175).

[0078] Volatiles from lipid oxidation associated with“stale cardboard” flavor in beer is believed to be associated with liberation or formation of the unsaturated aldehyde like trans-2- nonenal and others listed above (Nyborg et ak, Journal of the American Society of Brewing Chemists, 1999. 57(1): p. 24-28). However, non-oxidative routes have also been found

(Lermusieau, G., et ak, Journal of the American Society of Brewing Chemists, 1999. 57(1): p. 29-33). Efforts to reduced staling in beer led to a focus on the free radical formation and content of malts. Several research groups reported that highly roasted dark malts and highly colored additives, especially this with high levels of melanoidins such as caramel color, and heavy metals led to the stale flavor attributes, and increase in radicals and lipid oxidation volatiles, such as E-2-octenal, E-2-nonenal, £',Z-2,6-nonadienal and £',£'-2,4-decadienal a decrease in stability and shelf-life (Npddekaer, T.V. and M.L. Andersen; Ochiai et ak, Journal of Chromatography A, 2003. 986(1): p. 101-110; Jehle et al., Food Chemistry, 2011. 125(2): p. 380-387; Wunderlich et al., European Food Research and Technology, 2013. 237(2): p. 137- 148). In fact, the temperature during the withering and kilning steps in malt production was shown to have a direct influence on the generation of stable organic radicals in the finished malt, whereby higher temperatures resulted in greater radical concentrations (Cortes, N., et al., Journal of the American Society of Brewing Chemists, 2010. 68(2): p. 107-113). Many attempts were made to block these reactions, such as adding polyphenols or un-malted barley (Guido, et al., Journal of Agricultural and Food Chemistry, 2007. 55(3): p. 728-733; Kunz et al., Brewing Science, 2011. 64(7-8): p. 75-82; Kunz et al., Journal of the Institute of Brewing, 2012. 118(1): p. 32-39).

[0079] In one aspect, the present invention relates to methods for brewing beer having reduced“stale”,“cabbagy”,“musty”,“metallic”, “sour”, and/or“catty” attributes. In some embodiments, the method of brewing beer includes brewing a beer using a malt which has relatively low levels of radicals, volatiles from lipid oxidation, and/or Strecker aldehydes. In some embodiments, the method of brewing beer includes brewing a beer using a malt which is capable of producing a beer having relatively low levels of radicals, volatiles from lipid oxidation, and/or Strecker aldehydes.

[0080] In one aspect, the beer has an improved flavor profile compared to beers brewed with a different malt. In some embodiments, the beer brewed using the methods described herein has a flavor profile with lower“malty’ notes, e.g., resulting from the beer having lower levels of 2-methyl- and/or 3-methylbutanal. In some embodiments, the beer brewed using the methods described herein has a flavor profile with, lower“green” notes, e.g., resulting from the beer having lower levels of t-2-nonenal and/or t-2decanal. In some embodiments, the beer brewed using the methods described herein has a flavor profile with higher“sugary”,“caramel” and/or “toasty” notes. In some embodiments, the beer is shelf stable. In some embodiments, the beer is shelf stable for at least 4, 6, 8, 10, 12, 16, or 20 weeks. In some embodiments, the beer has a lesser amount of Strecker aldehydes than a beer brewed without the malt of the present invention. In some embodiments, the beer has a lesser amount of decanal than a beer brewed without the malt of the present invention.

[0081 ] In one aspect, the beer has an improved, i.e., more intense color profile compared to beers brewed with a different malt. In one aspect, the beer has a higher chromophore content than beers brewed with a different malt or brewed using different conditions. [0082] While not wishing to be bound by theory, some chromophores present in the beer are generated from reactions of furanones and furfuyl aldehydes. The formation of higher molecular weight chromophores can also be produced when the reducing end of

monosaccharides, such as hexoses or pentoses, or di-, oligo- or poly-saccharides, using the conditions described herein, react to form higher molecular weight furfurals on terminal reduction sugars that can react with amino acids, peptides, and/or proteins.

[0083] In one aspect, the method of brewing a beer includes the steps of: obtaining a malted barley kernel according to a malting process described herein; milling the malted barley kernel to form a milled malt; mashing the milled barley time to provide a mash wort, separating husks from the mash wort to provide a brew wort; boiling the brew wort to provide a brew; adding yeast to the brew to provide a brew mixture; and fermenting the brew mixture to provide a beer, wherein the beer has a yellow, orange, or red color.

[0084] Conditions, such as temperature and time, for these steps are well known in the art. In some embodiments, one or more of the above steps can be performed at a higher temperature than is typically used, which can drive the reaction of chromophore precursors to form chromophores.

[0085] In some embodiments, one or more grains are combined with the malted barley kernel prior to or during the mashing step. In such embodiments, the malted barley kernel can be combined with other malted grains (produced by the malting process described herein or produced by any other malting process) and/or adjuncts. In some embodiments, different malts produced by different versions of the malting process of the present invention can also be used in the form of a blend for a mash formula. Table A below lists non-limiting examples of a grain bill or mash formula that can be used in the beer brewing process. It is to be understood that the malt produced by the methods described herein can be used with different grist blend compositions and/or adjuncts than those listed in the table.

Table A: Embodiments of grain bills for producing a beer having a red, orange, and/or yellow color

using the Standard Reference Method (SRM), which is well known in the art.

[0086] In one aspect, the beer has a red or yellow-orange color. In one aspect, the color of the beer can be measured using methods established by the International Commission on Illumination (CIE), i.e., CIE L*a*b* color measurement. The a* parameter corresponds to the red and green color spectrum. Positive a* values indicate red color while negative a* values indicate green color. Accordingly, an a* value of a beer (or malt) which is greater than the a* value for a control sample indicates that the color of the beer (or malt) is more red than the color of the control sample. In some embodiments, the a* parameter of the beer is at least 20 percent, 25 percent, 30 percent, 35 percent, or 40 percent greater than a beer produced from a different malt composition, i.e., a malt produced by a method other than the method of the present invention, but that otherwise is produced using the same materials and brewing conditions as the beer of the present invention.

[0087] A non-limiting example of a method for producing a beer is as follows. Barley malt (5 kg) is filled into a mashing kettle and mashed with warm water (4 L/kg malt). Additional water (20 L) is filled in the steam boiler and the resulting steam is introduced via tubing on the bottom of the mashing kettle. After closing the steam boiler, the mashing procedure is started.

[0088] An automatic brewing computer program directs the brewing system to heat up the mash to 55°C, and maintain at this temperature for 5 minutes. Then, the temperature is increased to 64 °C and maintained for 30 min. In a third step, the mash is heated up to 73 °C and kept for 30 min. Finally, the temperature of the mash is increased to 75 °C and held for an additional 10 min. The still hot mash is added slowly to a vessel (e.g., a bucket) and washed with water (5 L, approx. 75 °C).

Then the mash is added to the emptied steam boiler (the steam boiler can also be used as wort kettle). During the wort boiling the mash is heated up to l20°C and maintained for 120 min. Protein flakes are removed by whirlpool procedure (95 °C, 4 min) and at this point 20 g of hop are added, completing the brewing process. After cooling down the wort to 20 °C, the gravity of the wort is determined (14.5 %) and diluted to 13 % which will result in a final alcohol content of 6-7 vol. %. An aliquot of the wort (1 L) is stored at 4-6°C for the bottle fermentation. The wort is added to a fermentation tank and yeast is added. The primary fermentation is carried out at r.t. and is finished after 3-5 days. Now, the unfermented wort is added to a fermentation tank and carefully mixed. The“green beer” is added carefully to bottles and stored for another week at r.t., followed by storage for additional 3 weeks at 4°C, at which time the beer is ready to drink.

Methods for Producing a Malt Extract

[0089] In one aspect, the present invention relates to methods for making a malt extract using a malt produced by the malting process described herein. In one aspect, the malt extract has better stability and/or has more color than other malt extracts. In some embodiments, the malt extract of the present invention can be used to increase the red, yellow, and/or orange color of a food or beverage

[0090] In some embodiments, the method for producing a malt extract includes the step of mixing a malted grain with water; heating the malted grain/water mixture; optionally mashing and/or milling the malted grain; and filtering the malted grain/water mixture to form a malt extract. In some embodiments, the malt extract is concentrated by evaporation. In some embodiments, the malt extract is dried to form a dry malt extract. The drying step can be performed by additional evaporation; centrifugation to separate the water from the malt and/or by using any technique for drying a powder known in the art.

[0091 ] Mixing a malted grain with water allows the enzyme in the malted grain to break down the starch and proteinaceous material of the malted seed. While using water for extraction is preferred, it is to be understood that other solvents suitable for food applications, such as ethanol, can be used instead of or in combination with water. The filtering step removes insoluble materials such as fiber, resulting in a sugary liquid. Instead of being fermented into beer, this liquid is concentrated to make a viscous, stable liquid sweetener or is dried to make a powder.

[0092] The liquid or dried malt extract can be used to brew beer, or it can be used as a sweetener or flavor modifying ingredient in a food or beverage. For example, barley malt syrup is a malt extract produced by extracting malted barley. It is about half as sweet as refined white sugar. Barley malt syrup is sometimes used in combination with other natural sweeteners to lend a malt flavor to a food or beverage. Malt extracts can have similar carbohydrate profiles to high maltose syrups. However, they also contain protein (typically about 6%) as well as free amino acids, vitamins, and minerals. Accordingly, malt extracts can be used as a nutritive sweetener. EXPERIMENTAL EXAMPLES

[0093] These examples are provided for purposes of illustration only, and should not be construed as limiting in any way.

Example 1: Isolation and Structure Determination of a Maillard-type Chromophore Isolated from Barley Malt

[0094] The development of the typical brown color during thermal treatment of foods, such as coffee, bread crust or kiln-dried malt, mainly originates from reactions between reducing carbohydrates and amino compounds, known as the Maillard reaction. Depending on their molecular weight, colored compounds may be differentiated in two classes, the so-called melanoidins, which are defined as macromolecular, nitrogenous, brown-colored Maillard reaction end-products, and low molecular weight colored compounds. Due to the high precursor levels of 4-hydroxy-5-methylfuran-3(2H)-one as well as 5-hydroxymethyl-2-furanaldehyde, and the Maillard-supporting reaction conditions during the kiln process, barley malt may be a suitable candidate for isolation of low molecular weight colorants.

[0095] A chromophore and related isomer is isolated from barley malt and identified. The compound is identified as (Z)-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5- methyl furan-3(2//)-one and its (^-isomer. Synthesis of the compound from precursor molecules 4-hydroxy-5-methylfuran-3(2//)-one and 5-hydroxymethyl-2-furanaldehyde is also performed. The compound is also identified and quantified in beer.

MATERIALS AND METHODS

[0096] Chemicals. All chemicals are purchased from Sigma- Aldrich (Steinheim,

Germany), solvents are obtained in HPLC grade from Merck (Darmstadt, Germany), water for chromatographic separations is purified with a Milli-Q Gradient A10 system (Millipore, Schwalbach, Germany), deuterated solvents are obtained from eurisotop (Saarbriicken,

Germany) and barley malt is provided by the industry.

[0097] Isolation of (Z)-4-hvdroxy-2-(5-(hvdroxymethyl)furan-2-ylmethylene)-5- methylfuran-3 -one. Barley malt (300 g) is frozen with liquid nitrogen and then ground by

means of an ultracentrifuge mill (Retsch, Haan, Germany) equipped with a sieve (2 mm pore diameter). Aliquots (50 g) of the powder are then extracted with a mixture of methanol/ water (70/30, v/v, 300 mL) in an Erlenmeyer flask for l2h with a magnetic stirrer. Then the extract is filtered, centrifuged and the solvent is reduced under vacuum. The residue is diluted with water (200 mL) and then extracted with ethylacetate (5 x 250 mL). The combined organic extracts are freed from solvent to a final volume of 10 mL, and then fractionated by column chromatography on silica gel (silica 60, 0.063-200 mm, Darmstadt, Germany) adjusted to a water content of 6 %. After application of the raw material onto the column (450 x 35 mm) preconditioned with ethyl acetate, isocratic chromatography is performed using ethyl acetate as the mobile phase, and the individual fractions are collected visually, fraction 0 (300 mL), fraction 1 (40 mL), fraction 2 (100 mL), fraction 3 (130 mL) and fraction 4 (300 mL). The yellow-orange fraction 2 containing the title compound is further fractionated by solid phase extraction (SPE) on RP-18 material (Chromabond, C18 ec, l0g/70 mL, Macherey & Nagel, Diiren, Germany) activated with acetonitrile (140 mL) and conditioned with formic acid (0.1 %, 210 mL). Fraction 2 is freed from solvent in vacuo, dissolved in water/acetonitrile (95/5, v/v, 1 mL) and applied onto the column. After flushing with formic aid (0.1 %)/acetonitrile (90/10, v/v, 60 mL), the yellow- orange compound is eluted with acetonitrile (100 %, 60 mL). After removing of the solvent in vacuum, the colorant is obtained in a purity of more than 95 %. (Z)-4-hydroxy-2-(5- (hydroxymethyl)furan-2-ylmethylene)-5-methylfuran-3(277)-one (la; Figure 1): (+) HRESIMS, m z 223.0594 [M + H] ⁺ (calcd for CnHnOs 223.0606); UV-Vis (H ₂ 0/ACN, 8:2, v/v) _max = 376 nm, e = 0.61 x 104 L mol-l cm-l; ¹ H-NMR and ¹³ C-NMR data of 1 are given in Tables 1 and 2.

Table 1. Assignment of ¹ H-NMR Signals (400 MHz, DMSQ-dr,, 25 °C) of (Z)-4-hydroxy-2-

(5-( hydroxymethyl )furan-2-yl methylene )-5-methylfuran-3 -one

aH at relevant ^b 5 [ppm] ^C I ^C M ^C J [Hz] ^d gs-COSY

C atom (Z) (E)

CH ₃ (11) 227 223 3 s

H-C(l) 4.46 4.46 2 d 5.5 HO-C(l)

HO-C(l) 5.47 5.40 1 t 5.5 H-C(l)

H-C(3) 6.53 6.54 1 d 3.4 H-C(4)

H-C(6) 6.57 6.86 1

H-C(4) 7.02 8.10 1 d 3.4 H-C(3)

HO-C(9) 8.90 8.79 1 bs

aNumbering of carbon atoms refers to formula 1 in Figure 1. ^b The chemical shifts (ppm) are given in relation to DMSO-de. ^c Determined from 1D spectrum. Observed homonuclear H,H correlations. Table 2. Assignment of ^-NMR Signals (100 MHz. DMSO-d _fi, 25 °C) of 4-hvdroxy-2-(5- (hvdroxymethyl)furan-2-v1methylene)-5-methylfuran-3 -one

cHeteronuclear H,C correlations aC-Atom ^b 5 [ppm] '7 ; _. ii via HSQC ^2,3,4 /C,H via HMBC

(Z) (E)

C(ll) CH ₃ 12.6 12.6 H-C(l l)

C(l) CH ₂ 56.2 56.3 H-C(l) HO-C(l)

C(6) CH 100.2 108.7 H-C(6)

C(3) CH 111.1 110.7 H-C(3) H-C(l), H-C(4)

C(4) CH 119.2 117.8 H-C(4) H-C(3), H-C(6)

C(l0) C 136.0 136.6 H-C(l l)

C(7) C 142.2 143.4 H-C(6)

C(5) C 147.5 147.5 H-C(3), H-C(4)

C(2) C 159.6 159.6 H-C(3), H-C(4),H-C(l), HO- C(l)

C(9) C 162.4 161.2 H-C(l l)

C(8) C 181.0 178.7 H-C(6), H-C(ll)

aNumbering of carbon atoms refers to formula 1 in Figure 1. ^b The chemical shifts (ppm) are given in relation to DMSO-de. Observed heteronuclear H,C correlations.

[0098] Synthesis of (E)/(Z)-4-hvdroxy-2-(5-(hvdroxymethyl)furan-2-ylmethylene)-5 - methylfuran-3(2H)-one. A solution of 4-hydroxy-5-methylfuran-3(2H)-one (10 mmol), 5- Hydroxymethyl-2-furanaldehyde (10 mmol), piperidine (0.2 mL) and acetic acid (0.2 mL) dissolved in an ethanol/water mixture (1:1, v/v; 30 mL) is heated for 30 min at 50°C. After evaporation of the ethanol under vacuo (40 °C, 20 mbar), the reaction mixture is adjusted to pH 5.0 with hydrochloric acid (0.1 mol/L) and then extracted with ethyl acetate (5 x 20 mL). The combined organic layers are dried over Na ₂ S0 ₄ , filtered and concentrated in vacuo to approx. 1 mL. Storing at -26 °C affords orange crystals showing identical spectroscopic data as colorant 1 isolated from the barley malt (2.7 mmol, 27% in yield). The mother liquor is separated on a silica gel column flushing with ethyl acetate as the mobile phase as described for the isolation of colorant 1. In fraction 1, (£ ^' )-4-hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5 - methylfuran-3(2H)-one (lb; Figure 1) is obtained in a purity > 95 %: (+) HRESIMS, m/z 223.0592 [M + H]+ (calcd for CiiHnOs 223.0606); UV-Vis (H2O/ACN, 8:2, v/v) k _max = 359 nm, e = 0.58 x 104 L mol-l cm-l; ¹ H-NMR and ¹³ C-NMR data of 1 are given in Tables 1 and 2.

[0099] Mass Spectrometry. High Resolution Mass spectra of colorant 1 and 2 are measured on a Waters Synapt G2 S HDMS mass spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA, USA). Prior to HPLC-MS/MS analysis of the beer, the samples are degassed by ultrasonification for 5 min and then directly injected. Barley malt (20 g) is frozen with liquid nitrogen, ground by a laboratory mill (A 10, IKA, Staufen, Germany) and then water (200 mL) is added and stirred for 3h at room

temperature. After filtration the aqueous phase is extracted with EtOAc (3 x 250 ml). The combined organic extracts are dried over Na ₂ S0 ₄ (50 g), filtered and freed from solvent in vacuum. The residue is dissolved in ACN/H2O for MS analyses. The Quantitation of colorant 1 a/b in beer and barley malt samples is performed using an UPLC-ECHO-ESI-MS/MS system consisting of an Acquity I Class Core System (Waters Bedford, MA, USA) equipped with an Acquity I-Class UPLC Binary Solvent Manager (Waters, Bedford, MA, USA), an Acquity UPLC Sample Manager FTN (Waters Bedford, MA, USA), and an Acquity UPLC High

Temperature Column Heater (Waters, Bedford, MA, USA) coupled to a Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters, Manchester, UK). Chromatographic separation is carried out using an Acquity BEH Cl 8 column (2.1 x 150 mm, l.7pm particle size) at 40°C with a mobile phase flow rate of 0.4ml/min. The injection volumes of sample and ECHO-standard are 2pl respectively. The elution is conducted with water (solvent A) and acetonitrile (solvent B) both containing 0.1% formic acid. Gradient elution starts at 100% A maintained for 1 min, then B is increased to 50% over 4 min, held isocratic for 1 min and increased to 100% B in 1 min, held isocratic for 1 min, followed by a return to starting conditions and re-equilibration for 3min. The ECHO standard is injected 2.5 min after injection of the sample. The Xevo TQS mass spectrometer is operated upon the following settings: ESI source capillary voltage +2.90 kV, cone voltage -155 V; Source Temperature is set to l50°C, desolvation temperature to 400°C; Desolvation gas flow is at 800 l/hr nitrogen, collision gas flow at 0.15 ml/min argon. Data acquisition is performed using MassLynx 4.1 (Waters, Bedford, MA, USA) software.

Calibration is performed by diluting a 7.9 mmol/1 stock solution, its purity and the concentration is verified by qHNMR experiments, to a total of eight standard solution ranging from 15.8 pmol/l to 1.58 nmol/l. The ECHO-standard is diluted to 79 nmol/l. Obtained results are analyzed using TargetLynx 4.1 (Waters, Bedford, MA, USA). Calculation of individual concentrations are carried out by plotting the calibration curve of the response ratio between the area under curve of the most intense analyte transition and the corresponding ECHO-standard transition, in relation to its corresponding analyte concentration (linear equitation y=l.0272x-0.ll l5).

[00100] Spectrometric data of compound 1: 223.223 m/z (ESI+), quantitation fragment 205.152 m/z (cone voltage setting 2.0, collision energy setting 18.0), qualifying fragment 121.049 m/z (cone voltage setting 2.0, collision energy setting 8.0), minor fragment 135.024 (cone voltage setting 2.0, collision energy setting 18.0), dwell time 25.0 ms.

[00101] High Performance Liquid Chromatography (HPLC). The HPLC apparatus (Jasco, GroB-Umstadt, Germany) consists of two PU 2087 type pumps, a Rheodyne injector with a 100 pL loop, and a MD 2010 pus type DAD detector, monitoring the effluent between 220 and 500 nm. Separations are performed on a RP-C18 column (Kinetex 150 x 10 mm, 5 pm, Phenomenex, Aschaffenburg, Germany) operated with a flow rate of 4.7 mL/ min. The chromatographic separation of the barley extract is performed starting with an isocratic step with a mixture of aqueous formic acid (0.1%) and acetonitrile (95/5, v/v) for four minutes, thereafter increasing the acetonitrile content to 10% within 6 min, and maintaining this content for four minutes.

Then, the acetonitrile content is increased to 45% within 16 min and then this solvent composition is maintained for additional 2 min.

[00102] Nuclear Magnetic Resonance Spectroscopy (NMR). NMR spectroscopy is performed on a Bruker AVANCE III 400 MHz System equipped with a BBFOplus Probe (Bruker Rheinstetten, Germany) at 298.15 K. Chemical shifts are referenced to the solvent signal and data processing is performed by using TopSpin version 3.2 (Bruker, Rheinstetten, Germany). Quantitative 1H-NMR spectroscopy (qHNMR) for determination of the

concentration of stock solutions of the colorants is performed according to methods known in the art.

[00103] Calculations of minimized energy and spatial distance. Calculations are performed with Spartan (Ί4 v 1.1.8, Wavefunction, Inc.).

RESULTS

[00104] Aimed at evaluating the potential of barley to generate low molecular weight chromophores upon malt preparation, commercial available barley malt (EBC 28 - 32) which is used to generate red ale type beers, is extracted by sequential solvent extraction. Ground barley malt is extracted with a mixture of methanol/water, and after removing the organic solvent, the remaining aqueous phase is extracted with ethyl acetate affording a yellow-orange colored organic phase. To detect the key chromophores within the ethyl acetate soluble compounds, the organic extract is separated by means of RP-HPLC and the peaks exhibiting UV-Vis absorption above 360 nm are detected. Other than some minor compounds, surprisingly just one major peak is detectable at the selected wavelength, exhibiting an absorption maximum [k _m ax] at 376 nm (Figure 2). The UPLC-TOF/ MS screening of the ethyl acetate fraction shows a

pseudomolecular ion [M+H]+ at m/z 223.0594 for the compound representing the major peak in the chromatogram and the same mass to charge ratio as well as the same elemental composition of CiiHnOs for a later eluting minor compound, indicating the presence of an isomer.

[00105] Structure Determination of colorant 1. After chromatographic separation of the ethyl acetate extract on silica gel, two yellow-orange fractions are isolated (high/ low intensity). Both are further purified by solid phase extraction (SPE) on RP-18 material and the colored fractions are analyzed again by UPLC-TOF/ MS. Each of the fractions show a compound with a pseudomolecular ion [M+H]+ at m/z 223.059 and an identical elemental composition of C11H11O5 just differing in retention time and l _{ITI 1c} of the individual UV-Vis spectrum. A rearrangement of the minor to the major compound during purification and sample preparation is observed. This indicates the presence of isomers, with the minor isomer being less stable. A rearrangement of the major to the minor isomer is not observed, which is in the line with the higher abundance of the more stable (major) form.

[00106] The structure determination of the major compound is performed by ID- and 2D NMR spectroscopy. The ¹ H NMR spectrum of the colorant measured in DMSO-de shows 7 resonance signals, among which one signal integrated for three protons, one for two protons and five signals for just one proton (Table 1). In the aliphatic shift region, the singlet resonating at 2.27 ppm (3H) is most likely corresponding to a methyl group, whereas, the methylene group at 4.46 ppm seems to be connected to a heteroatom like oxygen. In the chemical shift range between 6.5 and 7.1 three aromatic -/olefinic signals are obtained. Two of them showed a strong coupling in the gs-COSY with a coupling constant of 3.4 Hz which is typical for the protons in furan-moieties (Figure 3). The heteronuclear single quantum coherence (HSQC) spectrum optimized for Ucn coupling constants shows that out of the seven resonance signals only five were connected to carbon atoms (Table 2). The resonance lines at 5.47 and 8.90 ppm disappear due to the H/D exchange by adding two drops of D2O to the sample, indicating, in combination with the results of the mass spectroscopic analyses that two OH groups are incorporated in the molecule. The determination of the constitution of the colorant is achieved by heteronuclear multiple bond correlation spectroscopy (HMBC) optimized for ^2,3 /C,H coupling constants. The

HMBC experiment shows correlations between the methyl group H-C(l l) and the quaternary carbon atoms C(8), C(9) and C(l0) well in the line with the 2H-furan-3-one moiety in the structure. Couplings of the quaternary carbon atom C(5) with the protons H-C(3) and H-C(4) as well as the correlations of C(2) with H-C(l), HO-C(l), H-C(3) or H-C(4) confirm the 5- hydroxymethylfuran moiety in the molecule. The connection between the 5-hydroxymethylfuran and the 2H-furan-3-one motive via a methylidene bridge can be verified by the correlation signals of H-C(6) with the quaternary carbon atoms C(7), C(8) and C(4).

[00107] Taking all of the spectroscopic data into consideration, the major yellow-orange colorant isolated from the barley malt is identified as 4-hydroxy-2-(5-(hydroxymethyl)furan-2- ylmethylene)-5-methylfuran-3(2H)-one. The existence of (E)/(Z) isomers is suggested by the occurrence of a second compound in the barley with the same mass to charge ratio, the identical elemental composition, the same color, and the fact that the minor compound shows a rearrangement to the major compound.

[00108] To confirm the structure of the major compound and to verify the existence of its isomer, the colorant is synthesized by reaction of expected precursor molecules, i.e., heating an equimolar mixture of 4-hydroxy-5-methylfuran-3(2H)-one and 5-hydroxymethyl-2- furanaldehyde in an ethanol/water mixture in the presence of piperidine and acetic acid at 50 °C for 30 min. After a few minutes the color of the reaction mixture turns into orange and then into red. Crystallization shows an orange compound having the identical UV/Vis, MS and NMR data as colorant 1 (major isomer) isolated from barley. Aside from colorant 1, the ¹ H NMR spectrum shows a minor compound with the same signal pattern just differing in chemical shifts. The isolation of the minor compound in sufficient yield and purity for structure elucidation is achieved by silica gel column chromatography of the mother liquor of the chemical synthesis, due to a better ratio of minor to major compound compared to the extract of the barley.

[00109] The most notable differences in the ¹ H NMR spectrum can be observed for the protons H-C(6) showing 6.57 ppm for the major and 6.86 ppm for the minor compound as well as H-C(4) where a shift difference of more than 1 ppm between the colorants can be detected. For all other proton signals, comparatively small chemical shift differences can be observed (Table 1). The most pronounced differences for the carbon shifts are detected for C(6) and the carbonyl group C(8) (Table 2). On the basis of the chemical shifts, and the fact that all HMBC correlations are identical for both compounds, colorant 1 is identified as (E)/(Z) isomers of 4- hydroxy-2-(5-(hydroxymethyl)furan-2-ylmethylene)-5-methylfur an-3(277)-one.

[00110] The (£) -configuration (lb) is assigned on the basis that H-C(4) is very close to the carbonyl group C(8) (2.1 A) and would therefore strongly de-shielded to give a signal at lower field 8.10 ppm compared to the (Z)-isomer (la) 7.02 ppm were the distance between C(8) and H-C(4) is approx. 5.5 A. These chemical shifts are well in the line with a similar class of naturally occurring a,b-unsaturated furan-3-ones - the Aurones - where the aromatic protons H- C(2’) and H-C(6’) are close to the carbonyl-group showing the same strong de-shielding in the (E)-isomer, whereas the same protons in the (Z)-form are nearly unaffected. The same trend can be observed for the Proton H-C(6), which shows a chemical shift difference of approx. 0.3 ppm when comparing the (E)- (6.86 ppm) with the (Z)-isomer (6.57 ppm) (Table 1). Another aspect to confirm the stereochemistry of the double bond are the chemical shifts of the carbonyl-group C(8). For the (Z)-isomer of the colorant 181.0 ppm can be observed whereas the (E)-isomer showed 178.7 ppm. This difference of 2.3 ppm is comparable to the carbonyl-shifts of the different (E)- and (Z)-Aurone derivatives, where (Z)-isomers showed resonances between 184.1- 184.5 ppm and the (E)-isomers 182.5-182.8 (Table 2).

[00111] Colorant 1 a/b can be shown to form via a condensation reaction of the

carbohydrate degradation products 4-hydroxy-5-methylfuran-3(2H)-one and 5-hydroxymethyl- 2-furanaldehyde. As shown in Figure 1, the amino acid induced dehydration of hexoses as well as of pentoses via the Amadori product (Ia/Ib; R2 = amino acid) results initially in the formation of the 3,4-dideoxy-l,2-hexodiulose (Ila) and the l-deoxy-2,3-pentodiulose (lib). Ring closure followed by water elimination then give rise to 5-hydroxymethyl-2-furanaldehyde (Ilia) in case of the hexose and 4-hydroxy-5-methylfuran-3(2H)-one (Mb) for the pentose. A condensation reaction between the carbonyl function of Ilia and the methylene active intermediate Illb leads to the formation of colorant 1 (Figure 1).

[00112] Identification of colorant 1 in barley malt and beer. To verify the occurrence of the colorants 1 a/b in a real food sample, barley malt as well as beer is screened by means of

LC/MS/MS (ESI+) operating in the MRM mode. Prior to analysis, characteristic mass transitions are selected for the colorant in tuning runs. Thereafter, the retention time as well as the characteristic mass transition in barley malt and in beer are compared to those of the reference compounds. As shown in Figure 4 for the mass transition m/z 223.223— > 205.152, the target compounds can be identified in beer by means of HPLC-MS/MS (MRM). The influence of matrix effects is well accepted and requires the use of internal standards for an accurate quantitative analysis of target compounds. The synthesis of stable isotope-labeled analogues of colorant 1 as a suitable internal standard for quantitative purposes is challenging. Therefore, the so-called ECHO technique is used to compensate the effect of co-eluted matrix compounds in

LC-MS/MS analysis by using a non-labeled target compound as an internal standard, which is injected into the HPLC-MS system after a short time period as the“echo” of the analyte. For reliable quantitative purposes a signal/ noise ratio of > 10 is required. With the exception of the barley samples YMP C60 and 1400036, none of the other samples exceed this ratio, and colorant lb can be identified but not quantified (Table 3).

[00113] Notably, two of the malt samples show comparatively high amounts of la/b whereas in the other two samples only small amounts are detected. This indicates that the pretreatment and the processing conditions of the malt can play an important role for the formation of the chromophores. By comparing the color of the beer with the concentration of colorant la, it can be seen that a higher amount of la is not necessarily correlated to the color intensity. For example, Benediktiner (EBC 10) with a gold color showed higher amounts of chromophore la (0.043 pmol/L) compared to the much darker Duckstein (EBC 30) with a concentration of just 0.024 pmol/L, showing a reddish color. The highest concentration of chromophore la in all investigated beers was found in Salvator, whereas the darkest beer (Guiness) had one of the lowest concentrations of chromophore la.

Table 3. Concentrations of the colorants la/lb in commercial malt and beer samples

color sample cone. [pmol/L] ^b cone. [pmol/g] ^b

[EBC] ^a la lb la lb

Munich Malt 0.03 n.d.

YMP C60 2.60 0.19 2RC10 0.01 n.d.

14-00036 3.76 0.17

7 Augustiner 0.005 n.d

8 Konig Ludwig hell 0.015 n.d

8 Warsteiner 0.005 n.d

8 Becks 0.014 n.d

10 Bitburger 0.0 0.017 n.d

10 Benediktiner 0.043 n.d

11 Weltenburger hell 0.012 n.d

12 Becks gold 0.012 n.d 25 Erdinger Urweise 0.018 n.d.

28 Becks Amber 0.094 n.d.

Lager

30 Duckstein 0.024 n.d.

35 Kilkenny 0.011 n.d.

36 Paulaner dunkel 0.037 n.d.

46 Salvator 0.077 n.d.

40 Konig Ludwig 0.029 n.d.

dunkel

> 60 Guiness 0.010 n.d. ^a Color of the beer was approximated by a beer-color-card. Std. ^b deviation of technical triplicates < 3.5%.