Malting and Wort Production Potential of the Novel Grain Kernza (Thinopyrum intermedium)

Abstract As the environmental impacts of beer are of increasing concern to maltsters, brewers, and consumers, perennial cereal crops can offer a more sustainable solution. One new cereal species of interest to the brewing industry is intermediate wheatgrass (Thinopyrum intermedium subsp. intermedium), developed by The Land Institute, in Salina, Kansas, U.S.A., under the branded name Kernza®. It has been touted as a more sustainable alternative to barley and wheat in its requirements for less water and nutrient additions and reduced tilling of agricultural fields. To date, no published research has been performed to assess the potential for this grain for the malt and beer industries as a barley replacement. Here, the “M5” variety Kernza grain was micromalted and the finished malt was compared with both the raw grain and to a reference raw and malted barley (Hordeum vulgare L. var. Copeland). All samples were analyzed for starch gelatinization temperatures via differential scanning calorimetry, amino acid composition, alpha-amylase, diastatic power (DP), total and soluble nitrogen and protein, Kolbach Index (KI), extract, wort free alpha-amino nitrogen (FAN), wort β-glucan, wort color, pH, and clarity. The Kernza malt produced sufficient extract and FAN for typical fermentations with low β-glucan content. However, extract and FAN in the Kernza malts were lower than in the barley reference, and the Kernza worts exhibited higher levels of undesirable soluble protein and haze. Interestingly, the DP for both the raw and malted Kernza were similarly high, indicating that both of the raw grains exhibit high enzymatic activity, which malting did not increase substantially. Results support that Kernza could be an acceptable candidate for malting and subsequent wort production; however, specific techniques may be required to utilize this malt most effectively. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2022.2026662 .


Introduction
As the craft beer industry has grown, many brewers have sought to distinguish themselves by responding to consumer demands for not only novelty and flavor, but also localness and environmental sustainability. [1] By expanding the availability of alternatives for malt production beyond barley to more sustainable grains, the malt and beer industries have the potential to reduce their environmental costs while simultaneously engaging consumers with novel products. Kernza (Thinopyrum intermedium subsp. intermedium), a recently-domesticated perennial cereal grass, presents a novel opportunity to utilize a new grain for malt and wort production for beer brewing that has been touted as a more agriculturally sustainable alternative to barley and wheat.
Kernza plants differ most importantly from other domesticated cereal grains in their agricultural growth habits, and it is here that the environmental benefits are gained. Wheat, barley, and other annual cereals typically exhibit a life cycle of only one season, whereas perennial Kernza plants have the ability to grow over multiple years with grain produced and harvested each year. With this multi-year growth, Kernza plants are able to develop a deeply-penetrating root system. While the roots of wheat plants generally reach only 50-300 cm, [2] Kernza roots have been shown to grow to depths greater than three meters, and at a much greater density than those of wheat. [3] As a result, Kernza fields function as a carbon sink, with the top meter of soil in Kernza fields containing fifteen-fold the amount of organic carbon compared with untilled annual wheat fields. [3] This densely-packed root system is also ideal for more efficient water usage [4] and access to deeper and more stable water stores, both advantageous in climates with low or intermittent rainfall or unpredictable weather patterns. [5] Kernza has also been shown to not only require fewer nitrogen additions when compared with wheat, but can actually reduce nitrogen already leached into the groundwater from surrounding fields. [6] crop and for erosion control. It was chosen for breeding based on its potential for regenerative agriculture to be developed as an edible seed crop starting in the 1980s. [7] The Land Institute (Salina, Kansas, U.S.A.) has since trademarked the grain with the trade name Kernza ® , [8] and a cultivar "MN-Clearwater" was recently approved for sale to the public with human food as its primary intended use. [9] Kernza has been shown to share substantial genetic similarity to other grass species in the Triticeae tribe, such as domesticated wheat (Triticum aestivum L.), rye (Secale cereale L.), and somewhat less so to barley (Hordeum vulgare L.). [10] Its seeds present in a similar fashion to wheat and barley on two-rowed spikes and yield a hulled kernel after threshing. However, current varieties of Kernza produce kernels of roughly 10 mg each (wet basis), [9] much smaller than the typical 40 mg per seed weight for wheat or the 30-50 mg for barley. [11] Kernza kernels contain a carbohydrate content of roughly 67% and a starch content of 45-50% (w/w), lower than the 65% starch found in whole grain hard red wheat [12] and the 58-65% in barley varieties used for malting. [13] Kernza also contains a protein content of greater than 17% (w/w), significantly higher than the 9-15% found in wheat or barley. [14,15] From a malt perspective, Kernza grain has physiological characteristics that are encouraging for its potential as an alternative to barley. Kernza contains a similar carbohydrate content to barley and wheat, [16,17] with amylose and amylopectin of comparable quantity, and with size and branching structures as oats, barley, and rice. [18] Furthermore, many grains such as rice, corn, and sorghum contain starches that will not gelatinize at typical mash temperatures, and they require a cooking step prior their addition to the mash, which inactivates many of the endogenous malt enzymes. [19] Breeding lines of T. intermedium have been shown to contain starches that exhibit peak gelatinization at 60.2-62.4 °C, [18] similar to those in wheat and barley, at 58-64 °C and 51-60 °C, respectively. [11] This indicates that a Kernza mash may be used to contribute starch to the mash without requiring higher temperatures for starch gelatinization that would necessitate exogenous enzyme additions.
Kernza does still exhibit some impediments to successful malting or as a principal component of the mash. For example, the current breeding lines of Kernza are not completely dehulled, nor is the hull firmly attached, and thus mechanical methods are required to remove the hull from the grain for homogeneity. [8] Current methods [20] result in excessive germ damage that negatively impacts germination quality. [21] If properly dehulled, Kernza may require malting techniques similar to wheat and rye, which do not have an attached hull, and it will require careful management of the tightly-packed grain beds during germination to mitigate heat and carbon dioxide buildup due to lack of airflow. [22] Additionally, without the structural reinforcement provided by the hulls in the brewery mash, the wort will not percolate through the grain bed as efficiently. [23] An addition of rice hulls to the mash is frequently used to reduce the risk of filtration issues, as is the employment of a mash filter. [19] For a 100% Kernza beer, the hulls removed prior to malting might be added back to the mash and function much like the rice hulls.
To date, Kernza has only been used sparingly in food and beer, and mainly as a raw grain. [24] Thus, the aim of this study was to evaluate the potential of Kernza to produce malt intended for wort production for beer. Through the malting of small lots of this grain, alongside a barley reference, both the malt and laboratory scale worts were analyzed using a variety of methods to provide a holistic view of the functionality of this grain as a malt.

Raw grain selection
Samples of raw, sorted, and dehulled "M5" variety Kernza were obtained from Perennial Pantry (Burnsville, MN, U.S.A.) in October of 2020. This grain was harvested in Lake Benton, MN, U.S.A. in 2019 and dehulled using an impact dehuller. Samples of raw "Copeland" barley, a two-row malting variety, were obtained from Admiral Maltings (Alameda, CA, U.S.A.) in November of 2020. This grain was harvested in Tulelake, CA, U.S.A. in 2019.

Amino acid composition
The amino acid compositions of the raw Kernza and barley grains were determined using an L-8800a (Hitachi High-Tech America; Santa Clara, CA, U.S.A.) dedicated amino acid analyzer, which utilizes ion-exchange chromatography to separate amino acids followed by a post-column ninhydrin reaction detection system. Eluants and diluent were sourced from Pickering Labs (Mountain View, CA, U.S.A.), and a Concise Separations Analytical Column (San Jose, CA, U.S.A.) was used. Raw grains were milled to a fine flour, and roughly 5 mg was hydrolyzed with 200 μL of 6 N HCl under vacuum at 110 °C for 24 h. Prior to this, a portion was oxidized separately overnight with freshly prepared performic acid at 2 °C, dried, then hydrolyzed as previously described. A third portion was also hydrolyzed separately with 4.2 N NaOH under vacuum at 110 °C for 24 h, neutralized with 200 μL of 4.2 N HCl (for Trp measurement), and 600 μL of Na diluent was added. From each separate hydrolysis condition, a 50 μL sample was injected on the Hitachi L-8800a amino acid analyzer. Amino acids were separated by strong cation exchange, followed by a secondary reaction with ninhydrin and observed in the visible range at both 570 nm and 440 nm. The concentrations of each amino acid in the injection (in nmol) were calculated using the response factors generated from a known standard run in the same sequence. NorLeu was added to the diluent at 40 nmol/mL, as an internal standard, to monitor auto-sampler performance. Individual amino acid concentrations are reported as both a percentage of all amino acids (mg/100 mg protein), as well as a percentage of the total mass on a dry basis (mg/100 mg flour).

Germinative energy, germinative capacity, and water sensitivity
Prior to malting, germinative capacity, germinative energy, and water sensitivity for both Kernza and barley grains were assessed following the American Society of Brewing Chemists (ASBC) Method Barley-3: Germination. [25] Here, kernels from each species were transferred separately to two 90 mm polystyrene Petri dishes (Falcon Plastics, Los Angeles, CA, U.S.A.), each containing 100 kernels and either 4 or 8 mL of deionized water and two No. 1 filter papers (Whatman, Germany), in duplicate. The dishes were closed and held at room temperature for 72 h, with the chitted kernels removed every 24 h. After the 72 h, the remaining ungerminated kernels were counted, and 2 mL of a solution of 0.75% (w/v) hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, U.S.A.) was added to the Petri dish, which was held at room temperature for an additional 48 h before counting ungerminated kernels again. All values are reported per the ASBC method.

Micromalting
Kernza kernels were sifted and sorted to remove smaller and broken kernels, foreign husks, and foreign plant material using USA Standard Testing Sieves (Fisher Scientific, Waltham, MA, U.S.A.). The portion retained between No. 10 (1.4 mm) and No. 14 (1.00 mm) sieves, represented approximately 75% of the supplied material with the remaining 25% smaller, was used for malting. The barley kernels had been sorted previously and did not require additional cleaning. Both grains were portioned into four individual 400 g sets and malted at the on an automated dual tank 2SG Steep Germinator Curio Malting unit (Milton Keynes, U.K.) and kilned in a two-unit 2 K Curio Malting MMK kiln. The Kernza was divided into four treatments (Table  1): long, mid, and short steep times with air rests, all were finished at a low kiln temperature of 55 °C for 22 h, and a mid-steep time with air rests with a high temperature step-wise kilning ranging from 55 °C to 85 °C for 22 h. All Kernza treatments were germinated at 15 °C for 96 h. Barley was divided into two treatments: a low kiln temperature of 22 h at 55 °C, and a step-wise (high) kiln temperature ranging from 55 °C to 85 °C, over a total of 22 h. Steep times and temperatures for barley were identical for all treatments, consisting of 8 h submerged ("wet"), 14 h exposed to air ("dry"), 6 h wet, and 12 h dry, all at 14 °C. Both barley treatments were germinated at 15 °C for 96 h.
Raw grain and finished malt samples were stored in Ziploc (Racine, WI, U.S.A.) plastic bags and held in a −20 °C freezer until used. For all analyses requiring milled flour, kernels from each grain were milled to a fine flour with a benchtop MIAG burr mill (Braunschweig, Germany) directly before analysis and following the standards outlined in the associated method.

Starch gelatinization
Starch gelatinization temperatures in the raw grains and following highest-extract malt samples were measured via differential scanning calorimetry (DSC) using a TA Instruments DSC250 (New Castle, DE, U.S.A.), in duplicate, as described previously. [26] To the supplied aluminum pan, 1.5 mg of finely-ground flour and 2.5 μL of deionized water were added (i.e., 3:5 m:v), after which the pan was hermetically sealed with an aluminum lid and allowed to equilibrate to room temperature. Exothermal scans were performed while equilibrating the sample at 20 °C for 3 min, and while heating the sample from 40 to 100 °C at a rate of 5 °C per min, with an empty pan used as a reference. Onset (T o ), and peak (T p ) starch gelatinization temperatures were calculated using the supplied TA software based on the endothermic transition peaks.

Moisture content
Moisture contents for raw grains and selected malt samples were measured in duplicate following ASBC Method Malt-3: Moisture. [27] Here, 5.0 g of finely-ground flour was desiccated in a Shel Lab SMO1 Forced-Air Oven (Cornelius, Oregon, U.S.A.) at 102 °C for 3 h, with the resultant change in mass attributed to evaporative water loss. Moisture is reported as a percentage of the total weight of the grain or malt including water, or "wet basis."

Friability
Friability of the two barley malt treatments was calculated following ASBC Method Malt-12: Friability [28] using a Pfeuffer Friabilimeter (Kitzingen, Germany), in duplicate. A large portion of the Kernza grains were too small, and unground kernels were able to pass through the drum sieve used here, which would have resulted in a potential over-estimation of modification. Thus, this method was not used to assess the degree of modification for the Kernza malts produced, and it was only used for verification of the degree of modification of the barley references.

Alpha-amylase activity and diastatic power
Starch-hydrolyzing enzymatic activity for all selected raw grain and malt treatments were assessed following a protocol modified from ASBC Malt-7: Alpha-Amylase [29] and Malt-6: Diastatic Power. [30] Enzymes were first extracted from 12.5 g of finely-ground flour using 250 mL of a 0.5% sodium chloride solution and held at 20 °C for 2.5 h with intermittent mixing. Following the infusion, the mixture was filtered using a fluted paper filter, returning the first 50 mL and collecting for a maximum of 30 min, then diluted to a ratio of 1: Alpha-amylase activity in the final dilution was measured using a Thermo Fisher Gallery Plus Beermaster Discrete Analyzer (Waltham, MA, U.S.A.) using supplied reagents. Diastatic power was assessed from an aliquot of the same extraction following Kiviluoma et al., [31] again using the Thermo Fisher Gallery Plus Beermaster Discrete Analyzer and a 1% (w/w) special soluble starch solution (ASBC, St. Paul, MN, U.S.A.) substrate.

Total nitrogen and total protein
Total nitrogen was measured on the finely-ground raw grain and malts using the Dumas combustion method following ASBC Method Malt-8: Protein [32] using a LECO FP-528 Elemental Analyzer (St. Joseph, MI, U.S.A.). A nitrogen-to-protein conversion factor of 6.25 was used for the barley samples [33] and the wheat conversion factor of 5.70 [34] was used to for Kernza, as it is more closely related. Both total nitrogen and total protein as a percentage by weight in dry matter are reported.

Extract
Following malting, all malt treatments and raw grains from both the Kernza and the barley were used to produce worts (in triplicate) using a method adapted from ASBC Malt-4: Extract [35] as follows. Kernels were ground to a fine flour on a MIAG Benchtop Burr Mill (Dresden, Germany), portioned by 25 g into mash tins, and mashed in an automated, temperature-controlled mash bath (IEC, Melbourne, Australia) with 100 mL of deionized water at 65 °C for 60 min. Following the mash, deionized water at 65 °C was added to bring the final mass to 250 g (i.e., 1:8 Grist: Liquor). The slurry was passed through a Whatman grade 2555 ½ fluted filter (Germany), with the first 50 mL poured back over the grain bed and allowed to continue filtering until either 200 mL was collected or 2 h had elapsed, whichever came first. Density of the extracted wort in Brix was measured using a Reichert Brix/RI-Check Digital Refractometer (Depew, NY, U.S.A.), and pH was measured using a Mettler Toledo SevenCompact pH/Conductivity Meter with an InLab Ultra Micro pH Electrode (Columbus, OH, U.S.A.). Brix values were converted to degrees Plato (°P) and extract values were calculated and reported as fine grind, dry basis (FGDB) according to the equations described in ASBC Malt-4: Extract. [35] From the Kernza treatments, the triplicate worts produced with (1) the raw grain, (2) the malt from the low temperature kiln and modified steep treatment with the previously measured highest extract value, and (3) the malt from the high temperature kiln treatment, and these were all retained for additional analyses. The triplicate worts from (1) the raw barley and both (2) the low and (3) high temperature kiln treatments were retained for additional analysis.

Soluble nitrogen and soluble protein
Soluble nitrogen was measured on the aforementioned worts produced via the Dumas Combustion Method following EBC-Analytica 4.9.3 -Soluble Nitrogen of Malt: Dumas Combustion Method [36] on a LECO FP-528 Elemental Analyzer (St. Joseph, MI, U.S.A.). The soluble nitrogen measured in the worts was then used to calculate the amount of soluble nitrogen in the raw grain or malt used to produce those worts. Nitrogen-to-protein conversion factors of 6.25 for barley and 5.70 for Kernza were used again as previously noted to calculate the soluble protein content in the malt or grain. Soluble nitrogen and soluble protein are both reported as a percentage by weight in dry matter.

Free alpha-amino nitrogen, wort β-glucan, color, pH, and clarity
On the selected retained worts, free alpha-amino nitrogen (FAN), wort β-glucan, and color were measured on a Thermo Fisher Gallery Plus Beermaster Discrete Analyzer (Waltham, MA, U.S.A.) using supplied reagents and standard programs with sample dilutions as needed. Two technical replicates were analyzed for all samples. Here, FAN was quantified by measuring the absorbance at 340 nm resulting from the reaction of alpha-amino nitrogen on primary amines with o-phthaldialdehyde (OPA). This method excludes proline residues from quantification, as the alpha-amino nitrogen in this amino acid is bound in a ring structure. [37] The β-glucan content was calculated from the formation of a photometric calcofluor-β-glucan complex chromophore. The absorbance of the resultant solution was measured at 405 nm to quantify the concentration of β-glucan in solution. Both FAN and β-glucan are reported as concentrations in mg/L of the wort. Color is reported in SRM based on absorbance at 430 nm.
Wort pH was measured with a Mettler Toledo SevenCompact pH/Conductivity Meter with an InLab Ultra Micro pH Electrode (Columbus, OH, U.S.A.). Clarity of the worts was assessed visually and reported qualitatively.

Statistical analysis
All data was collected and analyzed using Microsoft® Excel 2019, Version 16. 16.27 (201012). Mean values and standard deviations were calculated for all biological and technical replicates as previously noted. Two-tailed t-tests were performed to identify statistical similarities (p > 0.05) among all treatments and species for each of the results as noted.

Germination
Prior to the start of any malting run, the viability and germination potential of the raw grains must be assessed to ensure the likelihood of proper malt modification. The barley samples used in this study generated both germinative energy and capacity (i.e., viability) values greater than 99%, indicating that this lot was likely to generate consistent malt data ( Table 2). The Kernza samples, however, only exhibited a germinative energy of 77 and 80% for the 4 and 8 mL treatments, respectively, indicating that a significant portion was unlikely to germinate during the subsequent malting runs. Furthermore, a germinative capacity of 78% indicates that those seeds are likely not dormant, but dead or irreversibly damaged, and would never germinate. Similar low germination values have been anecdotally observed with mechanically dehulled Kernza and the result of damage to the embryo during the dehulling process was hypothesized. [21] Continued improved breeding at the Land Institute is focused on decreasing seed shatter and improving free-threshing, [9] which may improve germination rates post-dehulling. Conversely, maltsters may consider using hulled Kernza to reduce the risk of damaging kernels, although an excessive proportion of detached hulls may be found in the grain beds while steeping and malting as the hulls are insecurely adhered. Comparative research into hulled and dehulled Kernza malt should be performed. The water sensitivity assay for the Kernza resulted in a negative value, −3%, implying that a longer steep time might be advantageous for higher germination rates as more kernels germinated in the greater volume of water, and informed the steeping times used in this study.

Amino acid composition
Kernza and barley showed strong similarities regarding most of the amino acid relationships measured here (Table 3). Proline (Pro) comprised greater than 12% of total protein by weight for both grains, and Glx, a combination of glutamic acid and glutamine, was above 20% for both as well. However, Kernza Glx values are markedly higher, at 27%, compared with 20% in barley. The third-most represented amino acid for both grains was glycine (Gly), at 6.9% for Kernza and 7.4% for barley. Although the relative amounts of glutamic composition is expressed as a molar percentage of each amino acid to total protein (g/100 g protein) and as a dry weight percentage of the grain (% w/w). glx denotes a combined value of glutamic acid and glutamine. asx denotes a combined value for aspartic acid and asparagine. reported values are the averages of biological replicates as per aSBc method guidelines. all barley grains germinated under every treatment, indicating this lot could produce a reliable barley malt reference. germinative energy and capacity for the Kernza were considerably lower and much less consistent, indicating a portion of the kernels were unable to sprout and might not produce a homogenously-modified malt. the negative water sensitivity value indicates that the Kernza germinated better in the treatment with excessive water and increased steep times might be advantageous.
acid and glutamine are not clear based on this assay; both are the most prevalent amino acids in prolamin proteins, along with glycine and proline. [38] As prolamins are the predominant proteins in many other cereal grains, [11] including those closely related Kernza, [10] this may indicate that Kernza is similarly rich in storage proteins similar to the barley hordeins and wheat glutenins. However, specific proteomic research is necessary to identify the prolamin content of Kernza.
The barley values reported here are consistent with previously-reported amino acid composition data. [39] Barley is known to be limiting in lysine and threonine, [11] and as such, Kernza can be considered limiting in these amino acids as well, as these two amino acids are lower in the Kernza sample compared with the barley sample. Additionally, when the amino acids are viewed relative to the total dry weight of the grain (Table 3), Kernza is more abundant in all amino acids due to its higher overall protein content, with the difference in Glx and proline content pronounced here as well.

Starch gelatinization
The starch gelatinization onset and peak temperatures for malted Kernza measured here were similar to the barley reference (Table 4), at 58.3 and 64 °C, respectively, and within the range previously reported on other breeding lines of T. intermedium. [18] The starches found in the endosperms of barley and wheat typically exhibit gelatinization temperatures ranging from 51 to 60 °C and 58 to 64 °C, respectively. [11] Endogenous amylases are also active at these temperatures [40] and thus historical mashing techniques have targeted this range for brewing using malts and raw grains from these cereals. Therefore, the gelatinization temperatures for the starches in Kernza are ideal for mash additions and do not necessitate a cereal cooking step, unlike corn, sorghum, and rice, which contain starches with higher gelatinization temperatures than those typically used in the mash. [11] Malted Kernza displayed onset and peak temperatures of roughly 0.5 °C greater than raw Kernza, implying similar efficacy in the mash. The increase in gelatinization temperatures from raw to malt was exhibited by barley as well, in which peak gelatinization increased by 1.2 °C after malting. In previous research on barley, [41] rice, [42] and quinoa, [43] increased starch gelatinization temperatures have been observed in germinated and malted grains relative to the raw form. This change was postulated to be a result of a combination of increased soluble sugar content, thermal starch degradation during kilning, and enzymatic degradation during germination. Further research into the changes in starch structure during malting should be performed to shed light on this phenomenon.

Friability
Following germination and/or kilning, the maltster typically performs a friability test to determine the extent of modification. As previously mentioned, the individual Kernza malt kernels, however, were too small for the equipment used for this method (Figure 1), as some of the smaller whole kernels and only partially milled unmodified portions were able to pass through the drum sieve and biased the calculated degree of modification. Modification for the Kernza, therefore, was estimated using the Kolbach index (KI) of the malts. The barley malt references used here were deemed well-modified, with friability values of 85.9 and 82.7% for the lower and higher temperature kilned samples, respectively (Table 5), as well-modified barley malts typically exhibit a friability of >80%. [44]

Alpha-amylase activity and diastatic power
Alpha-amylase activity showed a marked increase in the Kernza by malting, from 2.1 dextrinizing units (DU) in the raw grain to 18.2 DU in the lower temperature kilned malt and 13.4 DU in the higher temperature kilned malt (Table  5). Both the increase in α-amylase activity during malting and the activity measured in the malt were substantially lower than exhibited by the barley reference, which measured at 1.3 DU in the raw kernels and 84.3 DU in the lower-temperature kilned malt, and 68.0 DU in the higher-kilned barley malt. The decreased activity exhibited for both species in the higher temperature kilned malts compared with the lower temperature kilned malt is likely attributable to the inactivation of α-amylase at the higher kiln temperatures. [45] The DP of the barley malt reference also increased significantly as expected from raw grain, at 34°L, to 154°L in the lower-temperature kilned malt and 108°L in the higher-temperature kilned barley malt. Interestingly, the raw, unmalted Kernza exhibited high DP values of 102°L, which only slightly increased after malting to 112°L in the lower temperature kilned malt and 90°L in higher temperature kilned malt. The high starting DP value pre-malting, coupled with an apparent lack of meaningful change in DP in both malting treatments indicates that the raw Kernza grains in this sample already had a high level of overall starch-degrading enzymatic activity, which was retained and relatively unaltered by malting. In barley, β-amylase is already present in the endosperm of mature seeds in an inactivated form bound to insoluble proteins and released during malting. [22] It may be that Kernza contains most of its β-amylase in an unbound and active form prior to germination, unlike barley. However, further investigation into specific β-amylase activity and composition, as well as the activity of other starch-degrading enzymes, may elucidate this further. For this reason, brewers should exercise caution when considering an addition of raw Kernza to the grist, as additional untended conversion may occur in the mash.

Total nitrogen and total protein
The raw Kernza contained a protein content of 16.42% (w/w) (2.88% nitrogen, N), slightly lower than the 17.11% (3.00% N) of the lower temperature kilned malt and 16.86% (2.96% N) of the higher temperature kilned malt (Table 5). This is similar to the range of 6-27% protein found in wheat, though most commercial varieties measure between 8 and 16%. [11] The total protein content of the barley was substantially lower, but within typical ranges for malting barley, at 11.07% (1.17% nitrogen) in the raw grain, 11.65% (1.86% N) in the lower temperature kilned malt, and 11.66% (1.87% N) in the higher temperature kilned malt. Malting barley varieties of good quality typically exhibit protein contents ranging from 10.0 to 13.5%, [46] and grain outside of this range is often rejected and diverted to animal feed. For barley malt, higher protein concentrations are generally associated with slower water uptake during steeping [47] and lower relative amounts of starch, but higher enzymatic activity, specifically β-amylase, [48] which may prove to be true for Kernza as well. Higher levels of malt protein are also associated with increased formation of protein-polyphenol complexes during brewing that result in downstream quality issues including haze and filtration challenges, [49] and thus appropriate mitigation techniques should be considered when using Kernza if a clear final product is desired.  reported values are the averages of biological and technical replicates as defined previously in the methods section, with their associated standard deviations, as appropriate. Superscripts of the same letter indicate statistical similarity (p > 0.05) to other values in the same row via two-tailed t-tests. "DB" indicates "dry basis" and "wB" indicates "wet basis." "fgDB" indicates "fine grind dry basis." Dashes indicate values were not measured. additional data visualizations can be found in the online Supplementary material.

Soluble nitrogen, soluble protein, and Kolbach index (KI)
The soluble protein content of the Kernza was calculated to be 3.42% (w/w) (0.60% N) of the dry weight in the unmalted samples, which increased to 7.87% (1.38% N) and 6.93% (1.22% N) in the low and high temperature kilned malts, respectively ( Table 5). The soluble protein in the barley reference was calculated to be 2.49% (0.40% N) of the dry weight in the unmalted grain, and 5.36% (0.86% N) and 5.28% (0.84% N) in the low and high temperature kilned malts. On their own, some solubilized proteins contribute positively to foam quality and to the mouthfeel of the finished beer, [50,51] but others may reduce overall quality in the form of haze. [52] Additionally, low levels of soluble protein are correlated with beers that are thin and insipid. [53] The Kernza worts here contained ample solubilized proteins, and Kernza worts generally should be expected to produce abundant foam, body, and haze; yet beers must be brewed with these malts first to confirm. The soluble nitrogen content of the finished malt is also used to assess the extent of modification the grain has undergone during malting, and thus the effectiveness of the malting process, by calculating the ratio of soluble nitrogen to total nitrogen (S/T, or Kolbach index, KI). The Kernza malt treatments exhibited KIs of 46.0 and 41.1% for the low and high temperature kilned treatments (Table 5). Malts are generally considered to have undergone a satisfactory level of modification if 40-47% of the proteins have become solubilized, [46] thus indicating that the Kernza malt samples here are sufficiently modified using this methodology. Similarly, the barley malt references exhibited KIs of 46.0 and 45.3% for low and high temperature kilned treatments, respectively, and thus are appropriate references to represent typical well-modified malts.

Extract
The extract value (FGDB) for the barley references used here increased from 32.5% in the raw grain to 84.4 and 82.4% in the lower and higher temperature kilned malts, respectively (Table 5), an increase of over 150% for both barley malts. The Kernza samples, however, did not exhibit a similar increase. Extract measured 63.6% in the raw Kernza grain, compared with 72.4% in the lower temperature kilned malt and 67.1% in the higher temperature kilned malt, an increase of less than 15% for both Kernza malts. The absence of a substantial increase in extract value by malting Kernza may be due to the aforementioned high diastatic power present in raw Kernza, which was not markedly increased by malting. Extract values are representative of the combined effect of both the amount of available starch and the activity of the endogenous starch-degrading enzymes under standardized mash conditions (in addition to proteins, non-starch carbohydrates, and other dissolved substances), and are represented by calculating the proportion of these in the dry weight of the malt. Although starch is not synthesized during malting, it is made accessible to the starch-degrading enzymes during the malting process via enzymatic degradation of the proteins and non-starch polysaccharides that encase the starch molecules in the endosperm of unmalted kernels.
The extract values for both Kernza malts found here were below the standard acceptable range of 78-84% for a well-modified barley malt [46,47] and were likely due to the combined effects from number of sources. Although not measured here, Kernza of other varieties have been shown to contain somewhat smaller amounts of carbohydrates than wheat or barley. [54] Furthermore, the structural polymers in the endosperm that the starch is embedded within may not have been degraded fully to release the starches in the malt. Additionally, as both DP and α-amylase activity (previously mentioned) were lower than in the barley reference, the available starch may not have been fully degraded by the end of the mash. Additional inquiry into the composition of the carbohydrates in the raw and malted Kernza, as well as the amounts of other structural non-starch polysaccharides in the endosperm beyond β-glucan, such as arabinoxylan, may elucidate this deficiency. Furthermore, the aforementioned propensity for germ damage during the dehulling of Kernza may have decreased the overall finished malt quality.

Wort β-glucan
The β-glucan content is frequently measured and reported as the amount extracted into wort under standardized mashing conditions, as this is a more reliable predictor of how the malt will function in a brewery. [55] Here, the reference barley produced a wort with a HMW β-glucan content of 660 mg/L with unmalted grain that decreased to 125 and 98 mg/L in the low and high kiln temperature malts, respectively ( Table 5). The β-glucan content of the Kernza worts tracked similarly, with unmalted grain resulting in 354 mg/L HMW wort β-glucan, decreasing to 33 and 49 mg/L with the low and high temperature kilned malts, respectively. The American Malting Barley Association (AMBA) recommends that malts produce worts with a maximum β-glucan content of 100 mg/L; [46] although worts frequently contain β-glucan in excess of 200 mg/L without issue. [56] Thus, as before, these results indicate that wort produced with Kernza should not require a β-glucan rest, yet it does show that β-glucan was reduced via malting.

Wort FAN
The worts produced with Kernza exhibited a substantial increase in FAN by malting, from 46.8 mg/L with raw grain to 173 and 139 mg/L with the low and high temperature kilned malts, respectively (Table 5), or a relative increase of over 175% for both. The reference barley increased wort FAN by 200% by malting, from 62.7 mg/L with the raw grain to 206.9 and 189.3 mg/L using the low and high temperature kilned malts, respectively. FAN is an another measure of the degree of protein hydrolysis (i.e., modification) that has occurred during malting, and to a lesser degree during mashing. [57] In barley malt, 88% of the total FAN present in wort is estimated to have been produced during malting, while only 12% is produced during mashing. [57] FAN values have been recognized for their importance for some time, as nitrogen in this form is essential for yeast health during fermentation, especially during the growth phase. [58] Worts with a FAN level below 130 mg/L prior to pitching yeast [59] may result in decreased fermentation efficiency and can negatively impact final beer quality and stability. [60] The amount of FAN in the Kernza worts appears to be sufficient for yeast health and fermentation in an all-Kernza malt grist.
FAN is not, in actuality, only a measure of free amino acids, as it also includes all peptides and soluble proteins with exposed alpha-amino nitrogen, [61] and it does not include free proline or those with proline residues due to the inability of the o-phthaldialdehyde reagent to react with the alpha-amino nitrogen bound in a ring structure. [37] Although proline is by far the most abundant free amino acid found in barley worts, [62] it is not assimilated by brewing yeasts under normal fermentation conditions due to its structure, and thus its absence from the FAN measurement is appropriate for brewing applications. [63] Interestingly, though, increased FAN is generally associated with higher levels of malt protein, [64] which was not seen here when the higher protein content of the Kernza was taken into account. This suggests that the Kernza malts may require additional modification to reach higher FAN levels, or that the amino acid profiles of these free amino acids may be more heavily weighted with proline than barley malt worts, as this amino acid is not accounted for in the FAN measurement. Further investigation into the amino acid profiles of these worts may elucidate this disparity further.

Color and haze
Additional notes on wort quality include a slight increased color value of both Kernza malts and the observation of haze on both of the malted Kernza worts ( Table 5). The observed wort haze indicates that turbidity-mitigation strategies should be considered when brewing with Kernza malts if clear beers are desired; although measurement of more quantitative values may be required. The worts produced with both Kernza malts were below 4 SRM, acceptable for very light-colored beers. It should be noted that the scope of this study did not allow for fermentation, and thus further research is required as to the color and haze qualities of beers brewed with Kernza malt.

Conclusions
As the malt industry has been almost completely dominated by barley for centuries, investigations into other species may provide opportunities to address some of the environmental issues associated with barley agriculture and the malt and beer industries as a whole. The recent and ongoing domestication of the more environmentally sustainable Kernza grain may disrupt this myopic industry view. In this study, Kernza grain was micromalted and deemed sufficiently modified based on the soluble to total nitrogen ratio, or Kolbach Index. As the industry standard method for modification assessment, friability, was not possible due to the small grain size, further investigation into other methods for malt modification for this grain should be investigated, with a focus on the release of starch from the endosperm matrix.
The resultant Kernza malt produced wort with a high FAN level and low wort β-glucan content, meeting or exceeding barley malt quality standards. However, although the extract obtained in worts produced using Kernza malt were high, they were lower here than the barley malt reference, and slightly below typical malt standards. Starch-hydrolyzing enzymatic activity, paramount to the conversion of starch to sugars in the mash for later fermentation by yeasts into alcohol, was increased in the Kernza by malting, and the diastatic power in the final malt was of levels sufficient to completely convert an all-malt mash. Alpha-amylase activity increased seven-fold in the Kernza malt, yet it was still considerably lacking compared with the of the barley malt reference. Notably, the high diastatic power in the raw Kernza grain was not substantially increased by malting, as it did with the barley reference, indicating that β-amylase and other starch-hydrolyzing enzyme activity is also high in unmalted, raw Kernza. This indicates that Kernza may also have a potential use as an unmalted diastatic adjunct, which would further reduce the overall water usage at the malthouse. Also, although germination was slower and less pronounced than the barley reference here, the common practice of additions of gibberellic acid may aid to hasten modification.
These results taken together indicate that Kernza is poised for recognition by the malt and beer industries. Although the Kernza malt did not perform as well as the industry standard barley in all regards, many of the measured values were only slightly inferior, and when the environmental impact of these grains is considered, the relative value of this malt is greatly enhanced. Additionally, these results may aid the ongoing breeding programs and future malting optimization of this relatively new grain to better support these applications.