1. Introduction
Perennial grain crops which can be established once and harvested for numerous years without replanting have been proposed to address a wide array of challenges to agricultural production, including soil degradation, water contamination, and habitat loss [
1]. Through reduced soil disturbance and large root systems, perennial grain crops are expected to accumulate soil carbon, which could make a significant contribution to carbon sequestration and greenhouse gas mitigation [
2,
3]. Additionally, perennial grain crops have been shown to improve soil quality [
4,
5,
6,
7], and water quality via reduced nitrate leaching [
4,
8,
9,
10]. Producers benefit from reduced tillage requirements, equipment use, and input costs [
1,
11]. Perenniality and diversity can contribute to greater stability in production systems [
12]. The feasibility of perennial grain crops was recently demonstrated with the development of a perennial rice variety that has yields equivalent to annual rice while improving farmers’ incomes and increasing soil carbon content [
13]. Because perennial grasses with high yields of edible grains are not found in nature, an effort has been underway to domesticate wild perennials such as intermediate wheatgrass since the 1980s [
14].
The identification of
Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (common name intermediate wheatgrass, IWG) as a promising perennial grain candidate emerged from an evaluation of nearly 100 perennial grass species led by the Rodale Institute (Kutztown, PA, USA) [
15,
16]. For context, two cycles (generations) of selection were conducted by The Rodale Institute before The Land Institute (TLI; Salina, KS, USA) initiated an IWG breeding program, followed by three cycles of selection and the start of a University of Minnesota (UMN; St. Paul, MN, USA) breeding program. Additional breeding cycles have been performed in Kansas and Minnesota, with new breeding programs initiated in Utah, Canada, and Sweden [
14]. The work presented herein will focus on diverse source materials that were developed by The Land Institute prior to distribution to other programs and subsequent genetic differentiation. Therefore, the core objective is to provide baseline values for the new crop, based on evaluations of genetically diverse materials as a reference point prior to subsequent changes that are expected due to breeding in diverse environments.
IWG is the first widely available commercial perennial grain crop, sold under the trade name Kernza (
Figure 1) [
17,
18]. Consequently, IWG grain has been most extensively researched with regards to nutritional quality, food functionality, and performance in food products. This information informs end use, guiding the placement of Kernza in the marketplace.
Becker et al. (1991) provided the earliest insights into the compositional, nutritional, and functional properties of IWG [
19]. They also evaluated its performance in food products, sensory attributes, and consumer acceptance. IWG generally performed well in the different products, with muffins receiving higher scores relative to the other products. The study included stone milled wheat and commercial whole wheat flour for comparisons. Numerous studies in food science have since evaluated IWG. Fewer studies have investigated consumer acceptance and preferences, with preliminary insights indicating that extensive trialing of varying inclusion rates is necessary to produce a product that meets functional and sensory expectations. Consumer demand remains low compared to annual grains, and consumer education is a major barrier to acceptance. Bharathi et al. (2022) provide a comprehensive summary of progress in IWG breeding from a food science perspective [
20]. A particular focus has been on the protein composition of IWG in the context of breadmaking (
Figure 2). Overall, the body of literature provides broad, fundamental knowledge following progressive cycles of domestication and breeding to develop improved varieties of IWG for human consumption.
With breeding ongoing to further domesticate IWG, the chemical composition is expected to change. This is primarily due to breeding and selection for greater yields and seed size, which is likely to alter the storage of macronutrients in seed structures such as the starchy endosperm, germ, and bran. For example, protein is unevenly distributed in wheat kernels. The highest amount of protein is concentrated in the endosperm, while protein content decreases toward the outer layers of the kernel [
21]. Furthermore, phytochemicals that confer human health benefits, such as dietary fiber, minerals, and vitamins, are concentrated in the bran [
22]. Continuous evaluation of IWG varieties developed for human food use is critical to understanding nutritional quality, functionality, and potential impacts on human health. We present the results from two studies investigating the chemical composition of IWG from early cycles of selection, to strengthen the limited body of evidence currently available for macronutrients, dietary fiber, carotenoids, antioxidants, and antioxidant activity. We also provide novel insights into vitamin and mineral contents and amino acid profiles, which are currently lacking in the literature. Additionally, these studies evaluated samples produced in Kansas, a notably hotter and drier production environment compared to the upper Midwest, which has been the focus of most previous studies. Our aim is to report these findings so they may be useful to future studies investigating IWG nutritional quality, and to compare the chemical composition of IWG to annual wheat to determine where significant differences exist. An array of analytes is compared between IWG and whole wheat flour as reported in the United States Department of Agriculture (USDA) FoodData Central database, and the amino acid content of IWG is compared to annual wheat as reported in the literature.
2. Materials and Methods
2.1. Germplasm
Six IWG samples were tested in two separate studies. In the first study, the IWG samples were identified as Rodale1 and TLIC1. This study also included an annual wheat check (cv. Jagger) [
23]. The IWG samples in the second study were identified as TLIC3, TLIC4, TLIC5, and EllsworthC5.
The IWG samples represent different cycles of selection during early-stage perennial grain crop domestication and breeding. Rodale1 has its genetic origin in the population created by a joint breeding program between the United States Department of Agriculture (USDA) Big Flats Plants Materials Center (Corning, NY, USA) and the Rodale Institute (Kutztown, PA, USA). Rodale1 represents seeds from their first set of selected plants. The Land Institute obtained and planted this seed in 2001. After one cycle of selection primarily for yield and seed size, an IWG breeding nursery was planted at The Land Institute in 2005. TLIC1 was harvested from this breeding nursery. TLIC3, TLIC4, and TLIC5 were derived from the third, fourth, and fifth cycles of breeding at TLI, respectively. These three populations represent a broad base of genetic diversity out of which other breeding programs have begun to make selections. Programs to develop IWG varieties for diverse environments were mostly initiated using these materials [
14]. For instance, MN-Clearwater, the first and currently most widely grown IWG variety for grain, is a synthetic variety whose seven parents are all TLIC3 individuals [
24]. TLIC5 was the first widely distributed seed source used to produce Kernza perennial grain, with some of its harvested grain still being used in products. EllsworthC5 represents TLIC5 material grown by a producer on-farm. Detailed breeding methods applied in each cycle of selection are described by DeHaan et al. [
18].
2.2. Grain Production
For the first study, all seed was harvested in 2007. Wheat was harvested in June and IWG was harvested in August. TLIC1 was produced at 38.771° N/97.592° W on a Hord silt loam soil. Rodale1 and Jagger wheat were produced in the same field, separate from TLIC1, at approximately 38.766° N/97.572° W on a McCook silt loam soil. Both the IWG plots and the wheat were fertilized with urea at a rate of 100 kg ha−1 N. Additionally, the IWG stands were of different ages. Rodale1 was planted in fall 2001 and TLIC1 was planted in fall 2005. The plot sizes were larger than 0.2 Ha. Rodale1 was drilled in rows with 19 cm spacing and maintained as a solid stand. TLIC1 was planted in rows at 91.4 cm apart with weeds controlled through regular interrow tillage. Seed weights were determined based on the mass of 100 dehulled seeds, calculated as mg per seed.
For the second study, TLIC3, TLIC4, and TLIC5 were harvested from fields located at 38.774° N/97.592° W, 38.774° N/97.591° W, and 38.7697° N/97.596° W, respectively. All fields were larger than 0.5 Ha in size. Planting was in spring 2009, fall 2011, and fall 2013 with seed harvests in 2011, 2013, and 2015 for TLIC3, TLIC4, and TLIC5, respectively. Fields were managed without any application of pesticides or herbicides. Plants were established at an interrow spacing of 91.4 cm with regular cultivation for weed control. These fields received an application of nitrogen in the form of urea at a rate of 78.5 kg N ha
−1 in either November or December. EllsworthC5 was produced on-farm in a field larger than 5 Ha using organic practices, and harvest occurred during the transitional period prior to complete certification. Establishment was via drilling in rows spaced 19 cm apart, and the field was subsequently managed as a solid stand. No chemical fertilizers were applied. The EllsworthC5 field was grazed with cattle in early spring of the production year and again following grain harvest in late summer. The EllsworthC5 field was harvested in 2018 in Ellsworth County, Kansas, but exact coordinates and soil composition information were not available. Seed weights were determined based on the mass of 20 hulless seeds, calculated as mg per seed. The sample name, growing location, year of production, and seed weight are provided for each sample in
Table 1.
2.3. Chemical Analyses
Across the two studies, analytical testing was performed by separate fee-for-service laboratories using official methods of analysis. Medallion Laboratories (Minneapolis, MN, USA) provided testing services to analyze TLIC3, TLIC4, TLIC5, and EllsworthC5. Anresco Laboratories (San Francisco, CA, USA) provided testing services to analyze Rodale1, TLIC1, and Jagger wheat. Briefly, official methods of analysis were used, such as those of the Association of Official Agricultural Chemists (AOAC). Metals (i.e., heavy metals and minerals) were quantified by either dynamic mechanical analysis, inductively coupled plasma mass spectrometry (ICP-MS), or inductively coupled plasma optical emission spectrometry (ICP-OES). Analytical Laboratories, Inc. (Anaheim, CA, USA) performed betaine analysis (ALC518A) for Rodale1, TLIC1, and Jagger wheat. Brunswick Laboratories (Norton, MA, USA) conducted an oxygen radical absorbance capacity (ORAC) assay and ferric reducing antioxidant power (FRAP) assay [
25] and determined the phenolics and ferulic acid (liquid chromatography mass spectroscopy) for Rodale1, TLIC1, and Jagger wheat.
Supplementary Table S1 includes method references for the analytes, as provided by the laboratories.
To compare the essential amino acid content of IWG to adult daily requirements on a mg of amino acid per gram of protein basis, each essential amino acid (milligrams) was divided by the sum of all amino acids (grams). Therefore, the total amino acid content was used to represent the total protein content, rather than using crude protein values. The amino acids included histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, aspartic acid, cystine, glutamic acid, glycine, proline, serine, and tyrosine. The contents of sulfur amino acids (SAAs) and aromatic amino acids (AAAs) were represented by the sums of methionine and cystine and of phenylalanine and tyrosine, respectively.
Protein and amino acids values for TLIC1 were excluded, as the protein content and total amino acid content disagreed and the exact reason for this discrepancy could not be discerned. Energy was calculated using the following equation:
2.4. Statistical Analysis
Google Sheets (Google, Mountain View, CA, USA) was used to compile data into tables and perform basic statistical analyses.
Common analytes between the IWG samples and samples of whole wheat flour from the USDA FoodData Central were compared using a two-sample t-test in the R statistical software (v4.1.2; R Core Team 2021), to test the null hypothesis that no difference exists between IWG and whole wheat flour sample means. Specifically, Welch’s two-sample t-test was performed assuming unequal variances. Common analytes included energy, carbohydrate, fat, protein, ash, total dietary fiber, calcium, iron, magnesium, phosphorous, potassium, sodium, copper, manganese, selenium, thiamine, riboflavin, niacin, and folate. Statistical significance (p < 0.05) was determined according to p-values adjusted by the false discovery rate to control for Type I errors.
The amino acid content of IWG samples was compared to the amino acid content of annual wheat as reported in the literature. Studies were selected if units were expressed as grams per 100 g of sample or could be adjusted to such units as needed. Additionally, studies were selected to represent diverse environments and wheat varieties. These studies included those conducted by Tanács et al. (1995), Jiang et al. (2014), Tarkowski and Wojcik (1974), Hospodarenko et al. (2018), Shoup et al. (1966), Siddiqi et al. (2020), and Tomičić et al. (2022) [
26,
27,
28,
29,
30,
31,
32]. The amino acid content, expressed on an as-is basis, was compared using a two-sample
t-test in the R statistical software, to test the null hypothesis that no difference existed between the IWG and wheat sample means. Specifically, Welch’s two sample
t-test was performed assuming unequal variances. Seventeen amino acids were compared in total. Tryptophan was excluded from the analysis due to a lack of sufficient data. Statistical significance (
p < 0.05) was determined according to
p-values adjusted by the false discovery rate to control for Type I errors.
5. Conclusions
Two studies were performed to characterize the nutritional quality of early-generation Kernza (Thinopyrum intermedium, IWG) breeding program material. The results were compared to previously published values for whole wheat flour and wheat samples of diverse origins. The IWG chemical composition significantly differed from whole wheat flour in its key properties. IWG had 50% higher protein, 129% higher dietary fiber, and 65% higher ash contents than the reference whole wheat flour. Calcium and selenium were 267% and 492% higher, respectively, in IWG than whole wheat flour. Riboflavin and folate were 43% and 447% higher, respectively, and niacin was 74% lower in IWG versus whole wheat flour. Like wheat and other cereals, the IWG samples had a limiting lysine content. However, due to the higher total protein, IWG had 33% more lysine than whole wheat flour. The antioxidant capacity of IWG appeared to be higher than that of wheat and was associated with greater carotenoid and antioxidant contents. These studies evaluated IWG samples produced in Kansas, a notably hotter and drier environment compared to the upper Midwest, which has been the focus of most previous studies. The evaluated material represents the basis from which other Kernza breeding programs have been initiated, providing a baseline for comparisons of nutritional quality while providing novel insights into vitamin and amino acid contents.