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Agricultural And Food Sciences
Biology

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Untargeted Metabolomics Profiling of Bioactive Compounds under Varying Digestate Storage Conditions: Assessment of Antioxidant and Antifungal Activity

The rapid development of biogas plants in China has generated large quantities of digestate. The disparity between the continuity of biogas plant operation and the seasonality of digestate utilization has led to the need to store digestate. Therefore, untargeted profiling of bioactive compounds in the digestate stored under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was likewise assessed. The results delineated that digestate storage under varying conditions brought about the degradation of organic acids, alkenes, aldehydes, alcohols, ketones, ethers, amino acids and their derivatives, and esters, leading to the stabilization of digestate components. Together, these new data revealed that digestate storage for up to 20 days under aerobic conditions promotes glycine, serine, and threonine degradation pathways and enhances biotin and vitamins production. In contrast, anaerobic storage enhances the taurine and hypotaurine metabolic pathways and increases the derivation of antimicrobial substances, such as indole alkaloids. Moreover, digestate storage under anaerobic conditions promotes antioxidant and antifungal activity more than storage under aerobic conditions. These findings can contribute to the future development of high-value agricultural products from digestate and the sustainability of biogas plants. Further studies are required for the untargeted metabolomic of digestate under storage to explore the underlying mechanisms of promoting disease resistance by the digestate upon land application.

Introduction
In recent decades, the demand for animal-derived food has risen dramatically due to continuing population growth and a shift in diet toward meat consumption [1]. Increased protein-based food consumption has resulted in rapid, intensive livestock farming industrialization [2]. This quick industrialization of animal husbandry has prompted the generation of huge amounts of animal manure, posing a serious threat to the soil and water environment [3,4]. The anaerobic digestion (AD) process and its mild operating conditions of complex organic carbon distinguish it from conventional energy technologies, making it a highly appealing and sustainable green energy technology for manure treatment, producing methane-rich biogas and nutrient-rich digestate [5,6]. Since it is high in readily available macro-and micronutrients, anaerobic digestate is a beneficial soil amendment [7].
Despite its well-known agronomic benefits, the use of digestate as an organic fertilizer or soil amendment in farmlands is dependent on plant and crop seasonality. Besides this, the oversupply and improper distribution of digestate, especially in the regions of intensive biogas plants and its application at an inappropriate time, enhance air and water pollution [8][9][10]. Consequently, before favorable agronomic applications in adjacent or remote farmlands, digestate had to be stored onsite for a certain period (up to 10 months) [11]. However, the storage and transport of digestate would bring about gaseous emissions and nutrient leaching, resulting in various environmental concerns, such as global warming and eutrophication [12,13].
Digestate is usually stored in open and closed reservoirs after solid-liquid separation. Digestate storage in open tanks leads to various problems, such as gaseous and odor emissions, the production of toxic compounds, and pathogen regrowth, thus limiting its potential fertilizing value [14]. Few studies have already been conducted for evaluating gaseous emission, antibiotics, and heavy metals removal from digestate during storage. For example, Vergote et al. [15] observed that nitrous oxide and methane emissions increased with digestate storage volume and temperature increase. In contrast, Mehta et al. [16] concluded that around 76% of doxycycline was removed with the storage of digestate under anaerobic conditions. Similarly, Li et al. (2018) reported an increase in organic matter, nutrients, and heavy metals during the storage of digestate openly under mesophilic conditions [17]. Studies on variations in the contents and type of bioactive compounds in the digestate during storage are scarce. Under anaerobic conditions, the degradation of organic compounds results in recalcitrant fractions, like biopolymers, steroids, and lignin [18]. Bioactive compounds, such as fatty acid derivatives, alkaloids, flavonoids, and terpenoids generated during the digestion of organic matter, are responsible for the disease resistance property of the digestate [19,20]. Thus, profiling bioactive compounds in the digestate during storage is necessary for evaluating antimicrobial and antioxidant activity and the underlying mechanism of promoting disease resistance by the digestate upon land application.
Therefore, to address this knowledge gap, the current study was undertaken for untargeted metabolomics profiling and the meta-analysis of biochemical changes that occur during digestate storage at different temperatures (4 • C, 20 • C, and 30 • C) under aerobic and anaerobic conditions based on the cloud-based mass spectrometric data analysis software (XCMS) integrated with the MetaboAnalyst program. Furthermore, the contents and type of bioactive compounds in the digestate preserved under aerobic and anaerobic conditions were compared. The antifungal and antioxidant activity of the digestate under varying storage conditions was also evaluated.

Experimental Material
Anaerobic digestate was collected from a continuous;y stirred tank reactor fed with swine manure at Donghuashan Biogas Plant (40.2 • N, 116.9 • E) in Beijing, China. Basic physicochemical characteristics of the digestate are given in Table 1.

Experimental Set-Up
To evaluate the variations in untargeted metabolomics profiling of bioactive compounds, about 2 L of digestate was placed in amber glass bottles and incubated for three months at 4 ± 1 • C, 20 ± 1 • C, and 30 ± 1 • C under aerobic and anaerobic conditions. An-4, Ae-4, An-20, Ae-20, An-30, and Ae-30 were the names given to the digestate held under aerobic and anaerobic conditions at the formerly mentioned temperatures. Each treatment was repeated three times, with digestate samples obtained weekly from each treatment.

Antioxidant Activity of Digestate
The antioxidant activity of digestate was determined by the DPPH (2,2 -diphenyl-1picrylhydrazyl) method and then converted into the corresponding Trolox equivalents. Firstly, 7.856 mg of DPPH was weighed and dissolved in 200 mL of methanol to make a DPPH solution. After that, 1 mL of the digestate from different storage modes was mixed thoroughly with 4.5 mL of DPPH solution, left in the dark at room temperature for 30 min, and then the absorbance was measured at 517 nm (A test ). Around 5.006 mg of Trolox was dissolved in distilled water to obtain a 200 µmol Trolox standard stock solution. A total of 1 mL of Trolox standard solution was then mixed with 4.5 mL DPPH solution and used as a control (A control ). A mixture of 1 mL of methanol and 4.5 mL of DPPH solution was used as a blank (A blank ) [21]. The antioxidant inhibition rate of the digestate stored under different conditions was calculated using the following equation: A series of working standards from 20 to 60 µmol L −1 was prepared by diluting the Trolox standard stock solution using distilled water. To convert the DPHA radical scavenging rate into Trolox equivalents, a standard curve was drawn, with clearance (x) as the independent variable and Trolox equivalent (y) as the dependent variable.
The regression equation was as follows: where x is DPPH radical scavenging rate (%), and y is the Trolox equivalent (µmol/L).

Antifungal Activity of Digestate
The antifungal activity of digestate from different storage modes was measured against the pathogenic fungus (Fusarium oxysporum), which was procured from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China. The fungi were inoculated into a potato dextrose-agar (PDA) (Beijing Auboxing Biotechnology Co., Ltd., Beijing, China) medium on culture plates for 4 to 5 days for rejuvenation and then used as an inoculum. The PDA medium was sterilized by autoclaving at 121 • C for 20 min. When the autoclave temperature was reduced to 40-50 • C, the extracts (dichloromethane: methanol, 3:1) from the digestate in different storage modes were added to the melted PDA medium at a concentration of 5%. After thorough mixing, the medium was poured into petri dishes. The rejuvenated fungal colonies were taken out of the incubator for inoculation. Mycelial pads (5 mm diameter) were taken from the leading edge of actively growing colonies using stainless-steel forceps and put upside-down in the middle of the petri dish. The inoculated plates were then incubated at 25 ± 2 • C for one week. In addition, a mixture of pure extractant (Dichloromethane: Methanol = 3:1) and PDA was used as a blank control. The antifungal test was carried out in triplicate. The diameter of the colonies was measured every 24 h for one week using Vernier calipers. The relative inhibition growth (RIG) rate was calculated as described by Tao et al. [22].
D 0 = colony diameter mixed with pure extractant (dichloromethane: methanol = 3:1) as the medium (cm), and D = colony diameter as a mixture of different storage modes of methane (cm) as a medium (cm).

Sample Preparation for GC-MS
The digestate (20 mL) from each storage mode was blended with an equal amount of extractant (dichloromethane: methanol = 3:1) in a covered test tube. After that, 10 g of sodium chloride was added, and the mixture was agitated on an oscillator for one hour. An ultrasonic instrument was used to assist with the extraction process (KH300SP, 25 kHz, 300 W, Kunshan Ultrasonic Instruments Co., Ltd., Kunshan, China). The samples were first centrifuged for 30 min at 4032× g. After that, the supernatant layer was collected, and the rest was discarded. The extract obtained from the previous test was evaporated to 1.5 mL using a rotary evaporator (Heidolph Laborota 4001, Schwabach, Germany) at 40 • C and under a vacuum at 96 kPa.

GC-MS Analysis
Digestate extract analysis was conducted using a GC-MS (Agilent 7890/5975, Santa Clara, CA, USA), equipped with a DB-5MS capillary column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness) and a quadrupole analyzer, operated in electron impact (70 eV) mode, with an m/z ranging from 35 to 600. The column was programmed so that the temperature increased at a rate of 5 • C min −1 , from 60 • C to 180 • C; then, the rate was increased to 10 • C min −1 until the column reached 280 • C. The temperature was then maintained at 280 • C for 10 min. About 1.5 µL of the extract was then injected into the GC-MS using a micro-syringe, and scanning was performed for 50 min. After separation and detection, the compounds were eluted from the column and identified by comparing their mass spectra using the NIST08 data library [23].

Data Processing and Statistical Analysis
The raw GC-MS data were uploaded to the XCMS online program for peak identification, peak filtering, and peak alignment to obtain a data matrix including the nucleus to mass ratio and retention time and peak area. The processed data were subjected to a multivariate statistical analysis using SIMCA-P (version 14.1; Umetrics, Umea, Sweden). An unsupervised multivariate pattern recognition method, namely a principal component analysis (PCA), was performed to examine the intrinsic variation in the data set. The supervised projection of orthogonal partial least squares discriminant analysis (OPLS-DA)) was performed for class discrimination. The quality of the OPLS-DA model was then assessed using 200 permutation tests. Variables were selected based on their effect on projection (VIP), reflecting the correlation between different storage conditions and the digestate composition.
To confirm the metabolic substances significant for digestate under varying storage conditions, all ions were sorted in descending order according to their VIP values. Potential metabolic markers were selected based on VIP values >1 and critical p-values from t-tests. The effect of aerobic or anaerobic storage on the most critical metabolites in the digestate was evaluated under varying storage temperatures, with a similarity index (SI) of >70% for the variables.

Pathway Analysis
To discover the differential metabolites that significantly changed in the experimental groups, a pathway analysis was performed using MetaboAnalyst 4.0, a web-based metabolomics data analysis software. An enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database among these differential metabolites. Pathways with impact values > 0 and -log (p) > 2 (p < 0.01) were considered to be the most significant derivative pathways for the generation of metabolites.

Digestate Storage under Varying Conditions Changes Its Metabolites Profile
Based on the frameworks installed in the materials and methods and the multi-group analysis, the pre-processing of raw GC-MS data from digestate under varying storage conditions was conducted using the XCMS-Online software. XCMS-generated cloud plots allow an effective representation of GC-MS-based metabolomics data by providing information including the p-value, the directional fold change, the retention time, and the mass-to-charge ratio of metabolic features within a defined threshold [24,25]. The calibration procedure used for visualization and quality control, including an overlay of all chromatograms acquired before and after Rt. calibration, is shown in Figures S1 and S2. Based on the above pre-processing, around 1014 statistically significant features (p ≤ 0.05 *) were exported from the raw GC-MS data (Supplementary Materials). Moreover, the Non-Metric Multidimensional Scaling (NMDS) biplot provides a more straightforward visualization of authenticated samples, representing dissimilarities as a function of distance ( Figure S3).
In addition, unsupervised principal component analysis (PCA) models were constructed on datasets after preliminary processing to get the intuitive metabolic distribution in the digestate under different storage modes ( Figure 1). The PCA scores of PC1 and PC2 from the variable's covariance matrix were 46% + 13%. The depicted clusters of samples in the same group proved the accuracy of the analytical methodology. A certain degree of separation was observed between the digestate samples from different storage modes. This proved that the differences between samples and sample sets were generated by actual biological differences, rather than instrumental effects. In addition, there was a clear separation among the digestate at different storage temperatures and durations. Moreover, with increased storage time, the location of samples in the PCA score plot moved slowly from left to right, indicating a gradual change in the composition of the digestate. Furthermore, the storage temperature strongly affected the distribution of plots. The effect of storage temperature on the composition of the digestate was explicitly shown by the slow movement of the samples from the bottom to the top of the PCA scoring plot. However, when the storage temperature was ≥20 • C, the samples aggregated as a cluster after a storage duration of >40 days, especially at 80 days. Therefore, within 40 days of storage, the metabolite spectrum of digestate was significantly affected by the storage temperature. The composition of the digestate tended to be consistent after 80 days of storage. In addition, the PCA plot showed a separation in the metabolite distribution patterns of the digestate stored under anaerobic and aerobic conditions at 20 • C, which illustrates that air conditions affected the chemical composition of digestate at 20 • C under the first 40 days of storage. The chemical compositions of digestate under storage at 4 • C for more than 40 days were independent of the stored oxygen content.
When comparing aerobic and anaerobic storage conditions, the metabolites tanospermine, Methional diethyl acetyl, and Nor-nitrogen mustard were observed in digestate stored at 4 • C under aerobic conditions, while functional metabolites including 3-Hydroxypyridine and Latanoprost were observed in digestate stored under anaerobic conditions at 4 • C. In contrast, D-threonic acid, Lys Lys Arg, Nor-nitrogen mustard, and Vinylidene fluoride and 2,3-diphenyl-2-propenoic acid, Castanospermine, Cystanospermine, Cysteamine, 2-Hydroxyethanesulfonate, 4-Chlorophenylacetic acid, and N-Acetyltranylcypromine were observed for digestate stored at 20 • C under aerobic and anaerobic conditions, respectively ( Figure 2b). Conversely, metabolites 3-Hydroxypyridine and Arachidonoylmorpholine were found in the digestate stored at 30 • C under aerobic conditions, and Castanospermine, 2,3-diphenyl-2-propenoic acid, 5-Methyl-2-thiophene carboxaldehyde, Benzydamine, Bis (chloromethyl) ether, and Glyoxylic acid were observed for digestate stored at the formerly mentioned temperature under anaerobic conditions (Figure 2c). Furthermore, it was observed that the rate of degradation increased with incremented digestate storage duration, and the maximum degradation of components, including some organic acids, alkenes, aldehydes, alcohols, ketones, ethers, amino acids and their derivatives, and esters, occurred after 80 days of storage, resulting in the stabilization of the digestate composition. When comparing significant metabolites detected after 80 days of digestate storage at different temperatures, similar metabolites were observed for digestate storage at 4 • C and 30 • C under aerobic and anaerobic conditions; however, the metabolites 2,3-diphenyl-2-propenoic acid, Castanospermine, Taurine, Cysteamine, Tryptamine Cysteamine, 2-Hydroxyethanesulfonate, 4-Chlorophenylacetic acid, aand Acetyltranylcypromine were detected in the digestate stored at 20 • C under anaerobic conditions, while D-threonic acid, Hydroxy-pyruvate, Glyoxylate, Biotin, Uera, Lys Lys Arg, Nor-nitrogen mustard, and Vinylidene fluoride were detected in the digestate stored under aerobic conditions at the former temperature. These findings were consistent with the PCA analysis results, which represented a more significant contribution of oxygen content to the composition of the digestate at 20 • C storage.

Changes in the Metabolite's Derivative Pathways under Varying Digestate Storage Conditions
To compare the metabolic changes in the digestate under aerobic and anaerobic storage conditions, an SMPDB enrichment analysis was performed by importing exclusive metabolites (p < 0.05) into MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/faces/ModuleView. xhtml (accessed on 10 March 2022)) [26]. As shown in Tables 2 and 3, 40 and 49 derivative pathways were observed for the digestate stored at 20 • C under aerobic and anaerobic conditions, respectively. Based on the analysis of p-values and impact values, the nine most significant metabolic pathways enriched in the digestate stored at 20 • C under anaerobic and aerobic conditions were selected (Figure 3). The specific metabolite derivative pathways in the digestate at 20 • C under aerobic conditions included steroid hormone biosynthesis; purine metabolism; cysteine and methionine metabolism; biotin metabolism; galactose metabolism; pyruvate metabolism; purine metabolism; glyoxylate metabolism; and glycine, serine, and threonine metabolism. In contrast, cyanoamino acid metabolism; tryptophan metabolism; tryptophan biosynthesis; cysteine and methionine metabolism; tyrosine metabolism; glutathione metabolism; cyanoamino acid metabolism; glycine, serine, and threonine metabolism; and methane metabolism were found to be responsible for metabolite generation in the digestate stored under anaerobic conditions at the formerly mentioned temperature.    The color of the circles changes from white to dark red as the −log(p) value increases, and the size of the circles is shown visually as the impact value increases from small to large, Impact > 0.05.).
Some of the metabolic pathways in the digestate were disturbed by different storage modes, leading to an impact on the active components therein, for example, a decrease in the metabolism of vitamin substances and an increase in the purine substance-derived pathway under aerobic storage conditions. An increase in indole acetic acid production by tryptophan metabolism occurred mainly in anaerobic storage.

Aerobic Storage of Digestate Promotes the Biotin Metabolic Pathway for Vitamin Production
Biotin is a water-soluble vitamin with antioxidant properties derived from the breakdown of volatile fatty acids, promoting plant root growth [27]. Biotin was detected in digestate stored under aerobic conditions (Figure 4c). Biotin metabolism starts with fatty acids biosynthesis and then methylates to form pimeloyl-ACP methyl ester and Pimeloyl-ACP. The products are then converted to biotin following a biotin bicyclic assembly step (Figure 5a). Biotin is catabolized by beta-oxidation of the valeric acid side chain or oxidation of sulfur in the heterocyclic ring [28]. At the same time, an inverse relationship was observed between storage temperature and biotin in aerobic storage. Therefore, the biotin metabolism in the digestate is boosted in aerobic storage, and the antioxidant properties of the digestate reduce with an incremented temperature and storage duration.  Digestate storage under aerobic conditions promoted the metabolism of glycine, serine, threonine, and purine metabolism. The transamination reaction between serine and glycine to form hydroxyl pyruvate and glycine is catalyzed by glyoxylate aminotransferase and requires oxygen's involvement ( Figure 4b). As the temperature increased, glyoxylate gradually increased with the transamination reaction between serine and glyoxylate. Glyoxylate has a significant antimicrobial ability and is a target product for developing many antimicrobial agents [29]. In addition, under the aerobic storage of digestate, the purine metabolic pathway was enhanced with further conversion to urea, explaining the reduced antioxidant properties under high-temperature aerobic storage conditions.

Anaerobic Storage Enhances the Tryptophan and Lysine Metabolic Pathway to Produce Indole Alkaloids
The metabolism of tryptophan and lysine during anaerobic storage of the digestate resulted in the production of castanospermine and tryptamine (Figure 4e,f). It means the anaerobic environment facilitated the conversion of these readily catabolized amino acids to indole alkaloids. Moreover, the relative abundance of castanospermine and tryptamine gradually increased with an increasing temperature. The highest relative levels of castanospermine and tryptamine were observed at 20 • C and after 20 days of storage, with a relative abundance of 19,886 ± 4986 and 24,975 ± 1654, respectively, indicating an enhanced tryptophan metabolism. Castanospermine and Tryptamine are indolizidine alkaloids that have been identified in several synthetic antimicrobial agents as well as natural antibacterial ingredients. Castanospermine has been identified as having antibacterial, antifungal, and antitumour effects [30]. Tryptamine has antibacterial and anti-inflammatory effects [31,32].

Anaerobic Storage Promotes the Taurine and Hypotaurine Metabolism Pathways to Produce Antioxidants
Taurine and hypotaurine metabolism pathways were detected in the digestate stored under anaerobic conditions, with the maximum relative abundance observed at 20 • C storage (Figures 4h and 5b). Taurine in digestate is derived from cysteamine. The anaerobic condition activates anaerobic genes and promotes the production of oxygen-sensing enzymes, such as cysteamine oxygenase. These oxygen-sensing enzymes can control anoxia-dependent processes. For example, oxygen-sensing enzymes act on mutations of amino acids in their active sites, catalyze oxygen-dependent oxidation, and drive the addition of two oxygen atoms to free cysteamine to form taurine. They can also act on individual donors by incorporating molecular oxygen (oxygenase) to form taurine. Taurine can scavenge free radicals and attenuate lipid peroxidation; therefore, it can be used as an antioxidant to stabilize biofilms and applied in agriculture to enhance nutrient uptake by plants [33,34].

Antioxidant and Antifungal Properties of Digestate under Different Storage Modes
To identify the effect of different storage modes on the antioxidant properties, digestate's ability of digestate under varying storage conditions to scavenge DPPH radicals was determined ( Figure 6). The antioxidant capacity of the digestate in both aerobic and anaerobic storage modes first increased in short-term storage (0-40 days) and then decreased with long-term storage (40-80 days). The maximum antioxidant capacity of digestate stored at 4 • C, 20 • C, and 30 • C under aerobic conditions was obtained up to 10 day's storage and was approximately 65.03 ± 9.27, 76.00 ± 7.30, and 69.01 ± 8.20 µmol/L Trolox, respectively. The highest antioxidant capacity of the digestate stored at 4 • C, 20 • C, and 30 • C under anaerobic conditions was around 77.23 ± 7.04, 82.15 ± 9.5, and 73.27 ± 0.20 µmol/L Trolox, respectively. In comparison, the minimum antioxidant activity (<50 µmol/L Trolox) was obtained after 60 days of storage under different modes. The maximum antifungal activity of digestate against Fusarium oxysporum was observed for digestate stored at 20 • C under aerobic and anaerobic conditions (83.3 ± 7.37% and 89.8 ± 5.13%, respectively) ( Figure 6b). Conversely, the antifungal activity was minimum for the digestate stored at 4 • C under aerobic and anaerobic conditions (76.7 ± 4.04% and 78.9 ± 3.37%, respectively). Moreover, it was perceived that the antifungal activity decreased with an increase in storage duration. For low-temperature storage at 4 • C, the digestate could be stored for up to 40 days, regardless of oxygen content. Storage at 20 • C in an anaerobic state contributed to the best antioxidant and antifungal properties of the digestate. Storage of digestate at 30 • C for merely 10 days resulted in a significant reduction in antimicrobial activity. The short storage duration of the digestate helped to maintain the relative stability of the digestate, probably due to the conversion of proteins, amino acids, and sugars and fats into secondary derivatives in the digestate.
The increased antioxidant and antifungal activity in the digestate stored under anaerobic conditions was because of the accumulation of indolizidine alkaloids from ricin and tryptamine. The accumulation of cysteamine and taurine in the digestate stored under anaerobic conditions further promoted antioxidant and antifungal activity (Figures 4 and 5). Conversely, the accumulation of glyoxylic acid in the digestate stored under aerobic conditions was responsible for the antioxidant activity of the digestate (Figures 4 and 5). Contrasted with the current study, Ramli et al. (2021) evaluated extracts from a Durio zibethinus peel and seed for their antioxidant and antimicrobial activity [35]. They reported that the extract from peels contained higher contents of total phenolics and flavonoids than the seed. They further observed that both the peel and seed exhibited antioxidant activity.

Conclusions
Untargeted metabolomics profiling in digestate stored at varying temperatures (4 • C, 20 • C, and 30 • C) and time (0 to 80 days) under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was also evaluated. The results showed that the degradation rate increased with an increase in storage duration. The maximum number of metabolites was detected after 80 days of storage, irrespective of the temperature and mode (aerobic and anaerobic). Moreover, around 49 metabolic pathways of metabolite generation were observed for digestate stored under anaerobic conditions, while 40 metabolic pathways were detected in digestate stored under aerobic conditions. The maximum antioxidant and antifungal activity (82.15 ± 9.5 µmol/L Trolox and 89.81 ± 5.13%, respectively) were recorded for digestate stored at 20 • C for 20 days under aerobic conditions. This study also showed that metabolomics can be an innovative and promising approach to elucidate the derivation pathways of metabolites and provide new perspectives for a further analysis of digestate under varying storage conditions. More studies are needed for exploring untargeted metabolomic profiling and the antifungal activity of digestate under varying storage conditions to evaluate its disease resistance potential upon land application.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19084923/s1, Figure S1: The calibration procedure used for visualization and quality control, including an overlay of all total ion chromatograms acquired before Rt. Calibration; Figure S2: The calibration procedure used for visualization and quality control, including an overlay of all total ion chromatograms acquired after Rt. Calibration; Figure S3


provenance: AgriculturalAndFoodSciences,2022-2022-Agricultural And Food Sciences-2022.jsonl.gz.gz

text:
Performance Evaluation of Onion Varieties at Biyo Awale Cluster in Diredawa Administration

Onion (Allium cepa L.) is an important crop worldwide. It is important in the daily Ethiopian diet. It is widely produced by small farmers and commercial growers throughout the year for local use and export market. No strong study has been conducted to evaluate the suitability of onion cultivation in Biyo Awale district of Diredawa. Hence, the present experiment was conducted to study the performance of improved varieties of onion for their suitability for cultivation in Biyo Awale district of Diredawa. The study was carried out during 20182019 cropping season to test the performance of onion varieties. Five high yielding onion varieties were planted in complete randomized block design experiment with three replications including local check at farmer’s field. The quantitative data on bulb yield, plant height, bulb size and number of leaves of onion varieties were collected. Harvesting was done and weighed when all onion varieties reach maturity (90% tops down). The results revealed that the tested onion varieties differ significantly for leaf length, leaf number, neck diameter, bulb diameter, average bulb weight and bulb yield. Among the varieties Bombay red and Melkam produced higher bulb yield (25.56 and 21.13 t ha) and gave 140.68% and 98.96% yield advantage over local check varieties, respectively. Therefore, Bombay Red and Melkam varieties were recommended to the study area and similar agro-ecology.

Introduction
Onion (Allium cepa L.), is an important vegetable belonging to family Alliaceae and widely grown herbaceous biennial vegetable crop with cross-pollinated and monocotyledonous behavior having diploid chromosomes number 2n=16 (Bassett, 1986). The production of fruits and vegetables has a comparative advantage particularly under conditions where arable land is scarce, labor is abundant and markets are accessible (Lumpkin et al., 2005). The production of horticultural products offers opportunities for poverty alleviation, because it is usually more labor intensive than the production of staple food crops. Hence, the generation of additional employment opportunities in rural areas where labor is abundant is made possible. Increasing horticultural production contributes to commercialization of the rural economy and creates many off-farm jobs (Lumpkin et al., 2005). Vegetables took up about 0.98 % of the area under all crops at national level. Onion cultivated on 31,673.21 ha with a total production of 2,938,875.85 quintals (CSA, 2017). It is recently introduction and rapidly becoming popular among producers and consumers. It is widely produced by small farmers and commercial growers throughout the year for local use and export market. Onion is valued for its distinct pungency and form essential ingredients for flavoring varieties of dishes, sauces, soup, sandwiches, and snacks as onion rings. It is popular over the local shallot because of its high yield potential per unit area, availability of desirable cultivars for various uses, ease of propagation by seed, high domestic (bulb and seed) and export (bulb, cut flowers) markets in fresh and processed forms. The method of cultivation is via direct seeding or transplanting in the field growing. The weight of thousand seeds is about 3.6 g that can be produced by open Pollination via honey bees. Regarding seed production, it is necessary to plant maternal bulb in field in the due time. Otherwise, cold weather can influence seedlings which resulted in the induction of flowering. On the other hand, the proper time for planting maternal bulb is of value because of the optimum use of the land seed favorite production both quantitatively and qualitatively. Also, the bulb size is one of the most important factors in seed production (Nehra et al., 1988;Navab et al., 1998;Khodadadi, 2009). Therefore, planting bulbs with the proper diameter as well as high seed production, have been caused cost saving in the amount of consumption per hectare and, thus can be reduced the production cost (Basol et al., 1997).
Onion contributes substantially to the national economy, apart from overcoming local demands. Products like bulbs and cut flowers are exported to different countries of the world. Currently many farmers have changed their life in onion development program. With the growing irrigated agriculture in the country, there is a great potential for extensive onion seed and dry bulb in different production belt in the country. Unfortunately, some parts of the Diredawa ecology, particularly Biyo Awale cluster are unable to produce enough onion for local consumption due to non-availability of improved planting materials. The available local variety in production has low yield, with poor quality bulbs, short shelf life due to very high water content of the bulbs and high susceptibility to insect pest and diseases. However, the knowledge of the types of variety that will yield best in a Advances in Life Science and Technology www.iiste.org ISSN 2224-7181 (Paper) ISSN 2225-062X (Online) Vol.90, 2021 given environment is important for good crop management decisions. Therefore, this paper was initiated with the objective to identify high yielding and disease tolerant variety of onion with a view of recommending it for Diredawa rural areas of Biyo Awale

Materials and Methods Description of the Study Area
The experiment was conducted in Diredawa Administration Council at Adada and Bishan Baye village during the rainy season from early July to early November, 2017. Geographically, Diredawa Administration Council is located in the eastern part of Ethiopia between 9027'N and 9049'N latitude and 41038`E and 42019`E longitude. The mean annual temperature is about 24.8°C with a range of 31.4°C and 18.2°C average maximum and minimum temperatures, respectively. The rainfall pattern of the Administration has a bimodal characteristic. The small rainy season is from March to April; while the big rainy season extends from August to mid-September with average annual rainfall of the Administration is over 604mm (Diredawa Administration Meteorological Station)

Experimental material and Design
Four improved varieties of onion introduced from Melkasa Agriculture Research Center, Melkam, Nafis, Bombay red, Adama red with local check collected from "Adada and Bishan baye" peasant associations were used in this study. The experiment was laid out in RCB design with three replications. The size of each experimental plot was 5.4m 2 (3m wide and 1.8m long). The onion varieties were randomly assigned to the experimental plots. The inter row spacing used was 20cm for bulb. Seeds of each onion variety were sown in rows of 15cm on well prepared seed bed of 1x5m and covered with light soil and mulching grasses until emergence. The beds were watered with watering can as supplementary irrigation. The seedlings were thinned at 3cm spacing within rows, followed by proper weeding and watering. Land preparation was done in advance to reduce diseases and insect pest incidence. Finally, hardened, healthy and uniform seedlings of pencil size were transplanted at an age of 30 days (MoARD, 2009). The transplanted seedlings were watered and inorganic fertilizers (NPS) and urea was applied at the recommended rate. All the phosphate fertilizer were applied at transplanting whereas nitrogen was given at two equal splits (at transplanting and 30 days after transplanting) as basal application (EARO, 2004). Plots were irrigated when there is insufficient rainfall as supplementary irrigation. Weeding, cultivation, watering, staking, chemical spray etc. were done as recommended.

Data collected
Data of Bulb diameter, number of leaves per plant, leaf length, average weight of bulb, neck diameter, bulb yield, disease and insect pest were collected. Analysis of variance was carried out using GenStat discovery 15 th edition software for the parameters studied following the standard procedures outlined by Gomez and Gomez (1984).The means were separated using the Fisher's Protected Least Significant Difference (LSD) test at 5% level of probability.

Results and Discussions
Analysis of variance (ANOVA) indicated that there was significance difference among onion varieties for leaf number, leaf length, Neck diameter, bulb diameter, average bulb weight and Bulb yield tested at 5% probability level. However, no significance difference was observed between varieties for days to maturity and plant height. This might be due to genetic makeup and environment that agrees with findings of Jilani, M.S. and Ghafoor, A. (2003) reported that various cultivars of the same species grown in the same environment give different yields as the performance of a cultivar mainly depends on the interaction of genetic makeup and environment.

Leaf Number, Leaf length and Neck Diameter
Analysis of variance revealed that there was significance difference (P< 0.05) among varieties for leaf number, leaf length and neck diameter (table 1). The highest leaf number (12.92) was recorded from Adama red variety, while the lowest (8.54) from Nafis. On the other hand the highest leaf length (40.02 cm) was recorded from Melkam varieties while the lowest (34.68 cm) from Nafis variety. The result also showed the highest neck diameter (1.72cm) gained from Nafis while the lowest (0.94cm) was from Bombay red variety.

Bulb Diameter and Average Bulb weight
Analysis of variance revealed that there was significance difference (P< 0.05) among varieties for bulb diameter and average bulb weight (Table 1). The bulb diameter was range between 5.99 and 4.75cm. The highest bulb diameter was recorded from Melkam (5.99) while the lowest was from Nafis (4.75cm). The current result also revealed the highest average bulb weight (92.79 g) was from Bombay red while the lowest (42 g) was from local cultivar.

Bulb yield
Analysis of variance revealed that there was significance difference (P< 0.05) among varieties for bulb yield ( Table 1). The bulb yield ranged from 25.56 t ha -1 to 10.62 t ha -1 . The highest bulb yield (25.56 t ha -1 ) was obtained from Bombay red variety followed by the varieties Melkam (21.13 t ha -1 ), Adama red (16.32 t ha -1 ) and Nafis (13.23 t ha -1 ) while the lowest yield (10.62 t ha -1 ) was obtained from local check. Bombay red and Melkam gave 140.68% and 98.96% yield advantage over local check respectively. Statistically, significance difference was not observed among Adama red, Nafis and local check for bulb yield. The variation in bulb yield of the tested varieties showed the difference in adaptability of these varieties to the study areas. The highly performed varieties revealed that the most adaptability to this environment. These results are in agreement with the findings of (Jilani andGhofer, 2003, Kimani et al, 1993) reported that various cultivars of the same species grown in the same environment give different yields as the performance of a cultivar mainly depends on the interaction of genetic makeup and environment. These results are disagree with the findings of (Mitiku et al, 2017) It implies that the onion varieties do not responded to the climatic conditions and reflected non-significantly to their productivity performance.

Conclusion and Recommendation
After conducting for two consecutive years in the Diredawa, Biyo Awale area, varieties showed significant difference for leaf number, leaf length, neck diameter, bulb diameter, average bulb weight and bulb yield. Accordingly Bombay red gaves the highest average bulb weight and bulb yield followed by Melkam as compared to local check and other varieties. Furthermore, Bombay red and Melkam varieties showed 140.68% and 98.96% yield advantage over the local check, respectively. Therefore, Bombay red and Melkam varieties were recommended to the study area and similar agro-ecologies.


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Biocontrol of Alternaria alternata YZU, a causal of stem end rot disease on pitaya, with soil phosphate solubilizing bacteria

. Stem end rot is the most destructive disease caused by Alternaria alternata YZU in pitaya-growing regions of Vietnam. This study was conducted to characterize antagonistic phosphate-solubilizing bacteria (PSB) from rhizosphere soil for their biocontrol activities against A. alternata YZU and evaluate the effect of temperature, pH, and water activity on that antagonism. Among seven PSB isolated from 45 rhizosphere soil samples, PSB31 (identified as Bacillus sp . strain IMAU61039, Accession number: MF803700.1) exhibited the highest antagonistic activity against A. alternata YZU with an average inhibition diameter of 0.65 ± 0.05 cm. The results also show that the strain PSB31 controlled the mycelial growth of A. alternata YZU by secreting antifungal metabolites. The most potent inhibitory activity was identified under in vitro conditions of 25 °C, pH 7, and a w 1. The isolated PSB31 could be a potential biological control agent against A. alternata YZU.

Introduction
Pitaya (Hylocereus Undatus) is an important fruit crop grown well in Binh Thuan, Vietnam. In this region, however, there is a risk of fungal pathogens, particularly stem end rot caused by Alternaria alternata YZU, resulting in severe economic losses [1]. The control of fungal diseases attacking pitaya has been primarily performed by using chemical pesticides that are hazardous to human health, pollute the environment, and induce the resistance of phytopathogenic agents [2]. Hence, developing alternative methods to control this disease is crucial. Biological control with soil bacteria has received much attention as one of the non-hazardous pest management techniques against different plant pathogens [3].

Evaluating the antagonistic activity of isolated PSB
The potential for biological control of seven where R and r are the radial distance of the fungal pathogen growth for the control and the dual culture (mm) [1].  [13].

Effect of water activity
The water activity (aw) is defined as the ratio of the to PDA [13]. Glycerol attaches to a part of water, making it unusable for microorganisms. The dual culture was carried out. The PIRG was calculated after seven days of incubation in the dark at 25 °C.
The PDA medium was buffered to the desired pH with different buffers and adjusted by adding HCl or NaOH. The inoculated dishes were incubated at 25 °C. The PIRG was calculated after seven days of incubation.

Statistical analysis
All experiments were conducted in triplicates. The data were subjected to analysis of variance with the software STATISTICA for Windows v.6. The statistical significance of the results was determined by performing a test of Duncan's multiple ranges (p < 0.05). The results were expressed as mean ± standard deviation.

Antagonistic activity of bacterial strains
In  and selected for further studies.

Antifungal activity of PSB31 culture filtrates
The strain PSB31 significantly inhibited the mycelial growth of A. alternata YZU (

Effect of temperature on antagonistic activity of PSB31
All studied temperatures exhibited significant inhibition of PSB31, with the highest one (54.44%) at 25 °C (Fig. 2).

Effect of water activity on antagonistic activity of PSB31
The water exhibited significant inhibition against the mycelial growth of A. alternata YZU. The highest inhibition (71.23%) was found at aw equal to 1. At lower water activity values, the inhibition was also lower, indicating that PSB31 plays an essential role in inhibiting the mycelial growth of A. alternata YZU, and radial growth inhibition of the phytopathogenic agent increases with water activities (Fig. 3).

Effect of pH on antagonistic activity of PSB31
The in vitro assay of pH revealed that the pH significantly affected the antagonistic potential against A. alternata YZU (Fig. 4). pH 7 had the highest inhibitory effect on the growth of A. alternata YZU (51.14%); and the lowest effect (around 21%) was observed at pH 4, 4.5, and 9.

Discussion
Biological products for controlling plant diseases have been replacing fungicides. Stem end rot caused by A.
alternata YZU is one of the most destructive diseases that threaten pitaya production in Vietnam and worldwide. In this study, isolated rhizosphere PSB were evaluated for biocontrol activities against A. alternata.
Numerous studies have been carried out to identify different PSB strains and their efficacy in phosphate solubilization and to exploit the advantages of PSB for biocontrol activities and biofertilizers [16,17]. In addition, our data also showed the culture filtrate from the PSB31 strain significantly interfered with the growth of A. alternata up to 62.92%. A similar property was observed in B. siamensis strains LZ88 [15] and B. megaterium [19]. The culture filtrate of these strains was reported to inhibit mycelial growth, spore germination, and spore production in A. alternata [15,19]. This indicates the secretion of antifungal metabolites by the bacterial antagonist, such as hydrolytic enzymes, bacteriocins, antibiotics, or some other secondary metabolites [15,20].
On the other hand, it is well known that bacteria may also be good  [7]. Hence, our study provides evidence that the PSB31 strain influences the survival of A. alternata.
Biological control is essential in controlling the phytopathogen in sustainable agriculture [21]. Bacillus sp., such as B. subtilis and B. amyloliquefaciens, have been used in commercial products for biocontrol of plant disease because of their potential for biocontrol and high stability under harsh environmental conditions caused by spore forms [22]. In this study, we found that antagonists and host plants [24,25].  [7,14]. In this study, the bacteria antagonist showed the inhibitory effect at all temperatures studied, and the most potent inhibition in the fungal radial growth was observed at 25 °C.
Hence, the results agreed with those of previous studies [7,14].
Regarding water availability, the results showed that water activity significantly affected mycelial growth, which was optimal between 0.95 and 1. It is also consistent with some previous studies on Bacillus sp. For example, the optimal growth of B.
amyloliquefaciens was at a water activity of 0.960 and 37 °C [13], but it inhibited the growth of A. flavus and F.
verticillioides at a water activity of 0.99, 0.97, 0.95, and 0.93 [26] and also inhibited the growth and aflatoxin B1 production by Aspergillus section Flavi at a water activity of 0.982 [27]. The results suggested that changes in water activity might be useful for improving the environmental competence of the microorganism in the environment.
As for the influence of pH on the biocontrol, the results revealed that the pH of the growth medium played a crucial role in the inhibition competence of organisms, which was low at acid pHs, increased by increasing pH values to 7 (neutral pHs), and then decreased in alkaline pHs. Previous studies demonstrated that pH 6 to 7 was optimum for the growth and inhibition ability of the majority of bacterial strains [28,29]. We found that pH 6.5 was required for the optimum growth of PSB31. Therefore, the inhibitory effect of PSB31 against A. alternata YZU was investigated at pH 4.0-9.0. At pH 7.0, more than a 50% inhibition of the radial growth of A. alternata YZU was obtained, which decreased nearly to thirty per cent at pH 8.0 ( Figure 4). Hence, the result indicated that the optimal pH for antagonistic activity was 7. A higher or lower pH considerably reduced the growth and the antagonistic activity of PSB31 against A. alternata YZU.

Conclusion
Seven PSB isolated from rhizosphere soil samples could inhibit the mycelial growth of Alternaria alternata. The


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Analyses of Fatty Acids, Proteins, Ascorbic Acid, Bioactive Phenolic Compounds and Antioxidant Activity of Canadian Barley Cultivars and Elite Germplasm

Barley (Hordeum vulgare L.) grain is rich in fiber and antioxidant phytochemicals, including fatty acids, proteins, phenolic compounds, vitamins, and minerals, that offer various health benefits. Research on identifying different barley genotypes based on their health attributes is very limited. In this study, we performed an analysis of fatty acids, proteins, ascorbic acid, phenolic compounds, and antioxidant activity of several Canadian barley cultivars and elite breeding lines. Linoleic acid (C18:2) was the predominant fatty acid present in the tested barley cultivars. The cultivar CDC Bold contained the highest amount of ascorbic acid, while the highest protein content was in CDC Mindon. An assessment of the free and bound phenolic compounds of barley grains showed quantitative changes among different genotypes of Canadian barley. Catechin is the most abundant molecule in free phenolics, while ferulic acid and para-coumeric acid are the most abundant in bound phenolics. Ferulic acid and vanillic acid were molecules detected in the soluble free fraction of all genotypes. Para-coumeric acid was detected only in genotypes such as CDC Copeland, CDC Bold, Lowe, and elite breeding Line 5 of both free and bound fractions of barley. Breeding Line 5 had the lowest antioxidant activity. An analysis of the above molecules and parameters of Canadian barley would help to uncover potential biomarkers in order to distinguish individual barley genotypes.

Introduction
Barley (Hordeum vulgare L.) ranks fourth in global cereal production after maize, wheat, and rice [1]. The global production volume of barley was about 147.05 million metric tons in the 2021-2022 crop year, which was a decrease from around 160.53 million metric tons in 2020-2021 [2]. Barley is rich in several health-boosting components, such as dietary fiber, essential fatty acids, vitamins, and phytochemicals, including several phenolic compounds [3]. Over 60% of the total phenolic content found in beer comes from barley [4]. These barley phenolic compounds play an important role in beer quality, shelf-life, taste, and flavor [5].
Barley is a versatile crop grown for malting, feed, forage (general purpose), and food across the Canadian prairie provinces. Canada is the fourth largest producer of barley and the second largest exporter of malt barley [6]. Western Canada is a major contributor to the production and use of Canadian barley [7]. In 2021, a total of 3.257 million hectares were

Comparison of Fatty Acid Composition in Selected Genotypes of Canadian Barley Grains
A percentage of fatty acid methyl esters (FAMEs) showed variation among the 14 barley genotypes ( Figure 1 and Table S1). About 15 fatty acids were detected as FAMEs and quantified as a percentage using the GC/MS analysis. For example, methyl linoleate/linoleic acid (C18:2) was the predominant constituent (47-54%) in barley, while Harrington, CDC Bow, and elite breeding Lines 2, 3, and 4 showed a significant reduction of the C18:2 content compared with the other genotypes ( Figure 1 and Table S1). The content of C18:2 in barley seeds was more than five times higher than methyl oleate/oleic acid (C18:1) content and about 10 times higher than methyl linolenate/linolenic acid (C18: 3) content. Methyl palmitate/palmitic acid (C16:0) represented 24-28% composition in fatty acids and varied among the genotypes. Two compounds, citric acid trimethyl ester and methyl 9 (z), 11 (E), 13 (E) octadecatrienoate or conjugated linolenic acid (CLnA), were detected in trace amounts ( Figure S1).

Comparative Analysis of Protein Content in Barley Genotypes
Protein content in barley showed genotypic differences. Overall, the malting barley cultivar Lowe showed the lowest content of proteins. The highest protein content was observed in the general purpose cultivar CDC Mindon ( Figure 2).

Assessment of Ascorbic Acid Content
The ascorbic acid content was detected in the soluble extract. Genotypes of CDC Bold, CDC Copeland, CDC Mindon, Harrington, Lowe, and elite germplasm Line 4 showed significantly higher constitutive ascorbic acid content compared with most of the other genotypes analyzed (Table 2).

Assessment of Phenolic Compounds in Grains of Selected Canadian Barley Genotypes
Assessment of free and bound phenolic compounds of barley grains showed quantitative changes among different genotypes of barley included in the testing in this study (Tables 2 and 3). Cultivars such as CDC Bold and CDC Bow have a relatively higher amount of free phenolic compounds than the other cultivars. Para-coumeric acid (PCA) was quantified in only four of the cultivars, CDC Bold, CDC Copeland, Lowe, and elite breeding Line 1, as it was below the limit of quantification (LOQ) in other genotypes. The cultivar Lowe showed the highest content of PCA. Free sinapic acid (SIN) contents fluctuated among the genotypes, and it was not detected in elite breeding Line 5. The cultivar CDC Bold showed relatively the highest catechin (CAT) content compared with other genotypes. Overall, free PCA, SIN, and CAT compounds were not detected in elite breeding Line 5 (Table 3 and Figure 3A).  The analysis of the bound part of phenolic compounds showed that PCA content was low in genotypes such as elite breeding Line 5 ( Figure 3B), elite breeding Line 2, and CDC Bold. Caffeic acid (CAF) content was higher in CDC Mindon than in the other genotypes, such as CDC Bow, CDC Bold, and elite breeding Lines 3, 4 and 5. Sinapic acid content was significantly higher in elite breeding Line 1 and CDC Mindon compared with others such as AC Metcalfe, Lowe, and CDC Bold. Ferulic acid content was significantly higher in the Harrington genotype compared with CDC Bow, CDC Bold, and elite breeding Lines 2, 3, 4, and 5. No significant differences in the isoferulic acid (Iso FA) content were found among most of the genotypes analyzed. The elite breeding Line 5 showed significantly lower CAT content compared to those of Lowe and CDC Copeland cultivars. The content of 4-hydroxy benzoic acid (4HBA) was higher in cultivars such as AAC Synergy, AAC Goldman, and AC Metcalfe. AC Metcalfe contained high vanillic acid content, while elite breeding Line 2 and CDC Bold had the lowest. The highest VANILLIN content was in Harrington, while the lowest content was in CDC Bow and CDC Bold. Compounds such as caffeic acid, iso-ferulic acid, 4 hydroxy benzoic acid (4HBA), and vanillic acid were detected only in bound barley fractions (Table 4). A principal component analysis of free phenolics showed that the first three principal components provided a reasonable summary of the data and explained more than 75% of the total variations of the free form of phenolic compounds in barley genotypes ( Figure 4).   Cluster analysis generated a dendrogram, as illustrated in Figure 4D. The analysis is based on the mean values of the five measured phenolic compound concentrations in free form within 14 barley genotypes into clusters based on a similarity level of 70%. Each cluster was markedly different from the other clusters and consisted of various types of barley genotypes. The dendrogram shows there is a similarity between varieties AAC Synergy and AAC Goldman at a level of 87.41% similarity in cluster 1, and the cluster centroids show that it is because of the presence of CAT, FA, and VANILLIN. Cluster 4 has another two genotypes, CDC Copeland and CDC Mindon, which have a similarity level of 79.72% due to the positive relationship between CAT and FA. There is a 76.99% similarity level between AC Metcalfe and CDC Bow in cluster 3 based on CAT and FA. Even though cluster 3 is grouped based on the CAT and FA, the similarity is stronger in cluster 4 compared with cluster 2 ( Figure 4D). Based on these analyses, AAC Synergy and AAC Goldman genotypes are highly similar, followed by genotypes CDC Copeland, CDC Mindon, AC Metcalfe, and CDC Bow.
A principal component analysis of bound phenolic compounds showed that the first three principal components provided a reasonable summary of the data and explained more than 88% of the total variations ( Figure 5). The fact that variance in the dataset was distributed in the first three variables was likely due to the high correlation between the bound phenolic compounds in barley varieties which are responsible for the first three principal components.   In the dendrogram, it shows there is a similarity between AAC Goldman and Line 1 shown in red colour at a level of 96.54% similarity in cluster 2, and the cluster centroids show that it is because of the presence of PCA, FA and CAT. Cluster 3 which is shown in purple colour has another two genotypes; CDC Bow and Line 3 that have a similarity level of 95.46% due to the positive relationship of CAT, PCA and FA. There is a 92.74% similarity level between CDC Bow and Line 4 in cluster 3 based on CAT and FA. Based on the bound phenolic compound analysis, AAC Synergy, AC Metcalfe, and CDC Copeland can be grouped as one, whereas AAC Goldman, Line 1, and CDC Mindon can be grouped as another group of genotypes. The genotypes CDC Bow, Line 3, Line 4, and Line 5 also belong to one group having more than 80% similarity level.

Antioxidant Activity Assay Analysis
The antioxidant activity of barley was comparatively high and was not significantly different between genotypes, although the lowest activity was observed in elite breeding Line 5 ( Figure 6). There was also no significant correlation between the antioxidant activity measured by ABTS assay and any of the relevant individual phenolic compounds in the extract (Table 5). However, the correlation matrix showed a strong relationship between some compounds, such as caffeic acid and isoferulic acid, as well as ferulic acid and isoferulic acid.

Discussion
Barley is a staple food in some countries, and the nutritional constituents of barley include different types of phytochemicals such as phenolic acids, flavonoids, lignans, vitamin E, sterols, and folates [24]. These bioactive phytochemicals are known to have health-promoting benefits [3]. It has been suggested that the total phenolic compound content of barley grains is concentrated in the bran rather than the endosperm [25]. The content of bound insoluble phenolics was higher than those of soluble conjugate and free phenolic extractions, similar to the results observed in our study. The content of barley phenolics may also vary based on the extraction method and the genotype or cultivar. For example, protocatechuic acid, chlorogenic acid, gallic acid, and syringic acid were not detected in any of our tested hulled Canadian genotypes, though they were detected in previous studies [26] when hulless, colored (blue) barley varieties from the Qinghai-Tibet Plateau were assessed. A comparison of phenolic compounds in various barley genotypes showed that CAT is the major phenolic compound in free phenolics of barley, similar to that observed in previous studies [26,27]. Similarly, FA and PCA were the two major polyphenols representing more than 70% of the total bound phenolics in all genotypes studied, while caffeic acid, sinapic acid, isoferulic acid, catechin, 4HBA, vanillic acid, and vanillin were detected in smaller quantities in our study.
The contents of barley lipids could be changed based on the cultivars used [13]. Indeed, the FAMEs analysis of our study showed a variation among the tested 14 Canadian barley genotypes. Linoleic acid, linolenic acid, and oleic acid were among the unsaturated fatty acids involved in reducing cardiovascular risk by decreasing the LDL-cholesterol level [14]. Linoleic acid content (C18:2) in barley grains is the highest and represents about 50%, followed by oleic acid (C18:1) with about 11% and linolenic acid (C18:3) with about 4%, which is similar to that reported previously [13]. Citric acid trimethyl ester and methyl 9(z), 11 (E), 13 (E) octadecatrienoate or conjugated linolenic acid (CLnA), which have anticancer activity, were detected in trace amounts by GC-MS analysis [28]. The existence of citric acid trimethyl ester was only reported in only a few studies in barley [29,30].
Besides growth and environmental conditions, the barley protein content could vary based on the cultivar [12]. A previous study [31] revealed that the protein content in the barley samples fluctuated from 10-17%. However, based on another study [24], the total protein content was from 12 to 16%. Interestingly, the protein content of the germplasm tested in our study was slightly lower, ranging from 7 to 12%. Ascorbic acid was only detected in soluble free barley extracts. The antioxidant capacities of barley were highly similar to that reported in previous research [32], although there were no significant differences between the genotypes. It may be useful to use several assays with different modes of action to evaluate the antioxidant activity of the extract in vitro. The relationships between some phenolic compounds, such as caffeic acid and isoferulic acid or ferulic acid and isoferulic acid, might be due to synergistic effects in their response to environmental conditions that may need further investigation.
Overall, the results of our study showed varying quantities of phenolics, fatty acids, proteins, ascorbic acid content, and antioxidant activity in tested Canadian barley genotypes. The outcome of this study could be used as a potential genotype distinguishing factor(s).

Materials and Methods
Grains of several two-row spring hulled barley cultivars and elite breeding lines (malting and general purpose) that were grown during the 2020 crop season in the field of the Brandon Research and Development Centre, Brandon, MB, Canada, were used in this study (Table 1).

Preparation of Samples for the Extraction and Analysis
Grains of each of the genotypes included in the study were milled by a Perten Labmill 3100, PerkinElmer Inc. (Woodbridge, ON, Canada), and subsamples of the obtained homogenous powder was subjected to oven drying at 60 • C for 24 h to alleviate any fluctuations in moisture before GC/MS and high-performance liquid chromatography (HPLC) analysis.

Extraction and Analysis of FAMEs by GC/MS
Fatty acid analysis was performed using a one-step extraction/methylation method adapted from a previous study [33] with some modifications. Accurately weighed 200 mg of whole meal barley flour was placed in Pyrex glass tubes. Two milliliters of 3N methanolic-HCl were added, and the tubes were incubated in a digital dry bath (Boekel Scientific, PA, USA) at 80 • C for 45 min. Four milliliters of hexane were added to extract the fatty acids methyl esters (FAMEs), vortexed and kept overnight in dark conditions. The hexane layer was transferred using a glass Pasteur pipette to new glass, disposable tubes and evaporated under a vacuum to dryness (60 • C for 40 min) using Eppendorf Vacufuge Plus (Eppendorf, Germany). The extract was suspended in 700 µL hexane and vortexed for 30 s, and transferred to GC vials prior to analysis. An analysis of the FAMEs was performed using Bruker 436-GC equipped with EVOQ-TQ-MS (Bruker Daltonics, Germany). FAMEs were separated on an Rt-2560 capillary column (100 m × 250 mm ID × 0.20 um df) (Restek, Bellefonte, PA, USA). The carrier gas was ultrapure Helium with an initial flow rate of 0.8 mL/min. The temperature for the inlet was 250 • C, and the GC temperature program was set at 100 • C for 4 min ramping up to 250 • C at a rate of 3 • C/min and holding for 8 min. The MS was operated in the electron ionization mode at 70 eV in the full scan mode in the range of 50 to 500 amu using MSWS software. FAMEs were identified based on comparing their retention times with the retention times of a standard mix as well as searching the NIST library. FAMEs were represented in tables as a percentage relative to the total percentage.

Protein Composition Analysis
The grain protein of the samples was measured by a combustion nitrogen analysis (CNA) using a LECO Model FP-628 CNA (St. Joseph, MI, USA) analyzer calibrated by ethylenediamine tetraacetic acid (EDTA). Prior to that, the samples were ground on a UDY Cyclone Sample Mill fitted with a 0.5-mm screen (UDY Corporation, Fort Collins, CO, USA), and a moisture analysis was performed. The results were reported on a dry matter basis (ASBC Barley 7C).

Determination of Ascorbic Acid (Vitamin C)
The HPLC method employed a simultaneous analysis of free phenolics and ascorbic acid, an organic acid, in the same run. Extraction of ascorbic acid was performed by ethanol 90% solution using the same protocol for extracting the free phenolics as described earlier. Quantification of ascorbic acid was based on an external standard calibration method. A serial dilution of concentrations ranging from 24.1 to 386 µg/mL in methanol was prepared and analyzed by HPLC, and a standard curve was established between the concentrations and corresponding peak areas. The resulting equation was used to determine the concentration of ascorbic acid in sample extracts.

Analysis of Free Phenolic Compounds in Barley Using HPLC
Free phenolic molecules from the barley whole meal flour were extracted from adapted protocols of previously published studies using an ethanol 90% solution with modifications [27,34]. One gram of powder was extracted with 15 mL of ethanol 90% and sonicated in a water bath at 70 • C for 1 h with periodical inverting of tubes. The supernatant was filtered, using Whatman#4 filter paper, into new tubes and concentrated under a vacuum at 60 • C for 2 h using Eppendorf Vacufuge Plus (Eppendorf, Germany). The residue was suspended in 500 µL HPLC-grade methanol, then sonicated for 5 min at 50 • C until complete solubility. This methanolic extract was centrifuged at 12,000× g rpm for 20 min, and 140 µL aliquot was transferred into vial inserts prior to HPLC analysis.

Analysis of Bound Phenolic Compounds in Barley Using HPLC
Extraction of bound phenolic compounds from barley whole meal flour was also adopted from a previous study [35] with modifications and scaling down to be more environmentally friendly. It was based on successive acid and alkaline hydrolysis. Accurately weighed 0.1 g powder was weighed in 2 mL Eppendorf tubes and subjected to acid hydrolysis (1 mL, 2 N HCl). Tubes were inverted several times and sonicated in a water bath for 30 min at 60 • C. After cooling down, tubes were centrifuged at 12,000× g rpm for 15 min (Sorvall Biofuge Primo (Thermo Scientific, Waltham, MA, USA) and the supernatant was collected and discarded. Tubes containing the solid residues were let dry in a fume hood for 30 min then 1 mL of ddH 2 O was added to remove the acid residue. Tubes were centrifuged at 12,000× g rpm for 15 min, and the supernatant was discarded while the solid residues were left overnight to dry. Alkaline hydrolysis was performed by adding 600 µL 2N NaOH to tubes, vortexed and exposed to a sonication water bath for 30 min at 60 • C and tubes were inverted periodically. After cooling down, the pH of the tubes was adjusted to 2 with concentrated HCl. Extraction of bound phenolics was performed after adding 800 µL ethyl acetate to each tube, vortexing for 30 s and centrifuging at 12,000× g rpm for 15 min, and the supernatant was collected in new Eppendorf tubes. The second extraction was performed with 500 µL ethyl acetate, and the supernatants were pooled and concentrated under vacuum at 60 • C for 25 min using Eppendorf Vacufuge Plus (Eppendorf, Germany) until complete dryness. Two hundred microliters of HPLC-grade methanol were added to each tube, vortexed, and centrifuged at 12,000× g rpm for 20 min. An aliquot of 100 µL was placed in a vial insert and analyzed with HPLC. An analysis of compounds was performed using HPLC Dionex 3000 Ultimate (Thermo Scientific) equipped with a C18 reversed-phase column (Acclaim 120, 4.6 × 250 mm, 5 µm). Gradient elution was used to separate the compounds, where mobile phase A consisted of acidified water (0.1% phosphoric acid) and mobile phase B was acetonitrile. This gradient started with 10% B increasing to 90% in 38 min, and then equilibrated to the initial concentration before the next injection with a flow rate of 1 mL/min and injection volume of 20 µL. Compounds were acquired at different wavelengths, but the quantification of cinnamic acid derivatives was performed at 325 nm, while benzoic acid derivatives and flavonoids were quantified at 280 nm. The method of external standard calibration was used for the quantification of each compound after establishing the calibration curves covering at least 5 concentration points. The regression equation was used to quantify each compound in the unknown sample, and the results were expressed as µg/mg dry weight. Figure 7 shows the elution order of a mix of standards detected in barley extract.

Antioxidant Activity Assay in Barley
The barley extracts used in the bound phenolic compound analysis study were diluted one time by methanol. The analysis was performed according to a previous study [36]. After the addition of a reaction mixture (supplied in ABTS Antioxidant Assay Kit from amsbio, Cambridge, MA, USA), samples were incubated on a plate shaker at room temperature and read absorbance using a plate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany) at a wavelength of 405 nm. Data were expressed as Trolox equivalents (TE) per gram of starting sample (i.e., µM TE/g). All assays were run in triplicates.

Statistical Analysis
Quantitative data were subjected to one-way ANOVA using OriginPro 2022 (OriginLab Corporation, Northampton, MA, USA). Tukey's test was used to compare the means between cultivars or elite germplasm for each compound at p ≤ 0.05 level. A principal component analysis (PCA) was used to examine the contents of phenolic compounds in each genotype, followed by a cluster analysis.

Conclusions
Out of the genotypes tested in this study, CDC Copeland and CDC Bold are the genotypes that contained high values of phenolic compounds, fatty acids, proteins, ascorbic acid, and antioxidant activity. Elite breeding Line 5 has the lowest free phenolics, lowest antioxidant activity, and the second highest protein content. Due to the diversity and the quantitative differences of the parameters tested in these genotypes, we suggest that the contents of phenolic compounds, fatty acids, proteins, ascorbic acid, and antioxidant activity could be used as potential barley cultivar distinguishing factors. They will be beneficial to the breeders within their breeding programs and farmers during the cultivation of these cultivars. Moreover, they could serve for the assurance of purity during commercialization and even maybe for cultivar registration and protection. While our results are very interesting, further investigation on a larger number of barley cultivars and elite breeding lines is needed as well as across various growing conditions and years of testing.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27227852/s1; Table S1: Fatty acid methyl ester (FAMEs) percentage (%) in the ground meal of various barley cultivars analyzed by GCMS; Figure S1. GC/MS chromatogram of CDC Copeland grain sample showing the major peaks. C. Enlarged insert (blue square in A) of the chromatogram, Rt 55.69 is Citric acid trimethyl ester; Rt 56.261 is Methyl 9 cis, 11 trans, 13 trans-Octadecatrienoate.