Barley

The FFQ, originally developed for the TLGS, was a Willett-format questionnaire modified based on Iranian food items²⁵ and contains questions about average consumption and frequency for 168 food items during the past year.⁷ The food items were chosen according to the most frequently consumed items in the national food consumption survey in Iran.²⁵ Because different recipes are used for food preparation, the FFQ was based on food items rather than dishes, eg, beans, different meats and oils, and rice. Subjects indicated their food consumption frequencies on a daily basis (eg, for bread), weekly basis (eg, for rice and meat), monthly basis (eg, for fish), yearly basis (eg, for organ meats), or a never/seldom basis according to portion sizes that were provided in the FFQ. For each food item on the FFQ, a portion size was specified using USDA serving sizes (eg, bread, 1 slice; apple, 1 medium; dairy, 1 cup) whenever possible; if this was not possible, household measures (eg, beans, 1 tablespoon; chicken meat, 1 leg, breast, or wing; rice, 1 large, medium, or small plate) were chosen. Table 1shows food items and portion sizes used in the FFQ. Trained dietary interviewers with at least 3 of experience in the Nationwide Food Consumption Survey project²⁵ or TLGS^{26 (link)} administered the FFQs and 24-hour DRs during face-to-face interviews. The interviewer read out the food items on the FFQ, and recorded their serving size and frequency. The interview session took about 45 minutes. The interviewer for FFQ1 and FFQ2 was the same for each participant. Daily intakes of each food item were determined based on the consumption frequency multiplied by the portion size or household measure for each food item.²⁷ The weight of seasonal foods, like some fruits, was estimated according to the number of seasons when each food was available.
Dietary data were also collected monthly by means of twelve 24-hour DRs that lasted for 20 minutes on average. For all subjects, 2 formal weekend day (Thursday and Friday in Iran) and 10 weekdays were recalled. All recall interviews were performed at subjects’ homes to better estimate the commonly used household measures and to limit the number of missing subjects. Detailed information about food preparation methods and recipe ingredients were considered by interviewers. To prevent subjects from intentionally altering their regular diets, participants were informed of the recall meetings with dietitians during the evening before the interview. All recalls were checked by investigators, and ambiguities were resolved with the subjects. Mixed dishes in 24-hour DRs were converted into their ingredients according to the subjects’ report on the amount of the food item consumed, thus taking into account variations in meal preparation recipes. For instance, broth or soup ingredients—usually vegetables (carrot or green beans), noodles, barley, etc.—differed according to subjects’ meal preparation. Because the only available Iranian food composition table (FCT)²⁸ analyzes a very limited number of raw food items and nutrients, we used the USDA FCT²⁹ as the main FCT; the Iranian FCT was used as an alternative for traditional Iranian food items, like kashk, which are not included in the USDA FCT.
The food items on the FFQ and DR were grouped according to their nutrient contents, based on other studies,^{30 (link)} and modified according to our dietary patterns. Seventeen food groups were thus obtained, as follows: 1) whole grains, 2) refined grains, 3) potatoes, 4) dairy products, 5) vegetables, 6) fruits, 7) legumes, 8) meats, 9) nuts and seeds, 10) solid fat, 11) liquid oil, 12) tea and coffee, 13) salty snacks, 14) simple sugars, 15) honey and jams, 16) soft drinks, and 17) desserts and snacks (Table 1). The 168 food items on the FFQ were allocated to these 17 food groups, and the amounts in grams of each item were summed to obtain the daily intake of each food group.

Fourteen Angus × Herford heifers, approximately 8 months of age with an initial body weight of 290 ± 25 kg, were sourced from a research farm that was established in 1984 and free of the following pathogens: the herd was tested annually for bovine viral diarrhea virus, bovine herpes virus-1, Leptospira (serovars Canicola, Pomona, Hardjo, Grippotyphosa, and Copenhageni), Anaplasma phagocytophilum, bluetongue virus, and Brucella abortus, biannually for Mycobacterium avium subspecies paratuberculosis and bovine leukosis virus, and every five years for Mycobacterium bovis. Cattle positive for any of the above disease agents were removed from the herd. None of the cattle used were administered antimicrobials or vaccines prior to or during the study. Calves were weaned 41 d prior to study enrollment (day −41) and were bunk-fed an alfalfa-barley silage mixed diet in pens. On day 0, calves were transported to the feedlot (distance of 20 km).
Upon arrival at the feedlot, the heifers were not mixed with cattle from other sources and were fed alfalfa-barley silage mixed diets similar to the ones at the disease-free farm. Nasopharyngeal samples were collected from each calf in the study on days 0 (at the disease-free farm prior to shipment), 2, 7, and 14 according to Timsit et al. [3 (link)]. Prior to sampling, the nostril was wiped clean with 70% ethanol. Extended guarded swabs (27 cm) with a rayon bud (MW 124, Medical Wire & Equipment, Corsham, England) were used for sampling (Additional file 1: Fig. S1) and swabs were transported to the lab on ice for processing, within one hour of collection. Animals used in this study were cared for according to the guidelines set by the Canadian Council on Animal Care [13 ] and all experimental procedures involving cattle were approved by the Animal Care Committee of the Lethbridge Research Centre.

OSX, BiWX, Avicel PH-101, xylo-oligosaccharides (DP1-5) and β-(1 → 4)-linked gluco-oligosaccharides (DP1-5) were obtained from Sigma-Aldrich (Steinheim, Germany). WAX (medium viscosity), β-(1 → 3, 1 → 4)-linked glucan from barley (medium viscosity) and oat spelt (medium viscosity) were purchased from Megazyme (Bray, Ireland). Xyloglucan (XG; from tamarind seed) was obtained from Dainippon Sumitomo Pharma (Osaka, Japan). Regenerated amorphous cellulose (RAC) was prepared from Avicel PH-101 by adopting a method described elsewhere [42 (link)]. Briefly, Avicel PH-101 (100 mg) was moistened with 0.6 mL water. Next, 10 mL 86.2% (w/v) ortho-phosphoric acid was slowly added followed by rigorous stirring for 30 min until the cellulose was completely dissolved. The dissolved cellulose precipitated during stepwise addition of 40 mL of water. After centrifugation (4,000g, 12 min, 4°C), the pellet obtained was washed twice with water and neutralized (pH 7.0) with 2 M sodium carbonate. The pellet was washed again with water (three times) and the final pellet was suspended in water to a dry matter content of 1.4 ± 0.1% (w/w) RAC suspension.

Dietary fiber is derived from plant sources, especially agro-waste. These dietary fibers are an excellent source of natural antioxidants and may serve to improve the shelf life of meat products when incorporated into them. Antioxidants are an additive used in meat products that could be substituted with natural antioxidants obtained from DF. These substances prevent the oxidation of lipids, proteins, and pigments in foods, extending their shelf life and maintaining the food’s original color, texture, aroma, flavor, and overall quality (Damodaran & Parkin, 2017 (link)). Many fruit and vegetable wastes are the attractive source of DF. When these wastes included in different meat products, the DF content was noticed as in one of the studies, Mosambi (sweet lime) peel powder was incorporated in sausages and patties at various levels. After incorporation, 7.33% and 6.24% of total DF were found in sausage and patties, respectively (Younis, Ahmad & Malik, 2021 (link)). Similarly, in other study, 3% DF was found by incorporating the upper stem of white cauliflower (7.5%) in beef sausage (Abul-Fadl, 2012 ). While, in another study, 2.94% DF was found when 40% oyster mushroom was added in chicken sausage (Ahmad et al., 2020 (link)) as shown in Fig. 1. The inclusion of dietary fiber in meat products also helps in health management. Waste from fruits and vegetables includes phytic acid (Jayadev, 2017 (link); Ani & Abel, 2018 (link)), which acts as a cation exchange component and a mineral chelator for positively charged ions like cadmium, calcium, zinc, and copper (Ekholm et al., 2003 (link)). Chelating agents can influence metal toxicity by mobilizing the toxic metal mainly into the urine (Flora & Pachauri, 2010 (link)).
Dietary phytochemicals are substances derived from plants that are not considered nutrients but have been connected with a lower risk of developing certain chronic diseases (Liu, 2004 (link)). When these phytochemicals are ingested consistently through food, they even have the potential to protect against some malignancies and cardiovascular illnesses (Okarter & Liu, 2010 (link)). Soluble dietary fiber, such as barley and oats, protects against heart-related disorders and certain malignancies, and lowers total cholesterol and low-density lipoprotein cholesterol (Pins, 2006 (link)). Insoluble dietary fiber, such that found in wheat bran, has been demonstrated to reduce the risk of colon cancer, as well as the risk of developing other cancer, obesity, and gastro-intestinal issues (Stevenson et al., 2012 (link)).

For scanning electron microscopy, immature barley spikes were fixed in 50 mM cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and 2% formaldehyde at 4°C. Samples were washed with distilled water and dehydrated in an ascending ethanol series and point-dried in a Bal-Tec critical point dryer (https://leica-microsystems.com). Dried specimens were gold-coated in an Edwards S150B sputter coater (http://edwardsvacuum.com) and examined in a Zeiss Gemini30 scanning electron microscope (https://zeiss.de) at an acceleration voltage of 10 kV. For TEM, samples were embedded in Spurr’s resin. Ultrathin sections (70 nm) were cut with a microtome (Leica Ultracut, Leica Microsystems, Bensheim, Germany) and subjected to TEM (Tecnai Sphera G2, FEI, Eindhoven, The Netherlands) at 120 kV. For confocal laser scanning microscopy (CLSM) observation of chlorophyll, immature spikes were dissected and embedded in 8% agarose placed in a flat-bottomed mold. After concreting, agarose blocks were removed from the mold and cut into 80 μm with a microtome (Leica VT1000 S vibrating blade microtome). Autofluorescence was recorded under the LSM780 CLSM (Carl Zeiss MicroImaging) with a 640-nm laser line and 650- to 720-nm emission. For measuring the vascular area, transverse sections through all rachis of a barley spike were recorded by CLSM with autofluorescence of a 405-nm laser line and 406- to 715-nm emission. The surface area of rachis internodes and vasculature was measured with the open source Fiji software (67 (link)). The cross-sectional surface area of individual veins was determined as the area surrounded by a bundle sheath. The distinction between central, lateral, and peripheral bundles followed (68 ). Because of their aberrant distribution and different functions in metabolite transportation (68 , 69 ), peripheral bundles were not included in the present investigation, which thus involves lateral and central bundles only.

Before immunolabeling, coverslips were washed twice with 1xPBS keep in one line for 5 min at room temperature (RT) and incubated with blocking solution (5% BSA, 0.03% Triton X-100, 1×PBS) for 1.5 h at RT. Peptide Topo IIα (rb12 and gp13) (Kubalová et al. 2021b (link)) and rabbit anti-grassCENH3 (Nagaki et al. 2004 (link); Houben et al. 2007 (link)) antibodies were diluted 1:100 and 1:10,000, respectively, in antibody solution (1% BSA, 0.01% Triton X-100, 1 × PBS), and incubated overnight at 4 °C. Grass-CENH3 antibodies detect both α and βCENH3 of barley (Ishii et al. 2015 (link)).
Next, coverslips were washed with 1×PBS (three times, 5 min each) at RT and incubated with secondary donkey anti-rabbit Alexa488 (1:200, #711-545-152 Jackson ImmunoResearch) and goat anti-guinea pig Alexa488 (1:200, # A11073 Invitrogen) antibodies for 1 h at 37 °C. For colocalization with Topo IIα, CENH3 was labeled with Cy3-conjugated anti-rabbit IgG (Dianova). Subsequently, coverslips were washed in 1×PBS (three times, 5 min each) at RT and immediately dehydrated in an ethanol series (70%, 85%, and 100%), each step 2 min. Afterward, the coverslips were air-dried and subjected to microscopy.