2.5. Wort Quality Tests
Wort fermentable sugars were determined according to ASBC Method, Wort-14B [19
] with modifications. Ten microliters of wort were mixed with 200 mg AG 501-X8 resin and shaken for 15 min. After filtering through a 0.45 µm NLY filter (Whatman Inc., Sanford, ME, USA), the sample was analyzed by HPLC (HP 1050, Agilent, San Diego, CA, USA) with a refractive index detector (HP 1047A) in the range of 1/64 × 10−5
RIU/F.S. Glucose, fructose, maltose and maltotriose were separated on a Hi-Plex Ca (Duo) column (6.5 mm × 300 mm, 8 µm (p/n PL1F70-6850), Agilent, Santa Clara, CA, USA) at 85 °C, with 100% DI H2
O as mobile phase. The flow rate was 0.4 mL/min, and the injection volume was 5 µL. Wort β-glucan content was determined according to ASBC Method, Wort-18B [19
] using a Waters 515 HPLC pump (Waters Corporation, Milford, MA, USA) a Waters 717 autosampler and a Waters 474 scanning fluorescence detector. A 20 µL sample was injected into a capillary (4.37 m × 0.54 mm) mixing column. The flow rate was 3.0 mL/min, the detector was set to 365 nm excitation and 420 nm emission wavelengths.
The arabinoxylan content of wort was determined by the modified method of Carpita and Shea [23
]. A 100 µL aliquot of each rye wort sample was hydrolyzed with 2.5 mol/L trifluoroacetic acid (200 µL) by heating at 121 °C for 1 h, with an added internal standard of inositol (10 mg/mL). The hydrolysates were dried at 55 °C under nitrogen. The hydrolyzed sample was reduced by ammonium hydroxide (1 mol/L, 100 µL) and sodium borohydride in DMSO (20 mg/mL, 500 µL), and neutralized by glacial acetic acid after heating at 40 °C for 90 min. Then, 100 µL of 1-methylimidazol and 500 µL of acetic anhydride were added into each tube for acetylation and the reaction was stopped after 10 min with 4 mL of water. The acetylates were extracted with 1 mL of methylene chloride two times, the combined methylene chloride fractions were dried at 45 °C under nitrogen [24
], and finally re-dissolved in 1 mL of acetone for GC analysis.
The derivatized arabinose and xylose were analyzed with an Agilent 7890 gas chromatograph (GC) with flame ionization detector (Agilent technologies, Santa Clara, CA, USA). Separation was performed on a Supelco SP-2380 fused silica capillary column (30 m × 0.25 mm × 2 µm) (Supelco Bellefonte, PA, USA) with an injection volume of 5 µL. The system parameters were as follows: flow rate, 0.8 mL/min; flow pressure, 82,737 Pa; oven temperature, 100 °C; detector temperature, 250 °C; and injector temperature, 230 °C. The carrier gas was helium. A standard curve was prepared that contained monosaccharide standards in the concentrations of 0, 250, 500, 750 and 1000 ng/µL and inositol was added at 750 ng/µL as an internal standard. Arabinoxylan content in acetone was calculated as 0.88 × (Arabinose + Xylose). These results were multiplied by 10 to get yield the concentration of arabinoxylan in wort (mg/L). A/X was the ratio of total contents of arabinose and xylose [25
The phenolic acid content of wort was determined according to a modification of method reported by McMurrough et al. [26
]. After adjusting pH to 2.0–2.5, 5 mL of the wort was extracted three times with ethyl ether/ethyl acetate (5/5, v
). The extracts were combined and dried under nitrogen gas at 40 °C. Acetonitile (0.5 mL) was added to dissolve the phenolics and extracts were then filtered through a 0.2 μm Nylon membrane (Pall Life Sciences, Ann Arbor, MI, USA) into an amber vial. These concentrated samples were then analyzed on an Agilient 1290 series liquid chromatography with a 6540 UHD Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA).
Separation was performed on a ZORBAX SB-C18 column (1.8 µm, 2.1 mm × 50 mm, Agilent) at 30 °C. The mobile phase consisted of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Gradient conditions were as follows: 0–1 min isocratic with 3% B; then a linear increase from 3 to 97% B for 1–10 min; followed by an isocratic washout step for 5 min and shifting back to initial setting for 2 min. Flow rate was 0.4 mL/min, and injection volume was 2.0 µL.
The selecting reference wavelengths in DAD were 260.0 nm, 275.0 nm, 294.0 nm and 324.0 nm with the band width of 2.0 nm, respectively. The AJS electrospray ionization interface (ESI) was used in the positive mode, and the absorbance threshold of the data storage was 200 (Rel. 0.01%). The stop time was 15 min and cycle time 0.5 s. Source parameters were set as the following: drying gas at the temperature of 300 °C with the flow rate of 10 L/min; 30 psig nebulizer gas at 300 °C and 7 L/min; and 125 V fragmentor energy. The mass range (m/z) of TOF Spectra was 100–1000, and the acquisition rate and time were 2 spectra/s and 5 ms/spectrum, respectively. The m/z of reference masses were 121.0509 and 922.0098. Ferulic acid, p-coumaric acid, vanillic acid, sinapinic acid, caffeic acid, catechin, syringic acid and gallic acid were quantitated with their hydrogen adduct of m/z 195.0654, 165.0545, 169.0494, 225.0756, 181.0494, 291.0882, 199.0600 and 171.0287, respectively. Calibration curves were prepared by spiking phenolic acids standards into the extract, and the response area was calculated by deducting that in control extract. The limit of detection (LOD) and quantification (LOQ) for Ferulic acid, p-Coumaric acid, Sinapinic acid, Caffeic acid and Catechin were 0.02 µg/mL wort and 0.1 µg/mL wort, respectively. For Vanillic acid, Syringic acid and Gallic acid, LOD and LOQ were 0.05 µg/mL wort and 0.2 µg/mL wort, respectively.
2.6. Experimental Design and Statistical Analysis
This study was designed according to a randomized complete block design (RCBD) with five factors in a factorial arrangement. The factors were 2 levels of samples, three levels of steep moisture (40%, 45% and 48%), four levels of germination days (3, 4, 5, and 6 days), and two levels of kernel size (plump and medium). Malting was replicated (n = 2). Data was analyzed by Analysis of Variance (ANOVA), performed with procedures of the Statistical Analysis System (version 9.3, SAS Institute, Cary, NC, USA), and analyzed using interactions, with mean separation by F-protected LSD (p = 0.05).
Main effects and interactions were evaluated using the general linear models (GLM) procedure. Duncan’s multiple range test was used to compare treatment means. Stepwise regression was used to evaluate how much variability could be explained by each independent variable (e.g., sample, kernel size, germination moisture, days of germination) for the dependent variable (e.g., malting loss, extract, DP, α-amylase, soluble protein, FAN, wort viscosity, AX, β-glucans, fermentable sugars, and phenolics).