1260 infinity 2

Free amino acid was extracted and measured by the method described by Chen and Zhang [19 (link)] using a high performance liquid chromatograph (HPLC; 1260 Infinity II, Agilent, Santa Clara, CA, USA).
Three hydrolysis methods were used to determine total amino acids contents. The determination of methionine and cystine was based on the methods of oxidation with performic acid and hydrolysis [20 (link)]. The determination of tryptophan was based on the method of alkaline hydrolysis [21 (link)]. The determination of other amino acids was based on the method of acid hydrolysis [3 (link)]. Except tryptophan, the other amino acids were analyzed by an amino acid analyzer (S433D, SYKAM, Munich, Germany). The tryptophan was analyzed by HPLC (1260 Infinity II, Agilent, Santa Clara, CA, USA).
On the basis of the adult essential amino acid requirements, with respect to the WHO/FAO/UNU [22 (link)]. The requirement of each essential amino is histidine 15, isoleucine 30, leucine 59, lysine 45, methionine + cysteine 22, phenylalanine + tyrosine 38, threonine 23, valine 39, tryptophan 6 mg/g protein, respectively), essential amino acid score (EAAS) was calculated using the following formula: $$\mathrm{EAAS}=100\times \frac{\mathrm{essential}\mathrm{amino}\mathrm{acid}\mathrm{content}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{essential}\mathrm{amino}\mathrm{acid}\mathrm{requirement}\mathrm{for}\mathrm{adult}}$$
where the amino acid content was expressed as mg individual amino acid per g total protein.

In order to analyze tocopherol and carotenoid profile, oil samples were saponified and the unsaponifiable fraction was collected, then the lipophilic compounds were extracted according to a protocol described previously by Fromm et al. [15 (link)]. Compounds were separated on a carotenoid column (YMC, 3.0 µm 150 mm × 3.0 mm, BCS Bujno Chemicals, Warsaw, Poland) connected to HPLC Agilent Technologies 1260 Infinity II. Elution was carried out with the following solvents: line A—acetonitrile: methanol: water (15:81:4 v/v); line B—acetonitrile: methanol (50:50 v/v); flow rate of the mobile phase—1 mL/min; gradient from line A for 45 min to 100% of line B. Identification of tocopherols and carotenoids was made by comparing their retention times and spectra with the retention times and spectra of the respective standards and based on external standard curves in the concentration range of 0.01–0.2 mg/mL individual standards.

The sugars, organic acids, glycerol, and ethanol of wort and beer samples were quantitated by an HPLC-refractive index detection (RID) system (1260 Infinity II; Agilent, Santa Clara, CA, USA) equipped with a Rezex ROA-organic acid H⁺ (8%) column (300 by 7.8 mm; Phenomenex, Torrance, CA, USA).
Sample preparation and analysis were carried out as previously described (61 (link)) with a few modifications. Fermentation samples were centrifuged at 15,000 rpm for 5 min, followed by filtration of supernatant through a 0.45-μm nylon membrane filter. The filtrate obtained was loaded for sugar, organic acid, glycerol, and ethanol analysis using a mobile phase of 0.005 N H₂SO₄ at 60°C with a flow rate of 0.5 ml/min for 35 min. Serial standard solutions of compounds of interest were prepared and analyzed to establish standard calibration curves.

HPLC (High performance liquid chromatography, 1260 Infinity II, Agilent) equipped with a Raptor ARC-18 for LC-UV column (150 mm × 4.6 mm ID, pore size 2.7 µm) was used to analyze phytocannabinoids content in each fraction as described previously (Peeri et al., 2021 (link); Shalev et al., 2022 (link)). For chemical analysis of terpenes, 1 μL of each sample was analyzed by a gas chromatography-mass spectrometer (GC8860-MS5977B Agilent) equipped with 30 m, 0.25 mm ID, 5% cross-linked phenylmethyl siloxane capillary column (HP-5MS) with 0.25-μm film thickness, was used as described in (Peeri et al., 2021 (link)).

The sugar content in the enzymatic hydrolysate was determined using high-performance liquid chromatography (HPLC, Agilent 1260, USA) equipped with refractive index detectors (RID, 1260 Infinity II, Agilent, USA). The chromatographic column was an HPX-87P, and the mobile phase and column temperature were 5 mM H₂SO₄ and 80 °C, respectively. The flow rate and injection volume were 0.6 mL min⁻¹ and 10 µL, respectively. To ensure the accuracy of the test, all samples were diluted with ultrapure water before testing. The standard curves of glucose and xylose were measured. The enzymatic hydrolysis efficiency (EHE) was calculated according to the following formula:

HPLC–MS experiments were performed on the modular system Agilent Technologies 1260 Infinity II with a quaternary pump, vial sampler, column thermostat, diode-array detector, and mass spectrometer (Agilent Technologies, Inc., Santa Clara, USA) or on the Agilent Technologies 1260 HPLC system coupled to an amaZon iontrap mass spectrometer (Bruker, Bremen, Germany). A Synergi MAX-RP 80 Å column (150 × 4.60 mm, 4 micron) from Phenomenex (Aschaffenburg, Germany) was used as a stationary phase. The mobile phase comprised water (A) and acetonitrile with 0.1% formic acid (B). Elution was performed in gradient mode (0 min: 10% B, 3 min: 50% B, 5 min: 90% B, 7 min: 99% B, 11 min: 99% B, 11.1 min: 10% B, followed by 4 min of re-equilibration with 10% B). The DAD was set to a detection wavelength of λ = 430 nm, and flow rate, sample volume, and column temperature were adjusted to Q = 1.0 mL/min, V = 10 µL, and T = 40 °C, respectively. HPLC–DAD analyses were carried out on a Shimadzu LC-20AD XR system (Shimadzu Europa GmbH, Duisburg, Germany) or on an Agilent Technologies 1100 HPLC system, both equipped with a DAD detector, autosampler, and column thermostat. The chromatographic conditions were as described before. Extract components were identified using authentic reference samples [8 ] by evaluating compound-specific properties, such as retention time and mass (SI: Chapter 2.1.1).