Carrez 2

The profile of milk fermentation was assessed using high performance liquid chromatography (HPLC) as previously described before [22 (link)]. The procedure started by mixing 1 mL of filtered fermented milk with 10 μL Carrez I and 10 μL Carrez II (Sigma-Aldrich; St. Louis, MO, USA). The mixture was centrifuged at 14,000× g for 10 min at 4 °C then filtered (0.45 μm pore diameter) and the supernatant was stored till analysis at −20 °C. Directly before analysis, the samples were diluted 1:25 (v/v) with 0.0085 N sulfuric acid for assessment of the milk fermentation profile of lactose, lactate, and acetate.

The physical-chemical properties of the honey samples were determined according to standards and methods established by Codex Alimentarious [8 ], International Honey Commission [9 ], and the Association of Official Analytical Chemists [10 ]. The honey properties studied were pH, moisture, TSS, free acidity, lactonic acidity, total acidity, EC, Redox potential, HMF, and ash content. The chemical reagents used were standard hydrochloric acid (HCl) solution at 0.05 N, standard sodium hydroxide (NaOH) solution at 0.05 N, deionized water, acetone, buffer solutions, sodium bisulfite (Fermont, Canada), and the reagents Carrez I and Carrez II (Sigma-Aldrich, Saint Louis, MO, USA); all of them of analytical grade. A detailed description of the procedure for obtaining each physical-chemical property is given below.

Patulin (PAT; ≥97%) was purchased from Romer Labs Diagnostic GmbH (Tulln, Austria). Bisphenol A (BPA; ≥99%; Toronto Research Chemicals Inc., North York, Ontario, Canada) was provided by Dr. Noorfatimah Binti Yahaya (Advanced Medical and Dental Institute, Penang, Malaysia). Ochratoxin A (OTA; ≥98%), 5-hydroxymethylfurfural (HMF; ≥99%), formic acid, Carrez I, potassium hexacyanoferrate (III) (~99%), and Carrez II, zinc acetate dihydrate (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile (ACN) and ethyl acetate (EA) were purchased from Elite Advanced Materials Sdn. Bhd. (Selangor, Malaysia). Methanol (HPLC grade) was purchased from Fisher Scientific (Waltham, MA, USA). Chloroform was supplied by Merck (Darmstadt, Germany). Ultrapure water (resistivity: 18.2 MΩ cm⁻¹) was produced using a Milli-Q system (Millipore, Burlington, MA, USA) and was used throughout the study. A nylon membrane filter (0.45 µm) was purchased from LabServ Sdn Bhd (Selangor, Malaysia). Sodium hydroxide pellets were obtained from Bendosen Laboratory Chemicals (Bendosen, Norway).