Ferulic acid

The total phenolic compounds (TPCs) were extracted from 1 g of whole meal flour of dry chickpea seeds with 10 mL of an aqueous-methanol solution (20:80 v/v). The suspension, put in centrifuge tubes, was stirred for 2 h in the dark, then centrifuged at 12,000× g for 3 min to recover the supernatant, which was subjected to Folin-Ciocalteu reaction in the conditions reported in Pasqualone et al. [54 (link)] and subsequent spectrophotometric measurement at 765 nm by a Cary 60 UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The content of TPC was expressed as mg g⁻¹ of ferulic acid on dry matter (d.m.), considering a calibration curve previously prepared with ferulic acid (Merck KGaA, Darmstadt, Germany) at different concentrations. The analysis was carried out in triplicate.

The water bath used for storage of the co-pigmented samples was previously described elsewhere [35 (link)]. Ferulic acid (Sigma Aldrich, Denmark) was selected as model co-pigment based on previous studies on co-pigmentation of cyanidins obtained from blueberry wine pomace and mulberry juice [36 (link),37 (link)].
Anthocyanin dry extracts produced by adsorption–desorption were re-dissolved in acidified water (pH 3.0), and solid Ferulic acid was added in order to reach four different anthocyanin: co-pigment molar ratios, namely 1:23, 1:48, 1:86, and 1:172. A sample without co-pigment addition was used as control.
Absorption spectra of the samples were recorded using a spectrophotometer DR 3900 (Hach, Düsseldorf, Germany) after 60 min, one week, two weeks, and one month in storage (5 °C, darkness).
The magnitude of the co-pigmentation effect was measured as the increase in absorption maximum wavelength (bathochromic shift, Equation (1)) and percentage increase of absorption intensity (hyperchromic shift, Equation (2)).
$$\Delta {\lambda }_{max}={\lambda }_{max}-{\lambda }_{max,0}$$
$$\Delta A (\%)=\left[\frac{A_{max}-A_{max,0}}{A_{max,0}}\right]·100$$
where λ_max and λ_max,0 are the maximum absorption wavelengths (nm) of the co-pigmented and control samples at a given time, respectively; A_max and A_max,0 are the maximum absorbance intensities (mAu) of the co-pigmented and control samples at a given time, respectively.