Characterization of maritime pine bark extracts 1. Quantification of total phenolic compounds

Total phenolic compounds in pine bark extracts and in commercial tannin-rich products were quantified according to the Folin-Ciocalteu’s method, following the procedure proposed by Singleton and Rossi (1965) with some modifications based on Cheung et al. (2003).

Synthetic tannin product was not considered for this analysis. Extracts solutions were prepared using ethanol (10%, v/v) and up to 0.5 mL aliquots were reacted with the Folin-Ciocalteu’s reagent. Saturated Na2CO3 (~17%) and distilled water were added and the reaction was kept in the dark for 90 min, after which absorbances were recorded at 725 nm using a UV/VIS spectrophotometer (Jasco V-530, Japan). Results were expressed as gallic acid equivalents (GAE), in percentage (mg GAE/mg extract × 100 in a dry basis, d.b.).

7.3.4.2. Quantification of condensed tannins

The vanillin-H2SO4 methodology (Sun et al., 1998) was used to determine condensed tannins contents in pine bark extracts and in commercial tannin-rich products. Synthetic tannin was not considered for this analysis. Samples were diluted in methanol, centrifuged during 5 minutes at 3000 rpm, and the supernatant analyzed. Aliquots of each sample were reacted with 1.25 mL of the vanillin reagent (1% (w/v) vanillin in methanol) followed by 1.25 mL of the acid solution (25% (v/v) H2SO4 in methanol) at 303 K.

A non-vanillin-containing sample was run for each sample. Absorbances were recorded at 500 nm after 15 min, and results were expressed as (+)-catechin monohydrate equivalents (CME), in percentage (mg CME/mg extract × 100, d.b.).

7.3.4.3. Quantification of hydrolysable tannins

Hydrolysable tannins were assayed by reaction with KIO3, after a methanolysis step to release methyl gallate moieties, as described by Hartzfeld et al. (2002). Samples were reacted at 85 °C for 20 h in methanol and sulfuric acid, centrifuged, and the supernatant was analyzed. Ethanolamine was added and pH was adjusted to 5.5, followed by oxidation of the methyl gallate by KIO3 at 30 °C. Absorbances at 525 nm were recorded and results were expressed as methyl gallate equivalents (MGE), in percentage (mg MGE/mg extract × 100, d.b.).

7.3.4.4. Quantification of total tannins: hide powder assay

The experimental procedure followed for the quantification of total tannins by the hide powder assay was based on a method standardization development performed in the scope of the Cyted Project, in which several Ibero-American laboratories participated. It started with the preparation of 250 mL of aqueous solutions of pine bark extracts and tannin-rich products.

Total solids and soluble solids in these samples were determined gravimetrically by drying (at

~373 K) the original solutions (5 mL) and previously vacuum filtered solutions (25 mL - using a 0.45 µm cellulose acetate filter) until achieving a constant weight. The hide powder (2.0 g per sample) was placed inside a white cotton fabric and covered with distilled water.

Afterwards, it was mixed, allowed to stand for 15 minutes and squeezed. This process was repeated two more times. Sample solutions (33 mL) were added to the hide powder and the suspension was stirred (50-60 rpm) for 30 minutes, and finally vacuum filtered through a regular Whatman filter paper. The filtrate was evaporated in order to determine the non-tannins. Total tannins (%) were calculated as the difference between soluble solids (%) and non-tannins (%). If the tannin concentration in the initial aqueous extract solution was not between 3.75 and 4.25 g/L, the analysis was repeated, adjusting the extract concentration.

7.3.4.5. pH and rheological characterization

Aqueous solutions of pine bark extracts and of commercial tannin-rich products were prepared at two different concentrations, namely 20 and 50% (w/v), to cover the concentrations applied in their most common industrial applications (Garnier et al., 2001).

Mixtures were moderately stirred at room temperature using a magnetic stirrer. A Meterlab PHM210 standard pH Meter (Radiometer Analytical, France) was used to measure the pH of the 20% solutions. Rheological tests were carried out in a controlled stress rheometer (Model RS1, Haake, Germany) at 293 K. A cone-plate sensor (60 mm diameter) at a gap of 0.052 mm was used for all extract solutions, except for the solutions of P. pinaster bark extracts obtained with NaOH as the solvent additive at 50% concentration, given their higher viscosities. For these samples a plate sensor (20 mm diameter) at a gap of 1 mm was used. Flow tests were performed in triplicate with upward ramps and limit viscosities at high shear rates were reported.

7.3.4.6. Color measurements

Color (CIE L*, a*, and b*) values of pine extracts and of the commercial tannin-rich products considered in this study were measured using a hand-held Minolta CR-200b

colorimeter (Osaka, Japan). Chroma (c*) and hue angle (h*) values were then calculated. The reported results are the average of at least ten measurements.

7.3.5. Calculation procedures

The mean geometric diameter of P. pinaster bark particles was calculated according to the American Society of Agricultural Engineers ASAE S319.2 method (ASAE, 1993). Extraction yields were calculated as the ratio between the total extract mass and the raw material mass, on a dry basis. The results reported in this study are the averages of at least three measurements, and the coefficients of variation, expressed as the ratio between standard deviations and mean values (in %), were found to be less than 10% in all cases.

7.4. Results and discussion 7.4.1. Extraction yields

P. pinaster bark particles of mean geometric diameter of 0.81 mm, containing 5.5±0.4%

(w/w, d.b.) of humidity, were used in the extraction experiments. Figure 7.2 represents the extraction yields (%, d.b.) obtained from the two-hour long extractions, using a solid-to-solvent ratio of 1:10 and modified aqueous solid-to-solvents with NaOH, HCOOH (both at 0.5-1.5%) and EtOH (5-15%), either or not with 1.0% of Na2SO3. The presence of NaOH in the extraction medium was favorable in terms of the extraction yield, resulting in 21.5-38.5%, while for HCOOH and EtOH values were in the 5-7% range. An increase of the extraction yield with the additive amount was observed when NaOH and HCOOH were used, though this effect was considerably more evident for the alkaline additive. In contrast, extraction yield was not significantly affected by the amount of organic additive applied (for the tested concentrations).

NaOH is typically employed in the extraction of formaldehyde condensable phenolic compounds from pine bark, resulting in extraction yields usually above 15%, depending on several factors like particle size distribution, extraction temperature, solid-to-solvent ratio and NaOH concentration (Jorge et al., 2001; Vázquez et al., 2001). Vázquez et al. (2001) also observed an extraction yield increase with the NaOH concentration, which was attributed to an increase in the polysaccharides extraction yield, essentially hemicelluloses (Vázquez et al., 1987c), that may be credited to an increased decoupling of lignin-carbohydrate ester linkages (Sjöström, 1993). Moreover, lignin is relatively soluble in alkaline solutions (Kurth, 1947),

and significant amounts of lignin (~23%) were already detected in P. pinaster bark extracts obtained with NaOH aqueous solutions (Fradinho et al., 2002).

Figure 7.2. P. pinaster bark extraction yields (%, d.b.) obtained by two-hour long aqueous extractions at the solvent boiling point, using a 1:10 solid-to-solvent ratio. Additive amount: 0.5% for NaOH/HCOOH and 5% for EtOH; 1.0% for NaOH/HCOOH and 10% for EtOH; 1.5% for NaOH/HCOOH and 15% for EtOH; Na₂SO₃ was always applied at a 1.0% proportion.

For the 1.0% Na2SO3 extraction experiments, enhanced yields were obtained, in particular for the acidic and the organic additives, resulting in extraction yields ~1.4 fold higher for NaOH, 2.3-3.1 fold higher for HCOOH, and ~2.7 fold higher for EtOH, when compared to the ones performed without sulfite addition (Figure 7.2). This extraction yield increase may be explained by the increase in the amount of lignin and of carbohydrates in obtained extracts, due to decoupling of lignin-carbohydrate ester linkages, in an analogy to the acid sulfite process of wood delignification (Sjöström, 1993).