2.1. Characterization of Cell Wall Material
Ash, lipid content, proteins, uronic acid, soluble polysaccharides, cellulose, Klason lignin, and non-cellulosic glucose of all the different CWMs isolated were determined (Table 1
In all cases, the ash content was lower than 5%. This agrees with previous characterization of CWM from different cultivars [17
] as the main components of CWM are cellulose, pectin, hemicellulose and lignin. CWM4 and CWM5 had slightly higher protein content due to the absence of the phenol wash during the isolation process. CWM2 exhibited a higher protein content than CWM1 and CWM3 potentially due to the linking of oligosaccharides to proteins [18
]. CWM1 and CWM3, although lower than the other CWM preparations, still contained a significant amount of protein due the fact that the phenol buffer treatment only removes cytoplasmic proteins. Other types of proteins may exist within the structure of the CWM matrix such as glycoproteins and wall proteins [19
]. Regarding lipid concentration, CWM3 and CWM5 presented the highest values due to the absence of the MeOH/chloroform extraction during the isolation process. As for soluble polysaccharide content, the CWM that were extracted with HEPES buffer (CWM1, CWM3, and CWM4) presented very low amounts. The small amounts found could be explained by the existence of glycolipids and glycoproteins on the CWM that were not removed during the different washes [20
]. The difference in soluble polysaccharides between CWM2 and CWM5 may be the result of consecutive washings of CWM2 to remove other CWM components resulting in inadvertent removal of polysaccharides. The amounts of Klason lignin, cellulosic glucose and non-cellulosic glucose as well as uronic acids are comparable for all types of CWM analyzed, as they are not influenced by any of the extraction solvents used during the different isolation steps. The total polyphenolic content was less than 5% in all cases.
Our findings are in agreement with previous studies that characterized grape CWM [4
] although this study is the only known investigation of the compositional impact of each CWM cleaning step.
2.2. Adsorption Kinetics
shows the adsorption kinetics of anthocyanins during experiments performed with different types of CWM at low temperature (15 °C) in the presence of alcohol (15% EtOH). Under these conditions, the percentage of anthocyanin molecules adsorbed onto CWM varied from 28% ± 2% to 48% ± 3%. Even though each type of CWM reached a different maximum adsorption percentage, the time to this maximum adsorption was comparable for all, reaching a plateau after 60 min. Small adsorption changes between 60 and 120 min were found to be significant for all treatments. Preliminary experiments were carried out over 420 min but no significant changes were observed after 120 min (data not shown). Previous studies investigating the binding of polyphenols to different cell wall components found similar trends with the most binding occurring in the first 30 min to 1 h of contact [12
The adsorption capacity of CWM1 was larger than all others potentially due to the presence of more binding sites available for the anthocyanin molecules (Figure 1
). Conversely, CWM5 showed the lowest percentage of adsorption of anthocyanins possibly attributable to the blocking of the binding sites by the different macromolecules that would have been removed with the additional cleaning steps present in other types of CWM. The composition of the different types of CWM suggests that proteins and polysaccharides have a larger impact on the adsorption process, potentially due to their larger concentration compared to that of lipids. In addition, polysaccharides and proteins are generally larger molecules than lipids. This may explain the adsorption differences found among CWM2, CWM3 and CWM4, with CWM3 exhibiting a larger anthocyanin adsorption ratio than CWM2 and CWM4. Larger molecules would likely occupy more space within the CWM matrix effectively decreasing the accessibility to the binding sites for anthocyanin molecules resulting in lower maximum adsorption percentages. This trend was consistently found for all the experiments performed. These results are in agreement with previous findings where polyphenols were found to be bound less to CWM in the presence of proteins due to potential blocking of binding sites [12
The results showed that both temperature and EtOH concentration impacted anthocyanin adsorption. For each type of CWM, an increase in EtOH concentration and/or temperature increased the association of anthocyanin with molecules in solution rather than binding sites on the CWM. The large difference found between CWM1 and CWM 2–5 in the presence of alcohol and higher temperature (15% EtOH, 30 °C) could be attributed to expansion of the CWM matrix caused by the temperature and EtOH presence [23
]. It has also been probed that changes on temperature and ethanol can modify the interactions occurring between CWM and anthocyanins such as hydrogen bonds and coulombic interactions [10
]. The absence of the macromolecules interwoven with the CWM network (CWM1) makes this CWM more sensitive to EtOH and temperature changes leading to an opening up of the structure making more binding sites available.
Regarding the type of anthocyanin, no differences in binding were found between non-acetylated and acetylated anthocyanin. Nevertheless, the detailed anthocyanin profile showed that delphinidin-3-glucoside and petunidin-3-glucoside had a larger percentage adsorbed compared to the rest of the molecules analyzed at all the conditions analyzed. The adsorption percentage of delphinidin 3-glucoside ranged from 20% to 85% depending on the experimental conditions, and malvidin 3-glucoside adsorption percentage ranged from 10% to 70%. This trend was found for all the types of CWM suggesting that the presence of hydroxyl groups on the anthocyanin contribute to the potential hydrogen bonding between the anthocyanin molecules and the CWM polysaccharides that influence adsorption kinetics. Similar to these findings, previous studies found that non-acetylated and acetylated anthocyanin showed similar behavior in the presence of skin CWM [12
]. Additionally, Vasserot et al. obtained similar results regarding polarity (hydroxylation on the B ring) on the study of adsorption of five monoglycoside anthocyanins onto yeast CWM in the presence of alcohol [6
]. Table 2
shows the percentage of adsorption of individual anthocyanin species onto CWM1 under all the conditions analyzed. In the absence of EtOH the order of anthocyanins was: delphinidin-3-glucoside, petunidin-3-glucoside, malvidin-3-glucoside, malvidin-3-acetyl-glucoside and peonidin-3-glucoside. However, when EtOH concentration increases to 15%, the order of anthocyanins changes to delphinidin-3-glucoside, petunidin-3-glucoside, peonidin-3-glucoside, malvidin-3-glucoside and malvidin-3-acetyl-glucoside. Adsorption fluctuations could be due to the disruption of hydrogen bonds by EtOH [15
]. Additionally, the decrease in polarity of the solution in the presence of EtOH increased the concentration of the less polar molecules in solution. The presence of EtOH did not have a large impact on the adsorption process, this could be potentially due to the fact that the maximum concentration tested was 15%. Furthermore, the order of anthocyanin adsorption was not impacted by temperature changes between 15 and 30 ˚C. This trend was observed for all the different CWM matrixes tested.
In order to consider the ratio of the CWM to anthocyanin molecules in solution, weight per weight calculations were performed. Figure 2
shows the amount of anthocyanins (mg) adsorbed per mg of CWM after 120 min. The results indicate that for all the conditions CWM1 presented the highest anthocyanin adsorption value, while CWM5 showed the lowest. As stated before, CWM3 tended to reach a larger adsorption ratio than CWM2 and CWM4 likely due to the smaller size of the lipids and the absence of larger molecules blocking the binding sites, although it was not significant in all cases. Moreover, lipids are more significantly influenced by temperature and EtOH concentrations, modifying their fluidity and likely making binding sites more available when the EtOH content or the temperature increases compared to other macromolecules [25
Significant differences in the binding response between anthocyanin and the CWM at different conditions were determined from triplicate experiments using a multi-way analysis of variance (MANOVA). The results indicated that all the variables (temperature, cell wall composition and EtOH concentration) have a significant impact on the adsorption process (p < 0.001).
It has been observed that anthocyanin molecules can undergo thermal degradation by breaking the O
-glycosidic bond [26
]. In this study, the potential presence of break-down products produced by the degradation of anthocyanins was investigated by means of LC-DAD-MS/MS. In all samples, all screened break-down compounds fell below the LOD indicating changes in anthocyanin concentration were due to adsorption. This could be due to the fact that 30 °C is a low temperature to breakdown the short time period of the experiment (2 h).
2.3. Desorption Kinetics
Desorption assays were performed under the same sets of temperature and EtOH as those for the adsorption experiments. The rates of the desorption process were faster than adsorption reaching a plateau within the first 30 min. Figure 3
shows the kinetics of desorption for CWM2 at all the conditions tested. As can be observed, the desorption kinetics depended not only on the conditions of the experiment but also on the amount of anthocyanin initially adsorbed onto the CWM. Concerning the type of anthocyanin, delphinidin-3-glucoside and petunidin-3-glucoside showed the lowest percentage of desorption suggesting the breakdown of hydrophobic interactions by the solvent prior to hydrogen bonds. Similar trends were found for the other CWMs studied.
shows the amount of anthocyanin molecules adsorbed at the beginning of the desorption experiment, the amount released after 120 min and the percentage desorbed after 120 min for each of the experiments performed. At low temperature, the presence of alcohol resulted in an increase in the desorption percentage likely due to the disruption of the hydrophobic interactions [29
] or an increase in the solubility of anthocyanins in solution. A similar trend was observed when the temperature was increased in the absence of alcohol. However, at a higher temperature in the presence of EtOH (15% v
) this trend was not noted (increase on the desorption), potentially due to the expansion of the CWM [23
] and the low amount adsorbed of anthocyanin adsorbed under these conditions.
Follow up experiments performed with CWM with the same amount of anthocyanin adsorbed indicated that both, temperature and EtOH concentration increase the desorption rate with temperature having a larger impact (data not shown).
To better understand the types of interactions taking place during adsorption it is important to point out that at the working pH, approximately 18% of the anthocyanin molecules were positively charged [30
], while the CWM fibers (mainly cellulose, hemicellulose and pectin derivates) have been shown to have a negative surface charge. The results suggest the presence of different types of interactions between the CWM and the anthocyanin molecules. The existence of a base layer with the strongest interactions (coulombic interactions) between the anthocyanin and the CWM cellulose/pectin network, that will increase at higher temperatures due to the expansion of the CWM fibers, has been previously reported [10
]. Additionally, hydrogen bonding between the hydroxyl groups of anthocyanins and the oxygen atoms of the cross-linked ether bonds of sugars present in the CW polysaccharides [24
] as well as hydrophobic interactions take place. Moreover, π-π interactions between anthocyanin molecules can form anthocyanin self-association complexes, which can potentially stack on to the CWM. CWM is also a complex porous structure, which may trap molecules in solution.
The desorption results suggest that the amount of anthocyanin molecules released from the CWM depends on the strength of their interactions. Unlike the adsorption experiments, no clear trends on the desorption process were found depending on the CWM composition, although the quantity of molecules adsorbed was found to be dependent on CWM composition. The presence of the different macromolecules on the CWM modify the availability of the binding sites thus controlling the amount of anthocyanin adsorbed. Adsorption results suggested that anthocyanins do not interact with the macromolecules (soluble polysaccharides, cytoplasmic proteins and lipids) within the CW network and only with the polysaccharide network (primarily cellulose, pectin and hemicellulose) itself. Thus, the macromolecule composition of the CWM does not have a direct impact on the desorption process, explaining similar desorption from the different types of CWM.