Phenolic content of BSG extracts and antioxidant capacity of BSG extracts 41

The phenolic content of BSG extracts was determined by Folin-Ciocalteu and the antioxidant potential by ABTS and ORAC. These methods have been widely used in the determination of the antioxidant potential and the total phenolic content of plant extracts.

As it can be seen in Table 6, the content of total phenolics (TPC) varied from 1.83 mg gallic acid/g BSG, when ethanol was used, to 13.26 mg gallic acid/g BSG for the 60% ethanol:water extract, which represents a 0.9 fold increase in the extraction yield. It is worth highlighting that the highest amount of TPC obtained in the present work (13.26 mg gallic acid/g BSG) was higher than the values previously reported by McCarthy et al. (2013) (1.26 - 4.53 mg gallic acid/g BSG) by alkaline hydrolysis.

It is also possible to see that the extraction with 60% ethanol:water was more efficient than the one performed with 100% ethanol. According to Spigno et al. (2007) the addition of water to organic solvents leads to the increase of the polarity of medium, which facilitates the phenolic compounds extraction. The results obtained in the present work (Table 6) also showed that the extraction using only water was not as efficient as when using ethanolic mixtures as solvents, since phenolic compounds have higher affinity for less polar medium, such as ethanol (Kim & Lee, 2002).

Chapter 2 – Bioactive extracts from brewer’s spent grain

Table 6 – Total phenolic content and antioxidant capacity of the BSG extracts (Mean±S.D).

The data is given in mean ± S.D (standard deviation). For each column, equals letters it means that the difference is not significant at p > 0.005.

Water 100% ethanol 80% ethanol:water 60% ethanol:water

Folin-Ciocalteu (mg gallic acid/g

BSG) 10.28±0.07^a 1.83±0.11^b 11.25±0.09^c 13.26±0.19^d

ABTS (mg ascorbic acid/g BSG) 5.79±0.02^a 4.79±0.08^b 27.89±0.45^c 33.88±0.35^d

ORAC (mg Trolox/g BSG) 18.20±3.27^a 1.72±1.10^b 24.80±2.20^c 18.69±1.98^a

Chapter 2 – Bioactive extracts from brewer’s spent grain

Regarding ABTS and ORAC, from the results obtained it was possible to see (Table 6) that all the extracts showed antioxidant potential in both methods used, being the 100% ethanol extraction the one with the lowest antioxidant capacity. This value is much lower compared to the 60% ethanolic extract since it showed ca.7 times less activity in the case of ABTS method and ca. 10 times less than in the ORAC method. These results are similar to the ones observed by Dorta et al. (2012), who reported that using mixtures of ethanol with water produces higher antioxidant capacity than the extracts obtained using individually water or ethanol as solvents. The highest values of ABTS assay were obtained with 60% ethanolic extract, which was 5 times higher than the water extract and very close to the 80% ethanolic extract.

Using the ORAC method the extract with lowest activity was also the 100% ethanol (1.72 mg Trolox/g BSG). On the other hand, the extract with the highest antioxidant capacity was observed for the 80% ethanolic extract (24.80 mg Trolox/g BSG). Observing Table 2, the water extract (18.20 mg Trolox/g BSG) and 60% ethanolic one (18.69 mg Trolox/g BSG) had the second highest values of antioxidant activity, with no statistically difference between the results (p>0.05).

Comparing ORAC and ABTS assays, the ethanolic extract was consistently the one with the lowest antioxidant capacity, while the BSG extract with the highest antioxidant capacity differed between assays: for ABTS it was the extract with 60% ethanolic extract and for ORAC assay it was the 80%

ethanolic extract. This was an expected result due to the fact that ABTS method quantifies both water and lipid soluble antioxidants in extracts containing them (C. M. Oliveira et al., 2015), as the ORAC assay is a method that uses a radical source more biologically relevant (Prior et al., 2003).

This study is the first using ORAC as a method to quantify antioxidant capacity of extracts of BSG. Kitryte et al. (2015), using supercritical carbon dioxide extraction obtained phenolic compounds with antioxidant capacity from BSG. In this study hydrophilic and lipophilic ORAC methods were applied and the values of the antioxidant capacity varied from 9.01 to 17.41 mg Trolox/g BSG (H-ORAC) and 212.3 mg Trolox/g BSG (L-ORAC).

Socaci et al. (2018) used different concentrations of different solvents (methanol, ethanol, acetone ethyl acetate and hexane) with water to extract polyphenols from BSG. Regarding the ABTS assays, the ethanolic extracts had better antioxidant activities (1.35 mM Trolox/100 g DW for single ethanol and 1.09 mM Trolox/100 g DW for 40% ethanol water). When observing the results obtained by Socaci et al. (2018), it is possible to observe that, and contrary to what happened in this study, the extract with higher antioxidant capacity was the extract using 100% ethanol. Although there are more studies (Dorta et al., 2012, Mussatto et al., 2011)on BSG using ORAC to quantify antioxidant capacity,

Chapter 2 – Bioactive extracts from brewer’s spent grain

these are not comparable as they use extractions methodologies completely different as the one used in this study.

As phenolic compounds are soluble in polar organic solvents, mixtures with water, normally, are used to extract phenolic compounds in order to obtain a higher yield of these compounds.

2.3.2.2. Phenolic compounds

Phenolic compounds should be present in good quantities in BSG due to its composition as these compounds can be found generally in foods from vegetables sources. The phenolic content depends on the type of malt used, but typically BSG has high concentrations of p-coumaric and ferulic acids (Ikram et al., 2017). Beyond these phenolic compounds, other molecules could be identified in BSG, such as catechin, vanillin and vanillic, gallic, and syringic acids, among others (Ktenioudaki et al., 2015, Mussatto et al., 2007).

Table 7 – Phenolic compounds identified in BSG by HPLC (Mean±S.D).

Phenolic compounds

(g/g BSG) Water 100% ethanol 80%

ethanol:water 60% ethanol:water Mean±S.D. Mean±S.D. Mean±S.D. Mean±S.D.

4-hydroxybenzoic acid 20.30.8^a 42.30.7^b 59.20.7^c 104.60.2^d

Vanillin 33.91.1^a N.D. 79.21.7^b 109.20.5^c

Catechin 65.10.8^a N.D. 169.93.8^b 223.61.9^c

Vanillic acid 12.43.6^a N.D. N.D. 122.04.4^b

p-coumaric acid 7.20.1^a N.D. 42.90.3^b 25.80.3^c

Syringic acid N.D. 0.8720.7 N.D. N.D.

Protocatechuic acid 26.60.6^a N.D. 43.40.4^b N.D.

Ferulic acid N.D. N.D. 3.90.1^a 19.40.1^b

Total 100.4 43.2 394.6 604.6

The data is given in mean ± S.D (standard deviation). For each column, equals letters it means that the difference is not significant at p > 0.05. N.D. – Not detected.

By observing Table 7, the phenolic content varies depending on the extract, as expected. In water extract several phenolics were identified - 4-hydroxybenzoic acid, p-coumaric and protocatechuic acid (hydroxybenzoic acids), vanillin (benzaldehyde), catechin (flavanol), vanillic acid (hydroxycinnamic acid) – with catechin (65.1 g/g BSG) and vanillin (33.9 g/g BSG) being the compounds present at higher concentrations using this extraction solvent. Besides that, the total sum of polyphenols analysed

Chapter 2 – Bioactive extracts from brewer’s spent grain

respectively. On the other hand, when applying only ethanol as a solvent, only two phenolic compounds were identified, 4-hydroxybenzoic and syringic acids (hydroxybenzoic acids) with a very low total amount of polyphenols analysed (43.2 g/g BSG).

Vanillin, p-coumaric acid, 4-hydroxybenzoic, catechin, ferulic acid and protocatechuic acid were detected for the extract of 80% ethanolic extract. On the other hand, in the 60% ethanolic extract, catechin (223.6 g/g BSG) and vanillic acid (122.0 g/g BSG) were the main constituent of the extract, with 4-hydroxybenzoic (104.6 g/BSG), vanillin and p-coumaric acid being also identified and quantified.

Ikram et al. (2017) described that BSG different extracts were rich mainly in hydroxycinnamic acids, such as ferulic and p-coumaric. Contrary to what was expected, ferulic and p-coumaric acids were not the most abundant phenolic compounds in any of the extracts. Ferulic acid was only identified in the extraction using 80% and 60% ethanolic mixtures and it was the phenolic with the lowest concentration in both extracts. p-coumaric acid was identified in all the extractions with the exception of the 100% ethanolic extract and it was one of the phenolics with lowest concentration. The phenolic compounds identified in this study belong mainly to two groups of polyphenolic compounds (phenolic acids and flavanols) and their solubility in water increases with the increase of hydroxyl groups present in the molecules structure (Galanakis, 2018b). Hernanz et al. (2001), while doing an alkaline hydrolysis, observed that ferulic acid content in BSG can range from 1860 to 1948 g/g BSG and p-coumaric can range from 565 to 794 g/g BSG. In this work, the phenolics with highest concentration were vanillin, catechin and vanillic acid.

The differences in the phenolic profile presented in this study when compared to other phenolic profiles from BSG is probably linked to the type of cereal and brewing process that generate the BSG, type of solvents applied, as well as the methodology used to perform the extraction of phenolic compounds. The solubility of phenolic compounds depends on the chemical structure of the compounds, as described previously. Although, normally, all phenolic compounds are soluble in organic polar solvents, their solubility will depend on whether they are completely esterified, etherified or glycosylated (Bucić-Kojić et al., 2009, Galanakis, 2018). Still, in the present work the phenolic compounds profile and amount could be improved by the increasing of temperature in the extraction, as described by Bucić-Kojić et al. (2009) who observed that 80 ºC was the temperature with the highest extractability of phenolic compounds when compared to 25 ºC extraction, however this increases energy costs, and some more sensitive polyphenols can be destroyed with temperature.

Chapter 2 – Bioactive extracts from brewer’s spent grain

Comparing the polyphenol results (Table 7) with ORAC and ABTS results (Table 6), it was possible to see that the content of the different phenolic compounds quantified led to differences in the ORAC and ABTS results. Normally, flavonoids have a higher affinity with organic polar solvents such as ethanol and methanol. As stated above, the solubility of these compounds increases with the increase in the of hydroxyl groups in the compound structure, which can be observed when looking at Table 3 with the recovery of catechin, a flavonoid, in the water extract and in the ethanol:water mixtures.

However, the water extract presents a lower antioxidant activity when using ABTS to evaluate it, and when comparing the antioxidant activity of the water extract with the one of the hydroethanolic extracts, where the last have higher antioxidant activity.