The brewery industry generates waste that could be used to yield a natural extract containing bioactive phenolic compounds. We compared two methods of purifying the crude extract—solid-phase extraction (SPE) and supercritical fluid extraction (SFE)—with the aim of improving the quality of the final extract for potential use as safe food additive, functional food ingredient, or nutraceutical. The predominant fractions yielded by SPE were the most active, and the fraction eluted with 30% (v/v) of methanol displayed the highest antioxidant activity (0.20 g L−1), similar to that of BHA. The most active fraction yielded by SFE (EC50 of 0.23 g L−1) was obtained under the following conditions: temperature 40°C, pressure 140 bar, extraction time 30 minutes, ethanol (6%) as a modifier, and modifier flow 0.2 mL min−1. Finally, we found that SFE is the most suitable procedure for purifying the crude extracts and improves the organoleptic characteristics of the product: the final extract was odourless, did not contain solvent residues, and was not strongly coloured. Therefore, natural extracts obtained from the residual stream and purified by SFE can be used as natural antioxidants with potential applications in the food, cosmetic, and pharmaceutical industries.
Bioactive phenolic compounds are widely distributed in nature and are the most abundant antioxidants in the diet, being common components of fruits, vegetables, and beverages [
Beer production is an extensively studied biotechnological process that generates various by-products. The most common byproducts are generated from the main raw materials used to make beer, that is, barley malt, hop, and yeast. These by-products can be used in biotechnological processes, such as fermentative processes for the production of value-added compounds (e.g., xylitol, ethanol) as substrates for culturing microorganisms and as raw material for extraction of compounds such as antioxidants [
Beer contains a large variety of phenolic compounds which are derived from the biotechnological fermentation of barley malt (70%) and hop (30%) and which are responsible for the overall antioxidant activity of the beverage [
The composition of the extract will depend on the solvent used and also on the quality of the original material, its composition, genetic factors, environmental conditions, storage conditions, and any prior treatment. In order to obtain a high quality extract with antioxidant activity that is suitable for use in the food, cosmetic, and pharmaceutical industries, the extract must be purified to remove all inert and undesirable components, so as to improve the antioxidant activity of the extract and minimize any odour, taste, and colour [
A purification process that removes fractions with limited antioxidant activity enables a good level of antioxidant activity to be obtained from relatively small amounts of the original natural extract. Moreover, it is also important to obtain pure extracts to ensure the identity and safety of antioxidant compounds to be used as food additives [
In the present study, we evaluated two methods of purifying the crude extract—solid-phase extraction (SPE) and supercritical fluid extraction (SFE). SPE has been widely used for clean-up and purification of extracts as well as preconcentration of juices, wines, and beer. Phenolic compounds are readily fractionated by several formats of SPE in different materials of natural origin; elution with methanol on reverse-phase columns is the most popular method of separating these compounds [
Extraction and recovery of valuable compounds are the most common uses for SFE, which operate at low temperatures, in the absence of oxygen, and typically use CO2 as extraction solvent (SC-CO2). These features make SFE an ideal technique for extracting bioactive compounds [
The aims of the present study were (i) to evaluate the efficiency of the SPE and SFE techniques to purify natural antioxidants obtained from brewery waste and (ii) to determine the recovery yield and the radical-scavenging activity of the fractions obtained. Chemical analysis of the fractions by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to a diode array detector (DAD) was carried out to identify and quantify the polyphenols responsible for the antioxidant activity.
Ethyl acetate (GR for analysis), methanol (≥ 99.9%), absolute ethanol, hydrochloric acid (37%), glacial acetic acid, and acetonitrile (ACN, HPLC grade) were obtained from Merck (Darmstadt, Germany). Ultrapure water was prepared using a Milli-Q filter system (Millipore, Bedford, MA, USA). 2,2-diphenyl-1-picrylhydrazyl (DPPH, ≥ 85%) and gallic acid (≥ 98%) were supplied by Fluka Chemie AG (Buchs, Switzerland). 2,6-Di-tert-buthyl-4-methylphenol (BHT, 99.0%) and 2(3)-tert-butyl-4-hydroxyanisole (BHA, 98%) were provided by Sigma-Aldrich (Steinhein, Germany). Supercritical carbon dioxide, CO2 SCF (purity : 99.998%), was supplied by Air Liquide (Spain).
Polyphenol standards were supplied as follows: protocatechuic acid (≥ 97.0%), caffeic acid (≥ 98.0%), (−)-epicatechin (≥ 90%), acetosyringone (97%), resveratrol (≥ 99%), (±)-naringenin (95%), epigallocatechin (≥90%), (+)-catechin hydrate (98%), ferulic acid (99%), quercetin (≥ 98%), kaempferol (≥97.0%), gallocatechin (≥ 98%), p-coumaric acid (≥ 98.0%), and apigenin (≥ 97%) by Sigma-Aldrich (Steinhein, Germany); gallic acid (≥ 98.0%), syringic acid (≥ 97%), isoquercetin, and salicylic acid (≥ 99.0%) by Fluka Chemie AG (Buchs, Switzerland); and homovanillic acid (98%), 4-hydroxybenzoic acid (99%), and acetovanillone (98%) by Alfa Aesar (Karlsruhe, Germany).
In beer production, a clarification step is essential to improve beer stability. As a result of this process, a PVPP sludge is obtained in the brewing industry. The PVPP sludge loaded with polyphenolic compounds was washed with a NaOH solution (2% w/w) at room temperature. After the NaOH-PVPP was filtered, a cleaned PVPP resin and a PVPP washing solution (PVPP-WS) containing phenolic compounds were obtained (see Figure
Schematic representation of the brewing process, extraction, and purification of the PVPP-WS extract containing bioactive compounds.
The PVPP-WS (1000 L) was acidified to pH 1.5 with HCl (37%), and polyphenolic compounds were extracted with ethyl acetate (2000 L) by stirring for 30 minutes at room temperature. The organic and aqueous phases were separated by decantation, and the organic phase was collected and evaporated to dryness at 40°C. The residual water was removed from the extract by lyophilisation before the recovery yield was determined gravimetrically, and the dry extract was used in fractionation experiments (see Figure
SPE was performed with super clean cartridges (LC-18 20 mL, from Supelco, Germany) and 5 g of reversed-phase sorbent (modified silica with octadecyl groups). The crude extract (50 mg) was dissolved in 10 mL of water and loaded on the cartridge. The natural extract was eluted with different percentages of methanol (v/v): 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and finally 100% of methanol, so that eleven separate fractions were obtained at the end of the process. All fractions were evaporated to dryness, under vacuum at 40°C, in a rotary evaporator, and finally redissolved in methanol for further analysis. The recovery yield of each fraction was determined gravimetrically, and the antioxidant activity of each fraction was measured by the DPPH radical-scavenging test. The phenolic compounds responsible for the antioxidant activity were determined by HPLC-DAD.
The crude extract was fractionated using a supercritical fluid SCF R100 system (Thar Technologies, Inc.) equipped with a 5 mL SFE cell (Thar Technologies, Inc.).
Different extraction conditions were tested in three different assays. In each assay, 1 g of sample was submitted to the fractionation procedure. The assay conditions are shown in Table
SFE operational conditions tested.
Assay |
|
Time (min) | CO2 flow (g min−1) | Pressure (bar) | Modifier | Modifier (%) | Modifier flow (mL min−1) | Fractions obtained |
---|---|---|---|---|---|---|---|---|
None | None | None | A100, A120, A140, A160, A200, A250, A300 | |||||
1 | 40 | 30 | —* | 100, 120, 140, 160, 200, 250, 300 | Ethanol | —* | 0.1 | B100, B120, B140, B160, B200, B250, B300 |
—* | 0.2 | C100, C120, C140, C160, C200, C250, C300 | ||||||
| ||||||||
2 | 40 | 30 | 3 | 100, 120, 140, 160, 200, 250, 300 | Ethanol | 3 |
0.1 |
D100, D120, D140, D160, D200, D250, D300 |
| ||||||||
3 | 40 | 30 | 3 | 140 | Ethanol |
0, 0.5, 1, 1.5, 2, 2.5, 3 | 0.0, 0.02, 0.03, 0.05, 0.06, 0.08, 0.1 | F0, F0.5, F1, F1.5, F2, F2.5, F3 |
Single extracts obtained under the three different test conditions were evaporated to dryness under nitrogen steam. The recovery yield of each fraction was determined gravimetrically. Fractions were characterized by HPLC-DAD, and the antioxidant activity of each was determined by the free radical method DPPH.
Chromatographic analysis was performed on an HPLC system model 1200 HP (Hewlett-Packard, Waldbronn, Germany), equipped with a diode array detector (DAD) and controlled by HP Chemstation chromatographic software.
Chromatographic separation of polyphenols was carried out on a reverse phase Kromasil C18 column (250 × 3.2 mm internal diameter, 5
Individual phenolic compounds were identified by comparing their retention time and their UV spectrum with those obtained by injecting standards in the same HPLC conditions. Phenolic acids were monitored and quantified at 225 nm, flavan-3-ols, flavanones, flavones, and acetophenone derivates at 280 nm, hydroxycinnamic acids and resveratrol at 325 nm, and flavonols at 372 nm.
The antioxidant activity of phenolics in the crude extract and its fractions, obtained during the purification processes, were determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method described by von Gadow et al. (1997) with slight modifications [
Standard solutions of the different antioxidant fractions and of two synthetic compounds with antioxidant properties, BHA and BHT, which are commonly used in the food industry, were prepared in methanol. An aliquot of antioxidant (50
The concentration of antioxidant compound or fraction required to achieve 50% inhibition of the radical DPPH (equivalent concentration = EC50) was determined from the linear regression curve obtained by plotting the different concentrations of antioxidant compound or fraction used (within the range 0.1 to 3.5 g L−1) against the inhibition percentage of the DPPH (IP).
In this study, a new by-product (PVPP-WS) was considered as a natural source of antioxidant-rich bioactive compounds with several potential applications.
The extraction procedure used to obtain the crude extract has already been tested at laboratory scale and pilot plant scale in previous studies. With the overall aim of enabling the brewing industry to implement this extraction process in industrial plants, the present study investigated the scaling-up of the extraction process and the purification of the bioactive phenolic compound extracted. The extraction yield was approximately 0.1%. In the brewery industry, around one litre of this waste stream (PVPP-WS) can be generated from every 138 L of beer produced. Approximately 403 million hectolitres of beer were produced in Europe in 2010, which means that up to 400 tons of this crude extract could be obtained in Europe every year [
The crude extract must be processed (by purification and fractionation) as its brown colour would hinder its use as a food additive. In addition, more information about the composition of the crude extract in bioactive phenolic compounds could be obtained from different fractions to determine the correlation between the antioxidant activity and the phenolic compounds or group of phenolic compounds present in the fractions.
Solid-phase extraction is generally used for sample clean-up, fractionation, purification and/or preconcentration of natural extracts. In this study, eleven differently coloured fractions containing phenolic compounds were obtained (see Figure
The recovery yield and the radical scavenging activity were determined for each fraction obtained at each solvent ratio applied to the cartridge. The results are shown in Table
Recovery yield, radical scavenging activity (DPPH), and colouration of each fraction (Fr.) obtained by SPE with different % of methanol.
Sample | % of methanol | Recovery yield (% w/w)a | DPPH (EC50)b | Colour |
---|---|---|---|---|
Fr. 1 | 0 | 10.4 | 0.44 | Wheat |
Fr. 2 | 10 | 18.1 | 0.30 | Burnt orange |
Fr. 3 | 20 | 16.2 | 0.27 | Brown |
Fr. 4 | 30 | 19.4 | 0.20 | Maroon |
Fr. 5 | 40 | 18.3 | 0.26 | Dark brown |
Fr. 6 | 50 | 8.19 | 0.23 | Dark brown |
Fr. 7 | 60 | 6.71 | 0.89 | Ochre |
Fr. 8 | 70 | 1.59 | 7.02 | Colourless |
Fr. 9 | 80 | 0.324 | 6.64 | Colourless |
Fr. 10 | 90 | 0.615 | 8.25 | Colourless |
Fr. 11 | 100 | 0.194 | 14.3 | Colourless |
Crude extract | — | — | 0.32 | Dark brown |
BHA | — | — | 0.24 | — |
BHT | — | — | 2.67 | — |
aValues expressed as % of dry crude extract.
bValues expressed as g L−1 of extract (fraction).
The most active fractions and the best yields obtained in the SPE process corresponded to the first seven fractions eluted with a solvent mixture from 0%–60% of methanol. This showed that the natural extract and the polyphenolic compounds are water soluble but the addition of methanol yielded the most active fractions. Therefore, the fraction obtained with 30% (v/v) of methanol exhibited the highest antioxidant activity. All the fractions that display a notable level of antioxidant activity (fractions 1–7) were coloured, particularly fractions Fr. 4, Fr. 5, and Fr. 6, which also displayed the highest degree of antioxidant activity against the free radical DPPH. This is consistent with the results described by Woffendem et al. in a study evaluating the relationship between antioxidant activity and colour of crystal malt extracts [
The EC50 values of Fr. 3, Fr. 4, Fr. 5, and Fr. 6 were similar to that of the synthetic antioxidant BHA used in food industry. Except for the last 4 fractions yielded, all the fractions obtained from the crude extract showed a higher DPPH radical scavenging capacity than the antioxidant BHT, also commonly used in food industry. In this study, the antioxidant capacity of the crude extract (EC50 = 0.32 g L−1) was also calculated: (a) to compare the capacity of this extract and the fractions obtained; and (b) to evaluate whether the antioxidant activity increased as a result of the purification process. Purification of the crude extract yielded fractions with higher antioxidant activity than the crude extract.
The results of the identification and quantification of major polyphenolic compounds present in each fraction obtained in the fractionation process (SPE) by HPLC-DAD-UV are shown in Table
Phenolic compounds present in the different fractions, obtained with a LC-18 column, identified and quantified by HPLC-DAD.
Fraction | Crude extract | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Antioxidant compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | — |
mg g−1 of fraction | mg g−1 | |||||||||||
Gallic acid | 193 | — | — | — | — | — | — | — | — | — | — | 20.1 |
Gallocatechin | 178 | 641 | — | — | — | — | — | — | — | — | — | 132 |
Protocatechuic acid | — | 72.9 | 9.51 | — | — | — | — | — | — | — | — | 15.5 |
Epigallocatechin | 86.2 | 96.4 | 30.6 | |||||||||
Catechin | — | 114 | 75.5 | — | — | — | — | — | — | — | — | 29.9 |
4-Hydroxybenzoic acid | — | 6.51 | 7.49 | — | — | — | — | — | — | — | — | 2.54 |
Caffeic acid | — | 13.7 | 66.0 | — | — | — | — | — | — | — | — | 14.1 |
Epicatechin | — | — | 132 | — | — | — | — | — | — | — | — | 21.4 |
p-coumaric acid | — | — | 73.9 | 1.83 | — | — | — | — | — | — | — | 11.4 |
Isoquercetin | — | — | 166 | — | — | — | — | — | — | — | — | 28.3 |
Ferulic acid | — | — | 29.6 | 138 | — | — | — | — | — | — | — | 33.7 |
Acetosyringone | — | — | 35.2 | 50.9 | — | — | — | — | — | — | — | 14.6 |
Resveratrol | — | — | — | — | — | 51.6 | 14.5 | — | — | — | — | 5.35 |
Quercetin | — | — | — | — | 31.7 | 117 | — | — | — | — | — | 14.5 |
Apigenin | — | — | — | — | — | 58.8 | 59.7 | — | — | — | — | 8.10 |
Kaempferol | — | — | — | — | — | 25.3 | 23.4 | 120 | — | — | — | 6.06 |
Naringenin | — | — | — | — | — | 38.7 | 171 | 11.8 | — | — | — | 14.6 |
| ||||||||||||
Total (mg g−1) | 371 | 934 | 692 | 191 | 31.7 | 292 | 269 | 132 | — | — | — | 403 |
The different ratios of solvents yielded different fractions. These fractions displayed different levels of antioxidant activity because the polyphenolic content varies considerably with solubility.
The first fractions, which exhibited the highest level of antioxidant activity (see Table
Fraction 4 displayed the highest level of antioxidant activity, mainly due to the high content of ferulic acid, a phenolic compound. However, Fraction 3, which displayed a similar level of antioxidant activity to Fraction 4, also contains large amounts of antioxidant compounds such as epigallocatechin, caffeic acid, p-coumaric acid, and isoquercetin [
The recovery yields and the phenolic contents of fractions 6 and 7 were lower than those of the other fractions. However, both of these fractions displayed some antioxidant activity, mainly due to the flavonols. Fractions 6 and 7 contain the flavonols quercetin and kaempferol at similar concentrations. Fractions 8, 9, 10, and 11 were extracted using high contents of methanol, and no phenolic compounds were detected in the fractions.
SC-CO2 has been used successfully to purify crude extracts by concentrating the bioactive compounds (e.g., antioxidants) in the extract and also by removing contaminants. In the present study, several fractions were obtained from the SFE fractionation assay under different test conditions (see Figure
The extraction yield of the different antioxidant fractions obtained under the different operational conditions (see Table
(a) SFE yield determined gravimetrically (w/w) and colour of fractions A, B, and C. (b) SFE yield determined gravimetrically (w/w) and colour of fractions D and E. (c) SFE yield determined gravimetrically (w/w) and colour of fractions F and G.
|
A | B | C | |||
---|---|---|---|---|---|---|
Yield (%) | Colour | Yield (%) | Colour | Yield (%) | Colour | |
|
0.02 | Colourless | 0.24 | Wheat | 0.02 | Colourless |
|
0.02 | Colourless | 7.71 | Dark brown | 23.04 | Dark brown |
|
0.02 | Colourless | 4.27 | Burnt orange | 6.39 | Brown |
|
0.00 | Colourless | 2.77 | Burnt orange | 0.06 | Wheat |
|
0.02 | Colourless | 12.7 | Maroon | 0.01 | Colourless |
|
0.02 | Colourless | 8.09 | Dark brown | 0.07 | Ochre |
|
0.02 | Colourless | 3.29 | Maroon | 0.03 | Colourless |
| ||||||
Total | 0.12% | 39.1% | 29.6% |
|
D | E | ||
---|---|---|---|---|
Yield (%) | Colour | Yield (%) | Colour | |
|
0.89 | Brown | 3.27 | Burnt orange |
|
0.73 | Brown | 19.83 | Dark brown |
|
0.76 | Brown orange | 5.67 | Burnt orange |
|
1.42 | Burnt orange | 3.65 | Burnt orange |
|
1.13 | Burnt orange | 3.84 | Burnt orange |
|
0.79 | Burnt orange | 2.56 | Burnt orange |
|
0.98 | Burnt orange | 2.64 | Burnt orange |
| ||||
Total | 6.7% | 41.5% |
% Modifier | F | G | ||
---|---|---|---|---|
Yield (%) | Colour | Yield (%) | Colour | |
|
0.04 | Orange | 0.13 | Wheat |
|
0.07 | Coral | 0.08 | Wheat |
|
0.33 | Wheat | 0.31 | Wheat |
|
0.58 | Wheat | 0.28 | Wheat |
|
0.44 | Wheat | 0.58 | Wheat |
|
0.61 | Wheat | 0.41 | Wheat |
|
1.08 | Wheat | 0.59 | Wheat |
| ||||
Total | 3.15% | 2.38% |
Results showed that the increase in the percentage of modifier increases the amount of extract (see Table
Antioxidant activity of SFE extracts determined by DPPH. Results are expressed as EC50 (g L−1).
Fraction | |||||||||
---|---|---|---|---|---|---|---|---|---|
|
Pressure mode |
|
Flow mode | Flow mode | |||||
A | B | C | D | E | % modification | F | G | ||
|
— | — | 0.23 |
|
0.20 | 0.22 |
|
0.26 | 0.20 |
|
n.d. | 2.65 | 37.73 |
|
0.33 | 0.74 |
|
21.99 | 0.64 |
|
n.d. | 0.36 | 0.41 |
|
0.51 | 0.36 |
|
1.57 | 0.68 |
|
n.d. | 0.27 | 0.25 |
|
0.62 | 0.23 |
|
0.59 | 3.52 |
|
n.d. | 0.29 | 0.28 |
|
0.46 | 0.21 |
|
0.97 | 1.42 |
|
n.d. | 0.34 | 1.00 |
|
0.74 | 0.22 |
|
0.88 | 0.80 |
|
n.d. | 0.21 | 0.20 |
|
0.34 | 0.23 |
|
0.64 | 0.67 |
|
n.d. | 0.10 | 3.12 |
|
0.42 | 0.34 |
|
0.54 | 0.42 |
The darkest fractions, which contained more phenolic compounds per weight of extract fraction (see Tables
In the present study, methanol was also evaluated as a modifier in test G (see Table
The fractions obtained by SFE were analysed by HPLC-DAD-UV to the characterization of bioactive compounds in the crude extract.
Figure
Chromatograms acquired at 280 nm by HPLC-DAD-UV. (a) Chromatogram of the crude extract without fractionation process. (b) Chromatogram of the residue of the crude extract that remains in the extraction cell after the fractionation process (
The phenolic compounds present in the fractions obtained under optimal conditions in this study (fractionation E) are shown in Table
Phenolic profile of each fraction obtained under SFE conditions of test E: temperature 40°C, extraction time 30 minutes, modifier ethanol (6%), and modifier flow 0.2 mL min−1. Phenolic compounds are expressed as mg g−1 of fraction.
Peak no. | Phenolic compound | SFE fractions (mg g−1) | |||||||
---|---|---|---|---|---|---|---|---|---|
ECell | E100 | E120 | E140 | E160 | E200 | E250 | E300 | ||
1 | Gallic acid | 1.04 | 0.24 | 1.05 | 1.68 | 2.61 | 3.02 | 3.44 | 3.07 |
2 | Gallocatechin | 171 | 10.7 | 14.8 | 33.9 | 41.0 | 33.3 | 29.8 | 20.9 |
3 | Protocatechuic acid | — | 1.99 | 4.12 | 9.19 | 12.8 | 14.9 | 16.7 | 16.0 |
4 | Epigallocatechin | 1.43 | 10.7 | 10.2 | — | — | — | — | — |
5 | Catechin | 6.10 | 3.40 | 3.36 | 6.88 | 11.8 | 11.7 | 15.7 | 16.7 |
6 | 4-Hydroxybeinzoic acid | — | 3.58 | 1.96 | 4.78 | 4.22 | 2.72 | 2.36 | 1.55 |
7 | Caffeic acid | — | 7.29 | 7.38 | 17.1 | 19.65 | 14.8 | 13.3 | 9.52 |
8 | Epicatechin | — | — | — | — | 0.23 | 0.14 | 0.17 | 0.40 |
9 | p-coumaric acid | — | 7.97 | 5.89 | 13.5 | 13.9 | 9.66 | 8.45 | 6.26 |
10 | Isoquercetin | 2.51 | — | — | — | — | 3.71 | 3.75 | 1.43 |
11 | Ferulic acid | — | 35.7 | 27.6 | 69.0 | 70.1 | 47.0 | 38.9 | 22.0 |
12 | Acetosyringone | — | 2.92 | 0.83 | 3.62 | 2.07 | 1.18 | 3.10 | 2.55 |
13 | Resveratrol | — | — | 0.54 | 1.68 | 0.37 | 0.46 | 0.60 | 0.80 |
14 | Quercetin | 0.57 | 0.65 | 0.98 | 1.91 | 2.61 | 2.83 | 3.13 | 2.96 |
15 | Apigenin | — | — | 0.04 | 0.66 | 0.78 | 0.72 | 0.79 | 0.93 |
16 | Kaempferol | 1.48 | 0.94 | 1.65 | 3.17 | 4.42 | 3.18 | 6.24 | 6.64 |
17 | Naringenin | 0.39 | 0.40 | 0.78 | 1.69 | 2.45 | 2.75 | 3.26 | 3.52 |
| |||||||||
Total (mg g−1) | 11.0 | 88.9 | 82.5 | 172.7 | 192.8 | 155.9 | 152.3 | 117.0 |
The phenolic compounds were not successfully separated by the SFE fractionation procedure as in the SPE fractionation (LC-18 column). However, the purity of the fractions yielded by the SFE was greater than that of the fractions yielded by SPE. Moreover, large amounts of pure fractions containing the main bioactive phenolic compounds were obtained by SFE, which makes this technique the most promising for purification of the crude extract.
Regarding the antioxidant activity, the fractions with the highest contents of polyphenolic compounds are those with the highest antioxidant activity. However, the antioxidant activities of the different fractions did not differ significantly because the composition of bioactive compounds was also very similar.
Industrial extraction of a natural extract containing bioactive compounds was successful and could be implemented in the brewing industry to recover a residue with added value. The dried ethyl acetate crude extract was purified by two alternative procedures to improve its quality (antioxidant activity and organoleptic properties) for potential use as a food additive.
Both purification procedures yielded fractions with better organoleptic properties (odour and colour) and with higher antioxidant activity than the crude extract. The fractions that display strong antioxidant activity may be suitable for use as food additives (to increase the shelf life of food by preventing lipid peroxidation and protecting from oxidative spoilage during storage). Moreover, these bioactive compounds may be a good source of compounds with several applications in the food industry, as food ingredients and nutraceuticals, in the cosmetics, and in pharmaceutical industries.
The authors declare that they have no conflict of interests.
This work was financially supported by the Consorcios Estratégicos Nacionales en Investigación Técnica (CENIT) of Ministerio de Ciencia y Inovación (CENIT-2007-2016-FUTURAL). The authors are grateful to the Mahou-San Miguel Group and FUTURAL project (Ingenio Program-CDTI). The authors express their sincere thanks to Ms. Patricia Blanco Carro, Ms. Cristina Casal Romero, and Mr. Gonzalo Hermelo Vidal for their excellent technical assistance.