Antioxidant Activity of Polyphenols Extracted From Hop Used in Craft Beer

ANTIOXIDANT ACTIVITY OF POLYPHENOLS EXTRACTED FROM HOP USED IN CRAFT BEER

9

Jorge Carlos Ruiz-Ruiz*, Gabriela del Carmen Esapadas Aldana†, Alma Irene Corona Cruz†, Maira Rubí Segura-Campos† ⁎

School of Nutrition, Health Sciences Division, Anáhuac-Mayab University, Mérida, Yucatán, México, †Faculty of Chemical Engineering, Autonomous University of Yucatan, Mérida, México

9.1 Introduction Phenolic acids and flavonoids are the most abundant antioxidants in diet, being the common components of fruits, vegetables, and its derivatives (Martins et  al., 2011). Recent studies have correlated the consumption of foods rich in phenolic compounds, with the prevention of noncommunicable diseases, such as cardiovascular diseases, certain types of cancer, and diseases related to aging (Scalbert et al., 2005). The biological effects derived from phenolic compounds have been attributed to their antioxidant activity (Balasundram et al., 2006). Beer production is a process that generates by-products, such as barley malt, hop, and yeast. These by-products can be used in biotechnological processes for the production of value-added compounds as substrates for culturing microorganisms and as raw material for extraction of bioactive compounds (Mussatto, 2009). Beer contains a large variety of phenolic compounds, which are derived from the fermentation of barley malt (70%) and hop (30%). Only 15% of the hops constituents end up in the beer, 85% will become spent hop material (Callemien and Collin, 2007). Chemical composition of hop cones (Humulus lupulus) includes 0.3%–1% volatile oil, resins, bitter principles, 2%–4% tannins, flavones, and flavanones (izoxanthohumol). Hop is mainly used in brewery but also has applications in pharmaceutical purposes. In traditional medicine it is used to treat abdominal cramps, anemia, bacterial infections, dermatitis, diarrhea, dysmenorrhea, leucorrhea, migraine, and e­ demas Biotechnological Progress and Beverage Consumption. https://doi.org/10.1016/B978-0-12-816678-9.00009-6 © 2020 Elsevier Inc. All rights reserved.

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(Bubueanu et  al., 2015). Hop polyphenols had the same or higher ­antioxidant activity than tea polyphenols. Hop polyphenols might be useful as natural antioxidants and antimutagens (Wang et al., 2014).

9.2  Food Processing Wastes In the food industry, the convectional significance of processing is associated with transformation of the initial raw material into a safe, nutritious, and high-quality food product. However, in a modern biobased society, food processing should also provide viable alternative models that combine food production with valorization of waste and by-product, minimization of energy consumption, and environmental protection (Lin et al., 2014). Food wastes are produced throughout all the food life cycle, 42% of them are produced by households, 38% occurs during food processing, and 20% is distributed along the whole productive chain. Food industry wastes are derived from the processing of raw vegetable and animal materials into foodstuffs, which generally consists of the extraction or separation of the nutritional portion from the remains having low nutritional value or inedible components (Oreopoulou and Russ, 2007). Most food waste is derived from the drink industry (26%), followed by the dairy industry (21.3%), the production and preservation of fruits and vegetables (14.8%), the manufacture of grain and starch products (12.9%), the production, processing, and preservation of meat products (8%), the manufacture of vegetable and animal oils and fats (3.9%), and the production and preservation of fish and fish products (0.4%) (Laufenberg et al., 2003). The potentially marketable components present in food wastes need to be separated from the matrix through combined approaches for selective extraction and modification of the selected components and changed into products of higher value (Baiano et al., 2014a, b). These operations must be performed avoiding microbiological hazards and ensuring that the final products comply with the existing food regulations and meet consumer liking. Acceptability is a very important characteristic since the potential customers could perceive the use of coproduct valuable components as a reduction of the food quality. The optimization of the extraction conditions using conventional and emerging techniques and the use of the recovered biomolecules and by-products have been the object of several studies (Baiano, 2014). Examples include the use of combined conventional and microwave assisted extraction of antioxidants from vegetable solid wastes (Baiano et al., 2014a) or the replacement of flour with spent biomass derived from the production of beers in order to produce functional breads (Baiano et al., 2014b). The extraction of the high-value components must be economically feasible to perform. This objective can be achieved by separating the components of interests through individual and/or combined physical and biochemical approaches so as to provide a range of

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c­ omponents, from high value to low value, all of which would contribute to achieving whole-waste exploitation. In the case of coproducts unsuitable for food exploitation, they must be used as energy sources (Waldron, 2009). These statements clearly highlight that the valorization of food wastes can be achieved through an integrated biorefinery approach, in order to produce bioactive molecules for pharmaceutical, cosmetic, food, and nonfood applications (Baiano, 2014).

9.3  Wastes From Beer Production: Chemical Composition and Uses The brewing industries produce millions of tons of residues, which represent a management issue from both ecological and economical point of view. The accumulation of huge amounts of this biomass every year leads to environmental degradation and especially to significant loss of valuable material that could otherwise be exploited as food, fuels, and a great variety of additives. The valorization of brewing by-products can be achieved through the extraction of high-value components, such as proteins, polysaccharides, fibers, flavor compounds, and phytochemicals, which can be reused as nutritionally and pharmacologically functional ingredients (Mussatto, 2014). Brewery waste is a typical example of such unexploited potential. The most common by-products are spent grain, spent hops, and surplus yeast, which are generated from the main raw material used for beer elaboration. They represent large potential resources for use in biotechnological processes, as for example, in fermentative processes for the production of value-added compounds (ethanol, xylitol, lactic acid, among others) as substrate for microorganism cultivation, or simply as raw materials for extraction of compounds, such as proteins, sugars, acids, and antioxidants (Fărcas et al., 2013). Brewery waste shows significant and rich chemical composition, whose main components are summarized in Table 9.1. Taking into account the modern model of breweries, which purchase the barley malt from malting industries, barley malt or malt bagasse is the first solid waste to be generated through the brewery process. This is the residue that is generated in greater quantity and the one that has the highest nutritional value (Table 9.1). For every 100 kg of malt, between 120 and 135 kg of malt bagasse are generated, the above indicates that for each 100 L of beer, 14–20 kg of malt bagasse are generated (Aliyu and Bala, 2011). This bagasse contains between 15% and 26% protein and 70% fiber, of which 15%–25% is cellulose, 28%–35% is hemicellulose, and 28% is lignin. The residue also contains lipids (3.9%–10%) and ash (2.5%–4.5%) (Robertson et al., 2010). This waste is regularly used for animal feed. However, a number of potential uses are suggested (Table 9.2). Potential uses include animal and human nutrition, energy production, charcoal production, adsorbent material, microorganism

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Table 9.1  Chemical Composition of Brewery Wastes Component

Malt Bagasse

Hot Trub

Yeast

Fibers Carbohydrates Protein Free amino acids Ash Vitamins Phenolic compounds Fatty acids

√ – √ √ √ √ √ –

– √ √ – √ – √ √

– √ √ √ √ √ – √

Source: Dos Santos-Mathias, T.R., Moretzsohn de Mello, P.P., Camporese-Sérvulo, E.F., 2014. Solid wastes in brewing process: a review. J. Brew. Distil. 5(1), 1-9.

Table 9.2  Potential Application of Malt Bagasse Application

Reference

Animal feed and human nutrition Energy and biogas production Protein concentrates Fermentation products: ethanol, lactic acid, gums, antibiotics, enzymes Support for cell immobilization

Gupta et al. (2013) Gopi and Sang (2013) Niemi et al. (2013) Gencheva et al. (2012) Dos Santos-Mathias et al. (2014) Dos Santos-Mathias et al. (2014)

cultivation, support for cell immobilization, and obtaining bio products by fermentation (Aliyu and Bala, 2011). The second solid residue generated during brewing process is the hot trub; precipitate that results from insoluble coagulation of proteins with high molecular weight, during the boil. Its composition includes proteins (50%–70%), bitter substances from hops (10%–20%), polyphenols (5%–10%), carbohydrates (4%–8%), and fatty acids (1%–2%). After removal by filtration or by centrifugation, this waste is mixed with malt bagasse for preparation of animal feed. However, this waste has the potential to be used in various bioprocesses, due to its high content of proteins and phenolic compounds (Dos Santos-Mathias et al., 2014). With respect to yeast, this waste is generated in amounts of 1.5–3.0 kg per 100 L of beer. The amount of biomass depends on the parameters such as aeration, temperature, pH, inoculum concentration, type of microorganism, and cell viability. Its composition includes protein

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Table 9.3  Potential Application of Residual Yeast Application

Reference

Production of flavoring agents Medias of supplementation for microorganisms Substrate for microalgae cultivation Biosorption of heavy metals for soil remediation Biogas production

Vieira et al. (2013), Ferreira et al. (2010) Ferreira et al. (2010) Byung-Gon et al. (2013) Chen and Wang (2008) Zupančič et al. (2012)

(35%–60%) with high biological value, carbohydrates (35%–40%), minerals (5%–7.5%), and lipids (4%–6%) (Pinto et al., 2013). The most common use for residual yeast is mixed with malt bagasse for formulation of animal feed. Some studies reported the use of residual yeast for obtaining compounds with high biological value (Table 9.3).

9.4  Recovery of High Value-Added Components From Food Wastes Food wastes are considered an important source of nutraceuticals and it could be utilized as an efficient measure to deal with the prospects of feeding fast growing population (Parfitt et al., 2010). Food wastes are residues of high organic load, which are derived during raw materials processing to foodstuff and result in liquid or solid form. The fact that these substances are removed from the production process as undesirable materials defines them as wastes. Nevertheless, discharging of wastes does not account the potentiality of reutilizing. For this reason, the term food by-products is increasingly used among the related scientists in order to notify that food wastes are substrates for the recapture of functional compounds and the development of new products (Galanakis, 2012). Recent research provides scientific evidence to support the hypotheses that phytochemicals recovered from agro-industrial wastes can provide a range of health benefits to the consumers. This fact has impacted the food and pharmaceutical industries, among others. The phytochemical extracts can be used either for their biological properties as ingredients for nutraceutical preparations, functional foods, or for their food-quality-related properties (Fărcas et al., 2013). The extraction, fractionation, and isolation of high value-added compounds from food wastes follow the principles of analytical chemistry: (a) maximizing the yield of the compounds, (b) suiting the demands of industrial processing, (c) clarifying the high value-added ingredients from impurities and toxic compounds, (d) avoiding deterioration and

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loss of functionality during processing, and (e) ensuring the food grade nature of the final product. Processing progresses from the macroscopic to the macromolecular level (Fig. 9.1) and afterwards to the extraction of specific micromolecules, prior to the purification and encapsulation of the target ones (Galanakis, 2012). This downstream scheme is selected if two different ingredients are recovered or the valuable component is a micromolecule (i.e., antioxidant). On the contrary when the target compound is a macromolecule (i.e., protein), the second stage may be omitted.

I. Macroscopic pretreatment Wet milling, thermal and/or vacuum concentration, mechanical pressing, freeze drying centrifugation and microfiltration

II. Macro- and micro-molecules separation Alcohol precipitation, ultrafiltration, isoelectric solubilization-precipitation, extrusion

III. Extraction Solvent, acid, alkali, microwave-assisted steam diffusion, hydrodistillation, supercritical fluid

IV. Isolation and purification Adsorption, chromatography, nanofiltration electrodialysis

V. Product formation Fig. 9.1  Recovery stages of high value-added components from food wastes (Galanakis, 2012).

Spray- and freeze-drying emulsions, extrusion

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Conventional technologies (i.e., ultrafiltration and alcohol precipitation) for macro- and micromolecules separation are considered both as safe and cheap.

9.5  Polyphenols of Hop The amount of hops required in beer production is significantly smaller. However, this minor ingredient has a crucial impact on beer quality. The complex chemistry associated with hop substances and their function has been the subject of extensive investigation. Dried cones of hop (Humulus lupulus L.) are a bulky product, containing only about 20% w/w of valuable brewing compounds (resins, essential oils, and polyphenols). The majority of hop for brewing is processed into hop pellets or extracts. Waste hop is a rich source of phenolic antioxidants, which could be reused for many industrial or pharmaceutical purposes. Hop polyphenols comprise up to 4% of the total weight of dried hop cones. The higher concentration of polyphenols is located in hop petals (Jelínek et al., 2014). About 20%–30% of the polyphenols found in the wort come from the hop material. The importance of the hop polyphenols in the brewing process is due to protein-polyphenol interaction of nonbiological haze, which limits the shelf life of bottled beers (Almaguer et  al., 2014). Polyphenols are a very heterogeneous group of substances that are built up by multiple phenol units and share a common structural element: an aromatic ring with at least two hydroxyl groups. Polyphenols composition in hop is conditioned by variety, cultivation area, harvesting technique, and degree of aging. Some studies reported that aged hops contain higher content of polyphenols than the fresh ones (Jelínek et  al., 2014). Studies using high-performance liquid chromatography-diode array detection indicated the presence of over 100 compounds in the polyphenol fraction of hops. Hop polyphenols can be classified into flavonols, flavan-3-ols, phenolic carboxylic acids, and other polyphenolic compounds (Table 9.4). Flavonols and flavan-3-ols are classified as flavonoids, a subgroup of polyphenols. Some polyphenols are unique to hops: multifidol glucosides and prenylflavonoids, such as xanthohumol, desmethylxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin (Biendl, 2009). Approximately 20% of the total hop polyphenols consist of low-molecular-weight substances or monomer substances, such as the phenolic carboxylic acids as well as the flavonoids and their glycosides. Flavonoids in hop are constituted by catechins and their polymers, proanthocyanidins, quercetin, and kaempferol (Almaguer et al., 2014). Catechin and epicatechin (flavan-3-ols) are the monomers of proanthocyanidins or condensed tannins, which may also contain

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Table 9.4  Composition and Concentration of Hop Polyphenols (Almaguer et al., 2014) Compounds

Concentration (%)

Phenolic carboxylic acids Benzoic acid derivatives Cinnamic acid derivatives Flavonoids Xanthohumol 8-,6-Prenylnaringenin Quercetin Kaempferol Catechins and epicatechins Oligomeric proanthocyanidins Acylphloroglucinol derivatives

_ <0.01 0.01–0.03 _ 0.20–1.70 <0.01 0.05–0.23 0.02–0.24 0.03–0.30 0.20–1.30 0.05–0.50

gallocatechin and epigallocatechin. The first molecules consist of up to eight monomer units (oligomers) and the second ones consist of a higher number of monomers. These are the most reactive substances of the polyphenol fraction (Biendl et al., 2012).

9.6  Extraction Technologies for Phenolic Compounds 9.6.1 Solid-Liquid Extraction This technique allows soluble components to be removed from solids using aqueous organic solvents. Solvents should be chosen carefully to avoid chemical or physical interference with the matrix. For this technique, parameters such as temperature, time, pH, solid–liquid ratio, particle size, stirring, and solvent polarity must be optimized in order to obtain high yields of recovery of the selected compound. Some disadvantages to the application of this technique include: cost, toxicity, and solvent flammability, as well as the long extraction times. Its main industrial application is to obtain compounds from herbal materials. Some trends in this extraction technique include the use of less expensive and toxic solvents and their use in combination with other extraction techniques (Baiano et  al., 2014a, b; Luthria, 2008).

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9.6.2 Soxhlet Extraction This technique of extraction consists of contacting the matrix with pure and hot solvent, repeatedly. In this way the solubilization of the compound to be extracted is greater. Due to its operational characteristics, this technique is economical in terms of time, energy, and reagents. Small-scale Soxhlet extraction takes place in batches; however, it can be adapted to operate continuously in industrial processes. It also has more advantages than novel techniques, such as ultrasoundassisted, microwave-assisted, supercritical fluid, and accelerated solvent extractions in terms of industrial applications, reproducibility, efficiency, and extract manipulation. The main disadvantage is the sensitivity of some compounds to the temperature conditions of extraction. Some variants of this technique are: high-pressure, ­automated, ultrasound-assisted, and microwave-assisted Soxhlet extraction (De Castro and Priego-Capote, 2010).

9.6.3  Pressurized Fluid Extraction and Supercritical Fluid Extraction The first extraction technique is similar to Soxhlet extraction, except that the solvents are used in constrictions near their supercritical region, so that the elevated temperature allows a greater diffusion and solubility of the solute to be extracted. While the high pressure applied to the system keeps the solvent below its boiling point, allowing a higher concentration of this in the matrix. These operating conditions allow the use of low solvent volumes and reduce extraction times. The second extraction technique consists of the separation of a compound (solid or liquid) from a matrix, using fluid as a solvent under supercritical conditions. Under supercritical conditions a fluid coexists in both vapor and liquid states. One of the most commonly used fluids is carbon dioxide (CO2), which is sometimes combined with ethanol to change its polarity. The advantages of CO2 as extraction fluid are: moderate supercritical conditions (31.1°C and 73.8 MPa), absence of toxicity, chemical stability, easy to recycle, and low-cost. The advantages of supercritical extraction are: extraction capacity similar to liquid organic solvents and the extracts are more pure. Industrial use of supercritical fluid extraction is limited since this technique has been developed in isolation of other processing steps that are generally necessary to obtain a product (Baiano et al., 2014a, b).

9.6.4  Ultrasound-Assisted Extraction Ultrasound-assisted extraction has been used to extract bioactive compounds, such as antioxidants, essential oils, steroids, and lipids from plants. The use of ultrasound improves the penetration of

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the solvent into cellular materials, facilitating mass transfer and the release of the compounds to be extracted. The frequency of ultrasound has a great influence on the yield and extraction kinetics. At frequencies >20 kHz sound waves generate expansion-compression cycles, in a liquid this results in the formation of bubbles that grow and collapse near the solid matrix, facilitating extraction (Khoddami et al., 2013).

9.6.5 Microwave-Assisted Extraction Microwaves are electromagnetic waves consisting of an electric field and a magnetic field that oscillate perpendicularly to each other at frequencies between 0.3 and 300 GHz. The microwave energy acts directly on the molecules by ionic conduction and dipole rotation, reason why only polar materials can be heated in this manner. The microwave-assisted extraction depends on the dielectric susceptibility of both solvent and matrix. Because the water inside the matrix absorbs microwaves, the disruption of the material is determined by an internal overheating, which also improves the recovery of the extracted compound. Microwave-assisted extraction is classified into closed and open systems. In a closed system, the extractions are carried out in a sealed vessel under uniform heating; in this system the high pressure and temperature allow rapid and efficient extraction. On the other hand, open systems are more suitable for extracting thermolabile compounds, since they operate under less extreme conditions (Chan et al., 2011).

9.6.6  Pulsed Electric Field Extraction In plant and animal tissues, cellular walls, and membranes act as barriers, which prevent the extraction of bioactive compounds. The application of a pulsed electric field above the transmembrane potential of the cell (1 V) generates pores (electroporation). The pores may form reversibly or irreversibly, depending on the intensity of the electrical pulses. When a low-intensity treatment is applied, the pores are small compared to the total area of the membrane and the electric breakdown is reversible. On the contrary, increasing the intensity and duration of the treatment, the permeability of the membrane is irreversible (Xue and Farid, 2015).

9.6.7 Enzyme-Assisted Extraction This technique is an alternative to conventional solvent-based extraction methods. It is based on the ability of enzymes to catalyze reactions with specificity and regioselectivity in aqueous solutions. Enzymes with hydrolytic activity on the components of cell

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­ embranes, such as pectinases, cellulases, hemicellulases, etc. inm creases cell wall permeability and the extraction yield of bioactive compounds, such as pigments, antioxidants, and compounds with pharmaceutical applications (Puri et al., 2012).

9.7  Quantification of Phenolic Compounds High-performance liquid chromatography (HPLC) and gas chromatography (GC), or their combinations, with mass spectrometry are the two most applied methods to quantify phenolic compounds. Other techniques include spectrophotometric assays (Khoddami et al., 2013).

9.7.1 Spectrophotometric Assays These techniques are relatively simple, most common, and include Folin-Denis and Folin–Ciocalteu. Both methods are based on a chemical reduction involving reagents containing tungsten and molybdenum (Stalikas, 2007). The products of this reaction absorb at a wavelength around 760 nm. However, the reactions are not specific for phenols but also with other substances, such as ascorbic acid, aromatic amines, and sugars (Khoddami et al., 2013). Total phenolic quantification, total flavonoids, proanthocyanidin (condensed tannin), and hydrolyzable tannin can be estimated by colorimetric methods (Fernandes et  al., 2012). Vanillin and dimethylaminocinnamaldehyde (DMCA) assays are employed to determine the degree of polymerization, the hydroxylation pattern, and stereochemistry of flavan-3-ol subunits in proanthocyanidins. The butanol-HCl and bovine serum albumin (BSA) methods are also used for proanthocyanidin determination (Abeynayake et  al., 2011). For hydrolyzable tannins quantification, the most common methods are iodate, rhodanine, and sodium nitrite (Jin and Mumper, 2010). Spectrophotometric techniques to determine phenolic are simple and economical but only provide an estimation and do not quantify phenolic individually. However, these techniques can be useful for quick and inexpensive screening of numerous samples (Ignat et al., 2011).

9.7.2  Gas Chromatography (GC) It is applied to separate, purify, and quantify phenolic acids, condensed tannins, and flavonoids (Martin et  al., 2012). For the quantification of phenolic compounds by GC previous stages of sample conditioning are necessary, remotion of lipids, release of phenolic compounds from the glycoside and ester bonds in enzymatic, alkaline, or acidic media and finally chemical modification to generate

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volatile derivatives (Khoddami et  al., 2013). As derivatizing agents the following reagents are used: ethyl chloroformate, methyl chloroformate, diazomethane, and dimethyl sulfoxide in combination with methyl iodate are used to obtain methyl or ethyl esters of phenolic compounds. New reagents, which have advantages for obtaining volatile compounds, are trifluoroacetamide, N-(tert-butyldimethylsilyl)N-methyltrifluoroacetamide, and trimethylsilyl derivatives. Silylation does not generate interfering by-products (Zadernowski et al., 2009). Flame ionization detector (FID) and mass spectroscopy (MS) are the most common methods to detect derivatives of phenolic compounds. GC provides more sensitivity and selectivity when combined with mass spectrometry (Ignat et al., 2011).

9.7.3 High-Performance Liquid Chromatography HPLC is the most used technique for separation and quantification of phenolic compounds. The factors influencing the analysis of phenol compounds by HPLC include: sample purification, mobile phase, column types, and detectors (Khoddami et  al., 2013). Usually the analysis of phenolic compounds using HPLC is carried out utilizing a reversed-phase C18 column (RP-C18), photo diode array detector (PDA) and polar acidified organic solvents (Ignat et al., 2011; Steinmann and Ganzera, 2011). HPLC sensitivity and detection is based on purification of phenolic compounds and preconcentration. These operations include removing the interfering compounds from the crude extract with partitionable solvents and using open column chromatography or an adsorption–desorption process (Steinmann and Ganzera, 2011). The most common materials for phenolic compounds purification are Sephadex LH-20, polyamide, Amberlite, solid phase extraction (SPE) cartridges, styrene-divinylbenzene, and acrylic resins (Ignat et al., 2011). Acetonitrile and methanol, or their aqueous forms, are the dominant mobile phases utilized in HPLC quantification of phenolic compounds. It is recommended to maintain the pH of the mobile phase in the range pH 2–4 to avoid the ionization of phenolic compounds during identification. Aqueous acidified mobile phases predominantly contain acetic acid but formic and phosphoric acids or phosphate, citrate, and ammonium acetate buffers at low pH are also reported. A gradient elution system provides greater resolution and separation than an isocratic elution system (Diagone et al., 2012). Different phenolic compounds can be detected using a normal phase C18 or reversed-phase (RP-C18) column 10–30 cm in length, 3.9–4.6 mm ID, and 3–10 μm particle size. New types of columns (monolithic and superficially porous particles columns) from 3 to 25 cm length, 1–4.6 mm ID, and 1.7–10 μm particle size are employed in phenolic detection by ultra-high pressure chromatography (UHPLC), high-temperature liquid chromatography (HTLC), and two-dimensional liquid c­ hromatography

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(2-D LC) (LC × LC) (Lopes-Lutz et al., 2010). Identification is made by UV–VIS and photodiode array (PDA) detectors at wavelengths 190– 380 nm. Other techniques include fluorimetric (FLD), colorimetric arrays, PDA coupled with fluorescence, and chemical reaction detection techniques (De Villiers et  al., 2010; Moze et  al., 2011). For structural characterization and confirmation of different phenolic classes the most common techniques are mass spectrometric (MS) detectors attached to HPCL (HPLC-MS) (Moze et al., 2011), electrospray ionization mass spectrometry (ESI-MS) (Do Amaral et al., 2012), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (Wei et al., 2010), fast atom bombardment mass spectrometry (FAB-MS), and electron impact mass spectrometry (Khoddami et al., 2013). The new trends in the analysis of phenolic compounds are hydrophilic interaction liquid chromatography (HILIC) as well as 2-D LC (Zeng et al., 2012).

9.8  Total Antioxidant Capacity (TAC) Assays Applied to Phenolic Compounds Polyphenols as secondary metabolites are not considered as nutrients although they are important components in human diet. The interest of its possible effects on health lies in its antioxidant and free radical scavenging activities observed in in vitro assays (Procházková et al., 2011). Antioxidant mechanisms of polyphenols include (1) suppression of reactive oxygen species (ROS) formation either by inhibition of enzymes or by chelating trace elements involved in free radical generation; (2) scavenging ROS; and (3) upregulation or protection of antioxidant defenses (Kumar and Pandey, 2013). Phenolic acids and flavonoids inhibit the enzymes microsomal monooxygenase, glutathione S-transferase, mitochondrial succinoxidase, and NADH oxidase, which are involved in ROS generation. Free metal ions enhance ROS formation by the reduction of hydrogen peroxide with generation of the highly reactive hydroxyl radical. Due to their lower redox potentials, flavonoids are thermodynamically able to reduce highly oxidizing free radicals. Oxidation on the B ring of flavonoids having catechol group of a fairly stable orthosemiquinone radical is formed which is a strong scavenger (Fig.  9.2) (Kumar and Pandey, 2013). Polyphenol compounds are antioxidants that when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. The potential health benefits of polyphenol compounds are getting more and more recognition, as reports indicate that these compounds inhibit the harmful effects of ROS, which act as oxidants, thus protecting ­macromolecules, such as proteins, lipids, and DNA from oxidative degradation (Jelínek et al., 2014).

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Fig. 9.2  (A) Scavenging of ROS (R∘) by flavonoids (Fl-OH) and (B) binding sites for trace metals where Men+ indicates metal ions.

The chemical diversity of phenolic antioxidants makes it difficult to quantify individual antioxidants from their matrix. The TAC as parameter of antioxidants present in a complex matrix is more useful to evaluate health beneficial effects because of the synergism of antioxidants (Ghiselli et al., 2000). Antioxidant activity should be established by applying multiple antioxidant test models. Several in  vitro test procedures are carried out for evaluating antioxidant activities with the samples of interest, in current practice. Another aspect is that antioxidant test models vary in different respects (Alam and Rafiquzzaman, 2013). Antioxidant assays may be broadly classified as electron transfer (ET), hydrogen atom transfer (HAT), and ROS scavenging (Prior et  al., 2005). HAT based assays measure the capability of an antioxidant to quench free radicals (generally peroxyl radicals) by H-atom donation. The HAT mechanisms of antioxidant action in which the hydrogen atom of a phenol (Ar-OH) is transferred to an ROO•: ROO• + AH / ArOH → ROOH + A • / ArO• where the aryloxy radical (ArO•) formed from the reaction of antioxidant phenol with peroxyl radical is stabilized by resonance. AH and ArOH species denote the protected biomolecules and antioxidants, respectively (Apak et  al., 2007). Oxygen radical absorbance capacity (ORAC) assay, is an HAT based assay that applies a competitive reaction scheme in which antioxidant and substrate kinetically compete for peroxyl radicals (thermally generated) through the decomposition of azo compounds, such as ABAP (2,2′-azobis(2-aminopropane) dihydrochloride) (Apak et al., 2007).

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ET mechanism of antioxidant action is based on the reactions: ROO• + AH / ArOH → ROO• + AH•+ / ArOH•+ AH•+ / ArOH•+ + H2 O ↔ A • / ArO• + H3 O+ ROO• + H3 O+ ↔ ROOH + H2 O These reactions are slower than those of HAT assays, and solvent- and pH dependent. The aryloxy radical (ArO•) is subsequently oxidized to the corresponding quinone (Ar = O) (Apak et  al., 2007). Spectrophotometric ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. Examples of this kind of assay are Trolox-equivalent antioxidant capacity (ABTS/TEAC), DPPH, Folin total phenols assay, ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC) (Ozyurt et al., 2007). For molecular probes used in the colorimetric/fluorometric detection of ROS, nitro blue tetrazolium (NBT) has been used for superoxide anion (O2•−), scopoletin for hydrogen peroxide (H2O2), deoxyribose/ thiobarbituric acid (TBA) or modified CUPRAC reagent for hydroxyl radicals (•OH), and tetra-tert-butylphtalocyanine for singlet oxygen (1O2) (Barbosa-Pereira et  al., 2014). According to Alam and Rafiquzzaman (2013) in vitro and in vivo methods are being used for antioxidant evaluation purpose. DPPH method is the most frequently used one for in vitro antioxidant activity evaluation while Lipid peroxidation assay (LPO) was found as the mostly used in vivo antioxidant assay.

9.9  Prevention and Treatment of Several Chronic Diseases With Antioxidant Phytochemicals Overproduction of oxidants in human body can cause an imbalance and lead to oxidative damage to large biomolecules, such as lipids, DNA, and proteins. This damage is responsible for the pathogenesis of several human diseases, including cardiovascular diseases (CVD), certain types of cancers, and aging. Thus, antioxidant phytochemicals could play an important role in the prevention and treatment of chronic diseases (Singh et al., 2014). Phytochemicals are demonstrated to have antioxidant abilities in human studies. For example, the total antioxidant capacity of serum was increased significantly following consumption of red wine, strawberries, or vitamin C, and the plasma vitamin C levels and serum urate levels also increased significantly. The additive and synergistic effects of phytochemicals could be responsible for their potent antioxidant activities (Zhang et al., 2015).

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9.9.1  Protective Action on Cardiovascular Diseases CVD is the leading cause of death and disability in developed countries. Epidemiological studies have shown that flavonoids are linked to reduced incidence or mortality from CVD among adults in Europe and the United States. The etiology of CVD is very complex, and overproduction of oxidants is one of the main pathogenic factors (Peterson et al., 2012). Oxidative damage can cause endothelial cell injuries and deleterious vasodilator effects. It has been shown that antioxidant polyphenols could modify molecular events toward an improvement in endothelial function, and therefore, play an important role in the prevention of CVD. Polyphenols could also protect the cardiovascular system, not only from oxidative stress but other damage because they possess other physiological effects, such as blood pressure reduction and inflammation decreasing action (Prahalathan et  al., 2012). Therefore, antioxidant phytochemicals such as polyphenols could be good candidates for preventing and treating CVD through direct antioxidant activity as well as their other bioactivities (such as antiinflammation and preventing platelet aggregation and adhesion).

9.9.2  Antiobesity Activity The incidence of obesity is rising and becoming a major public health burden with enormous economic costs. Obesity is often accompanied by an increased risk of mortality, and the quality of life is impaired owing to sleep apnea, respiratory problems, osteoarthritis, and infertility. In addition, antioxidant defenses of the obese are lower, which is inversely correlated to central adiposity (Savini et  al., 2013). Low-grade chronic inflammation induced by inflammatory factors, such as tumor necrosis factor-α, interleukin-6, and monocyte chemotactic protein-1, is another key factor in the pathogenesis of obesity, which may act synergistically with oxidative stress to induce obesity. Chronic inflammation begins in white adipose tissue and eventually becomes systemic in obesity and Type 2 diabetes (T2D) (Alves et al., 2014). Flavonoids showed potent antiobesity activity in vitro and in vivo. For example, genistein was reported to regulate adipocyte life cycle and lower obesity-related low-grade inflammation and oxidative stress (Chuang and McIntosh, 2011). The antiadipogenesis activity of quercetin may be mediated by the adenosine monophosphate-activated protein kinase (AMPK) and mitogen-activated protein kinases signaling pathways (MAPK), respectively in preadipocytes and mature adipocytes (Dong et al., 2014).

9.9.3  AntiDiabetic Activity Diabetes is a major worldwide health problem and characterized by chronic hyperglycemia which leads to a number of microvascular and macrovascular complications. There are two types of diabetes,

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type-1 diabetes (T1D) and T2D. Diabetes is usually accompanied by increased production of free radicals or oxidative stress due to hyperglycemia and hyperlipidemia. It was also demonstrated that in the course of diabetes and its complications, plasma antioxidants showed a significant decrease. Cohort studies showed that metabolic homeostasis was improved, and the development of T2D and its complications was delayed or prevented by frequent consumption of wholegrain foods (Belobrajdic and Bird, 2013). A study showed that antioxidant activities of Ascophyllum nodosum were correlated with the phenolic contents, while the α-glucosidase inhibitory activity exhibited a pattern similar to the phenolic contents observed in  vitro (Apostolidis et al., 2011). Phenolic compounds could prevent diabetes through regulating α-glucosidase and lipase activities, reducing postprandial glycemic level, antiinflammatory activity, improving pancreatic function, and synergistic action with hypoglycemic drugs.

9.9.4  Antiaging Activity Aging is related to functional decline of the organism and progressive deleterious alterations leading to increased risk of disease and death with advancing age. Aging also presents motor and cognitive deficits. Free radicals and oxidative stress have been considered as important factors in the biology of aging and in many age-associated degenerative diseases, since the antioxidant systems are under deterioration during aging (Garrido et al., 2013). Antioxidant phytochemicals showed antiaging activities by different mechanisms. Epigallocatechin gallate extended lifespan of healthy rats by reducing the damage of liver and kidney and improving age-associated inflammation through inhibiting NF-κB signaling (Niu et al., 2013). Another study found that allicin could significantly ameliorate cognitive dysfunction in aged mice through enhancing of nuclear factor-like 2 antioxidant signaling pathways (Li et  al., 2012). Curcumin, resveratrol, and proanthocyanidins protected age-related cognitive decline and depression by modulating hypothalamic-pituitary-adrenal axis activity, serotonergic transmission, and hippocampal neurogenesis (Ogle et  al., 2013). Phytochemicals could extend lifespan, ameliorate cognitive dysfunction, and improve age-associated inflammation and oxidative stress.

9.9.5  Protective Action on Alzheimer’s Disease Alzheimer’s disease (AD) is a degenerative neurological disorder characterized by cognitive decline and memory loss (Rasool et  al., 2014). The brain is believed to be particularly vulnerable to oxidative stress due to a relatively high concentration of oxygen free radicals without commensurate levels of antioxidative defenses. Oxidative stress may be involved in pathogenesis of dementia or AD in elderly (Kumar

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and Pandey, 2013). AD patients show a remarkable reduction of acetylcholine (ACh) levels in the hippocampus and cortex of the brain, which can cause memory deficits. Acetylcholinesterase inhibition is linked to amelioration of Alzheimer’s symptoms. The flavonoids (including naringenin, hesperetin, eriodictyol, and their derivatives) from Paulownia tomentosa fruit exhibited a significant inhibition of both acetylcholinesterase and butyrylcholinesterase (Cho et al., 2012). In addition, curcumin, catechins, and resveratrol showed neuroprotective ability in AD (Davinelli et al., 2012), and curcumin (200 and 400 mg/kg) treatment reduced levels of oxidative stress in a dose dependent manner as well as attenuated increased acetylcholinesterase in mice (Rinwa and Kumar, 2012). Furthermore, polyphenolic compounds of walnuts reduced the oxidant and inflammatory load on brain cells, improved interneuronal signaling, increased neurogenesis, as well as enhanced sequestration of insoluble toxic protein aggregates (Poulose et al., 2014), so walnuts could play a role in preventing AD. Therefore, phytochemicals could protect against AD by reduction of oxidant stress and acetylcholinesterase.

9.10  Bioavailability of Phenolic Compounds Bioavailability is defined as the rate and extent to which the therapeutic moiety is absorbed and becomes available to the site of drug action (Zhang et al., 2015). Blood of 15 healthy volunteers was collected at time 0 and 1, 2, 3, and 4 h after consumption of 144 g of raisins. A total of 17 phytochemicals including 16 phenolics and oleanolic acid were identified and quantified in volunteers’ plasma. The results indicated that phytochemicals in raisins were bioavailable (Kanellos et al., 2013). In healthy participants, the maximal plasma concentrations (Cmax) of quercetin was 0.16 μM after ingesting grape juice containing 10 mg quercetin aglycone, which represents only about 1.4% of the ingested dose. The bioavailability of phenolic compounds can also be improved using delivery systems (Semalty et al., 2010). Although some epidemiological studies showed a relationship between the low incidence of cancer and the intake of plant-based foods, at present there is no conclusive proof that high antioxidant activity would result in high anticancer activity (Wang et al., 2011).

9.11  Polyphenols Content and Antioxidant Activity of Extracts From Discarded Hop Craft brewery is defined as (1) Small: with an annual production of 6 million barrels of beer or less. (2) Independent: <25% of the craft brewery is owned or controlled by a beverage alcohol industry member that is not itself a craft brewer. (3) Traditional: a brewer that has a majority of its total beverage alcohol volume in beers whose flavor

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is derived from traditional or innovative brewing ingredients and their fermentation (Chapman, 2015). Despite their lower production, artisanal breweries face the challenge of handling the same types of waste as large companies. An alternative for the use of discarded hop is to use it for the extraction of compounds with biological activity. They are employed in the food and pharmaceutical industries, either as additives or for the design of nutraceuticals. The preliminary results of polyphenols quantification and the antioxidant capacity of discarded hop generated during the manufacture of artisanal beer are presented below.

9.12  Materials and Methods The hop discarded used in this work was provided by a craft brewery, which employs 100% processed malt. The material was freezedried at −47°C and 13 × 10−3 mbar and stored in polyethylene bags at −10°C until use. All chemicals were obtained from Sigma-Aldrich or Merck (Darmstadt, Germany).

9.13  Proximate Composition Proximate composition of lyophilized hop was determined using official AOAC procedures (AOAC, 1997): nitrogen (method 954.01); fat (920.39); ash (923.03); fiber (962.09), and moisture (925.09). Protein content was calculated as nitrogen × 6.25, and carbohydrate content was estimated as nitrogen-free extract (NFE).

9.14  Phosphorus Content For phosphorus determination, method 365.3 of the United States Environmental Protection Agency was used. This method is based on reactions that are specific for the orthophosphate ion. Thus, depending on the prescribed pretreatment of the sample, the various forms of phosphorus may be determined (NPDES, 1978). The content of phosphorus was calculated using the formula Procentaje de fósforo =

(C )( DF ) × 100 (w )(1000 )

where C = concentration mg of phosphorus in 5 mL of standard solution. DF = Dilution factor (100). 100 = Percentage. w = sample weight. 1000 = mg to g conversion factor.

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9.15  Preparation of Methanolic Extracts Ten gram samples were placed in a homogenizer with 10 mL of methanol and were thoroughly mixed for 1 min. Then, the samples were centrifuged at 2000 × g for 15 min at 4°C. The supernatant was collected and stored in air tight glass vials covered with aluminum foil and kept at −20°C.

9.16  Quantitative Phenolic Analysis The total phenolic content (TPC) was determined by a modified Folin-Ciocalteau method. To 1 mL of the extract, 4 mL of FolinCiocalteau reagent diluted in water (1:5 H2O) was added, and after 3 min, 5 mL of Na2CO3 (7.5%, w/v) was added. The contents were agitated and left to stand for 1 h for the reaction to take place and stabilize. The absorbance at 740 nm was determined in a spectrophotometer. The calibration curve was performed with gallic acid and the results are expressed as gallic acid equivalent in mg g−1 of hop product. Total flavonoid content was determined according to the method described by Lee et al. (2003). One milliliter of extract was mixed with 1 mL of 2% AlCl3 × 6H2O solution and incubated at room temperature for 10 min. Thereafter, absorbance at 430 nm was measured. Total flavonoid content was calculated as a quercetin equivalent in mg g−1 of hop product.

9.17 ABTS•+ Decolorization Assay The assays were carried out using an improved ABTS decolorization assay (Pukalskas et al., 2002). ABTS solution at 7 mmol L−1 in water and aqueous solution of K2S2O8 at 2.45 mmol L−1 were prepared. The two solutions were mixed in the volume ratio 1:1 and stored for 6 h in the dark at room temperature. During that time, ABTS radical was generated. The ABTS•+ solution was diluted to an absorbance of 0.7 at 734 nm. A 0.040 mL aliquot of extract was added to 1.8 mL of ABTS•+. The absorbance at 734 nm was measured at the beginning and after 5 min of reaction. The affinity of test material to quench ABTS free radical was evaluated according to the following equation: Scavenging ( % ) = [ AC − AA ]×100 where AC is the absorbance of control (solvent instead of extract) and AA is the absorbance of sample.

9.18  Results and Discussion The proximal composition of the hops manifests high contents of carbohydrates and proteins. The contents of fiber and minerals (ash) are also relatively high. The direct use of spent hops as feed supplement

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is not desirable due to the presence of 2-methyl-3-buten-2-ol, which is the product of bitter acid degradation and has hypnotic-sedative properties. However, it could also be used as a substrate for the cultivation of microorganisms of industrial interest, given its high content of carbon and nitrogen sources (Table 9.5). With respect to its application in animal and human nutrition, this residue could be processed by wet fractionation to obtain concentrates and protein isolates, which can be applied in sport and clinical nutrition. Likewise, protein concentrates and isolated could be modified by enzymatic hydrolysis for the production of peptides with biological activity, which are used for the formulation of nutraceuticals and functional foods. Discarded hop is an adequate source of carbon, nitrogen, and phosphorus (Table 9.5). According to its composition, discarded hop could be recycled into organic fertilizer. Food wastes can be decomposed in anaerobic digestion by microorganisms to break down food waste into smaller materials and make useful products. This process is carried out inside an enclosed system in the absence of oxygen. Methane gas produced can be collected and converted into biogas to transport fuels and produce electricity and heat (Okareh et al., 2014). Methanolic extract exhibited adequate amounts of polyphenols and flavonoids (Table 9.5), comparable with reports of other authors (Fărcas et  al., 2013; Almaguer et  al., 2014). These kind of secondary

Table 9.5  Proximate Composition (%), Phosphorus Content (%), Polyphenols Content, and Antioxidant Activity (%) of Hop Methanolic Extract Parameters

Discarded Dry Hop

Moisture Protein Fiber Fat Ash Carbohydrates Phosphorus Parameters Total phenols content (g GAE/kg of discarded hop) Flavonoids content (g CE/kg of discarded hop) Inhibition of ABTS radical (%) TEAC (mM of Tx)

4.52 22.10 7.36 1.08 6.02 58.92 16.96 Methanolic extract 1.54 0.61 4.24 0.0299

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metabolites are antioxidants that when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. These compounds could be employed for design nutraceuticals, formulate functional foods, or as food additives. In the present study, 1.0 mL of methanolic extract from hop discarded inhibited 4.24% of ABTS radical during assay. Trolox (Tx) Equivalent Antioxidant Capacity was 0.0299 mM of Tx. Both values were obtained at a concentration of 25 mg/mL. During the processing of hops to pellets, the raw material is exposed to high temperatures (up to 55°C) and air; thus, some oxygen-sensitive antioxidants may be destroyed. Nevertheless, studies by Krofta et al. (2008) indicated that the pelletizing process had no significant effect on the antioxidative status of hops. According to these authors, thermal treatments such as drying could cause loss of some antioxidant activity of hops, but the loss is very low and it does not usually exceed 5%. The polyphenol extraction yield and antioxidant activity of plant extracts depend highly on the solvent polarity, which determines both quantitatively and qualitatively the extracted antioxidant compounds. Results indicate that the use of methanol provided adequate extraction of phenolic compounds as has previously been reported for different food-plant materials (Inglett et al., 2010).

9.19 Perspectives Solid-liquid extraction is a reproducible and economical method of extracting phenolic compounds. However, it depends on the polarity of the solvent used; therefore, more studies will be necessary to determine which solvent may be more suitable to extract the largest amount of phenolic compounds from the discarded hops. Another strategy would be to perform solid-liquid extraction assisted by ultrasound, a technique that has given good results with other matrices rich in bioactive compounds. Regarding the evaluation of antioxidant activity by in vitro assays, it will be necessary to test other techniques that evaluate different antioxidant mechanisms, such as the chelating capacity of prooxidant metallic cations or the reducing power. It is also necessary to use tests compatible with the sample; the TEAC method used in present study is for soluble samples, it is necessary to test methods for hydrophobic samples such as decolorization of the DPPH radical or β-carotene assay. It would be relevant to carry out assays for other biological activities of interest, such as the ability to inhibit the Angiotensin Converting Enzyme (antihypertensive), inhibition of amylolitic enzymes (Antidiabetic), lipid-lowering activity, antiinflammatory activity, etc. Finally, these in vitro assays should be correlated with in vivo studies.

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These studies will allow giving a better use to a product that is discarded by the brewing industry, allowing the obtainment of chemical compounds of interest for the chemical, pharmaceutical, and food industries.

9.20 Conclusion Hop discarded from craft beer production has the potential to be used as a by-product, not as waste. Its chemical composition, protein (22.10%), fiber (7.36%), and carbohydrates (58.92%), raises its use in the formulation of feeding stuffs for animals, as organic fertilizer (16.96% of P) and in the production of biogas. Also through biotechnological processes, compounds with biological activity such as concentrates, isolates, and peptide protein hydrolysates could be obtained. These products can be used in the design of nutraceuticals and functional foods. Finally using extractive conventional methods, it is possible to obtain specific compounds with biological activity such as polyphenols (1.54 g GAE/kg of discarded hop), which are used by the food and pharmacological industries. Conventional extractive methods have many advantages, such as low infrastructural costs, maintenance, and reagent costs. In the future, more antioxidant phytochemicals in foods and medicinal plants should be separated and identified, and their bioactivities and the mechanism of action should be studied further. In addition, attention should be paid to the potential adverse effects of antioxidant phytochemicals for human beings. The use of this resource will allow artisanal breweries to obtain other value-added products, which can expand and diversify their markets and utilities.

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