Bioactives from Side Streams of Wine Processing - ACS Publications

Chapter 21

Downloaded by CENTRAL MICHIGAN UNIV on December 28, 2015 | http://pubs.acs.org Publication Date (Web): November 24, 2015 | doi: 10.1021/bk-2015-1203.ch021

Bioactives from Side Streams of Wine Processing P. Winterhalter,** Stefanie Kuhnert, and Philipp Ewald Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany *E-mail: [email protected].

Large amounts of waste streams are produced in viticulture and vinification. They consist inter alia of vine prunings, grape stalks, pomace, grape seeds and yeast lees. All of these side streams contain important functional polyphenols with diverse biological activities (e.g. antioxidant, anticancer, antimutagenic). This chapter will present novel approaches for the exploitation of under-utilized processing wastes and by-products of the wine industry by converting them into value-added products relevant to market demands. Examples will include the use of membrane technologies for the recovery of pure anthocyanin fractions, a novel rapid characterization of grape seed extracts on a diol HPLC phase, depolymerization of polymeric proanthocyanidins in order to enhance their bioavailability, and the search for bioactive oligomeric stilbenoids in vine prunings.

The world grape production in 2011 was 69 million tons including approximately 22 million tons of table grapes (1). Of the remaining 47 million tons used in wine making, one can estimate that roughly 9 million tons of grape pomace are obtained as by-product (2). Grape pomace consists mainly of skins, seeds and stalks and is used for the production of ethanol, tartaric and citric acid, grape seed oil, protein, dietary fiber, and polyphenols (3–5). Whereas red grape skins mainly contain anthocyanins which can be used as natural food colorants, grape seeds are rich in proanthocyanidins. The latter are increasingly used in functional food, dietary supplements and cosmetics due to their strong antioxidant © 2015 American Chemical Society In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

effects. In the following strategies for the fractionation of the complex mixture of polyphenols in grape pomace will be presented. Moreover, the use of vine prunings as a rich source of resveratrol and oligomeric resveratrol derivatives will be demonstrated.


Fractionation of Polyphenols From Grape Pomace Solvent extraction is frequently used to recover polyphenols from the solid matrix grape pomace. As solvents water and ethanol are widely applied often in combination with acids (e.g. citric acid) and SO2 ((6) and refs cited therein). Alternatively, extraction with subcritical water or subcritical sulfured water has been described (7). In order to enhance extraction yield, enzyme-treatment (pectinolytic and cellulytic enzymes) and pulsed-electric field treatment or high hydrostatic pressure have been applied (8, 9). After the extraction process crude phenolic extracts are obtained which are commercialized as food colorants or due to their antioxidant activities as nutraceuticals (red and white grape extracts). Our attempt was a further fractionation of these crude extracts by application of membrane technology (membrane adsorber Sartobind S) in combination with liquid-liquid extraction (ethyl acetate). This approach allows the separation of the crude extracts into the following three groups of polyphenols: anthocyanins, color-less copigments (e.g. flavonols, flavanols, cinnamates, stilbenes) and polymers. In a first step the crude phenolic extracts are applied onto an Amberlite XAD-7 resin (Sigma, St. Louis, MO). The column is washed with water and the polyphenols are eluted with methanol/acetic acid (19:1, v/v) (10, 11). After evaporation of the solvents and freeze-drying, the phenolic mixture is redissolved in ethanol/acetic acid (19:1, v/v) and pumped through the membrane adsorber Sartobind S 75 (Sartorius Stedim Biotech, Göttingen, Germany). The adsorber consists of a stabilized cellulose membrane with negatively charged sulfonic acid groups. Under acidic conditions only the positively charged flavylium cations are retarded (retentate fraction) on the membrane surface and the other phenolic compounds pass the adsorber unretained (permeate fraction). After rinsing of the adsorber with ethanol/acetic acid (19:1, v/v), elution of anthocyanins is performed with a mixture of 1 M aqueous NaCl solution/methanol (50:50, v/v). In this way, a pure anthocyanin fraction free of copigments can be obtained. This method has first been developed for bilberry anthocyanins and later been adapted to anthocyanins from grape pomace (12). Depending on the size of the membrane adsorber cartridge, separation in the 10 g to one kg-scale can be carried out. From the permeate fraction which contains the copigments and polymeric phenolics, the low-molecular phenolic constituents are easily liquid-liquid extracted with ethyl acetate. In this way, three clearly separated fractions, i.e. anthocyanins, copigments, and polymers, are obtained. Whereas anthocyanins and copigments can be directly used for nutraceutical or pharmaceutical purposes, the polymer fraction may require a further treatment, e.g. an acid-catalyzed depolymerization (see below) in order to increase its bioavailability (13). In case an isolation of individual phenolic constituents of the different fractions 338 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


is required, application of the support-free technique of countercurrent chromatography (CCC) is recommended. This all-liquid chromatographic technique is especially suited for polar constituents and its versatility and preparative capabilities are well documented (14). First separations of anthocyanins have already been reported in the year 2000 (10, 15). In the meantime novel instrumentation has been developed enabling separations in the 100 g to kg scale (16). Whereas anthocyanins and copigments are easily separated by CCC, fractionation of the intact polymers requires alternative techniques, such as size-exclusion chromatography or centrifugal precipitation chromatography (for details cf. (17)).

Rapid Characterization of Proanthocyanidins in Grape Seed Extracts A large proportion of grape pomace consists of grape seeds (approx. 200 kg per ton of pomace). Grape seeds contain a valuable vegetable oil (approx.15-20%) rich in linoleic acid which is widely used for cosmetics and culinary applications. In addition grape seeds contain high amounts of proanthocyanidins (4-6%). Phenolic grape seed extracts (GSE) are important dietary supplements with an average market share of 150 tons per year. So far characterization of GSE is mainly made by spectrophotometric assays, namely the vanillin (18) as well as the acid butanol assay (19). Although widely applied, both methods are not considered to be appropriate for the determination of the content of oligmeric proanthocyanidins, for details cf. (20, 21). For this reason we attempted to establish a more reliable determination of the proanthocyanidin (PA) content of GSE and developed a novel HPLC method for PA analysis using a diol stationary phase (MonoChrom diol column, Agilent, Waldbronn, Germany). As can be seen from Figure 1 the analysis is completed within 32 min and allows a differentiation of bioavailable short chain PA and non-bioavailable polymeric forms. The latter elute together in a quantifiable peak at the end of the chromatographic run. Authentic references are used for the the quantification of the individual groups of oligomers (dimers, galloylated dimers, trimers, tetramers, and pentamers). The newly developed HPLC method enables a verification of the labeling of the PA content in nutraceuticals (Figure 2). In several cases considerable deviations from the labeled PA value have been detected.

Depolymerization of Polymeric Proanthocyanidins Bioavailability of proanthocyanidins is restricted to oligomers with up to three flavan-3-ol units. In order to convert the polymeric PA into bioavailable ones, an acid-catalyzed depolymerization process has to be applied. Generally, there are two possibilities. The first one uses a random cleavage of the flavan-3-ol chain under acidic conditions and gives rise to short chain cleavage products. The second approach uses a more controlled degradation, the so-called semisynthesis (13) (cf. Figure 3). 339 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 1. Chromatogram of the HPLC separation of a grape seed extract on a diol stationary phase.

Figure 2. Results of the analysis of commercial nutraceuticals and comparison of the newly developed HPLC method with spectrophotometric assays, i.e. vanillin assay (18) and the acid butanol (Bate-Smith) assay (19). 340 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 3. Mechanism of the directed depolymerization of polymeric PA. For this purpose the GSE polymers are enriched by a simple cleanup step using solvent precipitation. The PA extract is dissolved in ethanol and increasing amounts of n-hexane as a nonpolar solvent are added. The precipitate obtained (polymeric PA) is then treated with 1 N methanolic HCl. During this process the interflavanoid linkage of polymeric PA is cleaved by releasing the flavan-3-ol moieties of the extension unit as carbocation together with an uncharged terminal unit. The liberated carbocation can immediately react with in excess added nucleophiles, such as (+)-catechin or (−)-epicatechin, giving rise to the formation of dimeric procyanidins. During this process the polymeric procyanidins are depolymerized and bioavailable dimeric procyanidins are formed. Depending on the nucleophiles chosen, the reaction products can be tailored in a simple way. Isolation of the dimeric reaction products is then performed by preparative countercurrent chromatography (13, 22).

Oligomeric Resveratrol Derivatives from Vine Prunings Vine pruning production is up to 5 tons per hectare and year. Traditional use is for compost or charcoal production, only in recent years vine shoots have been recognized as an important source of resveratrol derivatives (23). Resveratrol as a phytoalexin is known to occur in free and glycosidically bound form in the skins of grapes in amounts up to 50 mg/kg. The same is true for grape pomace. Resveratrol is known to exhibit a broad range of biological effects that include antioxidant and anticancer activity and it apparently also increases stress resistance and lifespan (24). Vine shoots or canes which are obtained during the annual pruning of grape vine contain in addition to trans-resveratrol also high levels of resveratrol oligomers consisting of two to eight stilbene units. The main oligomer is the dimer trans-ε-viniferin (cf. Figure 4) . 341 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 4. HPLC chromatogram of the stilbenes in vine shoots. Example: Spätburgunder (Column: Kromasil 100-5; C18; 5 µm; 250 mm × 4,6 mm i.d.).

The yield of resveratrol derivatives extracted from vine prunings can be as high as 7 g/kg dry weight and it has been estimated that the commercial value of these stilbene derivatives is around 2000-3000 US$ per kg (25, 26). In contrast to resveratrol, the biological effects of the oligomers have hardly been studied. We therefore performed an activity-guided isolation of stilbenes from the commercially available vine shoot extract Vineatrol®30. This is a standardized product with a total stilbene content of >30%. For the fractionation the preparative all-liquid chromatographic technique of low-speed rotary countercurrent chromatography has been applied and the separated fractions were submitted to in-vitro testing using the following cell lines: A-431 (human epidermoid carcinoma), LNCaP (human prostatic adenocarcinoma), SW 480 and HT-29 (colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), HepG2 (human liver hepatocellular carcinoma) (27, 28). The chemical structures of some of the active compounds identified are depicted in Figure 5. In Table 1 the IC50-values for trans-resveratrol, hopeaphenol and r-2-viniferin are shown. Importantly, hopeaphenol and r-2-viniferin inhibited the growth of human tumor cell lines more strongly than resveratrol itself. In a recent study, hopeaphenol and r-2-viniferin were furthermore found to inhibit the growth of a canine glioblastoma cell line and a canine histiocytic sarcoma cell line (29). Today there are quite a few data available concerning the stilbene content of vine prunings throughout the world. Recent publications indicated that the stilbene levels in vine prunings strongly depend on postharvest storage conditions. After a storage period of approximately 6 months highest stilbene concentrations were observed (30, 31). Interestingly there was so far no information about stilbene levels in vine shoots from Germany available. We therefore analyzed the stilbene profile of eight grape vine varieties. The shoots were obtained during pruning season 2013 and stored for 6 months at room temperature protected from light exposure. Prior to analysis the vine shoots were lyophilized, ground and extracted with ethanol/water (80:20, v/v) with the assistance of ultrasonication as described in (26). HPLC analysis was performed on a Kromasil 100-5; C18; 5 µm column (250 mm × 4.6 mm i.d.; Eka Chemicals AB, Bohus, Sweden). We found transresveratrol and trans-ε-viniferin to be the major stilbenes beside minor amounts of e.g. ampelopsin A, hopeaphenol, r-viniferin, r-2-viniferin and miyabenol C (Figure 6). 342 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 5. Chemical structures of stilbene derivatives from vine prunings exhibiting anti-proliferative effects.

Table 1. IC50-values for trans-Resveratrol, Hopeaphenol and r-2-Viniferin in Human Tumor Cell Lines IC50-Values (µM) A431

LNCaP

SW480

HT-29

MCF7

HepG2

Resveratrol

20

5

25

20

27

10

Hopeaphenol

4.3

2

4.5

0.8

4.3

3.8

r-2-Viniferin

4.7

4

5

2.7

2

1.4

343 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 6. trans-Resveratrol and trans-ε-viniferin content in prunings from Germany 2013.

The content of trans-resveratrol ranged between approx. 1500 and 3300 mg/ kg dw. While the lowest content was observed for the variety “Regent” which is known to exhibit resistance against fungal diseases, the highest content was found in the variety “Weißburgunder”. The content of trans-ε-viniferin was in a similar range (approx. 1000 – 3100 mg/kg dw). Again, the variety “Regent” was found to possess the lowest amount, whereas the variety “Sauvignon Blanc” exhibited the largest amount of the dimeric stilbene.

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345 In Advances in Wine Research; Ebeler, Susan B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Bioactives from Side Streams of Wine Processing - ACS Publications

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