Deliverables
The results, in accordance to previously published results, show that dietary polyphenols are highly sensitive to the mild alkaline conditions in the small intestine and that a good proportion of these compounds can be transformed into other unknown and/or undetected structural forms with different chemical properties and consequently different bioaccessibility, bioavailability and, biological activity.
Pectinase treatments clearly increased antimicrobial and antioxidant activity of the berry juices. Increased antimicrobial activity was most probably a consequence of the following facts:
- increased amount of phenolic compounds (e.g. anthocyanins),
- slightly decreased pH,
- structural changes in phenolics and 4. other potential changes in berry material.
Increase in radical scavenging activity was most probable due to increased amount of phenolic compounds, which are known to be strong antioxidants. High pressure and high power ultrasound treatment seemed to be promising new technologies in berry processing. However, more research is needed to determine their specific effects on antimicrobial and antioxidant activity.
Different types of mechanical, physical and physicochemical unit operations, i.e. ultrasound (US), high pressure (HP), and flash release(FR) treatments for cell wall modification were investigated. The objectives are
- to elucidate the suitability of selected unit operations for grape and berry processing with the aim of enhancing the conventional pressing process efficiency
- to elucidate the effect of these unit operations on cell wall degradation and bioactive components.
The methods are expected to facilitate the cell wall disruption and their effect can be further enhanced by combining them to enzymatic pre- or post-treatments. These methods can also be regarded as minimal processing methods with little or no negative impact expected on the sensory quality of the berry and grape products.
The aim of the high-pressure treatment was to induce mechanical disruption of the berry tissue and possible activation of both endogenous and exogenous cell wall degrading enzymes. Mashed bilberries or black currants were prior to juice pressing treated with both enzymes and HP to increase juice yield and the anthocyanin content of the juice. Two types of processes were investigated. In the first process, the enzyme treatment was carried out at elevated pressure (50 or 400 MPa, 35°C, 15 min). In the second process, the mash was HP treated (50 or 400 MPa,35°C,15 min) before enzyme addition and incubation at 45°C for 2 h. The juice yield or anthocyanin content of the juice were significantly improved by none of the HP treatments compared with similar treatments carried out at atmospheric pressure.
High power ultrasound is known to induce mechanical disruption of cell walls. However, in the studied berry juice experiments it caused pectin gelatinization and decreased juice yield when enzyme incubation was not performed. On the contrary, when the high power US treatment was performed after enzyme treatment, juice yields were slightly increased both with bilberries and black currants and the amount of phenolic compounds in juices was increased. The amount of anthocyanin glycosides was increased by more than 30% with bilberries and more than 10% with black currants. When the used enzyme had undesirable side effects which degraded bilberry anthocyanin glycosides into aglycons, the US treatment intensified the effect. It also intensified the effect of enzymes in increasing the antimicrobial activity against pathogenic bacteria and DPPH radical scavenging activity of bilberry mash and juice.In conclusion, the results indicate the potential of power ultrasound in juice processing when combined to enzyme incubation. The amount of enzyme needed can be reduced and the yield of extractable health-relevant compounds increased.
Flash release treatment is known to increase phenolic extraction into the wine during subsequent fermentation, presumably as a result of mechanical disruption of the berry tissue. It accelerated extraction of phenolic compounds and significantly increased pigment level in juice. Wines obtained after FR treatment contained higher levels of phenolics (especially flavonols (+50-100%), catechins (+25-100%) and tannins(+30-55%))than the control wines but were less enriched in anthocyanins (+4-30%). They were also enriched in polysaccharides extracted from grape cell walls. Increasing the duration of high temperature exposure before pressure release further increased phenolic extraction and accelerated conversion of grape anthocyanins to other molecular species. Combining FR with enzyme treatment induced no further effect on phenolic extraction but modified polysaccharide composition. None of the treatments modified the juice or wine yield. FR wines were perceived more fruity (strawberry, banana, fruit drop) and more astringent than the control wines which showed more gamy and empyreumatic flavours.
Cell wall polysaccharides were isolated and characterised in press cake of bilberries, black currants, white and red grapes. Differences of white and red wine manufacture could be seen back in the press cake. In red wine press cake pectin was degraded to a further extent. The cell wall polysaccharide composition of conventional and flash release treated press cake did not differ.
HPP processing decreased the degree of methyl esterification (DM), probably due to enhanced activity of endogenous pectin methyl esterase (PME). The decrease in DM led to easier extractable pectin, while the sugar composition of the cell wall polysaccharides did not change. To investigate the effect of enzyme treatment in combination with HPP, three different commercial enzyme mixtures were added to bilberry and black currant mash immediately before HPP. Pectic polysaccharides were further degraded when high pressure was used. Hemicelluloses, however, were only degraded at atmospheric pressure. Pectinolytic enzymes show an improved activity, but hemicellulases are inactivated after HPP.
Chemical analysis of phenolic compounds.
The chemical analyses were focused on polyphenolic compounds as present in grapes, musts and wines prepared by INRA in WP3-5. On account of different matrices (grapes, musts, wines) and the large number of samples (over 330), efforts were developed for speeding up extraction and analysis of polyphenols. Simple polyphenols (anthocyanins, flavonols, flavanol monomers and phenolic acids) were analysed by HPLC coupled to diode array detection (DAD) and electrospray mass spectrometry (ESI-SM), either directly or after solid phase extraction. Flavanol oligomers and polymers were characterized in terms of elementary constituent units and degree of polymerisation by HPLC after thiolysis.
The results on different trials allowed to conclude
- the flash release treatment resulted in faster extraction of all classes of phenolic compounds;
- FR wines contained larger amounts of flavonols, catechins and proanthocyanidins than the control and similar (or slightly larger) amounts of hydroxycinnamic acids and anthocyanins;
- longer exposure to high temeprature before pressure release further accelerated extraction of all classes of polyphenolic compounds (except proanthocyanidins) but hardy modified final concentration of anthocyanins, catechins and proanthocyanidins in wines; this treatment enhanced the conversion of anthocyanins to other pigment species (including flavanol-anthocyanin dimers);
- the liquid phase treated wines contained a lower amount of phenolic compounds (especially proanthocyanidins) than the other treatments due to the absence of extraction during the fermentation;
- the addition of enzyme had no effect on extraction of polyphenols;
- concentrations of hydroxycinnamic acids and tannins in wines did not change after 12-month;
- concentrations of anthocyanins, flavonols and catechins in wines decreased strongly after 12-month storage; (8) after 1 year, differences induced by flash release were maintained.
Cell wall polysaccharides from black currants and bilberries were characterised with respect to difference in tissues, extractability, and common juice processing. The main difference between bilberries and black currants is the dominant sugar residue in seeds: mannose for black currants and xylose for bilberries. Black currants contain more pectic sugars than bilberries. Consequently a commercial enzyme used during processing releases more pectic material into the juice.
Rhamnogalacturonan II (RG II) was identified (as the dimeric form) and quantified via its diagnostic sugars in bilberries and black currants. More RG II was present in juice than in press cake. RG II might also be an important cross linker of pectic molecules and could play an important role for the structure of the pectin network of the cell wall.
Structure of xyloglucan in bilberries and black currants was characterised. Black currants show a common xyloglucan structure of XXXG-type. Bilberries contain XXXG-type xyloglucan with 'O-xylose containing side chains (U) only reported recently for the first time in argon tree.
This basic knowledge was used to follow changes in cell wall polysaccharides during different processes.
The yield and composition of various lipophilics from berry and grape processing residues was analysed and quantified. Blackcurrant was shown to have a high content of valuable gammalinoleic acid (GLA) and tocopherols, which were readily available in press residue seeds. Bilberry had a high yield of polyunsaturated fatty acids together with considerable amount of tocotrienols. Grape press residues contained less oil that was rich with linoleic acid, but with lower content of fat-sobuble antioxidants.
Effect of novel treatments (enzymes and flash release) on the lipophilic yield was shown to be negligible, with the exception of vitamin E yield from the red grape press residue.
Cutin composition of blackcurrant and bilberry was investigated in detail. Surprisingly, the berry cutin was shown to consist mostly of non-ester linked polymers, as more than 90 % of the isolated cutin was resistant to ester-degrading depolymerisation; in contrast, depolymerisation of apple cutin with same methods yielded over 70 % cutin monomers. The negligible portion of ester-linked cutin in berry cutin may have influence on the berry skin resistance to enzyme and other treatments. The cutin monomers liberated were mostly hydroxy fatty acids, that have earlier been shown to be abundant in fruit cutins. Cutin forms part of the non-soluble dietary fiber, which may have a role e.g. in carciogenesis.
Monomers released from cutin by cutinases developed in the project were analysed, and their composition profile seemed to differ significantly from those obtained by chenmical depolymerisation.
Results consist a report that explored the factors that affect the consumer acceptability of new fruit and berry products with additional health benefits due to enhanced polyphenol content. The perceived benefits and disadvantages or risks consumers associate with these new products and acceptability of production technologies were the target factors.
Survey (N=914) from three countries (Finland N=303; France N=301; and Spain N=310) indicates that consumers in all three countries have neutral or mildly positive attitudes towards products with enriched polyphenol content regardless of the health benefit. From a range of products yoghurt and juice were perceived as most appropriate carriers for the health benefits.
When respondents were grouped according to their responses on willingness to buy the product descriptions there were four clusters that described different consumer groups' dispositions towards products with enhanced polyphenol content. The largest group (N=430) was only interested in price not being higher than the market price. The so-called anti-wine group (N=142) wine was not an acceptable carrier for polyphenols. In Finland the share of this group was bigger than in the other two countries. The third group (N=130), on the contrary, was a pro-wine group with higher percentage of Spanish respondents belonging to that group. The last group was sensitive to the processing method (N=141) showing high preference for conventional processing and avoidance to enzyme-aided processing with pressure aided processing regarded as neutral in between the other two processing methods.
In general consumers were neutral or slightly positive towards increasing the amount of polyphenols in fruit-based products. Increased price or alcohol beverage as a carrier can be major obstacles for acceptance of products with enhanced health effects. Most people do not worry about how the health benefits have been gained in the product, but there seems to be a relatively strong segment that put emphasis on processing method as well. These respondents favour conventional processing, pressure-aided processing is regarded as neutrally and enzyme-aided processing as negatively.
A novel rhamnogalacturonan II(RGII)-degrading enzyme was isolated from an unknown fungus, purified and sequenced. Several cutinases were screened and also cloned and expressed in Aspergillus, and one was patented for its ability to liberate polyphenols from berries. Three rhamnosidases were isolated cloned and produced in Aspergillus.
Enzymatic extraction methods were developed to extract antohcyanins and other phenolic compounds from berry and grape mashes and press residues both using commercial enzyme mixtures and cutinases, rhamnosidases and RG IIases isolated in the project.
The main results were:
- especially arabinosidase and galactosidase side activity should be avoided in berry processing so as not to degrade the anthocyanins to unstable aglycones,
- Rhamosidase treatment could be used to produce flavonoid glucosides from the corresponding rutinosides,
- By optimally selecting the pectinolytic enzyme mixture both yield of juice and its phenolic content was increased,
- Ultrasound treatment intensified the effect of pectinolytic enzymes and thus reduced the enzyme dosage needed,
- High pressure processing influenced the enzymatic hydrolysis pattern of cell walls, but the phenolic extraction was not enhanced
- Humicola cutinase significantly increased the yield of polyphenols when applied to pectinase treated black currant pomace,
- In wine processing, enhanced cell wall hydrolysis in enzyme-assisted flash release process did not enhance polyphenol extraction with the pectinolytic enzyme mixture used.
Postharvest treatment of grapes with different gas ozone concentrations and for times of 1, 3 and 5 hours induced an increase in phenolic metabolism (particularly stilbenoid biosynthesis) [trans-resveratrol, piceatannol and viniferins (resveratrol dehydro-dimmers and dehydro-trimers)] during storage of the harvested grapes at 22 degrees C and 95% relative humidity. The maximal resveratrol concentration in grape skins was found after two days of storage, and this amount was similar to that induced by optimized UV-C treatments (1 min, 510W, 40 cm).
Although similar resveratrol concentrations accumulated in grapes after both UV-C and O3 treatments (maximum ozone production and time), the ozone treatment was more efficient inducing resveratrol polymer accumulation in grape berries. A sequence in the biosynthesis of stilbenoids was observed, starting with the resveratrol monomer to continue with the resveratrol dehydrodimers to end with four different resveratrol dehydrotrimers. These trimers were different from alpha-viniferin, a trimer previously reported to be induced in grapes after biotic and abiotic stresses.
Two alpha-viniferin isomers were also detected in the ozone-treated grapes although at very low concentrations that prevented their quantification. The structure of the new trimers produced is similar to those of the dimers already known in grapes. The ozone gas treatment decreased the sensory quality of the white grapes and prevented their use for consumption as fresh table grapes, but these could be used for the preparation of stilbenoid enriched grape musts and nutraceutical grape polyphenol extracts.
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