(191t) Concentration of Polyphenols from Blueberry Pomace Extract Using Nanofiltration

Polyphenols extracted from blueberry (Vaccinium corymbosum) pomace were concentrated using nanofiltration. Crossflow filtration was shown to be a feasible method for concentrating the polyphenols present in dilute aqueous solutions. High-performance liquid chromatography was employed for the determination of total anthocyanins, total flavonols and chlorogenic acid in the hot water extract. Both nanofiltration membranes (NF245 and NF270) showed complete rejection of phenolic compounds at good permeances, whereas crossflow mode of filtration was found to reduce membrane fouling considerably. Furthermore, a suitable protocol was developed for clean-in-place of the used membranes. After repeated filtrations followed by the cleaning protocol, the rejection performance was preserved unaltered and the relative permeance was recovered up to 73% for NF245 membrane and more than 99% for NF270 membrane.

For commercial purposes, an ample amount of the blueberries is processed either into juice or juice concentrate and then the remaining solid residue (pomace) is generally treated as a waste product. The skins of blueberry fruits contain most of anthocyanins by weight so that, depending on the complexity of the juice extraction method, the pomace is left with a substantial amount of valuable polyphenolics. Therefore, there is an incentive to further process the blueberry pomace and extract those remaining polyphenolics for applications as natural colorants, encapsulated supplements or added nutraceuticals.

Most of the pomace extraction methods lead to dilute aqueous juice fractions and processing them into concentrates facilitates storage and transportation. Particularly, volume reduction and separation techniques are highly employed to produce juice concentrates and fractionate the dilute extracts. Multiple techniques have been developed aiming at the production of stable, nutrient-rich concentrated streams. Freeze concentration (cryoconcentration), osmotic distillation, membrane filtration and other multi-stage evaporation techniques are commonly used. Anthocyanins are labile compounds that have been shown to easily degrade and lose biological activity under severe processing parameters such as high temperatures, UV radiation and cross reaction with other processing chemicals. Therefore, a careful consideration has to be given to the choice of processing techniques which should not only be economically feasible but also limit the deactivation and loss of the bioactive compounds. Membrane technology could be a promising technology for recovery of these fragile biologically active compounds without destroying the aroma, colour, taste and other nutrient quality.

Frozen blueberry pomace was allowed to thaw to 21^oC prior to extraction. A Dionex model 200 accelerated solvent extractor (ASE) system interfaced with a solvent controller (Dionex Corp., Sunnyvale, CA) was used to extract anthocyanins from blueberry pomace. Samples (0.5 g) were loaded into 22 mL stainless steel extraction cells with a cellulose paper filter inserted at the bottom of the cells. The ASE extraction was carried out using water as solvent: 68 bar, 120^oC, five extraction cycles, 70% flush volume, 90 sec nitrogen purge time (no static time and no preheat time). For each extraction cycle it took approximately 5-6 min for the water to heat to 120^oC for a total run time of 25-30 min. Approximately 22 mL of extract from each extraction cycle was pooled after passing through a large microporous sieve. Pressurized hot water extracts were stored at -20^oC prior to total anthocyanin analysis and nanofiltration testing. Non-prefiltered or prefiltered (0.22 µm and 0.45 µm) blueberry pomace hot water extracts are collectively described here as feed solution. The extract total anthocyanin concentration was found to naturally vary in the range 85-125 mg/L.

The HPLC data for total anthocyanins, flavonols and chlorogenic acid in the ASE extract, retentate and permeate revealed that the highest amount of polyphenols was due to total anthocyanins (with over 82% wt.), while the highest amount of sugars was due to fructose (with over 70% wt.) in the feed (ASE extract). The HPLC chromatograms of 16 anthocyanins and 7 flavonols were identified. The most prevalent ACYs were malvidin-3-galactoside (15% wt, 528.9 g/mol), delphinidin-3-galactoside (14% wt, 500.8 g/mol) and malvidin-3-glucoside (13% wt, 528.9 g/mol). The most prevalent FLA were quercetin-3-galactoside (37% wt, 464.4 g/mol), quercetin-3-glucoside (16% wt, 464.4 g/mol) and quercetin-3-acetyl rhamnoside (11% wt, 490.4 g/mol). Chlorogenic acid (354.3 g/mol) represented only 6% of total polyphenols. Both NF270 and NF245 exhibited complete retention of total polyphenols, and sucrose. Only small amounts of glucose and fructose were found in the permeate fractions so that the rejections were higher than 97%.

The nanofiltration membranes were tested at extended filtration times until the maximum amount of feed volume could be removed in a dead end mode. As permitted by the dead-end setup, a minimum of 20% v/v of initial feed should remain in the pressure vessel to not disrupt mixing. This analysis allowed to compare the performance of the two membranes in terms of permeance, rejection, anti-fouling properties, and also to investigate degradation of polyphenols at longer reactor residence time. NF270 showed better performance than NF245. NF270 required approximately 19 hours to remove 80% of the initial volume and the polyphenols content was concentrated by a factor of 4.6. In the same amount of time NF245 reduced the volume by 60% and concentrated the polyphenols by a factor of 2.2. It required almost 30 hours to reduce the feed volume to the same performance as with NF270, but the flux started to decrease considerably after 21 hours, due to increased fouling. The temperature of the feed and retentate was monitored at the start and at the end of each filtration and it was found to not change by more than ±1.0°C. Both membranes showed complete rejection of polyphenols and during analysis no polyphenol degradation was detected. However, both NF270 and NF245 showed adsorbed polyphenols on their surfaces, as observed from the dark purple color of the used membranes. This could be an effect of particle agglomeration and polyphenol adsorption as seen previously in unstirred systems.

Previously, the filtration performance in dead-end mode was tested under different experimental parameters and those findings were determinant in the design of the crossflow setup. The setup was constructed to keep the feed continuously stirred and the crossflow rate was set at a maximum flowrate of 57 mL/min. Then, the feed was passed through a 0.22 µm prefilter to remove larger aggregated particles. Total polyphenols were rejected completely and no polyphenol degradation was observed during analysis. The permeance was considerably higher with NF270, which started at 3.5 L m^-2 h^-1 bar^-1 and then reached approximately 2.0 L m^-2 h^-1 bar^-1 after 3 hours of filtration. For NF245 the permeance started at 1.5 L m^-2 h^-1 bar^-1 and then decreased to 0.6 L m^-2 h^-1 bar^-1 after the same filtration time.

For the purpose of membrane reconditioning, the used membranes were cleaned with the following protocol: (1) washed in deionized water for 24 hrs; (2) washed in 0.2% w/v HCl and (3) washed with 0.1% w/v NaOH. After steps (2) and (3) the membrane was cleaned-in-place with deionized water until the pH was constant. While establishing the wash protocol it was observed that step (2) leaves the previously purple stained membrane slightly pink in color, step (3) changes the staining to light brown and, for the membranes used in crossflow, the latter staining was eventually removed completely. The membranes used in dead-end maintained the light brown color and the fouling was irreversible. Only 37% of the initial water permeance could be recovered for NF270 and less than 20% for NF245.