Processing and Storage Effects on Blueberry (Vaccinium corymbosum L.) Polyphenolics

Blueberries are commonly consumed in processed forms, but limited information is available on how different processing methods and storage of processed products impact the polyphenolic content of the fruit. In this study, we determined how canning, puréeing and juicing of blueberries, as well as storage of processed products at 25°C, influenced the retention of chlorogenic acid, total flavonols, total anthocyanins, and total procyanidins. The retention of flavonols (5799%) and chlorogenic acid (64-100%) was greater than that of anthocyanins (4272%) and procyanidins (19-78%). The type of processing method impacted polyphenolic retention with canned products showing the greatest retention, followed by puréed products, and juices. Non-clarified juices retained higher levels of chlorogenic acid, total flavonols, and total anthocyanins than clarified juices, but clarified juices contained higher levels of total procyanidins. Significant losses of total anthocyanins and total procyanidins occurred in all processed products stored over six months at 25°C, which most likely was due to polymeric compounds formed as a result of condensation reactions among anthocyanins and procyanidins. In contrast, total flavonols and chlorogenic acid showed much greater retention during storage. Our results indicate that methods are needed to prevent polyphenolic losses during processing, especially in juices, and storage of processed products. INTRODUCTION Blueberries (Vaccinium corymbosum L.) contain a variety of polyphenolic compounds (anthocyanins, flavonols, procyanidins) in abundant quantities that are purported to have many health-promoting properties (Beattie et al., 2005). Blueberries, like other berries, are not only available fresh, but are available for consumption in several thermally processed forms (jellies, jams, juices, canned and purées). Several studies have investigated the effects of juice processing on blueberry polyphenolics (Skrede et al., 2000; Lee et al., 2002; Rossi et al., 2003; Srivastava et al., 2007), and the studies indicate that processing has a detrimental effect on polyphenolics. Besides juice processing, information is limited on how different processing methods and long term storage of processed products affect the nutritional quality of blueberries prepared from the same raw material. This information is needed for consumers who wish to incorporate higher levels of bioactive compounds into their diet, and processors who desire to retain, or possibly boost levels of bioactive compounds in their products. The objective of this study was to determine how different processing methods (juicing, canning, puréeing) and storage of processed products affected the retention of blueberry polyphenolics. MATERIALS AND METHODS Blueberry Samples Blueberries (cv. Bluecrop) harvested at the fully ripe stage were obtained from a commercial grower in Fayetteville, AR in June 2005. The fruit was stored at –20°C for less than one month prior to processing. Proc. IInd IS on Human Health Effects of F&V Ed.: B. Patil Acta Hort. 841, ISHS 2009 348 Juice Processing Frozen berries were simultaneously heated and mixed with a Mixco Batch mixer (Avon, NY) in a large steam kettle until the berry mash reached a temperature of 95°C. It was held at 95°C for 3 min and allowed to cool to 40°C. Depectinization of the mash was performed by adding 0.0827 ml/kg of Pectinex Smash® (Novozyme, Bagsvaerd, Denmark) and incubating the mash for 1h at 40°C. Negative alcohol precipitation test was used as an indication of complete depectinization. Following enzymatic treatment, the mash was pressed in a 25-L Enrossi bladder press (Enoagricol Rossi s.r.l., Calzolaro, Italy), and the juice and presscake were isolated. Half of the juice was clarified by centrifugation for 10 min at 6000 x g in a model CRU-5000 centrifuge (Damon/IEC Division, Needham, MA), while the other half received no clarification treatment. Both clarified and non-clarified juices were filled into 6 oz glass bottles and heated in a steam box (American Sterilizer Company, Erie, PA) until the juice temperature reached 90°C. The bottle caps were tightened and the juices were allowed to cool overnight. Juice samples were stored in the dark at 25°C. Canned-in-Water (CW) and Canned-in-Syrup (CS) Processing Blueberries were canned by the Downing (1996) method. Frozen berries (278 g) were added to 303 x 406 cans. Syrup was prepared by adding Sweetose 4300 corn syrup (Tate and Lyle, London, UK) to boiling water to reach a final brix reading of 40°. Boiling syrup (for CS cans) or water (for CW cans) were added to the cans to the brim and cans were exhausted for 4 min in a steam box (American Sterilizer Company, Erie, PA) at 87.8-93.3°C. The cans were then sealed, immersed in boiling water for 15 min, and stored at 25°C. Purée Processing Frozen berries were allowed to thaw and homogenized for 1 min on high speed using a commercial food processor. Blended berries were immediately added to the steam kettle and heated to a temperature of ~95°C. The purée was cooled and Sweetose 4300 corn syrup was added to the purée until 18° Brix was attained. The purée was subsequently heated to 92.8°C and added to 4 oz canning jars (Ball Corp., Muncie, IN). After sealing, the jars were immersed in boiling water for 15 min, cooled in cold water to 38°C, and stored in the dark at 25°C. Extraction of Polyphenolics Prior to the extraction of polyphenolics, the entire contents of canned samples (berries and cover solution) were blended for 1 min on high speed in a commercial food processor. Purée and juice samples required no pre-extraction step. The extraction of anthocyanins, flavonols and chlorogenic acid was performed as described by Cho et al. (2004). The concentrated extracts were filtered through 0.45 μm PTFE syringe filters prior to HPLC analysis. Procyanidins were extracted and purified according to the method of Prior et al. (2001). The concentrated extracts were filtered through 0.45 μm PTFE syringe filters prior to HPLC analysis. HPLC Analysis of Procyanidins The procyanidin analysis by HPLC was performed according to an adapted method of Kelm et al. (2006) with a 250 X 4.6 mm i.d., 5 μm Develosil diol (Phenomenex, Torrance, CA). The mobile phase consisted of a binary gradient of acetonitrile/acetic acid (98:2 v/v) (A) and methanol/water/acetic acid (95:3:2 v/v/v) (B). The flow rate was 0.8 ml/min with a linear gradient as follows: 0-35 min, 0-40% B, 35-45 min, 40% B, 45-47 min 50% B, 47-49 min 60% B, 49-50 min 100% B, 50-52 min, 100% B, 53-60 min, 0% B, followed by 5 min re-equilibration time. The procyanidin peaks were quantified by fluorescence detection with excitation at 276 nm and emission at 316 nm using a Waters Model 474 fluorescence detector (Milford, MA). Individual procyanidins with degrees of polymerization (DP) from DP1 through DP8 were