The effect of frozen storage on the phenolic compounds of Morus nigra L. (black mulberry) and Morus alba L. (white mulberry) fruit

Introduction. Morus nigra L. (black mulberry) and Morus alba L. (white 1 mulberry) display high concentrations of health-promoting compounds, particularly 2 phenolics. However, no published studies have addressed the changes in the 3 content of phenolic compounds during frozen storage, a widely used form of 4 preservation of these fruit in the Turkish countryside. This work was undertaken to 5 determine these alterations, if any, in order to assess whether the bioactive 6 properties of the produce may be altered significantly. Materials and methods. 7 Black and white mulberry fruit were collected at commercial maturity and frozen at 8 25 oC for up to 5 months. The content of selected phenolic acids and flavonoids was 9 analysed at harvest on fresh fruit and at monthly intervals on thawed samples by 10 High-Performance Liquid Chromatography with Diode-Array Detection (HPLC/DAD). 11 Results and discussion. Phenolic compound levels were higher in black than in 12 white mulberry fruit at harvest. Rutin and chlorogenic acid predominated 13 quantitatively in black mulberry, and decreased along frozen storage even though 14 some fluctuations were observed. Cathechin was the main compound detected in 15 white mulberry, and remained largely stable during the whole experimental period. 16 Conclusion. Although the concentration of the investigated phenolics varied to 17 different extents during frozen storage, fruit retained acceptable levels, which 18 suggests that this practice allows preserving satisfactorily the health-promoting 19 properties which characterise these fruit species. 20 21


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Mulberry (Morus sp.) fruit must be picked when fully ripe in order to attain their 21 delicate taste and flavour. Ripe mulberries are very soft and juicy, which causes the 22 fruit to be very prone to mechanical damage and to fungal rots. This means that 23 mulberry fruit are a highly perishable commodity, and shelf life at ambient 24 temperature may be as short as one day. All these aspects considerably limit the 25 commercial exploitation of mulberry species as a fresh produce, although mulberries 26 are used for the confection of a range of processed products, including juices, anti-27 obesity drinks, sauces, cakes, teas, wines, fruit powder or food colorants [1]. Yet, 28 extremely limited research efforts have been focused on the postharvest physiology 29 of these fruit, or on possible procedures for the extension of their keeping potential. 30 this study with the purpose of assessing the fate of some important phenolic acids 1 and flavonoids throughout frozen storage of mulberry fruit from two different species. 'Beyaz') were harvested from a family-led orchard in Alkamer (Iğdır, Turkey) at the 10 commercially ripe stage according to the usual indices in the producing area. Harvest 11 date was July 3, 2013, and fruit were collected from three trees of the same age per 12 species. Defect-and rot-free fruit samples were selected for uniform shape and 13 colour, and transported immediately to the laboratory. For each of both species, 14 samples were grouped into six batches, each of which was comprised of around 300 15 fruit (approximately 1 kg per batch). One batch per species was analysed 16 immediately after harvest as the fresh control. The five remaining batches were 17 introduced into food-grade polyethylene bags and stored at -25 ºC for up to five 18 months. One batch per species was removed monthly from the freezer and allowed 19 to thaw overnight at +4 ºC, after which samples remained at ambient temperature 20 during two hours before being analysed.

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Standard quality parameters were determined on 30 fruits per each species at 25 harvest. Fruit weight was determined with an electronic balance (0.01 g accuracy).

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Fruit width and length were measured with a digital calliper (0.01 mm accuracy).

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Titratable acidity (TA), pH, and soluble solids content (SSC) were assessed in juice 28 pressed from the whole fruit (10 fruit per replicate × 3 replicates). TA was determined 29 in 10 mL fruit juice by diluting in 10 mL distilled water and titrating with 0.1 N NaOH to 30 pH 8.1 [14], and expressed as g malic acid L -1 . A digital table refractometer (WAY-31 2S, Seoul, South Korea) was used for SSC assessment, and data given as ºBrix. The 32 pH of fruit juice was determined using a portable pH meter (Jenco Instruments Inc.,

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Phenolic compounds were extracted according to the method described in [15], 38 with some modifications. Approximately 200 g whole mulberries per species were 39 diced at each analysis date, and 5 g of this starting material were weighted and 40 sonicated for 10 min in 10 mL of 80% (v/v) acetone. The extract was centrifuged at 41 15,000 g and 4 ºC for 10 min, and the supernatant was collected. The insoluble 42 material re-extracted twice in 10 mL of 80% acetone, and the supernatants were  The phenolic extracts were analysed by High Performance Liquid 48 Chromatography (HPLC). The HPLC system included an LC-20 AT pump, a CTO-49 20A column oven, and a SPD-M20A prominence diode-array detector, and was 50 equipped with a SIL-20A HT auto sampler (Shimadzu Corp., Kyoto, Japan). The extract was filtered through a 0.45 µm nylon filter (Millipore Corp., Billerica, USA) 1 before injection. Chromatographic separations were performed on an Inertsil ® ODS-2 3V column (250 mm × 4.6 mm i.d., 5 µm particle size) (GL Sciences, Tokyo, Japan).

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Column temperature was 40 °C. The mobile phases were (A) acetic acid / water 4 (2:98, v/v), (B) 50% aqueous acetonitrile/0.5% aqueous acetic acid (1:1, v/v) and (C) 5 acetonitrile, delivered at a flow of 1.2 mL min -1 according to a gradient programme as 6 described in table I. The total running time per sample was 61 min. Individual 7 phenolic acids (chlorogenic acid, caffeic acid, syringic acid, o-coumaric acid, p-8 coumaric acid) and flavonoids (cathechin, myricetin, quercetin, rutin) were quantified 9 from regression curves calculated for authentic standards purchased from Sigma-Genotypic characteristics or climatic conditions may underlie these differences, as 1 phenolic-synthesising metabolic pathways are highly responsive to internal and 2 external factors.

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In this work, frozen storage at -25 ºC significantly affected the contents of 5 phenolic acids in both black and white mulberry fruit. Generally speaking, 6 concentrations of phenolic acids were lower in frozen fruit in comparison with freshly 7 harvested samples. However, some fluctuations were observed throughout frozen 8 storage. As an example, the content of chlorogenic acid decreased significantly 9 during the first 3 months of frozen storage in both black and white mulberry fruit, 10 followed by a late increase up to the end of the experimental period to similar levels 11 to those at harvest (figure 3). Although also displaying some fluctuations along 12 storage, caffeic acid content in black mulberries was lower than that at harvest at all 13 analysis dates, while no significant time-course differences were observed for white 14 mulberries (table III). For syringic and o-coumaric acids, different trends were 15 observed for each mulberry species: whereas their concentrations decreased 16 significantly in black mulberry with respect to harvest, the opposite was found for 17 white mulberry (table III). The content of p-coumaric acid in black mulberry showed a 18 transient increase after two months of frozen storage, but declined thereafter to 19 levels well below those at harvest.

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The content of selected flavonoids in black and white mulberry fruit after frozen 22 storage at -25 ºC was also determined. Similar cathechin levels were found at 23 harvest for both mulberry species considered (table IV) (table IV).
To the best of our knowledge, there have been no published studies on the 1 evolution of individual phenolics during frozen storage of mulberry fruit. Therefore, 2 results are discussed in comparison with other small fruits. Data indicate that all the 3 compounds considered in this work decreased to different extents during frozen 4 storage of black mulberry samples. In a similar study on wild blackberry (Rubus 5 ulmifolius Schott), it was found that total anthocyanins and phenolics decreased after 6 6 months of frozen storage at -24 ºC [21]. Significant anthocyanin loss also occurred 7 in blueberry (Vaccinium corymbosum L.) fruit after 6 months of frozen storage at -18 8 ºC [22], which was attributed to oxidation and/or condensation reactions with other 9 phenolic compounds. Häkkinen [23] reported that the effects of frozen storage for up 10 to 9 months on the content of flavonols and phenolic acids varied for different berries.

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For lingonberry (Vaccinium vitis-idaea L.) and bilberry (Vaccinium myrtillus L.) fruit, it 12 was found that myricetin levels decreased by 30% and 25%, respectively. In contrast, 13 no significant effects on total phenolics were found after frozen storage of raspberries

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In this work, the content of all the investigated phenolic compounds was higher in 35 black than in white mulberries at harvest. Generally speaking, frozen storage at -25 36 ºC significantly affected the levels of these compounds, but dissimilar trends were 37 observed for each species. In both black and white mulberry fruits, fluctuations in the 38 concentration of the selected phenolics were observed throughout the experimental 39 period, which may be attributed to their broad reactivity. However, although some 40 decrease was found in most cases in comparison with values at harvest, the most 41 prominent phenolics (particularly chlorogenic acid and rutin in black mulberry) 42 retained acceptable levels after 5 months of frozen storage.

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Since this work was formulated as a preliminary exploration of the effects of the 45 countryside practice of freezing mulberries on the contents of particular phenolics, no 46 additional anthocyanins were investigated. Given the strong effects on human health attributed to these compounds, and the finding that rutin content declined to some   Tables   Table I. Mobile phase gradient programme for HPLC analysis of phenolic extracts obtained from fresh and frozen samples of black and white mulberry fruit (Solution A: Acetic acid / water (2:98, v/v), Solution B: 50% aqueous acetonitrile / 0.5% aqueous acetic acid (1:1, v/v), Solution C: acetonitrile).     Values represent means of three replicates. Within each species, different letters represent significant differences along frozen storage.