Infusion of fruits with nutraceuticals and health regulatory components for enhanced functionality

Food Research International 45 (2012) 93–102

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Infusion of fruits with nutraceuticals and health regulatory components for enhanced functionality Jissy K. Jacob a, 1, Gopinadhan Paliyath b,⁎ a b

Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G2W1 Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G2W1

a r t i c l e

i n f o

Article history: Received 5 August 2011 Accepted 12 October 2011 Keywords: Antioxidant activity Cell structure Lecithin Phospholipids Fruit preservation Short chain fructooligosacharides Solids content

a b s t r a c t Enrichment of fruits with nutraceutical components for increased nutritional- and health-beneficial qualities is a major goal for the food processing industries. In the present study, we have investigated the benefits of an osmotic infusion process by means of which a fructo-oligosacharide (Nutraflora™) was used to partially substitute sucrose in the infusion medium. Fruits such as sweet cherry, mango and blueberry could be subjected to osmotic infusion, resulting in improved quality characteristics. During infusion, loss of anthocyanins from blueberry and cherry fruits was observed, but this did not reduce the visual appeal of infused fruits. In conjunction with Nutraflora, fruits could be infused with soy lecithin (phospholipids) and excised mango assimilated significant amounts of phospholipids. Structurally, Nutraflora infused cherry fruits showed preservation of tissue structure similar to that in a fresh fruit. As well, Nutraflora infused fruits showed higher levels of soluble and insoluble solids content. DPPH radical scavenging activity of fresh and infused fruit extracts did not differ significantly suggesting that the antioxidant activity of infused fruits is not impaired by the process. Thus, infusion of fruits with desired components having health benefits can provide nutritionally superior fruit products with improved functionality. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The principle of osmosis as a means of dehydrating fruits and vegetables has been utilized for a long time. Controlled application of osmotic treatments can be used to generate food products (for instance, intermediate moisture foods, IMF) with improved quality compared to conventionally dried materials. The treatment involves immersing foods in aqueous solutions of sufficiently high concentration of sugars (for fruits) or salts (for vegetables). As a result, water drains from the tissue into the solution and solute transfers from the solution into the tissue due to differences in osmotic pressure. Simultaneously, solutes from the tissue may also leach out into the solution. Hence, the water activity of infused fruits is decreased while maintaining most of the sensorial characteristics (color, aroma and texture) that are very similar to the fresh fruit (Hawkes & Flink, 1978; Heng, Guilbert, & Cuq, 1990). Current technologies employ sucrose for osmotic dehydration of fruits. However, in terms of health, consumption of such fruit products is not ideal because of the high glycemic index of sucrose and the increased chances of developing type II diabetes. Osmotic infusion of fruits can also be used as a pretreatment step prior to processing operations such as convection drying, or freezing,

⁎ Corresponding author at: Department of Plant Agriculture (E.C. Bovey building), University of Guelph, Guelph, Ontario, Canada, N1G2W1. Tel.: +1 519 824 4120x54856; fax: +1 519 767 0755. E-mail address: [email protected] (G. Paliyath). 1 Present address: Nestle PTC, Marysville, Ohio, USA. 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.10.017

thereby reducing the overall energy requirements in the dehydration process. In addition, it has been reported that sugars that are infused into the fruit tissue help retain the aroma in osmo-vacuum dehydrated fruits (Weinjes, 1968). Depending on the osmotic solute and processing conditions, significant water removal (40–70% of initial moisture) can be achieved (Matuska, Lenart, & Lazarides, 2006). The efficiency of the process and the quality of final product depend on factors such as; a) initial product characteristics viz., species, variety, maturity level, size, shape; b) application of pretreatments such as freeze/thawing or blanching; c) composition of the infusion medium (type of solute, its molecular weight); d) concentration of the infusion medium; e) process temperature; f) phase contact (agitation, solution/product mass ratio, contact between the food pieces and solution) and g) process duration (Rastogi, Raghava Rao, & Niranjan, 2005). In the present study, we have addressed two major issues facing the fruit sector; (1) continuous availability of high quality fruits year-round, and (2) improving the nutritional quality of fruits after harvest. Not all fruits contain all classes of health-beneficial components. Therefore, if strategies could be developed for enriching fruits with the required nutraceuticals and preserve them for a long duration to ensure year-round availability, it will be helpful to the consumers, growers and the food industry. Consequently, processed fruits and fruit ingredients with high quality have a high demand for use in many food formulations such as frozen desserts, yogurt, ice cream and fruit salads. Fruits used in such applications must preserve their natural color and flavor, be preferably free of preservatives, and possess

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acceptable texture. Fresh or IQF fruits can be processed to reduce their water content and lower the water activity (aw) so that they can be stored at room temperature for longer duration. The present study describes a novel protocol for dehydrating fruits with nutraceuticallyactive short chain fructo-oligosaccharides (sc FOS) to achieve an ideal water activity. Sc FOS has been reported to improve digestive and immune health. NutraFlora® is diabetic-safe and can bring exceptional functional value to foods formulated for glycemic control (www. gtcnutrition.com). NutraFlora® consists of molecules of which ~35% are oligosaccharides that are one glucose and two fructose units (GF2), 55% that are one glucose and three fructose units (GF3) and 10% that are one glucose and four fructose units (GF4). Bonds between fructose units are β (1 → 2) linked to the terminal D-glucose unit by α (1 → 2) linkage. As well, during this process, the fruits can be enriched with other nutraceutical components that are not normally present in fruits, but that can provide increased health benefits and protection from diseases (Sabater-Molina et al., 2009). 2. Materials and methods Bing cherries harvested at optimum maturity (days after bloom, acidity, °Brix, color, taste) were obtained from Vineland research station, Ontario. Fresh ripe mangoes and blueberries were purchased from a local grocery store. Fresh blueberries were quick frozen at −20 °C (18 h) prior to use. Sucrose was purchased from a local grocery store. NutraFlora® (GTC Nutrition) was supplied by Inovita Inc., Toronto. Soy lecithin (L-α-Phoshatidylcholine from soybean, Type II-S) and Vitamin C were purchased from Sigma. Wine polyphenols, Provinols®, was procured from Givaudan-Lavirotte, France. 2.1. Infusion of fruits 2.1.1. Pretreatment of fruits Fresh harvested cherries were stored in the cold room at 5 °C until use. Cherries were washed, blot dried and pitted. Fresh mangoes at an intermediate ripe stage were peeled and stones removed. Fresh high bush blueberries (Vaccinium cyanococcus) were quickly frozen at −20 °C for 18 h before use. 2.1.2. Preparation of infusion medium The compositions of the media used for infusion are given in Table 1. The common ingredients used in the preparation of Infusion medium were sucrose syrup (25%), NutraFlora® and soy lecithin (1%). Wine polyphenols (Provinols, 1%) were included in the infusion medium for cherry and blueberry. The media also contained vitamin C (0.25%). The medium used for infusion of mango slices included 0.1% of mango flavor. The ratio of the volume of fruit to volume of infusion medium was approximately 1:1. Lecithin was dissolved in an adequate amount of hot water (10 g/50 ml) to form a thick slurry. After cooling to room temperature, this was added to sucrose syrup with thorough mixing. Finally NutraFlora® (a thick liquid with approximately 80% solids) was added and the solution was mixed Table 1 Composition of infusion medium used for the infusion of cherry, mango and blueberry. Ingredients

NutraFlora® Sucrose Lecithin Wine polyphenols (Provinols®) Vitamin C Mango flavor Water

Quantity required to make 1 l of infusion medium Cherry

Mango

Blueberry

500 ml 250 g 10 g 10 g

500 ml 250 g 10 g –

500 ml 250 g 10 g 10 g

2.5 g – To make up to 1 l

2.5 g 1 ml To make up to 1 l

2.5 g – To make up to 1 l

well. These ingredients when dissolved in water gave final solids concentration between 70 and 75°Brix. Infusion medium sufficient to conduct three cycles of infusion were prepared and stored at room temperature protected from light. 2.1.3. Infusion process Pitted, weighed cherries were taken in a container and infusion medium was added at a ratio of 1:1(w/v) to completely immerse the cherries. The container was then loosely covered with aluminum foil and placed in a vacuum apparatus under 35–40 mm Hg vacuum for about 2 h. The vacuum was released and infusion was continued at room temperature for 24 h (1st cycle). At the end of the first cycle, the infusion medium was drained using a strainer and fresh medium was added to the fruits. The 2nd cycle continued for another 24 h at room temperature followed similarly by a 3rd cycle. At the end of the 3rd cycle, fruits were drained, blot dried on absorbent paper and weighed. The infused fruits were then dried in trays in a precision mechanical convection oven (Model STM 135) at 45 °C for 6–7 h. After cooling to room temperature, the infused and dried fruits were packed in Ziploc bags and stored under refrigeration (4 °C) until further use. Samples were also stored at room temperature to observe their shelf stability. 3. Analytical methods 3.1. Moisture content determination Moisture content of fresh, infused and oven dried fruits was determined using a laboratory oven. Samples were accurately weighed in an aluminum dish and kept in the oven set at 50 °C for several hours until there was no further change in weight. Moisture content, expressed as % wet basis is obtained as below: % Moisture ¼ ðInitial weight of fruits − dried weight of fruitsÞ  100: Initial weight of fruits

Total solids content can be determined from moisture content as below: Total solids=Dry matter ¼ 100 − % Moisture:

3.2. Water loss (WL) and solids gain (SG) The amount of water loss and solids gain after infusion of fruits is calculated as follows: Water Lossð% Þ ¼

Solid Gainð% Þ ¼

ðM 0 −m0 Þ−ðM−mÞ  100 M0

ðm−m0 Þ  100: M0

Where, M0 M m0 m

Initial weight of the fruits before infusion (g) Weight of fruits after infusion (g) Dry matter content of untreated fruits (g) Dry matter content of infused fruits (g)

3.3. Water activity determination Water activity (aw) of fruit samples was determined using AquaLab™ Series 3 water activity meter (Decagon Devices Inc.). AquaLab™ uses

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the chilled-mirror dew point technique to measure aw of a sample. Measurements were conducted on three separate samples. 3.4. Soluble solids Total soluble solids were determined using a handheld refractometer (Fisherbrand®) with a measurement range of 0–25°Brix. Ten grams of untreated and/or infused fruits were homogenized in water (20 ml) using a Brinkman Homogenizer, fitted with a Polytron PTA 10 probe. The homogenate was centrifuged using a Sorvall RC-6 Plus centrifuge at 15,000 ×g for 15 min and the supernatant was collected. A drop of the supernatant was placed on the sample glass of the refractometer and soluble solids content was measured directly from the graduated scale by focusing the eyepiece. Double-distilled water was used to calibrate the refractometer. High Brix samples were suitably diluted with distilled water to obtain readings within the range of the instrument. The insoluble solids (wet weight) were measured by weighing the pellet obtained after centrifugation. 3.5. Isolation and estimation of total polyphenols content Ten grams of untreated and/or infused fruits were homogenized in water (20 ml) using a Brinkman Homogenizer, fitted with a Polytron PTA 10 probe. The homogenate was centrifuged using a Sorvall RC-6 Plus centrifuge at 15,000 ×g for 15 min and the supernatant was collected and used for total polyphenol estimation. Using the same procedure, fruit samples were also extracted using methanol and the supernatant was dried over a heating block at 40 °C under N2 to remove methanol and dissolved in water before polyphenol estimation. Total polyphenol content was estimated by using the Folin-Ciocalteau method (Singleton, Orthofer, & Lamuela-Raventos, 1999). To each 1 ml of sample, 5 ml distilled water was added along with 0.5 ml of Folin Ciocalteau reagent (2 N), vortexed and allowed to incubate for 5 min. Next, 1 ml of 5% (w/v) sodium carbonate solution was added to each sample, vortexed, and incubated in the dark for 1 h. After incubation, samples were vortexed and absorbance measured at λmax725 nm using a spectrophotometer. A standard curve was generated using catechin with concentrations ranging from 25 to 200 μg/ml. The total polyphenol concentrations were expressed as weight catechin equivalent. 3.6. Scanning Electron Microscopy (SEM) of fresh and infused fruits High resolution images of fresh and infused cherries were obtained using a Scanning Electron Microscope (Model S — 570, Hitachi, Tokyo). Cherry samples were sectioned using a razor blade and fixed in 2% glutaraldehyde in 0.07 M Phosphate buffer at pH 6.8 for 14 h at 4 °C. The fixed samples were washed with phosphate buffer three times (10 min cycles) to remove glutaraldehyde. The sections were dehydrated in four 15 min changes of 50, 70, 80 and 90% ethanol followed by three 15 min changes in 100% ethanol. The tissues were dried using the critical point drying technique, freeze fractured in liquid nitrogen and mounted on labeled aluminum specimen stubs. The samples were sputter-coated (Emitech K550 sputter coater) with a 1.8 nm thick layer of gold/palladium. The coated samples were viewed and photographed at an accelerating voltage of 20 kV. The images were obtained using Quartz PCI-7 image management system software.

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This methanol extract was gently heated on a heating block set at 40 °C purged with N2 gas to remove methanol. Samples were re-dissolved in distilled water and passed through a C18 Sep-pak column. The phenolic components were eluted using methanol and were used for LCMS analysis as described earlier (Jacob, Hakimuddin, Fisher, & Paliyath, 2008). 3.8. Sugar analysis of infused fruits by liquid chromatography Five gram each of untreated or infused fruits was homogenized in 15 ml methanol using a Brinkman Homogenizer, fitted with a Polytron PTA 10 probe. The homogenate was centrifuged using a Sorvall RC-6 Plus centrifuge at 15,000 rpm for 15 min and the supernatant was collected. This methanol extract was gently heated on a heating block set at 40 °C purged with N2 gas to remove methanol. Samples were re-dissolved in distilled water and passed through a C18 Sep-Pak column. The flow through containing the sugars was collected and HPLC (Waters 600E System Controller) performed using a Biorad Aminex column coupled to a refractive index detector (Waters 410) using acetonitrile:water (1:1) at a flow rate of 1 ml/min. 3.9. Thin layer chromatography to assess the uptake of lecithin in infused fruits Ten gram samples of untreated and infused fruits were homogenized in 15 ml chloroform using a Brinkman Homogenizer, fitted with a Polytron PTA 10 probe. After extraction, 0.5 ml of 4 N HCl was added followed by 5 ml of methanol and left covered overnight at room temperature. The chloroform:methanol layer was removed and filtered using glass wool and the filtrate was centrifuged at 300 rpm for 5 min in a Sorvall Legend RT centrifuge. Chloroform: methanol layer was evaporated under N2 using a heating block at 40 °C. The samples were then re-dissolved in 250 μl of chloroform: methanol (2:1). An appropriate aliquot (50 μl) of samples were spotted on the TLC plate (Whatman LKD) along with appropriate standards. After spotting, the plates were air dried at room temperature in the fume hood before subjecting to separation in solvent system 1 consisting of chloroform:acetic acid:methanol:water (80:20:2:1 v/v). When the solvent front reached about half the total height of the plate, the plate was removed and air dried in the fume hood before it was kept in solvent system 2 consisting of petroleum ether:diethyl ether:acetic acid (80:20:2 v/v). The plates were allowed to dry overnight and the separated lipids were detected by spraying with 25% H2SO4 and drying in a laboratory oven at 80 °C. The intensity of the charred area was compared to qualitatively assess the uptake of lecithin. 3.10. DPPH radical scavenging capacity of infused fruits The ability of polyphenols present in fruits (control/untreated versus infused) to scavenge free radicals was measured by using 1, 1diphenyl-2-picryl-hydrazyl (DPPH) (Molyneux, 2004). A 0.2 ml aliquot of each phenolic fraction (to give a final total phenol content of 0.25, 0.50, 1.0, 2.0, 5, 10 or 20 μg) was mixed with 0.8 ml of 0.1 mM ethanolic DPPH, vortexed well and incubated at room temperature for 30 min. The absorbance was then measured at λmax517 nm. The control sample was 0.2 ml of 95% ethanol. Antioxidant activity was expressed as % DPPH scavenging, calculated as [(control absorbance-quenched extract absorbance) / control absorbance] × 100.

3.7. Mass spectral analyses of phenolic components present in untreated and infused cherries

3.11. Statistical analysis

Ten grams of each sample was homogenized in 10 ml methanol using a Brinkman Homogenizer, fitted with a Polytron PTA 10 probe. The homogenate was centrifuged using a Sorvall RC-6 Plus centrifuge at 15,000 ×g for 15 min and the supernatant was collected.

The data were subjected to statistical analysis by using GraphPad Prism software, version 4 and multiple means were compared using one way ANOVA followed by “Tukey's test” to evaluate the level of significance. Significantly different means (p b 0.05) are denoted by

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different superscripts. All experiments were replicated three times or more and the results shown are the mean ± SD. 4. Results The infusion medium described in this study was used for osmotic dehydration/infusion of three seasonal fruits: cherry, mango and blueberry (Table 1, Fig. 1). The composition of the infusion medium was formulated in an attempt to minimize the uptake of sucrose into the tissue, while enhancing the incorporation of nutraceuticals such as NutraFlora®, lecithin, and wine polyphenols. Sucrose was partially replaced with NutraFlora® to achieve high osmotic concentration (65–70°Brix) in the infusion medium. After infusion, the fruits were dried in a laboratory model convection oven at 45 °C for 6–7 h to further reduce the moisture content and enhance the shelf stability. 4.1. Characteristics of infused fruits Untreated and infused cherries were oven dried at 45 °C for several hours to reduce water activity. Cherries were infused in infusion media containing sucrose + NutraFlora® + wine polyphenols + lecithin (Table 1). During convection drying of untreated and infused cherries, moisture loss was significantly higher in untreated cherries compared to the infused cherries. After drying for about 8–10 h, the untreated cherries shrunk considerably and were less flavorful compared to infused cherries (data not shown). Fig. 2 shows the physical appearance of cherry, mango and blueberry before and after the infusion process and convection oven drying for 7 h at 45 °C. There were no drastic differences in the physical characteristics of fruits. In general, the infused, dried cherry, mango and blueberry had an appealing flavor and texture. Moisture content, solids gain and water activity values for untreated and infused fruits — cherries, mangoes and blueberries are shown in Table 2a, 2b and 2c. The infusion process (72 h)

significantly (p b 0.05) reduced moisture content of fresh cherries (75% to 49%). A further reduction was achieved by convection drying resulting in a product with nearly half the moisture content of fresh cherries (Table 2a). Similarly, after infusion, mango pieces lost about 50% of its initial moisture (89% to 35%) which was the highest among the three fruits studied. However, subsequent oven drying did not significantly (p > 0.05) lower the moisture content of the infused fruits. This may be attributed to relatively high solids uptake (Table 2b) by infused mango slices, potentially blocking the surface layers of the product and creating an additional resistance to mass exchange in the convection drying step. This may also be due to increased retention of water molecules in the infused fruits that may provide added structural stabilization from the infused molecules. The gain in solids was relatively low in infused and oven-dried cherry and blueberry fruits (Table 2b). In the case of Blueberry, a reduction in moisture content of only about 15% could be attained by infusion process (81 to 67%) which may be attributed to the tough waxy skin surface (cuticle) that was mostly impermeable to the diffusion of water. Consequently, the oven drying step could not remove significant amounts of moisture from infused blueberries. The water activity (aw) of infused and oven dried fruits ranged from 0.857 for cherries, 0.792 for mangoes and 0.895 for blueberries (Table 2c) showing significant reduction (p b 0.05) from the fresh samples. The infused fruits could be stored at 4 °C for several months without any noticeable physical quality change.

4.2. Structural analysis of fresh and infused cherries Osmotic dehydration of fruits may result in drastic changes to the structure of the tissue. The objective of an ideal infusion/dehydration process is to preserve the integrity of the tissue as much as possible, while facilitating the uptake of the desired nutraceutical components. During osmotic dehydration of fruits using sucrose alone, the tissue structure collapsed by the removal of water and a large proportion of the nutritional ingredients. In commercially available sucrose-

Fig. 1. Flowchart for infusion process of cherries.

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Untreated

Infused

Infused & oven dried

Cherry

Mango

Blueberry

Fig. 2. Fresh, infused and oven dried cherry, mango and blueberry.

infused fruit products, the color and flavor are added back to provide a product that is quite distinct in taste, texture and quality from the original fruit. The structural features of cherry fruits infused in various media were analyzed by scanning electron microscopy to evaluate the effectiveness of this novel infusion process to maintain the tissue integrity of fruits. The tissue and cellular structural features of fresh and infused cherries are shown in Fig. 3 A, B, and C. The dehydrated tissues were fractured in liquid nitrogen to obtain better exposure to structural features before sputter-coating. Fig. 3A shows the tissue structure of a fresh cherry segment depicting the typical parenchymatous shape of the cell with well preserved cell walls. The cells are usually quite large in ripe fruits having a large central vacuole. Once these are ruptured during dehydration, the cell membrane adheres to the cell wall showing ridge like structures showing the impression of underlying cellulose microfibrils. Fig. 3B shows the microstructure of oven dried cherries infused in NutraFlora®+ sucrose where the microstructure of cells appears completely collapsed. Even though the water loss and water

Table 2a Moisture content (%) of untreated infused and oven dried cherries, mangoes and blueberries. The superscripts indicate the values showing significant differences; a- from untreated fruits; b- from infused fruits; and c- from infused, oven-dried fruits. Fruit

Cherry Mango Blueberry

Moisture content (%)

activity in fruits infused in sucrose and NutraFlora alone are comparable to those infused in other media, the cell structure appears to be totally altered. The smooth appearance of the membrane in fresh cherry segments appears to have been lost and is replaced by ridge like structures. However, for fruits infused in infusion media containing NutraFlora® +sucrose and lecithin, the structural integrity was very well maintained and indistinguishable from the tissue structure of the fresh cherry, and well preserved in infused fruits after several hours of convection oven drying (Fig. 3C). Structural integrity is directly related to physical and organoleptic properties of foods. The shape and size of infused fruits remain more close to the original fresh fruit. Similarly the eating quality of NutraFlora/lecithin infused cherries in terms of juiciness or ‘plumpness’ is more close to a natural fresh fruit than air-/sun-dried fruits which are commercially available. 4.3. Composition of polyphenols and sugars in infused cherries An objective of the infusion process is to maintain the nutritional quality of fruits as close as possible to the fresh fruit or improve the nutrient content during infusion. Consequently, the levels of polyphenols and sugars in infused fruits were analyzed to evaluate changes Table 2b Water loss and solids gain values for infused cherries, mangoes and blueberries.

Untreated

Infused

Infused and oven dried

Fruit

Water loss (%)

Solids gain (%)

75.7 ± 2.5b,c 89.3 ± 2.2b,c 81.8 ± 1.4b,c

49.6 ± 2.4a,c 35.0 ± 2.2a 67.5 ± 1.0a

38.1 ± 1.7a,b 31.3 ± 2.0a 62.6 ± 3.2a

Cherry Mango Blueberry

44.7 ± 3.3 62.1 ± 1.4 28.9 ± 0.3

7.0 ± 3.0 40.0 ± 3.4 7.3 ± 0.1

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Table 2c Water activity (aw) of untreated, infused and oven dried cherries, mangoes and blueberries. Fruit

Water activity (aw) Fresh

Cherry Mango Blueberry

Infused b,c

Infused and oven dried a

0.882 ± 0.034 0.924 ± 0.001a,c 0.938 ± 0.009a,c

0.967 ± 0.002 0.970 ± 0.015b,c 0.968 ± 0.005b,c

0.857 ± 0.021a 0.792 ± 0.007a,b 0.895 ± 0.014a,b

The superscripts indicate the values showing significant differences; a- from untreated fruits; b- from infused fruits; and c- from infused, oven-dried fruits.

that may have occurred during infusion. The fruits were subjected to aqueous and methanol extraction and the levels of polyphenols and sugars estimated. The total polyphenol contents of fresh and infused fruits are given in Table 3. Aqueous-extraction yielded much higher levels of polyphenols from fresh and infused cherries in comparison to methanol extraction. A drastic decline in the extractable polyphenols after infusion (745 μg to 343 μg, Table 3) suggests that the polyphenols may have leached out during the infusion process in cherries. The polyphenol levels in mango fruits were relatively low. Aqueous extraction of fresh and infused blueberries yielded nearly

A

similar amounts of polyphenols. However, methanol-extraction resulted in a much higher yield of polyphenols, especially from infused blueberries. Thus, different fruits appear to have different characteristics of retaining nutritional components during infusion. LC-MS analysis of methanol extracts of fresh and infused cherries in various media was conducted to understand the changes, if any, in the polyphenolic profile due to infusion. The major anthocyanins in sweet cherry include cyanindin-3-rutinoside and cyanidin-3-glucoside and the major hydroxycinnamates included neochlorogenic acid and p-coumaroyl quinic acid (Sharma, Jacob, Subramanian, & Paliyath, 2010). The polyphenolic profile is expressed in terms of total hydroxycinnamates and total anthocyanins (mg per 100 g dry matter content). Fig. 4 shows the differences in polyphenolic profile between fresh and infused cherries. There was no significant reduction (p> 0.05) in the amount of hydroxycinnamates present in fresh or NF+ S-infused or sucrose-infused cherries while NF+ S + L-infused cherries showed a significant (pb 0.05) reduction in hydroxycinnamates. In the case of anthocyanins, significant (pb 0.05) differences were observed between treatments. Sucrose-infused cherries retained significantly (pb 0.05) more polyphenols than those infused in the other formulations. The infusion process appears to leach out a considerable amount of anthocyanins, but not hydroxycinnamates from fresh cherries. This may

B

C

Fig. 3. Scanning electron micrograph of a cherry tissue segment subjected to infusion in various media. A—Fresh cherry; B—Cherry infused in sucrose medium; C—Cherry infused in NutraFlora, sucrose and lecithin containing medium. Infused fruits were oven dried for 12 h before analysis. CW—cell wall; M—membrane folds.

J.K. Jacob, G. Paliyath / Food Research International 45 (2012) 93–102 Table 3 Total polyphenol content of untreated and infused cherries, mangoes and blueberries, extracted using water and methanol. Fruit description

Untreated cherry Infused and oven dried cherry Untreated mango Infused and oven dried mango Untreated blueberry Infused and oven dried blueberry

Table 4 Sugar profile of untreated and infused cherry and mango, as analyzed using HPLC. Sugars

Total polyphenol content (μg/g fwt*) Water extraction

Methanol extraction

745 ± 45 343 ± 10 14 ± 5 71 ± 9 727 ± 35 706 ± 21

289 ± 16 211 ± 22 71 ± 10 147 ± 27 1210 ± 55 1556 ± 97

*fwt = fresh weight.

also be due to a decreased extraction efficiency of polyphenols from NutraFlora® infused fruits, since NutraFlora® may tightly bind the polyphenols. 4.4. Sugar levels in fresh and infused fruits Fruits are generally high in sucrose, fructose and glucose. Potential changes in sugar levels in fresh and infused fruits were analyzed by HPLC-refractive index detection. Known amounts of NutraFlora®, sucrose, fructose and glucose were first analyzed to confirm elution times and relative area percentages of sugar standards. During HPLC analysis, NutraFlora was eluted at 8.50 min, sucrose at 9.97 min, glucose at 12.19 min and fructose at 15.97 min. The composition of various sugars in fresh and infused cherries and mango slices is given in Table 4. Fresh cherries contained primarily glucose and fructose. During infusion, there was a significant uptake of NutraFlora (16 mg/g). Sucrose levels also increased in infused fruits. However, the levels of glucose and fructose were 15–20 times higher in infused fruits. It is likely that during infusion, some of the sucrose that is absorbed might have been enzymatically (invertase) broken down resulting in high tissue levels of glucose and fructose. By contrast to cherries, mango showed the highest level of uptake of NutraFlora® (283 mg/g). The sucrose level also increased five-fold; however, this was nearly half of the NutraFlora® level. Glucose and fructose levels remained almost similar in fresh and infused mango tissue. 4.5. Soluble and insoluble solids content in infused fruits The soluble and insoluble solids levels in fresh and infused fruits were analyzed to evaluate if the infusion process enhanced the dietary fiber content of fruits. Equal amounts of fresh and infused fruits

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Cherry (mg/g) Fresh

NutraFlora Sucrose Glucose Fructose

Mango (mg/g) Infused

b

0.0 ± 0.00 0.1 ± 0.01b 6.2 ± 0.04b 6.1 ± 0.03b

Fresh a

16.0 ± 0.46 5.0 ± 0.18a 108.0 ± 1.63a 121.0 ± 2.30a

Infused b

0.0 ± 0.00 29.5 ± 3.21b 5.9 ± 1.79b 31.6 ± 0.27b

283.0 ± 6.11a 143.0 ± 3.48a 9.0 ± 0.22a 39.0 ± 0.96a

The superscripts indicate that the values are significantly different; a- from fresh fruits, and b- from infused fruits.

(10 g) were extracted in equal volume of water (20 ml), centrifuged and the soluble solids level determined using a hand held refractometer. The infusion process decreased the soluble solids level in cherries by 45% (Table 5a). This may primarily be due to the loss of components other than sugars, since the infusion process increased the level of glucose and fructose in cherries (Table 4). The soluble solids level increased from 4% to 30% in infused, oven dried mango (Table 5a) which is more in line with the increase in NutraFlora® and sucrose in infused fruits (Table 4). There was a slight increase in the soluble solids content of infused blueberries (Table 5a). The content of insoluble solids (residue left over after centrifugation of the homogenate at 15,000 ×g) varied among fresh and infused fruits. In most cases, aqueous extraction yielded a higher insoluble solids content/g dry matter as compared to methanol extraction. This suggests that fiber composition in the infused fruits may tend to bind water and increase the volume of insoluble solids. The insoluble solids contents of fresh and infused fruits are given in Table 5b. Fresh cherries had similar levels of insoluble solids irrespective of the infusion medium. Infused cherries possessed a slightly higher amount of insoluble solids when extracted in an aqueous medium. Infused mango had the highest level of insoluble solids and when extracted in an aqueous medium, the levels were three times higher than that obtained from fresh mango. Infused blueberries showed a slightly lower level of insoluble solids than the fresh fruits (Table 5b). The physical appearance of the insoluble solids pellet is shown in Fig. 5. It was interesting to notice that the proportion of wet mass of insoluble solids fraction of commercially available sucrose infused samples of blueberry, sweet cherry, sour cherry and cranberry was considerably low compared to those isolated from NutraFlora/sucrose/lecithin infused fruits (Table 6). As well, the soluble solids level in the aqueous fraction was much higher in the sucrose infused fruits potentially due to a high level of sucrose uptake into the fruits.

4.6. Lipid composition of fresh and infused fruits Fruits normally contain very low levels of phospholipids due to extensive phospholipid catabolism that occur during the ripening process (Paliyath, Tiwari, Yuan, & Whitaker, 2008). Thus, enriching ripe fruits with nutritionally essential phospholipids is of extreme interest both in terms of quality and nutrition perspectives. To evaluate the changes in fruit lipid composition that may have resulted from the uptake of lecithin, total lipids were isolated from fresh and infused blueberry, cherry and mango fruits. Soy lecithin is enriched in Table 5a a: Total soluble solids content of water extracts of untreated and infused cherries, mangoes and blueberries.

Fig. 4. Polyphenol levels in cherries obtained by LCMS analysis (NF = NutraFlora; S = sucrose; L = lecithin).

Fruit description

Total soluble solids (Brix)

Untreated cherry Infused and oven dried cherry Untreated mango Infused and oven dried mango Untreated blueberry Infused and oven dried blueberry

18 10 4 30 6 10

100

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Table 5b b: Total insoluble solids content of untreated and infused cherries, mangoes and blueberries, extracted using water and methanol. Fruit description

Untreated cherry Infused and oven dried cherry Untreated mango Infused and oven dried mango Untreated blueberry Infused and oven dried blueberry

Table 6 Soluble and Insoluble solids from commercial samples of sucrose infused cranberry, blueberry, sweet cherry and sour cherry.

Total Insoluble solids (g/g DM⁎⁎)

Parameter

Water extraction

Methanol extraction

0.51 2.55 3.87 10.88 3.36 2.20

0.60 1.21 2.42 5.04 1.60 2.46

Wet weight of pellet(g) Aqueous-extraction 3.51 Methanol extraction 1.74

Cranberry

°Brix Aqueous-extraction Methanol extraction

⁎⁎ DM = Dry Matter.

phospholipids such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Both phosphatidylcholine and phosphatidylethanolamine are recognized as nutritionally important ingredients (National Academy of Sciences, 1998). Total lipids were isolated by homogenization of equal amounts of fruit tissue in chloroform:methanol (2:1 v/v) and purified. The lipids were subjected to thin layer chromatography using a dual solvent system for separating the phospholipids and the neutral lipids (Fig. 6). Phospholipids were generally low in the composition of lipids of fresh fruit. Phosphatidylcholine was present as a minor component in the lipid profiles of un-infused fruits. After infusion in lecithin/NutraFlora medium, the fruits accumulated a considerable amount of lipids. In general, the infused fruits contained much more phospholipids than the fresh fruits. The uptake was exceptionally prominent in mango, where extensive accumulation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine could be observed. In addition, there were notable increases in the 1,2- and 1,3-diacylglycerols and free fatty acids (bands corresponding to DG). Blueberries contained a higher level of anthocyanins that were separated during the chromatography. As well, blueberries and cherries also contained a high level of alkanes (band corresponding to HC). Since mangoes were sliced, extraction of surface waxes potentially did not occur. The results suggest that phospholipids from lecithin can be incorporated into phospholipid-deficient fruits to improve their nutritional quality.

31 33

Total polyphenols (μg/g) Aqueous-extraction 204 ± 3 Methanol extraction 274 ± 3

Blueberry

Sweet cherry

Sour cherry

6.38 2.06

4.35 1.93

2.23 1.32

27 35

29 35

30 35

300 ± 14 1530 ± 5

1440 ± 15 1949 ± 27

194 ± 5 524 ± 11

DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging potential has been reported to be an ideal method for the evaluation of free radical scavenging capacity of fruit extracts (Sanchez-Moreno, 2002).Therefore, the DPPH radical scavenging capacities of polyphenols isolated from fresh and infused cherry, mango and blueberry, were evaluated. Fig. 7 is a graphical representation of DPPH scavenging abilities of polyphenols in these fruits before and after infusion. The DPPH radical scavenging activity showed a dose dependent increase in all samples. Among all the fruit extracts tested, the mango extract showed the highest scavenging ability on a specific activity basis. However, there were no significant (pb 0.05) differences in the DPPH radical scavenging ability of extracts from fresh or infused fruits of cherry, mango or blueberry. 5. Discussion Fruits form an important food group in human diet, and contain several nutraceutical components with disease preventive function. The availability of some fruits such as the tender fruits (cherry, peach, plum, grape etc.) and berry fruits (strawberry, blueberry, raspberry etc.) is seasonal, as they have a very short ripening and marketing window. These fruits cannot be subjected to long-term controlled

BF

BI

CF

CI

MF

MI

4.7. Antioxidant potential of infused versus fresh fruits (DPPH assay)

HC An important aspect of any infusion process is the maintenance of nutritional quality in infused fruits. Fruit consumption is important for disease prevention and regulation of health owing to the antioxidant activity of several components in fruits. In technologies involving dehydration and processing, it is always a concern that the antioxidant activities may be negatively affected because of the loss of antioxidants by leaching or polymerization. In order to evaluate if the NutraFlora/ lecithin infusion process affected the antioxidant activity in infused fruits, the fruit extracts were evaluated for their free radical scavenging ability. The ability of polyphenols to scavenge free radicals has been reported in several studies (Bors, Heller, Michael, & Saran, 1990; Cotelle et al., 1996; Hanasaki, Ogawa, & Fukui, 1994; Jacob et al., 2008). The

ALD FFA DG1,3 DG1,2

PA

Infused blueberry

Fresh blueberry

Infused mango

Fresh mango

Infused cherry

Fresh cherry

PE / PS

Fig. 5. Sediment obtained after water extraction and centrifugation (15,000 rpm, 15 min) of fresh and infused fruits.

PC

Fig. 6. Thin layer chromatographic analysis of total lipids isolated from fresh and lecithin-infused fruits. (HC: hydrocarbons; ALD: aldehydes; FFA: free fatty acids; DG 1, 2: 1, 2-diacyl glycerols; PA: phosphatidic acid; PE: phosphatidyl ethanolamine; PS: phosphatidylserine, PC: phosphatidyl choline; BF: Fresh blueberry; BI: Infused blueberry; CF: Fresh cherry; CI: Infused cherry; MF: Fresh mango; MI: Infused mango).

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Fig. 7. DPPH radical scavenging capacity of polyphenols isolated from fresh and infused fruits.

atmosphere storage as they senesce very rapidly. Osmotic dehydration of fruits with sucrose is a common method, and detailed studies have been conducted on the process and quality aspects of osmotically dehydrated fruits (Shi & LeMaguer, 2000; Shi, Mazza, & Le Maguer, 2009). The existing technologies use gradual dehydration of fruits using sucrose or sugar syrups of high concentration which also removes the soluble nutritional ingredients as well as color. At present, the healthfulness of such products is not being scrutinized, however, the enhanced sucrose levels can lead to a product with a high glycemic index, and unhealthy for regular consumption. Infusion in the presence of short chain fructo-oligosaccharides (scFOS) such as NutraFlora® with demonstrated health benefits helps to alleviate some of the nutritional concerns of infused fruits. NutraFlora® is a highly concentrated, short chain fructo-oligosaccharide (Sc FOS; GTC Nutrition, Co, USA), a natural prebiotic fiber derived from beet or cane sugar. Non-digestible oligosaccharides (NDO) are complex carbohydrates

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which are resistant to hydrolysis by acid, and enzymes in the human digestive tract, by virtue of the configuration of their glycosidic bonds (anomeric C atom C1 or C2), and have no calorie value in the traditional sense. However, as they reach the colon and undergo fermentation, they have an energy contribution of about 1.5 kcal/g, similar to that of soluble dietary fiber. Most of the NDOs are hydrolyzed to small oligomers and monomers in the colon and are further metabolized by the anaerobic bacteria (Lee & Prosky, 1995). Fructo-oligosaccharides (FOS) is a soluble dietary fiber naturally found in a variety of fruits, vegetables, and grains, such as bananas, barley, garlic, honey, onion, rye, brown sugar, tomato and asparagus root. Water retention capacity of FOS is higher than that of sucrose, and they are stable at pH >3 and temperatures up to 140 °C (Bornet, 1994). NutraFlora® passes undigested through the mouth, stomach and small intestine to the colon where it is completely fermented by beneficial bacteria into short-chain fatty acids (Sc FA). This fermentation is accompanied by a significant change in the composition of the colonic micro-biota due to selective proliferation of bifidobacteria and a concomitant reduction in the number of other bacteria, such as fusobacteria and clostridia (Hartemink, Van Laere, & Rombouts, 1997; Bouhnik et al., 2007). The production of Sc FA lowers intestinal pH to an optimal level for keeping calcium and minerals in solution longer, thereby enhancing their absorption. (Van den Heuvel, Muvs, Van Dokkum, & Schaafsma, 1997). The principle of osmosis has been utilized for a long time in the dehydration processing of food products (Rastogi et al., 2005, Tortoe, 2010). Water from the cell sap of tissue diffuses out into the osmotic medium owing to difference in osmotic pressures. Osmotic dehydration can be used as a pre-treatment before convection drying as fruits and vegetables become more attractive for direct use due to their improved physico-chemical properties (Sunjka & Raghavan, 2004; Shi, Mazza, LeMaguer, 2009; Tortoe, 2010). Solute uptake largely depends upon the solute molecular size and processing conditions such as solution concentration and temperature (Lazarides, Katsanidis, & Nicolaidis, 1995). High molecular weight osmotic agents (e.g. low dextrose equivalent corn syrup solids) could decrease the solute intake during osmosis (Hughes, Chichester, & Sterling, 1958; Lazarides & Mavroudis, 1995). As well, this process may cause leaching of natural solute from the food leading to negative values of net solid gain. In the case of fruits and vegetables, convection drying at temperatures beyond a limit may have a negative impact on final product quality due to softening, browning, flavor loss etc. It has been reported that temperatures higher than 45 °C had adverse effects on both water loss and solute gain (Torreggiani, 1993; Dalla Rosa, Bressa, Mastrocola, & Pittia, 1995). Dried fruits are increasingly being used in several foods such as cereals, snacks, fruit bars etc. These fruits are produced by osmotic dehydration or in some cases, infusion of sucrose and other desirable materials such as pectin. The technology we have developed involves; 1) the use of cane sugar derived short chain fructooligosacharides (scFOS, NutraFlora®, GTC Nutrition, Colorado) that are below a DP of 5 to infuse the fruits; and 2) the use of lecithin as a stabilizing ingredient. The ripe fruits are naturally low in phospholipids that results from enhanced phospholipid degradation during ripening which decreases the stability, texture and shelf life of fruits. By replenishing the lost phospholipids, the composition of the fruits can be preserved better. As well, lecithin has several other health benefits and is approved for food use both as an ingredient as well as a nutraceutical component. Analysis of infused fruit properties, especially mango segments, revealed a significant amount of solids gain after infusion of fruits which suggested that NutraFlora and lecithin are incorporated into the fruit tissue during the infusion process. The sugar composition of fresh fruits was variable in blueberries, cherries and mangoes. Mango showed the highest level of incorporation of NutraFlora reaching a level of 283 mg/g tissue. Sucrose level in infused mango was nearly half that of NutraFlora. This suggests that a fruit product that

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is almost completely devoid of sucrose can be obtained just by using NutraFlora alone for infusion. The ratio of NutraFlora to sucrose more or less reflects the ratio of these components in the infusion medium having a proportion of 45% and 25% respectively. Fruits obtained after infusion and oven drying can be categorized as intermediate moisture foods (IMF). IMF normally range in water activity (aw) from 0.7 to 0.9 and in water content from 20 to 50% (Karel, 1973). They possess a plastic texture and sufficiently low aw to prevent bacterial growth. These foods, however, may be susceptible to mold growth enzymatic degradation, or non-enzymatic browning. The aw values of infused and oven dried fruits ranged from 0.857 for cherries, 0.792 for mangoes and 0.895 for blueberries (Table 2c) showing significant reduction (p b 0.05) from the fresh samples. Such water activity levels may not provide protection from microbial infection, as, most of the yeasts are inhibited below aw of 0.87, and most molds are inhibited below 0.80 (Rahman & Labuza, 2007). In general, the fruits retained good shape and texture despite dehydration and the moisture content could be lowered further to reduce microbial growth. The colors were also retained to a large degree. Just as NutraFlora, the infused fruits were enriched in phospholipids derived from lecithin. Thin layer chromatographic analysis of the total lipid fraction revealed a very high degree of enrichment of phosphatidylcholine and phosphatidylethanolamine in mango segments. Cherries and blueberries were enriched to a far lesser extent. To the best of our knowledge, this is the first attempt to infuse soy lecithin components into fruits. Just as the enrichment of fruit juice with lecithin (Oke, Jacob, & Paliyath, 2010), infusion of fruits with lecithin may serve as another avenue to enhance the nutritional value of fruits. The results conclusively demonstrate that the nutritional quality of fruits can be enhanced through lecithin enrichment. The present method has also explored the enrichment of fruit tissues with polyphenols (anthocyanins) to compensate for the anthocyanins lost during infusion. However, simple addition of polyphenols (provinols) to the infusion medium did not prevent the leaching of anthocyanins from cherries. Enrichment with other nutraceuticals (carotene, lycopene), and flavors can be successfully achieved, however, cost effective procedures need to be optimized. The present protocol for infusion needs further refinement in terms of the technology. The infusion is best achieved in fruits that have cut areas exposed as observed in cranberry fruits (Sunjka & Raghavan, 2004) and in mango segments. Physical barriers such as cuticle and skin tend to reduce the infiltration even in IQF fruits. As well, strategies for the infusion of vitamins, provitamins, anthocyanins, flavor components and other nutraceuticals of interest are under further optimization. Altogether, the present technology provides the basic framework for incorporating a variety of nutritional components into infused fruits to provide an array of fruit products targeted to various food industry sectors. Acknowledgment This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We thank Valsala Kallidumbil and Ramany Paliyath for technical assistance. We also thank Dr. Brian Cox, Inovita, for helpful discussions. References Bornet, R. J. (1994). Undigestible sugars in food products. The Journal of Clinical Nutrition, 59, 763S–769S. Bors, W., Heller, W., Michael, C., & Saran, M. (1990). Flavonoids as antioxidants: Determination of radical scavenging efficiencies. Methods in Enzymology, 186, 343–355.

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