Influence of Geographical Location of Orchards on Green Kiwifruit

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INFLUENCE OF GEOGRAPHICAL LOCATION OF ORCHARDS ON GREEN KIWIFRUIT BIOACTIVE COMPONENTS Ivana Giangrieco, Simona Proietti, Stefano Moscatello, Lisa Tuppo, Alberto Battistelli, Francesco La Cara, Maurizio Tamburrini, Franco Famiani, and MARIA ANTONIETTA CIARDIELLO J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03930 • Publication Date (Web): 10 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Journal of Agricultural and Food Chemistry

INFLUENCE OF GEOGRAPHICAL LOCATION OF ORCHARDS ON GREEN KIWIFRUIT BIOACTIVE COMPONENTS

Ivana Giangriecoa#, Simona Proiettib#, Stefano Moscatellob, Lisa Tuppoa Alberto Battistellib, Francesco La Carac, Maurizio Tamburrinia, Franco Famianid, Maria Antonietta Ciardielloa.

a

Istituto di Bioscienze e Biorisorse, C.N.R., Via Pietro Castellino 111, I-80131 Napoli, Italy

b

c

Istituto di Biologia Agro-ambientale e Forestale, CNR, V.le Marconi 2, 05010 Porano, Italy.

Istituto di Biologia Agro-ambientale e Forestale, CNR, Via Pietro Castellino 111, I-80131 Napoli,

Italy d

Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Via

Borgo XX Giugno 74, 06121, Perugia, Italy.

#

These authors equally contributed to the study

Corresponding author: Maria Antonietta Ciardiello, Tel: +39 081 6132573 Fax: +39 0816132646 Email: [email protected]

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ABSTRACT

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Italy is one of the major world kiwifruit producers and exporters with orchards located in different

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areas from the north to the south of the peninsula. In this study we sought to investigate for the first

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time the possible influence of the geographical location of kiwifruit orchards on some fruit

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components, selected because of their involvement in beneficial or negative effects on human

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health. The fruits harvested in 16 Italian areas were analyzed and the results obtained show that the

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observed variations of the relative amounts of total proteins, kiwellin, the major allergen actinidin,

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ascorbate, polyphenols and SOD-like activity seem not to be related to the geographical location of

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the orchards. In contrast, the high concentration of the nutraceutical peptide kissper seemsto have

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some relationship with the cultivation area. In fact, its amount is much higher in the fruits from the

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Lazio region, thus providing added value to these kiwifruits.

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Keywords:

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Kiwifruit, Italian cultivations, allergens, kissper, SOD-like activity, antioxidants.


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INTRODUCTION

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Kiwifruit is well known as a food that can have positive and/or negative effects on human health. In

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fact, it is considered both a health-promoting food, but also a source of allergenic molecules

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inducing reactions characterized by mild or severe symptoms. Green kiwifruit (Actinidia deliciosa –

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cultivar Hayward) is the most widespread and studied species/cultivar in the genus Actinidia.

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Therefore, most of the literature on kiwifruit is focused on this species/cultivar.

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Among the beneficial effects associated with the consumption of green kiwifruit, laxation

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activity is probably the most well-known nowadays.1 However, this food has been commonly used

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since ancient times in some Asian geographical areas where Chinese traditional medicine has

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treated even severe diseases, such as different types of cancer, using kiwi-therapy. More recent

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studies have suggested that kiwifruit is endowed with some additional health-promoting properties

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that may influence human wellness. For example, Abe and co-workers2 obtained results suggesting

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that green kiwifruit may exert beneficial effects against diabetes due to its ability to regulate

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adipocyte differentiation and function. In addition, the anti-inflammatory properties of extracts from

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gold and green kiwifruit in in vitro models comprising lipopolysaccharide (LPS)-stimulated

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macrophages or intestinal epithelial cells have been reported.3 Literature reports also describe

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cardiovascular protective properties4 and antimicrobial activities against human pathogens5 of green

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kiwifruit. High levels of in vitro anti-oxidant activity and protection against oxidative DNA damage

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or oxidative stress have also been described for green and gold kiwifruit.6,7

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The presence in kiwifruit of a high concentration of vitamin C and polyphenols has been

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associated to the significant anti-oxidant activity of the fruit berries. Ascorbate in plant has a

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scavenging role of reactive oxygen species (ROS), produced in response to biotic and abiotic

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oxidative stresses. Humans are unable to synthesize vitamin C endogenously, so it is an essential

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dietary component and its antioxidant capacity is able to prevent or delay the development of

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dangerous diseases, and degenerative illnesses connected to the aging processes.8,9 Polyphenolic

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compounds have also been reported as potential health-promoting molecules displaying a broad 3 ACS Paragon Plus Environment


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spectrum of effects correlated to their anti-oxidant activity,10,11 including anti-carcinogenic

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activities and neuroprotective effects. A contribution to the anti-oxidant power of kiwifruit also

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derives from enzymatic activities, such as superoxide dismutase (SOD).12,13

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In addition to the positive effects on human health, kiwifruit also contains proteins that can

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cause allergic reactions. Thirteen proteins of this food have been identified so far as allergens and

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registered by the World Health Organization and International Union of Immunological Societies

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(WHO-IUIS) Allergen Nomenclature Sub-Committee. The first allergen to be described was the

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protease actinidin (Act d 1), which is a very abundant protein in the green species, and is registered

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as a major allergen of this fruit.14 Afterwards, other allergens, listed in the Table S1 (see the Online

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Supplementary Material), were described: a thaumatin-like protein,15 a 40-kDa protein,16 a cysteine

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protease inhibitor,17 kiwellin,18 a pectin methylesterase and its inhibitor,19,20 a Bet v 1-like protein

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and kirola,21 a profilin,14 a lipid transfer protein,22 and two seed storage proteins.23 This list of

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allergens includes proteins that cause allergic reactions less frequently as well as those giving

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reactions more often. Among them, actnidin (Act d 1) is considered a major allergen because it

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causes allergic reactions with a high prevalence, sometimes with severe symptoms, and is also

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regarded as a marker allergen for genuine sensitization to kiwifruit.14 It is also worth noting that

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some kiwifruit allergens belong to pathogenesis-related (PR) protein families, involved in the plant

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defence mechanisms. For instance, the thaumatin-like protein, the Bet v 1-like protein and the 9k-

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LTP belong to the PR-5, PR-10 and PR-14 protein families, respectively. Furthermore, actinidin

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and phytocystatin were reported to have antimicrobial activity.24,25

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Kiwellin (Act d 5) is another abundant protein in kiwifruit but, unlike actinidin, it is

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considered a minor allergen.26 Kiwellin is a two-domain protein27 that may undergo in vivo and in

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vitro proteolytic processing catalyzed by the endogenous allergen Act d 1, thus producing KiTH

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and kissper, corresponding to the C-terminal and N-terminal domains, respectively.28,29 The amount

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of processed kiwellin is variable in green kiwifruit and it is affected by at least two factors, i.e. the

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ripening stage and the post-harvest treatments.30 4 ACS Paragon Plus Environment

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Kiwellin is especially associated with potential positive effects on human health, rather than

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with the negative allergenic function, because it is the precursor of the nutraceutical peptide kissper.

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This is a 39-residue peptide displaying pH-dependent and voltage-gated pore-forming activity, with

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anion selectivity and channeling in synthetic planar lipid membranes having a composition similar

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to that of the human intestine.28 This activity, that has been observed in in vitro experiments,

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suggests a possible in vivo beneficial effect of this peptide in diseases involving an insufficient

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transport of specific ions, such as cystic fibrosis and dry eye syndrome. In addition, kissper displays

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anti-inflammatory and anti-oxidant effects in human model systems consisting of cultured intestinal

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cells and ex vivo colonic tissues from subjects suffering from Crohn’s disease.31 Therefore, kissper

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appears to be a molecule contributing to the health-promoting effects of green kiwifruit. This means

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that the fruits having higher amounts of this peptide could be considered healthier than those

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containing lower amounts or even lacking it. In fact, the kissper concentration in green kiwifruit is

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very variable and the factors involved in modulating its production are not yet known.

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Italy is one of the main world kiwifruit producers together with New Zealand. In recent

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years the marketable production has reached about 500,000 tonnes, highlighting its great production

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potential. Italy exports most of its production to foreign markets, both to European countries and to

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other continents. In Italy this fruit is grown in different geographical areas, from northern to

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southern regions.

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It is well known that several factors, including the geographical location of orchards,

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growing and environmental conditions, pedoclimatic parameters and agronomic methods of

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cultivation, affect the nutritional quality of different fruits and vegetables largely used in the human

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diet. Kiwifruit is considered as a functional food and a large number of consumers is interested in

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knowing the amount, and possible variations, of bioactive components contained in the different

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batches of fruit available on the market. With the aim of collecting information on the possible

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influence of the geographical location of kiwifruit orchards on the fruit features, we performed a

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comparative analysis of some fruit components selected because of their association with beneficial 5 ACS Paragon Plus Environment


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or negative effects on human health. In particular, the amounts of (i) the major allergen actinidin (ii)

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the minor allergen kiwellin, (iii) the nutraceutical peptide kissper, indirectly estimated by the

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detection of the C-terminal domain (KiTH) of the precursor kiwellin, that is produced by in vivo

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proteolytic processing in a ratio 1:1 with respect to the N-terminal domain (kissper) and (iv) the

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antioxidant power represented by ascorbate, polyphenols, FRAP and SOD-like activity, have been

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analyzed in fruit samples collected in 16 Italian areas spanning from the north to the south of Italy.

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MATERIALS AND METHODS

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Plant material and sampling

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The study was carried out on kiwifruits (Actinidia deliciosa Planch.) cv. ‘Hayward’ harvested in

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2012.. Sixteen orchards located throughout the Italian peninsula, from 40° to 45° latitude North, and

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from 7° to 16° longitude East (Table 1), were considered as representative of the Italian kiwifruit

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production and selected for this study. The year 2012 was a regular year in terms of climatic

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seasonal pattern (i.e. temperatures and precipitations). Indeed, no unusual climatic stress conditions

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were recorded in any of the considered areas/orchards. All the orchards were constituted by adult

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vines and the kiwifruit production was obtained by a conventional agronomical management of fruit

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plants.

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In each orchard, fruits were harvested at the commercial ripening stage, that means when the

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total soluble solids reached about 7° Brix, and they were immediately delivered to the laboratory of

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IBAF in Porano (Terni) for subsequent analyses. For each feature under investigation, four samples

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per orchard, comprehensive of all fruit tissues and formed by longitudinal slices from 4 different

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fruits, were collected. The samples were immediately frozen in liquid nitrogen and laterground to a

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fine powder in a mortar and pestle. The frozen fruits and the fruits powder were stored at -80 °C

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until the use.

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Determination of ascorbic acid 6 ACS Paragon Plus Environment

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Ascorbic acid content was determined using the kiwifruit powder obtained after grounding the fresh

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fruits with liquid nitrogen. 100 mg of sample were extracted using a glass-glass homogeniser

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containing 1.5 mL of 10% (w/v) trichloroacetic acid (TCA), and then centrifuged at 12000 g for 15

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min. Ascorbate (AsA) and dehydroascorbate (DAsA) in the supernatant were measured using a

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colorimetric assay.32 Total ascorbate (AsA + DasA) was determined through the reduction of DAsA

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to AsA by 2 mM dithiothreitol (DTT). For AsA measurement, the assay solution (1 mL final

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volume) contained 0.1 mL of kiwifruit extract, 2.5% TCA, 0.8% 2,2'dipyridyl, 0.3% FeCl3. Then,

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the absorbance at 525 nm was recorded. For the total ascorbate (AsA + DAsA) measurement, the

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assay mixture (1mL final volume), containing 0.1mL of fruit extract, 2 mM DTT and 0.1% N-

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ethylmaleimide, was incubated at 42 °C for 15 min. After reduction of DAsA to AsA the same

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protocol described above for AsA measurement was applied and the absorbance was measured at

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525 nm. For each sample, DAsA was calculated as the difference between total ascorbate and

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reduced ascorbate concentrations, using a standard curve in the range of 0–80 nmol ascorbate.

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Determination of total phenolic content, and antioxidant activity by FRAP assay

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About 100 mg of frozen kiwifruit powder were extracted with 1 mL of methanol for 1 hour at 25 °C

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using an ultrasonic bath. The total polyphenols (TPC) in the extracts were measured using the

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Folin-Ciocalteau phenol reagent method according to Usenik et al.33 The method involves reduction

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of the reagent by phenolic compounds contained in the fruit extracts and subsequent formation of a

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blue color recorded at 765 nm. Extract (50 µL) were incubated at 25 °C for 8 min with 100 µL

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Folin-Ciocalteau reagent and then 300 µL of sodium carbonate (20% w/v) were added. The

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mixuture was mixed and incubated 30 min at 40 °C and then the absorbance at 765 nm was

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measured. On the basis of a standard calibration curve using the gallic acid in a range of 100-800

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mg/L, the total phenolic content was expressed as gallic equivalents (GAE) in mg/100 g fresh

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weight (FW) of kiwifruit.



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The methanolic extracts mentioned above were used also for the determination of

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antioxidant activity using the FRAP (Ferric ion Reducing Antioxidant Power) assay, according to

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Benzie and Strain.34 The method measures the iron-reducing capacity of different antioxidant

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molecules contained in the fruit extracts. It is a redox-linked colorimetric reaction where the

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antioxidants work as reductants. The FRAP solution included 300 mM acetate buffer pH 3.6, 10

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mM TPTZ (ferric-tripyridyl-S-triazine) solution in 40 mM HCl, and 20 mM FeCl3. The fresh FRAP

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solution was prepared using 10 volumes of acetate buffer, 1 volume of TPTZ and 1 volume of

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FeCl3. FRAP solution (900 µL) was added to appropriately diluted kiwifruit extracts (30 µL), and

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incubated 30 min at 37 °C. Then the absorbance at 593 nm was recorded to measure the blue color

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due to the reduction of TPTZ complex and formation of the ferrous form. The antioxidant capacity

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was expressed as µmol Trolox per 100 g fresh weight of kiwifruit on the basis of a standard

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calibration curve obtained using the Trolox in a range of 50-1500 µM.

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Protein extracts preparation

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For each geographical area, the protein extracts from four fruits were separately prepared and

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analyzed in triplicate. It is well known that an optimal extraction of different proteins would require

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different procedures. We chose a well-tested procedure showing a good efficiency in the extraction

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of many proteins.30

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Protein extracts were prepared from four kiwifruit samples collected in each of the 16

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selected farms. Briefly, 2 g of kiwifruit were homogenized after addition of 2 mL of 0.5 M NaCl.

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After three hours on ice, the samples were centrifuged at 10400 × g for 45 min. About 2 mL of

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supernatant representing the total extractable proteins, were collected from each kiwifruit sample

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consisting of 2 g of fruit pulp. The extracts were stored at -20 °C until use.

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Measurement of protein concentration


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Protein concentrations were determined by the Bio-Rad Protein Assay using calibration curves

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made with BSA. 10 µl of each 1:10 diluted protein extract was assayed in triplicate. The total

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protein content in the kiwifruit samples was expressed as the mean of the values obtained from the

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four different samples collected in each farm.

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Analysis of the protein components in the kiwifruit extracts

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The extracts were subjected to reducing 15% SDS-PAGE. 10 µL (deriving from 10 mg of fresh

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fruit) of each extract solution were precipitated by adding 100% trichloroacetic acid (1:10, v/v).

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After 15 min on ice, the samples were centrifuged at 12000 x g for 15 min. The pellets were

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resuspended in the SDS-PAGE loading buffer and then loaded on the gel. Following

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electrophoresis, proteins were either stained with Coomassie Brilliant Blue or transferred onto

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PVDF membranes.

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The proteins actinidin, kiwellin and KiTH were identified by N-terminal amino acid

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sequence analysis. Briefly, the transferred proteins were stained with Coomassie Brilliant Blue and

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the protein bands, after excision, were subjected to automated Edman degradation by an Applied

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Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, Foster City, CA).

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The amounts of actinidin, kiwellin and KiTH in the protein extracts were evaluated by the

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analysis of the band intensities after SDS-PAGE separation. Following the electrophoretic run, gels

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were photographed and scanned on a Chemi Doc instrument (Bio-Rad, Milan, Italy), and the

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images were analyzed with the Quantity One software package (Bio-Rad). Known amounts of

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actinidin, kiwellin and KiTH were run on the the same gel and used as a reference to estimate their

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amount in the analyzed protein extracts.

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Evaluation of SOD-like activity

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The SOD-like activity was measured in the protein extracts obtained from the four different samples

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collected in each farm, and expressed as the average of enzyme units per g of fresh fruit (U/g FW). 9 ACS Paragon Plus Environment


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Direct quantification of the SOD activity is impaired by the instability of the substrate, superoxide

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radical. Therefore, the quantification of the SOD activity is generally obtained by an indirect

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method based on the SOD-mediated inhibition of a redox reaction that involves superoxide. In this

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study an indirect coupled assay has been used to evaluate the activity, which is called SOD-like

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activity, due to the interfering factors contained in the raw protein extracts.

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Briefly, SOD-like activity of extracts was

tested by the “SOD determination kit”

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(SIGMA-Aldrich, Buchs, Switzerland), following the manufacturer’s instructions. This method

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employs xanthine and xanthine oxidase to generate superoxide radicals, which react with Dojindo’s

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highly water-soluble tetrazolium salt WST-1 (2-(4-iodophenyl)-3(4-nitrophenyl)-5-(2,4-disulfo-

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phenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye that is

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assayed spectrophotometrically at 450 nm. Inhibition of production of the chromogen is

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proportional to SOD activity in the sample.

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Briefly, 10 µL (deriving from 5 mg of fresh fruit) of each extract solution were tested in

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triplicate. Samples and controls were incubated at 37 °C for 20 min and then the optical density

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(O.D.) was measured at 450 nm using a nanodrop 1000 spectrophotometer (Thermo Fisher

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Scientific, DE, USA). SOD-like activity was calculated as inihibition rate (%) and expressed as

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units (U) per g of kiwifruit, using a reference inhibition curve made with different known

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concentrations of bovine SOD (SIGMA-Aldrich).

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Statistical analysis

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Statistical analysis was carried out using the STATISTICA 8 software package (StatSoft for

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Windows 1998). Protein contents and antioxidant parameters were analysed by One-Way analysis

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of variance (ANOVA) with the geographical areas of sampling and with different farms as

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separated factors. Differences between averages were tested by a LSD test for a significance level

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of P= 0.05. To better understand the trends and relationships among the investigated parameters of

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kiwifruit from different geographical areas, principal component analysis (PCA) was applied, using 10 ACS Paragon Plus Environment

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the software package STATISTICA 8. PCA was applied on the whole data set of the 16 farms,

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including data on the amounts of total proteins, SOD-like activity, ascorbic acid, polyphenols,

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FRAP, actinidin, kiwellin and KiTH, to model multifactorial relationships. Therefore, this analysis

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reduces the number of variables retaining the maximum amount of variability present in the data in

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order to provide a better visualization of the possible relationships between all the analyzed

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parameters.

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RESULTS

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Analysis of the protein concentration in the kiwifruit extracts

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The amount of total protein per g of fresh fruit weight (mg/g FW) ranged from 4.0 ± 0.23 (P1) to

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7.1 ± 0.53 (B1) mg (Figure 1A), which means that the highest value is 1.77 fold higher than the

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lowest one. It can be observed that farms of the same geographical area do not group together on

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the basis of their total protein concentration. An average value of 5.57 mg/g FW can be calculated

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for the overall Italian kiwifruit production.

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Analysis of SOD-like activity in the kiwifruit extracts

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Figure 1B shows significant differences in the amount of SOD-like activity when the

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analyzed farms are compared. In fact, values ranging from 12.6 ± 0.78 U/g FW (P1) to a value 2.5

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fold higher, 31.4 ± 6.57 U/g FW (B1), were measured. Two orchards, one located in the South (B1,

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Basilicata) and the other in the North (V1, Veneto), stand out for the higher activity. An average

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value of 22 U/g FW of SOD-like activity was calculated for the entire collection of fruits.

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Estimation of ascorbate, total polyphenols and radical scavenging activity (by FRAP assay)

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A high total ascorbate content (AsA + DAsA) was recorded in all the samples. In fact, an average

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amount of 59.0 ± 2 mg/100 g FW was calculated. The values recorded for the individual farms 11 ACS Paragon Plus Environment


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(Figure 2A) range from 42.0 ± 1.0 (B3) to 73.0 ± 2.0 (ER1) mg/100 g FW. High amounts of TPC,

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ranging from 57.23 ± 4.03 (B3) to 96.93 ± 8.60 mg/100 g FW (ER3), with an average value of 77

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mg/100 g FW, were measured (Figure 2B). Most of the farms produced fruits with a very similar

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concentration of TPC. Figure 2C reports also the antioxidant activitiy measured by the FRAP assay.

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In kiwifruits from the 16 farms under investigation, this activity ranges from 237.95 ± 25.71 (V2) to

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411.67 ± 31.84 (V3) µmol Trolox/100 g FW. It can be observed that high levels of activity were

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recorded and the farms displaying the highest and the lowest value, V2 and V3, respectively, are

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both located in the same geographical area.

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Analysis of the actinidin, kiwellin and KiTH content in the kiwifruit extracts

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Figure 3A, B shows the values of total actinidin and kiwellin, expressed as mg/g FW

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estimated for each farm. The amount of actinidin ranged from 1.2 ± 0.05 (P1) to 2.2 ± 0.05 (B1)

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mg/g FW (Figure 3A), that means a variation of 1.8 fold when the fruits of the 16 orchards were

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compared. Kiwellin ranged from 1.2 ± 0.10 (P1) to 1.8 ± 0.24 (B2) mg/g FW (Figure 3B),

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corresponding to a variation of 1.5 fold. In contrast, KiTH was present at lower concentrations and

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showed a higher variability, the values ranging from about 0.075 mg/g of fruit, in the samples P1

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and B2 to 0.5 ± 0.20 mg in the fruits from L1 (Figure 3C). Therefore, an important variation of 6.7

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fold was obtained. In addition, it can be observed that the highest amount of this protein, deriving

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from the in vivo processing of kiwellin, was observed in the fruits collected in the orchards L1-L4

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located in the Lazio region, and in a farm of Emilia Romagna (ER2).

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Principal Component Analysis (PCA)

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The results of PCA analysis (Figure 4) show that most of the farms fall almost in the same area of

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the figure on the basis of the amount of antioxidants, total proteins and the allergenic proteins

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actinidin and kiwellin. No clustering correlated to the geographical location of the cultivation, in the

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north, center or south of Italy, is observed. Conversely, PCA analysis groups some farms on the 12 ACS Paragon Plus Environment

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basis of the shared high content of KiTH. This cluster includes the fruits from the orchards located

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in Lazio and one in Emilia Romagna (ER2). In addition, only two farms (V2 and B3), one located

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in the north and one in the south, were placed in the area of Figure 4 characterized by a lower

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content of proteins, of KiTH and of antioxidants, compared to the other orchards.

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DISCUSSION

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The analyses of the green kiwifruit samples collected at the commercial harvesting stage suggest

281

that most of the analyzed features are not affected, or only slightly influenced, by the geographical

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location of the farms. In fact, as summarized by the modeling of the multifactorial relationships by

283

PCA analysis, the variation, that is generally low, in the content of proteins, allergens (actinidin and

284

kiwellin) and antioxidants is independent of the geographical location of the providing orchards.

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Indeed, the kiwellin processing, responsible for the release of the nutraceutical peptide kissper,

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seems to be the only exception. In fact, the highest values of processed kiwellin have been

287

associated with 5 farms located in central Italy, 4 in Lazio and 1 in Emilia Romagna. Anyhow,

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further studies are required to confirm, and better understand the observed correlation.

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Generally, values measured for parameters such as the amounts of total proteins, ascorbate,

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polyphenols, FRAP and SOD-like enzyme activity are in line with those reported in the available

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literature. For instance, the comparison with literature data highlights that the average amount of

292

SOD-like activity measured in the Italian kiwifruits is comparable to that found in the same fruit

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collected at the harvesting stage in very distant locations, such as New Zealand. The results of that

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study have been reported in a Master’s Thesis,12 carried out at the University of Canterbury, New

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Zealand. A more recent study has described the effects on the antioxidant enzyme activity,

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including the SOD-like one, of cold treatments after the harvesting of kiwifruits.35 However, the

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amounts of SOD-like activity measured by Yang and collaborators in fruits harvested from an

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orchard in Zhouzhi County, China, are not reported per g of fruit, and therefore it is not possible to

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compare their results with the amounts we have measured in the fruits produced in Italy. 13 ACS Paragon Plus Environment


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SOD activity contained in foods has been receiving increasing attention because it is reputed

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to have a protective role against different pathologies and aging processes associated with oxidative

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stress36 such as inflammation and neurodegeneration. In fact, this enzyme is a powerful antioxidant

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which is being increasingly proposed as a pharmaceutical composition for therapeutic uses, and also

304

as a dietary supplement or as a bioactive component included in some cosmetic products.37,38

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Concerning commercial dietary supplements, often the providers suggest the intake of 250-500 IU

306

of SOD per day as an optimal dosage to counteract oxidative stress. The study described in this

307

paper indicates that the average amount of SOD-like activity contained in kiwifruit from Italian

308

orchards is about 20 IU per g of fresh fruit. Since the weight of a kiwifruit is generally around 100

309

g, it means that each fruit contains an average amount of SOD-like activity of about 2000 IU, and

310

therefore the consumption of one fruit provides even more than the daily recommended dose.

311

Therefore, independently of the Italian area of cultivation, all the analyzed kiwifruit, even those

312

containing the lowest concentration of the enzyme activity, represent an excellent supplier of the

313

daily recommended amount of SOD-based antioxidant activity.

314

In agreement with the values reported in the literature,8,39,40 kiwifruit is also one of the best

315

sources of ascorbate. The fruits from the 16 Italian orchards display quite similar average amounts

316

of this vitamin and the consumption of 100-150 g of any of the kiwifruit sampled and analyzed in

317

this study would contain an amount of ascorbate sufficient to provide the entire daily dosage

318

recommended for the human diet, corresponding to about 75 mg/day. In addition to ascorbate, the

319

polyphenol amounts also appear comparable in the analyzed fruits, thus confirming that the

320

geographical location of the orchards does not affect, at least in a significant way, these features,

321

and in general the antioxidant power of kiwifruit.

322

The amount of three protein components of the kiwifruit extracts was analyzed by the

323

evaluation of the band intensities on SDS-PAGE. Actinidin and kiwellin are allergenic proteins

324

contained in high amounts in green kiwifruit. Kiwellin undergoes in vivo processing producing two

325

protein molecules, KiTH and kissper. Therefore, the determination of the amount of KiTH 14 ACS Paragon Plus Environment

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Page 15 of 29


326

represents an easy way of evaluating the level of kiwellin processing and the amount of kissper in

327

the fruit. In fact, kiwellin processing generates equimolar amounts of kissper and KiTH.

328

former has a molecular mass of 4 kDa and can be analyzed by SDS-PAGE only with difficulty,

329

whereas the latter is a 16 kDa protein, showing a band at 20 kDa on reducing SDS-PAGE.

330

Therefore, this band at the apparent molecular weight of 20 kDa has been used as a marker to

331

evaluate the kiwellin in vivo proteolytic processing. The function of kiwellin is still unknown,27 and

332

therefore it is not possible to ascribe a biological role in the natural source to this process.

333

Nevertheless, kiwellin processing also generates kissper, that represents a nutraceutical molecule

334

with interesting potential effects on human health, as experiments on ex vivo human intestinal

335

tissues suggest.31 Therefore, a high level of kiwellin processing can be regarded as an added value

336

contributing to increase the amount of the nutraceutical peptide kissper and therefore the beneficial

337

effects of this food.

29

The

338

As stated above, the concentration of kiwellin shows a little variation between the different

339

geographical areas. In contrast, the amount of processed kiwellin appears quite variable among the

340

different orchards. The highest level of processing is observed in the fruits harvested in the orchards

341

located in the Lazio region. Therefore, the kiwifruits from this region contain a much higher amount

342

of the nutraceutical peptide kissper that provides an added value to this crop. The results obtained

343

for the other cultivations do not allow the identification of a clear correlation between this

344

parameter and the geographical location of the farms, thus suggesting that probably more than one

345

factor may contribute to this variability. It is well known that different cultivars of the same fruit

346

species can have different patterns of proteins and allergens.41 Therefore, for this study a specific

347

kiwifruit cultivar, representing that widely used in Italy, has been selected and used. It is also

348

known that factors such as ripening stage and cold exposure contribute to the variation of the

349

protein patterns of fruits. In the case of kiwifruit, it was reported that the kiwellin processing is also

350

affected by these factors.30 However, in this study kiwifruit samples were carefully selected, all of

351

them were collected at the commercial ripening stage and immediately frozen. Therefore, it is 15 ACS Paragon Plus Environment


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352

assumed that the variations we observe have to be correlated to factors other than the cultivar, the

353

ripening stage and the storage conditions. What appears quite clear is that the kiwellin proteolytic

354

processing may be associated with specific biological requirements occurring in the natural source,

355

whereas the high concentration of this unprocessed molecule and of other proteins, such as

356

actinidin, appears to be a quite constant condition probably less affected by contingent factors.

357

In conclusion, it appears that the geographical location in the Italian peninsula of kiwifruit

358

orchards does not have an important effect at least on some features of the fruits, such as the content

359

of total proteins, SOD-like enzyme activity, vitamin C, polyphenols, overall antioxidant power,

360

kiwellin and the major allergen actinidin. Nevertheless, some variations between farms occur.

361

However, in line with literature reports, other factors, worth being investigated in the future, should

362

be involved in the modulation of those parameters. Certainly, the only feature that appears

363

correlated with the orchard location, that is the concentration of the nutraceutical peptide kissper,

364

could be the subject of further studies in the future to understand (and possibly copy) the

365

peculiarities, associated with that geographical location, able to produce that desirable effect.

366 367

Abbreviations used:

368

TCA, trichloroacetic acid; DTT, dithiothreitol; AsA, ascorbate; DAsA, dehydroascorbate; TPC,

369

total polyphenol content TPC; FRAP, Ferric ion Reducing Antioxidant Power; TPTZ, ferric-

370

tripyridyl-S-triazine; SOD, superoxide dismutase; BSA, bovine serum albumin.

371 372

Supporting Information. Allergenic proteins contained in green kiwifruit.

373 374

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375

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Funding

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All authors received funding from the Italian Ministry of Economy and Finance (MEF) Project

509

“CISIA-Conoscenze Integrate per la Sostenibilità e l’Innovazione del made in Italy

510

Agroalimentare”. The authors FLC, MT and MAC received funding from the Regione Campania

511

Project “BenTeN -Nuovi Processi e Prodotti per la Nutraceutica, la Cosmeceutica e la Nutrizione

512

umana” (P.O.R. 2007/2013, objectives 2.4).

513 514


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FIGURE CAPTIONS

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Figure 1. Total protein concentration (A) and SOD-like activity (B) in kiwifruits from the 16

517

analyzed orchards. Data are the mean of four replicates ± S.E. Values labelled with different letters

518

are statistically significant for P=0.05.

519 520

Figure 2. Ascorbic acid (A), TPC (B) and FRAP (C) in kiwifruits from the 16 analyzed orchards.

521

Data are the mean of four replicates ± S.E. Values labelled with different letters are statistically

522

significant for P=0.05.

523 524

Figure 3. Actinidin (A), kiwellin (B) and KiTH (C) in kiwifruits from 16 farms located in different

525

areas of Italy. Data are the mean of four replicates ± S.E.

526 527

Figure 4. PCA plot of kiwifruit from the 16 analyzed Italian orchards defined by the two first

528

principal components obtained with the investigated parameters.



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Table 1 Geographical Coordinates of the 16 Farms Providing the Fruits for this Study. Region Farm Latitude Longitude N E 45°38’00” 10°40’00” V1 45°22’22” 11°07’49” V2 Veneto 45°47’11” 11°55’22” V3 44°34’00” 7°29’00” P1 44°33’50” 7°21’17” P2 Piedmont 44°48’00” 7°20’00” P3 44°08’00” 12°14’00” ER1 Emilia 44°25’04” 12°11’58” ER2 Romagna 44°19’50” 12°08’11” ER3 41°40’00” 12°47’00” L1 41°40’00” 12°47’00” L2 Lazio 41°36’00” 12°50’00” L3 41°32’58” 12°50’05” L4 40°15’00” 16°42’00” B1 40°23’00” 16°33’00” B2 Basilicata 40°17’00” 16°34’00” B3 The farms are indicated with the first letter of the geographical region name and then numbered progressively. So, the farms located in Veneto, Piedmont, Emilia Romagna, Lazio and Basilicata, are indicated with the letters V, P, ER, L and B, respectively, and then given a number.


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Influence of Geographical Location of Orchards on Green Kiwifruit

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