Pecan (Carya illinoinensis (Wagenh.)
K. Koch) Nut Shell as an Accessible Polyphenol Source for Active Packaging
and Food Colorant Stabilization
Herein, the antioxidant and food stabilizing properties of a pecan nut shell (PNS) hydroalcoholic extract (PNSE) are reported. Chemical degradation of PNSE demonstrated the presence of condensed tannins as the main phenolic components. PNSE showed remarkable antioxidant properties in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (EC50 = 0.004 mg/mL). PNSE was initially tested as an inhibitor of mushroom tyrosinase, exhibiting a quite low IC50 value (0.055 mg/mL) against the enzyme diphenolase activity, suggesting its use in enzymatic browning inhibition. The anthocyanin stabilization properties were evaluated under accelerated aging conditions of both pure pigments and commercial fruit juices, and PNSE was found to be effective at concentrations (0.05 mg/mL) at which well-known stabilizers such as chlorogenic and ferulic acids proved to fail. PNSE also performed well in the stabilization of spray-dried anthocyanins for use as a food colorant, increasing the half-life of blackberry anthocyanins up to 20%. In order to explore the possibility of using PNSE as a functional additive for active packaging, polylactic acid (PLA) films containing PNSE were prepared by solvent casting, and no substantial alteration of the mechanical properties was found on addition of the extract up to 10% w/w. The films showed remarkable antioxidant properties (DDPH reduction >60% with a 3% w/w loading, at a dose of 1 mg/mL in the DPPH solution) and delayed the onset of browning of apple smoothies (ca. 30% inhibition with a 10% w/w loading). These results highlight the exploitation of PNS as a low-cost polyphenol source for food industry applications.
Introduction<!>Materials<!>Electron Paramagnetic Resonance (EPR) Analysis<!>Phloroglucinolysis<!>Thiolysis.37<!>Alkali Fusion.38<!>2,2-Diphenyl-1-picrylhydrazyl
(DPPH) Assay.39<!>Ferric Reducing/Antioxidant Power (FRAP)
Assay.40<!>Cell Viability Assays<!>Film Preparation and Characterization
by Scanning Electron Microscopy
(SEM)<!>DPPH Assay<!>FRAP Assay<!>Release of PNSE from PE and PLA Films in
Assay Media<!>Film Mechanical Properties Characterization<!>FT-IR Analysis and Oxygen Transmission Rate (OTR) Measurement<!>Mushroom Tyrosinase Inhibition Assay41,42<!>Apple Smoothie Browning
Inhibition Assay<!>Anthocyanin
Stabilization Assay<!>Storage Stability
of Spray-Dried Anthocyanins<!>Statistical Analysis<!>Structural Characterization
of PNSE<!><!>Structural Characterization
of PNSE<!><!>Antioxidant
Properties of PNSE<!>Biocompatibility of PNSE<!>Antioxidant Properties of PE and PLA Films Containing PNSE<!><!>Antioxidant Properties of PE and PLA Films Containing PNSE<!><!>Antioxidant Properties of PE and PLA Films Containing PNSE<!>Characterization of Solvent-Cast PLA Films
Containing PNSE<!><!>Characterization of Solvent-Cast PLA Films
Containing PNSE<!><!>Characterization of Solvent-Cast PLA Films
Containing PNSE<!><!>Characterization of Solvent-Cast PLA Films
Containing PNSE<!><!>Enzymatic Browning Inhibition Properties of PNSE<!><!>Enzymatic Browning Inhibition Properties of PNSE<!>Enzymatic Browning Inhibition Properties of Solvent-Cast PLA
Films Containing PNSE<!>Anthocyanin
Stabilization Properties of PNSE<!><!>Anthocyanin
Stabilization Properties of PNSE<!><!>Anthocyanin
Stabilization Properties of PNSE<!><!>Conclusions<!>
<p>The
search for natural and sustainably produced antioxidant additives
for use in health, food, or cosmetic applications has received considerable
attention in recent years, prompted by the increasing need for green
and sustainable approaches to novel functional materials.1−4 In this context, waste materials from agrifood industries represent
an easily accessible source of phenolic compounds that, apart from
their use as food supplements or as additives in functional foods,
have become increasingly attractive also from a technological point
of view due to their possible exploitation in materials science.5−9 Incorporation of antioxidant additives into polymers, both for stabilization
and functionalization purposes, is particularly relevant for active
food packaging. This latter represents an important sector of the
food industry to avoid or delay oxidative deterioration of food.10−15 Several approaches have been proposed to provide biocompatible polymers
with antioxidant functionality, the most sustainable of which being
based on the simple incorporation of the additive by extrusion or
solvent casting, without the need for chemicals to covalently link
the antioxidant to the polymer.16−20 Several articles have reported that polyphenols from agroindustry
byproducts, including grape pomace, spent coffee grounds, and orange
peels, are able to exert a powerful stabilizing action on several
polymers.6,20−23 Another noticeable example is
represented by pecan nut shell (PNS) hydroalcholic extract (PNSE),
which acted as a thermal and photo-oxidative stabilizer of both polyethylene
(PE) and polylactic acid (PLA).24 Acid
insoluble lignins obtained by sulfuric acid treatment of PNS were
also found to confer higher ductility to PLA biocomposites determining
an increase in stress and strain at break.25</p><p>The pecan (Carya illinoinensis (Wagenh.)
K. Koch) nut belongs to the Juglandaceae family and is native to the south of the United States and north
of Mexico. Pecan nut processing results in the production of a high
amount of shells, containing mainly fiber (cellulose, hemicellulose,
and lignin) as well as a significant amount of antioxidant phenolic
compounds,24,25 which are commonly used for infusion
in folk medicine due to their beneficial health effects. More recently,
the use of PNS as an adsorbent to remove toxic ions and organic pollutants
from aqueous solutions26−28 and as a reinforcing filler for polymers has been
described.25,29 The impact of an aqueous extract
of PNS on the oxidative stability of margarines during storage has
also been reported.30 The exploitation
of PNS as a carbon source for fungal or bacterial production of bioactives
is another emerging field of application of this multifaceted waste
material.31,32</p><p>Prompted by these observations, the
possible use of PNSE in food
industry applications, with particular reference to antioxidant active
packaging, enzymatic browning inhibition in fruits, and stabilization
of anthocyanins largely used as food colorants, was investigated.
The presence of condensed tannins as the main phenolic components
of PNSE was confirmed by chemical degradation methods.</p><!><p>PNSE was obtained as previously
described.24 Briefly, 1 g of PNS was extracted
at room temperature
with 2 × 10 mL of ethanol/water (6:4 v/v) for 30 min. 4042D PLA
(94% l-lactic acid) was from NatureWorks LLC. Unstabilized
linear low-density DJM1826 PE (2.5 g × 10 min–1 melt flow index) was purchased from Versalis. Anthocyanins were
extracted from red wine pomace, black currant, and blackberry juice
by adsorption on XAD7 HP (Sigma-Aldrich) and further purification
on membrane adsorber Sartobind S IEX 150 mL (Sartorius Stedim Biotech).33 Red Delicious apples and bilberry juice (>40%
fruit content) were purchased from local shops. All reagents and solvents
were of analytical grade (Sigma-Aldrich).</p><!><p>EPR
measurements were performed following an experimental procedure recently
set up.34,35 Samples were measured using an X-band (9
GHz) Bruker Elexys E-500 spectrometer equipped with a superhigh sensitivity
probe head. PNSE was introduced in a flame-sealed glass capillary
coaxially inserted in a standard 4 mm quartz sample tube. Measurements
were carried out at room temperature, with the following settings:
sweep width, 140 G; resolution, 1024 points; modulation frequency,
100 kHz; modulation amplitude, 1.0 G, and receiver gain, 60 dB. The
amplitude of the field modulation was preventively checked to be low
enough to avoid detectable signal overmodulation. A microwave power
of ∼0.6 mW was used to avoid microwave saturation of the resonance
absorption curve. To improve the signal-to-noise ratio, 16 scans were
accumulated. As concerning power saturation experiments, the microwave
intensity was gradually increased from 0.004 to 127 mW. The g value and the spin density were determined using Mn2+-doped MgO as an internal standard.35 Spin density values must be considered as order of magnitude estimates,
since sample hydration was not controlled.</p><!><p>Phloroglucinolysis was conducted
following a reported protocol36 with slight
modifications. Briefly, 5 mg of PNSE was dissolved in 1 mL of methanol.
One aliquot was used as the control to assess the content of free
flavanols. Another aliquot of 100 Î¼L was evaporated to dryness
under nitrogen. Subsequently, 100 Î¼L of the reaction solution
containing ascorbic acid (10 mg/mL) and phloroglucinol (50 mg/mL)
in 0.1 M methanolic HCl was added. After shaking for 20 min in a water
bath at 50 °C, the reaction was quenched by adding 500 μL
of aqueous sodium acetate (40 mM); after that, the mixture was filtered
and analyzed by UHPLC-ESI-MS. A Waters Acquity i-Class instrument
equipped with a binary pump, an autosampler (cooled to 10 °C,
injecting 5 μL, a column oven (40 °C), and a diode-array
detector was used. The column was a Nucleoshell phenyl-hexyl (2.0
mm × 150 mm, 2.7 μm, Macherey-Nagel), equipped with a security
guard cartridge of the same material (2.0 mm × 5 mm, 1.8 μm).
Eluant A was 0.1% formic acid in water, and eluant B was 0.1% formic
acid in acetonitrile. A gradient elution program at a flow rate of
0.4 mL/min was used as follows: 1% B, 0–3 min; from 1 to 20%
B, 3–22 min; from 20 to 100% B, 22–23 min; 100% B, 23–25
min. The mass spectrometer was a LTQ-XL ion trap system (Thermo Fisher
Scientific) operating under the following detection conditions: sheath
gas (N2), 52 arbitrary units; aux gas (N2),
1 unit; sweep gas (N2), 1 unit; ion spray voltage, 5 kV;
capillary temperature, 300 °C; capillary voltage, 35 V; collision
energy, 35 V.</p><p>The mean degree of polymerization (mDP) was calculated
according to eq 11</p><!><p>Here, 8 mg
of PNSE was treated with 2 mL of methanol, 20 Î¼L of 37% HCl,
and 50 μL of benzyl mercaptan (BM) at 40 °C under stirring.
After 1 h, the mixture was diluted in 5 mL of methanol/water 1:1 v/v
and directly analyzed by HPLC and LC-MS. HPLC analysis was run on
an instrument equipped with an Agilent G1314A UV–vis detector,
using a Phenomenex sphereclone ODS column (250 mm × 4.60 mm,
5 Î¼m) at a flow rate of 1.0 mL/min; a gradient elution was performed
with 0.1% formic acid (solvent A)/methanol (solvent B) as follows:
5% B, 0–10 min; from 5 to 80% B, 10–45 min; detection
wavelength was 254 nm. LC-MS analyses were performed in a positive
ionization mode on an Agilent LC-MS ESI-TOF 1260/6230DA instrument
with the following parameters: nebulizer pressure, 35 psig; drying
gas (nitrogen), 5 L/min; 325 °C; capillary voltage, 3500 V; fragmentor
voltage, 175 V. An Eclipse Plus C18 column (150 mm × 4.6 mm,
5 Î¼m) was used with the same mobile phase as above, at a flow
rate of 0.4 mL/min.</p><!><p>Here, 100
mg of KOH, 100 mg of NaOH, and 2 mg of Na2S2O4 were melted in a Pyrex tube at 240 °C. PNSE (20
mg) was then added, and the mixture was kept at 240 °C for a
further 10 min. After cooling to room temperature, a 1% sodium dithionite
solution (10 mL) was added. The resulting mixture was taken to pH
3 with acetic acid and then extracted with 3 × 15 mL of ethyl
acetate. The organic layers were anhydrified with sodium sulfate and
taken to dryness. The residue was dissolved up in methanol and analyzed
by HPLC and LC-MS under the conditions described for thiolysis.</p><!><p>Here, 20 Î¼L
of a 0.1–1 mg/mL PNSE
solution in dimethyl sulfoxide (DMSO) were added to a 0.2 mM ethanolic
solution of DPPH (2 mL), and the mixture was taken under stirring
at room temperature. After 10 min, the absorbance at 515 nm was measured
using an Agilent Hewlett Packard 8453 UV–vis spectrophotometer.
Trolox was used as reference antioxidant. Each experiment was run
in triplicate.</p><!><p>Here, 20 Î¼L of
a 0.05–0.5 mg/mL PNSE
solution in DMSO were added to a solution containing 1.7 mM FeCl3 and 0.83 mM 2,4,6-tris(2-pirydyl)-s-triazine
in a 0.3 M acetate buffer (pH 3.6) (2 mL). The resulting mixture was
vigorously stirred at room temperature, and the reduction of Fe3+ to Fe2+ was evaluated after 10 min by measurement
of the 593 nm absorbance. An Agilent Hewlett Packard 8453 spectrophotometer
was used. Each experiment was run in triplicate. Results were expressed
as Trolox equivalents.</p><!><p>Cytotoxic
effects on human hepatocarcinoma
(HepG2) cells were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) dye. HepG2 cells were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and grown at 37 °C
in a humidified incubator containing 5% CO2 in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal
bovine serum, 4 mM glutamine, 400 U/mL penicillin, and 0.1 mg/mL streptomycin.</p><p>Cells were plated on 96-well plates at a density of 5 × 103 per well in 100 μL of medium and incubated at 37 °C
with 5% CO2. The medium was then replaced with 100 Î¼L
of fresh media containing PNSE at 5–50 μg/mL, and cells
were incubated at 37 °C with 5% CO2. After 24–72
h incubation at 37 °C, the medium containing PNSE was withdrawn,
and fresh medium (100 Î¼L) containing 10% MTT was added to each
well. After that, cells were incubated in darkness at 37 °C for
4 h. Cell survival was reported as the relative absorbance, with respect
to control, of blue formazan measured at 570 nm with a Synergy Multi
Plate Reader. Cell viability was reported as mean ± SD percentage.</p><!><p>Extruded PE and PLA films were prepared as previously
described.24 Briefly, PNSE was mixed with
PE or PLA powder at 1%, 2%, or 3% w/w (grams of PNSE per grams of
polymer). Then, the powder mixture was extruded with a flat die single-screw
extruder, and films with an average thickness of 70 ± 10 μm
were obtained by calendering. Higher concentrations of PNSE yielded
visibly inhomogeneous films with poor mechanical properties.</p><p>Additionally, PLA films, neat and added with 3% or 10% w/w PNSE,
were prepared through solvent casting. A suitable amount of PNSE was
dissolved in 50 mL of a chloroform:methanol (8:2 v/v) solution by
sonication over 30 min. Then, 2 g of PLA pellets was added, and the
mixture was stirred for 120 min at 80 °C using a magnetic heater-stirrer.
The resulting solution was transferred to a 12 cm-diameter Petri dish,
and the solvent was allowed to evaporate slowly for 2 d to obtain
films of 180 ± 10 μm thickness. The solvent-casting technique
was not applicable to PE due to its extremely low solubility in most
solvents.</p><p>Scanning electron microscopy (SEM) characterization
of extruded
and solvent-cast films was carried out using a FEI Quanta 200 FEG
device. Before observation, the samples were coated with a Au–Pd
alloy layer of 18.0 ± 0.2 nm, using a MED 020 Bal-Tec AG coater.</p><!><p>Here, 2–10
PE or PLA (both extruded or solvent-cast) film sections of 1 cm2 total surface area (corresponding to ca. 20–100 mg
of material) were introduced in a vial containing 20 mL of a 55 Î¼M
ethanolic solution of DPPH and allowed to sit up to 7 d at room temperature.
Blank samples without the films were also prepared. The absorbance
of each solution at 515 nm was periodically analyzed. Each experiment
was performed in triplicate.</p><!><p>Here, 5 film sections
of 1 cm2 total surface area (corresponding to ca. 50 mg
of material) were introduced in a vial containing 20 mL of a FRAP
solution prepared as described above and allowed to sit for 4 d at
room temperature. The assay was performed on PLA (both extruded or
solvent-cast) films containing or not PNSE. Blank samples without
the films were also prepared. The absorbance of each solution at 593
nm was periodically analyzed. Each experiment was performed in triplicate.
Results were expressed as Trolox equivalents. Experiments were run
in triplicate.</p><!><p>Five PE or PLA (both extruded or solvent-cast)
film sections of 1
cm2 total surface area (corresponding to ca. 50 mg of material)
were introduced in a vial containing 20 mL of ethanol or 0.3 M acetate
buffer (pH 3.6) and allowed to sit for 4 d at room temperature. UV–vis
spectra of each solution were periodically recorded. The amount of
PNSE released was determined by means of a calibration curve obtained
with 0.05–0.2 mg/mL PNSE solutions in the same solvents. Experiments
were run in triplicate.</p><!><p>Tensile
tests were performed at 23 ± 2 °C and 45 ± 5% relative
humidity (RH) on solvent-cast PLA films using a dynamometer (Instron
5564) equipped with a 1 kN load cell at a 5 mm min–1 clamp displacement rate. Before testing, the dumbbell-shaped specimens
were conditioned at 25 °C and 50% RH for 48 h. At least 10 specimens
were tested for each formulation. Mechanical characterization of extruded
PE and PLA films has been already reported.24</p><!><p>FTIR-ATR spectra were recorded as an average of 16 scans in the
range of 4000–450 cm–1 (resolution of 4 cm–1) on a PerkinElmer Spectrum 100 spectrometer equipped
with a Universal ATR diamond crystal accessory. OTR was measured on
solvent-cast PLA films by using a ExtraSolution PermeO2 instrument working in a gas/membrane/gas configuration, using a
measuring surface of 50 mm at 50% RH, 23 ± 1 °C, and 1 atm
of pressure difference (ΔP) across the membrane.
The test was ended when the collected data reached an OTR accuracy
within 0.5%. Oxygen permeability was obtained from OTR as follows
(eq 2)2</p><!><p>Here, 10 Î¼L of a DMSO solution of PNSE was incubated
at room temperature in 2 mL (0.02–0.25 mg/mL PNSE final concentration)
of 50 mM phosphate buffer (pH 6.8) containing 20 U/mL of mushroom
tyrosinase. After 10 min, 20 Î¼L of a 100 mM l-DOPA
or l-tyrosine solution in 0.6 M HCl was added (1 mM final
concentration), and the absorbance at 475 nm was measured after an
additional 10 min. In blank experiments, the reaction was run in the
absence of PNSE. When required, the assay was carried out as described
above, but l-DOPA was added to the reaction mixture soon
after the addition of PNSE (0.06 mg/mL).</p><!><p>Red Delicious
apples were rapidly cut in small pieces after removing the peel, and
ca. 50 g were finely blended with a domestic mixer in the presence
of a 0.085% PNSE solution (prepared by dissolving 100 mg of PNSE in
15 mL of DMSO followed by addition of 100 mL of double-distilled water)
and transferred on a watch glass. Blank smoothie samples were prepared
in the absence of PNSE. Changes in color were periodically analyzed
with a Chroma Meter CR-400/410 (Konica Minolta) colorimeter (three
different measurements were taken during the same experiments, and
three different experiments were performed). The browning index (BI)
was calculated as follows (eq 3)433</p><p>Kojic acid and
ascorbic acid were used
as reference compounds in parallel experiments.</p><p>In other experiments,
smoothies prepared by blending 50 g of apple
pulp in 100 mL of double-distilled water were rapidly transferred
on a watch glass and immediately covered with solvent-cast PLA films,
neat or containing PNSE. The changes in color were periodically analyzed
with a colorimeter as above. Uncovered smoothies were used as blank
samples.</p><!><p>The assay was performed
as described.44 Briefly, to 5 mL of a 0.1
mg/mL red wine anthocyanin solution in a 0.3 M acetate buffer (pH
3.6), 40–250 μL of 1 mg/mL PNSE solution in DMSO was
added (corresponding to 0.008–0.05 mg/mL final concentration
of PNSE in the anthocyanin solution). The mixtures were taken at 90
°C and periodically analyzed by measuring the absorbance at 521
nm at 1 h intervals. Control mixtures containing the same amount of
DMSO (without extracts) and unheated samples were analyzed in the
same way. In other experiments, anthocyanins were incubated under
the same conditions with 250 Î¼L of a 1 mg/mL DMSO solution of
chlorogenic acid or ferulic acid as reference compounds (0.05 mg/mL
final concentration).</p><p>In another series of experiments, PNSE
(2–10 mg) was added to 5 mL of bilberry juice, and the mixtures
were taken at 90 °C and periodically analyzed as described above,
following a 1:40 v/v dilution in a 0.3 M acetate buffer (pH 3.6).</p><p>Three different measurements were taken during the same experiments,
and three different experiments were performed.</p><!><p>The spray-drying
procedure followed a previously published method,33 using a Büchi B-290 lab-scale drier. Feed solutions
contained 10 g/L of anthocyanins from blackberry or black currant
juice and either 50 g/L of maltodextrin 19 (DE-value 18–20)
for the control or 10 g/L of PNSE and 40 g/L of maltodextrin. All
components were solubilized in ultrapure water (100 mL), which was
taken in an ultrasound bath for 20 min. The solution was then filtered
using a 595 Whatman paper filter (GE Healthcare). The following parameters
were adopted: inlet temperature, 150 C; air-flow rate, 470 L/h; and
aspirator set at 100% (approximately 35 m3/h). The resulting
outlet temperature was kept between 96 and 99 °C by slightly
adjusting the flow rate (approximately 5 mL/min). The storage stability
of the resulting powders was determined by analysis of anthocyanin
degradation using UHPLC-DAD.33 The samples
were stored under UV light with or without exposure to oxygen at 35
°C for 10 weeks. Quantification of anthocyanins and calculation
of half-lives (t1/2) were performed as
previously reported.33 Samples were dissolved
in water/acetonitrile/formic acid (80/15/5 v/v/v) at a concentration
of 0.5 mg/mL. The CIELab color metrics were obtained as previosusly
reported.33 The samples were diluted to
a 1:2 ratio with water/acetonitrile/formic acid (80:15:5 v/v/v) after
that absorption spectra were recorded with a Jasco V-730 spectrophotometer
(Pfungstadt, Germany). The color parameters Chroma C and hue h and the resulting color difference ΔE were calculated as described previously.33</p><!><p>Data were processed
by one-way
analysis of variance using OriginPro 8.5. Significant differences
among the means were assessed using Tukey's test (P < 0.05).</p><!><p>HPLC analysis of
PNSE dissolved in DMSO and diluted in methanol showed very low amounts
of chromatographically defined compounds. A great chemical heterogeneity
was apparent also from 1H NMR analysis, showing very broad
signals in the aromatic region (not shown), whereas the UV–vis
spectrum featured an absorption maximum at 278 nm, with a shoulder
at around 300 nm (Figure 1a), indicative of the presence of phenolic moieties. EPR spectroscopy
revealed a signal typical of a phenolic polymer (Figure 1b), characterized by a g value
of 2.0035 ± 0.0002 due to carbon-centered radicals, a ΔB
value of 5.1 ± 0.2 G, and a spin density of 5.4 ± 0.5 ×
1014 spin/g. On the basis of the high (ca. 75%) percentage
of Lorentian line shape, this relatively large ΔB value would
be indicative of a low extent of π-electron conjugation across
the phenolic polymer moieties.35 The observed
slope change in the monotonously increasing trend of the normalized
power saturation profile (Figure 1c) suggested that the paramagnetic centers in PNSE
had a high extent of molecular heterogeneity.35,45</p><!><p>(a)
UV–vis spectrum of a 0.2 mg/mL PNSE methanolic solution,
(b) EPR spectrum of PNSE, and (c) EPR power saturation profile of
PNSE.</p><!><p>These results are in line with
literature data reporting condensed
tannins as the main phenolic components of PNS extracts.24,46−51 These are polymeric compounds constituted of terminal and multiple
extension moieties related to flavan-3-ols linked by carbon–carbon
bonds, which are commonly analyzed by chemical degradation treatments
based on acid-catalyzed depolymerization in the presence of a strong
nucleophile, followed by HPLC separation and quantification of individual
polymer subunits. In this case, HPLC analysis of mixtures obtained
by thiolysis, phloroglucinolysis, and alkali fusion revealed (epi)gallocatechin,
(epi)catechin, and hydroxybenzoic acids as the main cleavage products
(Table 1 and Figure S1). In particular, phloroglucinolysis
experiments revealed a rather high mDP value (Table 1) and (epi)gallocatechin as the most abundant
terminal and extension unit, suggestive of prodelphinidin-type tannins.</p><!><p>Calculated mean degree of polymerization
(mDP) is 20.1.</p><!><p>Several articles have
described PNS as a source of polyphenols using different extraction
procedures, and these polyphenols, mainly condensed tannins, have
been regarded as a viable alternative for food supplements and nutraceuticals
as well as for the treatment of cancer and other pathologies, given
their effective antioxidant properties.46−52 In this work, PNSE was obtained by treatment of nut shell with ethanol
and water (6:4 v/v) under the conditions previously developed.24 The antioxidant properties of PNSE were investigated
by two widely used assays, i.e., the DPPH assay, which determines
the electron donor capacity by the spectrophotometric monitoring at
515 nm of a DPPH solution containing the sample,39 and the FRAP assay, in which the ability of the sample
to reduce a Fe3+-tripyridyltriazine complex to a dark blue
Fe2+ complex is measured by monitoring the absorbance at
593 nm.40 A EC50 value of 4.00
± 0.01 μg/mL was determined in the DPPH assay, confirming
a very high efficiency of PNSE when compared to the standard antioxidant
Trolox, which under the same conditions exhibited a EC50 value of 6.00 ± 0.02 μg/mL. On the other hand, a value
of only 0.183 ± 0.007 Trolox equivalents was measured in the
FRAP assay, likely due to a lower PNSE solubility in the aqueous assay
medium.</p><!><p>To assess the biocompatibility
properties of PNSE, possible toxic effects on HepG2 cells were evaluated
by a MTT reduction assay at three different times of incubation (24,
48, and 72 h). HepG2 cell line is widely used as a model system of
the human liver to study the toxicity as well as the chemopreventive
potential of different substances.53 The
results are presented in Figure S2 and
show that PNSE does not exert significant toxic effects over a time
interval of 72 h at concentrations as high as 50 Î¼g/mL, which
can be considered physiological and realistic in the perspective of
using PNSE as a food additive.23 These
results well compare with literature data reporting no motor, gastric,
or toxicological alterations in mice treated with PNS extracts.48,54−57</p><!><p>In subsequent experiments, the possible use of PNSE as a functional
additive for active packaging was evaluated. To this aim, PLA or PE
films containing PNSE at 1%, 2%, or 3% w/w were prepared by extrusion
as described in a previous work24 and tested
in the DPPH assays in comparison with control films not containing
PNSE. The incorporation of the extract introduced an antioxidant functionality
in a dose-dependent manner in both polymers. This effect was particularly
marked in the case of PLA films, leading to more than 80% DPPH reduction
after 1 week with a 3% w/w loading of PNSE (Figure S3).</p><p>Remarkably higher antioxidant properties were observed
in the DPPH assay for solvent-cast PLA films compared to those obtained
by extrusion, since even at a dose as low as 1 mg/mL in the DPPH solution
they were able to induce a 60% reduction after only 30 min (Figure 2a).</p><!><p>(a) DPPH-reducing activity
of solvent-cast PLA films containing
PNSE. (b) Fe3+-reducing activity of solvent-cast PLA films
containing PNSE. Mean ± SD values of three experiments are reported.</p><!><p>The solvent-casting technique was not applicable
to PE due to its
extremely low solubility in most solvents.</p><p>The differences in
the antioxidant properties of the tested films
may be ascribed both to the nature of the polymer and to the technique
used to obtain the film. During extrusion, high temperature causes
the polymer to melt, while the mechanical shear due to screw rotation
allows for comminution of the additive and blending with the polymer
matrix. The subsequent roll calendering process results in the formation
of a thin film in which the finely dispersed additive particles are
confined inside the bulk of the film.</p><p>Figure 3a shows
the cross sections of extruded PE and PLA films containing PNSE, highlighting
the presence of submicrometer-sized PNSE particles (indicated by arrows)
well distributed in PE, while some particle agglomerates can be observed
in the case of PLA. Efficient particle embedding entails that migration
of PNSE from the bulk of the film into the solution is hampered, also
accounting for the poor antioxidant properties measured in the DPPH
assay.58,59 This applies in particular to PE, which
is a hydrophobic polymer with low wettability in polar solvents. Indeed,
release experiments showed no migration of PNSE in the DPPH assay
medium from the extruded films.</p><!><p>(a) SEM images of cross sections of extruded
PE + 3% PNSE (left)
and PLA + 3% PNSE (right) films; PNSE particles are indicated by arrows.
(b) SEM images of cross section (I and II), top surface (PNSE-rich)
(III), and bottom surface (PLA-rich) (IV) of solvent-cast PLA + 10%
PNSE films.</p><!><p>As far as solvent-cast PLA films
are concerned, the absence of
mechanical shear and the slow solvent evaporation cause the polymer
to settle on the bottom of the Petri dish, while the extract remains
on the top layer, leading eventually to a coating layer of about 30
Î¼m on the film surface (Figure 3b). Therefore, higher diffusion of PNSE from solvent-cast
PLA films is expected, explaining the higher antioxidant properties
determined in the DPPH assay. Actually, parallel experiments indicated
the release of almost all of PNSE from solvent-cast PLA films under
the conditions of the DPPH assay after 24 h, following different kinetics
depending on the percentage of incorporation of PNSE (Figure S4). In particular, a faster diffusion
of PNSE was observed in the case of a lower loading concentration,
likely as the result of a thinner and hence more permeable layer over
the surface of the film.</p><p>The antioxidant properties of the PLA
films were evaluated also
in an aqueous medium by the FRAP assay (Figure 2b). Trolox equivalent values of 0.78 ×
10–3 and 1.26 × 10–3 were
exhibited by solvent-cast PLA films with a PNSE loading of 3% and
10% w/w, respectively, at 96 h. In contrast, extruded PLA films containing
3% w/w PNSE exhibited a reducing activity which was not so different
from that of control extruded PLA films not containing PNSE (Figure S5). Notably, in this case, no significant
migration was observed either for solvent-cast or extruded films,
so it is unlikely that the differences found in the reducing properties
could be ascribed to a release of PNSE in the assay medium.</p><p>On the basis of the results of the antioxidant assays, indicating
a lower performance of extruded films compared to solvent-cast PLA
films in view of their potential use in the food industry, only the
latter were further investigated and characterized in the subsequent
experiments.</p><!><p>The PLA films containing PNSE showed a fairly
homogeneous dark
red coloration (Figure 4).</p><!><p>Digital photo of solvent-cast neat PLA film (left) and PLA film
containing 10% w/w PNSE (right).</p><!><p>The effect of the addition of PNSE on the structure of the solvent-cast
PLA film was assessed by FTIR spectroscopy (Figure 5a and Figure S6). The main spectral changes due to the presence of phenolic moieties
were the appearance of absorption at 3300 cm–1 (phenol
O–H stretching), 1613 cm–1 (aromatic C=C),
and 1538 and 1515 cm–1 (in-plane bending of phenyl
C–H bonds).17,24 Notably, no changes were observed
in the absorption frequency of the PLA carbonyl groups (1750 cm–1), indicating the absence of significant interactions
between the polymer and the additive.</p><!><p>(a) FTIR-ATR spectra and (b) OTR curves
of solvent-cast neat PLA
films and PLA films containing 10% w/w PNSE.</p><!><p>The mechanical properties of the solvent-cast films were measured
by tensile tests. Strain and stress at break and elastic modulus values
of PLA and PLA + 10% PNSE are shown in Table 2.</p><!><p>Average of three
determinations
± standard deviation.</p><p>Starred values in the same column
are significantly different (P < 0.05).</p><!><p>PNSE slightly, although not significantly,
affected the strain
at break value of PLA, which decreased from 41% to 32%. This apparent
drop in the mechanical properties can be attributed to the formation
of PNSE aggregates during solvent evaporation (Figure 6). These defects are able to initiate cracks
which accelerate the sample failure.24 On
the contrary, the addition of PNSE did not affect the mechanical performance
of solvent-cast PLA films in terms of modulus, considering that a
slight increase in modulus is generally expected in the presence of
natural additives or fillers.17,24,60 In other experiments, the oxygen permeability of the solvent-cast
films was determined based on the experimental OTR values (Figure 5b). Values of 8.33
± 0.46 × 10–16 and 8.26 ± 0.50 ×
10–16 g m/m2 s Pa were found for solvent-cast
neat PLA films and PLA films containing 10% w/w PNSE, respectively,
indicating that PNSE did not significantly affect the oxygen permeability
property of the film. This could be the result of the preferential
localization of PNSE on the film surface (Figure 3b) entailing that the film bulk is essentially
made of almost pure PLA, with a negligible influence on the gas permeability.
Further, the recorded values were in good agreement with literature
data61,62 and compatible with food packaging applications.63,64</p><!><p>SEM
images of cross section of solvent-cast PLA + 10% PNSE film,
highlighting the presence of PNSE aggregates.</p><!><p>To
further expand the possible applications of PNSE in the food sector,
in subsequent experiments, the inhibitory properties of PNSE toward
mushroom tyrosinase were investigated. Tyrosinase belongs to the enzyme
class of polyphenol oxidases, playing a critical role in the oxidative
deterioration of food products, particularly fruit and vegetables.
It is a copper-containing enzyme which catalyzes the hydroxylation
of monophenols to o-diphenols (monophenolase or cresolase
activity) and the oxidation of diphenols to quinones (diphenolase
or catecholase activity). Molecular oxygen acts as the electron acceptor
in both reactions. Quinones are highly reactive species and can undergo
nonenzymatic polymerization reactions ultimately leading to brown
pigments. Enzymatic browning in spoiled fruits and vegetables during
postharvest handling and processing represents an important problem
in the food industry and for the consumers, because it leads to undesirable,
potentially toxic products. Natural tyrosinase inhibitors have therefore
attracted strong interest in the food sector to control the rate of
this process.65 Tyrosinase inhibition is
an increasingly important target also for the cosmetic industry, as
this enzyme catalyzes melanogenesis key steps in humans, that is hydroxylation
and oxidation of l-tyrosine to dopaquinone; overproduction
of melanin is associated with several pigmentary disorders, whose
major medical and aesthetical consequences have led to the quest for
new nontoxic depigmenting agents.66,67</p><p>The
ability of PNSE to inhibit mushroom tyrosinase was preliminarily investigated
using l-dopa or l-tyrosine as the substrate. The
assays is based on the detection of dopachrome formation (Î»max 475 nm) following oxidative cyclization of dopaquinone
resulting from the tyrosinase-induced oxidation of the substrate,
with or without inhibitor.42 As reported
in Figure S7, quite low IC50 values were observed for both cresolase and particularly catecholase
activity of the enzyme. These values were comparable to those previously
reported for condensed tannins from Vigna angulariz seeds,68 cherimoya pericarp,69 fig leaves,70 and
longan bark.71 Under the same conditions,
the well-established depigmenting agent kojic acid66 exhibited a IC50 value for the catecholase activity
of ca. 50 Î¼M, that is, 7 Î¼g/mL.</p><p>On the basis of
these encouraging results, the enzymatic browning
inhibition properties of PNSE were then evaluated in apple smoothies.
Red Delicious apples were chosen for this study because of their high
susceptibility to browning.72 In particular,
apples were finely grinded with a hand blender in the presence or
absence of a 0.1% w/v PNSE solution, and the changes in color development
were periodically analyzed with a colorimeter. A significant inhibition
of browning was observed in the presence of PNSE, peaking to a value
of about 40% at 3 h (Figure 7a).</p><!><p>(a) Effect of PNSE on apple smoothie enzymatic browning at varying
times. Mean ± SD values of three experiments are reported (three
different measurements were taken during each experiment). (b) Browning
index calculated after 3 h for apple smoothies covered with solvent-cast
PLA films. Mean ± SD values of three experiments are reported
(three different measurements were taken during each experiment).
*P < 0.05 compared to control. (c) Apple smoothies
covered with solvent-cast neat PLA film (left) and PLA film containing
10% w/w PNSE (right).</p><!><p>In parallel experiments,
kojic acid and ascorbic acid were used
as reference compounds. As mentioned before, the first is a well-known
tyrosinase inhibitor acting through chelation of copper at the active
site,66 whereas ascorbic acid is an antioxidant
able to reduce quinonoid species thus preventing their polymerization;73 in addition, the lowering of the pH caused by
ascorbic acid leads to a decrease of the enzymatic activity, which
is maximal at pH 6–7. Under the same experimental conditions,
kojic acid was more effective than PNSE, leading to a ca. 60% inhibition
at 3 h. In contrast, ascorbic acid was able to induce an effect only
in the first 2 h, after that no browning inhibition was observed.</p><!><p>To assess the ability of PNSE to delay
enzymatic browning processes even when incorporated in PLA films,
in further experiments, finely grinded apples were rapidly transferred
on a watch glass and covered with the films, and the changes in color
development were analyzed as described above. Figure 7b displays the browning index calculated
after 3 h. Although PLA alone or with a loading of extract of 3% w/w
did not exert any effect on enzymatic browning in the presence of
10% w/w PNSE a 30% inhibition was observed (Figure 7c). On the basis of the findings reported
above, this effect cannot be ascribed to a change of the oxygen barrier
properties of the films due to the presence of PNSE.</p><!><p>In a final
set of experiments, the effect of PNSE on anthocyanin stability was
determined. Anthocyanins are natural pigments responsible for the
blue, purple, and red colors of many fruits and vegetables. They are
commonly used as food additives not only to impart specific color
but also in light of their health-beneficial properties, including
antioxidant, anti-inflammatory, antidiabetic, and anticancer activities.74−76 However, anthocyanins isolated from natural sources suffer from
extensive degradation with consequent color loss, depending on several
factors including light, pH, and temperature.77−79 For these reasons,
several studies have been recently devoted to the evaluation of the
effects of natural additives as stabilizers of anthocyanin color.80 Organic acids, metal ions, phenolic compounds,
and aromatic amino acids like tryptophan have been found to inhibit
anthocyanin degradation in model systems.81−83 In particular,
phenolic compounds such as hydroxycinnamic acids and tannins are able
to form noncovalent complexes with anthocyanins through π-stacking
interactions, and this "copigmentation" phenomenon prevents
water addition to the flavylium ion causing its degradation, with
an overall effect of color stabilization.84 Copigmentation has also been shown to improve color intensity.85 The use of copigments to increase stability
of anthocyanins in red wine or fruit- and berry-derived food and beverages
is now widely documented. Moreover, enhanced anthocyanin storage and
heat stability has been reported as a consequence of copigmentation,
too.86,87 Hence, a further objective of this study
was the evaluation of the effect of PNSE on red wine anthocyanin stability
at 90 °C, under literature-reported conditions.44 When added to a 0.1 mg/mL anthocyanin solution (PNSE final
concentration 0.008–0.05 mg/mL), no significant hyperchromic
or bathochromic effect was observed, ruling out the occurrence of
any copigmentation effect. However, PNSE was found to improve the
heat stability of the pigments even at a concentration as low as 0.02
mg/mL (Figure 8a).
Notably, at the same concentrations, no stabilizing effect was observed
with the well- known anthocyanin copigments ferulic acid and chlorogenic
acid (Figure 8b).88,89 Moreover, PNSE was found to be effective at lower concentrations
compared to other polyphenol-rich extracts.87</p><!><p>(a)
Effect of PNSE at different doses and (b) comparison with ferulic
acid and chlorogenic acid (all 0.05 mg/mL) on a red wine–anthocyanin
solution stability at 90 °C. Mean ± SD values of three experiments
are reported (three different measurements were taken during each
experiment). *P < 0.05 compared to control.</p><!><p>To determine the efficiency of PNSE in a more complex
food system,
the anthocyanin-stabilizing properties of the extract were then evaluated
in a commercial bilberry juice. As reported in Figure S8, a dose-dependent effect was observed also in this
case, with an almost 50% lower decrease in anthocyanin content with
respect to the control samples in the case of a PNSE dose of 2 mg/mL.</p><p>Future studies will be aimed at providing deeper insights into
the mechanisms involved in the anthocyanin stabilization induced by
PNSE.</p><p>Application of anthocyanins as natural food colorants
necessitates
their stabilization not only in solutions but also as a free-flowing
powder which could ensure safe and easy dosage during processing.
To this aim, spray drying has been applied in numerous studies, as
a mild method to dry anthocyanin extracts.90 Additional stabilization of the dried anthocyanins can be achieved
by supplementing the feed solution with other phenolic compounds that
act as copigments.33 In the present study,
two different anthocyanin extracts from black currant and blackberry
juice were spray dried with or without PNSE. Characterization of the
powders obtained by spray drying are reported in Table 3. The lower total yields observed
for the samples with PNSE might be explained by the low solubility
of PNSE in the aqueous solution, and the resulting decreased stability
of the feed solution emulsion.</p><!><p>Yield might be
overestimated due
to quantification as cyanidin 3-glucoside equivalents.</p><!><p>The resulting powders were then
stored under UV light in open or
evacuated and sealed Petri dishes at 35 °C for 10 weeks. The
anthocyanin profile of blackberry is dominated by cyanidin 3-glucoside,91 whereas in black currant delphinidin derivatives
are most abundant.92 The stability of the
black currant anthocyanins during spray drying was significantly higher
compared to the blackberry anthocyanins (Table 3), which is surprising because delphinidin
derivatives have been shown to be more heat sensitive than the respective
cyanidin derivatives.93,94</p><p>In the present study, a
higher susceptibility of delphinidin derivatives
was observed during storage, since the anthocyanins from blackberries
were retained in higher concentrations after storage than the black
currant anthocyanins (Figure 9). It was previously shown that addition of pigments could
improve the stability of blackberry anthocyanins during dry storage.33 This could be confirmed in the present study,
particularly when no oxygen was present.</p><!><p>(a) Loss of spray-dried
anthocyanins during storage and (b) calculated
half-life of spray-dried anthocyanins after storage under UV light
in open (O2) or sealed (no O2) Petri dishes.
Mean ± SD values of three experiments are reported.</p><!><p>In conclusion, in this article, the possible
exploitation of PNS
as a low cost polyphenol source for use in active packaging and food
preservation is reported. Apart from acting as an antioxidant, the
hydroalcoholic extract of PNS, consisting mainly of condensed tannins,
can efficiently delay enzymatic browning in fruit even when incorporated
in solvent-cast PLA films. These films exhibited also good antioxidant
properties both in organic and in aqueous solvents, therefore emerging
as potential packaging materials for oxidative stabilization of foods.
In particular, the low amounts of PNSE released from the films when
taken in aqueous media, reaching concentrations that were shown to
be of no toxicity for HepG2 cells, would suggest their applications
to water-rich foods. On the other hand, the more substantial migration
observed in organic solvents would warrant even higher oxidative stability
in the case of low water content foodstuffs, such as oils, butters,
and sauces. This is a result of considerable value in light of the
literature data indicating very low toxicity of PNSE in animal models.</p><p>Preliminary experiments showed that PNSE is also able to stabilize
anthocyanin pigments from thermal degradation in food, displaying
higher efficiency compared to well-known copigments. Application of
PNSE as a copigment during spray drying of anthocyanins showed promising
results, which might be further enhanced by adjustment of the composition
of the feed solution and optimization of the spray-drying parameters.</p><p>These results fulfill most of the 12 Principles of Green Chemistry,95 namely, (a) Less Hazardous Chemical Synthesis
and Designing Safer Chemicals (pecan nut shell and PLA are of natural
origin, and PNSE was found to be nontoxic in cellular assays), (b)
Safer Solvents and Auxiliaries (only water and ethanol are needed
to obtain PNSE, and only water was used to prepare the stabilized
spray-dried food colorant powder), (c) Use of Renewable Feedstocks
(PNSE is a food waste-derived product), and (d) Design for Degradation
(PLA is a degradable polymer).</p><p>Although still preliminary for
the purposes of a real application,
these results, coupled with previous evaluations demonstrating the
technical and economic feasibility of PNSE manufacturing,24 would make PNSE a highly convenient and competitive
functional additive for the food industry.</p><!><p>Elution profiles
of the chemical degradation mixtures
of PNSE, data from cell viability experiments, DPPH-reducing properties
of extruded films, release of PNSE from solvent-cast PLA films, Fe3+ reducing activity of extruded PLA films, close up view of
the FTIR spectra of solvent-cast PLA films, data from mushroom tyrosinase
inhibition assays, and effect of PNSE on anthocyanin stability in
bilberry juice (PDF)</p><p>sc0c00356_si_001.pdf</p><p>The authors declare no
competing financial interest.</p>