Effects of preheating and drying methods on pyridoxine, phenolic compounds, ginkgolic acids, and antioxidant capacity of Ginkgo biloba nuts
Abdou Madjid Olatounde Amoussa1,3 Lixia Zhang1 Camel Lagnika1
Asad Riaz1 Liuquan Zhang2 Xianjin Liu2 Trust Beta4
1 Research Institute of Agricultural Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, P. R. China
2 Research Institute of Agricultural Product Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu, P. R. China
3 Laboratory of Biochemistry and Bioactive Natural Substances, Faculty of Science and Technology, University of Abomey-Calavi, Cotonou, Benin
4 Department of Food & Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada
Dr. Lixia Zhang, Research Institute of
Agricultural Product Processing, Jiangsu
Academy of Agricultural Sciences, Zhong
Lingjie road No. 50, Nanjing, 210014,
Jiangsu, P. R. China.
Email: [email protected];
[email protected]
Dr. Xianjin Liu, Professor, Research Insti￾tute of Agricultural Product Safety and
Nutrition, Jiangsu Academy of Agricul￾tural Sciences, Zhong Lingjie road No. 50,
Nanjing, 210014, Jiangsu, P. R. China.
Email: [email protected]
Funding information
the Special Project of Agriculture Pro￾duce Quality Safety Risk Assessment,
Grant/Award Number: GJFP2019043;
Ministry of Agriculture and Rural
Affairs of China, and National Key R &
D project of China, Grant/Award Number:
Abstract: Although ginkgo nuts are very nutritious and loaded with numer￾ous bioactive compounds, the nuts contain significant levels of unwanted com￾pounds (ginkolic acids) which are toxic to consumption. To reduce or elimi￾nate these toxic compounds without impacting the nutritional value and the
bioactivity of the final product, an appropriate processing technology is needed.
Thus, the effect of preheating (90 and 120◦C) prior to drying (freeze drying:
FD, hot air drying: HAD, and HAD in tandem with FD: HAD-FD) was evalu￾ated on ginkgolic acids, pyridoxine analogues, phenolic compounds, and antiox￾idant properties of ginkgo nuts. Our results pointed out a significant decrease
(below 50%) of ginkgolic acids in ginkgo nuts samples processed at 90◦C com￾pared to the control. The major compounds found after treatments were respec￾tively, kaempferol (36.66-354.38 µg/g), quercetin (9.04-183.71 µg/g), and caffeic
acid (19.66-106.88 µg/g). Principal component analysis (PCA) revealed that pre￾heating at 90◦C prior to HAD-FD would be a proper and reasonable approach
for preserving the bioactive compounds and antioxidant capacity of ginkgo nuts
(EC50 ranged from 2.25 to 4.60 mg/mL) while significantly reducing their content
in toxic compounds.
antioxidant activity, drying, ginkgo nuts, ginkgolic acids, preheating, pyridoxine analogues
Nuts are rich in a variety of bioactive constituents with
beneficial health effects. Historically, nuts are sources of
regular constituent of humankind’s diet where they are
consumed in a variety of forms, such as snacks, desserts,
or are combined with a meal or eaten in whole (raw, fried,
and roasted) (Ghazzawi & Akash, 2019). The most popu￾lar edible nuts can be obtained from plant species, such as
Prunus amigdalis, Corylus avellana, Juglans regia, and Pis￾tachia vera (Ros, 2010). Nuts from Ginkgo biloba species are
important component for optimal diets. Ginkgo seeds are
J. Food Sci. 2021;86:4197–4208. © 2021 Institute of Food TechnologistsR 4197
rich in proteins, carbohydrates, vitamin C, minerals, and
phenolic compounds with significant antioxidant activities
(Ros, 2010). The seeds are also a source of other equally
important biologically active compounds such as vitamin
B6 and its vitamers (pyridoxine (PN), pyridoxal [PL], and
pyridoxamine [PM]). These forms of vitamin B6 are highly
bioaccessible, making their presence in food particularly
nourishing for children when incorporated in their meals,
all of which has positive outcome in vitality and growth
(Yaman & Mızrak, 2019). Ginkgo biloba seeds are used as
medicine or eaten as part of daily diet in China and other
parts of the world (Gong et al., 2019).
Ginkgo nuts are not suitable for consumption as raw
forms due to their toxic potential as well as subsequent
rejection by consumers (Wang & Zhang, 2019). Indeed,
ginkgolic acids (GAs) in ginkgo seeds have been reported
to be potentially toxic (Wang & Zhang, 2019). Furthermore,
the consumption of these nuts may cause allergies among
some consumers due to GAs. To solve this problem, some
thermal treatments are used either by direct heating or
coupling with a drying method involving the use of high
temperature to detoxify ginkgo nuts by reducing its GA
concentration (Yang et al., 2014). Traditionally, cooking
and roasting methods have been used to produce ginkgo
nuts. Unfortunately, excessive exposures of ginkgo nuts
to high temperatures could degrade their bioactive com￾pounds which subsequently affect their nutritional quality
(Calín-Sánchez et al., 2013). Therefore, it is important to
establish an effective degradation approach that can effec￾tively eliminate GAs while preserving the bioactive com￾pounds to ensure nutritional quality of the processed prod￾uct. Preheating followed by an appropriate drying method
could reduce or eliminate ginkgolic acids, extend the shelf
life of ginkgo nuts, and improve their nutritional quality.
Preheating offers many advantages, such as energy effi￾ciency, a significant reduction in the drying process time
as well as an improvement in the quality of the processed
product (da Silva et al., 2016). Freeze drying (FD) and hot
air drying (HAD) are two common drying methods for
expelling water from food. However, the degradation of
the bioactive constituents sensitive to heat as well as the
extensive drying duration and costs are all limiting fac￾tors coupled to the use of these two methods (FD, HAD).
Hybridization of these two methods would greatly reduce
drying time and cost resulting in high-quality dried prod￾ucts. To our knowledge, there is no scientific report on the
bioactive compounds, the nutritional quality, as well as on
the reduction of the ginkgolic acid contents of ginkgo nuts
following postharvest processing.
In the present work, the effects of a preheating treat￾ment combined with different drying methods (FD, HAD,
and combination drying) were studied to document the
changes in the concentration of GAs as well as, the con￾tent in PN analogues, phenolic compounds along with the
antioxidant properties of ginkgo nuts.
2.1 Chemicals reagents
All chemicals used in the experiments were of analytical
reagent grade. HPLC-grade standards, including GAs
(C15:1 and C17:1), phenolics (ferulic acid, chlorogenic acid,
caffeic acid, catechin, epicatechin, epicatechin gallate,
Kaempferol, luteolin, and Quercetin), PL, PM, and PN
with hydrochloride (≥98%) were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Folin–Ciocalteu reagent
(FCR), 2,2-diphenyl-1-picrylhydrazyl chloride (DPPH),
2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), and 6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid (Trolox) were also from Sigma–Aldrich.
HPLC-grade acetonitrile, methanol, phosphoric acid,
gallic acid, hyperoside, formic acid, luteolin, and sodium
pentanesulfonate were purchased from National Pharma￾ceutical Corporation (Beijing, China).
2.2 Experimental design
Fresh Ginkgo seeds were purchased from Taixing Agricul￾tural Trade Market, Taixing city Jiangsu in China. Ginkgo
biloba seeds were subjected firstly to a thermal pretreat￾ment step. The heat treatment conditions were set at 90
and 120◦C for 30 min using a ceramic plate (Midea RT2110).
Prior to drying process, the hulls and seeds coat of the
preheated samples were manually removed. Sample with￾out pretreatment was used as control. The preheating pro￾cess was aimed at reducing the potentially toxic com￾pounds, such as Gas, in the ginkgo nuts. Photograph of
ginkgo nuts pretreated have been illustrated in Figure 1.
After dehulling, sorting, and moistening by a water jet, the
heated and unheated samples ofG. biloba nuts (1.5 kg each)
were subjected to three drying methods such as Freeze
Drying (FD), Hot Air Drying (HAD), and combination of
freeze and HAD (HAD-FD). Drying processes were marked
by monitoring the moisture content to less than 5% (dry
Freeze Drying (FD): CHRIST freeze dryer (Alpha-1-2
LD Plus, Martin Christ, Gefriertrocknungsanlagen GmbH,
Germany) was used to dry the Ginkgo nuts. For the drying,
samples were placed on trays of the freeze dryer at −36◦C
with absolute pressure of 0.20 mbar for 36 h.
Hot Air Drying (HAD): During HAD, ginkgo nuts
were spread uniformly on the metal trays of an oven
dryer (DHG-9146A, Shanghai Jinghong Experimental
FIGURE 1 Photograph of ginkgo nuts pretreated (90 and
120◦C). Samples were dried under freeze drying (FD), hot air
(HAD), and combined method (HAD-FD) before analysis for
bioactives compounds and ginkgolic acids
Equipment Co., Ltd., Shanghai, China) and dried for 24 h.
The set air temperature and velocity were 70◦C and 0.1 m/s,
respectively. The limit of the maximum internal tempera￾ture of samples was kept constant at 70◦C.
Combination drying method (HAD-FD): For combina￾tion drying, the HAD predried samples were immediately
subjected to FD. Briefly, samples were dried in the first step
by HAD for 4 h and then continued to FD for 12 h in the sec￾ond step. The drying parameters for HAD and FD methods
were the same as described above.
These parameters were chosen based on preliminary
experiments. All dried samples of ginkgo nuts were
crushed using an electric grinder (Midea MJ-BL15U11,
Guangdong Mei’s Life Electrical Appliances Manufactur￾ing Co. Ltd. Guangdong, China) to pass through a 0.5-mm
screen and placed in aluminum-lined bags for storage at
4◦C until analyzed. All experiments were carried out in
2.3 HPLC-PAD analysis of pyridoxine
analogues in G. biloba nuts
Pyridoxine (PN), PM, and PL were extracted from pre￾heated and dried ginkgo nuts according to a modified
method by Gong et al. (2019). Waters Alliance e2695 HPLC
system consisted of the Windows based Empower 3 soft￾ware and Photodiode Array Detector (PAD, Waters 2998)
was used to determine PN analogues in ginkgo nut sam￾ples. The PN analogues were separated with a Waters
XBridge C18 column (250 × 4.6 mm, 5 µm) using 96%
of buffer solution (5 mM potassium phosphate containing
5 mM sodium pentane sulfonate adjusted to pH 2.5 with
phosphoric acid) and 4% of acetonitrile. The flow rate was
0.8 min/mL and the column temperature was set to 30◦C.
Quantification of PN analogues was achieved using stan￾dard chromatograms recorded at 290 nm and calibration
curves prepared at six points of concentration (PN, 0.10-
10 µg/mL, R2 = 0.998; PL, 0.10-10 µg/mL, R2 = 0.999, and
PM, 0.10-10 µg/mL, R2 = 0.997). The content of each PN
analogue was expressed as mg/kg on dry matter (dm) basis.
2.4 HPLC-PAD/ESI-MS analysis of
phenolic compounds
Before analysis of phenolic compounds, ginkgo nuts phe￾nolic extract (GPE) was prepared. Briefly, 5 g of ground
ginkgo nuts was mixed with 50 mL of 1% formic acid in
85% methanol. The mixtures were stirred for 2 h at 4◦C and
then centrifuged (7000 rpm, 5 min at 4◦C). After filtration
with Whatman N◦1 filter paper, the extracts were evapo￾rated in vacuum at 35◦C up till the volume of 5 mL. The
GPE obtained was subsequently used for analysis of phe￾nolic compounds, total phenol content, and antioxidant
capacity of ginkgo nuts.
GPE (3.5 mL) was evaporated under vacuum with a
rotary evaporator at 35◦C and were recovered with 1 mL
of methanol (85%). The recovered extracts were succes￾sively sonicated (3 min), vortexed (2 min), and then filtered
through Millipore membrane 0.45 µm filter before injec￾tion in Waters HPLC-PAD/ HPLC-ESI-MS system.
For the separation, a reverse-phase analytical column
was employed (XBridge C18, 250 × 4.6 mm, 5 µm) for the
analysis with a working temperature of 35◦C. The UV pho￾todiode array detector (PAD) was set at 280 nm for gal￾lic acid, ferulic acid, chlorogenic acid, caffeic acid, cat￾echin, epicatechin, epicatechin gallate, and 360 nm for
hyperoside, kaempferol, luteolin, and quercetin. The run￾ning time was 50 min. The mobile phase was a binary
gradient prepared from (A) formic acid solution (0.1%)
and (B) methanol. The elution started with a linear gra￾dient using 5% methanol (B), followed by 5-45% (B) for
40 min, and then isocratic elution at 45% (B) for 5 min
before ending with 5% (B) for 5 min. The flow rate was
0.8 mL/min and the injection volume was 10 µL. For
the identification of phenolic compounds, the mass spec￾trometer was operated using an electrospray ion source in
negative mode. Recording was done by repeatedly scan￾ning over the mass range m/z 200-400 using the following
parameters: dry gas N2 at 350◦C, and flow rate of 10 L/min,
capillary set at 4000 V, and nebulizer operated at 25 psi. The
chromatographic peaks corresponding to each phenolic
compound were identified by comparing their retention
times, UV spectra, and/or their m/z ratios with those of
phenolic standards. Quantitative determinations were per￾formed using calibration curves from the pure standards
analyzed and the content of each phenolic compound was
expressed as µg/g on dm basis.
2.5 Measurement of total phenol
content (TPC)
FCR was used to determine the total phenolic content
(TPC) following slightly modified method by Dadan et al.
(2018). An aliquot of suitable samples (GPEs) diluted
with methanol (0.125 mL) and deionized water (1.5 mL)
were transferred into test tubes and mixed with Folin–
Ciocalteu’s reagent (0.125 mL). Each test tube with its con￾tent was vortexed for 1 min and allowed to rest for 5 min
before addition of 1.25 mL of sodium carbonate solution
(7%). Subsequently, each mixture was vortexed again and
incubated in the dark at room temperature. After 90 min
of incubation, the absorbance versus prepared blank was
recorded at 760 nm. The TPC measurements in dried
ginkgo nuts were carried out in triplicates for each sam￾ple and the results were expressed as milligrams of gallic
acid equivalents per gram of dm (mg of GAE.g−1 of dm)
from the standard calibration curve prepared using gallic
acid (Sigma Aldrich). The calibration curve concentration
range was 0.08-0.100 mg/mL.
2.6 Antioxidant capacity determination
The antioxidant capacity of the samples was assayed spec￾trophotometrically using three antioxidant methods: 2,2′-
azinobis (ABTS), DPPH, and hydrogen peroxide scaveng￾ing (H2O2). The details of each method are described
2.6.1 ABTS radical scavenging assay
ABTS radical scavenging capacity of GPEs was mea￾sured spectrophotometrically (Nguyen et al., 2015). Briefly,
a stock solution was firstly prepared by mixing equal
amounts of 7 mM ABTS radical and 2.45 mM potassium
persulfate solution. The mixture was set to stand in the
dark at room temperature for 15 h to obtain the stable form
of ABTS radical cation. Before use, a working solution was
prepared by mixing 1 mL of stock solution with 71.8 mL
of methanol (90%) to obtain an absorbance of 0.7 ± 0.02
at 734 nm using a UV-VIS spectrophotometer (P5 UV-Vis
Spectrophotometer, MAPADA). GPEs (0.15 mL) or stan￾dard (Trolox) were let to react with 2.85 mL of the working Ginkgolic
solution (fresh blue-green ABTS radical solution) and the
resulting mixture incubated in the dark at room tempera￾ture for 2 h. The mixture absorbance was read at 734 nm.
Methanol (90%) and Trolox were used as control and stan￾dard, respectively. All samples were measured in triplicate.
Trolox equivalent (TE) antioxidant capacity of GPEs was
calculated from the calibration curve ranging 0-100 µM
Trolox concentrations. The results were expressed in mM
TE/kg dried sample.
2.6.2 DPPH radical scavenging assay
Scavenging activity of GPEs against DPPH radical was
measured using spectroscopic method (Dadan et al., 2018).
Briefly, 3 mL of methanolic solution of DPPH (0.1 mM) was
added to 1 mL of the methanolic preparation of GPEs (0.01-
1 mg/mL). The mixture was immediately vortexed and
stored in the dark for 30 min. Absorbance measurements
of the resulting solution were performed at 517 nm against
methanol using an UV/Vis Spectrophotometer (P5 UV￾Vis Spectrophotometer, Shanghai Mapada Instruments
Co. Ltd., China). The capacity of Ginkgo nuts to scav￾enge the DPPH radical was calculated using the following