LWT - Food Science and Technology 112 (2019) 108236

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Solid-state fermentation with Aspergillus niger to enhance the phenolic T

contents and antioxidative activity of Mexican mango seed: A promising
source of natural antioxidants
Cristian Torres-Leóna,c, Nathiely Ramírez-Guzmána,c, Juan Ascacio-Valdésa, Liliana Serna-Cockb,
Maria T. dos Santos Correiac, Juan C. Contreras-Esquivela, Cristóbal N. Aguilara,∗
a
Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, 25280, Saltillo, Coahuila, Mexico
b
School of Engineering and Administration, Universidad Nacional de Colombia, Street 32 Chapinero. Palmira, Valle del Cauca, Colombia
c
Centro de Ciências Biológicas e Departamento de Bioquímica, Universidade Federal de Pernambuco. Recife, Pernambuco, Brazil

ARTICLE INFO ABSTRACT

Keywords: This study examines changes in phenolic compounds and the antioxidant activity of Mexican mango seed in the
Agro-industrial byproducts bioprocess of solid-state fermentation (SSF) with the fungus Aspergillus niger GH1. The chemical composition and
Mango seed evaluation of mango seed as solid fermentation substrate support was also evaluated. Because of the chemical
Phenolic compounds composition and the rapid growth of the fungus, mango seed is a suitable material to be used in SSF. The results
Antioxidants
showed that SSF of mango seed mobilized the polyphenolic compounds and improved the nutraceutical prop-
Solid-state fermentation
erties. The total phenol content in ethanol extract increased (p < 0.05) from 984 mg GAE/100 g to 3288 mg
GAE/100 g at 20 h of fermentation. Total phenolic and HPLC-MS analysis of the free and bound fractions showed
that SSF is an effective method to release the bound phenols, suggesting that this strategy may help to enhance
potential antioxidant. Therefore, fermented mango seed extracts are particularly promising as natural anti-
oxidants.

1. Introduction of these compounds (Ediriweera, Tennekoon, & Samarakoon, 2017).

In recent years, the interest and demand for natural polyphenolic
compounds have increased. This is mainly attributed to their potential
beneficial health effects, recognized antioxidant properties (Lucci,
Saurina, & Núñez, 2016) and the negative effects reported in the syn-
thetic antioxidants as the butylated hydroxytoluene (BHT) (Sarafian,
Kouyoumjian, Tashkin, & Roth, 2002). Polyphenolic compounds exhibit
a wide range of functional properties such as anti-inflammatory, anti-
diabetic, anti-cancer, anti-microbial and anti-viral activities
(Balasundram, Sundram, & Samman, 2006; Del rio et al., 2013; Roleira
et al., 2015; Torres-León, Rojas, Serna-Cock, Belmares-Cerda, &
Aguilar, 2017; Torres-león, Ventura-Sobrevilla et al., 2017). Human
health benefits are explained by the incomparable abilities of poly-
phenol in quenching reactive oxygen species (ROS) in the human body
(Shaikh et al., 2016).
The polyphenolic compounds are widely available in the plants and
agro-industrial byproducts such as mango seed are a sustainable source
Mango seed had a high content of bioactive phytochemicals, mainly
polyphenolic compounds (Hoyos-Arbeláez, Blandón-Naranjo, Vázquez,
& Contreras-Calderón, 2018; Torres-León et al., 2016). Mango (Mangi-
fera) is the second most traded tropical fruit in the world and fifth in
total production (FAOSTAT, 2017). Although traditionally the fruit is
consumed fresh, a wide range of foods can be obtained with the pulp.
Since, of the 35–60% of the fruit is discarded after processing (O’Shea,
Arendt, & Gallagher, 2012) in tropical and subtropical countries mango
processing generates a high amount of waste. In Mexico for example, it
is estimated that the byproducts and waste of mango processing con-
stitute approximately 228,096 tons per year (FAOSTAT, 2017). These
byproducts and wastes generate high management costs and are a
source of environmental contamination (Torres-León et al., 2018).
The extraction of polyphenolic compounds is an alternative for the
valorization of mango seed. However, extraction methods currently
used have some disadvantages: traditional techniques where high
temperatures are used to generate negative effects on the biological

Corresponding author.
∗

E-mail addresses: ctorresleon@uadec.edu.mx (C. Torres-León), nathiely_ramirez@uadec.edu.mx (N. Ramírez-Guzmán),

alberto_ascaciovaldes@uadec.edu.mx (J. Ascacio-Valdés), lserna@unal.edu.co (L. Serna-Cock), mtscorreia@gmail.com (M.T. dos Santos Correia),
carlos.contreras@uadec.edu.mx (J.C. Contreras-Esquivel), cristobal.aguilar@uadec.edu.mx (C.N. Aguilar).

https://doi.org/10.1016/j.lwt.2019.06.003
Received 17 December 2018; Received in revised form 7 May 2019; Accepted 2 June 2019
Available online 07 June 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
C. Torres-León, et al.
activity of the extracts, the use of toxic solvents generates damage to
the environment and hinders the subsequent use of the extracts, alter-
native technologies (green) are expensive, complex and difficult to
implement in poor countries where the greatest amount of waste is
produced. Also, the techniques currently used produce an incomplete
release of the phenolic compounds, due to the impossibility of re-
covering the bound polyphenols (Dey, Chakraborty, Jain, Sharma, &
Kuhad, 2016), due to the strong interactions of these compounds with
the plant matrix. Although hydrolysis (alkaline, acidic or enzymatic)
can increase the release of polyphenols, these are not specific and can
cause degradation of the compounds (Domínguez-Rodríguez, Marina, &
Plaza, 2017). Also, the use of enzymes is limited by the instability and
high associated costs (Navarro-González, García-Valverde, García-
Alonso, & Periago, 2011). Therefore, a technology of high specificity,
low toxicity and high performance in the release of polyphenolic
compounds are necessary.
Solid-state fermentation (SSF) has great potential for obtaining
bioactive compounds (Martínez, Aguilera, Rodríguez, & Aguilar, 2012);
since, microorganisms such as fungi naturally produce enzymes that
degrade the cell wall, generating a hydrolysis (Jamal, Idris, & Alam,
2011) and mobilization of compounds towards the extraction solvent.
Additionally, this biotechnological process is economical and easy to
implement as it requires small equipment, lower capital and reduced
operating costs (SSF does not require stages of sterilization, aeration or
agitation) (Letti et al., 2017). SSF has also several environmental ad-
vantages since it allows the use of solid agro-industrial wastes as sub-
strate (residues which are also considered as a pollutant) and besides
lesser wastewater production. The use of agroindustrial waste also re-
duces production costs, which together with the high biological activ-
ities achieved in the final extracts guarantee the economic profitability
of the process. Fermentation is preferred instead of the utilization of
commercial enzymes to enhance the nutraceutical value of foods be-
cause it is relatively cheap (Acosta-estrada, Gutiérrez-uribe, & Serna-
saldívar, 2014). Although several studies have investigated the use of
SSF for the separation of phenolic compounds in foods and agro-in-
dustrial wastes (Dey et al., 2016), in the scientific literature there are
few studies where mango seed is used as a substrate support for SSF and
the reported studies were developed to increase nutritional values and
not to release bioactive compounds (Kayode, Sani, & Kolawole, 2010;
Kayode & Sani, 2008; Munishamanna, Suresha, Veena, & Subramanya,
2017). The main aim of this research was to investigate the change in
the content of phenolic compounds and the antioxidant activity of the
Ataulfo mango seed subjected to the bioprocess of SSF with the fungus
A. niger GH1.
2. Materials and methods
2.1. Chemicals and reagents
DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS⁺ (2,2′-azino-bis (3-
ethylbenzothiazoline-6-sulphonic acid)), Trolox, Folin-Ciocalteu re-
agent, Gallic Acid (GA) and BHT (butylated hydroxytoluene) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade
ethanol (C2H5OH), hexane (C6H14) and ethyl acetate (C4H8O2) were
purchased from Jalmek® (Nuevo Leon, Mexico). Mineral salts, sodium
hydroxide (NaOH), hydrochloric acid (HCl), sodium carbonate
(Na2CO3), formic acid (CH2O2) and acetonitrile (C2H3N) were pur-
chased from J.T. Baker® (Mexico City, Mexico). Potato dextrose agar
(PDA) from Solviosa ® (Jalisco, Mexico). Ultra-pure water was prepared
by Milli-Q system (Simplicity® UV, Millipore Corporation Bedford, MA,
U.S.A.)
2.2. Microorganism
A. niger GH1 strain from DIA/UAdeC (Departamento de
Investigación en Alimentos/Universidad Autonoma de Coahuila)
2
LWT - Food Science and Technology 112 (2019) 108236
collection (Fungal strains are crio-conserved at −50 °C) were used in
this study (Cruz-Hernandez, Contreras-Esquivel, Lara, Rodrıguez, &
Aguilar, 2005).
2.3. Plant materials
Mango seeds (Mangifera caesia, cv. Ataulfo) were obtained from a local
industry in Saltillo city (Mexico) in March 2017. Also, fresh fruits
(329.1 ± 25.07 g) of the same batch processed were studied before the
processing to identify the state of maturity of the fruit. The color parameters
were measured with a colorimeter (NR20XE, China); lightness (L∗), a*, b*
hue angle (h◦), and chromaticity (C∗) of mango peel or pulp were
70.5 ± 1.80 or 74.1 ± 1.10, 24.1 ± 0.99 or 22.5 ± 0.76, 58.7 ± 3.06
or 55.8 ± 2.04, or 67.5 ± 2.50 or 60.8 ± 1.09, 68.1 ± 1.26 or
68.6 ± 1.09, respectively. Fruit texture was measured as penetration
(Humboldt H-1200, USA), 88.5 ± 9.26 mm. Total soluble solids (Atago,
Japón) were 11.5 ± 2.52 °Brix.
The mango seed kernels (MSK) was manually separated from fibrous
endocarp and testa (MSET) by using a very sharp knife. MSK was cut
(1 × 1 cm). MSK and MSET were dried at 60 °C ± 5 in a convection
oven (Felisa, Mexico). The moisture was determined with a moisture
balance (Ohaus MB 23, USA) and water activity (aw) with an Aqua Lab
(Decagon Devices, 3TE, USA). Subsequently, MSK and MST were milled
using impact grinding (Pulvex-Mexico) and sieved throughout intense
vibration, to obtain samples of particle diameter > 600 μm (#30).
2.3.1. The chemical composition of MSK and MSET
The MSK and MSET were analyzed for its content in ash (A) at
550 °C for 8 h, crude protein content (CP) was conducted by the
Kjeldahl method with a conversion factor of 6.25, and lipids content (L)
(ether extract by the Soxhlet system) as described by the AOAC (AOAC
920.39, 1990). The content of fiber was evaluated by the gravimetric
enzymatic methods (AOAC 991.43, 1993). Cellulose, hemicellulose and
lignin contents were calculated by determining neutral detergent fiber
and acid detergent fiber's by the method of Van Soest. Reducing sugars
content was evaluated using the colorimetric method reported by Miller
(1989). Total carbohydrates (Tc) were calculated using the following
equation (1):
Tc = 100 % A + % CP + %L (1)
where Tc represents the total carbohydrates, %A represents ash con-
tent, %CP represents crude protein content, and %L lipids content.
2.4. Tests of support in solid state fermentation
Water absorption index (WAI), critical humidity point (CHP), water
activity (aw), pH and invasion capacity were evaluated to know the
potential use as a support in SSF of combinations of MKS and SMET
(100%, 75%, 50%, 25%, and 0%).
WAI was determined by referring to the method reported by Du,
Jiang, Yu, and Jane (2014). The samples (2.5 g) were dissolved in
30 mL of distilled water and cooked at 70 °C for 30 min in a water bath
(VWR, USA). The cooked paste was cooled to room temperature and
transferred to pre-weighed centrifuge tubes, and centrifuged at 3000 g
for 20 min. The supernatant was decanted, and the sediment was
weighed. WAI was calculated using the following equations:
Weight of sediment
WAI (g / g ) =
Weight of dry solids (2)
The supernatant obtained in the previous test was used for the de-
termination of CHP. Briefly, the CHP was calculated with a thermo-
balance (Ohaus MB 23, USA) identifying the transition point between
the periods of constant speed of drying and decreasing the speed of
drying. The drying kinetics was performed at 60 °C with 1 g of the
sample impregnated with saturated water (IAA result). The aw was
evaluated using an AquaLab (Decagon Devices, USA) to 25 ± 2 °C. The
C. Torres-León, et al.
pH was measured using a pH meter (Thermo Orion 420, USA), briefly,
1 g of sample was homogenized with twice its mass in distilled water in
a thermos bath at 30 °C (10min), pH was measured by direct immersion
of the electrode.
The Invasion capacity (fungal growth) was evaluated in Petri dishes,
where combinations of MKS and SMET (100%, 75%, 50%, 25%, and
0%) were moisturized (according to CHP) with the Czapek-Dox medium
[g/l: NaNO3 (7.65); KH2PO4 (3.04); MgSO4 (1.52); KCl (1.52)]. A. niger
GH1 was reactivated on PDA agar for 7 days at 30 °C. Subsequently,
explants (8 mm) were inoculated in the center of the plates, radial
growth was monitored kinetically (12 h). All these assays were in tri-
plicate and the invasion capacity was expressed as the growth rate in
mm/h.
2.5. Solid state fermentation (SSF)
Reactors of Petri dishes containing 10.42 g of sample and 11.58 mL
of Czapek Dox mineral medium inoculated with 2 × 107 spores per
gram of support were incubated at 30 °C. Samples were monitored ki-
netically for 60 h. Controls without inoculum were monitored under the
same fermentation conditions.
2.6. Extraction and separation of phenolic fraction
2.6.1. Free phenolic fraction
The sample (1 g) was extracted with 90% ethanol at a ratio of 1:30
(w/v). The mixture was kept in a 45 °C water bath for 1 h (in containers
protected from light). After, the extracts were filtered (11 μm). Finally,
the extracts were stored at −20 °C for subsequent analyses. The sedi-
ment from this extraction was saved for the determination of bound
phenols.
2.6.2. Bound phenolic fraction
The bound phenolic was extracted from the above residue according
to the methods described by Bei, Liu, Wang, Chen, and Wu (2017) with
some modifications. The residue was first digested with 50 mL of 2 M
sodium hydroxide at room temperature for 4 h. The mixture was then
acidified with concentrated hydrochloric acid to pH 2.0. Hexane was
used to extract lipids in the mixture. The remaining mixture was then
extracted three times with 70 mL of ethyl acetate via a liquid-liquid
partition. The ethyl acetate fractions were pooled and evaporated to
dryness. The bound phenolics were redissolved in 5 mL of 90% ethanol
(v/v). All phenolic extracts were stored at −20 °C before analysis.
2.7. Qualitative and quantitative analysis of phenolic extracts
2.7.1. Determination of total phenolic content (TPC)
Total phenolic content (TPC) was determined using Folin-
Ciocalteu's reagent and expressed as gallic acid equivalents (GAE) ac-
cording to the methods described by Makkar H. (2003), modified pro-
cedure to the microplate technique by Wong, Muñiz, Aguilar,
Rodríguez, and Aguilar (2014). Briefly, 20 μL of extract was mixed with
20 μL of Folin-Ciocalteu reagent in a well. After 5 min, 20 μL of sodium
carbonate (0.01 M) was added to each sample and allowed to stand for
5 min. Then, the solution was diluted with 125 μL of distilled water and
absorbance was read (790 nm) using a spectrophotometer microplate
reader (Epoch, BioTek Instruments, Inc.; Winooski, VT, USA) controlled
with the Gen5 Data Analysis software interface. All samples were in-
dependently analyzed in triplicate and results were expressed as mg
GAE/100 g DW.
2.8. Analytical RP-HPLC-ESI-MS
The phytochemicals were analyzed in mango seed extracts by RP-
HPLC-ESI-MS according to Torres-León, Rojas et al. (2017). Varian
3
LWT - Food Science and Technology 112 (2019) 108236
HPLC system including an autosampler (Varian Pro Star 410, USA), a
ternary pump (Varian Pro Star 230I, USA) and a PDA detector (Varian
Pro Star 330, USA). Briefly, 1.5 mL of extracts were filtered through a
0.45 μm membrane filter before injection in the HPLC (C18 column:
150 mm × 2.1 mm, 3 μm, Grace, USA). The eluents were as follows:
formic acid (0.2%, v/v; A solvent) and acetonitrile (B solvent). LC-ESI-
MS analysis was performed using a mass spectrometer (Varian 500-MS
IT Mass Spectrometer, USA) equipped with an electro-spray ionization
source (ESI) and operated in negative ion mode [M-H]−1. Nitrogen was
used as nebulizing gas and helium as damping gas Data were processed
using MS Workstation software (V 6.9).
2.9. Antioxidant activity assay
2.9.1. DPPH radical scavenging activity
The DPPH radical scavenging activity of the sample was analyzed
using the method reported by Molyneux (2004) and modified in our
laboratory. In brief, 7 μL of the phenolic extract was mixed with 193 μL
of freshly prepared 60 μM DPPH radical solution. The reaction mixtures
were incubated in darkness at room temperature for 30 min. After,
absorbance was measured at 517 nm using a spectrophotometer mi-
croplate reader (Epoch, BioTek, Instruments, Inc.) controlled with the
Gen5 Data Analysis software interface. In the characterization analyzes,
the free-radical-scavenging activity of extracts was expressed as a per-
centage of reduced DPPH and was calculated according to the equation:
DPPH inhibition (%) =
(Ac As)
(100)
Ac (3)
where Ac is the control absorbance and As is the sample absorbance.
In the fermentation kinetics, the results were expressed as mg of
Trolox equivalents (TE) per gram of extract (dw), using a standard
calibration curve of Trolox (r2 = 0.999). In the evaluation of the phe-
nolic fractions, the IC50 value was calculated as the concentration of the
compounds that caused a 50% inhibition of the DPPH radical (Equation
(3)). The IC50 values were obtained by interpolation or extrapolation
from the linear regression analysis.
2.9.2. ABTS radical cation scavenging activity
The ABTS radical cation (ABTS⁺) scavenging activity of the sample
was measured using the method reported by Re et al. (1999). Briefly,
ABTS⁺ was generated by oxidation of 7 mM ABTS with 2.45 mM po-
tassium persulfate after incubation in the dark (16 h). The freshly pre-
pared ABTS⁺ solution was diluted to obtain an absorbance of
0.70 ± 0.02 at 734 nm in a spectrophotometer (Genesys 20, USA).
1 mL of the ethanolic solution of ABTS⁺ was mixed with 10 μL of sample
in a quartz cell, and the absorbance was then recorded at 734 nm in a
spectrophotometer. In the evaluation of the phenolic fractions, the
analysis was carried out in a similar way to the radical DPPH.
2.10. Statistical analysis
The data presented in this paper were the average of three in-
dependent assays and were expressed as the mean ± standard devia-
tion (SD). The results were analyzed by analysis of variance (ANOVA).
Duncan's multiple range test or independent sample T-tets were used to
analyze the significant difference. Differences were considered to be
statistically significant when P values were less than 0.05. A tow-tailed
Pearson's correlation was performed to evaluate the correlations among
variables. Statistical analysis was performed using the Statistica 7.0
software ® (Statsoft, Tulsa Ok, USA).
C. Torres-León, et al. LWT - Food Science and Technology 112 (2019) 108236

Table 1 of the research.

The chemical composition of MSK and MSET.
Componenta MSK MSET 3.2. Tests of support in solid state fermentation

Moisture (Fresch) 40.01 ± 0.010 66.06 ± 6.672 Agro-industrial byproducts and wastes are dehydrated to guarantee
Moisture (Dry) 2.30 ± 0.984 4.76 ± 0.832
physicochemical and microbiological stability. The properties of de-
Dry matter 60.05 ± 0.015 33.90 ± 3.561
Ash 5.71 ± 0.444 1.72 ± 0.324 hydrated materials must be evaluated before the SSF process (partial
Protein 7.25 ± 0.191 3.96 ± 0.120 rehydration). The WAI is the quantity of water that can be absorbed by
Lipid 5.38 ± 0.071 0.77 ± 0.010 the support and CHP represents the water linked to the support, which
Carbohydrates 81.89 ± 0.080 93.55 ± 0.075
cannot be used by the microorganisms for their metabolic reactions.
Crude fiber 22.05 ± 0.582 52.59 ± 0.347
Hemicellulose 14.13 ± 0.021 9.60 ± 0.031
Results of the evaluation de WAI and CPH using MSK and MSET are
Cellulose 2.88 ± 0.389 22.42 ± 0.020 given in Table 2. The WAI values increased proportionally to the MSET
Lignin 2.03 ± 0.474 9.77 ± 0.50 content, the maximum value was achieved with 100% of MSET, this
Reducing sugarsb 14.81 ± 0.064 15.22 ± 0.364 phenomenon may be associated with high dietary fiber content and the
Antioxidant activityc 93.1 ± 2.241 59.23 ± 2.016
low lipid content calculated in the material (Table 1). Materials with
a
Results reported in % on a dry basis. high WAI levels are preferred since their moisture content can be
b
Mg Glucose/g. modified during solid-state culturing (Robledo et al., 2008). Similar
c
DPPH˙ radical scavenging activity (%). MSK: mango seed kernel, MSET: WAI values have been reported in biomaterials used to obtain bioactive
mango seed endocarp and testa. compounds by SSF (Orzua et al., 2009).
In SSF it is advisable to obtain low CHP values since this facilitates
3. Results and discussion the microbial culture (Mussatto, Aguilar, Rodrigues, & Teixeiraa, 2009),
a high CHP value represents a lower amount of water bound in the
3.1. The chemical composition of MSK and MSET material. The lowest CHP values were reached in high concentrations of
MSK. The CHP values calculated for MSK and MSET and their combi-
Chemical characterization and antioxidant content of MSK and nations are within the ranges reported for other agro-industrial residues
MSET are shown in Table 1. The ash content was higher than the range as pecan nutshell (50%), pistachio shell (48%), wheat bran (66%),
previously reported in mango kernels (1.46–3.2%) (Ashoush & apple pomace (35%) and bean residues (45%) (Orzua et al., 2009).
Gadallah, 2011; Elegbede, Achoba, & Richard, 1995; Zein, El-Bagoury, The optimum pH for the growth of A. niger is from 5.0 to 30 °C
& Kassab, 2005). The protein content is in accordance with the values (Aguilar et al., 2007), the results show that the increase of MSK in-
reported for mango kernels (6–7.76%) (Ashoush & Gadallah, 2011; creases the pH values, up to values close to the optimum pH
Odunsi, 2005). The lipid content was lower than that reported in other (4.8–30 °C). The results of the growth rate showed that the micro-
mango varieties (8.15–13.6%) (Ashoush & Gadallah, 2011; Odunsi, organism can grow in the evaluated support combinations. However,
2005). Differences in chemical composition can be attributed to factors the growth rate was higher in high MSK concentrations. The speed of
such as genetic characteristics, environment, stage of maturity, and growth was higher than that previously reported for A. niger GH1 in
agricultural practices (Torres-León et al., 2016). agro-industrial waste as pomegranate husk (0.4 mm/h), pomegranate
MSK presented higher levels of dry matter, ash, protein, and lipids seed (0.2 mm/h) (Robledo et al., 2008) and grape seeds (1.1 mm/h)
than MSET. The moisture content of the fresh material, carbohydrates, (Martínez et al., 2012). The good growth results can be attributed to the
and crude fiber were higher in MSET, the analysis of the components of enzymes excreted by A. niger that allow an excellent assimilation of the
the fiber showed that MSK has a higher content of hemicellulose and waste. The aw is the quantity of water available for the metabolic
MSET a higher content of cellulose and lignin. As previously was re- functions of microorganism on the support. The optimal value of aw for
ported by Mukherjee, Adhikari, and Rai (2008), and Robledo et al. A. niger is 0.96 (Robledo et al., 2008), the best value of aw was achieved
(2008), biomaterials with proximal contents similar to those found in with a combination MSK (75%) and MSET (25%). This value of aw is
the MSK and MSET are suitable to be used as support in solid fermen- suitable for optimal growth and is related to the high values of growth
tation. The reducing sugars were similar in the two evaluated bioma- velocity obtained. The results of humidity and antioxidant activity
terials, the reducing sugars are very important in the refining since they (97.1 ± 0.16) corroborate that the combination MSK (75%) and MSET
provide the carbon source needed for fungal growth and enzyme pro- (25%) is suitable as a substrate in SSF. In general, the characterization
duction (Buenrostro et al., 2017). Finally, DPPH˙ radical scavenging showed that mango byproducts are suitable as a substrate support for
activity (%) was higher in MSK, this parameter is very important since SSF. However, the combination of 75% MSK and 25% MSET is the best
the obtaining of extracts with high biological activity was the objective combination of materials, since it achieves a high growth rate and a
high antioxidant activity. According to the results obtained, the

Table 2
Characterization of MSK and MSET as support for SSF process.
Analsys MSK 100% 75% 50% 25% 0%

MSET 0% 25% 50% 75% 100%

WAI (g/g) 3.4 ± 0.09 3.5 ± 0.04 3.8 ± 0.05 3.9 ± 0.21 4.0 ± 0.07
CHP (%) 46.2 ± 2.47 46.5 ± 2.94 56.7 ± 0.04 55.8 ± 0.81 56.5 ± 5.02
pH 4.8 ± 0.04 4.4 ± 0.01 4.3 ± 0.01 4.1 ± 0.02 3.7 ± 0.01
Growth rate (mm/h) 1.4 ± 0.03 1.4 ± 0.07 1.4 ± 0.02 1.3 ± 0.13 1.3 ± 0.01
awa 0.98 ± 0.01 0.96 ± 0.01 0.98 ± 0.01 0.97 ± 0.01 0.97 ± 0.01
Humiditya (%) 47.3 ± 6.58 53.6 ± 1.63 55.8 ± 1.13 60.2 ± 1.27 68.2 ± 0.57
Antioxidant activitya,b (%) 95.4 ± 0.71 97.1 ± 0.16 85.8 ± 2.01 35.6 ± 9.20 27.1 ± 4.07

WAI: Water Absorption Index, CHP: Critical Humidity Point.

a
Measurement in the final time of radial growth.
b
DPPH˙ radical scavenging activity.

4
C. Torres-León, et al.
Fig. 1. Polyphenolic content and antioxidant activity during fermentation of
mango seed.
combination 75% MSK and 25% MSET will be used in the fermentation
kinetics. To facilitate the nomenclature in the text, the combination
75% MSK and 25% MSET will be identified as mango seed.
3.3. Solid state fermentation (SSF)
Mango seeds are an interesting source of polyphenols which are in
part responsible for their antioxidant activity (Torres-León et al., 2016).
The polyphenol content in mango seed and fermented mango seed was
determined. SSF of mango seed performed with A. niger GH1 led to a
significant (P ≤ 0.05) rise in TPC from 984 ± 32 mg GAE/100 g to
3288 ± 290 mg GAE/100 g at 20 h of fermentation (Fig. 1). These re-
sults were superior to those reported in mango seed extracted by con-
ventional methods (2190 mg GAE/100 g) (Abdel, Ashoush, & Nessrien,
2012). The results are consistent with the phenolic content reported in
lentil fermented by SSF with Bacillus subtilis (3400 mg GAE/100 g)
(Torino et al., 2013), and were superior to those reported in soybean
fermented by SSF with Bacillus strains (2266 mg GAE/100 g) (Juan &
Chou, 2010). Moreover, the polyphenolic contents were highly corre-
lated with DPPH scavenging activity (r = 0.97, p < 0.01), which
confirms that the increase in antioxidant activity is due to the increase
in phenolic content during fermentation. The increase in polyphenols is
attributed to the fact that fermentation induces the structural break-
down of plant cell walls, leading to the liberation or synthesis of various
antioxidant compounds (Jin, Yuan, Kim, Choi, & Kim, 2014). Pre-
viously, our research group led by co-author Dr. C. N. Aguilar has
identified that solid fermentation with A. niger GH1 is an adequate
process for improving the concentration of phenolic compounds.
In the present study, our results revealed the important effect of
fermentation time on the content of polyphenols and on the anti-
oxidative activity of the mango seed. As shown in Fig. 1, there is an
increase in the polyphenol content and antioxidant activity with a
maximum increase of 20 h of fermentation. However, after the twen-
tieth hour of fermentation, there is a reduction in the content of poly-
phenols and antioxidant activity. Which may be attributed to further
degradation of compounds by enzymes released from A. niger, the hy-
drolysis of phenolic compounds can lead to the loss of antioxidant ac-
tivity (Othman, Roblain, Chammen, Thonart, & Hamdi, 2009). Similar
results in the decrement after a fermentation time with A. niger have
been previously reported in fermented by-products (Zhang et al., 2017),
the authors suggest controlling the fermentation time so that an ex-
cessive fermentation does not reduce the biological activity.
3.4. Extraction and separation of phenolic fraction
Plants contain a high level of polyphenols. However, those compounds
are predominantly bound or conjugated form (covalently bound to cell wall
structural) as combined to cellulose, hemicellulose, lignin, pectin and rod-
shaped structural proteins (Acosta-estrada et al., 2014). These conjugations
inhibit the antioxidative activity of phenolic compounds by decreasing their
available hydroxyl groups (Ajila, Brar, Verma, Tyagi, & Valéro, 2011). As
5
LWT - Food Science and Technology 112 (2019) 108236
Fig. 2. Phenolic fractions in fermented and unfermented mango seed.
presented in section 3.3. Fermentation process could convert bound phe-
nolic compounds into a free form which increases TPC (Fig. 1), this bio-
conversion process also contributes to increasing the antioxidant activity.
The effect of fermentation on the release of bound polyphenols in
mango seed was investigated. As shown in Fig. 2, free phenolics in
unfermented mango seed were 713.6 ± 70 mg GAE/100 g of mango
seed, which increased dramatically to 2477.5 ± 140 mg GAE/100 g in
fermented mango seed. The content of bound phenols showed an in-
verse tendency, this was higher in the unfermented seed and decreased
with fermentation. The release of bound polyphenol compounds is due
to the action of enzymes released by the microorganism during fer-
mentation. Previously, Ascacio-Valdés et al. (2014), have reported that
A. niger GH1 has a high cellulase activity, xylanase, b-glucosydase,
polyphenoloxidase, tannase, enzymes that have good results in the
degradation of the cell wall (Jamal et al., 2011) and therefore, good
results in the release of polyphenol compounds (Fernández, Vega, &
Aspé, 2015). Similar results in the increase of free phenolic content
have been previously reported in biological substrates fermented by
SSF: Korean black raspberry (Kyoung et al., 2009), pancake (Shumoy,
Gabaza, Vandevelde, & Raes, 2017) and oat (Bei et al., 2017). Torino
et al. (2013) and Juan and Chou (2010) also reported that the bio-
conversion of the conjugated forms of phenolic compounds into their
free forms during fermentation improves their functionality.
3.5. Analytical RP-HPLC-ESI-MS
High-performance liquid chromatography (HPLC) fingerprint analysis
is widely used for scanning total compounds. Fig. 3A and 3B illustrate the
changes in the phenolic compounds present in the mango seed during
fermentation with A. niger GH1. At time 0 of fermentation, the major
compound appears in the retention time 30.4 min with a [M − H]− ion at
m/938.9 (peak 8) and was identified as penta-galloylglucose (PGG)
(Fig. 3A), this compound has been identified as the majority compound in
Ataúlfo mango seed (Torres-León, Rojas et al, 2017). Peaks 10 and
11 at m/z 1090.9 and 1242.8 were identified as hexa-galloylglucose and
hepta-galloylglucose, respectively. These results show that mango seed is a
good source of gallotannins with high molecular weight.
During the course of fermentation, the complex galotannins were
hydrolyzed to lower molecular but polyphenols following the hydro-
lyzable tannins route: hepta-galloylglucose (Peak 11); hexa-galloylglu-
cose (Peak 10); PGG (Peak 8); tetra-galloylglucose (Peak 6); tri-gal-
loylglucose (Peak 5); di-galloylglucose (Peak 3), galloylglucose (Peak 1)
and gallic acid (Peak 2), these last two compounds as well as ellagic
acid (Peak 7) and PGG were those that were found in greater proportion
after 20 h of fermentation (Fig. 3B). Complex polyphenols are hydro-
lyzed to other simpler and biologically more active compounds during
fermentation. This process of bioconversion may be due to the effect of
C. Torres-León, et al. LWT - Food Science and Technology 112 (2019) 108236

Fig. 3. HPLC-MS chromatograms of the

fermentation of the mango seed with A.
niger GH1 (A) at fermentation time 0 h and
(B) 20 h, and (C) bound phenolic fraction of
unfermented (BPFU), and (D) bound phe-
nolic fraction of fermented mango seed
(BPF); 1, galloylglucose; 2, gallic acid; 3, di-
galloylglucose; 4, digallic acid; 5, tri-gal-
loylglucose; 6, tetra-galloylglucose; 7, el-
lagic acid; 8, penta-O-galloyl-glucoside
(PGG); 9, hexa-galloylglucose; 10, rham-
netin-3-[600-2-butenoil-hexoside]; 11,
hepta-galloylglucose.

Fig. 4. ABTS⁺ radical caption scavenging prop-

erty (A) and DPPH radical caption scavenging
property (B) of phenolic fractions extract from
unfermented and fermented mango seed. FPFU:
free phenolic fraction in unfermented seed,
BPFU: bound phenolic fraction in unfermented
seed, FPF: free phenolic fraction in fermented
seed, BPF: bound phenolic fraction in fermented
seed. Values are expressed as the mean ± SD
(n = 3).

enzymes tannin acyl hydrolase that produces the fungus A. niger GH1 in (Torres-León, Rojas et al, 2017).
the presence of gallotannins (Kyoung et al., 2009). Peak 9 was identi- Fig. 3C and 3D shows the base peak chromatograms with the
fied as rhamnetin-3-[600-2-butenoil-hexoside] showing a [M − H]− identification of bound phenolic compounds in mango seed un-
ion at m/z 545, this compound was previously reported in mango seed fermented and fermented, respectively. Peaks 2 y 7 at m/z 169.0 and

6
C. Torres-León, et al.
Table 3
IC50 values (mg/mL) of phenolic fractions from unfermented and fermented mango seed.
Antioxidant method Trolox BHT
DPPH scavenging effect 0.06 ± 0.000a 0.70 ± 0.006e
ABTS+ scavenging effect 0.32 ± 0.008a 0.56 ± 0.022b
Values were presented as the mean ± SD (n = 3). Means with different small letters within a row were significantly different (p < 0.05). *** Significantly different
from unfermented sample (p < 0.05).s.
300.0 were identified as gallic acid and ellagic acid, respectively, these
compounds have recently been reported in polyphenolic fractions
bound in mango seeds from Spain (Gómez, Lopez, Verardo, Segura, &
Fernández, 2016). This work is one of the first studies where the phe-
nolic fractions bound in the mango seed are evaluated. Peak 4, at
19.58 min and m/z 321 was identified as digallic acid, this compound
has not been reported in bound fractions of mango seed. A remarkable
reduction in the area units of the bound phenolic compounds was ob-
served after fermentation, this phenomenon can be interpreted as an
effective release of bound phenolic compounds and is related to the
increase of phenolic compounds reported in Fig. 2. About it, Kyoung
et al. (2009), reported that the increase in the content of polyphenols
after fermentation could be due to the enzymatic hydrolysis or the
biodegradation of the ester-type phenolic acids bound.
3.6. Antioxidant activity assay
ABTS radical cation scavenging activity and DPPH radical scaven-
ging activity were used to investigate the antioxidant capacities of the
phenolic fraction extracts. The most common parameter for measuring
the antioxidant capacity of a substance is the concentration of anti-
oxidant required to reduce the initial concentration of radical (ABTS⁺
and DPPH) by 50% (IC50) (Bei et al., 2017); in this analysis, the lower
the IC50 value is, the higher is the antioxidant power. Fig. 4A and 4B
shows the dose-response curves on the antioxidant activity of BHT,
Trolox and the two phenolic fraction extracts from unfermented (FPFU:
free phenolic fraction in unfermented seed and BPFU: bound phenolic
fraction in unfermented seed) and fermented mango seed (FPF: free
phenolic fraction in fermented seed, BPF: bound phenolic fraction in
fermented seed) on the ABTS⁺ and DPPH radical scavenging activity.
Table 3 summarizes de IC50 values of each phenolic fractions. As the
result of fermentation, the IC50 values for the free phenolic fractions
significantly decreased in the analysis ABTS⁺ and DPPH from
0.34 ± 0.004 mg/mL (FPFU) to 0.29 ± 0.009 mg/mL (FPF) and
0.16 ± 0.001 mg/mL (FPFU) to 0.12 ± 0.001 mg/mL (FPF), respec-
tively; which expresses a significant increase in antioxidant activity.
The opposite effect was also observed in the bound fractions, the fer-
mentation process increased the IC50 values in the bound fractions in
the analysis ABTS⁺ and DPPH from 1.10 ± 0.069 mg/mL (BPFU) to
3.12 ± 0.255 mg/mL (BPF) and 0.46 ± 0.01 mg/mL (BPFU) to
2.76 ± 0.03 mg/mL (BPF), respectively. Therefore, a large reduction
in the antioxidant activity of the fractions bound after fermentation was
obtained, this behavior shows that the antioxidant activity is associated
with the bound phenolic compounds that are released and that pass to
the free fraction during fermentation. The highest radical-scavenging
activity was observed in the free fraction, from the fermented sample,
and the lowest activity was observed in the bound fraction, from the
fermented sample. The fermentation process increases the radical-
scavenging activity of the extracts, this increase in biological activity
was proportional to the increase in the phenolic content of the free
fraction, suggesting that phenolic compounds appear to contribute
antioxidant activity. The increase in radical-scavenging activity (ex-
pressed in the decrease of IC50) of free fractions as a result of fermen-
tation has been previously reported in the scientific literature (Bei et al.,
2017; Kyoung et al., 2009; Zhang et al., 2017).
LWT - Food Science and Technology 112 (2019) 108236
Free phenolic fraction Bound phenolic fraction
Unfermented Fermented Unfermented Fermented
0.16 ± 0.001c 0.12 ± 0.001b*** 0.46 ± 0.010d 2.76 ± 0.03f***
0.34 ± 0.004a 0.29 ± 0.009a*** 1.10 ± 0.069c 3.12 ± 0.255d***
The free phenolic fraction of the fermented seed exhibited a higher
ABTS⁺ scavenging activity (0.29 ± 0.009 mg/mL) than Trolox
(0.32 ± 0.008 mg/mL) and BHT (0.56 ± 0.022 mg/mL), also the DPPH
analysis showed a higher antioxidant activity than the BHT positive con-
trol (0.70 ± 0.007 mg/mL). Trolox is a commercial antioxidant that has
biochemical applications to reduce oxidative stress, it is widely used as a
standard in antioxidant analysis and BHT is a synthetic antioxidant used in
the food industry. The high antioxidant activity determined suggests that
the extracts have a wide use potential in the food, pharmaceutical, and
biotechnology industry. Health benefits such as anti-carcinogenic and anti-
inflammatory effects of bioactive compounds are based on their anti-
oxidative capacity (Torres-león, Ventura-Sobrevilla et al., 2017).
4. Conclusion
SSF with A. niger GH1 is an effective method that significantly enhances
the contents of polyphenolics in Ataulfo mango seed, at 20 h of fermenta-
tion. The fermentation process released the phenolic compounds that were
bound to the vegetable matrix, which is reflected in an increase in the
content of free phenols. The free phenolic fraction of the fermented mango
seed presented a higher antioxidant activity than the unfermented mango
seed. It is due to the high content of polyphenolic compounds mainly gal-
lotannins. The extracts of fermented mango seeds are particularly promising
as functional ingredients for the pharmaceutical and food industry.
Acknowledgments
C. Torres-León thanks the Mexican Council for Science and
Technology (CONACYT) for his post-graduate scholarship (No.
291137).. Authors are thankful to Ph.D. Janeth Ventura-Sobrevilla for
their valuable suggestions during this study.
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