molecules
Article
Valorization of Citrus Reticulata Peels for Flavonoids and
Antioxidant Enhancement by Solid-State Fermentation Using
Aspergillus niger CGMCC 3.6189
Daniel Mamy 1,2,3,4, Yuanyuan Huang 1,2,3, Nelson Dzidzorgbe Kwaku Akpabli-Tsigbe 1,5, Maurizio Battino 3,6
and Xiumin Chen 1,2,3,*
1 School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Jingkou District,
Zhenjiang 212013, China
2 Institute of Food Physical Processing, Jiangsu University, 301 Xuefu Road, Jingkou District,
Zhenjiang 212013, China
3 International Joint Research Laboratory of Intelligent Agriculture and Agri-Products Processing,
Jiangsu University, Zhenjiang 212013, China
4 Higher Institute of Sciences and Veterinary Medicine (ISSMV) of Dalaba, Dalaba-Tangama P.O. Box 09, Guinea
5 Department of Nutrition and Food Science, College of Basic and Applied Sciences, University of Ghana,
Legon P.O. Box LG 134, Ghana
6 Department of Clinical Sciences, Università Politecnica delle Marche, 60100 Ancona, Italy
* Correspondence: xmchen@ujs.edu.cn
Abstract: The bioactive components and bioactivities of citrus peel can be enhanced with microbial
fermentation. Accordingly, this study investigated the ability of Aspergillus niger CGMCC3.6189 to
accumulate flavonoids in Citrus reticulata peel powder (CRPP) by solid-state fermentation (SSF).
Under the optimal SSF conditions including 80% moisture, 30 ◦C, pH 4.0, 4 × 107 spores/g d.w.
CRPP, and 192 h, the total phenolic content (TPC), total flavonoid content (TFC), and 2,2′-azinobis-
Citation: Mamy, D.; Huang, Y.;
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH)
Akpabli-Tsigbe, N.D.K.; Battino, M.;
Chen, X. Valorization of Citrus scavenging activities of fermented CRPP significantly increased by 70.0, 26.8, 64.9, and 71.6%, re-
Reticulata Peels for Flavonoids and spectively. HPLC analysis showed that after fermentation, the contents of hesperidin, nobiletin, and
Antioxidant Enhancement by tangeretin were significantly increased from 19.36, 6.31, and 2.91 mg/g to 28.23, 7.78, and 3.49 mg/g,
Solid-State Fermentation Using respectively, while the contents of ferulic acid and narirutin were decreased under the optimal fermen-
Aspergillus niger CGMCC 3.6189. tation conditions. Fermentation time is one of the most important factors that affect the accumulation
Molecules 2022, 27, 8949. https:// of flavonoids and antioxidant activity; however, extended fermentation time increased the darkness
doi.org/10.3390/molecules27248949 of CRPP color. Therefore, our study provides a feasible and effective SSF method to increase the
Academic Editor: Francesco Cacciola bioactive components and the antioxidant activity of CRPP that may be used in food, nutraceutical,
and medicinal industries.
Received: 19 November 2022
Accepted: 12 December 2022 Keywords: citrus peels; solid-state fermentation; polymethoxylflavones; nobiletin; tangeretin;
Published: 15 December 2022
antioxidant properties
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Citrus is a group of the most abundant fruits in terms of production and growing
area. China is the largest tangerine (Citrus reticulate) producer worldwide, with an annual
Copyright: © 2022 by the authors. production of 23.12 million tonnes (MT) in 2020, accounting for about 60% of global
Licensee MDPI, Basel, Switzerland. production [1]. This production results in a significant accumulation of waste, which
This article is an open access article creates a major management challenge in developing countries [2].
distributed under the terms and The dried peel of the citrus fruit (Pericarpium citri reticulatae, PCR) known as Chenpi
conditions of the Creative Commons exhibits various beneficial functional properties such as anti-inflammatory, anti-diabetic,
Attribution (CC BY) license (https:// anticancer, anti-microbial, anti-viral, antioxidative, antimutagenic, and anti-glycemic ac-
creativecommons.org/licenses/by/ tivities due to their richness in polyphenols, carotenoids, vitamins, and fiber [3,4]. Over
4.0/). eighty flavonoids classified into flavones, flavonols, flavanones, polymethoxylflavones,
Molecules 2022, 27, 8949. https://doi.org/10.3390/molecules27248949 https://www.mdpi.com/journal/molecules
Molecules 2022, 27, 8949 2 of 15
and anthocyanins are found in citrus fruits, among which naringin, hesperidin, narirutin,
neo-hesperidin, nobiletin, tangeritin, hesperetin, and naringenin are most abundant in
citrus peels [5,6]. It is generally known that “the longer time Chenpi is stored, the better
health benefits it has”. Aging increases the accumulation of phenolics and flavonoids in
Chenpi [7], therefore promoting the health benefits of aged citrus peels. However, it is
not necessarily true that the longer the storage time, the higher the flavonoid composi-
tions. The PCR metabolite levels increased within 3–15 years of storage, while showing a
decrease trend to a stable state after storing for 15–30 years [8]. Yang et al. [9] found that
the antioxidant activity of Chenpi reached a maximum in the 5-year-old Chenpi and then
decreased with the extended storage time. Therefore, storage time is critical for preparing
high-quality Chenpi.
The natural aging process of Chenpi generally takes place in moisture-controlled conditions
for many years, during which flavonoids accumulate due to microbial biotransformation. The
genera of microbes identified in Chenpi include Penicillium citrinum, Penicillium milmonillium,
Penicillium common, Aspergillus flavus, Aspergillus niger, Penicillium minioluteum [7], Bacillus,
Lactococcus, Pseudomonas, Oceanobacillus, Pseudarthrobacter, Enterococcus, and Psychrobacter [10],
among which Bacillus and Lactococcus are the two main genera [10]. It is believed that
the microbes significantly improve the chemical quality of Chenpi. Currently, microbial
processes are being developed to biotransform steroids and flavonoids for direct use or
as precursors for new drugs and other beneficial compounds [11]. Various microbes have
been used to accelerate the biotransformation of flavonoids, among which A. niger is
one of the most used microorganisms [12]. For example, flavone was hydroxylated to
4’-hydroxyflavone and subsequently to 3’,4’-dihydroxyflavone by A. niger ATCC 43949.
A. niger NRRL 2295 and A. niger X172 also hydroxylated flavone to 4’-hydroxyflavone [11].
The inoculation of A. niger isolated from Citrus reticulata peel (Chenpi) using solid-
state fermentation (SSF) increased the total flavonoid content (TFC) and the flavonoid
aglycones such as hesperetin and naringenin, while the corresponding flavanone glyco-
sides (hesperidin and narirutin) were decreased in a much shorter period compared with
the natural aging process [7]. SSF is an effective, environmentally friendly, cost-effective,
and feasible approach that has been used to increase the concentration of bioactive com-
pounds and antioxidant activity in agro-industrial wastes and plant by-products [13]. It is
an effective technique to increase the concentration of phenols and flavonoids [14]. Con-
sequently, it could be an asset for the accumulation of flavonoids in Citrus reticulata peel
by A. niger strains. The biotransformation of flavonoids using A. niger has been widely
reported [12]; however, studies associated with the changes in the phytochemical profile,
color, and antioxidant activity of the citrus peels under the SSF by A. niger are scarce.
Accordingly, this study aimed to evaluate the potential of increasing flavonoid compounds
and antioxidant activity in Citrus reticulata peel by A. niger CGMCC 3.6189 under different
SSF conditions including pH, temperature, moisture content, inoculation concentration,
and fermentation time. In addition, the changes in the color of citrus peel were also assessed.
Our study provides valuable information for the valorization of citrus peel waste.
2. Results
2.1. Effect of pH on TPC, TFC, Antioxidant Activity, and Phytochemical Compositions in CRPP
Table 1 shows the effects of the inoculum pH on the TPC, TFC, and ABTS and DPPH
scavenging capacities of CRPP before and after SSF. The TPC, TFC, and ABTS and DPPH of
the unfermented CRPP were 10.77 ± 0.27 mg GAE/g, 4.78 ± 0.07 mg QE/g, 22.19 ± 0.97,
and 13.35 ± 0.71 µmol TE/g, respectively. As pH increased from 4.0 to 6.5, the TPC, TFC,
and ABTS scavenging capacity showed a decreasing trend, however, DPPH scavenging
capacity showed no significant change. At pH 4.0, the TPC and ABTS scavenging capacity
of the fermented CRPP significantly (p < 0.05) increased to 11.97 ± 0.26 mg GAE/g and
25.43 ± 1.89 µmol TE/g, respectively, while TFC and DPPH scavenging capacity slightly
(p > 0.05) increased to 4.83 ± 0.11 mg QE/g and 14.05 ± 1.12 µmol TE/g, respectively.
Molecules 2022, 27, 8949 3 of 15
Table 1. Effects of solid-state fermentation conditions on the TPC, TFC, and ABTS and DPPH
scavenging capacity of Citrus reticulata peel.
Sample Name Variables TPC (mg TFC (mg ABTS (µmol DPPH (µmolGAE/g) QE/g) TE/g) TE/g)
pH 1
Control - 10.77 ± 0.27 b 4.78 ± 0.07 a 22.19 ± 0.97 abc 13.35 ± 0.71 a
FC1a 4.0 11.97 ± 0.26 a 4.83 ± 0.11 a 25.43 ± 1.89 a 14.05 ± 1.12 a
FC1b 4.5 10.57 ± 0.18 b 4.28 ± 0.15 b 25.07 ± 1.09 ab 14.65 ± 0.49 a
FC1c 5.0 10.40 ± 0.08 b 4.06 ± 0.17 b 24.97 ± 1.99 ab 14.61 ± 0.82 a
FC1d 5.5 10.48 ± 0.09 b 4.07 ± 0.09 b 23.63 ± 0.20 ab 13.56 ± 0.46 a
FC1e 6.0 10.41 ± 0.30 b 3.67 ± 0.10 c 21.46 ± 1.91 bc 13.65 ± 0.90 a
FC1f 6.5 10.44 ± 0.36 b 3.51 ± 0.06 c 18.72 ± 0.17 c 13.61 ± 0.92 a
Incubation temperature (IT, ◦C) 2
Control - 10.77 ± 0.27 b 4.78 ± 0.07 a 22.19 ± 0.97 a 13.35 ± 0.71 a
FC2a 25 11.41 ± 0.35 ab 4.71 ± 0.18 a 24.56 ± 1.69 a 13.44 ± 0.69 a
FC2b 30 11.97 ± 0.26 a 4.83 ± 0.11 a 25.43 ± 1.89 a 14.05 ± 1.12 a
FC2c 35 12.05 ± 0.44 a 4.01 ± 0.14 b 22.49 ± 1.20 a 13.33 ± 1.44 a
Moisture content (MC,%, w.b.) 3
Control - 10.77 ± 0.27 c 4.78 ± 0.07 a 22.19 ± 0.97 b 13.35 ± 0.71 a
FC3a 70 11.97 ± 0.26 b 4.83 ± 0.11 a 25.43 ± 1.89 a 14.05 ± 1.12 a
FC3b 80 12.95 ± 0.59 a 4.90 ± 0.06 a 25.63 ± 0.47 a 15.00 ± 1.41 a
FC3c 90 13.43 ± 0.20 a 4.29 ± 0.10 b 24.73 ± 0.45 ab 14.08 ± 0.44 a
Spore concentration (SC, spores/g) 4
Control - 10.77 ± 0.27 b 4.78 ± 0.07 b 22.19 ± 0.97 c 13.35 ± 0.71 b
FC4a 4 × 106 12.95 ± 0.59 a 4.90 ± 0.06 ab 25.63 ± 0.47 b 15.00 ± 1.41 ab
FC4b 2 × 107 13.17 ± 0.40 a 5.04 ± 0.04 ab 26.00 ± 0.21 b 16.29 ± 1.83 ab
FC4c 4 × 107 13.47 ± 0.36 b 5.15 ± 0.23 a 27.53 ± 0.24 a 17.33 ± 1.40 a
Fermentation time (FT, h) 5
Control - 10.77 ± 0.27 d 4.78 ± 0.07 d 22.19 ± 0.97 c 13.35 ± 0.71 c
FC5a 60 13.47 ± 0.36 c 5.15 ± 0.23 c 27.53 ± 0.24 b 17.33 ± 1.40 bc
FC5b 96 13.77 ± 0.21 c 5.35 ± 0.20 bc 28.13 ± 1.36 b 17.36 ± 1.09 bc
FC5c 144 15.69 ± 0.33 b 5.69 ± 0.21 ab 29.37 ± 0.52 b 18.99 ± 1.55 ab
FC5d 192 18.31 ± 0.35 a 6.06 ± 0.11 a 36.60 ± 1.82 a 22.91 ± 3.45 a
Note: Means that do not share a letter are significantly different; data are expressed as mean ± SD (n = 3)
on a dry weight basis. 1 The SSF conditions are as follows: incubation temperature = 30 ◦C, moisture con-
tent = 40%, spore concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0, 4.5, 5.0, 5.5, and
6.0, respectively. 2 The SSF conditions are as follows: moisture content = 40%, spore concentration = 4 × 106
spores/g, fermentation time = 60 h, pH = 4.0, incubation temperature = 25, 30, and 35 ◦C, respectively. 3 The
SSF conditions are as follows: spore concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0,
incubation temperature = 30 ◦C, moisture content = 70, 80, and 90%, respectively. 4 The SSF conditions are as
follows: moisture content = 80%, fermentation time = 60 h, pH = 4.0, incubation temperature = 30 ◦C, spore
concentration = 4 × 106, 2 × 107, and 4 × 107 spores/g, respectively. 5 The SSF conditions are as follows: mois-
ture content = 80%, spore concentration = 4 × 107 spores/g, pH = 4.0, incubation temperature = 30, and 35 ◦C,
fermentation time = 60 h, 96, 144, and 192 respectively.
We further investigated the changes in the phytochemicals in CRPP fermented with
different pH. Figure 1 shows the HPLC chromatographs of the standard and CRPP sam-
ples and Table 2 presents the quantified contents in different samples. The results show
that chlorogenic acid, caffeic acid, p-coumaric acid, and naringenin (compounds 1–3, 7)
are not able to be quantified in CRPP, while the other six compounds including ferulic
acid, narirutin, hesperidin, hesperetin, nobiletin, and tangeretin (compounds 4–6, 8–10)
were identified and quantified. Hesperidin is the most abundant flavonoid in CRPP
with a concentration of 19.36 ± 0.47 mg/g, followed by nobiletin (6.31 ± 0.11 mg/g),
narirutin (4.97 ± 0.07 mg/g), and tangeretin (2.91 ± 0.04 mg/g). The contents of ferulic
acid and hesperetin are relatively low. After fermentation at pH 4.0, narirutin content
significantly (p < 0.05) increased to 5.53 ± 0.13 mg/g, while the other five compounds
remained unchanged. Further increasing the pH value to 6, the contents of all six com-
pounds either decreased or remained consistent. According to the results of TPC, TFC,
antioxidant activity, and phytochemical composition, pH 4 was selected for the following
fermentation experiments.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 17 
 
moisture content = 70%, spore concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0, 
incubation temperature = 25, 30, and 35 °C, respectively. 3 The SSF conditions are as follows: spore 
concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0, incubation temperature = 30 
°C, moisture content = 70, 80, and 90%, respectively. 4 The SSF conditions are as follows: moisture 
content = 80%, fermentation time = 60 h, pH = 4.0, incubation temperature = 30 °C, spore concen-
tration = 4 × 106, 2 × 107, and 4 × 107 spores/g, respectively. 5 The SSF conditions are as follows: 
Molecules 2022, 27, 8949 moisture content = 80%, spore concentration = 4 × 107 spores/g, pH = 4.0, incubation temperat4uroef =1 5
30, and 35 °C, fermentation time = 60 h, 96, 144, and 192, respectively. 
 
Figure 1. HPLC profile of Citrus reticulata peels powder extract at 330 nm. Note: HPLC chro-
Figure 1. HPLC profile of Citrus reticulata peels powder extract at 330 nm. Note: HPLC chromato-
matographs of mgirxaepdhs sotfa mnidxeadr dsta(nAda)rda n(Ad) athnde tehxe terxatrcatcto off Ciittrruus sretriectuilcautal apteael pfeeremlenfeterdm foern etiegdht fdoaryse (iBg)h. t
days (B). The numThbee nrusmobfecrso omf pcoomupnodunsdfsr ofrmom1 1 ttoo 1100 ccoorrrersepsopndo ntod thteo ttehsteedt epshteendolpich coemnoploiucncdos:m 1 cpholouron-ds:
1 ge2nic acid; 2 caffeic a3cid, 3 P-coumaric acid, 44 ferulic acid, 5 na5rirutin, 6 hespe6ridin, 7 chlorogenic acid; caffeic acid, P-coumaric acid, ferulic acid, narirutin, hespnearriidnginen, i7n,
8 
n arin-
hesperetin, 9 nobiletin, 10 tangeretin. 
genin, 8 hesperetin, 9 nobiletin, 10 tangeretin.
2.2. Effect of Fermentation Temperature on TPC, TFC, Antioxidant Activity, and Phytochemical 
Table 2. Effects oCfosmoploidsi-tsiotnast einf eCrRmPPen  tation conditions on the phenolic contents (mg/g) of Citrus
reticulata peels. Under pH 4.0, the CRPP was fermented at temperatures of 25, 30, and 35 °C, respec-
tively. The results show that ABTS and DPPH scavenging capacity did not change signif-
Samples VariabicleantlyF ewruhleinc  Athceid incubatNioanr itreumtipnerature inHcreesapseerdi dfirnom 25 toN 3o5b °iCle t(iTnable 1T).a Inngcerreeatsiinng 
the temperature from 25 to 35 °C caused a significant decrease in TFC from 4.71 ± 0.18 
1
µmol TE/g to 4.01a  ± 0.14 µmol TE
p/Hbg, while TPC signifiacantly increased from 11.41 ± 0.3Control - 0.46 ± 0.00 4.97 ± 0.07 19.36 ± 0.47 6.31 ± 0.11 a 2.91 ± 0.04 a
5 
µmol TE/g± to 12.a05 ± 0.44 µ±mol TEa /g. When in±cubateda  at 25 an±d 35 °Ca , CRPP ±also had FC1a 4.0 0.45 0.01 5.53 0.13 19.69 0.13 6.36 0.08 2.91 0.02 a
FC1b 4.5 lower hespNeDridin, nobil4e.t2i5n,± an0d.2 2tacngeretin13 c.8o3n±ten0t.s0 4thban th5e. 6u3n±fer0m.20enbted 2C.5R9P±P 0(T.0a9bble 
FC1c 5.0 2). TherefoNreD, in the furt4h.2e3r ±exp0.e0r3imc ents, th9e. 2f8er±m1e.n1t3atcion te5m.7p6e±rat0u.0r5e bwas 2s.e6t4 a±t 300. 0°2Cb.  
FC1d 5.5 ND 4.25 ± 0.04 c 7.46 ± 0.24 c 5.73 ± 0.03 b 2.67 ± 0.01 b
FC1e 6.0 2.3. Ef0fe.3ct1 o±f M0.o0i0stbure Co4n.t3e0nt± on0 .1T6PcC, TFC, 1A5n.3t9io±xid0a.n72t Ab ctivit5y.,7 7an±d P0.h0y5tobchem2.i6c6al± 0.02 b
FC1f 6.5 Compo0s.i2t4io±ns 0in.0 1CRc PP  4.17 ± 0.09 c 8.41 ± 0.96 c 5.56 ± 0.31 b 2.54 ± 0.14 b
Table 1 shows Itnhcautb iantciorenatseimngp ethraet umreoi(sItTu,r◦eC c)o2ntent (MC) from 70% to 80% resulted 
Control - in a sig0.n4i6fi±can0.t0 (0pa < 0.054) .i9n7c±rea0s.0e7 inbc TPC fro19m.3 161±.970 .±4 70.a26 to 162.3.915± ± 0..1519 amg G2.A91E±/g,0 w.0h4iale 
FC2a 25 TFC, a0n.4d3 A±B0T.0S2 aand DP4P.4H8 s±ca0v.3e3ngc ing cap1a5c.0it2ie±s d0.i3d6 nbot inc5r.e7a1s±e s0ig.0n5ifbican2t.l6y0 (±p >0 .00.305b). 
FC2b 30 a a a aHowe0v.e4r5, ±TF0C.0,1 and AB5T.5S3 a±nd0 .D13PaPbH scav1e9n.6g9in±g c0a.1p3acities 6d.e3c6r±ea0se.0d8 as th2e. 9M1C± f0u.r0t2her 
FC2c 35 a a b c cincrea0s.e4d1 t±o 09.00%3 . Simila5r.7ly6, ±fe0ru.3l0ic acid an1d6 .h4e1s±pe1r.i1d0in also 5si.4g5ni±fic0a.0n1tly inc2r.e5a2s±ed0 w.0h2en 
3
the MC increased frMomoi s7t0u troe c8o0n%te anntd(M thCe,n% d, ewc.rbe.)ased as the MC reached 90%. At the MC 
Control - 0.46 ± 0.00 b b c a aof 90%, nobiletin and t4a.n9g7e±re0ti.n07 also show19e.d36 a± d0e.c4r7easing 6tr.3e1nd±. 0T.1h1erefor2e.,9 1th±e M0.0C4 of 
FC3a 70 0.45 b a bc a a80% was s±ele0c.t0e1d for th5e. 5fo3ll±ow0.i1n3g experim19e.n69ts±.  0.13 6.36 ± 0.08 2.91 ± 0.02
FC3b 80 0.53 ± 0.01 a 5.41 ± 0.25 a 22.73 ± 0.38 a 6.27 ± 0.05 a 2.63 ± 0.03 b
 FC3c 90  0.39 ± 0.01 c 4.30 ± 0.14 c 20.42 ± 0.47 b 5.91 ± 0.15 b 2.57 ± 0.07 b
Spore concentration (SC, spores/g) 4
Control - 0.46 ± 0.00 b 4.97 ± 0.07 c 19.36 ± 0.47 c 6.31± 0.11 ab 2.91 ± 0.04 a
FC4a 4x106 0.53 ± 0.01 a 5.41 ± 0.25 ab 22.73 ± 0.38 b 6.27± 0.05 ab 2.63 ± 0.03 b
 FC4b 2x107 0.39 ± 0.0 c 5.69 ± 0.05 a 23.85 ± 0.69 ab 6.42± 0.04 ab 3.01 ± 0.05 a
FC4c 4x107 0.40 ± 0.01 c 5.14 ± 0.15 bc 24.89 ± 1.10 a 6.46 ± 0.02 a 3.05 ± 0.10 a
Fermentation time (FT, h) 5
Control - 0.46 ± 0.00 a 4.97 ± 0.07 c 19.36 ± 0.47 c 6.31 ± 0.11 b 2.91 ± 0.04 c
FC5a 60 0.40 ± 0.01 b 5.14 ± 0.15 c 24.89 ± 1.10 b 6.46 ± 0.02 b 3.05 ± 0.10 c
FC5b 96 0.33 ± 0.00d 5.56 ± 0.04 b 27.52 ± 0.55 b 7.91 ± 0.10 a 3.68 ± 0.05 a
FC5c 144 0.46 ± 0.00 a 6.63 ± 0.03 a 27.56 ± 0.77 b 7.83 ± 0.04 a 3.55± 0.02 ab
FC5d 192 0.37 ± 0.01 c 4.69 ± 0.09 d 28.23 ± 0.76 a 7.78 ± 0.07 a 3.49 ± 0.06 b
Note: ND: Not detected. Means that do not share a letter are significantly different, data are expressed as
mean ± SD (n = 3) on a dry weight basis. 1 The SSF conditions are as follows: incubation temperature = 30 ◦C,
moisture content = 70%, spore concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0, 4.5, 5.0, 5.5,
and 6.0, respectively. 2 The SSF conditions are as follows: moisture content = 70%, spore concentration = 4 × 106
spores/g, fermentation time = 60 h, pH = 4.0, incubation temperature = 25, 30, and 35 ◦C, respectively. 3 The SSF
conditions are as follows: spore concentration = 4 × 106 spores/g, fermentation time = 60 h, pH = 4.0, incubation
temperature = 30 ◦C, moisture content = 70, 80, and 90%, respectively. 4 The SSF conditions are as follows: moisture
content = 80%, fermentation time = 60 h, pH = 4.0, incubation temperature = 30 ◦C, spore concentration = 4 × 106,
2 × 107, and 4 × 107 spores/g, respectively. 5 The SSF conditions are as follows: moisture content = 80%, spore
concentration = 4 × 107 spores/g, pH = 4.0, incubation temperature = 30, and 35 ◦C, fermentation time = 60 h, 96,
144, and 192, respectively.
Molecules 2022, 27, 8949 5 of 15
2.2. Effect of Fermentation Temperature on TPC, TFC, Antioxidant Activity, and Phytochemical
Compositions in CRPP
Under pH 4.0, the CRPP was fermented at temperatures of 25, 30, and 35 ◦C, re-
spectively. The results show that ABTS and DPPH scavenging capacity did not change
significantly when the incubation temperature increased from 25 to 35 ◦C (Table 1). In-
creasing the temperature from 25 to 35 ◦C caused a significant decrease in TFC from
4.71 ± 0.18 µmol TE/g to 4.01 ± 0.14 µmol TE/g, while TPC significantly increased from
11.41 ± 0.35 µmol TE/g to 12.05 ± 0.44 µmol TE/g. When incubated at 25 and 35 ◦C, CRPP
also had lower hesperidin, nobiletin, and tangeretin contents than the unfermented CRPP
(Table 2). Therefore, in the further experiments, the fermentation temperature was set at
30 ◦C.
2.3. Effect of Moisture Content on TPC, TFC, Antioxidant Activity, and Phytochemical
Compositions in CRPP
Table 1 shows that increasing the moisture content (MC) from 70% to 80% resulted in
a significant (p < 0.05) increase in TPC from 11.97 ± 0.26 to 12.95 ± 0.59 mg GAE/g, while
TFC, and ABTS and DPPH scavenging capacities did not increase significantly (p > 0.05).
However, TFC, and ABTS and DPPH scavenging capacities decreased as the MC further
increased to 90%. Similarly, ferulic acid and hesperidin also significantly increased when
the MC increased from 70 to 80% and then decreased as the MC reached 90%. At the MC of
90%, nobiletin and tangeretin also showed a decreasing trend. Therefore, the MC of 80%
was selected for the following experiments.
2.4. Effect of Spore Concentration on TPC, TFC, Antioxidant Activity, and Phytochemical
Compositions in CRPP
Spore concentrations ranging from 4 × 106 to 4 × 107 spores/g CRPP were used to
inoculate CRPP. Compared to the control, TPC, TFC, and ABTS and DPPH scavenging
capacities increased significantly (p < 0.05) in the CRPP after fermentation with different
spore concentrations of A. niger CGMCC 3.6189 and showed an increasing trend when the
inoculum concentration increased from 4 × 106 to 4 × 107 spores/g (Table 1). At the spore
concentration of 4 × 107 spores/g, TPC, TFC, and ABTS and DPPH scavenging capacities
increased to 13.47 ± 0.36 mg GAE/g, 5.15 ± 0.23 mg QE/g, 27.53 ± 0.24 µmol TE/g, and
17.33 ± 1.40 µmol TE/g, respectively. The contents of hesperidin, nobiletin, and tangeretin
significantly increased with the increased spore concentrations, however, narirutin showed
a significant decrease when the spore concentration increased from 4 × 106 to 4 × 107. The
spore concentration of 4 × 107 spores/g was selected for the further fermentation.
2.5. Effect of Fermentation Times on TPC, TFC, Antioxidant Activity, and Phytochemical
Compositions in CRPP
Increasing the fermentation time resulted in significant increases (p < 0.05) in TPC,
TFC, and ABTS and DPPH scavenging capacities, which reached 18.31 ± 0.35 mg GAE/mg,
6.06 ± 0.11 mg QE/g, 36.60 ± 1.82 µmol TE/g, and 22.91 ± 3.45 µmol TE/g, respec-
tively, when CRPP was fermented for 192 h. Hesperidin also reached the maximum of
28.23 ± 0.76 mg/g after 192 h fermentation, however, nobiletin and tangeretin contents
were highest with 96 h fermentation. According to the experimental results, the opti-
mal SSF conditions for flavonoid accumulation and antioxidant activity improvement in
CRPP were pH 4.0, moisture content 80%, temperature 30 ◦C, inoculum concentration
4 × 107 spores/g, and fermentation time 192 h.
2.6. Effect of Fermentation Conditions on the Color of CRPP
Alongside the chemical and bioactivity changes in CRPP after fermentation, we also as-
sessed the impact of the fermentation conditions on the physical change of CRPP. Figure 2a
shows the changes in the color indexes of CRPP after fermentation under different con-
ditions. The results show that L*, a*, and b* values of CRPP significantly decreased with
different fermentation conditions compared with the unfermented CRPP, while the CCI
Molecules 2022, 27, 8949 6 of 15
values significantly increased. Among all these five factors, fermentation time was the
most important factor that affected the changes in the color parameters, while the pH of the
inoculum had the least effect. Increasing fermentation time significantly decreased L*, a*,
and b* values while increasing CCI and ∆E* values. Within the first 96 h fermentation, the
changes in the color indexes were mild, however, these color indexes changed dramatically
with the extended fermentation times, thereby producing much darker CRPP samples. It
is noted that color is also an important attribute that influences consumers’ choices. Al-
though increasing the fermentation time to 192 h significantly enhanced the accumulation
of flavonoids and the antioxidant activity of CRPP, the extended fermentation time tends to
Molecules 2022, 27, x FOR PEER REVIpErWod uce CRPP with an unpleasantly dark color (Figure 2b). Therefore, if considering7 boof t1h7 
 the bioactivity and the organoleptic quality of CRPPs, a fermentation time of 96 h should
be chosen.
 
FFiigguurree 22.. ((AA)) TThhee cchhaannggeess iinn tthhee ccoolloorr ppaarraammeetteerrss ((LL**,, aa**,, bb**,, ∆∆EE**,, aanndd CCCCII)) ooff CCiittrruuss rreettiiccuullaattaa ppeeeellss 
undeerr diiffffeerreennttf feerrmeennttaattioionnc coonndditiitoionnss: :a a= = pH,, b = ffeerrmeenttattiion tteempeerratturree,, cc = moiisstturree ccontteentt,, 
d = spore concceenttrraattiioonn,, ee == fefremrmenentattaitoino ntimtime.e (.B()B T)hTeh iemiamgaegs eosf oCfRCPRPP: aP :=a u=nfuernmfeernmtedn tCedRPCPR, PbP =, 
b60= h6 0fehrmfeernmtaetniotant,i ocn =, 9c6= h9 6fehrmfeernmtaetniotant,i odn =,  d14=4 1h4 f4ehrmf ernmtaetniotant,i oe n=, 1e9=2 1h9 f2erhmfernmtaetniotnat. ion.
22..77.. PPeeaarrssoonn CCoorrrreellaattiioonn bbeettwweeeenn SSSSFF CCoonnddiittiioonnss aanndd QQuuaalliittyy AAttttrriibbuutteess ooff CCRRPPPP  
TThhee PPeeaarrssoonn ccoorrrreellaattiioonn aannaallyyssiiss wwaass uusseedd ttoo ddeessccrriibbee tthhee rreellaattiioonnsshhiipp bbeettwweeeenn tthhee 
TPC, TFC, antioxidant activities (ABTS and DPPH), phytochemical compositions, color pa-
TPC, TFC, antioxidant activities (ABTS and DPPH), phytochemical compositions, color 
rameters, and fermentation conditions including pH, spore concentration, moisture content,
parameters, and fermentation conditions including pH, spore concentration, moisture 
incubation temperature, and the fermentation time. Figure 3 shows that the contents of
content, incubation temperature, and the fermentation time. Figure 3 shows that the con-
nobiletin, tangeretin, and hesperidin, the TPC, TFC, ABTS and DPPH scavenging capacities,
tents of nobiletin, tangeretin, and hesperidin, the TPC, TFC, ABTS and DPPH scavenging 
∆E*, and CCI were positively correlated (p < 0.05) with the fermentation time and spore
capacities, ∆E*, and CCI were positively correlated (p < 0.05) with the fermentation time 
and spore concentration, but were not correlated to the fermentation temperature. The 
contents of nobiletin and hesperidin and the TPC, TFC, and ABTS and DPPH scavenging 
capacities were also positively (p < 0.05) correlated with the initial moisture content. More-
over, the contents of hesperidin, nobiletin, and tangeretin, the TPC, and TFC were posi-
tively (p < 0.05) correlated with ABTS and DPPH scavenging capacities of CRPP, suggest-
ing that these flavonoids and phenolic compounds are closely associated with the antiox-
idant activity of citrus peel. The ∆E* and CCI showed a positive (p < 0.05) correlation with 
the contents of nobiletin and tangeretin, TPC, TFC, and ABTS and DPPH scavenging ca-
pacities, while the color indexes L*, a*, and b* showed negative correlation to these six 
attributes, indicating that color parameters can be good indicators for assessing the anti-
oxidant activity of citrus peel that has undergone fermentation. The pH was also nega-
tively correlated with the contents of ferulic acid and hesperidin. Our results suggested 
 
Molecules 2022, 27, 8949 7 of 15
concentration, but were not correlated to the fermentation temperature. The contents of no-
biletin and hesperidin and the TPC, TFC, and ABTS and DPPH scavenging capacities were
also positively (p < 0.05) correlated with the initial moisture content. Moreover, the contents
of hesperidin, nobiletin, and tangeretin, the TPC, and TFC were positively (p < 0.05) corre-
lated with ABTS and DPPH scavenging capacities of CRPP, suggesting that these flavonoids
and phenolic compounds are closely associated with the antioxidant activity of citrus peel.
The ∆E* and CCI showed a positive (p < 0.05) correlation with the contents of nobiletin and
tangeretin, TPC, TFC, and ABTS and DPPH scavenging capacities, while the color indexes
L*, a*, and b* showed negative correlation to these six attributes, indicating that color
Molecules 2022, 27, x FOR PEER REVIEW 8 of 17 
 parameters can be good indicators for assessing the antioxidant activity of citrus peel that
has undergone fermentation. The pH was also negatively correlated with the contents of
ferulic acid and hesperidin. Our results suggested that fermentation temperature and spore
concentrationtahraet tfweromoefntthaetiomno tsetmimpperoarttuarnet afancdto srpsotrhea ctoanffceecntttrhaetiopnh eanreo ltiwc oan odf tflhaev monoositd important fac-
contents, the atonrtiso txhiadta anftfeactt itvhiety p, haesnwoelilcl asndth felacvoolonrooidf CcoRnPtePndtsu, rtihneg aSnStFio, xwidhailnetm acotiisvtiutyre, as well as the 
content has a csoiglonri fiocf aCnRt PimP pdaucrtinogn SthSFe,b wiohaicleti mveocisotmurpeo cnoenntetsnat nhdasb ai osaigcntiivfictya,nbt uimt npoatct hoen the bioactive 
color of CRPPc.oFmerpmoennetnattsi oandti mbieo acntdivintyit, ibaul tp nHoth tahvee cmoluocrh olfe CssRiPnPfl.u Feenrcmeeonntatthioenq utiamliety and initial pH 
attributes andhtahveeo mrguacnho lepsst iicncflhuaernacete orins ttihcse oqfuCalRitPyP a. tWtreibaulsteosf aonudn dthteh aotrgthaenofllaevpotnico cidhsaracteristics of 
such as hespeCriRdPinP,. nWobe ialelstion f,oaunnddt athnagte trheeti nfl avsowneolildass stuhceh caosl ohresinpdereixdeisn,( Ln*o,bai*le, tbin*,, ∆anEd*, tangeretin as 
and CCI) are wgoeolld asc htheem ciocalolra innddepxheyss (iLca*,l ain*,d bic*,a ∆toEr*s, faonrdt hCeCfIe) ramree ngoteodd CchRePmPicwailt ahnhdi gph ysical indica-
antioxidant acttoirvsi tfyo.r the fermented CRPP with high antioxidant activity. 
 
Figure 3. PearsoFnigcuorrer e3la. tPioeanrbsoetnw ceoernretlhaetifoenrm beetnwtaeteionn thcoen fderitmioennstaatnidont hceonqudaitliiotynsa tatnridb uthtees qoufaCliittyru asttributes of Cit-
reticulata peel ruunsd reertiscoulliadt-as tpaetel fuernmdern staotliodn-s.taNteo tfe:rmCeCnIta=ticointr.u Nsoctoel:o Cr CinId =e cxi,trFuTs =cofleorrm inednetaxt, iFoTn = fermentation 
temperature, MtCem=pmeroaitsuturer,e McoCn t=e nmt,oSisCtu=res pconretecnotn, cSeCn t=r astpionre,  Fcton=cfeenrtmraetniotant,i oFnt =ti mferems.e*nAtasttieornis tkimes. * Asterisk 
denotes significdaenntodtiefsfe sriegnncieficaatnpt< d0if.f0e5r.ence at p < 0.05.  
2.8. Principal C2o.8m. pPorninencitpAaln Caloymsipsoonfetnhte AQnuaalylistiys Aoft ttrhieb uQtuesaloiftyC ARPttPributes of CRPP  
We performedWfuer tpheerrfoprrminecdip faul rctohmerp ponriennctipanala lcyosmisp(PonCeAn)t taonvailsyusaisli z(PeCthAe)r etola vtiiosnusahliizpe the relation-
between the osbhsiper bveattwioenesn athned ovbasreiravbalteiso.nAs atnodta vlaorfiatbwleesn. tAy tsoatmalp olfe tswinencltuy dsianmgpnleins eintecelunding nineteen 
fermented anfderomneenutendf earnmde onnteed unsafemrmpleenwteder seaamnpalley zweedr,ea anndaltyhzeedre, saunldts thaere resshuolwts narien shown in Fig-
Figure 4. Theufirres t4.a Tnhdes eficrostn danPdC seccoonntrdi bPuCte ctoont6r7ib.4u4t%e taon 6d71.454.6%4 %anodf 1th5.e64to%ta olfv tahreia tnoctael, variance, re-
respectively. Tshpeecrteisvuelltys. hTohwe srethsuatltF sCh1oaw, Fs ct2haa,t FFCC21ba,, FFCc22ca,, FFCC32ab,, FFCC32bc,, FFCC43aa,, FFCC43bb,, FFCC44ca,, FC4b, FC4c, 
and FC5a were more similar to the control and were characterized by similar L*, a*, and 
b* values. The samples including FC1b, FC1c, FC1d, FC1e, FC1f, FC2a, and FC3c were 
identified by low ferulic acid, narirutin, and hesperidin contents, while FC5b, FC5c, and 
FC5d were distinguished by high contents of TPC, TFC, nobiletin, and tangeretin, ABTS 
and DPPH scavenging capacities, ∆E*, and CCI, especially for the FC5d CRPP.  
 
Molecules 2022, 27, 8949 8 of 15
and FC5a were more similar to the control and were characterized by similar L*, a*, and
b* values. The samples including FC1b, FC1c, FC1d, FC1e, FC1f, FC2a, and FC3c were
identified by low ferulic acid, narirutin, and hesperidin contents, while FC5b, FC5c, and
Molecules 2022, 27, x FOR PEER REVIEW 
 FC5d were distinguished by high contents of TPC, TFC, nobiletin, and tangeretin,
9A oBfT 1S7 
and DPPH scavenging capacities, ∆E*, and CCI, especially for the FC5d CRPP.
 
FFiigguurree 44.. PPrriinncciippaall ccoommppoonneenntt aannaallyyssiiss ooff CCiittrruuss rreettiiccuullaattaa ppeeeellss ppoowwddeerr uunnddeerr AAssppeerrggiilllluuss nniiggeerr 
GGCCMMCCCC 33..66118899 ssoolliidd--ssttaattee ffeerrmmeennttaattiioonn:: ((AA)) SSccoorree pplloottss ((BB)) llooaaddiinngg pplloott.. TTaabblleess 11 aanndd 22 ccoonnttaaiinn aa 
detailed description of the sample names. 
detailed description of the sample names.
33.. Diissccussssiion 
SSF iiss a llow--moiisstturre fferrmenttattiion ttechniique tthatt hass been ussed ffeassiiblly and eco--
nomiicalllly ffor llarge--scalle biioconversiion and biiodegradattiion off agrii--ffood wastte or by--
produccttss [[15]].. Fermentation conditions such as pH,, temperature,, moisture content,, miicro-
bial concentration, and fermentation time are critical factors that affect microbial growth 
during SSF,, tthuss iinflflueenciing tthe ccheemiicall,, biiollogiical, and organoleptic qualities of the 
prroduccttss.. pH is a determining factor for the growth of microorganisms due to its inflfluence 
on enzyme activity,, cceellllullarr prroocceesssses,, aand ccompllex physiiollogical phenomena such as 
membrane permeability and morphology [16].. Previous research showed that A. niigeer 
grown at a pH ranging from 5 to 6 accelerated the accumulation of ffllavonoiids [17,18]. 
Initial media pH values between 6..5 and 7..5 were optimal for flflavonoid accumulation by 
A.. niiger iin IIsattiis ttiincttorriia L.. haiiry roott [[19]].. Usiing A.. niiger B1b,, Ahmed ett all.. [[20]] ffound tthe 
opttiimallpH off 8..5 fforr phenolliic accumullaattiioonn.. Siince iitt iiss harrd tto meassurre tthe pH off tthe ssolliid 
ssubssttrraattee iin tthee SSF,, tthee pH off tthee iiniittiiaall iinoccullum waass usseed tto aasssseessss tthee eeffffeecctt off pH on 
tthhee aaccccumullaattiioonn ooff ffllaavvoonnooiidss.. IInn ththee ppreresseennt tstsutuddyy, ,wwee fofouunndd ththata tththe einioncoucluulmum wwithit ha 
apHpH ofo 4f.40. 0wwasa tshteh ebebset sftofro frlaflvaovnoonidoi dacaccucmuumlautliaotnio, nw, hwichhic mh amya bye breelraetleadte tdo ttohet heeffeefcfte octf 
opfHp Hon oennzeynmzyem aectaivcittiyv.i tpy.Hp hHash baesebne eshnoswhno wton htaovhea av ediaredcitr eecffteecftf eocnt tohne tahcetivacittyiv oitfy eonf-
eznyzmyems.e ps.Hp iHnfliuneflnuceensc tehset ihoeniizoantiizoant ioofn thoef  tchoemcpoomnpenotnse innt sthien gthroewgtrho wmtehdmia eadnida tahned styhne-
sthyenstihse osifs eonfzeynmzeysm [2e1s][. 2R1u].tiRnuostiindoassied, ansaer,inngairninagsein, ahsees,pheerisdpienraidsei,n aa-sLe-,rah-aLm-rnhoasmidnaossei,d paesce-,
ptiencatsine,a scee,llcuellalusela, stea,ntnanasnea, spe,hpyhtaystea,s eβ,-βgl-ugcluocsoidsaidsea,s ea,nadn dlipliapsaes ehhavave ebbeeeenn uusseedd iinn cciittrruuss 
pprroodduuccttss flfalavvoonnooididb bioiotrtarnansfsofromrmataitoinonu nudnedrefre rfemrmenetnattiaotniocno ncodnitdioitniosnosf poHf pfHro mfro3m.5 t3o.57 .t5o, 
t ◦7e.m5, pteemraptuerreatfurorme f3ro0mto 3700 toC 7, 0a°nCd, ianncdub iantciuonbattimione  ftriomme 2frtoom1 220 tho [162]0.  Ah s[t6u].d Ay  osftuthdeye offfe tchtse 
oefffpecHtso onf tphHe p ohne nthoeli cpsh, eflnaovloicnso, ifdlas,vaonndoiadnst, ifaunndg aanl taicfutinvgitayl ianctthiveitlyiq iuni dthceu lltiuqrueidm ceudlituumre 
fmeremdieunmte dfewrmithenAte.dta wmaitrhii Are.v teaamleadriit hreavt epaHle5d wthaast ipdHea l5[ 1w6a].s Tidheeayl a[l1s6o].s hTohweye daltshoa sthsotrwonegd 
athciadt icst(rpoHng= a3c)i,dnice u(ptrHal ,=a n3)d, bnaesuitcra(pl,H an=d9 baansdic 1(1p)Hco =n d9i taionnds 1s1ig) nciofincdaintitolynsd escigrenaifsiecdantthley 
TdPecCreaansdedT tFhCe. THPoCw aenvde rT, FthCe. sHtaorwtinevgepr,H thoef s7tawrtainsgt hpeHb eosft 7t owaacsc uthme ubleastte tfloa avcocnuomiduslaitne 
the root of Isatis tinctoria L. fermented by immobilized A. niger 3.3883 [19]. In contrast to
flavonoids in the root of Isatis tinctoria L. fermented by immobilized A. niger 3.3883 [19]. 
our study, the root of Isatis tinctoria L. was submerged in the liquid culture media and
In contrast to our study, the root of Isatis tinctoria L. was submerged in the liquid culture 
A. niger was immobilized in Ca-alginate gel beads. Therefore, it is important to screen the
media and A. niger was immobilized in Ca-alginate gel beads. Therefore, it is important 
to screen the optimal pH for the accumulation of flavonoids in the fermentation systems 
with different conditions, substrates, and microorganisms. 
Fermentation temperature affects the heat and mass transfer as well as microbial 
growth and activity. Low temperature limits microbial growth and production of bioac-
tive compounds, while high temperature also disturbs the growth of or even kills the mi-
crobes, and, thus, inhibits the formation of products. The temperature tolerance of A. niger 
 
Molecules 2022, 27, 8949 9 of 15
optimal pH for the accumulation of flavonoids in the fermentation systems with different
conditions, substrates, and microorganisms.
Fermentation temperature affects the heat and mass transfer as well as microbial
growth and activity. Low temperature limits microbial growth and production of bioactive
compounds, while high temperature also disturbs the growth of or even kills the microbes,
and, thus, inhibits the formation of products. The temperature tolerance of A. niger isolated
from the Himalayan soil was in a range of 9–42 ◦C, with an optimal growing temperature
at 28 ◦C [22]. We also found that the optimal temperature for A. niger to accumulate
the phenolics and flavonoids in citrus peel was 30 ◦C. A similar result was found by
Jiao et al. [19], who observed the highest flavonoid production in the roots of Isatis tinctoria
L. fermented by immobilized A. niger at 30 ◦C. Another optimal temperature for flavonoid
accumulation was also reported. For example, Bose et al. [16] found that A. tamarii grown at
35 ◦C produced the highest TPC and TFC, while phenolics and flavonoids were produced
at a much lower level when grown at 15 and 45 ◦C. Liu et al. [23] also reported that the
optimal temperature to accumulate flavonoids in dandelion during SSF was 35 ◦C. The
optimal temperature for flavonoid accumulation may be due to the different substrates and
microorganisms used.
During fermentation, the substrates must contain enough moisture to enable microbial
development [24]. In SSF, the moisture content of the substrate usually ranges from 30
to 85% [25]. The heat applied and produced in SSF causes the low-moisture sample to
dry out, resulting in the poor growth of microorganisms [15]. Low moisture content
also reduces the solubility of nutrients in the substrate, causing reduced availability of
nutrients for microbial growth [24]. However, high moisture reduces the porosity of the
solid matrix and leads to the aggregation of substrate particles, thereby limiting the oxygen
transfer [24], and thus inhibiting microbial growth. Therefore, to maximize the growth of
microbes and the accumulation of flavonoids, appropriate moisture content needs to be
selected. The initial moisture contents of 60–90% were used to ferment citrus by-products
to produce multi-enzymes using different fungi and the results showed that the optimal
moisture content for A. niger BTL was 90% [24]. In our study, we found that phenolics
and flavonoid accumulation followed a trend of first increasing and then decreasing as
the moisture content increased from 70 to 90%, with a maximum accumulation at the
initial moisture content of 80%. A similar trend was also observed in citric acid production
in citrus peel using A. niger CECT-2090 [26]. For the fermentation of dandelion by the
mixture of L. plantarum and S. cerevisiae in solid state, a moisture content of about 53%
was best to accumulate the flavonoids [23]. Inoculum concentration is an important factor
that promotes microbial growth and metabolite production in SSF. Flavonoid content
increased when the spore concentration of immobilized A. niger was increased from 10 to
104 spore/mL [19]. Liu et al. [23] reported that as the inoculum concentration increased,
the flavonoid content first increased and then decreased, reaching a maximum at the
inoculum concentration of 1.2 × 107 spores/g. A concentration of 2.5 × 105 spores/g
of A. niger 3.13901 improved the flavonoid accumulation in Citrus reticulata peel [27].
Cai et al. [28] used 106 spores/g A. oryzae and A. niger to increase the TPC and TFC in
fermented oats. We found that the highest accumulation of flavonoids and phenolics
was in the CRPPs inoculated with 4 × 107 spores/g. It is known that the increase in the
inoculum concentration can shorten the fermentation time and limit the growth of other
microorganisms [29]. However, high inoculum level increased the crowdedness of the
microorganisms, leading to the enhanced consumption of sugar, and thus resulting in the
reduction in bioactive productivity [30].
Fermentation time is also a critical factor that affects the TPC, TFC, antioxidant activity,
and the biotransformation of flavonoids in citrus peels. The fermentation time is determined
by the nature of the medium, the fermenting organisms, the concentration of nutrients,
and the physiological parameters of the process [18]. Ahmed et al. found that the phenolic
compounds were the highest with fermentation of A. niger B1b for 9 days [20]. Pérez-
Nájera et al. [31] showed that TPC, TFC, and antioxidant activity of lime peels fermented
Molecules 2022, 27, 8949 10 of 15
by A. saitoi remained unchanged within the first 5 days of fermentation, while significantly
increased to 8.66, 5.14, and 5.8 times, respectively, after 6 days fermentation, followed by a
dramatic decrease when fermentation time extended to 7 days. The maximum hesperidin
content was observed in the lime peels fermented for 2 days. The much higher increases in
the TPC, TFC, and antioxidant activity of lime peels compared to our results may be due to
the different fermentation conditions, substrates, and microorganisms used. We found that
fermentation time was the most important factor that affects the accumulation of phenolics
and flavonoid compositions in citrus peels. TPC, TFC, and antioxidant activity significantly
increased as the fermentation time extended. A similar result was reported by Liu et al. [23],
who found that fermentation time was the only factor that significantly affect the flavonoid
content in dandelion fermented by a mixture of L. plantarum and S. cerevisiae using a four-
factor response surface methodology design. They found the maximum flavonoid content
was obtained when dandelion was fermented for 52 h. Metabolomics analysis further
showed that in the fermented dandelion, 27 flavonoids were upregulated and 30 flavonoids
were downregulated. Santos et al. [32] showed that TPC peaked at 48 h of fermentation;
however, the TFC did not reach this peak even at 168 h of fermentation in Passiflora ligularis
seed. We also found that the times for obtaining the maximum phenolic and flavonoid
contents were different. For example, the ferulic acid and narirutin reached their maximum
at a fermentation time of 144 h, hesperidin peaked at 192 h, while nobiletin and tangeretin
were the highest after 96 h fermentation. This result may be related to the different enzyme
activities that are needed for the biosynthesis of flavonoids.
The increases in TPC, TFC, and antioxidant activity in the fermented CRPP can be
attributed to the enzymes involved in the biosynthesis of flavonoids, as well as the hydro-
lases. In plants, polyphenolic compounds exist in bounded and free forms. Cellulases,
xylanases, and ligninases can release bounded polyphenolic compounds from the cell
wall through disruption of the hemicellulose, cellulose, and lignin, thus increasing the
free phenolic compounds. β-glucosidase hydrolyzes phenolic glycosides to release free
phenolics and tannases catalyze the breaking of ester bonds and depside linkage of the
polyphenol complexes to produce smaller phenolic compounds with higher antioxidant
activity [32]. Moreover, other enzymatic reactions such as hydroxylation, dihydroxyla-
tion, dehydrogenation, methylation, oxidation, and reduction reactions occurring during
microbial fermentation may also contribute to the increased antioxidant activity of citrus
peels after fermentation due to the production of compounds with higher antioxidant activ-
ity [33,34]. Cyclization of chalcones or transformation of other compounds can also increase
flavonoid accumulation and antioxidant activity [35]. It is well known that antioxidant
activity is closely associated with the phenolics and flavonoids in the plant extract. We
found that ABTS and DPPH scavenging capacities of fermented CRPP were positively
correlated with the TPC, TFC, and contents of hesperidin, hesperetin, and nobiletin. Similar
results were found by Long et al. [36] and Guo et al. [37], who also reported a positive
correlation between ABTS and DPPH scavenging ability of Citrus sinensis extract or citrus
peel extract and TPC, TFC, and nobiletin content.
Aside from the nutritional value of food products, color is another important quality
attribute that influences the acceptability of foods. The color of food products is closely
associated with the physical, chemical, biochemical, and microbial reactions during the
postharvest storage or processing of food products [38]. Therefore, color changes can be
used to predict the changes in other quality attributes of food products. The color indexes
a* (redness (+) or greenness (–), b* (yellowness (+) or blueness (–), L* (brightness (100)
or darkness (0), and ∆E (the total color difference) are generally used to assess products’
changes of color quantitatively [39]. The citrus color index (CCI) is specifically used to
measure the variable of color parameters of citrus products and by-products. CCI ≤ −5
indicates dark green color, −5 < CCI ≤ 0 indicates green color, 0 < CCI ≤ 3 corresponds to
yellowish green color, 3 < CCI ≤ 6 indicates greenish yellow color, 6 < CCI ≤ 8 represents
yellowish orange color, 8 < CCI ≤ 10 indicates orange color, and CCI > 10 corresponds
to dark orange color [40]. Similar to the changes in the TPC, TFC, antioxidant activity,
Molecules 2022, 27, 8949 11 of 15
and flavonoid compositions in CRPP during SSF, the color of CRPP was also significantly
affected by the fermentation conditions, among which fermentation time was also the most
significant influencing factor. Compared with the unfermented CRPP, the fermented CRPP
has lower a*, b*, and L* values, indicating that the redness, yellowness, and brightness
of CRPP decreased after fermentation. Similar results were also observed in the tempe, a
nutritious food prepared from the fermentation of soybeans by Rhizopus spp. [38]. One
possible explanation for this is that high fermentation temperatures promote fungal growth,
resulting in an early formation of spores and affecting the color of the fermented product.
For example, black spores of A. niger were only observed after 72 h at 34 ◦C and after 96 h at
31 ◦C, but not after 120 h for 25 and 28 ◦C [41]. Moreover, high temperatures can accelerate
the degradation of chlorophyll, causing caramelization and the Maillard reaction, which
produce browning [14].
4. Materials and Methods
4.1. Materials and Chemical
The fruits of Citrus Reticulate Blanco ‘Chachiennsis’ (Chachi) were harvested from
an orchard in Xinhui, Guangdong Province, China, on 8 November 2020. The peels were
collected and sun-dried for 5 days followed by vacuum sealing in plastic bags. The samples
were stored in a desiccator at room temperature. Aspergillus niger CGMCC 3.6189 was
purchased from China General Microbiological Culture Collection Center (Beijing, China).
The Folin–Ciocalteu, potato dextrose agar (PDA), and yeast powder were provided by
Solarbio® Science and Technology Co., Ltd. (Beijing, China). The ABTS and 1, 1-DPPH
were bought from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). The HPLC
standards (chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, narirutin, hesperidin,
naringenin, hesperetin, nobiletin, and tangeretin) were purchased from Chengdu-Must
Technology Co., Ltd. (Chengdu, China). The other chemicals were all of analytical grades
and were acquired from Sinopharm Chemical Reagent Co., Ltd. (Zhenjiang, China).
4.2. Samples Preparation
The peels of the “Chachi” fruits were sun-dried until the moisture content reached 11%
(w.b., wet basis). The peels were ground into fine powders and sieved through a 50-mesh
stainless steel sieve. The Citrus Reticulate peel powders (CRPP) were stored at 4 ◦C for the
following experiments.
4.3. Preparation of the Growth Curve and the Inoculum of Aspergillus niger CGMCC 3.6189
Aspergillus niger CGMCC 3.6189 grown on the PDA (pH 5.6 ± 0.2) was inoculated
in a culture medium containing 10 g/L glucose and 20 g/L yeast extract, and the initial
concentration of the inoculum was adjusted to OD 600 of 0.1. The inoculum was grown
in an incubator (LHS-100CL, Shanghai Yiheng Technology Co. Ltd., Shanghai, China) at
30 ◦C and 100% relative humidity (RH). The spore suspension was taken every hour until
32 h. The absorbance at 600 nm was recorded. The concentration of the spore solution was
determined using the method described by Bastidas [42]. Briefly, 0.5 mL of 106 diluted
spore solutions was mixed with 0.5 mL of methylene blue (1%) and then 10 µL of the
mixture was read under a microscope using a hemocytometer. The concentration of the
spores was calculated using the following equation:
Number o f cells× 10, 000
Spore concentration = (1)
Number o f square× times o f dilution
The growth curve was plotted using the culture time and the logarithm of the spore
number. The spores grown at mid-log phase (after 15 h growing in the culture medium)
were used to inoculate CRPP.
Molecules 2022, 27, 8949 12 of 15
4.4. Solid-State Fermentation (SSF)
SSF was conducted using the method described by Wang et al. [7] with minor modifi-
cations. Briefly, 1.5 g of CRPP was placed in a Petri dish and sterilized for 30 min on each
side before the addition of the mid-log phase spores’ suspension, followed by incubation
in an incubator (LHS-100CL, Shanghai Yiheng Technology Co. Ltd., Shanghai, China)
under 100% RH for different times. The experimental factors included pH (4.0, 4.5, 5.0,
5.5, 6.0, and 6.5), temperature (25, 30, and 35 ◦C), moisture content (70, 80, and 90% w.b.),
inoculum concentration (4 × 106, 2 × 107, and 4 × 107 spores of A. niger/g of CRPP), and
fermentation time (60, 96, 144, and 192 h).
4.5. Extraction of CRPP
The CRPP was extracted using a modified ultrasound-assisted method described by
Luo et al. [43]. Briefly, the unfermented (control) and fermented samples were freeze-dried
and extracted with 80% methanol at a solid-to-solvent ratio of 1: 30 (w/v) for 20 min under
ultrasonication. The extract was centrifuged at 5000 rpm, 4 ◦C for 20 min, filtered through
a 0.22 µm filter membrane, and the supernatant was stored at 4 ◦C for further analyses.
4.6. Analysis of Total Phenolic Content (TPC)
The TPC of CRPP extracts was determined using the Folin–Ciocalteu method described
by Chen et al. [44]. Briefly, 20 µL of diluted CRPP extracts or the standard solution
(0.1 mg/mL gallic acid) were mixed with 100 µL of Folin–Ciocalteu solution (10 times
diluted) and incubated in darkness for 1 min, followed by the addition of 80 µL of Na2CO3
(75 mg/mL) and further incubation for 30 min. Absorbance was measured at 765 nm using
a Spark® 10M multimode microplate reader (Tecan, MA, USA). The results were expressed
as mg Gallic acid equivalents (GAE)/g of CRPP (d.w).
4.7. Analysis of Total Flavonoid Content (TFC)
The TFC of CRPP extracts was determined using a spectrophotometric method accord-
ing to Shraim et al. [45] with some modifications. Briefly, in a 15 mL glass test tube, 1000 µL
appropriated diluted CRPP extracts or quercetin standard solution (0.2 mg/mL) was mixed
with 60 µL NaNO2 (5%). The mixture was allowed to stay in the dark for 5 min. Thereafter,
60 µL AlCl3 (10%) was added, followed by the addition of 400 µL NaOH (1.0 mol/L). After
6 min incubation in darkness, all the solutions were vortexed and the absorbance was
recorded at 510 nm against methanol 80% as blank using a 96-well microplate reader (Tecan,
MA, USA). The results were expressed as mg quercetin equivalents (QE)/g of CRPP (d.w.).
4.8. Analysis of ABTS Radical Scavenging Capacity
The ABTS scavenging capacity was evaluated according to Chen et al. [46], with slight
modifications. Briefly, 20 µL of CRPP extract or Trolox standard (0.25 mmol/L) was reacted
with 180 µL of ABTS working solution. After 10 min incubation in darkness, the absorbance
intensity was measured at 734 nm. The results were expressed in µmol Trolox equivalents
(TE)/g CRPP (d.w.).
4.9. Analysis of DPPH Radical Scavenging Capacity
The DPPH scavenging capacity was assessed according to the method described by
Chen et al. [46], with minor modifications. An aliquot of 180 µL of methanol-diluted
CRPP extract or Trolox standard (0.2 mmol/L) was reacted with 20 µL of DPPH reagent
(0.394 g/L). After 10 min incubation in darkness, the absorbance was read at 519 nm. The
DPPH radical scavenging capacity was expressed as µmol Trolox equivalents (TE)/g of
CRPP (d.w.).
4.10. Analysis of Phytochemicals Using HPLC
The phytochemicals in CRPP extracts were determined using HPLC according to the
method of Gao et al. [47]. An LC-20AD HPLC instrument (Shimadzu L.C., Kyoto, Japan)
Molecules 2022, 27, 8949 13 of 15
equipped with a diode array detector, and a Phenomenex Kinetex C18 (100× 4.8 mm, 5 µm)
column (Phenomenex, Torrance, CA, USA) were used and the temperature of the column
was set at 25 ◦C. The CRPP extract was eluted with 0.1% TFA (solvent A) and acetonitrile
(solvent B) at a flow rate of 1.0 mL/min. The elution gradient includes: 0–5 min, 15–20%
B; 5 −10 min, 20% B; 10 −16 min, 20–25% B; 16−17 min, 25–26% B; 17–25 min, 26–27% B;
25–28 min, 27–30% B; 28–33 min, 30–40% B; 33–40 min, 40–65% B; 40–45 min, 65–15% B; and
45–50min, 15% B. Chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, nobiletin, and
tangeretin were detected at 330 nm. Narirutin, hesperidin, hesperetin, and naringenin were
detected at 280 nm. The phytochemical content was expressed as mg/g of CRPP (d.w.).
4.11. Determination of Color Parameters
The color parameters (L*, a*, and b*) of the CRPP were evaluated using a colorimeter
(WS-2300, iWAVE Co. Ltd., Zibo, China). The citrus color index (CCI) value and the total
color difference (∆E*) were determined according to Arzam et al. [41] and Sun et al. [40]
using the following equations:
100× a∗
√ CCI = L ∗ × (2)b∗
∆E ∗ = (a∗ − a∗ 2 ∗ ∗ 2 ∗ ∗ 20) + (b − b0) + (L − L0) (3)
where L*, a*, and b* are the color index of the fermented CRPP and L0*, a0*, and b0* are the
color index of the unfermented CRPP (control).
4.12. Statistical Analysis
The data are expressed as mean ± SD. One-way ANOVA with Tukey’s test was used
to evaluate the significant differences between CRPP samples using MINITAB 18 (Minitab
Ltd., State College, PA, USA). A p < 0.05 represents a significant difference. Pearson
correlation analysis and principal component analysis (PCA) were performed using Origin
9.9 software (OriginLab Co., Northampton, MA, USA).
5. Conclusions
In the present study, we found that A. niger CGMCC 3.6189 can increase the phe-
nolic and flavonoid contents and the antioxidant activity of citrus peel in SSF when the
fermentation conditions are appropriately controlled. Hesperidin, nobiletin, narirutin,
and tangeretin are four of the major flavonoids in CRPPs, all of which were significantly
increased after SSF by A. niger. The maximum flavonoid accumulation conditions were pH
4.0, temperature 30 ◦C, moisture content 80%, and spore concentration 4 × 107 spores/g
d.w. for 192 h. Among these five factors, fermentation time, spore concentration, and
moisture content are the three most important factors that affect flavonoid accumulation,
antioxidant activity, and the color of CRPP, while the fermentation temperature has the least
impact. Although long-time fermentation significantly increased the flavonoid contents
and antioxidant activity, it also caused the production of CRPP with a much darker color.
Therefore, in consideration of both bioactive components and the organoleptic charac-
teristics of CRPP, a fermentation time of 96 h is a better choice. Thus, we recommended
fermentation by A. niger CGMCC 3.6189 as an alternative method for bioactive compound
accumulation in Citrus reticulata peel. Nevertheless, future investigations on energy source
effects are needed to reduce the fermentation time that affected the color of the peel powder
and to help in the optimization of the flavonoid accumulation.
Author Contributions: Conceptualization, X.C.; methodology, D.M. and Y.H.; software, D.M.; formal
analysis, D.M.; investigation, D.M. and Y.H.; writing—original draft preparation, D.M. and X.C.;
writing—review and editing, X.C., N.D.K.A.-T. and M.B.; supervision, X.C.; project administra-
tion, X.C.; funding acquisition, X.C. All authors have read and agreed to the published version of
the manuscript.
Molecules 2022, 27, 8949 14 of 15
Funding: This research was funded by the Jiangsu Specially Appointed Professor Program (19TPJS-
002) and the Senior Talent Startup Fund of Jiangsu University (4111360002) to X. Chen.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets generated during and/or analyzed during the current
study are available from the corresponding author on reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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