Steroids 136 (2018) 40–46

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Steroids
journal homepage: www.elsevier.com/locate/steroids

Eco-friendly microbial production of diosgenin from saponins in Dioscorea T

zingiberensis tubers in the presence of Aspergillus awamori
⁎
Yu Chenc, Yi Donga,b, Yuanlong Chia,b, Qiang Hea,b, Hui Wuc, Yao Rena,b,
a
College of Light Industry, Textile and Food Engineering and Healthy Food Evaluation Research Center, Sichuan University, Chengdu 610065, PR China
b
The Key Laboratory of Food Science and Technology of Ministry of Education of Sichuan Province, Sichuan University, Chengdu 610065, PR China
c
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: A novel microbial procedure was proposed for diosgenin production from Dioscorea zingiberensis C.H. Wright
Dioscorea zingiberensis (DZW) tubers via employing Aspergillus awamori for the first time. The optimal conditions of fermenter culti-
Diosgenin vation were established as inoculation dosage of 8%, fermentation temperature of 30 °C, cultivation time of
Aspergillus awamori 8 days, initial pH of 7.0 and a stirring rate of 180 rpm when the converted diosgenin content reached a peak
Biotransformations
value of 74.26 ± 3.23 mg/g substrate. The product was purified by silica gel column and then confirmed as
Clean production
diosgenin (purity: 96.9 ± 2.42%) by nuclear magnetic resonance (NMR). Compared with traditional acid hy-
drolysis, this new process generated indeed less wastewater with lower chemical oxygen demand (COD) reduced
to 500 mg/L from 10,000 mg/L and absence of acid and alkali. This research provided definitely an environ-
mental and high-efficiency microbial technology for diosgenin production.

1. Introduction achieve diosgenin, various enzymes (cellulose β-glucosidase and pec-

It has been reported that diosgenin has various biological con-
tribution in anti- cardiovascular disease, anti-diabetic and anti-tumor
etc. and was also an important steroidal precursor for the synthesis of
steroid hormones and contraceptives [1–5]. Currently, 60% of steroidal
drugs and contraceptives have been produced from diosgenin, and as a
result, the product diosgenin is in a great demand and its production
has caught wide attention [6,7]. In nature, diosgenin is mainly stored in
the form of saponins which take especially a high level content in
Dioscorea zingiberensis C.H. Wright (DZW) tubers, and exist primarily in
the joint type of glycoside form [8]. Therefore, removing glycoside off
saponins is a crucial step for preparing diosgenin (Scheme 1).
So far, large processes have been implemented for diosgenin pro-
duction, most of which focused on cutting off glycosidic linkages with
acid hydrolysis [9,10]. Unfortunately, a large quantity of wastewater
with low pH and high level of chemical oxygen demand (COD) are
discharged during the industrial process, thus leading to serious en-
vironmental pollution and surely becoming a major problem [5].
Nevertheless, it is well known that biotransformation is a specific and
environmentally friendly process. Compared with traditional acid hy-
drolysis, biotransformation possesses remarkable predominance due to
their high selectivity and mild reaction condition [10,11]. Some scho-
lars focused on the direct enzymatic conversion [12]. In order to
tinase) were selected and applied in the procedure converting saponin
to diosgenin. Although some good results were reported, enzymatic
approach is uneconomic as a result of expensive enzymes used, which
limit largely its application in diosgenin industry [13,14].
Thus, another biotransformation method, namely microbial fer-
mentation, has been explored lately [15–17]. Microorganisms have a
peculiarity that they can secrete rapidly enzymes via metabolism for
survival responding to the ambient environment and reduce product
inhibition effects. It was more economical than direct enzymatic con-
version when microbial treatment was implemented [18]. In addition,
the starch in DZW tubers can be fully utilized by microorganisms under
acid conditions, which reduces greatly the emissions of high levels of
COD. Therefore, microbial treatment is suggested to be a stronger ap-
plicability than direct enzymatic treatment and acid hydrolysis in the
conversion of saponins to diosgenin. To our best knowledge, there are
few reports on screening of particular microorganism with a high bio-
transformation rate of diosgenin. Several previous researches revealed
successful bioconversions of steroidal saponins to diosgenin, but the
yields cannot be compared together due to the difference of the sum of
diosgenin units in per gram of DZW tubers [19,20].
In the present study, our objective was to employ efficient micro-
organisms for producing cleanly diosgenin with a high biotransforma-
tion rate from the extracted food steroid saponins in DZW tubers.

⁎
Corresponding author at: College of Light Industry, Textile and Food Engineering, Sichuan University, NO.24 South Section 1, First Ring Road, Chengdu 610065, China.
E-mail address: reny@scu.edu.cn (Y. Ren).

https://doi.org/10.1016/j.steroids.2018.05.005
Received 31 January 2018; Received in revised form 10 April 2018; Accepted 7 May 2018
Available online 09 May 2018
0039-128X/ © 2018 Elsevier Inc. All rights reserved.
Y. Chen et al.
Several crucial fermentation parameters of the target strain were opti-
mized, including inoculation dosage, fermentation temperature and
cultivation time. The crude fermentation products were purified by si-
lica gel column chromatography, then quantified by high performance
liquid chromatography (HPLC) and confirmed using nuclear magnetic
resonance (NMR). The new microbial approach and traditional acid
hydrolysis were finally compared in cleaner production.
2. Experimental
2.1. Materials and chemicals
DZW tubers were obtained from Airport Chinese Medicinal
Materials Market (Shanxi, China) and authenticated by South China
Institute of Botany. Diosgenin (≥95%) was purchased from Sigma Co.,
USA. Acetonitrile and methanol (chromatographic grade) were bought
from Merck Co., Germany. Sulfuric acid (analytical grade) was pur-
chased from Development Center of Kemiou Chemical Reagents
(Tianjin, China). Ultrapure water was acquired by a Milli-Q Water
Purification System (18.2 M < OMEGA > *cm at 25 °C, Millipore, MA,
USA). Taq Platinum Blue was purchased from Invitrogen Co., USA.
Hydrophilic and oleophilic organic microporous membranes (0.45 μm)
were purchased respectively from New Asia Purifier factory (Shanghai,
China). Silica gel (80–100, 300–400 mesh) was purchased from
Oriental Silica Gel Co. Ltd. (Shanghai, China). All solutions and samples
prepared for chromatographic analysis were filtered through 0.45-μm
membrane before injection into HPLC.
2.2. Microorganisms
Rhizopus oryzae ATCC96382 was preserved in American Type
Culture Collection (ATCC). Aspergillus awamori CCTCC M2013639 was
preserved in China Center for Type Culture Collection (CCTCC) in
Wuhan University. Geotrichum candidum GIM2.12, Rhizopus chinensis
GIM3.144, Rhizomucor miehei GIM3.510, Bacillus subtilis GIM1.135, and
Pseudomonas cepacia GIM1.139 were preserved in Guangdong
Microbiology Culture Center (GIMCC). Candida parapsilosis ACCC20221
was preserved in Agricultural Culture Collection of China (ACCC).
2.3. Extraction of total saponins in DZW tubers
Total saponins in DZW tubers were sufficiently prepared in ac-
cordance with the optimal microwave-assisted extraction conditions of
employing 75% ethanol as solvent, a ratio of solid/liquid of 1: 20 (g/
mL), a temperature of 75 °C, an irradiation power of 600 W and three
extraction cycles of 6 min each [8]. The reaction mixture was filtered
with a Buchner funnel, and extracted saponins was isolated and dried
by rotary evaporation and vacuum freezing successively.
2.4. Fermentation of extracted total saponins
2.4.1. Culture medium
Plate screen medium (g/L): NaNO3 3.0, KH2PO4 1.0, MgSO4·7H2O
0.5, KCl 0.5, Fe2(SO4)·7H2O 3.0, extracted saponins 3.0 and agar
powder 20.0.
Agarslant activation medium (g/L): NaNO3 3.0, KH2PO4 1.0,
41
Steroids 136 (2018) 40–46
Scheme 1. Conversion of steroidal saponins to dios-
genin under microorganism strain fermentation. R
represents sugar chain.
MgSO4·7H2O 0.5, KCl 0.5, FeSO4·7H2O 3.0, extracted saponins 3.0, and
agar powder 20.0.
Fermentation medium (g/L): NaNO3 3.0, KH2PO4 1.0, MgSO4·7H2O
0.5, KCl 0.5, FeSO4·7H2O 3.0, and extracted saponins 30.
2.4.2. Strain screening
A spot of single colony was picked and added into 10 mL of sterile
saline, mixing adequately on an oscillator (Ruipu precision instrument
Co., Ltd, China). A 4 mL dosage of suspension was put into fermentation
medium in a conical flask, then incubated for 5 d at 30 °C with a rotate
speed of 180 r/min in a shaker table (HWEI-29, Jinke precision in-
strument Co., Ltd, China). Simultaneously, 4 mL of suspension was re-
placed with the same volume of sterile saline as a negative control.
After 5-d fermentation, the precipitate was collected through suction
filtration, and dispersed in 30 mL of methanol with a 2-h supersonic
treatment (200 W). Ultimately, 200 μL of the supernatant was diluted to
1 mL in methanol for the determination of the diosgenin content using
the below-mentioned HPLC procedure. The target strain that can de-
grade extracted saponins to diosgenin was screened out, inoculated on a
fresh agarslant activation medium, and stored at 4 °C for further assay.
2.4.3. Fermentation conditions
Seed liquid: fresh agarslant activation medium was inoculated with
a spot of target strain lawn and cultivated at 28 °C. After 5 d, a ring of
the lawn was scraped out by an inoculating loop and dispersed in sterile
saline. And seed liquid was prepared successfully after a shake.
Shaker cultivation: 100 mL of fermentation medium was placed into
a “500 mL” conical flask, followed by fermentation cultivation in a
rotary speed of 180 rpm for 5 d. The prepared seed liquid with different
inoculation dosages of 2%, 4%, 6%, 8%, 10% and 12% were added in
fermentation medium, respectively, when fermentation pH and tem-
perature were preliminarily set as 6.0 and 30 °C. The diosgenin content
of fermentation mixture was determined per 12 h. 8% of inoculation
dosage was added into 100 mL of fermentation medium with the initial
pH of 6.0, and the temperatures were controlled as 23 °C, 26.5 °C, 30 °C,
33.5 °C, 37 °C and 40.5 °C. The diosgenin content of fermentation
mixture was also determined per 12 h.
Fermenter cultivation: A dosage of seed liquid was added into a
“5 L” fermentor (Biostat Aplus, Sartorius Co., Ltd, Germany), and the
cultivation conditions were given that stirring rate of 180 r/min, ven-
tilatory volume of 1.0 m3/min, pressure of 0.7 kg/m2, cultivation time
of 10 d. In addition, inoculation dosage, fermentation temperature and
initial pH were in according with the optimal values in shaker culti-
vation. The pH value, biomass, residual sugar and diosgenin content of
fermentation mixture were detected in the whole process. When the
biotransformation process was terminated, biomass concentration in
fermentation medium was measured indirectly by intracellular protein
concentration [21]. Residual sugar concentration was measured by 3,5-
dinitrosalicylic acid (DNS) method [22].
2.5. Determination of diosgenin by HPLC
A 20 mg aliquot of diosgenin standard was dissolved in 5 mL of
methanol (chromatographic grade) to prepare eight fractions with dif-
ferent concentrations of 10, 80, 160, 240, 320, 400, 480, and 560 μg/
mL, which were determined under the following chromatographic
Y. Chen et al.
conditions. Standard equation is y = 24,513x − 131,703, where
R2 = 0.9967, y is the peak area and x is the diosgenin concentration,
and a good linearity with the range of 10 μg/mL–560 μg/mL. The
sample of 1 mL was injected into the HPLC system after being filtered
through a 0.45-μm membrane to determine the diosgenin content under
the same chromatographic conditions, which were as follows: high
performance liquid chromatography analyzer (Waters 2996 diode-array
detector), C18 column: Xterra® MS reversed-phase column
(4.6 × 150 mm, 5 μm), mobile phase: acetonitrile: water = 8: 2, V/V.
Flow rate: 0.9 mL/min, column temperature: 32 °C, detection wave-
length: 200 nm and injection volume: 20 μL. Yields of diosgenin (Y)
were calculated as follows:
M Statistical calculation was performed by one-way analysis of var-
Y=
W (1)
where M is the weight of diosgenin prepared by fermentation cultiva-
tion, mg, and W is the weight of extracted saponins, g.
2.6. 18S rDNA sequence
DNA sequence of strain was obtained using 18S rDNA sequencing
method with a modification [23]. The genomic DNA of the strain was
obtained with SanPrep Mini DNA extraction Kit (Sangon Biotech Co.,
Ltd, China). Polymerase chain reaction (PCR) was applied with 8.5 µL
of Taq Platinum Blue, followed with 3 pmol of each primer (forward
and reverse) and 30 ng of target DNA genes were amplified with pri-
mers targeting NS1 (GTAGTCATATGCTTGTCTC) and NS6 (GCATCAC
AGA C CTGTTATTGCCTC) structural genes. The employed thermal
cycling was given that denaturation at 94 °C for 4 min, annealing at
94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, with a total of 30 cycles,
extension at 72 °C for 10 min, and termination at 4 °C. The PCR pro-
ducts were analyzed in 1% (w/v) agarose gel and purified using Illustra
GFX PCR DNA and gel band purification (GE Life Science), and then
sequenced using ABI 3730 DNA Analyzer (Applied Biosystems, USA).
Sequence was uploaded and compared with these available in the Basic
Local Alignment Search Tool (BLAST) search program of the National
Center for Biotechnology Information (NCBI).
2.7. Purification through silica gel column
A 2.5 g dosage of fermentation product in 2.3.4 dissolved in 300 mL
of petroleum ether with a 20-min ultrasound assist, and was absorbed
adequately in 10 g of silica gel (80–100 mesh). After a air-drying in a
fume hood, the sample gel was prepared successfully. A 200 g aliquot of
silica gel (300–400 mesh) was loaded onto a chromatography column
(2.6 × 60 cm) followed tightly by the addition of prepared sample gel.
First of all, 500 mL of petroleum ether was added completely through
the column, and then a solvent mixture of petroleum ether and ethyl
acetate (v/v = 8: 2) was poured as an elution at a flow rate of 2 mL/
min. Each fraction of 100 mL was concentrated by rotary evaporation,
frozen drying and collected for the determination of diosgenin content
and further identification. The distilled solvent mixture could be re-
cycled with a sufficient utilization.
2.8. NMR analysis
The structure of purified product in above-mentioned procedure
was further confirmed with NMR. 1H (600 MHz) and 13C (150 MHz)
NMR experiments were carried out on a spectrometer (Bruker Avance
III HD 600, Bruker, Germany). 1H-NMR (600 MHz, CDCl3) δ 1.83 (1H,
m, H-1), 1.10 (1H, m, H-1), 1.86 (1H, m, H-2), 1.65 (1H, m, H-2), 3.37
(1H, t, H-3), 2.23 (2H, m, H-4), 5.34 (1H, d, J = 6.0 Hz, H-6), 1.98(1H,
m, H-7), 1.63(1H, m, H-7), 1.61 (1H, m, H-8), 1.10 (1H, m, H-9), 1.57
(1H, m, H-11), 1.46 (1H, m, H-11), 1.77 (1H, m, H-12), 1.12 (1H, m, H-
12), 1.28 (1H, m, H-14), 2.29 (1H, m, H-15), 1.52 (1H, m, H-15), 4.41
(1H, q, H-16), 1.85 (1H, m, H-17), 0.79 (3H, t, H-18), 1.03 (3H, s, H-
42
Steroids 136 (2018) 40–46
19), 1.98 (1H, m, H-20), 0.97 (3H, d, J = 6.0 Hz, H-21), 1.85 (1H, m, H-
23), 1.48 (1H, m, H-23), 1.64 (1H, m, H-24), 1.46 (1H, m, H-24), 1.67
(1H, m, H-25), 3.52 (2H, m, H-26), 0.79 (3H, t, H-27). 13C-NMR
(150 MHz, CDCl3) δ 37.2 (C-1), 30.3 (C-2), 80.0 (C-3), 39.8 (C-4),
140.8 (C-5), 121.4 (C-6), 32.1 (C-7), 31.6 (C-8), 50.1 (C-9), 37.2 (C-10),
20.9 (C-11), 39.8 (C-12), 40.3 (C-13), 56.5 (C-14), 31.9 (C-15), 80.8 (C-
16), 62.1 (C-17), 16.3 (C-18), 19.4 (C-19), 41.6 (C-20), 14.5 (C-21),
109.3 (C-22), 31.4 (C-23), 28.8 (C-24), 31.5 (C-25), 66.9 (C-26), 17.1
(C-27).
2.9. Statistical analysis
iance (ANOVA) using SPSS, version 19.0 (SPSS Inc., Chicago, IL). Data
were expressed as the mean ± standard deviation (SD) of triplicate
determinations. Differences were considered to be significant at
p < 0.05 or 0.01.
3. Results
3.1. Screening and characterization of target strain
After strains culture and products test, only the microorganism
strain Aspergillus awamori CCTCC M2013639 that could convert total
saponins to diosgenin with a high yield was screened out, while the
above-mentioned other seven experimental strains in “2.2” lacked this
capacity. It was observed in Fig. 1A and B that the target strain in slant
preservation presented brown-black, flocky with no exudate, while it
formed plentiful furvous globules during fermentation cultivation as a
result of the shaking in table concentrator. As shown in Fig. 1C and D,
irregular radial conidial head and long tubular conidiophore of the
strain were also observed clearly with a magnification of 400 times
under a microscope (BDS200, OPTEC Co., Ltd, China). The conidial
head was spherical with a diameter of 70–80 μm, while the con-
idiophore, whose top capsule was also spherical with a diameter of
30–40 μm, appeared on the substrate [24,25]. Furthermore, the DNA
sequence of this microorganism was listed in Fig. 2 and its GenBank
accession number was provided as KX138070. BLAST analysis indicated
the homology between the present DNA sequence and that of Aspergillus
awamori in GenBank is 99.7%.
3.2. Optimization of fermentation conditions
A suitable inoculation dosage should be given to guarantee that the
target strain Aspergillus awamori can make full use of the total saponins
in fermentation medium. As shown in Fig. 3A, the diosgenin content
had a climbing tendency with the increase of inoculation dosage of seed
liquid from 2% to 12% during given 5-d cultivation time. However, the
content had no a significant difference among 8%, 10% and 12% of
inoculation dosages (p > 0.05). Therefore, to save seed liquid, 8% was
considered as the optimal inoculation dosage for the following fer-
menter cultivation. It was speculated that the increscent Aspergillus
awamori produced abundant metabolites, especially enzymes which
could hydrolyze the glycosyl off the ring of steroid saponins for sur-
vival. When the inoculation dosage reached 8%, the metabolic enzymes
secreted had been sufficient to cut off the glycosyl. Thus, 10% and 12%
of inoculation dosages couldn’t cause a significant increase (p > 0.05).
For any microorganism survival, ambient temperature is an essen-
tial factor that influences directly the growth of microorganism and the
release of metabolic enzymes with high bioactivities. As shown clearly
in Fig. 3A, the diosgenin contents presented different under various
cultivation temperatures. It is more suitable for Aspergillus awamori to
complete the biotransformation at the special temperature range from
30 °C to 37 °C, during which the contents of converted diosgenin had no
a significant difference (p > 0.05). When the fermentation tempera-
ture reached 40.5 °C, the diosgenin content had a remarkable decline
Y. Chen et al. Steroids 136 (2018) 40–46

Fig. 1. Photograph of Aspergillus awamori CCTCC M2013639 strain in slant preservation (A), shaker cultivation (B) and micrograph of conidial head (C), con-
idiophore (D) of Aspergillus awamori strain with a magnification of 400 times.

(p < 0.05), which signified it was out of optimal temperature scope.
And it was concluded that the temperature range of less than 30 °C or
more than 37 °C was not adverse for the invertase releasing of Asper-
gillus awamori. In order to economize energy, 30 °C was confirmed as
the optimal temperature for the 5-d fermentation. Besides, the initial
pH and stirring rate were not further discussed in present research.
3.3. Fermenter cultivation
After both the inoculation dosage and fermentation temperature
were determined in according with the above-mentioned results, fer-
menter cultivation of 5 L was initiated, and the consecutive results
during 10 d were recorded in Fig. 3B. After a 8% dosage of seed liquid
of Aspergillus awamori was inoculated into the medium, the fermenta-
tion process was initiated. Meanwhile, the biomass of Aspergillus awa-
mori presented an increasing tendency. On the 2nd day, the pH sud-
denly dropped to 5.61 from the initial 7.00 due to the release of the
metabolites, and after one day climbed up to 6.60. Until the 8th day,
both the diosgenin content and the biomass concentration reached their
peak value of 74.26 ± 3.23 mg/g and 8.34 ± 0.79 g/L followed by a
stabilization of the diosgenin and a decline of the biomass, indicating
that the rest of saponins would not be taken advantage of by Aspergillus
awamori spores sequentially. Therefore, the total diosgenin releasing
after 8-d fermentation cultivation reached a maximum.

Fig. 2. DNA sequence list (sequence accession number: KX138070) of the target strain isolate with a high biotransformation rate of steroid saponins to diosgenin
obtained by the amplification of NS1 and NS6 structural gene.

43
Y. Chen et al.
Fig. 3. (A) Effect of inoculation dosage and cultivation temperature on the
diosgenin content in shaker cultivation, and (B) evolution profile of pH, bio-
mass, residual sugar and diosgenin content with the cultivation time in fer-
menter cultivation. Data are expressed as the mean value ( ± SD) of three in-
dependent experiments. Means with different letters are significantly different
(p < 0.05). Upper case letters indicated the significant difference of diosgenin
contents due to different inoculation dosages, while lower case letters indicated
the significant difference of those due to different temperature.
3.4. Purification and confirmation of fermentation product
To well achieve diosgenin with a high purity, the fermentation
product from above-mentioned cultivation was purified using repeated
silica gel column chromatography. The products with various polarities
after 8-d fermentation cultivation were absorbed in silica gel at various
degrees. The contained diosgenin could be eluted easily down with the
solvent mixture of petroleum ether and ethyl acetate (v/v = 8:2) due to
the close polarity. HPLC spectrums of fermentation product before and
after the purification were shown in Fig. 4. Compared to the standard
spectrum in Fig. 4A, some other saponins in fermentation product were
also exhibited at 11.75 min besides the diosgenin in Fig. 4B. Compared
to that in Fig. 4B, a small portion of saponins couldn’t be transformed
and was removed through the purification procedure in Fig. 4C. A large
elution fraction was collected, frozen drying and recrystallized with a
preparation of 172.45 ± 5.26 mg of diosgenin. HPLC analysis revealed
the purity of this diosgenin product was 96.9 ± 2.42%, whose struc-
ture would be further confirmed using NMR.
The structure of the recrystallized product, dry white powder, was
identified as diosgenin by comparison of its NMR spectra (see
44
Steroids 136 (2018) 40–46
Fig. 4. HPLC chromatograms for diosgenin standard (A), fermentation products
from the fermenter before and after the purification through silica gel column
chromatography (B, C).
experimental part) with those described in previous reports [26,27].
Furthermore, not any sugar chain signals was observed in these spectra.
4. Discussion
The target strain that could convert effectively saponins in DZW
tubers to diosgenin was successfully screened out as Aspergillus awamori
CCTCC M2013639. The present strain enlarged the strains family for
biotransformation of saponins to diosgenin [14,18,19]. Although these
microbial transformations were eco-friendly, the yields were expected
to reach higher value by diosgenin manufacturer thereby substituting
thoroughly traditional acid hydrolysis in Fig. 5. The present study
worked out that 30 °C was the optimal temperature for the bio-
transformation during the whole fermentation. It is noteworthy that
Trichoderma harzianum was also employed for the conversion at the
same temperature [19]. It is assumed that these strains that have been
confirmed to be capable to convert saponins to diosgenin at the similar
circumstance can generate potentially a synergistic effect when they are
applied together.
Compared to shaker cultivation, fermenter cultivation can be con-
veniently controlled and the whole process can be traced and recorded,
so is more suitable in industrial production. During the fermenter cul-
tivation, residual sugar from extracted total saponins in medium was
firstly utilized by Aspergillus awamori as a carbon source. On the 2nd
day, as a result of the shortage of residual sugar, the strain had to se-
crete glycosidase to cut residual sugar off the steroid saponins for sur-
vival with the release of the metabolites which caused a decline and
successive rise of pH in Results. At this stage, a mass of diosgenin began
to assemble quickly around Aspergillus awamori globules. Interestingly,
the residual sugar storage demonstrated a conspicuous fluctuation be-
cause of continual hydrolysis of glycosidase and simultaneous con-
sumption from Aspergillus awamori spores. Until the 8th day, the peak
values of both the diosgenin content and the biomass concentration
indicated that the remaining saponins couldn’t be utilized efficiently by
Aspergillus awamori spores like before, which was observed in Fig. 3B.
Y. Chen et al. Steroids 136 (2018) 40–46

Fig. 5. Flow chart of microbial treatment (A) and traditional acid hydrolysis (B) proposed for diosgenin production.

When the microorganism strain developed a series of metabolic actions water. In acid hydrolysis process, besides the saponins, cellulose and
taking extracted total saponins as carbon source, some unknown medial starch were also degraded into low molecular weight sugars to various
steroid saponins with higher polarity than diosgenin were released extents. These degradation products were soluble in wastewater with
firstly. And then the microorganism cut glycosyls sequentially off these other soluble compounds and were discharged into the environment as
intermediate products thereby producing diosgenin. Theoretically, any organic carbon, which led to a high COD level of 10,000 mg/L. In new
microorganism is incapable of converting absolutely all the saponins in process, most compounds (cellulose, starch etc.) that can cause high
DZW tubers to diosgenin. Meanwhile, different strains can release COD level in wastewater were taken out before microbial treatment.
various invertase for utilizing saponins [12]. Thus, combining fermen- Moreover, the only sugars mixture remained in extracted saponins were
tation of two or more than two strains for clean diosgenin production is continually consumed by the microorganism strain as carbon source. As
very significant and will be explored and discussed in future. a consequence, the COD level in the wastewater was reduced sub-
The traditional acid hydrolysis and new microbial treatment pro- stantially to 500 mg/L from 10,000 mg/L.
posed for diosgenin production were compared. Flow chart and some
vital parameters were observed and discussed in Fig. 5 and Table 1.
4.2. Acid and alkali in wastewater
4.1. Reducing of COD in wastewater
In traditional diosgenin manufacturing industry, sulfuric acid or
hydrochloric acid that can contaminate severely the environment is
DZW tubers composed mainly of cellulose, starch, saponins and
frequently employed for degrading saponins. As a result, the pH value
of the wastewater is less than 1 and has to be neutralized by the ad-
Table 1
dition of sodium hydroxide before being discharged. However, the
Comparison of parameters for the traditional acid hydrolysis and the new mi-
crobial process. chloride ion (Cl−) still can’t be precipitated thereby affecting badly the
activated sludge treatment. Under this situation, Cl− concentration in
Parameters Traditional acid New microbial process the wastewater can excess 10,000 mg/L and is very difficult and costly
hydrolysis (t/t diosgenin) (t/t diosgenin)
to be lowered [28]. Furthermore, 8 t of 98% sulfuric acid and 1 t of 10%
98% Sulfuric acid 8 0 sodium hydroxide are employed when 1 t of diosgenin is produced.
consumed Especially, acid, alkali and high Cl− content are also adverse for pro-
10% Sodium hydroxide 1 0 duction apparatus. Fortunately, acid and alkali aren’t used during the
consumed
Wastewater 560 230
whole Aspergillus awamori treatment process. Most of organic solvents
COD 10,000a 500a (ethanol, methanol used in microbial process and gasoline used in
traditional process) aren’t discussed in detail due to their recycle in the
The aforementioned results were given for production of per 1.0 t of diosgenin. process.
Unit of the data marked with ‘‘a” is mg/L.

45
Y. Chen et al.
4.3. Recycle of residue
If the residue isn’t properly used, it will become waste, then con-
taminant. In traditional acid hydrolysis process, the organic carbon
compounds in DZW tubers were degraded into target product dios-
genin, soluble sugars mixture and insoluble residue. Due to a mass of
acid and organic solvent, the residue in factory is very difficult to be
well reused and has to be abandoned. About 1 t of residues are re-
mained per 10 t of tubers material treatment in diosgenin production.
However, the residue, composing mainly of cellulose and starch, iso-
lated before the microbial treatment can be well reused. (1) Fuel. The
residue is an efficient source for the production of heat with co-firing
coal in the factory processing [9]. (2) Activated carbon. The residue can
be also manufactured into activated carbon that possesses superior
adsorption ability than the commercially available ones [29]. (3) Pro-
duction for ethanol. To increase the benefit, the residue can be sold
directly to fermentation enterprises for producing ethanol.
In traditional acid hydrolysis, DZW tubers, water, sulfuric acid
(H2SO4), sodium hydroxide (NaOH), gasoline and coal were employed
and consumed. The most of used water was mainly consumed in
washing the acid hydrolysate, and the energy consumption (coal)
mainly focused on the acid hydrolysis process which was implemented
in high temperature (121 °C) for 3 h. Contrastively, in microbial treat-
ment, DZW tubers, water, fermentation medium, ethanol and methanol
were employed and consumed. The most of used water was mainly
applied in bioreactor, and the energy was mainly consumed in fer-
mentation process (30 °C, 8 d). In general, new microbial approach
consumed less water, 0% of H2SO4, NaOH and gasoline, employing no
wastewater processor and could sell the residue containing starch and
cellulose. Thus, it was speculated that it could be applied more eco-
nomical in diosgenin production.
Although high profit exists in diosgenin industry, a mass of plants
were forced to shut down due to the excessive pollution. The pollution
rather than profit limited observably the sustainable development of
this field. This new microbial hydrolysis could not only decrease the
discharge of contaminant, but also save cost potentially. Thus, it is a
promising procedure in clean industrial production of diosgenin.
5. Conclusion
A clean economical fermentation approach was applied successfully
to convert steroidal saponins in DZW tubers to diosgenin instead of
traditional acid hydrolysis. The present strain was screened out as
Aspergillus awamori CCTCC M2013639 with its sequence accession
number of KX138070, and its fermentation conditions were optimized.
Under the optimal fermentation circumstance, the diosgenin content
demonstrated a high value of 74.26 ± 3.23 mg/g substrate. The pur-
ified product with a purity of 96.9 ± 2.42% was finally confirmed as
diosgenin by NMR. This is the first report on employing Aspergillus
awamori to prepare diosgenin, which represents an eco-friendly ap-
proach with less wastewater with lower COD level reduced to 500 mg/L
from 10,000 mg/L and absence of acid and alkali. Combining fermen-
tation of two or more than two microorganism strains for cleaner and
more efficient diosgenin production will be developed in future.
Acknowledgements
We thank Dr. Yanbiao Guo for helpful discussion throughout and
technical expertise.
The authors are grateful to the National Science Foundation for
Young Scientists of China (No.31701565), the National Science
Foundation for Young Scientists of China (No.31601442), China
Postdoctoral Science Foundation Funded Project (No. 2017M623035)
and Scientific Research Foundation for Young Teachers of Sichuan
University (No.2016SCU11035) for the financial supports.
The authors have declared no conflict of interest.
46
Steroids 136 (2018) 40–46
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