Biocatalysis of platycoside E and platycodin D3 using fungal extracellular β-glucosidase responsible for rapid platycodin D production

Article abstract of DOI:10.3390/ijms19092671

Platycodi radix (i.e., Platycodon grandiflorum root) products (e.g., tea, cosmetics, and herbal supplements) are popular in East Asian nutraceutical markets due to their reported health benefits and positive consumer perceptions. Platycosides are the key

Full text of DOI:10.3390/ijms19092671

International Journal of
Molecular Sciences
Article
Biocatalysis of Platycoside E and Platycodin D3 Using
Fungal Extracellular β-Glucosidase Responsible for
Rapid Platycodin D Production
Hyung Jin Ahn 1, Hyun Ju You 2, Myung Su Park 3, Tony V. Johnston 4 , Seockmo Ku 4,
*
and Geun Eog Ji 1,5,
*
1
Department of Food and Nutrition, Research Institute of Human Ecology, Seoul National University,
Seoul 08826, Korea; ahnjin2000@snu.ac.kr
Center for Human and Environmental Microbiome, Institute of Health and Environment,
Seoul National University, Seoul 08826, Korea; dhlover1@snu.ac.kr
Department of Hotel Culinary Arts, Yeonsung University, Anyang 14001, Korea; mspark@yeonsung.ac.kr
Fermentation Science Program, School of Agriculture, College of Basic and Applied Sciences, Middle
Tennessee State University, Murfreesboro, TN 37132, USA; tony.johnston@mtsu.edu
Research Center, BIFIDO Co., Ltd., Hongcheon 25117, Korea
2
3
4
5
*
Correspondence: seockmo.ku@mtsu.edu (S.K.); geji@snu.ac.kr (G.E.J.);
Tel.: +1-615-904-8290 (S.K.); +82-2-880-6282 (G.E.J.); Fax: +1-615-898-5169 (S.K.); +82-2-884-0305 (G.E.J.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 July 2018; Accepted: 5 September 2018; Published: 8 September 2018
Abstract: Platycodi radix (i.e., Platycodon grandiflorum root) products (e.g., tea, cosmetics, and herbal
supplements) are popular in East Asian nutraceutical markets due to their reported health benefits and
positive consumer perceptions. Platycosides are the key drivers of Platycodi radixes’ biofunctional
effects; their nutraceutical and pharmaceutical activities are primarily related to the number
and varieties of sugar side-chains. Among the various platycosides, platycodin D is a major
saponin that demonstrates various nutraceutical activities. Therefore, the development of a novel
technology to increase the total platycodin D content in Platycodi radix extract is important, not only
for consumers’ health benefits but also producers’ commercial applications and manufacturing
cost reduction. It has been reported that hydrolysis of platycoside sugar moieties significantly
modifies the compound’s biofunctionality. Platycodi radix extract naturally contains two major
platycodin D precursors (platycoside E and platycodin D3) which can be enzymatically converted
to platycodin D via β-D-glucosidase hydrolysis. Despite evidence that platycodin D precursors
can be changed to platycodin D in the Platycodi radix plant, there is little research on increasing
platycodin D concentrations during processing. In this work, platycodin D levels in Platycodi
radix extracts were significantly increased via extracellular Aspergillus usamii
p < 0.001). To increase the extracellular -D-glucosidase activity, A. usamii was cultivated in a culture
media containing cellobiose as its major carbon source. The optimal pH and temperature of the
fungal -D-glucosidase
-D-glucosidase were 6.0 and 40.0 ◦C, respectively. Extracellular A. usamii
β-D-glucosidase (n = 3,
β
β
β
successfully converted more than 99.9% (w/v, n = 3, p < 0.001) of platycoside E and platycodin D3
into platycodin D within 2 h under optimal conditions. The maximum level of platycodin D was
0.4 mM. Following the biotransformation process, the platycodin D was recovered using preparatory
High Performance Liquid Chromatography (HPLC) and applied to in vitro assays to evaluate its
quality. Platycodin D separated from the Platycodi radix immediately following the bioconversion
process showed significant anti-inflammatory effects from the Lipopolysaccharide (LPS)-induced
macrophage inflammatory responses with decreased nitrite and IL-6 production (n = 3, p < 0.001).
Taken together, these results provide evidence that biocatalysis of Platycodi radix extracts with
A. usamii may be used as an efficient method of platycodin D-enriched extract production and novel
Platycodi radix products may thereby be created.
Int. J. Mol. Sci. 2018, 19, 2671; doi:10.3390/ijms19092671
www.mdpi.com/journal/ijms

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Keywords: platycodi radix; platycodon grandiflorum; biotransformation; biocatalysis; aspergillus usamii;
β-glucosidase; phytochemicals; platycoside E; platycodin D3; platycodin D
1. Introduction
Platycodi radix (PR), the Platycodon grandiflorum root, has traditionally been used in popular
herbal medicine for the prevention and treatment of cold-related symptoms (e.g., coughs, phlegm,
tonsillitis, asthma and bronchitis) in various East Asian countries [
and food scientists have verified that triterpenoid saponins known as platycosides are the major
phytochemicals of PR’s bio-functionality as a plant medicine [ ]. About 69 platycoside varieties have
been identified as key functional substances of PR [ ]. According to Ha et al. [ ], platycosides account
for only 2% of PR; however, the total platycoside content of PR may be dependent on Platycodon
grandiflorum’s soil, weather, cultivation method, and years of growth [ ]. PR’s nutraceutical and
1,2]. Modern analytical chemists
3
4
5
6
pharmaceutical properties vary widely according to the type, position, and number of sugars on the
saponin molecules [7].
Among the various platycosides, platycodin D (PD, chemical formula: C57H92O28; formal name: 3
-D-glucopyranosyloxy)-2 ,16 ,23,24-tetrahydroxy-O-D-apio- -D-furanosyl-(1 3)-O- -D-xylopyranosyl-
4)-O-6-deoxy-olean-12-en-28-oic acid; Chemical abstracts service number: 58479-68-8; Molecular
weight: 1225.34), consisting of a main oleanane type backbone with two R groups (i.e., 3-Glc,
and 28-O-Api-Xyl-Rha-Ara), is regarded as the key bio-active molecule of PR [ ]. Previous research
β-
(
(1
β
β
α
β
→
β
→
8,9
has reported that platycodin D reduced the risk of inflammation [10], obesity [11], Hepatitis C viral
infection [12], allergy-related diseases [13], tumorigenicity [14] and the development of cancers
(i.e., liver, ovarian, breast, gastric and leukemia) [15–19]. These platycodin D studies demonstrated
that platycodin D-enriched products can be used not only to alleviate cold-related symptoms but
also to prevent chronic and fatal diseases. The platycodin D level in commercial products is the key
factor used to appraise product quality and meet consumers’ needs [
with the potential to transform into platycodin D are present in PR [
1
]. Multiple chemical precursors
]. Theoretically, a technique
3
that efficiently transforms the chemical structures of these precursors without degrading the existing
platycodin D would increase the total platycodin D content of PR products. Such a technique would
ultimately contribute to improving the quality of the product and its pharmacological effects.
Enzymatic bio-conversion has been extensively utilized to structurally transform plant saponins
(e.g., ginsenoside, wogonoside, daidzin and genistin) [20–24]. Unconjugated forms of glycosides
(also known as aglycones and deglycosylated saponins) are most desirable, and the use of
an edible microbial enzyme to execute the bioconversion provides for stereospecific and sustainable
reactions and may offer eco-friendly and time-cost effective processing attributes [25]. Two major
platycodin D precursors (i.e., platycoside E (PE) and platycodin D3 (PD3)) can be converted to PD
using
β-D-glucosidase (EC 3.2.1.21;
β-D-glucopyranoside glucohydrolase; gentiobiase; cellobiase)
hydrolysis [5,26,27]. Despite the existence of two major PR precursors in the source plant, there is
little research on increasing PD production during processing. Accordingly, the principal objective
of our research is the development of a novel protocol for rapid PD production using fungal
biocatalysis of PD precursors during processing. After PD production, we conducted qualitative
(liquid chromatography/mass spectrometry (LC/MS) and cell inflammatory assays) and quantitative
(high-performance liquid chromatography (HPLC)) analysis to demonstrate the practicality of our
protocol, which allowed us to consistently obtain high PD yields via PE and PD3 conversion.

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2. Results and Discussions
2.1. Microbial Screening
Among the various food-grade edible microorganisms, Aspergillus, Lactobacillus, and
Bifidobacterium spp. are considered by the food industry as effective -D-glucosidase producers [28].
Many research groups and individual researchers have tried to cultivate microorganisms in media
using glucose as the major carbon source for active proliferation [23 29 30]. Glucose is one of the
β
,
,
simplest nutrients used by microorganisms to support rapid microbial growth [23]. In the vast majority
of documented cases, the most favored microbial sugar source is glucose. Glucose serves in industry as
a useful metabolic precursor for obtaining large quantities of cell biomass [23]. However, if producers
aim to produce enzymes rather than cell biomass, their microbial culture strategy must be changed.
The addition of glucose to microbial media can significantly reduce the production of the desired
microbial enzyme (i.e.,
using in-house media containing cellobiose as a carbon source showed that some microorganisms have
the potential to express substantial levels of -D-glucosidase [22]. Therefore, in this study, six probiotics
(Lactobacillus and Bifidobacterium spp.) and five Aspergillus strains were cultured in glucose-free culture
media. Cellobiose was added as the major carbon source to enhance microbial -D-glucosidase activity.
Bacterial and fungal cell extracts and media samples were used to evaluate their
-D-glucosidase-producing abilities via p-nitrophenyl- -D-glucopyranoside (pNP, artificial substrate)
β-D-glucosidase) due to catabolic repression [31]. In contrast, other research
β
β
β
β
and platycoside catalysis. Crude microbial enzyme preparations exhibiting substantial catalytic
activity demonstrate a light or dark yellow color (450 nm) during enzymatic hydrolysis due
to the release of p-nitrophenol molecules from the pNP substrate [32
evaluated with semi-purified (crude) enzyme extracts from either cell lysis or the microbial media.
-D-glucosidase from the five Aspergillus strains was identified in the cell culture supernatant.
However, the -D-glucosidase activities of the six probiotics were detected in the cell-free extract
(intracellular enzyme) produced by cell disruption. -D-glucosidase activities were not detected
without cell breakage processes or from cell culture supernatants. These results are similar to our
previous reports [33 35]. Chi et al. [35] and You et al. [33] found -D-glucosidase activities in cell-free
–34]. β-D-glucosidase was
β
β
β
,
β
extracts of Bifidobacterium bifidum, Lactobacillus delbrueckii, and Lactobacillus cacei. A sonication cell
disruption process was critical to exhibit β-D-glucosidase’s activities.
The cultures used in this study showed different total and specific
β
-D-glucosidase activities,
which ranged from 0.3 to 145.1 M pNP (min M pNP (min
µ
·
mL)−1 and 0.5 to 150.0
µ
·
mL)−1
·
(mg of
protein)−1. Nine microbial extracts—Lactobacillus delbrueckii ssp. delbrueckii KCTC 1047 (LD1047),
Lactobacillus delbrueckii subsp. bulgaricus KCTC 3188 (LD3188), Lactobacillus cacei KCTC 3109 (LC3109),
Bifidobacterium sp. SJ32 (SJ32), Bifidobacterium bifidum BGN4 (BGN4), Aspergillus oryzae KFRI 888
(AO888), Aspergillus oryzae KFRI 954 (AO954), Aspergillus awamori KFRI 899 (AA899) and Aspergillus
awamori KFRI 984 (AA984)—failed to perform the desired reaction. Two isolates of Bifidobacterium sp.
SH5 (SH5) and Aspergillus usamii KFRI 1004 (AU1004) successfully performed the desired reaction.
Figure 1 shows the β-D-glucosidase activities of the tested groups.
The enzyme activities extracted from the nine strains (i.e., LD1047, LD3188, LC3109, SJ32, BGN4,
AO888, AO954, AA899 and AA984) mentioned above were not statistically different (n = 3 p > 0.05).
The β-D-glucosidase activities of AU1004 and SH5 were significantly higher than the other groups
(n = 3, p < 0.001). AU1004 had the highest total and specific activity values (n = 3, p < 0.001) compared
to the others. Accordingly, the fungus AU1004 was utilized in subsequent experiments.

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Figure 1. Total (ꢀ) and specific (ꢁ) β-D-glucosidase activities of 11 cell strains. One-way ANOVA
followed by a Tukey’s post hoc test statistical analyses were performed. Treatments with different
letters (where A > B > C and a > b > c > d) are significantly different at p < 0.05.
2.2. Enzyme Induction
We further examined the effect of cellobiose as an enzyme inducer via enzyme activity analysis
when AU1004 was cultured in basal medium (BM, cellobiose-free condition) and the experimental
condition. As indicated in Figure 2, using cellobiose as the major carbon source in AU1004 cultivation
significantly enhanced the β-D-glucosidase activities of AU1004 (n = 3, p < 0.001).
Figure 2. The significantly enhanced total (ꢀ) and specific (ꢁ) β-D-glucosidase activities via cellobiose
treatment of the culture media (n = 3). Bars with *** represent statistically significant differences
between groups at p < 0.001.
β-glucosidase breaks down the
β
-glucosidic bonds between aglycons and glucose molecules.
-glucosidic bond hydrolysis [36].
-D-glucosidase is produced by multiple bacterial and fungal species [37]. The induction of increased
-D-glucosidase may occur when probiotic bacterial and fungal strains are grown in a culture media
-D-glucosidase to convert the cellobiose
contained in the medium into an energy source. Multiple groups have reported that carboxymethyl
cellulose, cellobiose, and Solka-Floc were effective inductors of enzyme biosynthesis [22 38 40].
Our previous work showed that Lactobacillus delbrueckii Rh2 (cultivated in media containing galactose
without glucose) significantly increased -glucuronidase activity [20]. Lactobacillus rhamnosus GG
This enzyme also catalyzes cellobiose into two glucose molecules via
β
β
β
supplemented with cellobiose. Microorganisms may produce
β
,
–
β

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cultivated in media containing 2% cellobiose (w/v) as a key carbon source showed significantly
enhanced β-D-glucosidase activity compared to media containing glucose [22].
Microorganisms’ sugar ingestion methods vary based on the proteins and enzymes involved
in sugar hydrolysis [41]. Such diverse mechanisms support the cell’s effective uptake of multiple
sugars to maintain their metabolism, in particular for microbial proliferation under low-glucose
environmental conditions [42]. The microorganism’s catabolite repression system of sugar substances
is a physiologically-controlled mechanism for the efficient exploitation of carbon sources for microbial
growth. This repression system acts as an autoregulatory tool that screens favored substrates
(i.e., monosaccharides) within extracellular fluids. Specifically, the utility of various sugars in
culture media is regulated by carbohydrate catabolic genes and/or operons at the level of carbon
source-specific induction [33,43,44]. In this system, sugars act as inducers and/or repressor of gene
expression. The induction-repression mechanism turns on the sugar-specific catabolic genes required
to grow in particular conditions.
Previous studies have used nutrient-dependent enzyme induction systems that provide various
carbohydrates in glucose-free culture media. According to Saloheimo et al. [45], glucose media
supplements reduced cellulase gene (i.e., cbhl, cbh2, egll, eg12 and egl5) expression of Trichoderma
reesei. Suto and Tomita [46] summarized the glycosylase induction and catabolic repression models
of Hypocrea jecorina and Penicillium purpurogenum. These two fungal species regulated and produced
glycosylases at the transcriptional level to feed carbon sources, multiply in nature, and produce
glycosylases. The production steps are as follows: (i) expression at a basal level that mainly
produce cellulose to degrade complex plant cellulose near fungal hyphae into cellooligosaccharides;
(ii) active cellulase gene transcription by sophorose and gentiobiose that act as cellulase inducers;
(iii) full-scale cellulase secretion outside of hyphae via sophorose and glucose production; (iv) active
cellulose degradation results in increased cellooligosaccharides and glucose levels; and (v) catabolite
repression caused by glucose and decreased cellulase production. According to Ku et al. [21],
α
-L-arabinofuranosidase and α-L-arabinopyranosidase activity increased simultaneously when
Bifidobacterium longum RD47 was cultured in a medium supplemented with 2% ascorbic acid (w/v).
The enhanced enzymes were used to catalyze ginsenosides. Bifidobacterium longum RD47 also showed
increased α- and β-galactosidase productivity via soybean oligosaccharide treatment [47].
2.3. Biotransformation of Platycoside E and Platycodin D3 into Platycodin D
PR extracts contain about 2% platycosides [
PD3, and PD are approximately 8.3, 15.4 and 20.9%, respectively [48]. These three platycoside types are
distinguished by their glucose units present at the C-3 position of PD [ ]. Therefore, the transformation
5]. Among the various platycosides, the ratios of PE,
5
of the two major platycosides (i.e., PE and PD3), which account for more than 20% of all platycosides,
into a more bioactive platycoside such as PD has significant nutraceutical and pharmaceutical
applications. It has been reported that
β
-D-glucosidase converted PE and PD3, which removed
]. Specifically, -D-glucosidase hydrolyzed PE along the
PD. PD3 was also transformed to PD via the removal of one glucose
one and two glucose molecules at C-3 of PD [
5
β
following pathway: PE
molecule (Figure 3).
→
PD3
→
However, a few reports incorporated enzymatic biocatalysis in the preparation of PD [5,26,27].
Prior to our work, various commercially-available enzymes extracted from almonds, microorganisms,
and snail digestive tracts were utilized to increase the PD level in PR extracts. However, these biocatalytic
processes were time-consuming (1–3 days) or yielded low conversion rates. Specifically, Ha et al. [
attempted to transform PE and PD3 in PR extracts using two types of commercially-available,
almond-derived -D-glucosidase and one fungal cellulase to increase platycodin D production.
Despite -D-glucosidase treatment for 24 h, most of the platycoside E and platycodin D3 glycosidic
5]
β
β
linkages at C-3 residues remained intact. The platycodin D content increased by less than 10% after
24 h. They also utilized commercially available fungal cellulase to hydrolyze platycoside E and
platycodin D3. Cellulase from Trichoderma reesei successfully cut the β-1-6 glucose bonds of platycoside

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E and platycodin D3 and converted all platycoside E and platycodin D3 (100%) in Platycodi radix
extracts into platycodin D after 24 h incubation. Li et al. [26] also reported the transformation of
platycoside E and platycodin D3 to platycodin D by commercially available snailase (i.e., a mixture
of cellulase, hemicellulase, pectinase and β-glucuronidase) treatment. While their snailase presented
notable transformation rates (about 100%), a lengthy incubation process (24 h) was necessary to
achieve 100% platycoside E and platycodin D3 transformation. Recently, Lee et al. [27] collected crude
enzymes from Cyberlindnera fabianii and Trichosporon loubieri after cell screening about 200 Korean nuruk
microorganisms and attempted to produce PD from Platycodi radix extracts. Their three-day enzyme
reaction increased the total amount of PD by between 12% and 43% and featured low selectivity.
β-D-glucosidase
Platycoside E
-Glucose
β-D-glucosidase
Platycodin D3
-Glucose
Platycodin D
Figure 3. Biosynthetic platycodin D (PD) pathway from platycoside E (PE) and platycodin D3 (PD3) in
Platycodi radix (PR). PD and PD3 undergo glycosylation by β-D-glucosidase and transform into PE.
To determine whether our screened extracellular enzyme stimulates the cleaving of platycoside
glycosidic bonds, we applied the AU1004 enzyme to PR extracts containing natural substrates
(i.e., platycoside E and platycodin D3). The enzymatic reactions of each key point were examined
via HPLC analysis; chromatographic data are shown in Figure 4a,b. Within 2 h, >99.9% of PE and
PD3 was converted to PD. PE and PD3, which were present at concentrations of 0.08 and 0.12 mM,
respectively (n = 3, p < 0.001) (Figure 4c), were successfully converted to PD within 2 h by AU1004
enzyme incubation. As a result, the PD present at 0.20 mM concentration in the original PR extract
increased to 0.40 mM (n = 3, p < 0.001) (Figure 4d).
The PD generated via bioconversion was further verified using LC/MS. Specifically, the chemical
structure of PD existing in compound peak fractions that were collected and recovered by
prep-HPLC was analyzed by Eletrospray ionization (ESI)-Triple Quadrupole LC/MS according to the
manufacturer’s instructions. Initially, the ESI-MS experiments were performed in the negative mode

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to determine molecular weight. The ESI mass spectra of transformed PE, PD3, and standard PD in
negative ion mode displayed sharp and distinguished peaks at m/z 1223.5 and 1319.5, which represent
the deproto−nated ion [M
−
H]− of PD (Figure 5a,b). The value 1319.5 is the adduct sulfate ion
[M–H+SO4] . These results confirm that platycodin D is rapidly generated by the enzymatic hydrolysis
of the 1-6 glycosidic linkage at the C-6 of glucose within 2 h.
Figure 4. Typical High Performance Liquid Chromatography (HPLC) metabolic profiles of 3 mg crude
platycosides separated from PR extracts (
a
,
c
) after a 2 h incubation with AU1004 crude
). A significantly altered biotransformation profile, with increased PD ( ) and reduced PE and PD3
), was observed following the enzyme treatment ( ). Bars with *** represent statistically significant
β-D-glucosidase
(b
,
d
ꢁ
(ꢀ
c,d
differences between groups at p < 0.001 (n = 3, c and d).
Figure 5. LC/MS profiles in the ESI negative mode of commercially-available, standard PD (
a) and
PD produced by our protocol ( ). The m/z ratios of biotransformed PE and PD3 (PD) were 1223.5 and
b
1319.5 (m/z), respectively, the same as that of PD. The value of 1319.5 is the resulting adduct sulfate ion
−
[M–H+SO4] .
2.4. The Inhibitory Effects of Platycodin D on LPS-Induced IL-6 and Nitrite Production in RAW 264.7
(KCLB 40071) Mouse Macrophages
To evaluate LPS-induced macrophage inflammatory responses, we investigated the inhibitory
effects of platycodin D on LPS-induced IL-6 and nitrite production in RAW 264.7 (KCLB 40071) mouse
macrophage cells. Nitrite and IL-6 are commonly used endogenous markers for the characterization of
inflammatory diseases, so the measurement of nitrite and IL-6 metabolites in cell lines is considered to

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be a useful in vitro inflammation monitoring measure [49]. The RAW 264.7 cell lines were incubated
with multiple levels of PD produced by our protocol in the presence and/or absence of Escherichia coli
O127:B8 LPS. The released extracellular nitrite and IL-6 were examined.
Figure 6a shows the effect of PD on the LPS-induced macrophage inflammatory response.
The mouse macrophages were treated with the indicated levels of PD for 2 h, and then treated
with LPS 0.1 µg/mL for 18 h. The PD-free group was used as an inflammatory positive control.
LPS significantly activated RAW 264.7, which secretes inflammatory substances (i.e., IL-6 and nitrite),
as shown in Figure 6a (n = 3, p < 0.001). However, the PD pretreatment prominently downregulated
LPS-induced IL-6 and nitrite release in a dose-dependent manner. LPS and PD were not added to the
control group. Significantly reduced concentrations of IL-6 and nitrite generated from macrophages by
LPS are presented as histograms in Figure 6a (n = 3, p < 0,001).
Figure 6. PD treatment regulation of IL-6 and nitrite in macrophage cells. (
that PD inhibited the production of LPS-induced IL-6 ( ) and nitrite ( ) in a dose-dependent manner.
The significantly decreased extracellular IL-6 and nitrite levels confirmed the anti-inflammatory effect
of PD. The values presented are the mean SD of three independent experiments. Bars labeled by
letters (where A > B > C >D and a > b > c > d) are statistically significant from each other (p < 0.05),
which was determined using a one-way ANOVA followed by Tukey’s post hoc test; ( ) Cell viability
a) The results indicate
ꢁ
ꢀ
±
b
( ) was determined via MTT assay. “n.s.” indicates no statistically significant difference among the
three treatment groups with p > 0.05 (n = 3). Bars labeled by letters (A, AB, B)
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results showed no
obvious cytotoxicity of PD (up to 10 µM) toward mouse macrophage cells in the presence of LPS
although a trend toward decreased cell viability was observed (Figure 6b (n = 3, p > 0.05)). The findings
of this work concur with the previous data and suggest that PD inhibited inflammatory marker
production in RAW 264.7 cells [49–53].
3. Materials and Methods
3.1. Chemicals and Reagents
PE, PD3, and PD were generously donated by BNC Biofarm (Seoul, Korea). The analytical reagents
and solvents used in this work were obtained from Samchun Chemical Co., Ltd. (Seoul., Korea) unless
mentioned otherwise. All macrophage cultivation materials, including the serum-free cell culture
media, fetal bovine serum, and related culture solutions, were GIBCO products from Thermo Fisher
Scientific (Waltham, MA, USA). HPLC-grade mobile phases (e.g., acetonitrile, methanol, and water)
were purchased from Fisher Scientific (Pittsburgh, PA, USA).
3.2. Preparation of Bacterial Crude Enzymes
Lactobacillus delbrueckii ssp. delbrueckii KCTC 1047 (LD1047), Lactobacillus delbrueckii
subsp. bulgaricus KCTC3188 (LD3188), Lactobacillus cacei KCTC 3109 (LC3109), Bifidobacterium

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sp. SJ32 (SJ32), Bifidobacterium sp. SH5 (SH5), and Bifidobacterium bifidum BGN4 (BGN4) were
generously donated by BIFIDO LTD (Hongcheon, Korea). The six probiotic strains were incubated
in Man–Rosa–Sharpe (MRS) medium (BD Difco
(w/v) L-cysteine under strict anaerobic culture conditions for 24 h at 37 ◦C and subcultured in 50 mL
of MRS broth. Cultured probiotic cells were recovered via centrifugation (3000
g for 30 min at 4 ◦C).
™, Franklin Lakes, NJ, USA) broth containing 0.05%
×
The harvested cells (8 log colony-forming unit (CFU)/mL) were washed twice with 50 mM phosphate
buffer (PB, pH 6.0, 0.02 M) to remove the media ingredients from the cells. The washed cell samples
were then resuspended in 10 mL of phosphate buffer. β-D-glucosidase from the above six probiotic
strains was considered as a cytosolic (intracellular) enzyme due to the absence of enzyme activities in
the cell-free media supernatant. All probiotic strains were disrupted v◦ia cell sonication (Sonics and
Materials, Inc., Newtown, CT, USA) at 45 amplitude for 30 min at 4 C (1 s burst and 1 s cooling
intervals) to extract intracellular enzymes for the purpose of producing a crude enzyme solution as
described in You et al. [54].
3.3. Preparation of Fungal Crude Enzymes
All of the experimental Aspergillus strains (Aspergillus oryzae KFRI 888, Aspergillus oryzae KFRI
954, Aspergillus usamii KFRI 1004, Aspergillus awamori KFRI 899 and Aspergillus awamori KFRI 984) were
purchased from the Korea Food Research Institute (Seongnam, Korea). Aspergillus crude enzymes
were prepared following the methods of You et al. [33] Specifically, the spores were harvested
from a subcultured, commercially available potato dextrose agar (glucose 20 g/L, agar 15 g/L
and potato extract 4 g/L; Sigma-Aldrich, St. Louis, MO, USA) plate by scraping, followed by
suspension of the fungal spores (0.9% NaCl solution with 0.005% polysorbate 80). A hemocytometer
(Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) was used to count the Aspergillus
spores. The recovered fungal spores were spiked at 6 log spores/mL (9 log cell spores/L) into the BM
with cellobiose (5 g/L). The BM composition was identical to that used in our previous study (You et al.):
sodium nitrate (CAT 7631-99-4, 0.5 g/L), dipotassium phosphate (CAT 7758-11-4, 1.0 g/L), magnesium
sulfate heptahydrate (CAT 10034-99-8, 0.5 g/L), iron(II) sulfate heptahydrate (CAT 7782-63-0, 0.01 g/L)
and 0.5% (w/v) casamino acid (65072-00-6), with a pH adjusted to 7.0.
According to Decker et al. [55], Aspergillus strains produce extracellular enzymes, so we attempted
the concentration and recovery of extracellular
strains were incubated in 1 L culture broth at 30 C for seven days under aerobic conditions with
shaking at 180 rpm (SWB-03, JEIO TECH, Anyang, Korea). The mycelia were removed from the
β
-D-glucosidase from the culture media. All Aspergillus
◦
culture broth via microfiltration using a 1.6 µm cut-off GF/A borosilicate microfiltration membrane
(Z242128, Whatman®, Kent, UK). The filtrate-reserving extracellular enzymes were concentrated
via a centrifugal membrane module (Amicon Ultra, 30 K molecular weight cut-off, UFC903008,
Millipore Korea, Seoul, Korea) at 4000
×
g for 30 min at 4 ◦C. The concentrated and recovered filtrates
were washed and rinsed with the same volume of PB (0.02 M, pH 6.0) to purify the enzymes and
remove the media ingredients with a pH adjustment. The washed sample was concentrated a second
time using centrifugation with Amicon Ultra centrifugal filter units. The concentrated crude enzyme
sample was resuspended in 50 mL of PB.
3.4. Assay of β-D-Glucosidase Activity
The
media for the fungal extracellular enzyme and in the disrupted cell suspensions of six probiotic strains
for the intracellular enzyme. The total bacterial and fungal -D-glucosidase activities were determined
as described by Wie et al. [34]. Specifically, to enumerate the microbial -D-glucosidase activities,
20 L of crude enzyme extracts were added to 20 L of the artificial substrate (5 mM, pNP, # N7006,
Sigma-Aldrich, St. Louis, MO, USA) with 60 L of PB (0.02 M, pH 6.0). The crude enzyme and pNP
substrate mixtures were incubated at 37 ◦C for 8 min with shaking. The enzyme hydrolysis reaction
was stopped by adding 100 L Na2CO3 (0.5 M). The released p-nitrophenol (pNP) was measured at
β-D-glucosidase activity was determined in the supernatant of the five Aspergillus culture
β
β
µ
µ
µ
µ

Int. J. Mol. Sci. 2018, 19, 2671
10 of 15
450 nm (96 cell microplate reader, Bio-Rad Laboratories, Philadelphia, PA, USA). Specific activity was
determined per mg protein. The specific activity (U/mg) of -D-glucosidase was calculated as units of
β
enzyme activity per mg of protein. The level of protein was evaluated using a commercially available
BCA Protein Assay Kit (Pierce™, CAT 23225, Waltham, MA, USA).
3.5. Extraction of Crude Platycosides from Platycodi Radix
Platycodon grandiflorum root (Platycodi radix, 3 years) powder was purchased from a local grocery
market (Seoul, Korea). To extract the platycosides, we used the protocol of Ha et al. [5]. Specifically,
100 g of Platycodi radix powder was added to 1 L of 70% methanol at held at 70 ◦C in a waterbath
shaking at 100 rpm for 4 h. The powder was then removed by filtration (Whatman® Grade 3, Z240478,
Maidston, UK). After a dead-end filtration process, the collected filterates were evaporated using
an Eyela rotary evaporator N-100 (Rikakikai Co., Ltd., Tokyo, Japan) and 100 mL of deionized water
was added to the samples.
According to Nyakudya et al., Platycodi radix is composed of approximately 90% sugar
(i.e., glucose, fructose, saccharose, kestose, inulin and platycodin), 2.4% protein, 2% platycosides,
0.1% lipid and 1.5% ash [1]. It was therefore assumed that the collected sample contains not
only platycosides but also other molecules (e.g., soluble sugars, dietary fibers and proteins).
A purification process was conducted via absorption chromatography to remove the unwanted
compounds in the sample. Specifically, the sample was loaded onto a column (100 cm
×
5 cm)
packed with non-polar copolymer styrene-divinylbenzene adsorbent resin (Diaion® HP-20, 9052-95-3,
Sigma-Aldrich, St. Louis, MO, USA) and sequentially eluted with water, 30, 70, and 100% methanol so
that the platycosides and other molecules were separated by polarity differences. According to Ha et
al. [5], most platycosides exist in the 100% methanol fractions, so we only used the sample collected
from the 100% methanol fraction for this study. Using a rotary evaporator, the 100% methanol fraction
was evaporated and the sample residues (powder form) were used as crude platycoside samples [5].
3.6. Biotransformation of Platycosides Using β-D-Glucosidase of A. usamii
The biotransformation of platycosides was conducted based on the procedures of You et al. [33].
Three milligrams of crude platycosides were resuspended in 1 mL of A. usamii crude enzyme extract
and incubated at optimal pH and temperature conditions (6.0 and 40.0 ◦C) in a waterbath shaking at
180 rpm for 2 h. The optimal pH and temperature of crude β-D-glucosidase of A. usamii by pNP-assay
was used for the rapid bioconversion process. The Aspergillus enzyme activities in each experiment
were evaluated by measuring the product level (i.e., free p-nitrophenol molecules). pNP was used
as a substrate, and the level of released free p-nitrophenol molecules was estimated by reading the
absorbance at 405 nm using a microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA).
The biotransformation process was terminated by boiling (100 ◦C) for 10 min. The collected samples
were freeze-dried and stored at
for high-performance liquid chromatography (HPLC) with an evaporative light scattering detector
(ELSD) [33,46].
−
20 ◦C before use. The residue was dissolved in 3 mL of methanol
3.7. Analysis of PE, PD3 and PD via HPLC-ELSD
The platycoside-methanol solution was centrifuged for phase separation. The supernatant was
collected and microfiltered by a Millex LCR syringe filter membrane (0.45 µm cut off, Millipore, Bedford,
MA, USA). Twenty microliters of the filtered sample were loaded on a HPLC system. HPLC analysis
was conducted with a Dionex P680 HPLC (Dionex Corporation, Sunnyvale, CA, USA) equipped
with a ZORBAX® SB C18 reverse phase 5
µm, 4.6 mm
×
250 mm column (Agilent Technologies,
Santa Clara, CA, USA) and PL-ELS 2100 ELSD (Dionex Corporation, Sunnyvale, CA, USA). A solvent
gradient consisting of water with a pH of 2.5 (0.1% trifluoroacetic acid) (A) and acetonitrile (B) flowing
at 1.0 mL/min under the following profile was used: 0–10 min (25–30% B), 10–20 min (30–45% B),
20–40 min (45–100% B), and then equilibrated with 100% B for 10 min. The ELSD was set to a probe

Int. J. Mol. Sci. 2018, 19, 2671
11 of 15
◦
temperature of 70 C, a gain of 7, and the nebulizer gas nitrogen adjusted to 2.5 bar. The chromatogram
peaks were assigned by comparing to the retention times of PE, PD3, and PD standard compounds
under the same conditions.
3.8. Preparation of Platycodin D Using Preparative-HPLC
To separate and recover the purified platycodin D molecules from the platycoside-methanol
solution, preparative HPLC (Young Lin Acme 9000, Younglin Instrument Co., Ltd., Anyang,
Korea) equipped with a semi-preparative ZORBOX SB-C18 5
µ
m, 9.4 mm
×
250 mm column
(Agilent Technologies, Santa Clara, CA, USA) was utilized. The mobile phase consisted of solvent
A (0.1% (v/v) trifluoroacetic acid in HPLC grade water, pH 2.5) and solvent B (methanol) with the
following gradient: 0–20 min, linear gradient from 55% to 65% B; 20–35 min, 65–70% B, 35–50 min,
70–100% B. The injection volume of the samples was 1 mL, the flow rate was 5 mL/min, and the
absorbance was measured using a UV detector (UV VIS detector, Younglin Instrument Co., Ltd.,
Anyang, Korea) at a wavelength of 210 nm.
3.9. Chromatographic and Mass Spectrometric Conditions
The sample molecular weights were determined using an Agilent 6410A triple quadrupole LC/MS
system (Agilent Technologies, MA, USA) equipped with a ZORBAX® SB C18 column 5
µm, 4.6 mm
×
250 mm column (Agilent Technologies, Santa Clara, CA, USA) to qualitatively confirm that the
platycodin D molecules were purified in the samples collected after the preparative-HPLC process.
Mass spectrometric analysis of the platycodin D in the sample was carried out at the Central Laboratory
for Instrumental Analysis at Kyung Hee University Global Campus (Youngin, Korea).
3.10. Evaluation of Anti-Inflammatory Effects of Platycodin D
We conducted an in vitro assay using macrophages to proceed with the qualitative evaluation and
confirmation of functionality of our platycodin D. Commercially available mouse macrophage cell lines
(RAW 264.7, KCLB 40071) were used to evaluate the anti-inflammatory effects of platycodin D via nitrite
and IL-6 assays. Mouse Macrophage Cell Lines (RAW 264.7, KCLB 40071) were purchased from the
Korean Cell Line Bank (Seoul, Korea). The cell lines were cultured under identical culture conditions,
and the cell bioavailability was maintained by MTT assay according to the manufacturer’s instructions.
The cell lines were subcultured in Dulbecco’s modified Eagle’s medium (GIBCO, 12491-023, Carlsbad,
CA, USA) with 10% (v/v) fetal bovine serum (GIBCO, 12483-020, Carlsbad, CA, USA) and 1% (v/v)
antibiotic-antimycotic solution (GIBCO, R25005, Carlsbad, CA, USA) at 37 ◦C in a humidified
atmosphere of 95% air and 5% CO2. Cell enumeration was conducted using a hemocytometer
(Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany); the number of viable cells
was determined using trypan blue dye (Sigma-Aldrich, T8154, St. Louis, MO, USA) exclusion.
To evaluate the anti-inflammatory effects of platycodin D, we intentionally treated commercially
available lipopolysaccharide (LPS) extracted from Escherichia coli O127:B8 (Sigma-Aldrich, L4516, St.
Louis, MO, USA) into cell lines with platycodin D molecules and evaluated the secretion levels of nitrite
and IL-6. These evaluation protocols were adopted from Kim et al. [56]. Specifically, the macrophage
cells were seeded in polystyrene tissue culture-treated, 96-well plates (Corning® 96 Well, #3596,
Corning, NY, USA) at 5
at 37 ◦C for 24 h. The cell lines were treated and incubated for 2 h with 5, 10, or 20
D extracted and purified from our samples. To evaluate the LPS-induced macrophage inflammatory
responses, LPS (0.1 g/mL) was treated and incubated for 18 h. Sample centrifugation (Hanil Scientific
Inc., Hanil Union 32R PLUS, Kimpo, Korea) was carried out at 100
g for 5 min at 4 ◦C to obtain the
medium supernatant from which the macrophages had been removed. The supernatants were used for
the subsequent nitrate and IL-6 assays. The nitrite and IL-6 supernatant concentrations (100 L/well)
in the medium were measured using a commercially available colorimetric nitrite/nitrate assay kit
(Sigma-Aldrich, #23479-1KT-F, St. Louis, MO, USA) and IL-6 assay kit (BD OptEIA Mouse IL-6 ELISA
×
104 cells/well with 200
µ
L of complete cell culture media and incubated
µM of platycodin
µ
×
µ
™

Int. J. Mol. Sci. 2018, 19, 2671
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Kit, 550950, BD Pharmingen, San Diego, CA, USA). The analysis process was conducted according to
the manufacturer’s instructions. The nitrite and IL-6 levels were measured at 540 and 450 nm using
a microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA), respectively [47]. Cell viability
was determined using the MTT Cell Proliferation Kit I (11465007001 ROCHE, #11465007001, Basel,
Switzerland) according to the manufacturer’s instructions.
4. Conclusions
The objective of this work was to use edible bacterial and fungal strains (i.e., Bifidobacterium,
Lactobacillus and Aspergillus spp.) to biotransform platycoside-enriched PR extracts and increase the
level of PD, which likely confers biofunctionality to consumers. The results of this work indicate
that among the 11 microorganism strains tested, AU1004 had the highest β-D-glucosidase activity
and active platycoside E and platycodin D3 bioconversion properties, with a transformation rate of
>99.9% within 2 h. As the PD precursors were converted, the PD content increased proportionally.
Thus, biocatalysis via AU1004 extracellular enzymes could provide a means to overcome the major
factors (e.g., low recovery percentage and time requirements) that limit the development of novel,
platycodin D-enriched products. Our method may cut production costs by eliminating the heating,
and acid hydrolysis of PD production. Moreover, our study contributes to further investigation of the
large-scale production of value-added PR products.
Author Contributions: H.J.A. initiated this work in partial fulfillment of his Ph.D. degree at Seoul National
University under the supervision of G.E.J. and the mentorship of S.K., H.J.Y., and M.S.P. H.J.A. performed the
enzyme, microbiology and cell line experiments under the mentorship of H.J.Y. H.J.A. and S.K. collaboratively
wrote the manuscript and performed the literature review. S.K. and T.V.J. edited and revised the manuscript
based on a non-disclosure research agreement between Middle Tennessee State University and BIFIDO Co., Ltd.
All authors discussed the drafts and approved the final manuscript for publication.
Acknowledgments: This work was carried out with support from the National Research Foundation of
Korea (NRF) grant (No.2017R1A2B2012390) funded by the Korea government (MSIP), High Value-added Food
Technology Development Program (No. 317043-3), Korea Institute of Planning and Evaluation for Technology
in Food, Agriculture, Forestry and Fisheries (IPET), Ministry of Agriculture, Food and Rural Affairs (MAFRA),
and the Faculty Research and Creative Activity Committee (FRCAC) grant (No. 221745) funded by Middle
Tennessee State University (MTSU). The authors wish to thank Lauren B. Mallet at Purdue University for her
review and feedback.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
PR
Platycodi radix
PE
Platycoside E
PD3
PD
Platycodin D3
Platycodin D
Glc
Api
Xyl
β-D-Glucopyranose
β-D-Apiofuranose
β-D-Xylopyranose
Rha
Ara
pNP
BM
LPS
NO
IL
α-L-Rhamnopyranose
α-L-Arabinopyranose
para-Nitrophenol
Basal medium
Lipopolysaccharide
Nitrite
Interleukin
ELISA
MTT
Enzyme-linked immunosorbent assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Int. J. Mol. Sci. 2018, 19, 2671
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