International Journal of Bio-Technology

and Research (IJBTR)

ISSN(P):2249-6858; ISSN(E):2249-796X
Vol. 7, Issue 1, Feb 2017, 19-24
TJPRC Pvt. Ltd.

POTENTIAL OF RESVERATROL PRODUCTION WITH

BIOTRANSFORMATION BY YEAST USAGE

HSIAO-PING KUO, YI-SHENG LIN, JINN-TSYY LAI & SHYUE-TSONG HUANG

Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan
ABSTRACT

Resveratrol, a phytoalexin, excreted naturally by several plants such as grape, Japanese knotweed and peanut.
Also, many of them contain some kinds of glycosides such as piceid which shows less bioactivity. -Glucosidase is an
enzyme that hydrolyzes -glycosidic bonds between the reducing side of glucose and an aryl, alkyl aglycone or
oligosaccharide. Therefore, -glucosidase could be used for deglycosylation of piceid into resveratrol for a commercial
application. In this research, hydrolysis of piceid in Polygonum cuspidatum to resveratrol by a patented microorganism
Dekkera bruxellensis (BCRC 920084), secreting -glucosidase was investigated. In addition, -glucosidase for
enhancing biotransformation efficiency was multiple used within whole cells under condition of liquid suspension.
Then, a series of enlarging experiments were followed by 50-mL, 3-L and 10-L operation volume and the conversion rate
was average 92.55 9.66% after 5 times reused efficiency.

Original Article
KEYWORDS: Resveratrol, Polygonum cuspidatum & Yeast

Received: Dec 21, 2016; Accepted: Jan 06, 2017; Published: Feb 02, 2017; Paper Id.: IJBTRFEB20173

INTRODUCTION
Background

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenolic phytoalexin produced primary for the defensing

system of certain plants against environmental stressors (Burns et al., 2002), was first isolated and identified by
Takaoka in 1940 from the root of white hellebore (Veratrum grandiflorum O. Loes). Later, resveratrol was also
being found in a traditional Chinese herb, P. cuspidatum and other plant species including grapes, peanuts, cocoa,
pomegranates, jackfruit, mulberry, and blueberry (Burns 2002, Bauret al.,2006, Counet et al., 2006, Manach et al.,
2004). Resveratrol might be bound to sugar like piceid for stable purpose and hydrolysized to release resveratrol
against the environmental pressure in plant. Like P. cuspidatum, glycoside was even higher than resveratrol by
four times (Shan et al., 2008). Not surprisingly, Japanese knotweed became most potential material for producing
resveratrol.

-Glucosidase, a kind of cellulase, widely existed in microorganisms and plant fruits. It is known to
hydrolyze glucose from various aglycone structures for enhancing the pharmacological effects of various chemical
compounds, including resveratrol, norisoprenoids, monoterpenes, sesquiterpenes, aliphatic alcohols, volatile
phenols, and benzyl derivatives (Bhatia et al., 2002, Todaro et al., 2008, Gonzaler-Pombo et al., 2011). In this
study, -glucosidase could catalyze the hydrolysis of terminal non-reducing -D-glucose residues in piceid with
the release of -D-glucose. Our patented strain, D. bruxellensis, secreted -glucosidase which possesses more
specific at piceid than other -glucosidase from almond in our previous study.

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20 Hsiao-Ping Kuo, Yi-Sheng Lin, Jinn-Tsyy Lai & Shyue-Tsong Huang

According to that research, glycosides were hydrolyzed by different methods including acid hydrolysis, heating,
enzymatic and microbial conversion (Chen et al., 2014). The results exhibited enzymatic hydrolysis was most effective
compared with microbial conversion, acid or heating hydrolysis. However, pure enzyme is expensive and difficult to make
recovery; as a result, -glucosidase was immobilized for multiple reused. Normally, immobilized enzyme needed very pure
material and immobilized process caused enzyme activity loss and change. Research indicated that many microbes were
capable conversing piceid including fungi, bacterial and yeast. Microbial conversion could be applied by microorganism,
crude and pure extracted enzyme, cell fixation or extraction (Basholli-Salihu et al., 2016). These researches were limited in
laboratory scale and difficult to mass production. In our research, microbial conversion was achieved by a patented
microorganism without -glucosidase purification and was multiple used within whole cells under condition of liquid
suspension without microorganism fixation. This conversion process was accomplished through a series of scale up for
further evaluation of industrial mass production.

METHODS

Strains and Chemical Materials: D.bruxellensis (BCRC 920084) was obtained from the Bioresource Collection
and Research Center, Hsinchu, Taiwan. Standard of resveratrol and piceid were from Sigma-Aldrich. YPD medium was
from Becton Dickinson. Chemicals, including acetonitrile, ethanol, sodium acetate and acetic acid, were from Merck. P.
cuspidatum was purchased from market.

Strain Cultivation and Biotransformation: Yeast was activated in YPD broth at 25 on a shaker until OD 600 10
from 1.2 mL Cryotube and then incubated in Japanese knotweed medium (P. cuspidatum powder 2% in 20 mM pH5
sodium aceate buffer) of 5% inoculation for bioconversion. After 48 hours, we collected the sediment and transferred
whole cell suspension into new Japanese knotweed matrix at the same bioconversion condition. After repeating the
bioconversion step with five times and collecting freeze dried samples. The Japanese knotweed was further extracted and
analyzed the content of resveratrol and piceid.

Piceid and Resveratrol Analysis: Piceid and resveratrol standards were prepared into 0.5-175 mg/L in 50%
ethanol for standard curve establishment. Samples collected from fermentation were freeze dried and extracted piceid and
resveratrol with 50% of ethanol at 70 for 30 min. The extracted samples were diluted and filtered with 0.22 m
membrane before injection (20 L). C18 column (5 m, 4.6 mm x 250 mm, Waters, Milford, MA, USA) was used to
separate compounds and detect signals were set at 307 nm. Contents of resveratrol and piceid were calculated using peak
area relative to standard curves. The multistep gradient method was applied using mixed mobile phases of water with 0.1%
of acetic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 1.5 mL / min. The mobile phase started with 5% of B
and gradually increased to 55% at 25.3 min, and reached 100% from 25.3 to 31 min (Mark et al., 2005).

RESULTS

Bioconversion was demonstrated by using D. Bruxellensis to focus on P. cuspidatumto transfer piceid into
resveratrol in a 250-mL flask (50-mL working volume). After five times multiple use of D. bruxellensis, piceid was
effectively converted to resveratrol and bioconversion efficiency was higher than 96%. Residual piceid content of
P. cuspidatum was lower than 2 mole/g and resveratrol content higher than 31 mole/g after conversion (Figure 1).
The bioconversion efficiency was almost 100% in the first four rounds; as a result, we speculated on the possibility of
different stilbeneglucosides (ex. piceid, resveratroloside and piceatannol glucoside) in P. cuspidatum (Vastanoet al., 2000).

Impact Factor (JCC): 3.4273 NAAS Rating: 3.80

Potential of Resveratrol Production with Biotransformation by Yeast Usage 21

These glucosides were also possible converted to resveratrol; however, piceid was only one glucoside with obvious
bioconversion rate calculation.

According to preliminary study in flask volume test, multiple use of D. bruxellensis has great potential, especially
in P. cuspidatum bioconversion in after a series of enlarge from 5-L
5 glass bottle (3-L
L working volume) to 20-L
20 PP bucket
(10-L
L working volume) was carried out. After five times transferring, piceid was also effectively converted to resveratrol
by multiple use of D. bruxellensis in P. cuspidatumin
cuspidatum expanded scale. Bioconversion rates were 90% and 78% in 5-L
5 glass
bottle (Figure 2) and 20-L
L PP bucket (Figure
(Fig 3) which showed similar trend to flask test. In 5-L
5 glass bottle, bioconversion
rate was almost 100% in the first four repeats and 90% in the last one. Bioconversion rates were
w average about 80% in 20-
L PP bucket.

The more repeats usage of D. bruxellensis,

bruxellensis the more industrial potential. However, cost effectiveness analysis
should be considered. In our research, when increasing repeat usage of D. bruxellensis,, more turbid and glutinous of
conversion medium caused difficult to precipitate of P. cuspidatum. Therefore, the final supernatant was less than 70% and
hard to transfer to a new substrate in the flask test in the fifth time. However, the situation will be
b improved with scale
enlarge. On the basis of conversion rate in each scales of the fifth transfer, more than five repeats usage of D. bruxellensis
should be considered.

The five times bioconversion data in 250-mL

250 and 20-L
L scale showed no significant difference
diff after statistical
analysis (Table 1). But, in a 5-L
L scale, the final repeat significant lower than others (P>0.05). This result showed the
stability of the final repeat should be considered. However, the phenomenon was only found in 5-L
5 scale, and it might be
due to experimental deviation. As a result of series experiment, the further repeats use of D. bruxellensis
(more than 5 times) should be evaluated.

Microbial conversion was accomplished by D. bruxellensis without enzyme purification and was multiple used
within whole yeast cells without microorganism fixation. Our patented yeast secreted -glucosidase
glucosidase which hydrolyzed
piceid in P. cuspidatum to resveratrol. The bioconversion repeats were fulfilled five times, and conversion rates were
average higher than 92% in a series of enlarge scale. However, after statistical analysis of bioconversion date, the repeats
used (more than 5 times) of D. bruxellensis,
bruxellensis due to mass production, should be further considered.

Formula and Equation

R0: Resveratrol content (mole/g)

mole/g) in P. cuspidatum before bioconversion.

R1: Resveratrol content (mole/g)

mole/g) in P. cuspidatum after bioconversion.

P0: Piceid content (mole/g) in P. cuspidatum before bioconversion.

P1: Piceid content (mole/g) in P. cuspidatum after bioconversion.

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22 Hsiao-Ping Kuo, Yi-Sheng Lin, Jinn--Tsyy Lai & Shyue-Tsong Huang

Figures and Tables

Figure 1: Concentration of Resveratrol and Piceid with of P. cuspidatum

uspidatum and
Bioconversion Rate Along with Five Times Repeat in a 250-mL
250 Flask Testing

Figure 2: Concentration of Resveratrol and Piceid with of P. cuspidatum

uspidatum and
Bioconversion Rate along with Five Times Repeat in a 5-LL Glass Bottle Testing

Figure 3: Concentration of Resveratrol and Piceid with of P. cuspidatum

uspidatum and
Bioconversion Rate along with Five Times Repeat in a 20-L L PP Bucket Testing

Impact Factor (JCC): 3.4273 NAAS Rating: 3.80

Potential of Resveratrol Production with Biotransformation by Yeast Usage 23

Table 1: Resveratrol Concentration (mole/g, MeanSD, n=3) of Different

Bioconversion Repeats under Different Scale Operation
Repeat Times 250mL Shake 5L Glass Bottle 20L PP Buckets
R1 37.899.56 31.453.36 30.345.07
R2 40.887.12 36.853.32 33.644.53
R3 38.811.52 37.792.54 35.521.00
R4 43.165.29 37.304.53 34.095.37
R5 31.0411.34 28.861.37* 28.911.03
Significant difference from R2 (p < 0.05).

Bold number is statistical basis for comparison among different scale.

CONCLUSIONS

Resveratrol, a polyphenolic phytoalexin produced primary for the defensing system of certain plants against
environmental stressors. In P. cuspidatum that piceid was four times higher than resveratrol.

-Glucosidase could be used for deglycosylation of piceid into resveratrol for a commercial application. In this
research, our patented yeast secreted -glucosidase which hydrolyzed piceid in P. cuspidatum to resveratrol. The whole
cells can be effectively transformed after repeated used. And bioconversion repeats were fulfilled five times, and
conversion rates were average higher than 92% in a series of enlarge scale. However, after statistical analysis of
bioconversion date, the repeats used (more than 5 times) of D. bruxellensis, due to mass production, should be further
considered.

ACKNOWLEDGMENTS

This research was supported by the Ministry of Economic Affairs of Taiwan (105-EC-17-A-22-0525).

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Impact Factor (JCC): 3.4273 NAAS Rating: 3.80