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Survey the Polyphenol Extraction from Artocarpus Altilis of Vietnam by

Enzyme method

Conference Paper · May 2014

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Proceeding of 2nd AFSSA Conference on Food Safety and Food Security
August 15 – 17, 2014
Asian Food Safety And Security Association
ISSN: 2306-2150

Survey the Polyphenol Extraction from Artocarpus

Altilis of Vietnam by Enzymatic Method
Dam Sao Mai1, Truong Hoang Duy1, Ngo Ke Suong2, Tran Thi Mai Anh1, Nguyen Thi Mai
Huong1
1
Institute of Biotechnology and Food technology – Industrial University of Ho Chi Minh City, Vietnam
2
Dong Nai University of Technology, Bui Trong Nghia, Trang Dai Ward, Bien Hoa city, Vietnam

Email address:
damsaomai@gmail.com (Dam Sao Mai)

Abstract:

Polyphenols has potential applications asantioxidant component that obtained good application in the
food processing, medicine and cosmetic. Artocarpus altilis is a very common plant in Asian country and in
Vietnam. The leaves of this plant contain high polyphenols. The polyphenols extraction was surveyed, with
pectinase and cellulase using. The experiment was conducted according to a central composite design, with
the following variables: with pectinase, solvent pH (pH-P, 3.0–5.0), the concentration of pectinse (C-P, 0.1-
0.3%), solvent temperature (ST-P, 30–50 °C) and extraction time (ET-P, 20–40 min); with cellulase, solvent
pH (pH-C, 4.0–6.0), the concentration of cellulase (C-C, 0.1-0.3%), solvent temperature (ST-C, 40–60 °C)
and extraction time (ET-C, 20–40 min).
Artocarpus altiliswere ground with the solvent, vacuum filtered, centrifuge in 20 min with 6000 rpm and
evaluated for polyphenol contents. A highly significant model was obtained for polyphenol extraction
efficiency (PEE), which was positively affected by ST-P (ST-C) and C-P (C-C), and inversely affected by
pH. The most extraction conditions with pectinase were pH, 4.0; C-P, 0.1%; ST-P, 40 °C and ET-P, 25 min;
at such conditon, 68.607 mg/g raw material was extracted. The most extraction conditions with cellulase
were pH, 5.0; C-C, 0.2%; ST-C, 50 °C and ET-C, 25 min; at such condition, 131.927 mg/g raw material was
extracted.

Keywords: Artocarpus altilis, polyphenols, pectinase, cellulase, antioxidant

prevent the risk of cancer, high blood pressure, heart

1. Introduction
Sake (Artocarpus altilis) is under the
Moraceae, grown in the tropical area of Southeast
Asia and the Pacific Islands. Sake is the big trees
with the high about 15 - 20m [1], [3], [7].
Pradhan et al. (2013) [5] have published about
the presence of phenolic compounds and terpenoids
in sake via using ethyl acetate and mathanol to
extract.
Polyphenols are known as an antioxidant,
found in onions, cocoa, tea, apples,… According to
the American Cancer Society, polyphenols help
disease and diabetes. By chromatography and
spectroscopic methods, scientists have isolated and
identified the compounds in sake, such as
flavonoids, triterpene, chalcone, stilbene ... [2].
Flavonoids are an important group of polyphenol
compounds and biological activity. Therefore, the
study of extraction of phenolic compounds with
biological activity in food applications will be
highly beneficial.
Some recent studies have also published
polyphenol extraction by using pectinase and
cellulase. [4].

14
 In this study, we extracted polyphenols from enzyme changes in temperature: 300C, 350C, 400C,
the leaf of sake by using pectinase and cellulase. 450C, 500C (enzyme cellulase: 400C, 450C, 500C,
550C, 600C). The results was shown in Figure 1.
2. Materials and Methods The velocity of the enzyme reaction will
increase with temperature, but it increases only to a
Materials: Sake leaves that were used in this study certain limit.
were collected from Vinh Loc Industrial Park, Binh
Chanh, Vietnam The highest polyphenol content at 400C when
using the enzyme pectinase and 500C with cellulase
Basic extraction: Material was extracted in water enzyme.
with enzyme pectinase/cellulase, solution was
vacuum filtered, centrifugal in 20 min with 3.2. Effect of the extracted time on the extraction:
6000rpm/min. Fixed pH 4.5 (pectinase), pH 5 (cellulase),
Survey the effect of pectinase enzyme on the 0:15% enzyme concentration, the ratio of raw
extraction: four surveys were done: extraction material/water solvent: 1:8, temperature extract of
temperature (30, 35, 40, 45, 500C); extraction time pectinase enzyme 400C (500C cellulase enzyme).
(20, 25, 30, 35, 40 minutes); pH (3, 3.5, 4, 4.5, 5); Changing time of 20 minutes, 25 minutes, 30
enzyme concentrations (0.1, 0.15, 0.2, 0.25, 0.3%). minutes, 35 minutes, 40 minutes. The results were
shown in Figure 2.
Survey the effect of cellulase enzyme on the
extraction: four surveys were done: extraction Increasing extraction time, the reaction rate
temperature (40, 45, 50, 55, 600C); extraction time increases. Polyphenol content increased slightly
(20, 25, 30, 35, 40 minutes); pH (4, 4.5, 5, 5.5, 6); from 25 to 30 minutes. Polyphenols are only stable
enzyme concentrations (0.1, 0.15, 0.2, 0.25, 0.3%) in acid environment, and unstable in base
environment. So there should be appropriate
Methods of analysis: the quantification of total extraction time for enzyme reaction.
polyphenols by the Folin - Denis method [6]
3.3. Effect of the extracted pH on the extraction
Data analysis: All experiments were repeated 3
times. Data is processed by ANOVA - one way using Fixed time of 25 minutes, 0:15%
statistical software Statgraphics Centurion XV to concentration of enzyme, the ratio of raw
test differences between pairs of samples with a material/water solvent: 1:8, temperature extract of
significance level of 5%. pectinase enzyme 400C (500C cellulase enzyme), pH
changes: 3; 3.5; 4; 4.5; 5 (cellulase: 4, 4.5, 5, 5.5, 6).
The results were shown in Figure 3.
3. Results and Discussion
pH affects to reaction of the enzyme and
3.1. Effect of temperature on the extraction durability. Polyphenol content between different pH
Fixed time of 20 min, pH 4.5 (pectinase), pH values is not much difference. The highest
5 (cellulase), 0.15% enzyme concentration, the ratio polyphenol content at pH 4 (enzyme pectinase), pH
of raw material/water solvent: 1:8. Pectinase 5 (enzyme cellulase).
5   29.7   29.0   2.4   11.5  
27.3  
Polyphenol  contents  

11.4  
Polyphenol  contents  

26.3   26.7  
4   2.3   11.0  
2.2   10.7  
(mg/g)  

(mg/g)  

3  
2.1   10.1  
2  
2  
1   1.9  
0   1.8  
40   45   50   55   60   30   35   40   45   50  
Extraction  temperature  ( C)  
0
Extraction  temperature  (eC)  
(a) (b)
Fig 1. Effect of temperature to extract polyphenol. a) enzyme cellulase; b) enzyme pectinase)

15
 5  

Polyphenol  contents  
Polyphenol  contents   3  
37.0   38.0   14.6   15.1  
4   34.3   34.3   33.2   14.0   14.3  
12.4  

(mg/g)  
(mg/g)  

3   2  

2  
1  
1  

0  
20   25   30   35   40   0  
20   25   30   35   40  
Extraction  time  (min)  
Extraction  time  (min)  
(a) (b)
Fig 2. Effect of extraction time to extract polyphenol. a) enzyme cellulase; b) enzyme pectinase)

3  
Polyphenol  contents  

Polyphenol  contents  
5   44.1   20.1   21.3  
43.4   43.1   19.4  
39.7   2.5  
4   35.7   15.25   15.26  
(mg/g)  

2  

(mg/g)  
3   1.5  
2   1  
0.5  
1  
0  
0   3   3.5   4   4.5   5  
4   4.5   5   5.5   6  
Extraction  pH   Extraction  pH  
(a) (b)
Fig 3. Effect of pH to extract polyphenol. a) enzyme cellulase; b) enzyme pectinase)

3.5. Effect of the concentration of enzyme on the

When excess substrate, the reaction rate
extraction
increased with increasing concentrations of enzyme
Fixed time of 25 minutes, pH 4 (pectinase), but when the concentration of enzyme saturation
pH 5 (cellulase), the ratio of raw material/water with substrate concentration, reaction rate did not
solvent: 1:8, temperature extract of enzyme change or not increased with increasing enzyme
pectinase 400C (500C enzyme cellulase), Changing concentration.
enzyme concentration of 0.1%, 0.15%, 0.2%,
3.6. Effect of the extracted time on the extraction
0.25%, 0.3%. The results was shown in Figure 4.
Total 4 times extraction using pectinase
Extractable polyphenol content corresponding
enzyme, polyphenol content obtained is
to the enzyme concentration is not much difference.
68.607mg/g, and when using enzyme cellulase with
Increasing concentrations of 0.1 - 0.2%, the
4 times extraction, polyphenol content obtained is
concentration of polyphenols increased and
131.927mg/g.
decreased slightly at the next level.
5   2.5   23.1   21.9  
Polyphenol  contents  
Polyphenol  contents  

21.1   21.3  
48.3   46.3   19.1  
4   44.1   44.6   2  
38.7  
(mg/g)  
(mg/g)  

3   1.5  

2   1  

1   0.5  
0   0  
0.1   0.15   0.2   0.25   0.3   0.1   0.15   0.2   0.25   0.3  
Concentration  of  enzyme  (%  v/m)   Concentration  of  enzyme  (%  v/m)  

(a) (b)
Fig 4. Effect of concentration of enzyme to extract polyphenol. a) enzyme cellulase; b) enzyme pectinase)

16

4. Conclusion
In this study we found that temperature, time,
pH, enzyme concentration are the factors that is
affecting the extraction process.
The extraction method using enzyme
pectinase after 4 times extraction, total obtained
polyphenol content is 68.607mg/g at 400C
temperature, time of 25 minutes, the ratio of raw
material/water solvent 1:8, pH 4, concentration of
enzyme 0.2%. And when using enzyme cellulase
after 4 times extraction, the total obtained
polyphenol content is 131.927mg/g at 500C
temperature, time of 25 minutes, the ratio of raw
material/water solvent 1:8, pH 5, concentration of
enzyme 0.2%.
So using cellulase in this case will give more
affection on the polyphenol extraction from sake
leaves. The researched method is easy to apply in
the industry. The product can be used in food
industry
Acknowledgements
Thank to the Institute of Biotechnology and
Food technology – Industrial University of Ho Chi
Minh City, Vietnam where we completed this study.
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