Isolation of Prebiotics from

Artocarpus integer’s Seed

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 Team Members
Joel-Ching-Jue Wong , Siew-Ling Hii , and Chen-Chung Koh

Centre for Research of Innovation & Sustainable Development (CRISD),

School of Engineering and Technology,
University of Technology Sarawak, 96000 Sibu, Sarawak, Malaysia.

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 Outline
• Introduction
• Methodology
• Results and Discussion
• Conclusions
• Publication
• References

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Introduction

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• Sarawak carries abundant biodiversity and has endemic flora and
fauna that are different from other regions of the country.
• Examples: terung asam, dabai, engkala, belimbing hutan.
• Underutilized plant species only receive little investment through
formal research and development.

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• Most of the underutilized plant species are reported as natural
sources of bioactive compounds and possess the potential to be
further developed into functional foods.

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Prebiotics
• A substrate that is selectively utilized by host microorganisms
conferring a health benefit (Gibson et al., 2017).
• Examples: inulin and fructooligosaccharides.

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Artocarpus integer
• Greek words “artos” (bread) and “karpos” (fruit).
• Locally known as buah cempedak (Malay), jackfruit (English), and
bolomi (Mandarin).
• Originates in forests countries in the Indian sub-continent and
Southeast Asia countries.

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Photo 1. The fruit of
A. integer

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• Extraction represents the primary procedure in the studies of plants
because it is a prerequisite to extract the desired bioactive
compounds from the plants.

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Microwave-assisted Extraction (MAE)
• Innovative extraction technique.
• Reduced extraction time and solvent volume; improved analyte
recovery and reproducibility but appropriate conditions are required
to prevent thermal degradation (Kaufmann & Christen, 2002).

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 Objectives of Study
1. To study the various parameters affecting the microwave-assisted
extraction of prebiotics from underutilised plant species Artocarpus
integer.
2. To determine the in vitro resistance of isolated prebiotic materials
to human gastric acid and enzymatic digestion.
3. To evaluate in vitro fermentability of isolated prebiotic materials by
selected pure cultures of probiotics and gut pathogen.

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 Significance of Study
1. To create awareness of the underutilised plant resources of
Sarawak.
2. To identify new sources of prebiotic materials to be used as
functional food ingredient.

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Methodology

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 Figure 1. A. integer’s Fibrous Powder Preparation

Wet milling
Fruits Seeds Pomace
(1 seed: 1 water)

Washing
Drying
(5 minutes, 1 Filtration Grinding
(50°C, 18 hours)
pomace: 2 water)

Sieving A. integer Fibrous

(250 m) Powder

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 Experimental design

• Type of design: Response surface methodology

• Independent variables:
Microwave power (watt)
Extraction time (second)
Solvent-sample ratio
• Response:
Total carbohydrate content (mg/L)

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 Figure 2. Microwave-assisted Extraction

Mixing with Microwave-

Centrifugation Deproteinisation
Deionised Water assisted Extraction

Dialysis
Centrifugation (against Deionised Storage at 4 °C
Water for 24
hours)

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 Figure 5.
In vitro Digestion by Artificial Gastric Juice

Preparation of pH Adjustment Mixing (gastric juice +

Artificial Gastric Juice (pH 1, 2, 3, 4) sample/inulin)

Incubation Determination of
Percentage of
Total Carbohydrate
(37 °C for 5 hours) Hydrolysis
and Reducing Sugar

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 Figure 6.
In vitro Enzymatic digestion

Preparation of α- Incubation
Mixing (α-amylase +
amylase in Sodium
sample/inulin) (37 °C for 5 hours)
Phosphate Buffer

Determination of
Percentage of
Total Carbohydrate
Hydrolysis
and Reducing Sugar

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 In Vitro Fermentation by Selected Bacterial Strains

• Selected probiotic strains:

 Lactobacillus acidophilus DSM20079
 Lactobacillus casei DSM20011

• Selected gut pathogen:

 Escherichia coli DSM1103

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Figure 7. In Vitro Fermentation by Selected Bacterial Strains

Replacement of
glucose in MRS and
Inoculation
TS broth by
sample/inulin

Viable count
Incubation
(at 0, 24, 48, 72
(37 °C up to 72 hours)
hours)

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 Data Analysis

• Design-Expert® Version 10
• IBM SPSS Version 23
• One-way Analysis of Variance
• Tukey test

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Results and Discussion

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• Optimized extraction parameters:
Microwave Power – 1500 W
Extraction Time – 180 s
Solvent-sample Ratio – 1000:1

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 Table 9.
Hydrolysis of A. integer and Inulin by Artificial Gastric Juice

% Hydrolysis
pH
A. integer Extract Inulin
1 6.14±0.71a 0.00±0.00a
2 7.12±0.36ab 0.72±0.01b
3 8.98±0.71bc 0.90±0.06c
4 10.23±0.36c 1.16±0.02d

All results are expressed as mean ± standard deviation of three replicates. Results of the same column
followed by different superscripts were significantly different (Tukey test, p < 0.05).

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 Table 10.
Hydrolysis of A. integer and Inulin by α-amylase at pH 7.

% Hydrolysis
A. integer extract 0.16 ± 0.12a
Inulin 0.09 0.04a

All results are expressed as mean ± standard deviation of three replicates.

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Figure 8: Growth profile of L. acidophilus in media containing A. integer extract and inulin.

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Figure 9: Growth profile of L. casei in media containing A. integer extract and inulin.

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Figure 10: Growth profile of E. coli in media containing A. integer extract and inulin.

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• The presence of significant growth of L. acidophilus and L. casei in
media containing A. integer extract and inulin indicated that L.
acidophilus and L. casei were able to utilise them as carbon sources.
• Both A. integer extract and inulin can also support the growth of E.
coli rather than inhibiting its growth at earlier stages.

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• Other reported studies have also demonstrated the utilization of
prebiotics by pathogenic bacteria:
• The growth of Clostridia and Enterobacteria in arabinoxylan substituted
media (Vardakou et al., 2008)
• The growth of Clostridia in oligosaccharide-substituted media (Rada et al.,
2008)
• The growth of Salmonella in media containing Gigantochloa levis extract
(Azmi et al., 2012)

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Conclusions

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• A. integer extract showed prebiotic properties such as:
Resistant to gastric juice digestion
Resistant to enzymatic digestion
Support the growth of probiotics such as L. acidophilus and L. casei

• A. integer extract has the potential to be used as an ingredient to

develop functional foods.

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Publication

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Acknowledgments

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• Research permit granted by the Sarawak Biodiversity Centre (Permit
No.: SBC-2018-RDP-17-KCC)
• University of Technology Sarawak (UCTS/RESEARCH/1/2018/10;
UCTS/RESEARCH/1/2019/03)

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 References
Azmi, A. F. M. N., Mustafa, S., Hashim, D. M. & Manap, Y. A. (2012). Prebiotic
Activity of Polysaccharides extracted from Gigantochloa levis (Buluh beting)
shoots,” Molecules, 17(2), 1635–1651.
Gibson, G.R., Hutkins, R., Sanders, M.E., et al. (2017). Expert Consensus
Document: the International Scientific Association for Probiotics and Prebiotics
(ISAPP) Consensus Statement on the Definition and Scope of Prebiotics. Nature
Reviews Gastroenterology & Hepatology, 14, 491–502.
Invasive Species Compendium (2019). Artocarpus integer (Champedak). [Online]
Available at: <https://www.cabi.org/isc/datasheet/1834> [Accessed 6 April 2019].

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Kaufmann, B. & Christen, P. (2002). Recent Extraction Techniques for Natural
Products: Microwave-assisted Extraction and Pressurized Solvent Extraction.
Phytochemical Analysis, 13, 105-113.
Rada, V., Nevoral, J., Trojanová, I., Tománková, E., Šmehilová, M. & Killer, J.
(2008). Growth of Infant Faecal Bifidobacterial and Clostridia on Prebiotic
Oligosaccharides in in vitro Conditions. Anaerobe, 14(4), 205–208.
Vardakou, M., Palop, C. N., Christakopoulos, P., Faulds, C. B., Gasson, M. A. &
Narbad, A. (2008). Evaluation of the Prebiotic Properties of Wheat Arabinoxylan
Fractions and Induction of Hydrolase Activity in Gut Microflora. International
Journal of Food Microbiology, 123(1-2), 166–170.

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End of Presentation

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Questions and Answers

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