 *Corresponding author.

Email: mkhairi@umt.edu.my

eISSN: 2550-2166 / © 2020 The Authors. Published by Rynnye Lyan Resources

  Food Research 4 (4) : 1303 - 1311 (August 2020)
  Journal homepage: http://www.myfoodresearch.com

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Ramification of pH in pectinase-assisted extraction on the antioxidant capacity

of Arabica spent coffee ground (SCG) extract

1Shukri, N.A., 1Mohd Zin, Z.,1MohdMaidin, N., 2Hasmadi, M. and 1,*Zainol, M.K.

1Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, Mengabang Telipot, 21030,

Kuala Nerus, Terengganu, Malaysia
2Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia

Article history:

Received: 15 February 2020

Received in revised form: 26

March 2020

Accepted: 1 April 2020

Available Online: 22 April

2020

Keywords:

Spent coffee ground (SCG),

Pectinase assisted extraction,

Antioxidative activities,

Antimicrobial activities

DOI:
https://doi.org/10.26656/fr.2017.4(4).070

Abstract

Spent coffee ground (SCG) is the by-product of coffee processing that is produced up to

45% in coffee beverage and instant coffee processing and is believed to contain high

amounts of antioxidants. Despite the fact that SCG has exerted many advantages, the

information on obtaining the antioxidant values using enzymes are still scarce. The

objective of the study was therefore to determine the total phenolic and flavonoid content

of antioxidant activity and antimicrobial activity in Arabica SCG extracted using pectinase

at different pH values. Arabica SCG was extracted using pectinase at pH 3, pH 4, pH 5,

pH 6 and pH 7 and analyzed for its anti-microbial activity and antioxidant properties

(DPPH, FRAP, total phenolic and total flavonoid content along with individual flavonoids

using HPLC). Arabica SCG was extracted using pectinase at pH 3, pH 4, pH 5, pH 6 and

pH 7 and analyzed for its antimicrobial activity and antioxidant properties (DPPH, FRAP,

total phenolic and total flavonoid content along with individual flavonoids using HPLC).

The result showed that the antioxidant capacity of the SCG extract at pH 4 exhibited

higher DPPH and FRAP values. The total phenolic and flavonoid content exhibited in

(1.38±4.42, mg GAE/g sample and 22.57±0.27 mg QE/g of dry sample). Flavonoids

namely quercetin, kaempferol, rutin, gallic acid, catechin, epigallocatechin gallate, p-

coumaric acid and myricetin were present in all samples at various levels. The SCG in pH

7 extract showed the highest concentrations of the individual flavonoid compound in the

sample and the highest inhibition zone on Pseudomonas aeruginosa and Staphylococcus

aureus. Thus, the optimum pH of the pectinase which is pH 4-5 had provided the highest

yield in antioxidant capacity and activity of the Arabica SCG extract with this extraction

method that can be used for food preservation as well as in the nutraceutical industry.

1. Introduction

Coffee is a brewed drink that was introduced in New

York City in the mid-1600s and is one of the most

widely consumed drinks made from roasted coffee beans

around the world (Zhou and Hyppönen, 2019). The high

and growing demand for coffee has led to large

quantities of by-products such as silver-skin coffee, spent

ground coffee (SCG), coffee husk and coffee-cut stem to

the environment. SCG is the by-product or residue in the

process of coffee powder treated with steam or hot water

in the preparation of instant coffee. SCG is obtained

from coffee processing where 45% of this by-product is

generated in coffee beverage and instant coffee

manufacturing (Cruz et al., 2014).

Coffee by-products such as SCG still contain a

considerable amount of antioxidants and phenolic

compounds such as rutin, catechin, quercetin, and others.

The major polyphenol in coffee is known as chlorogenic

acid (Wungrath et al., 2016). The antioxidant content

and nutrient composition of the coffee and by-products

vary depending on the processing, preparation and

storage (Soares et al., 2015). A considerable amount of

antioxidants remains in SCG after brewing and more

attention has to pay in recycling the SCG for healthier

development (Choi and Koh, 2017). The antioxidant

plays roles to neutralize excess free radicals for

preventing cells against free radical damage and

contributing to the prevention of disease (Lobo et al.,

2010). Yet, the antioxidant can be extracted in many

other different techniques and one of them is enzyme-

assisted extraction using pectinase, cellulase and others

https://orcid.org/0000-0003-3629-586X

https://orcid.org/0000-0002-1344-8109

https://orcid.org/0000-0001-8637-5257

https://orcid.org/0000-0002-6387-9927

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(Jooste et al., 2013).

Enzyme-assisted extraction seems to be a potential

alternative to traditional solvent extraction methods and

is gaining greater attention because it is an effective,

sustainable and environmentally friendly extraction

technology (Puri et al., 2012; Nadar et al., 2018). This

method is typically used in leather making, skincare

products, degumming of soybeans oil, juice processing

and beer clarification (Puri et al., 2012). The role of the

enzyme in the extraction is to destroy the cell wall matrix

of the sample and especially to clarify the cloudy food

product. The common enzyme that is used in this

assisted extraction is pectinase, cellulase and

hemicellulose (Jooste et al., 2013). Pectinases were

known to be stable at pH 2.5-8.0 and active at a

temperature below 50°C (Acufia-Argfielles et al., 1995).

The pH can affect the efficiency of extraction and the

greatest yield extraction is always at the optimum pH

value (Maimulyanti and Prihadi, 2014).  Thus, pectinase

contributes a lot in the food industry where it acts as

juice clarifier, colour and yield improver in fruit mash

treatment and used in degumming fibre crops (Singh et

al., 2019).

The residues from the coffee industry are graded as

highly pollutant due to the presence of organic material,

specifically tannins, caffeine and polyphenols (Chanakya

and Alwis, 2004). The major challenges of conventional

extraction are time consuming and use a large amount of

solvent (Ajila et al., 2011). Therefore, the purpose of this

study was to determine the antioxidant activities and to

measure the total phenolic and total flavonoid content

along with the evaluation of antimicrobial activity in

Arabica SCG extracted using pectinase at different pH

values.

2. Materials and methods

Local Arabica (Coffea arabica L.) coffee was

purchased from Kilang Kopi Fama, Banting (Selangor,

Malaysia). The preparation of SCG was conducted using

a French press coffee maker. The roasted coffee beans

freshly were grounded using a coffee grinder for 90 s to

a course-size ground. Hot water (98°C) of 500 mL was

added to 40 g of coffee ground in the French coffee press

before slowly pushed down the plunger after brewing for

2 mins. The coffee was poured out and the spent coffee

ground (SCG) was collected to be dried out on the tray in

the cabinet dryer for 24 hrs at 60°C. The collected dried

SCGs were placed in ambient and dark containers in an

airtight container prior to the extraction process.

2.1 Pectinase-assisted extraction

Arabica SCG of 8 g was added to a solution

containing 0.8 mL of 0.2 M commercial pectinase in the

liquid form and 120 mL of 200mM sodium acetate

buffer in every pH 3, 4, 5, 6 and 7. The mixture was

shaken gently in a water bath shaker at 37°C for 1 hr.

The solution is then immediately placed in a water bath

at 90°C for 5 min to stop enzymatic hydrolysis. Then,

the solution was filtered using Whatman paper (no. 4) by

vacuum pump to obtain the liquid. The liquid was

concentrated in a rotary evaporator (Buchi Rotavapor R-

200, New Hampshire, USA) at 60°C. The extract

obtained was then kept in amber bottles and store in the

chiller prior to further analysis (Zohdi and Amid, 2013).

2.2 Total phenolic content

The total phenolic compounds in SCG extracts was

determined using Folin-Ciocalteu reagent according to

the colourimetric method described by Ng et al. (2019).

An aliquot (1 mL) of every sample was diluted into 50

mL of stock solution. From the stock solution, 1 mL was

added to 17.9 mL of distilled water in and 0.5 mL of

Folin-Ciocalteu reagent and left to stand for 1 min. Then,

1.5 mL of 20% sodium carbonate was added to the

mixture. The sample prepared was then left at room

temperature for 2 hrs in dark. The absorbance values

were taken at 765 nm, resulting in mg GAE per gram of

sample extract (mg GAE/g) expressed as gallic acid

equivalent.

2.3 Total flavonoid content

The content of flavonoids in the extracts was

determined using Hau et al. (2018) with slight

modifications. Sample of 50 mg were mixed with 1.5 mL

methanol, 0.1 mL 10% aluminium chloride, 0.1 mL 1 M

potassium acetate and 2.8 mL distilled water. The

mixture was then incubated at room temperature for 30

mins. The absorbance of the reaction mixture taken at

415 nm. The result was expressed as mg of quercetin

equivalent (QE) per gram of sample extract (mg QE/g).

2.4 Determination of antioxidative properties

2.4.1 Antioxidant activity based on 2,2-diphenyl-2-

picrylhydrazyl (DPPH) radical scavenging assay

The capacity of trapping of free radical DPPH was

evaluated according to the method described by Malik et

al. (2017) with slight modification. DPPH solution at the

concentration of 6.1 × 10-5 M was prepared in ethanol.

Diluted extract of 75 µL was mixed with 3 mL of the

DPPH solution. After 1 hr, the absorbance was recorded

at 515 nm using methanol with DPPH as negative

control while quercetin as a positive control. All

operations or conducted in dark or dim light. The

inhibition percentage (IP) of the DPPH by the extract

was calculated according to this formula:

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IP (%) = [(A0 min - A60 min) / A0 min] x 100

Where A0 min is the absorbance of the blank at t = 0

min, and A60 min is the absorbance of samples at 60

mins. The result expressed as μmol Trolox equivalent

(TE) per gram of sample on a dry basis, through a dose-

response curve for Trolox (0-350 μM).

2.4.2 Ferric reducing antioxidant power (FRAP)

analysis

The total antioxidant potential of a sample was

determined using the ferric reducing ability of plasma

FRAP assay by Chong et al. (2018) as a measure of

antioxidant power. A potential antioxidant will reduce

the ferric ion (Fe3+) to the ferrous ion (Fe2+); the latter

forms a blue complex (Fe2+/TPTZ), which increases the

absorption at 593 nm. Briefly, the FRAP reagent was

prepared by mixing acetate buffer (300 mM, pH 3.6), a

solution of 10 mM TPTZ in 40 mM HCl, and 20 mM

FeCl3 at 10:1:1 (v/v/v). Three millilitres of the FRAP

reagent was added to each test tube containing 0.1 mL

diluted sample and mixed thoroughly. The absorbance

value of the reacted mixture was observed at 593 nm

after 0 min. The results were expressed as μmol trolox

equivalent/g dried weight.

2.5 Antimicrobial activity

The well-diffusion method was used to determine the

antimicrobial activity of SCG extract extracted with

different pH. The SCG extract was tested on Muller-

Hilton Agar (MHA) that has been swab with Bacillus

subtilis, Staphylococcus aureus, Escherichia coli and

Pseudomonas aeruginosa. The well was prepared by

punching the agar with a sterile micropipette tip (6 mm).

Fifty microliters of the diluted extract at the

concentration of 300mg/mL was introduced into the well

and let it diffuse at room temperature. Gentamicin

antibiotics were used as positive control while sterile

distilled water was used as a negative control. The plates

were incubated at 37°C for 24 hrs and the diameter of the

inhibition zone was measured in centimetres (cm)

(Duangjai et al., 2016).

2.6 Determination of individual flavonoids

The flavonoid standards that were used in HPLC

determination were flavonol (quercetin, myricetin,

kaempferol), catechin, flavones (apigenin, luteolin) and

flavanone (naringin). Firstly, 5 mg of the samples were

refluxed in 6 M HCl at 90°C for 2 hrs with 60% (v/v)

aqueous methanol (Mohd Zainol et al., 2009). HPLC

analyses were performed using an  Analytical High -

Performance Liquid Chromatography (HPLC)

(Shimadzu, Japan) with 4 solvent delivery system

quaternary pump (LPG 3400 SD) equipped with a diode

array detector (DAD 3000) with 5 cm flow cell, a

manual sample injection valve equipped with a 20 µl

loop and Chromeleon 6.8 system manager as data

processor. The separation was achieved by a reversed-

phase Acclaim TM 120 C18 column (5 µm particle size,

i.d. 4.6 x 250 mm). The stock solution of concentration

40 ppm was prepared by dissolving phenolic acid and

flavonoids in 0.5mL HPLC-grade methanol (60%)

followed by sonication for 10 min and the resulting

volume was made up to one millimetre with the solvent

for the mobile phase (acetonitrile and 1% aqueous acetic

acid 1:9). The standard and working solutions were

filtered through a 0.45 µm PVDR-syringe filter and the

mobile phase was degassed before the injection of the

solutions. The injection process in flow rate was adjusted

to 0.7 mL/min with the column was thermostatically

controlled at 28°C and the injection volume was kept at

20 µL Total running time was 31 mins and pre-

equilibrium time was allowed at 5 mins. HPLC

chromatograms were detected using a photodiode array

UV detector at three different wavelengths (272, 280 and

310 nm) according to the absorption maxima of analysed

compounds. Each compound was identified by its

retention time and by spiking with standards under the

same conditions (Seal, 2016; Shin et al., 2019).

2.7 Statistical analysis

The statistical comparison was performed with one-

way analysis (ANOVA) using Fisher's Least Significant

Difference (LSD) test and values of p < 0.05 were

considered significant. The statistical software used to

analyze the data is Minitab 19.

3. Results and discussion

3.1 Extraction yield

Figure 1 shows that sample extract at pH 7 and pH 6

have a higher yield which is 52.96±1.88% and

51.48±5.99%, respectively. The yield of Arabica SCG

extracts at pH 3 and pH 4 decreases due to the unfolding

and decomposition of the enzyme structure in low acidic

medium (Zohdi and Amid, 2013).  The enzymes contain

ionic groups where the active site is in a stable form.

Therefore, the variation of a medium such as pH can

change its ionic form that affects the reaction rate and the

enzyme activity. Enzyme activity decreased at lower

buffer pH when homogenizing with the sample which

affects less enzyme solubilization in crude extract (Chen

et al., 2006). In addition, the results obtained by Bravo et

al. (2013) showed a similar trend to the study in which

the yield of SCG extract increased as the pH increased.

In pectin hydrolysis, pectinases group which consists of

polygalacturonases and pectin lyase playing a relevant

role in the hydrolysis.

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3.2 Total phenolic content

Table 1 showed that SCG extracted using pectinase-

assisted at pH 3, pH 4 and pH 5 had higher TPC value

than that of pH 6 and pH 7. Since phenolic compounds

were less vulnerable to oxidation at lower pH due to

proton-protected hydroxyl groups, total phenolic

compounds should be increased from pH 6 to pH 3

(Nayik and Nanda, 2016). The findings contrasted with

the prediction that the enzymatic synthesis of

polyphenolic compounds may result from the hydrolytic

removal of polysaccharides from the plant cell wall,

which retains phenolic compounds in the polysaccharide

and lignin chains (Heemann et al., 2019). However,

higher phenolic content in extracts could be associated

with increased disruption and maceration due to

enzymatic pre-treatment, which contributes to the release

of bound phenolic (Mushtaq et al., 2015).

3.3 Total flavonoid content

Table 1 also showed that the total flavonoid content

(TPC) of SCG extracted using pectinase-assisted pH 5

had significantly (p<0.05) the highest TPC (22.75 ± 0.27

mg QE / g). This data corresponds with the study made

on saffron honey that showed the value of TFC was

higher at pH 4.5 mg QE/ 100 g and dropped at pH 6 to

9.46 mg QE/100 g (Nayik and Nanda, 2016). Then, the

author also claimed that the de-protonation of the

hydroxyl group caused the decreased oxidation of honey

polyphenols at low pH in TFC analysis. This was also

supported by Haile and Kang (2019), as they expressed

the fermentation of coffee influence the flavonoid

content in the coffee.

3.4 Antioxidant activities

3.4.1 DPPH radical scavenging assay

SCG samples extracted using pectinase-assisted

technique at pH 4 showed the strongest inhibition of 2.2-

diphenyl-2-picrylydrazyl (DPPH) radicals, which was

significantly higher than other samples (Table 1). The

finding is in concert with the study by Zhu et al. (1997)

who stated that green tea catechin is very stable at pH <

4 for 18 hrs while unstable at pH > 8 which degraded

completely in a few minutes. They also stated that the

green tea catechin at pH 6.5 to 7.5 was less stable

compared to the acidic medium. Low pH is the main

aspect of extraction efficiency as the flavanol is stable

below pH 5. The structural alteration of the flavonoid

decreases with the decreasing pH that leads to the

opening of structures that are normally inaccessible

(Zimmermann and Gleichenhagen, 2011). MEBP

powder samples encapsulated with 8% GA exhibited the

best radical scavenging activity among all those which,

due to the good condition or non-clotting state of the

microencapsulated powder, were able to fully

demonstrate its antioxidant capacity.

3.4.2 Ferric reducing antioxidant power (FRAP)

Table 1 also illustrates that all pectinase extract with

different pH values had a higher value than the negative

control (distilled water). Moreover, extraction at pH 3,

pH 4, pH 5 and pH 6 showed higher inhibition compared

to pH 7. Owusu-Apenten (2015) postulated that at pH

higher than 7.0) or lower than 3, would  decreased the

rate of reduction of Fe (III) resulting in an incomplete

response At high pH values, the ascorbic acid or high

antioxidant sample act to iron (III)-OH formation and

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Figure 1. The yield of the Arabica SCG extract prepared from

the SCG extracted using pectinase at different pH. Bars with

the same letters are not significantly different (P<0.05).

Samples
TPC

(mg GAE/g sample)
TFC of dried plant
material (mg QE/g)

DPPH radical inhibition
(%)

Ferric reducing antioxidant
power (FRAP) assay

pH 3 61.38±5.06a 15.10±3.11c 40.56±2.21d 0.04±0.001ab

pH 4 42.75±1.26b 18.62±1.11bc 55.37±1.36b 0.05±0.001a

pH 5 54.13±5.22a 22.76±5.22a 34.84±2.42d 0.05±0.001a

pH 6 32.38±2.04c 19.53±1.14ab 45.06±3.31c 0.04±0.001ab

pH 7 37.54±2.68b 21.60±2.23ab 34.76±2.57e  0.03±0.001b

blank -  9.78±0.57f 0.01±0.001c

Trolox -  68.74±3.17a -

Values represent the mean±standard deviation. Values with the same superscript letters within the same column are not

significantly different (P<0.05).

Table 1. Total phenolic content (TPC), total flavonoid content (TFC), FRAP assay and DPPH inhibition of the SCG extracted

using pectinase at different pH

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decreasing the rate of reduction for the Fe (III) hydroxyl

complex while at lower pH value, the protonation of

ferrozine occurred as the reduction of Fe (III) decreasing

with pH (Jaselskis and Nelapaty, 1972). Most

researchers used pH 4 and pH 5 with 50°C and 40°C as

the optimal pH and temperature in preparation for

enzyme - assisted extraction (Nguyen et al., 2014; Wang

et al., 2017; Ghandahari Yazdi et al., 2019). The

antioxidant capacity obtained is highly dependent on the

concentration of phenolic compounds of the samples, the

type of enzymes and type of extraction, respectively.

3.5  Antimicrobial activities

 There was no significant difference in the growth of

the inhibition zone for B. subtilis, yet S. aureus and P.

aeruginosa showed somewhat significant difference

between the samples especially at pH 7 (Table 2). Table

2 also tabulates that Arabica SCG extract at pH 7 has the

most significant inhibition effect than gentamicin

(antibiotic) on S. aureus and P. aeruginosa which are

2.17±0.15 cm and 2.77±1.10 cm while the antibiotics

only 1.23±0.25 cm and 1.9±0.0 cm, respectively.

S. aureus, a gram-positive and P. aeruginosa, a gram

-negative bacteria were more susceptible to the SCG

extract especially at pH 7 where a bigger region of

inhibition zone on the agar. The alteration of pH can

affect the growth of bacteria where most of the optimum

pH of most bacteria is at pH 7. Meanwhile, E. coli shows

not susceptible to every sample except for gentamicin

might because these bacteria have a structure of the

bacterial envelope or this concentration of the extract is

not strong enough to against the bacteria. This is in

accordance with the study by Klangpetch (2017) that E.

coli showed an inhibition zone only when the SCG

extracted with 60% and 100% of ethanol. The caffeine

concentration of more than 1% is excellent in lowering

or inhibiting the growth of E. coli, below that there is no

effect on bacterial growth (Ramanaviciene et al., 2003).

Radha Krishnan et al. (2014) stated that the

antimicrobial activity related to the antioxidant activity

like phenolic compounds in plant sources could inhibit

the pathogenic bacteria. This compound can break down

the cell wall of bacteria, disrupt the cytoplasmic

membrane, and disturb protein translocation and others

(Shan et al., 2007).

3.6 Determination of individual flavonoid using high-

performance liquid chromatography (HPLC)

Figure 2 shows the standard flavonoid HPLC

chromatogram identified by its retention time and by

spiking under the same conditions with standards. Figure

3 shows that every peak on the chromatogram indicates

the presence of individual flavonoids present during

specific retention time. This can be detected if more time

and flavonoid standard is allowed to run the sample

(Gandhiraja et al., 2009). Table 3 shows that most of the

flavonoids identified in the study are the same as the

rutin, myricetin, kaempferol, gallic acid, catechin, gallate

epigallocatechin, quercetin and p-coumaric acid used.

The epigallocatechin gallate exhibited the highest

concentration in every sample ranging from 1638±406 to

5293±6805 mg/100g while the lowest flavonoid

concentration in every sample is gallic acid that ranging

from 91.22±2.21 to 760.8±98.8 mg/100 g.

The results also showed that SCG extract in pH 7

had the highest concentration of total individual

flavonoid while pH 4 shows the lowest concentration of

total individual flavonoid between samples. The data

obtained had supported the result from the antimicrobial

activity where large inhibition occurs at SCG in pH 7

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Sample
Inhibition Zone (cm)

B. subtilis S. aureus P. aeruginosa E. coli

pH 3 1.08±0.07b 1.93±0.10b 1.87± 0.23b

No inhibition
zone

pH 4 0.7±0.6b 1.55±0.06c 1.67±0.21b

pH 5 1.07±0.05b 1.63±0.09c 2.00± 0.1ab

pH 6 1.07±0.15b 2.05±0.05ab 2.23±0.21ab

pH 7 1.1±0.1b 2.17±0.15a 2.77±1.10a

Gentamicin 2.23±0.25a 1.23±0.25d 1.9± 0.00b 2.00± 0.02a

Values represent the mean±standard deviation. Values with the same superscript letters within the same column are not

significantly different (P<0.05). Gentamicin was used as the standard antibiotic for comparison.

Table 2. Inhibition effect of Arabica SCG extracted using pectinase at different pH against pathogenic bacteria in antimicrobial

activity

Figure 2. HPLC Chromatogram of flavonoids of the standard

mixture

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because containing a high amount of individual

flavonoid. Flavonoids such as quercetin and catechin

may be the compounds responsible for antiviral,

antiallergic, antitumor, antibacterial, anti-fungal, and

antithrombotic activity expressing anti-infective and anti

-replicative characteristics against the herpes virus,

parainfluenza virus as well as inhibiting the growth of P.

aeruginosa and S. aureus (Middleton, 1996; Kaurinovic

and Vastag, 2019). The amount of gallic acid (Table 3)

found in this study was in line with Ghandahari Yazdi et

al. (2019) who reported gallic acid found in pistachio

green hull using pectinase and cellulase assisted

extraction is lower than 5.70±0.02 and 4.69±0.01 mg/g

dry extract powder respectively as the pectinolytic and

cellulolytic enzyme treatment had affected the phenolic

composition and antioxidant activity of the food product.

4. Conclusion

The extraction of Arabica SCG using pectinase at

different pH had a significant effect on antioxidant and

antimicrobial properties. Low pH showed better

antioxidant properties, while higher pH showed better

antimicrobial properties. The SCG sample extracted at

pH 4 showed the best antioxidant properties in DPPH

and FRAP. Extraction at pH 7 clearly showed better

antimicrobial activity with the largest inhibition zone for

P. aerunogisa and S. aureus. The HPLC analysis showed

the presence of flavonoids, namely quercetin,

kaempferol, rutin, gallic acid, catechin, epigallocatechin

gallate, p-coumaric acid and myricetin, in all samples

with different concentrations. The study showed that the

optimum pH for the extraction of Arabica SCG was at

pH 4 to 5 which provided the highest yield in antioxidant

capacity and activity that could be used for food

preservation as well as in the pharmaceutical industry.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgments

This research was supported by Universiti Malaysia

Terengganu. The authors would like to thank the central

lab of UMT and the Faculty of Fisheries and Food

Science (FPSM) for the facilities provided to conduct the

study.

References

Acufia-Argfielles, M.E., Guti6rrez-Rojas. M., Viniegra-

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Flavonoid compound
The concentration of the individual flavonoid in every sample (mg/100 g)

pH 3 pH 4 pH 5 pH 6 pH 7

Catechin 641.07±44.52a 1151.87±88.76ab 1874.67±153.53ab 2044.98±227.54a 5112.76±66.43a

Epigallocatechin Gallate 5293.67±68.05a 1911.32±112.21a 1638.56±40.56ab 1776.54±171.92a 1936.72±78.54b

Gallic Acid 120.16±11.54a 91.22±2.21c 182.7±12.41ab 104.4±9.81 a 760.84±98.83c

Kaempferol 585.98±19.28a 1092.95±25.55abc 1611.40±464.34ab 1800.43±24.67a 570.88±24.05c

Myricetin 897.09±24.93a 1696.77±47.78a 923.36±84.9ab 790.48±43.19a 1552.54±30.84b

p-Coumaric acid 299.31±5.42a 502.33±47.43bc 262.61±76.62ab 535.22±31.64a 671.63±33.54c

Quercetin 975.67±83.70a 501.08±70.73bc 1480.89±128.19ab 407.52±37.29a 558.62±14.28c

Rutin 1555.68±49.69a 1659.26±32.41a 2310.75±576.54a 1864.57±121.06a 948.22±19.52c

Values represent the mean±standard deviation. Values with the same superscript letters within the same row are not significantly

different (P<0.05).

Table 3. The concentration of individual flavonoid in Arabica SCG extracted using pectinase at different pH identified by the

HPLC system

Figure 3. HPLC chromatogram of the SCG extracted using

pectinase-assisted extraction at pH 3, pH 4, pH 5, pH 6, pH 7.

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