8
III. RESEARCH METHODOLOGY
A. MATERIALS AND INSTRUMENTS 1. Materials
The main ingredient used in this research was coffee ground residues CGR that were collected from 10 coffee shops in Chiang Rai Province, Thailand. The microorganisms used for
the antimicrobial activity assay were Stapphylococcus aureus ATCC25923, Pseudomonas aeruginosa ATCC27853 purchased from the American Type Culture Collection; Escherichia coli
TISTR780, Bacillus subtilis TISTR008, Salmonella Typhimurium TISTR292 purchased from the Thailand Institute of Scientific and Technological Research; and Listeria monocytogenes
DMST17303 purchased from the Department of Medical Sciences Thailand. Chemicals used in phytochemical screening were HCl 2 N, ammonia, FeCl
3
, gelatin and NaCl. While those for the bioactive compounds assay were Folin-Ciocalteu, gallic acid, caffeine, chlorogenic acid, 72
H
2
SO
4
, 7.5 Na
2
CO
3
, and dichloromethane. Chemical that used in antioxidant activity assay are trolox ±-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid , DPPH 2,2-diphenyl-1-
picryhydrazyl, ascorbic acid, 1 K
3
FeCN
6
, 10 trichloroacetic acid, and FeCl
3
. Chemicals used in antimicrobial activity assay were BD DifcoTM Muller Hinton Agar MHA, BD DifcoTM
Muller Hinton Broth MHB, ampicillin, and polymicin B sulphate.
2. Instruments
Instruments that used in this research were microwaveconvention oven, UV-Vis Spectrofotometer, rotary evaporator, homogenizer, separatory funnel, laminar, vacuum suction,
and glassware.
B. EXPERIMENTAL DESIGN
This research was divided into four parts. The first part was phytochemical screening of CGR and lignin content assay as preliminary research. The second part was extraction of CGR
with hot water and MAE method. The third part was bioactive compounds such as phenolic compounds, chlorogenic acid, caffeine, and anthocyanin assay in CGR. The last part was
antioxidant and antimicrobial assay in three best CGR samples with highest bioactive contents. The diagram of the whole research is shown in Figure 4.
1. Preliminary Research
Coffee ground residue CGR collected from 10 coffee shop in Chiang Rai Province, Thailand was dried in the oven at temperature of 60
o
C until less than 13 moisture content was obtained. Then, the samples were kept in the desiccator until used.
Moisture content of the samples were determined by drying samples ~1 g for 24 h at 105 ± 0.5
⁰C to a constant mass AOAC, 1995. The average moisture content in dry basis, expressed in percents, is calculated using the following equation:
where: m
1
= mass of samples before drying m
2
= mass of samples after drying
1.1. Phytochemical Screening Fransworth, 1966
Phytochemical screening was done in order to check qualitatively the dominated-bioactive compounds in CGR with several methods. First, sample 50 mg was mixed with 50 mL distilled
water in a beaker. Then, the mixture was homogenized for 2 min and placed in the water bath 60- 65
o
C for 15 min. After that, it was cooled in the room temperature then the extract was used for phytochemical screening. The phytochemical screening was done by several methods as follows:
1. Ferric Chloride Test Phenolic compounds and Gallic Acid
The extract 2mL was transferred in the tube. Then, 2-3 drops of FeCl
3
were added into the tube. The extract with precipitates having blue, dark blue, blue purple, green, or green-blue
color, indicates the presence of phenolic compounds and gallic acid.
9
PRELIMINARY RESEARCH
MAIN RESEARCH
Figure 4 Research diagram
Drying in the oven at 60
o
C for ± 2 days
Coffee Ground Residue
Phytochemical screening assay
Lignin assay
FeCl
3
test Pew and Base reaction test
pH test Leucoanthocyanidin test
Gelatin test
CGR sample
Total Phenolic TPC Chlorogenic Acid
Caffeine Anthocyanin
Bioactive compounds assay
Antimicrobial Activity
Agar-well diffusion
Broth dilution Choosing three samples with the highest
of bioactive contents
Antioxidant activity
DPPH FRAP
CGR sample
Hot water extraction
Condition: Wattage : 800 W
Extraction time: 3,4, and 5 min
Condition: Temp: 90
o
C Time: 5 min
Microwave-assisted extraction MAE
10
2. Pew Test and Base Reaction Flavonoids
Flavonoids compounds were determined by two different methods, as follows: Pew Test
One milliliter of extract was transferred to a test tube. Then, 0.5 g of Zn powder and 2 drops of 2 N HCl were added in the tube and the mixture was vortexed for 1 min. After that, 10
drops of 37 HCl were added in the mixture. The dark red color solution indicates the presence of flavanonol, and flavonol -3- glycoside, while the light color of solution indicates flavanone,
and flavonol.
Base Reaction One milliliter of extract was transferred to a test tube. Then, 5 drops of 9.5-10.5
ammonia solutions were added in the mixture. The yellow, orange-red, red-brown, or orange- brown color solutions indicated the presences of flavones, flavanol, and xanthone, flavanone,
chalcone, ourone and flavanonol.
3. pH Test Anthocyanins
Briefly, one milliliter of the extract was transferred in a test tube then a drop of 2N HCl was added in the mixture. After that, 5 drops of 9.5-10.5 ammonia solutions were added in the
mixture. The red color solution appeared after HCl addition and changed into blue color after ammonia addition indicated the presence of anthocyanin.
4. Leucoanthocyanidin Test Leucoanthocyanidin and catechin
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