ANTIMICROBIAL ACTIVITY LITERATURE REVIEW

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F. ANTIMICROBIAL ACTIVITY

Food is a good vehicle for dispersion of harmful agents such as biological pathogen, toxin, heavy metals, parasites, radio-isotopes and other illness-causing agents. Pathogen contamination is a major concerning problem in food safety. Food-borne pathogen is pathogen present in food, which may cause human diseases or illness through consumption of food contaminated with pathogen andor biological products produced by the pathogen Codex Alimentarius Comission CAC, 2011. There are some efforts to prevent food-borne pathogen growth in food products such as food process controlling, GMP Good Manufacturing PracticesGAP Good Agricultural Practices and HACCP system Karaca, 2011. The processes usually are insufficient to prevent contamination in food products. Consequently, addition of antimicrobial agent must be included in the processes. Antimicrobial agent is a substances of natural, semi-synthetic or synthetic origin which kills or inhibits microbial growth yet results in minimal or no damage to the host Prescott et al.,2000. Increasing of consumer demand for foods with lesser chemicalsynthesized food addictives, have been impacted increasing utilization of antimicrobial agent derived from plants as natural substitutes for chemical food preservatives Roller and Lusengo, 1997. Nowadays, phenolic compounds which contained in plant materials has been reported to have antimicrobial activity against several pathogen bacteria such as Staphylococcus aureus, Salmonella, Escherichia coli, and etc Cueva et al., 2010. Besides preservative function, the use of phenolic compounds as antimicrobial agent in foods provides health benefits. Generally, there are four major modes of antimicrobial agent mechanism for bacterial infections, for instance, 1 interference with cell wall synthesis, 2 inhibition of protein synthesis, 3 interference with nucleic acid synthesis, 4 inhibition of metabolic pathways Neu, 1992. Antimicrobial action of phenolic compounds mostly is related to inactivation of cellular enzymes of microorganisms and changing membrane permeability Moreno et al., 2006. Therefore, effectiveness of antimicrobial action of phenolic compounds is depended to cell surfaces structure of the microorganism. Generally, Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, are more susceptible to the action of phenolic compounds than Gram- negative bacteria such as Escherichia coli and Salmonella sp. Karaca, 2011. This is because of the complexity of cell surfaces structure of Gram-Negative Bacteria comparing with Gram- positive bacteria. Phenolic compounds derived from various plants have been intensively studied as antimicrobial agent. The phenolic compounds could be potentially natural substitutes for chemical food preservatives and develop new healthy food ingredients, medical compounds, and pharmaceuticals. Antimicrobial action of phenolic compounds was related with increasing of membrane permeability where compounds may disturb membranes and cause a loss of cellular integrity Moreno et al., 2006. The antimicrobial potential of the phenolic acids against microorganisms were influenced by the number and position of substitutions in the benzene ring of the phenolic acids and the saturated side-chain length Cueva et al., 2010. Most of phenolic compounds have been proved their antimicrobial activity. Tannin and anthocyanin in pomegranate peels extracts both in vitro agar diffusion and in situ chilled fish could inhibit the growth of Listeria monocytogenes Al-zoreky, 2009. Bacillus sp. and Escherichia coli showed the highest susceptibility to flavonoids from mango extracts Kanwal et al., 2009. Phenolic compounds in coffee such as chlorogenic acid, caffeine and tannin also have been studied as antimicrobial agent. Chlorogenic acid 5-CQA and caffeine caffeic acids from coffee exert inhibitory activity against Streptococcus mutan cell-growth and Streptococcus mutan biofilm formation Antonio et al., 2010. The addition of coffee extract into pasteurized milk could extend the storage life of milk Fardiaz, 1995. Caffeine contributed to the antibacterial activity of coffee extracts against some bacterial such as Lactobacillus bulgaricus, Bacillus cereus and Staphylococcus aureus Fardiaz, 1995. 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.