Consumption Of Coffee Is Associated With Protective Role Commerce Essay

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Studies have found that consumption of coffee is associated with protective role due to the numerous antioxidant components found in coffee, while some studies have also opposed the fact that coffee has cardio-protective roles. Chemical composition of different commercially available ground coffee from different geographical areas and different brands were investigated by gas chromatography-mass spectroscopy (GC-MS) by using different methods of analysis including direct liquid injection, solvent extraction and solid-phase microextraction. Antioxidant activities of different commercially available coffees were also investigated by the ferric reducing ability of plasma (FRAP) assay using ferric- tripyridyltriazine (Fe3+-TPTZ) complex and ascorbic acid was used as an antioxidant. The main purpose was to investigate the relationship between the chemical compounds both volatile and less volatile components and the antioxidant activities of each coffee sample, compare the finding and relate the results to the contribution of the geographical area to the varying results and conclude on which coffee sample will have a better protective role in human health. According to the GC-MS methods, caffeine, furfural, furfuryl alcohol, furfuryl acetate was common components in all the coffees but at different percentage levels. The FRAP assay showed variable results on the antioxidant properties of filtered and unfiltered coffee samples so the filtered results were selected. The FRAP assay show that Kenyan coffee has the highest antioxidant activity while Guatemalan Antiguan coffee has the least antioxidant activity.

Introduction

Coffee is one of the most popular beverages in the world1, being appreciated for its characteristic taste and aroma and, more recently, for its potential beneficial effects on human health. Coffee is consumed worldwide for its physiological effects1, metabolic activity and its peculiar sensory properties i.e. enhance perception and reducing fatigue. Coffee is prepared by extraction of the soluble material from roasted grounds by adding boiling water. Three species of the coffee plant, Coffea arabica L., Coffea robusta L., Coffea iberica L., have commercial value2. Coffea arabica L is mostly used because of its better quality compared to the other types of coffee plants.

Typical compounds in coffee that are relevant for taste, aroma, flavor and/or antioxidant activities are caffeine, trigonelline, nicotinic acid, chlorogenic acid, furans and other polyphenols3. The chemical compounds present are said to have antioxidant properties, these antioxidants reduce oxidant damage to the biomolecules, thereby suggested to have cardio-protective role, and also protect against cerebrovascular disease4, neurodigestive diseases, cancer2 and diabetes which are all associated with oxidant damage5.

Coffee has been researched to lower cholesterol level6, enhances gastric secretion and urine production, and reduces serum uric acid concentrations therefore may reduce the risk of developing gallstone disease4. Heat treatment of coffee is said to contribute heat- induced antioxidant to the natural antioxidant present5. Over the years, more than 800 volatile compounds have been detected in brewed and grounds coffee by mainly using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) 7. These compounds are released during the grinding and heating process of coffee, therefore many of them are lost before sampling but adequate amount of them were identified7.

In this study, the different coffee samples from eight countries were identified and their antioxidant activities were determined. The coffee samples are from Kenya, Colombia, Pico Duarte (Dominican Republic), Guatemala Antigua, Panama, Mexico, Costa Rica and Papua New Guinea. The conditions seen in all these countries are there high level of volcanic soil which in effect provided a nutrient rich soil for coffee beans to grow. The location and other factors such as chemistry of the soil, the weather, altitude, amount of sunshine and wetness are believed to affect the different compounds such as antioxidants present in coffee8

Different methods were carried out to identify the volatile and less volatile compounds present and these are direct injection of coffee extract, solvent extraction and SPME coupled with the GC-MS. The antioxidant activities of the coffee samples were also determined by using ferric reducing ability of plasma (FRAP) assay. This method works at low pH to reduce ferric ions to ferrous ion i.e. ferric-tripyridyltriazine (Fe3+-TPTZ) complex in the reagent is reduced to ferrous (Fe2+) formed as a measure of the antioxidant activity present in the coffee; this produces an intense blue colour. The absorbance of the coloured product can be measured using a spectrophotometer9. The intensity of colour that develops in the FRAP assay corresponds to the concentration of the reductant (antioxidant) present in the test solution (i.e. coffee).

The aim of this project was to identify the different compounds in these coffees using different analytical methods and relate relevant components to the antioxidant activities of the coffees. The objective was to determine which country's coffee will have a protective role to human health.

Materials and Methods

Materials

Chemicals

2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 20mmol/L ferric chloride and1000μmol/L ferrous sulphate were from Sigma-Aldrich Chemicals (Poole, Dorset, UK). 300mmol/L Sodium acetate buffer and 1000μmol/L ascorbic acid were also used. Dichloromethane, n-hexane and ethyl acetate (more than 99% HPLC grade) were from Fisher Scientific UK Ltd (Leicestershire, UK).

Coffee samples

Eight varieties of ground medium roast coffee were selected. These coffee samples were of different geographical origin and were grounded by using a coffee grinder and also manually. Five were purchased from Whittard of Chelsea (Kenyan, Colombian, Pico Duarte, Guatemalan and panama) and the other three from Sainsbury's supermarket (Mexican, Costa Rica and Papua New Guinea). Coffee samples used are all Arabica coffee from Coffea arabica L. coffee plant.

GC-MS parameters

Analysis was carried out on an Agilent Technologies 7890A GC coupled with Agilent Technologies MS (Manchester, UK). The GC used was fitted with a fused capillary column (5% phenyl methyl siloxane 30 m x 250 µm i.d., 0.25 µm film thickness). Carrier gas was helium (1 ml/min); oven temperature was programmed from 500C to 3000C at 100C/min.

SPME device

The SPME fibre (polyacrylate with 85 µm film thickness) was from Supelco Co Ltd. (Poole, UK).

Methods

Ferric reducing ability of plasma assay

Preparation of standard solutions

The standard solution was made up in duplicate according Table 1 below.

Table 1 A total of 1000 µl of the standard solution was prepared for different concentrations (Refer to Supplementary information for the calculation used to determine the volume of water and ferrous sulphate used).

Concentration (µmol/L)

1000

750

500

300

100

Deionised H2O (µL)

-

250

500

700

900

FeSO4(µl)

1000

750

500

300

100

Total volume (µl)

1000

1000

1000

1000

1000

100 µl of solution for each concentration and deionised water (blank) was transferred into fresh test tubes in duplicate. 1 ml of working solution (Refer to Supplementary information for the preparation of working solution) was added to the entire samples and the blank. Ascorbic acid was treated liked a sample and used as a control to establish sample and standard curve range. The test tubes were agitated for adequate mixture and placed in the in a water bath for 10 min at 37oC.

Preparation of coffee samples.

Eight different coffee samples from Kenya, Colombia, Pico Duarte (Dominican Republic), Guatemala Antigua, Panama, Mexico, Costa Rica and Papua New Guinea were used. 100 mg of each coffee samples were weighed and 200 ml of deionised water were added to each Pyrex beaker. A magnetic stirrer was added and the coffee solutions were stirred and heated on a hotplate at 3000C for approximately 10 min.

100 µl of each coffee solution was transferred to clean test tubes in triplicate and 1 ml of working solution was added to each test tube and agitated for adequate mixture. The coffee solution is then placed in a water bath for 10 min at 370C.

The absorbance of the standard solution and the coffee solutions were read at 593 nm after the spectrophotometer has been zeroed with the blank.

Gas chromatography-mass spectrometry

Coffee sample preparation

Direct injection method: 500 mg of each coffee sample was weighed made up to 10 ml with ethyl acetate in a 10 ml volumetric flask. The coffee sample was dissolved by placing the labelled volumetric flasks in a sonicator for 5 min. Each sample was transferred to eight different sterile glass 20 ml bottles and was centrifuged at 2000 rpm for 5 min. The supernatant was transferred into 1 ml vial and each sample was analysed by the GC-MS.

Solvent extraction method: a similar process was carried out as the direct injection, the only difference was 5 ml of hot deionised water was added to the coffee granules and sonicated then 5 ml of ethyl acetate was then added to separate the coffee extract. The extract was at the upper layer while the deionised water and undissolved granules were at the bottom layer. After centrifuging in the sterile glass bottle, the top layer was analysed as before.

SPME method: One g of each coffee sample was place in a tightly closed vial and heated at 1000C for 30 min. The fibre was then exposed to the coffee for another 30 min. The fibre was immediately placed into the GC-MS injection port and thermally desorbed for 32 min at 3000C.

Statistical analysis

ANOVA analysis was carried out on SPSS 15 to determine the significant differences in the results obtained from the coffee sample also to determine if there is significant difference between each coffee type. Scheffe post hoc test was chosen because of unequal sample sizes for each coffee type.

Results

FRAP assay

In order to observe and determine the antioxidant power of coffee samples, standard had to be determined to plot a standard curve so as to determine the concentration of the eight coffee samples. The absorbance at 593 nm was plotted against five different concentrations (Fig. 1).

Fig. 1 Standard curve for ferrous sulphate solution with H2O, TPTZ and ferric chloride as the standard at different concentration (Refer to Supplementary information for the absorbance values of the standards).

The concentrations of the coffee samples were calculated by using the equation of the standard curve. For example:

y = 0.0017x

x = 0.788/0.0017

x = 464 µmol/L

Table 1: Calculated concentration of eight filtered coffee samples from different origins using the FRAP assay method. (Refer to Supplementary information for the absorbance values.)

Coffee origin

Antioxidant concentration expressed as concentration of ferrous ion (µmol/L)

Kenyan

485

Colombian

407

Pico Duarte (Dominican republic)

437

Guatemala (Antigua

357

Panama

442

Mexico

439

Papua New Guinea

415

Costa Rica

453

Direct injection of coffee extracts method and solvent extraction method.

The direct injection of coffee extract and solvent extraction were both carried out using ethyl acetate solvent to extract the less volatile components of eight different coffee samples from different origins. Examples of the GC-MS chromatograms obtained for both methods are shown in Fig. 2 and Fig. 3.

Compounds

Kenya

Colombia

Pico Duarte

Guatemala Antigua

Panama

Mexico

Papua New Guinea

Costa Rica

Furfuryl alcohol

-

0.18

0.37

0.11

0.56

-

-

-

Caffeine

12.45

16.77

14.50

20.68

17.29

5.59

3.44

3.53

Furfuryl acetate

-

0.08

-

-

-

-

-

-

Palmitic acid

2.05

2.99

1.31

-

-

-

-

-

2-Methyl-Z,Z-3,13 octadecadienol

-

-

0.19

0.23

0.13

0.17

0.22

1.10

13-Octadecenal

-

0.23

-

0.22

-

-

-

-

Oleic acid

-

1.03

-

-

--

-

0.06

0.04

Linoleic acid chloride

0.29

-

-

0.64

0.67

-

0.40

2.93

Cyclopentadecanone, 2-hydroxy

0.69

0.42

0.38

1.07

-

5.71

-

-

Cyclopropaneoctanal, 2-octyl-

0.13

0.26

0.82

0.36

0.13

-

-

-

Cis-11-Hexadecenal

-

0.10

0.55

0.38

0.38

-

-

-

Methyl linoleate

1.20

-

-

0.51

1.99

1.08

-

-

7,11-Hexadecadienal

-

-

-

-

0.24

-

0.08

-

9,17-Octadecadienal

-

-

-

0.13

1.32

2.42

3.75

3.47

Gossyplure (Z,Z)-

0.96

-

-

-

1.42

-

-

-

Linoleic acid

0.20

0.50

0.52

1.09

0.64

0.16

1.30

1.80

1,2-Benzisothiazole,3-(hexahydro-iH-azepin-1-yl)-,1,1-dioxide

0.70

0.63

-

-

13.92

3.65

1.10

2.94

7-Pentadecyne

0.53

0.37

-

-

-

-

-

-

Cis, cis-7,10-hexadecadienal

0.06

-

-

-

-

-

0.42

2-Heptadecenal

-

0.50

-

-

0.07

-

-

-

Methyl pentadecyl ether

0.65

-

-

-

-

-

-

-

1,5,9,13-Tetradecatetraene

-

-

-

-

-

1.69

0.38

4.08

1,2,5,16-Diepoxyhexadecane

-

-

-

-

-

1.76

1.26

-

Epoxycyclodenane

-

-

-

0.09

-

-

1.49

2.66

Table 2: Compounds identified from chromatographic peaks by comparison of their mass spectroscopy spectra in eight ground coffee samples from different origins with those in the NIST library. The percentage composition of each sample is illustrated below.

B

A

Figure 2 Guatemala Antigua as an example of GC-MS chromatogram obtained using the direct injection of coffee. Key: A = caffeine, B = palmitic acid.

Compounds

Kenya

Colombia

Pico Duarte

Guatemala Antigua

Panama

Mexico

Papua New Guinea

Costa Rica

Furfuryl alcohol

1.19

1.37

1.90

2.02

1.57

0.84

0.91

0.67

Furfural

0.29

0.08

-

-

0.14

0.10

0.13

0.10

5-Methylfurfural

0.24

0.16

0.14

-

0.22

0.15

0.17

0.14

Furfural acetate

0.07

0.12

0.13

0.21

0.11

0.05

-

0.04

Caffeine

20.69

16.77

21.65

28.16

18.09

9.09

12.12

8.11

Tridecanoic acid

1.64

-

-

-

-

-

-

-

Palmitic acid

-

3.06

-

2.72

2.99

-

-

-

2-methoxy-4-vinylphenol

-

-

0.22

-

-

0.09

-

0.11

Myristic acid

-

-

2.28

-

-

-

-

-

2-Isopropylbenzenthiol

-

-

-

0.14

-

-

-

-

4-Hydroxy-3-methylacetophenone

-

-

-

-

0.23

-

-

-

Linoleic acid

1.10

0.61

0.30

0.48

1.17

0.04

0.11

0.03

2-Methyl-Z,Z-3,13 octadecadienol

-

0.11

0.06

-

-

0.23

-

0.56

9-Eicosene,(E)-

-

-

0.03

-

-

-

-

-

Oleic acid

0.56

0.96

-

0.03

0.29

0.01

-

0.11

1,2-Benzisothiazole,3-(hexahydro-1H-azepin-1-yl)-,1,1-dioxide

0.83

0.22

0.04

-

0.27

5.20

2.85

2.07

Estriol

2.59

-

-

-

-

-

-

-

7,11-Hexadecadienal

0.41

-

-

-

-

-

-

-

7-Pentadecyne

0.53

0.53

0.26

1.12

-

-

-

-

Cyclohexene,4-(4-ethylcyclohexyl)-1-pentyl

0.02

-

-

-

-

-

-

-

11-Hexadecenal

0.31

0.43

0.30

-

-

-

-

-

Linoleic acid chloride

2.77

0.74

-

-

-

0.16

1.28

3.83

Methyl pentadecyl ethyl

0.02

-

-

-

-

-

-

-

(R)-(-)-14-Methyl-8-hexadecyn-1-ol

0.02

-

-

-

-

-

-

-

Pyridine,2-ethyl-4,6-dimethyl-

2.12

-

-

-

-

-

-

-

Cyclopropaneoctanal,2-octyl-

1.32

0.23

0.48

-

0.62

-

-

-

Olealdehyde

0.33

0.45

-

-

-

-

-

-

Table 3: shows the Compounds identified from chromatographic peaks by comparison of their mass spectroscopy spectra in eight ground coffee samples from different origins with those in the NIST library. The percentage composition of each sample is illustrated below.

Table 3 continued

Compounds

Kenya

Colombia

Pico Duarte

Guatemala Antigua

Panama

Mexico

Papua New Guinea

Costa Rica

Gossyplure (Z,Z)-

0.58

-

-

-

0.56

0.12

-

0.90

Methyl linoleate

0.45

0.53

-

-

-

-

-

-

13-Octadecenal

0.30

-

0.63

-

-

-

-

0.01

10,13-Octadecadienoic acid methyl ester

-

-

2.06

1.66

-

-

-

-

Dimesol

-

-

-

10.86

-

12.08

-

-

Cis, cis-7,10,-Hexadecadienal

1.03

0.36

-

-

-

-

-

-

6-Octadecenoic acid, (Z)-

0.63

-

-

-

-

-

-

-

2-Heptadecenal

-

1.16

-

0.02

-

-

-

-

Z,Z-10,12-Hexadecadien-1-ol acetate

-

-

-

-

0.34

-

-

-

4-Nitro-3-carbethoxy-2-methyl-1,7 trimethyleneindole

-

2.98

-

4.18

-

-

-

-

Linolelaidic acid, methyl ester

-

-

0.13

-

-

-

-

-

1,5,9,13-Tetradecatetraene

-

-

-

-

-

-

-

1.56

*Table 3 Components present in different coffee samples by using the solvent extraction method on the GC-MS.

B

A

D

C

Figure 3 Guatemala Antigua as an example of GC-MS chromatogram obtained using the solvent extraction. Key: A = furfuryl alcohol, B = caffeine, C = palmitic acid and D = 4-Nitro-3-carbethoxy-2-methyl-1,7-trimethyleneindole.

Compounds

Kenya

Colombia

Pico Duarte

Guatemala Antigua

Panama

Mexico

Papua New Guinea

Costa Rica

Furfuryl alcohol

-

0.40

0.17

0.30

1.82

0.72

5.28

-

Pyridine

-

-

-

-

0.50

-

1.49

-

β-Terpinen

-

2.94

-

-

-

-

-

-

L-Terpinen-4-ol

11.93

1.70

3.15

-

-

-

-

-

Safrol

1.07

-

0.49

-

-

-

-

-

Limonene

5.66

-

-

-

-

-

-

-

p-Cymene

7.55

3.93

-

-

-

-

-

-

Cis-β-terpineol

2.45

-

-

-

-

-

-

-

Acetic acid, bornyl ester

0.26

-

-

-

-

-

-

-

Elemicine

2.51

0.20

1.63

-

-

-

-

-

γ-Terpinen

1.06

-

-

0.10

-

-

-

-

(+)-4-Carene

0.34

-

-

-

-

-

-

-

α-Cubebene

0.78

0.22

-

-

-

-

-

-

Copaene

1.87

-

0.73

-

-

-

-

-

Eugenol methyl

0.67

-

0.33

-

-

-

-

-

Myristicine

4.75

0.47

3.20

-

-

-

-

-

7-Pentadecye

-

-

0.05

-

-

-

-

-

Table 4 Compounds identified from chromatographic peaks by comparison of their mass spectroscopy spectra in eight ground coffee samples from different origins with those in the NIST library. The percentage composition of each sample is illustrated below.

*Table 4 shows the volatile components present in different coffee samples by using the SPME coupled with GC-MS.

F

E

D

C

B

A

Figure 4 Kenyan coffee as an example of GC-MS chromatogram obtained using SPME coupled with GC-MS. Key: A= (R)-α-Pinene, B = (-)-β-Pinene, C = limonene, D = β-terpinen, E = Myristicine and F = Elemicine

Discussion

The antioxidant concentration of coffee from different geographical origins were calculated in Table 1 and expressed as concentration of ferrous ion formed in the FRAP assay. In order of decreasing activity, antioxidant concentration expressed as concentration of ferrous ion was, Kenyan > Costa Rican > panama > Mexican > Pico Duarte > Papua New Guinea > Colombia > Guatemala Antiguan. Kenyan coffee demonstrated the highest level of reducing ability therefore it had the antioxidant content according to the FRAP assay. The differences in the antioxidant activity in the different geographical areas selected in this project were apparent. The values generated are not definite because they were generated at different condition for example, Bunsen burner was initially used to prepare coffee and hotplate was later used, this could have added heat induced compounds or vaporised the antioxidant content the coffee preparation thereby showing a higher or lower levels of antioxidant activities in the different origins.

The method used also considered the antioxidant activity of unfiltered coffee and the result was very unstable and did not repeat well but when filtered a correlation was observed in the antioxidant activities in the different region. This means that the coffee extract from heating was at its maximum and when the absorbance was measured, the particle present in the unfiltered coffee concluded to affected the intensity of the blue solution formed in the FRAP assay and affected the absorbance measurement.

ANOVA was carried out to analyse if there was a significant difference in the different origins. It showed that there was significant difference between the coffee samples with p< 0.05. The ANOVA analysis showed a significant difference between Kenyan coffee and Colombian coffee p = 0.019 (p< 0.05) and Kenyan coffee and Guatemalan Antiguan coffee, p = 0.000 (p< 0.05). A significant difference was observed between Pico Duarte coffee and Guatemalan Antiguan coffee, p = 0.038 (p< 0.05) and there was a significant difference between panama coffee and Guatemalan Antiguan coffee, p = 0.031 (p< 0.05).

The analyses of coffee from different geographical areas were obtained by using three different methods of extraction. The direct liquid injection and solvent extraction methods separated less volatile compounds and SPME analyse the more volatile compounds in coffee. A total of fifty-five less volatile and volatile components were analysed with more than twice of this compounds with less than 85 % in quality, which is why they were not, included in Tables 2, 3 and 4 as they could be impurities from the GC-MS column.

The direct liquid injection method showed a very low percentage composition of all the compounds compared to solvent extraction method. This indicated that the direct injection method was unable to extraction the components in the coffee samples. The solvent extraction showed higher percentage composition of almost all the compounds identified and clear difference can be observed in these two methods. This is because water was first used to extract the components i.e. water-soluble and the ethyl acetate completed the extraction of the other components identified. This can be compared to normal circumstances when coffee is consumed as ground coffee, which is normally extracted by using water, and the benefits can still be observed.

The SPME identified numerous volatile components such as p-cymene, limonene, terpinens, furfuryl alcohol, pyridine and copaene which have already been identified in coffee in numerous studies7, 10. These compounds contribute to the aroma and tastes observed in different coffee samples and are more predominant in the Kenyan coffee compared to the other coffee sample of which some volatile compounds were completely absent.

The Kenyan coffee had the highest number of compounds in all the analytical techniques used while the other coffee samples vary in compounds present.

The solvent extraction method identified compounds such as furans (furfural, furfural acetate, 5-methyl furfural and furfuryl alcohol), caffeine, palmitic acid, oleic acid, linoleic acid, myristic acid, 2-isopropylbenzenthiol, 4-hydroxy-3-methylacetophenone, estriol, 11-hexadecenal, (Z,Z)-gossyplure, methyl linoleate, Cyclohexene and 4-(4-ethylcyclohexyl)-1-pentyl. These compounds have also been associated with different taste and aroma perceived in coffee.

The highest percentage composition of caffeine, furfuryl alcohol and furfural acetate are present in Guatemalan Antiguan, Pico Duarte, Kenyan coffee and the lowest percentage composition is identified in the Costa Rican coffee.

Caffeine, which is a xanthine compound4, furans, limonene, pyridine, identified in the samples have being studied and they are believed to have antioxidant effect in the body, such compounds intercepts free radical damaging activity to the biomolecules11.

Guatemalan Antigua coffee has the highest percentage composition of caffeine (table 3) followed by Kenyan coffee and Pico Duarte coffee. It can be deduced that Guatemalan Antiguan coffee will be most appropriate for reducing fatigue and increase perception.

In comparison to the FRAP assay, there is a conflict between Kenyan, Pico Duarte and Guatemalan Antigua coffee. This is because, the GC-MS showed that Guatemalan Antigua coffee had higher percentage composition of key compounds (caffeine, furans) while the Kenyan coffee had more compounds present and is supported by the FRAP assay.

The GC-MS method is a highly selective method12 therefore peaks of some very low level compounds might have been ignore while the FRAP assay is a very sensitive and selective method13 therefore, can represent the antioxidant activity in the samples. With this information, Kenyan coffee is concluded to be the coffee with the most beneficial role to the human health compared to the other countries analysed in this study.

Conclusion

In conclusion, the objective of this project was reached as the coffee with the best possible protective role was identified as the Kenyan coffee.

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