EFFECT OF COFFEE ROASTI�G A�D ADDITIVES O� HEAD

SPACE VOLATILES OF COFFEE BREWS

BY

WASEEM TAHIR

A dissertation submitted for the partial fulfilment of the requirements for the degree

of MSc Food Science and Microbiology

SUPERVISOR

DR. JOH� R. PIGGOTT

DEPARTME�T OF BIOSCIE�CE

FACTULTY OF SCIE�CE

U�IVERSTIY OF STRATHCLYDE,GLASGOW

AUGUST, 2004

CO�TE�TS

Acknowledgements ...................................................................................................... i

Abstract ....................................................................................................................... ii

CHAPTER-1 INTRODUCTION ................................................................................. 1

1.1 Coffee and coffee beverage .................................................................... 1

1.2 Coffee processing ................................................................................... 2

1.2.1 Dry processing method .......................................................................... 2

1.2.2 Wet processing method .......................................................................... 2

1.2.3 Curing and storage ................................................................................. 3

1.3 Coffee roasting . ..................................................................................... 5

1.3.1 Chemical and physical changes during roasting .................................... 6

1.4 Coffee flavour: aroma and volatiles ....................................................... 7

1.4.1 Coffee roasting and its effect on volatile composition ........................... 10

1.4.2 Effect of adding milk and coffee additives on coffee volatiles .............. 11

1.4.3 Screening/differentiation of coffee by volatile composition .................. 12

1.5 Techniques for analyzing coffee aroma ................................................. 15

1.5.1 Sample preparation and isolation techniques ......................................... 15

1.5.2 Measurement techniques .. ..................................................................... 19

1.5.3 Instrumental and human measurements ................................................. 19

1.6 Scope of the study .................................................................................. 19

CHAPTER-2 MATERIALS AND METHODS ........................................................... 20

2.1 Materials ............................................................................................... 20

2.1.1 Coffee ............................. ..................................................................... 20

2.1.2 Milk/milk products and additives .......................................................... 21

2.2 Methods ............................................................................................... 22

2.2.1 Coffee brew .. ......................................................................................... 22

CHAPTER-3 RESULTS AND DISCUSSION ............................................................ 25

3.1 SPME GC-peak area of black coffee brews and with additives ............ 25

3.2 Analysis of variance (ANOVA). ............................................................ 27

3.3 Principal component analysis (PCA) .................................................... 27

CHAPTER-4 SUMMARY AND CONCLUSION ....................................................... 32

4.1 Recommendations .................................................................................. 33

REFERENCES ....................................................................................... 34

List of Tables .. ....................................................................................... 37

List of Figures ....................................................................................... 38

i

ACK�OWLEDGEME�TS

All praise to almighty Allah. I am extremely thankful to my supervisor Dr. John

R. Piggott.

Thanks are also due to Dr. Eduarda Cristovam from Matthew algie for providing

the coffee samples and valuable suggestions. I am thankful to Lorrain Allen for

practical guidance in laboratory.

I would also like to mention Vanessa Braganza and Samuel Imathiu for their

support and help.

Waseem TahirWaseem TahirWaseem TahirWaseem Tahir

ii

ABSTRACT

The effect of 6 different roasting gradients and addition of UHT milk, sweetened

condensed milk and non-milk coffee creamer in coffee brew were investigated by using

head space solid phase microextraction technique. Headspace volatiles were separated

by gas chromatography (GC) and Principal component analysis (PCA). Coffees with

different roasting gradients were found different from each other. It was possible to

clearly differentiate the coffee on the basis of variability obtained only from GC peak

areas into distinct groups with combined HS SPME-GC/PCA technique. The technique

did not require identifying the volatile components. Coffee brews with additives were

separated having decreased coffee aroma.

1

Chapter-1

I�TRODUCTIO�

1.1 Coffee and Coffee Beverage

Coffee is the name of seeds of the coffee plant as well as a beverage produced from

cleaned and roasted coffee beans. Coffee drink is established worldwide and is

particularly common in Europe. Coffee plants are grown in different parts of the world.

The history of coffee plant dates back to the thirteenth century, when Arabs took the

plant to Arabian Peninsula. Europeans discovered this drink during journeys in the

Middle East in the sixteenth century. In 1700 coffee plantations started in Java, around

1800 in South and Central America and in the nineteenth century in Africa. Brazil,

Colombia, Indonesia and Guatemala are important coffee producing countries.

The two varieties of coffee beans important in coffee trade, are Arabica (Coffea

arabica ) and Robusta (Coffea canephora). Arabica accounts for 75% of world

production.. Robusta is produced in Indonesia, Africa, South America and Asia Pacific.

It accounts for 25% of the world production.

The two varieties can be differentiated as Robusta coffee will grow at relatively low

altitudes, can tolerate higher temperatures and heavier rainfall and requires high soil

humus than Arabica. It is much more resistant to disease. Arabica is pale green in colour

and oval in shape, robusta tends to be rounder and may be brownish rather than green.

Arabica species produces the type of coffees appreciated by the coffee drinker. It is

further subdivided according to its processing at the origin. Arabicas prepared by the

wet process are considered to be of better quality, the best will have an acidy cup

character, aromatic flavour and good full body.

Robusta coffees can assist in blend with full-bodied base but do not contribute to the

fine coffee flavour (Clark and Macreae, 1985).

2

1.2 Coffee Processing

Harvested coffee beans can be processed in two ways: the wet or the dry methods.

1.2.1 Dry Processing Method

In dry processing the fruits are spread out on large drying floors and dried in sun for 2

or 3 weeks. Sometimes beans are artificially dried and take a shorter time. Dried and

shrivelled berries are peeled to remove pulp and parchment.

Dry treatment is also used for 90% of Brazilian Arabica coffees. The method is

convenient but the final coffee brew is hard (Clark and Macrae, 1987). The berries are

spread out in a thin layer of 30-40 mm, frequent raking is needed to avoid moisture

penetration that can cause mould growth at a susceptible area of beans. Fungus

(Aspergillus, Penicillium or Rhizopus sp.) yeasts (Toroula, Saccharomyces etc) and

bacteria can easily develop. Climatic conditions, maturity of beans and their sizes also

affect the drying process. Approximately 12% moisture remains after drying.

Artificial drying is also practiced due to better control of drying factors. Drying

temperatures are limiting factor in artificial dryings as it is known that high

temperatures will develop stinker beans. Control over humidity, air flow, moisture of

beans and time of drying determine the drying efficiency. Several types of drying

equipment (static, rotary, horizontal and vertical) are available with combination of heat

sources.

1.2.2 Wet Processing Method

Wet processing yields a high quality product. The process is not labour intensive but

requires processing equipment for cleaning, classification, fermentation, washing and

drying. It requires a continuous supply of water and ripe fruits only. Ripe fruits are

harvested and transported quickly to the processing site to avoid field heat damage.

Berries are unloaded in receiving tanks that feed to the pulpers. Pulpers remove the

exocarp (outer skin) and mesocarp of the fruits. The operation is carried out under

running water. Various types of pulpers including Disc pulpers, Drum pulpers, Raoeng

pulper, Pulper-Repasser system and Roller pulper etc. are available. Berries are again

3

differentiated by draining, use of fermentation tanks or Aagaard densimetric grader

(Clark and Macrae, 1987).

Pulp is fermented to hydrolyse the mucilage and then washed. The hydrolysis of pectin

by pectinase is accelerated by microorganisms. Prolonged fermentation and

development of harmful moulds is avoided. Acid produced is controlled by controlling

pH. The length of fermentation varies according to the climatic conditions from 16 hrs

to 48 hrs. The mucilage is washed manually or mechanically. It is drained and moisture

of parchment is reduced from 60% to 53%. The parchment coffee is dried to 30%

moisture by a stage called “wet stage” and hygroscopic stage to below 30%.

1.2.3 Curing and Storage

Wet or dry processed coffee beans are further prepared for consumption by curing into

green bean condition. Coffee beans are further dried to 11% moisture content which

enables easy removal of husk and parchment.

The impurities are removed by air-float separators that separate the stones and

extraneous material on the basis of density. Hulling is done by using Huller to remove

the dried parchment layer surrounding bean. Dry processed coffee beans further need

polisher to remove hull. After hulling the coffee beans are size graded by different

methods including gravimetric, pneumatic sorting. Discoloured beans are also removed

manually or electronically. Coffee at this stage can be stored as dried cherries, dry

parchment coffee or cured green coffee. The moisture content is recommended not to be

more than 11%. Beans are stored in traditional bulk storage. Steps involved in dry and

wet processing are shown in Fig-1.1.

4

Fig-1.1: Flow sheet showing stages of wet and dry processing (Clark and Macrae, 1987)

RECEPTION

PULPING

FLOATATION CLEANING

FERMENTATION

WASHING

DRYING DRYING

HULLING

SIZE GRADING

SORTING

(density/colorimetric)

CLEANING

STORAGE

Bagging off

DRY PROCESSI�G WET PROCESSI�G

Harvested

Coffee Berries

Dried Cherry coffee Dry Parchment coffee

Parchments

(Hulls)

Oversize

Triage / Waste

HUSKS

Triage / Waste

CURING

Stones/Dirt

Pulp

Mucilage

Green coffee

(flat beans, peaberries)

Floaters

5

1.3 Coffee Roasting

Raw green coffee beans are sorted, packaged and transported to the consuming country.

Raw coffee beans are not aromatic. Coffee aroma is developed during roasting. Beans

of different kinds are blended together to achieve unified characteristics. Blending also

reduces the variations of raw coffee. There are various household and commercial

practices for roasting coffee beans. The principles are the same in both types of roasting.

Green coffee beans are heated inducing physical and chemical changes in the beans.

There is loss of dry matter as gaseous Carbon dioxide, water and volatile compounds of

pyrolysis. The roasted whole beans are characterised by the degree of roast. This is

measured from their external colour, flavour, and other chemical changes. This can be

light, medium or dark colour roasts. Roasting is either a batch or continuous process.

Roasters offering different mechanical systems are used for processing. Horizontal

rotating drum are common, vertical static drum, vertical rotating bowl, fluidised bed and

pressure roasting are other choices.

Roasting is a time and temperature controlled process. Different types of roasters can

now automatically control temperature, humidity, recirculate roaster gases and control

residence time of beans. Development of optimum flavour in various blends depends on

roasting steps.

Roasting stages are described as in the first stage of roasting beans are slowly dried to

become yellow in colour and smells like toast or popcorn (Davids, 1996). Second step

called first crack occurs at 205oC. The bean size become double, light brown in colour

and lose 5% of weight. In the third step with increase in temperature from 205oC to

220oC, colour changes to medium brown and 13% weight loss occurs. Carbon dioxide

is released during pyrolysis. Second pyrolysis occurs between 225-230oC and roast

colour is medium-dark brown.

6

1.3.1 Chemical and Physical changes during Roasting

Chemical changes in green coffee beans during roasting develop the characteristic

aroma and flavour of roasted coffee. The compositional factors of interest are the dry

matter loss, CO2 evolution and soluble solids content. Roasted coffee beans contain the

following compounds (Catsberg and Dommelen, 1990).

Table-1.1: Compounds in roasted beans

No Compound Percentage

1 Water 2.7%

2 Protein 13.3%

3 Fat (Coffee oil) 12.8%

4 Carbohydrates 67%

5 Minerals (mainly potassium) 4.1%

6 Caffeine 1-2.5%

7 Chlorogenic acid 4.1%

(Source: Catsberg and Dommelen, 1990)

The dry matter loss is according to the degree of roast. Moisture content of green beans

is variable typically it is around 12% and can fall to 8%. Percentages of dry mass loss

are given Table 1.2.

Table-1.2: Approximate % dry mass loss for different degrees for roast

No Degree of Roast Percentage dry mass loss

1 Light 1-5

2 Medium 5-8

3 Dark 8-12

4 Very Dark > 12

(Source: Clark and Macrae, 1987)

Carbon dioxide evolved during pyrolysis is entrapped. The amount depends on blend

type and degree of roast. After roasting whole beans contain a quantity of 2-5 ml CO2

per gram of roast coffee.

7

1.4 Coffee Flavour: Aroma and Volatiles

Coffee flavour and aroma are important quality attributes; coffee volatiles with different

potency and concentrations contribute to the aroma quality. Volatiles develop from non-

volatile components of green coffee beans and the breakdown of components during

pyrolysis, interactions of sugars, amino acids, organic acids and phenolic compounds

develop coffee flavour. Final aroma composition depends on different factors from bean

variety, growth conditions, storage, harvesting, roasting, packaging and preparation of

beverage thus including all the processing steps.

Chemical processes and mechanisms of aroma development from green coffee to

roasted coffee have been well researched. Aroma precursors are degraded products from

the Maillard reaction, formation of pyrazines and oxazoles, degradation of trigonelline,

phenolic acids, lipids, sugars, sulphur amino acids, hydroxy amino acids and praline

(Clark and Macrae, 1985). Table 1.3 shows the main components of green and roasted

coffee.

Table-1.3: Composition of green and roasted coffee

No Component Green coffee (%) Roasted coffee (%)

1 Cellulose 36 37

2 Legnin 5.6 5.8

3 Fat 11.4 11.9

4 Ash 3.8 4.0

5 Sucrose 7.3 0.3

6 Chlorogenic acid 7.6 3.5

7 Protein 11.6 3.1

(Source: Clark and Macrae, 1985)

8

Major nine pathways have been identified with respect to volatile compositional

changes (Clark and Macrae, 1985), (Illy and Viani, 1995).

� Maillard or non-enzymatic browning reaction between nitrogen containing

substances, amino acids, proteins, as well as trigonelline, serotonine, and

carbohydrates, hydroxy-acids and phenols.

� Strecker degradation.

� Degradation of individual amino acids, particularly, sulfur amino acids,

hydroxy amino acids.

� Degradation of trigonelline.

� Degradation of sugar.

� Degradation of phenolic acids, particularly quinic acid.

� Minor lipid degradation.

� Degradation of proline and hydroxyproline.

� Interaction between intermediate decomposition products

Volatile compounds identified in coffee are well over 800 and the number increases

each year with further research. Major classes of volatiles include sulphur compounds,

pyrazines, pyridines, pyrroles, oxazoles, furans, aldehydes, ketones, phenols and

miscellaneous other groups. Coffee aroma is particularly affected by approximately 30

volatiles termed as “potent odorants” (Grosh, 1998). Table 1.4 shows a few of the

important volatiles in coffee.

9

Table-1.4: Important compounds in coffee aroma

No Volatile Conc.

(mg/L)

Aroma

Description

1 (E)-ß-Damascenone 1.95x10-1 honey-like, fruity

2 2-Furfurylthiol 1.08 roasty (coffee)

3 3-Mercapto- 3-methylbutylformate 1.30x10-1 catty, roasty

4 3-Methyl-2-buten-1-thiol 8.20x10-3 amine-like

5 2-Isobutyl-3-methoxypyrazine 8.30x10-2 earthy

6 5-Ethyl-4-hydroxy- 2-methyl-3(2H)-furanone 1.73x101

7 Guaiacol 4.20 phenolic, spicy

8 2,3-Butanedione (diacetyl) 5.08x101 buttery

9 4-Vinylguaiacol 6.48x101 spicy

10 2,3-Pentanedione 3.96x101 buttery

11 Methional 2.40x10-1 potato-like, sweet

12 2-Isopropyl-3-methoxypyrazine 3.30x10-3 earthy, roasty

13 Vanillin 4.80 vanilla

14 4-Hydroxy-2,5-dimethyl- 3(2H)-furanone 1.09x102 caramel-like

15 2-Ethyl-3,5-dimethylpyrazine 3.30x10-1 earthy, roasty

16 2,3-Diethyl-5-methylpyrazine 9.50x10-2 earthy, roasty

17 3-Hydroxy-4,5-dimethyl- 2(5H)-furanone 1.47 seasoning-like

18 4-Ethylguaiacol 1.63 spicy

19 5-Ethyl-3-hydroxy-4-methyl- 2(5H)-furanone 1.60x10-1 seasoning-like

(Source: Grosch, 1995)

10

Furans with caramel like odours predominate in coffee aromatics and are the principal

degradation products of monosaccharides and higher sugars. Pyrazines are the second

most abundant compounds contributing to coffee aroma with roasted, walnut, cereal,

cracker or toast like flavour which along with thiazoles contribute to coffee aroma.

Pyrroles are responsible for sweet, caramel, mushroom like aroma (Clark and Macrae,

1985).

In a study by Mayer et. al. (2000) potent odorants quantified in medium roasted Arabica

coffee brew showed a large proportion (>75%) of acetaldehydes, 2,3 butanedione, 2,3-

pentanedione, vanillin and furanones. Aroma was caused by 2-furfurylthiol, methional

and 3-mercapto-3-methylbutyl. High methional and low aroma activity of 4-

vinylguaiacol were in contrast to previous findings. Also, potent odourants for earthy

notes in coffee by aroma extract concentration analysis (AECA) lead to the

identification of two odourants classified as alkypyrazines. The results suggested

presence of ethenyl group pyrazines in roasted coffee.

A high impact aroma compound in coffee causing its major “burned-roasted” is furfuryl

mercaptan that was established in reconstitution studies (Row, 2002). Derivatives of

furfuryl mercaptan are also important; disulphide (contributes mild notes) and

monosulphide (mild earthy notes, mushroom notes contributing to “earthy” aroma

character). Aroma chemical responsible for the smoky flavour was 2-methoxy-4-vinyl

phenol. Another group of coffee volatiles including prenyl mercaptan were related to

“prenal” and were termed as Prenoids. These were regarded as contributors to “fresh

roast” aroma rather than taste (Taylor and Mottram, 1996).

1.4.1 Coffee roasting and its effect on volatile composition

It is argued that only bioactive volatiles called key volatiles are responsible for coffee

aroma. Study of coffee volatiles is a constant subject of investigations to differentiate

variations in coffee, types of roast, analytical techniques or to identify key odorants.

Volatile composition is affected by roasting temperature, time, methods, roaster types

and degree of roast. Prolonged roasting time increases volatiles’ concentration, few

volatiles decrease while some fluctuate in concentration during prolonged roasting due

to break down of two or more precursors.

11

Studies found that pyrazines tended to decrease in roasting, pyrroles, phenols and

pyridines increased in concentration; furfuryl alcohol was correlated with the bitter

flavour of dark coffee. There is an increase in phenols and bitter, burnt flavour,

aldehydes increased through out, while pyrazines, formation increased above 100oC

(Clark and Macrae, 1985).

Roasting of coffee from light to dark increased roasty/sulphury, earthy and smoky notes

that may be caused by 3-methyl-2-butenthiol (63% increase) and 2-furfurylthiol (108%

increase). In coffee brew the impact changed due to shift in concentrations, that yielded

higher than 70% of thiol, pyrazine, furanones, guaiacol, vanillin and diones, lower than

25% for some pyrazines and β-damascenone (Grosch, 1998).

Maeztu, et. al. (2001) identified 77 compounds in Espresso coffee prepared from 3

different roast types, 13 key odorants were quantified and coffees were discriminated to

classify samples by the aroma profiles. Among pyrazines associated with roasted

aromas, 3 were quantified as key odorants. 2-ethyl-3,5-dimethyl pyrazine was correlated

with woody/papery, roasty burnt and earthy/musty flavours.

A non-empirical approach was considered to investigate the degradation of compounds

to form aroma. The microscopic mechanism of radical formation during degradation of

coffee aroma components was studied. Radicals were classified according to their

thermodynamic stability. The “thermodynamic” classification identified six radicals as

most probable: two on thiols, two on 2-methoxy phenols and two on 4-hydroxy 2,5

dimethyl-3(2H)-furanone. The results and procedures suggested a new programme for

relevance of unforeseen compounds in deterioration of fresh coffee aroma (Munro et.

al,. 2003).

1.4.2 Effect of milk addition and coffee additives on coffee volatiles

Studies on coffee aroma compounds have concentrated on roasted coffee powder or

black coffee brew. Addition of milk or coffee creamer to coffee is a common practice,

but only three investigations have been found on these areas.

Kim et. al. (1996) investigated the effect of creamer/milk addition on the aroma

retention in coffee beverages and found that the headspace volatile concentration in

12

coffee decreased with milk or creamer addition. The purpose of these additions was to

achieve desirable colour, body and to reduce bitter and sour tastes. Commercial whole

milk, non fat milk solid and milk fat were used as additives. Comparison with black

coffee revealed decreased headspace aroma in the order of Black coffee, coffee with

reduced fat non dairy creamer, with milk, non fat milk solid and coffee with milk fat.

Fat content in creamer and both protein as well as fat in milk products increased aroma

retention.

Bucking and Steinhart (2002) investigated the effect of eight milk and dairy creamer

products and one non-dairy creamer coffee whitener on aroma of coffee beverages.

Headspace Gas Chromatography-Mass Spectrometer (GC-MS) analysis was used with

specially developed external static headspace device. Milk and dairy creamer products

were chosen having different combination of lipid, carbohydrates and protein. All milk

and vegetable additives reduced the perception of major aroma components. Products

with high fat content particularly influenced the concentration of volatiles. Sucrose

added with creamer also showed a significant effect on aroma release.

Further studies (Bucking et. al, 2004) concentrated on the structural characteristics of

aroma retention. Volatile compounds were shown to be affected by saliva when

researching the change in odour profile by artificial or human saliva. The release of

volatiles in the oral cavity of volunteers was measured by oral vapour gas

chromatography. Investigations indicated that coffee beverages with milk or vegetable

additives reduced typical coffee odour profile.

1.4.3 Screening/Differentiation of Coffee by volatile composition

Coffee volatiles are conventionally used to differentiate various coffee types originating

from a variety of sources and processes. Research has been concentrated on isolation,

detection and identification of volatile compounds and their correlation with sensory

perceptions. It is argued that to differentiate different types of coffee exhaustive,

laborious and expensive procedures should be converted in easy to handle and

inexpensive procedures. The rationale exists supported by established principles

combining variations in volatiles, techniques of aroma analysis and sensory perception.

13

As a technique gas sensor array (Freitas et. al., 2001) differentiated Arabica and

Robusta coffee into two distinct groups and also on the basis of geographic origin. This

technique was compared with Headspace Solid Phase Microextraction Gas

Chromatography-Mass Spectrometer (HS SPME GC-MS) and results treated by

Principal Component analysis (PCA). The SPME GC-MS did not show any

differentiation on geographic basis. The sensor array technique was faster then GC-MS

analysis taking only 7 minutes as compared to 1 h for GC-MS.

According to Rocha et. al. (2003) a clear product differentiation is possible by HS

SPME GC-MS without the need to identify the volatile components. In an investigation

of volatile profiles of espresso and plunger coffees, it was possible to screen the coffee

using the variability provided solely by the GC peak areas and retention times in

combination with PCA. This provided a methodology with no need for identification of

volatile components by mass spectrometry (MS).

Bicchi, et. al. (1997) used HS SPME GC-MS to characterise roasted coffee and coffee

beverages; different coffee samples originating from different blends and treatment

were studied to illustrate the discrimination capability of this technique.

Sanz, et. al. (2001) studied optimisation of headspace temperature and time sampling.

Three equilibrium temperatures of 60, 80 and 90oC were studied. One hundred and

twenty two volatiles were identified in ground roasted Arabica coffee by HS-GC

analysis. It was concluded that although the maximum number of volatiles will elute at

high temperatures, the optimum time and temperature should be considered according

to the volatile compounds of interest. (Sanz et. al., 2002) compared potent odorants in a

filtered coffee brew (FCB) and in an instant coffee beverage by aroma extract dilution

method (AEDA). In FCB, 40 odour active compounds were identified of which (E)-β-

damascenone, methional, 3-mercapto-3-methylbutyl formate, 5 ethyl-3hydroxy-4-

methyl-2(5H)-furanone, 4-hydroxy-2,5-dimehtyl-3(2H)-furanone and several phenols

were detected as the most intense odorants. The difference was determined by some

sulphur-containing odorants, 2-methoxyphenol, 4-ethyl-2-methoxyphenol, 4-vinyl-2-

methoxyphenol and vanillin.

14

Maeztu, et. al. (2001) characterized and classified Espresso coffee (EC) from different

botanical varieties and types of roast by foam, taste and mouth feel using multivariate

methods of PCA. The coffee samples were successfully discriminated. Kumazaw and

Masuda (2003) investigated headspace volatiles to differentiate between coffee drinks

during heat processing. Gas chromatography-olfactometry of headspace (GCO-H) was

used as the technique and 12 odour active peaks were detected; 8 potent odorants were

identified. Methanthiol (putrid), acetic acid (sour), 3-methyl butanoic acid (sour), 2

furfuryl methyl disulfide (meaty) and 4-hydroxy 2,5-dimethyl-3(2H)-furanone

(caramel-like) increased after heating of the coffee sample, while 2 furfuryl thiol

(roasty), methional (potato-like) and 3-mercapto-3-methyl butyl formate (roasty)

decreased as compared with the coffee sample before heat treatment.

15

1.5 Techniques for Analysing Coffee Aroma

Aroma components are related to the volatiles of food. Their preliminary isolation is

necessary before analysis. Classic methods of isolating headspace volatiles have lead to

Head Space Solid Phase Microextraction (HS SPME) coupled with GC and GC-MS.

New developments in electronic devices are promising but need careful comparison

with human perception. The success of electronic devices depends on their correlation

to human senses.

Aroma analysis can be divided into different techniques applicable and suitable for

various stages of analysis (Sides et. al,. 2000).

1. Sample preparation and Isolation techniques.

2. Measurement techniques

3. Identification, quantification and relation of instrumental results with human

perception.

1.5.1 Sample preparation and isolation techniques

Sample preparation according to analyte type is important in aroma analysis.

Limitations with techniques are the destruction or decrease in concentration of aroma

volatiles and it needs to provide isolation close to human consumption. Different

isolation techniques include solvent extraction, HS analysis, Super critical fluid

extraction, Solid Phase analysis, SPME in combination with measuring techniques of

GC-MS and sophisticated multivariate statistical analysis.

Solid Phase Microextraction (SPME)

SPME was first developed by Pawliszyn and co-worker in 1990. It has been routinely

used in combination with GC-MS for flavour and aroma analysis (Kataoka, 2000).

SPME Components and sampling procedure

SPME uses a short, thin solid rod of fused silica (typically 1cm long and 0.11mm outer

diameter), coated with an absorbent polymer. The fibre is the same as GC columns,

inert and stable at high temperatures (Wercinski, 1999). Coated fused silica (fibre) is

attached to a metal rod protected by a metal sheath covering the fibre. The whole

apparatus is enclosed in a fibre holder, shaping in to a modified syringe (Fig-1.2 ).

16

(Source: Kataoka, et. al. 2000)

Fig-1.2: Commercial SPME device

Solid phase microextraction process

The SPME extraction consists of two stages.

1. The analytes partition between sample (headspace) and the fibre coating.

2. The concentrated analyte is desorbed from fiber to an analytical instrument.

The process of SPME with fiber is shown in figure 1.3. The sample is placed in a vial

and sealed with a septum. The needle is injected through the septum into the sample

matrix. The Plunger is pushed down, thus exposing the fiber to the sample matrix. It can

be used to extract analytes from head space (HS-SPME) as well as direct immersion

(DI-SPME). In headspace the fibre is only exposed to the vapour phase above a gaseous,

liquid or solid sample. In DI-SPME the fiber is directly immersed in the liquid sample.

17

(Source: Kataoka, et. al. 2000)

Fig-1.3: Extraction process by headspace and immersion fibre SPME, desorption

systems for GC and HPLC analysis.

After a suitable extraction time, the plunger is retrieved and ready for the second stage

of desorption directly into the injection port of the GC. The technique is used with GC

or GC-MS. The desorption is performed by heating the fibre in the injection port, and

thus the analytes are then ransferred to GC column for analysis.

SPME theory and practical approach is discussed in detail by Wercinski (1999),

Kataoka et. al., 2000, Theodoridis et. al., 2000 and Wilkes et. al., 2000.

Applications

Studies on coffee headspace volatiles report the use of SPME to characterise roasted

coffee and coffee beverage with GC and PCA (Bicchi et. al,. 1997). Freitas et. al. (2001)

compared HS-SPME GC-MS with electronic aroma sensing device to assess the coffee

18

differentiation. HS-SPME was used for screening and distinction of coffee brews with

GC-PCA (Rocha et. al., 2003). Akiyama et. al. (2003) used a dynamic SPME method

for sample isolation of fresh headspace volatile compounds released during grinding of

roasted coffee beans coupled with GC-MS and GC-Olfactometry and compared it with

a static HS-SPME technique.

Advantages

SPME is a solvent free technique. It offers rapid sampling, low cost, sensitivity and is

easy to operate. There is no interference from sample matrix components. SPME is

established for close correlation to human perception of food aroma.

Limitations

SPME is very sensitive to experimental conditions. Headspace or liquid volume, pH,

time and temperature are important for reproducibility. The technique may still be time

consuming as compared to electronic devices (nose) in differentiation of coffee brews

on the basis of headspace volatiles (Freitas et. al., 2001).

19

1.5.2 Measurement Techniques

Chromatographic measurements are the major part of measurement techniques.

Chromatography coupled with mass spectrometry (MS) is ideal for identification and

elucidation of compounds.

1.5.3 Instrumental and human measurements

Identification of all aroma components is not feasible or desirable as only potent high

impact aroma volatiles are responsible for aroma. Not all peaks in a chromatogram are

of aroma active compounds. In GC, retention indices (e.g Kovats) are useful for aroma

studies in comparison with mass spectral data. Headspace techniques have provided a

system of measuring aroma related compounds and their simple correlation with

sensory perceptions. Complex volatile components require further sophisticated

multivariate techniques to determine which peak on a chromatogram is highly

correlated to sensory results.

1.6 Scope of the Study

Studies on head space volatiles of coffee brews are mostly focused on ground coffee or

coffee brews from different processes and origins. Available literature is scarce on the

effect of adding milk, milk products and non-dairy coffee additives (creamers) on

headspace volatiles of coffee brews.

The main objective of the study was:

� To investigate the effect of different roasting gradients on head space volatiles

of coffee brews.

� To investigate the effect of adding milk/milk products and coffee creamer on

changes in head space volatiles of coffee brews.

20

Chapter-2

MATERIALS A�D METHODS

2.1 MATERIALS

2.1.1 Coffees

The roast gradients determined by the coffee manufacturer provided 6 gradients with two

duplicate roasts generating a total of 12 products. Samples were analysed for volatiles by

Headspace solid phase microextraction gas-chromatography mass spectrometry (HS

SPME GC/MS).

A blend of Colombia (Arabica, wet processed), Uganda (Robusta standard, dry

processed), India (Arabica, wet processed), Sumatra, Java Lintong (Arabica, semi-wet

processed), and Ethiopia (Arabica, wet processed) coffees with 6 different roasting

gradients were supplied by Matthew Algie & Co Ltd, Glasgow, UK. Coffee samples

were supplied sealed in laminated bags with uni-directional valve. Roast gradients were

determined by the coffee manufacturer (Table 2.1).

Table 2.1: Coffee samples according to roasting gradients

S.�o Sample Roast Gradients

1 Control 1/Control 2 Control 3/Control 4

Phase Bean temp Burner temp Air flow

IN 210 530 42%

1 170 530 48%

2 180 530 50%

3 195 530 58%

4 209 530 100%

2 A1/A2

Phase Bean temp Burner temp Air flow

IN 210 530 42%

1 180 530 48%

2 190 530 50%

3 200 530 58%

4 209 530 100%

3 B1/B2

Phase Bean temp Burner temp Air flow

IN 210 530 42%

1 170 530 50%

2 187 530 58%

3 200 530 65%

4 209 530 100%

21

4 D1/D2

Phase Bean temp Burner temp Air flow

IN 210 530 42%

1 170 530 45%

2 180 530 50%

3 185 530 65%

4 209 530 100%

5 G1/G2

Phase Bean temp Burner temp Air flow

IN 210 450 42%

1 170 450 48%

2 180 450 50%

3 195 450 58%

4 217 450 100%

6 H1/H2

Phase Bean temp Burner temp Air flow

IN 210 390 42%

1 170 390 48%

2 180 390 50%

3 195 390 58%

4 217 390 100%

2.1.2 Milk/milk products and additives

Milk (Ultra High Temperature -UHT Standardised Whole Milk) from Dairy Gate, (New

Century House-Manchester), Coffee Creamer and Sweetened Condensed Milk as

typical coffee additives from Nestle (UK) were purchased from a local super market.

The ingredients of the milk/milk products and coffee creamer are shown in Table 2.2.

Table-2.2: Fat, Carbohydrate and Protein content of coffee additives in g per 100g

S.�o

.

Milk/Milk products Fat Carbohydrates Protein

Carbohydrates Sugar

1. UHT Standardised Whole Milk 3.6g 4.7g -- 3.2g

2. Sweetened Condensed Milk

(CM)

9.0g 55g 55g 8.0g

3. Coffee Creamer (CC) 29.5g 59.7g 9.0g 2.1g

22

Retention Index Standard - a mixture of aliphatic hydrocarbons dissolved in hexane

from Sigma Aldrich Co. Ltd, Poole, UK was used to calculate retention indices.

StableFlex Divinylbenzene/Carboxen/PDMS 50/30µm (Supelco, Poole) fibre was used;

water used for coffee brews was purified by MilliU10 system (Millipore, Watfor UK),

Glass vials 20ml fitted with PTFE lined silicone septa in plastic screw caps (Waters Ltd,

Elstree UK) and Bunn-o- Matic Corporation coffee grinder, Spring field-Illinois USA

was provided by Matthew Algie, Glasgow.

2.2 METHODS

2.2.1 Coffee Brew / Extract

Coffee brews were prepared from freshly ground coffee; sealed sample bags were

opened just prior to grinding. Water (400ml) at 80°C was used to brew fresh ground

coffee (30g) in a cafetiere (Premier House wares-Glasgow) and after 3min the plunger

was pushed down. Coffee brew (30ml) was filtered through Watman no. 1 filter paper.

The filtrate (5 ml) was placed in a 20ml-vial and sealed. For samples with milk additives

4ml of additive was added to 21ml of filtered coffee brew (16%-V/V) and 5ml was

directly taken in a 20ml-vial for analysis.

Coffee Head Space Method Using Solid Phase Microextraction Method (SPME)

Headspace sampling

Sealed vials were put into a water bath at 50oC with the fibre exposed to the headspace

for 30min during equilibration. Immediately after sampling, the fibre was inserted into

the gas chromatograph injector for 10min at 230oC. Only one injection was made per

vial and samples analyzed in duplicate on a Carlo Erba Mega series gas chromatograph

(CE Instrument Ltd, Crawley) using a flame ionisation detector at 250oC. A CP-WAX

52 CB fused silica capillary column 50m x 0.25mm id., df = 0.2µm was used with

helium carrier gas at 20 psi. The column was held at 40oC for 6min then increased to

240oC at 3

oCmin

-1. Peak areas were calculated using Chromperfect integration software

(Justice Innovations, Mountain View, California, USA).

23

The average retention times of volatiles were used to calculate retention indices using

retention times. GC peaks were manually integrated and peak areas were processed in

Excel.

Table-2.3 : Sample coding and description

S.�o. Sample Additive

1. (Control) Co-1 Nil (Black coffee brew )

2. (Control) Co-3 Nil

3. A 1 Nil

4. B 1 Nil

5. D 1 Nil

6. G 1 Nil

7. H 1 Nil

8. Co2-1 UHT Milk

9. A2-1 UHT Milk

10. B2-1 UHT Milk

11. D2-1 UHT Milk

12. Co2-2 Condensed Milk (CM)

13. A2-2 Condensed Milk (CM)

14. B2-2 Condensed Milk (CM)

15. D2-2 Condensed Milk (CM)

16. Co2-3 Coffee Creamer (CC)

17. A2-3 Coffee Creamer (CC)

18. B2-3 Coffee Creamer (CC)

19. D2-3 Coffee Creamer (CC)

24

Statistical Analysis

The data was taken from variations of GC peak areas and analysed with Analysis of

Variance (ANOVA) for differences between coffee types and milk additives as factors,

yielding peaks as response at 95% level of confidence (alpha 0.05) using statistical

software Minitab Version 12/13.

Principal component analysis (PCA) was applied to differentiate GC peaks and samples,

using software Unscrambler V7.0.

25

Total Peak Area of Coffee Brews

Black

UHT Milk

Condensed Milk

Coffee Creamer

Chapter-3

RESULTS A�D DISCUSSIO�

3.1 SPME GC-Peak area of black coffee brews and with additives

SPME GC analysis differentiated the coffees through 269 reproducible peaks. The

chromatographic areas obtained for black coffee brews were higher than brews with

additives (Fig 3.1).

Fig-3.1: Total peak area of different coffee brews

Total peak areas of black coffee brews shows that the samples G and H have maximum

peak area. These samples were roasted for longer time. It suggests that the total peak area

indicates high increase in volatiles. (Fig 3.2).

Black coffee - Total peak areas

0

10000000

20000000

30000000

40000000

50000000

60000000

Co A B D G H

Black coffee samples

Total Peak area

Black

Fig-3.2: Total peak area of black coffee brew samples

26

Total Peak Area of different coffee brews

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

Black UHT Milk Condensed

Milk

Coffee

Creamer

Additives

Area

Co

A

B

D

Total peak areas of black coffee brews were clearly differentiated from the coffee brews

with added coffee creamer, while coffee brews with addition of UHT milk and condensed

milk were difficult to differentiate (Fig 3.3). The total headspace aroma of all samples

decreased as found by previous studies (Kim et. al., 1995), Bucking and Steinhart (2002)

found the same results, addition of whipping cream or coffee creamer with a high fat

content particularly influenced the concentration of volatiles. The same pattern can be

seen with the coffee creamer used with high fat content 29.5% and carbohydrate

(vegetable sources 59.7%), sweetened condensed milk and UHT milk were not highly

different.

Fig-3.3: Total peak area of different types of coffee brews

Total peak areas could only differentiate between 10 peaks; a maximum of 80 peaks were

differentiated out of 269 peaks (Fig-3.4).

27

Total Peak Area by peaks

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

1 18 35 52 69 86 103 120 137 154 171 188 205 222 239

Peak Number

Area

Fig-3.4: Peak area of different peaks

3.2 Analysis of Variance (A�OVA) results

ANOVA showed the majority of peaks (>200) as highly significant (p<0.05) in

differentiating between all the coffee products. Few peaks (<20) were not significant in

differentiating the products.

ANOVA for peaks on coffee type and milk also showed the same highly significant

difference for majority of peaks.

3.3 Principal component analysis (PCA) of Coffee types and Peaks

PCA was used to study the source of variation between the different coffee brews and to

see the relation between different coffee types and addition of additives.

Standardized PCA of black coffee samples have clearly differentiated between black

samples. It shows that all the samples were different from each other. Sample H and G

are in a distinct position from other samples these were the samples with high roaster

temperatures. The position of each black sample on consensus space prove that the

different roasting gradients have significant effect on head space volatiles of coffee

28

-10

-8

-6

-4

-2

0

2

4

6

8

-15 -10 -5 0 5 10

RESULT1, X-expl: 49%,11%

Co1

Co1

A1

A1

B1

B1

D1D1

G1

G1

H1

H1

PC1

PC2 Scores

-0.20

-0.15

-0.10

-0.05

0

0.05

0.10

0.15

0.20

-0.10 -0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.10

RESULT1, X-expl: 49%,11%

17

18

19

20

21

22

23

24

25

26

27

28

29

3032

36

38

39

40

444648

50

51

5253

54

55

56

5758

59

60

61

62

63

64

66

67

68

69

70

73

74

75

76

77

79

82

83

84

85

89

92

93

94

9596

9899

100

101

102

103

105

107108109

110

111

112

113114

115

116

117

118

119

120

121

122123

124

125

126

127

128

129

130

131

132

133

134

135136

137

138

139

140

141

142143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170171

173

174

175

176

177

178

179

180

181182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199200

201

202

203

204

205

206

207

208

209

210

211

212

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237238

239

240

241

242

243

244

245

246

248

249

250251

252

253254

255

256257

258 259

260

261

262

263

264

265

266

267

268269

PC1

PC2 X-loadings

brews. There is a significant difference among sample A and D which are distributed at

the opposite ends. Control and sample B1 are placed showing similarity between them.

Fig-3.5: PCA of black coffee samples

Fig-3.6: Scree plot of Peak for Black coffee brew

The loading plot of peaks shows some peaks associated with sample G and H, these

peaks are important in differentiating the coffee samples which were roasted at high

29

temperatures. This suggests that roasting to high temperature must have caused elution

of new volatiles different from the other samples. Peaks associated with these samples

can be further investigated and identified through mass spectrometry (MS) and the

volatiles responsible. These results when correlated with the sensory results will

establish there role in aroma. Maeztu et. al., (2001) characterized Espresso coffee aroma

by identifying few key odorants through head space GC-MS and sensory flavour

profiles. Higher degree of roast cause roasty/sulphury, earthy and smokey notes.

Analysis of key odorants indicate that this may be caused by 3-methyl-2-butenthiol and

2-furfuryl-thiol. Phenols 4-ethylguaiacol and guaiacol increase strongly during roasting

(Grosch, 1988).

PCA of standardised variables showed that all the 6 principal components to be highly

significant and accounts for more than 70% of variation. The coffee brews are closely

grouped in to black coffee samples and coffee with added coffee creamer on (PC1

negative and PC2 negative).

Coffee with addition of UHT milk and condensed milk are grouped together for

similarity but separated from others.

Fig-3.7: Standardized PCA scatter plot of areas of different coffee brews (PC1vs

PC2)

-15

-10

-5

0

5

10

-15 -10 -5 0 5 10 15 20

standardised pca, X-expl: 33%,20%

Co1Co1 A1A1

B1

B1

D1D1

Co2.1Co2.1

A2.1A2.1

B2.1B2.1

D2.1

D2.1Co2.2

Co2.2A2.2

A2.2B2.2

B2.2

D2.2D2.2

Co2.3Co2.3A2.3A2.3B2.3B2.3D2.3D2.3

Co3

Co3

G1

G1H1H1

PC1

PC2 Scores

30

Standardized screen plot for peaks is difficult to interpret as there is no clear

discrimination possible Fig 3.8.

Fig-3.8: Standardized PCA scores scatter plot of chromatographic areas of Peaks

Scree plot for total consensus and residual variance per dimension shows that the

variability can be explained by adding more dimensions, as the first two dimensions

cannot explain the variability.

-0.15

-0.10

-0.05

0

0.05

0.10

0.15

-0.10 -0.05 0 0.05 0.10 0.15

standardised pca, X-expl: 33%,20%

17

18

19

20

21

22

23

24

25

26

27

28

29

30

32

33

3435

36

37

38

39

4041

42

4344

45

46

4748

49

50

51

52

5354

5556

5758

59

60

61

62

63

64

65

66

67

6869

70

72

7374

75

76

77

78

79

80

82

83

84

8586

87

88

89

90

91

9293

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138139

140

141

142

143

144

145

146

147

148

149150

151

152

153

154

155

156

157158

159

160

161

162

163

164

165

166

167

168

169170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186187

188

189

190

191

192193

194

195

196

197

198199

200201

202

203

204

205

206

207

208

209

210

211

212

213

214215

216217218

219

220

221 222

223

224

225

226

227228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

248

249

250251252

253254

255

256

257258

259

260

261

262

263

264

265266

267

268

269

PC1

PC2 X-loadings

31

Fig-3.9: Screen plot of consensus and residual variance per dimension of

peak area analysis

Fig-3.10: Residual variance of different coffee samples

Standardized residual variance is high for coffee samples and that explains the lack of

consensus.

0

10

20

30

40

50

60

70

80

PC_00 PC_01 PC_02 PC_03 PC_04 PC_05 PC_06

standardised pca, Variable: c.Total v.Total

PCs

X-variance Explained Variance

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24

standardised pca, PC: 4,4

Co1

Co1

A1

A1

B1

B1

D1

D1

Co2.1Co2.1

A2.1

A2.1B2.1

B2.1D2.1

D2.1Co2.2 Co2.2 A2.2

A2.2

B2.2

B2.2

D2.2

D2.2

Co2.3 Co2.3

A2.3A2.3B2.3 B2.3

D2.3

D2.3

Co3

Co3

G1

G1

H1

H1

Leverage

Residual X-variance Influence

32

Chapter-4

SUMMARY A�D CO�CLUSIO�

Coffee is a widely consumed drink for its flavour and aroma. Coffee flavour is

influenced by the volatiles produced during coffee roasting. Studies on coffee aroma

have identified more than 800 volatile components responsible for coffee aroma. Coffee

aroma is affected by various factors including variety of coffee beans, processing and

preparation of drink.

Head Space Solid Phase Microextraction (HS SPME) is a developed analytical

technique offering clear advantages over other sample preparation techniques. Its use

for head space analysis and measuring of volatile flavour components coupled with Gas

chromatography mass spectrometry (GC-MS) has been used for identification of coffee

volatiles. The technique is extremely useful in screening differences among coffee

originating from different sources or processed through different processes.

Investigations on coffee volatiles has been limited to ground coffee or black coffee

brews. Addition of milk, milk products and non milk coffee additives is practiced by

coffee consumers. The objective of adding coffee additives is to obtain preferable

flavour and aroma. There are few studies relating to the flavour change in coffee brews

with additives.

The main objective of the study was to investigate the effect of roasting and coffee

additives on the coffee volatiles. Coffee roasted with high degree of roast were found

significantly different and separated from each other. The coffee processed with higher

degree of roast had higher peak areas. This is due to higher concentration of volatiles

produced. The coffee brews with added coffee additives showed decreased but typical

coffee aroma. Coffee samples originating from different roasting gradients and with

addition of milk, condensed milk and coffee creamer were clearly differentiated by the

HS SPME GC and using multivariate statistical analysis technique of Principal

Component Analysis (PCA).This established the previous findings that coffee can be

differentiated with out the necessary identification of coffee volatiles.

33

4.1 Recommendations

It is suggested that further investigations can be carried out on the peaks responsible for

the differentiation of coffee samples with different level of roasts.

Key odorants are already identified, this will provide with an easy technique to identify

the volatiles responsible for screening coffee samples.

Limited number of identified volatiles should be correlated with sensory results to

observe their effect on human perception.

34

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37

List of Tables

Table-1.1: Compounds in roasted beans

Table-1.2: Approximate % dry mass loss for different degrees for roast

Table-1.3: Composition of green and roasted coffee

Table-1.4: Important compounds in coffee aroma

Table 2.1: Coffee samples according to roasting gradients

Table-2.2: Fat, Carbohydrate and Protein content of coffee additives in g per 100g

Table-2.3: Sample coding and description

38

List of Figures

Fig-1.1: Flow sheet showing stages of wet and dry processing

Fig-1.2: Commercial SPME device

Fig-1.3: Extraction process by headspace and immersion fibre SPME,

desorption systems for GC and HPLC analysis.

Fig-3.1: Total peak area of different coffee brews

Fig-3.2: Total peak area of black coffee brew samples

Fig-3.3: Total peak area of different types of coffee brews

Fig-3.4: Peak area of different peaks

Fig-3.5: PCA of black coffee samples

Fig-3.6: Scree plot of Peak for Black coffee brew

Fig-3.7: Standardized PCA scatter plot of areas of different coffee brews (PC1vs

PC2)

Fig-3.8: Standardized PCA scores scatter plot of chromatographic areas of Peaks

Fig-3.9: Screen plot of consensus and residual variance per dimension of peak

area analysis

Fig-3.10: Residual variance of different coffee samples